EA043034B1 - METHOD FOR PROCESSING PROTEIN-RICH SEEDS TO INCREASE THEIR NUTRITIONAL VALUE - Google Patents

Description

The present invention relates to a method for treating protein-rich seeds to increase their nutritional value, in particular for animals.

Prior Art

The feeding of monogastric farm animals such as broilers and egg-laying poultry, pigs and ruminants is highly dependent on the inclusion of soy flour in diets. This raw material comes mainly from South America, which is not without challenges in terms of autonomy in the production of feed proteins and the stability of their supply, in particular due to the problems of increasing competitiveness due to significant social and environmental wishes.

Due to its high protein content, soybean meal has been widely established as a source of protein in animal feed formulations. At the moment, the soy flour used in France is mainly imported and contains genetically modified organisms (GMOs), and the possibility of its operational control is being questioned (Gourdouvelis et al., 2012).

At the same time, new public aspirations are emerging in France (with a focus on food diversity, food origin, environmental impact, food quality) and today the use of soy in animal husbandry is at risk of a new social crisis (Delanoue et al., 2015 ).

In animal husbandry, meeting protein needs is essential to achieve the goals of improving zootechnical performance. However, for animal feed, France imports almost half of the protein-rich raw materials, most of which correspond to more than 3 million tons of soybean meal per year (Bouvarel et al., 2014).

Due to the increase in the supply of products imported from Asia, farmers' incomes are affected by the relative scarcity and volatility of soybean prices. In this regard, one of the main challenges for livestock production is to become less dependent on the supply of soybean meal and therefore to find alternatives to reduce the need for imports (Bourin and Bouvarel, 2015), while ensuring the economic viability of farms, especially by striving to optimize the characteristics of their means of production, as well as to make greater use of their products.

In addition, with regard to pets such as dogs and cats, their owners are concerned about the quality of the food they buy for them, in particular in terms of giving them energy and protecting them from certain metabolic and digestive diseases or allergic reactions.

In Western countries, two-thirds of human protein requirements are covered by animal proteins. However, with the demand for new vegetarian products, the introduction of protein-rich seeds directly into the human diet is becoming an increasingly important criterion. Soybeans make up the bulk of this production, but for the same physiological reasons as animals, humans should be able to access plant protein sources in seed form with improved nutritional value. In view of this, what is described below for animals is also true for humans. In the description and claims below, it is to be understood that the human is part of an animal monogastric mammal.

France lacks a variety of crops, which are mainly cereals and whose protein yields are guaranteed to be achieved through the use of nitrogen fertilizers and pesticides (Messean et al., 2014). Therefore, farmers are looking for highly productive, environmentally sound and sustainable farming systems through the use of new inputs for crop rotation that consume fewer inputs (nitrogen fertilizers, pesticides) and generate profits, among other things.

There are many tasks on the agenda to meet the numerous wishes of consumers and the public, namely:

it should be sustainable food, characterized by nutritional benefits, closer proximity to natural food, biodiversity and full digestibility;

it must be agricultural and food products of local production;

production methods should not use GMOs;

emphasis should be placed on preserving the environment with lower greenhouse gas emissions, conducting phytosanitary control of products, and so on.

Finally, each link in the food chain reflects wishes, which can be summarized as follows:

farmers - to expand crop rotations, diversify crop rotation, make their production system more streamlined;

livestock breeders - there should be local production and use of protein in animal diets;

pet owners - have a balanced and healthy diet;

consumers - to consume locally produced, non-GMO products with a higher nutritional density.

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But there are other wishes that have great influence and are expressed:

institutions - to limit the import of soybeans, nitrogen fertilizers and pesticides and to increase the production of vegetable proteins in France and Europe;

companies throughout the food chain - to focus on differentiation and obtaining additional value in a tense economic situation;

companies working with the agro-industrial complex - to provide a wide network of services and products.

There are many ways in which partial solutions can be provided to meet these many wishes, including for the consumer, for example:

organizing the production of environmentally friendly products, etc., but this is not affordable enough to satisfy a very large number of consumers;

for livestock breeders, for example:

using grass or bean feed as a replacement for corn silage, but on many dairy farms this will only be a partial replacement;

the use of co-products such as grain or cake, which also provide additional protein, but to remain effective, their amount should be limited;

the use of synthetic amino acids, which is a different form of promotion and strategy, but which takes into account the priorities of farmers only, and not those of crop and livestock producers and consumers;

for farmers, for example:

in some regions other inputs such as beetroot, potatoes, flax, etc. are used for crop rotations other than cereals and oilseeds, but their use proves to be very inefficient to meet the wishes of individual French farmers.

These examples show that each link in the food chain individually can offer some solutions, but they remain partial in relation to many of the wishes mentioned above and, above all, not integrated up and down the chain, which does not allow adding value to French farmers' products. and livestock keepers, especially in terms of recognizing benefits for consumers and the public.

However, the proposed solution appears to have huge potential to meet their fields, feeders and plates.

Indeed, the use of high-protein crops or legumes can be an interesting strategy for diversifying crops (whether grown alone or with annual crops) while meeting the demand of farms and livestock areas for autonomy in the production of feed proteins.

The introduction of legumes into crop rotations is of agrotechnical interest in both traditional and biological farming systems, and contributes to mitigating the problem of global warming (Magrini et al., 2016; Schneider et al., 2017).

Indeed, they have the ability to fix atmospheric nitrogen in the soil thanks to the bacteria contained in the tubers of their root system, which reduces the use of nitrogen fertilizers, which are responsible for half of greenhouse gas emissions from agricultural activities.

On the other hand, legumes are also of agricultural and economic interest due to improved plant productivity and lower production costs (Magrini et al., 2016; Schneider et al., 2017). The introduction of leguminous crops favors the yield of grain crops sown after. Thus, limiting only the effects of yield and reduced N fertilization, the increase in gross profit from the sale of a wheat crop after growing a crop with a high protein content (what is known as wheat after protein) is of the order of +160 euros/ha compared to growing wheat after wheat (i.e. a case of wheat planting after a previous wheat planting) (Terres Univia, 2016; Magrini et al., 2016).

The protein-rich seeds are an important source of protein and energy, in the form of starch in the case of peas and common beans. Among other things, these proteins are richer in amino acids such as lysine than cereal proteins, which enhances their value in balanced animal diets.

The nutritional values for protein-rich pea seeds, common beans and lupine are shown in the table below.

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PEAS Ripe BOB Lupine white Lupine yellow Lupine blue Dry Matter (%) 86.4 86.5 88.1 88.8 90.3 Crude protein (%) 20.7 25.4 33.4 37.5 30.3 Raw fat (%) 1.0 1.3 8.4 4.8 5.4 Crude cellulose (%) 5.2 7.9 12.0 14.7 14.2 Raw ash (%) 3.0 3.3 3.5 4.1 3.2 Starch (%) 44.6 38.3 7.1 5.4 4.3 Lysine (%TN) 7.3 6.5 4.9 4.8 5.0 Methionine (%TN) 1.0 0.7 0.8 0.7 0.9 Calcium (g/kg) 1.1 1.4 2.7 3.0 2.6 Phosphorus (g/kg) 4.0 4.6 3.8 4.6 3.4 Gross energy (kcal/kg) 3770 3870 4460 4245 4380

TN means total nitrogen (Source: Table de composition et de valeur nutritive des matieres premieres destinees aux animaux d'elevage, INRA, 2002; Heuze V., Thiollet H., Iran G., Lessire M., Lebas F., 2018. Yellow lupin (Lupinus luteus) seeds Feedipedia, a program by INRA, CIRAD, AFZ and FAO https://www.feedipedia.org/node/23097 Last updated May 17, 2018 10:41 am)).

However, despite their promising nutritional potential, utilization of protein-rich seeds remains insufficient across species due to poor digestibility and the presence of numerous anti-nutritional factors.

By monitoring the energy intake from the protein of various seeds by monogastric animals and ruminants, one can easily see that there is an important untapped potential in terms of nutrition, on the order of 40-50% energy and 20% protein (according to INRA (French L'lnstitut national de la recherche agronomique; National Institute for Agricultural Research), 2002).

These protein-rich seeds contain many anti-nutritional factors that may limit their use. Their presence leads to restrictions on their inclusion in foodstuffs and to poor technical performance, which can be summarized as a real difficulty in terms of competitiveness of protein-rich seeds with other protein sources, especially for monogastric animal species.

The main anti-nutritional factors described are as follows.

Tannins.

Tannins are heat-labile phenolic compounds localized in the seed coat and are known to reduce protein digestibility in monogastric animals because they bind to proteins prior to degradation, forming insoluble complexes.

The content of tannins correlates with the color of the seed or even the fruit (Myer et al., 2001). The inclusion of tannin-rich beans results in lower in vitro degradation (Bond, 1976) and lower in vivo digestibility in poultry, while tannin-free varieties are characterized by greater protein and amino acid digestibility (Gatel, 1994; Crepon et al. , 2010). In monogastric birds, tannins impair the uptake of the nitrogen-containing fraction from the diet, resulting in decreased growth rate and feed efficiency (Carre and Brillouet 1986; Garrido et al., 1988), as well as reduced egg weight (Martin-Tanguy and others, 1977).

There are tannin-free varieties of common beans. However, their agrotechnical indicators are unsatisfactory, at the moment this makes their introduction into products irrelevant.

The content of tannins in common bean seeds varies greatly, with an average value of 0.49 g/100 g dry matter, up to a maximum of 1.70.

In the case of pea seeds, tannins are either absent or present at an average level of 3.9 g/100 g dry matter.

Vitsin i konvitsin.

The cotyledons or kernels of common bean seeds contain vicin and convicin. They are thermostable glycosides (Muduuli et al., 1982) that are responsible for favism in populations suffering from glucose-6-phosphate dehydrogenase deficiency.

Vicin and convicin have not been shown to affect bean digestibility in pigs, but there have been reports that they are responsible for reduced egg weight in laying hens (Lesser et al., 2005; Gatta et al., 2013) and egg production rate (Muduuli and others, 1981). From the very beginning, studies have shown that the presence of vicin and convicin reduced the performance of laying hens in terms of quantity and quality of eggs (Guillaume et al., 1977; Fru-Nji et al., 2007; Olaboro et al., 1981).

There are varieties that do not contain vicin and convicin. They have, in contrast to those that do not have tannins, the advantage of achieving quite adequate agrotechnical parameters (Duc et al., 1999).

Their levels found in common bean seeds range from 0.02 to 1.49 g/100 g (Khamassi et al., 2013).

antitriptic factors.

Trypsin and chymotrypsin inhibitors are protease (and amylase) inhibitors that bind to trypsin in the small intestine, preventing protein breakdown. Ingestion is accompanied in rats, pigs and, to a lesser extent, in chickens by increased loss of endogenous protein in the form of an inhibitor-enzyme complex rich in sulfur-containing amino acids, which is excreted from the body (Liener, 1979). This phenomenon exacerbates the lack of such amino acids in legume seeds, and then the growth of the animal slows down. A lower concentration of the enzyme in the small intestine leads to both hyperactivity and hypertrophy of the pancreas. These protein molecules are relatively thermolabile in nature.

In the case of peas, trypsin inhibitors are the main anti-nutritional factor, accounting for just under 2% of the protein content (untreated soybeans contain 8 times the amount listed). There are significant varietal differences in antitriptic factors. The trypsin-inhibiting activity of 33 European spring pea varieties ranged from 1.69 to 7.56 trypsin-inhibiting units (TIU), while for the wintering pea varieties it ranged from 7.34-11.24 TIU (Leterme et al., 1998). In the case of common beans, the content of trypsin inhibitors appears to be lower with an average value of 2.9 TIU (0.88-6.25).

Pectins.

This group of compounds, widespread in the plant kingdom, is very diverse. These compounds are heat-sensitive glycoproteins whose common feature is their affinity for sugars, which explains their properties to cause erythrocyte agglutination in vitro (lectins combine with glycosyl residues present on the walls of erythrocytes), to varying degrees depending on the types of lectins considered. and animal species (Liener, 1986). They act in the small intestine to interfere with the absorption of end products of digestion by binding to and disrupting the function of epithelial cells (Dixon et al., 1992). Their use leads to growth retardation, which is poorly explained today.

They are present at about 2.5% in the pea protein (Perrot, 1995) and from 2.0 to 13.0% in the bean protein.

Alkaloids.

Previously it was recognized that varieties of lupins are bitter. They contained a toxic alkaloid (lupanine) and were not recommended for use in animal feed without pretreatment. Thus, it is known that high levels of some lupine varieties containing these alkaloids can cause adverse effects, manifested in a decrease in growth rates and feed intake.

However, due to advances in genetics, sweet varieties (low in alkaloids) have been produced and there are no longer any palatability problems.

Oligosaccharides.

These small polymers are carbohydrate in nature and are thermostable. Farm animals and pets lack alpha-galactosidase, the enzyme needed to hydrolyze the bond between galactose and glucose, and between the two molecules of galactose. These molecules do not pass through the intestinal wall and therefore enter the colon intact, where they are metabolized by the microorganisms present, resulting in poor growth performance due to reduced ingestion of food. As a result of fermentation, discomfort in the gastrointestinal tract (flatulence, diarrhea) occurs, which can slow down food intake (Diaz et al., 2006, Kaysi and Melcion, 1992). This deterioration in growth is exacerbated by increasing the introduction of lupine. The results of experiments on pigs have shown that oligosaccharides have a negative effect on the true breakdown of proteins, fats and some minerals. In addition, large amounts of oligosaccharides increased the own weight of the small intestine. Since the tissues of organs such as the intestines are metabolically very active, more energy is needed to maintain the animal's own metabolism, leaving less energy for growth.

White lupine seeds contain 7-14% α-galactosides, the most predominant component among which is stachyose (2.8%), followed by sucrose (1.8%), raffinose (0.4%) and verbascose (0.3% ) (Zdunczyk et al., 1996; Saini, 1989). The level of oligosaccharide may depend on the variety, as well as on growing and harvesting conditions (Pisarikova et al., 2009). There is a relationship between stachyose and verbascose content and flatulence, with raffinose appearing to have a lesser effect. They are also present in the seeds of the common bean and pea, although to a lesser extent, as shown in the table below.

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g/100 g dry matter (DM) Stachyose Raffinose Verbascose Lupine 2.8-5.3 0.4-1.1 1.4-2.0 Peas 2.3-2.6 0.5-0.6 2.2-3.4 Bob ordinary 0.8-1.6 0.1-0.4 2.5-3.4

Opazo et al., 2012; Ezierny et al., 2010.

Phytic acid.

Phytic acid is the main storage form of phosphorus in the tissues of many plants. Phosphorus in this form is not absorbed by non-ruminants due to the absence of the enzyme phytase, which catalyzes the separation of phosphorus from phytic acid molecules.

Phytic acid is an important chelating agent for minerals such as calcium, magnesium, iron and zinc and therefore may contribute to the creation of mineral deficiencies. To a lesser extent, it also binds to protein and starch, resulting in reduced availability of these nutrients in the digestive tract.

The amount of phytic acid in common beans is on the order of 0.2-0.7% by dry weight.

The use of phytases is currently gaining popularity through incorporation into monogastric feed, and this is the main way to regulate this anti-nutritional factor.

Cellulose.

Typically, monogastric animals have a limited ability to digest fiber. The content of crude fiber in protein-rich seeds is relatively high and amounts to 5.2%, 7.9% and 11.4% in the seeds of peas, common beans and white lupine, respectively. Thus, this fiber also belongs to the category of anti-nutritional factors to which it is important to pay attention, in particular in the case of young monogastric farm animals such as chickens, piglets and fish, as well as in the case of young pets such as dogs and cats.

Not only is this fiber low in digestibility, but it also contributes to less digestibility of protein and other nutrients by acting as a shelter for animal digestive enzymes, limiting their availability, and as a clump in the gut, limiting nutrient intake.

As a result, based on the above, the combination of anti-nutritional factors present in protein-rich seeds should be taken into account in order to move towards a more limited use of these seeds in the diets of monogastric animals and towards improving their nutritional and metabolic value.

A technical issue that needs to be resolved.

Restrictions on the use of protein-rich seeds for animal feed are varied and related to technical and economic barriers.

From a technical point of view, the goal is to reduce the impact of anti-nutritional factors and to improve the energy and protein digestibility of these seeds.

And from an economic point of view, to date, no technical implementation has succeeded in meeting the challenge of providing economic feasibility, in view of the existence of examples of only extremely underdeveloped use of protein-rich seeds for monogastric animals, both in traditional and in biological farming systems.

There are two main approaches to improve the nutritional value of protein-rich seeds: plant breeding and seed treatment technologies.

For example, in terms of variety breeding, this approach has already been applied for some anti-nutritional factors, for some varieties successfully, that is, with the use of these varieties by farmers: this applies, in particular, to tannins in peas, vicins and convicins in common beans and alkaloids in lupins.

However, in the case of many antinutritional factors present in these plant species, the importance of a genetic approach for this area has not yet been proven. In addition, varieties depleted in some anti-nutritional factors are unproductive varieties.

In parallel with the breeding work on anti-nutritional factors, many technological methods have been tested to reduce or eliminate anti-nutritional factors and/or improve the nutritional value and digestibility of seeds.

The various methods tested to date are mechanical or thermal or thermomechanical or enzymatic approaches.

It should be noted that the data presented in the literature concerning the technological methods of processing protein-rich seeds are very heterogeneous, incomplete, not very informative, and most often outdated.

Many published studies have attempted to compare a single technology using an untreated control or other technologies with each other in a pairwise comparison or using often different in vitro evaluation methods and/or in vivo studies under different conditions.

Moreover, the individual results reported in the literature are outdated and have not kept up with the changes in technology that have taken place over the last 30 years: and they only present comparisons of technologies without really considering the optimization of these technologies or, moreover, combinations of technologies.

For this reason, the current literature does not in any way allow clear conclusions to be drawn regarding the technologies and the corresponding parameters to be used, in particular for industrial applications.

On the other hand, in vivo studies, which tend to be rather old, have mostly been conducted in animals with less productive genetics and with foods less adapted to modern food systems.

In fact, the genetic breeding method for animals that increase feed consumption by an average of 2.5% per year is carried out for global production using maize and soybean based feeds that are traditionally used (Schmidt et al., 2009; Zuidhof et al. ., 2014).

This does not allow optimal added value to be obtained with more diverse raw materials as energy and protein sources, such as protein-rich seeds.

Therefore, the influence of anti-nutritional factors (ANFs), plant varieties and technologies is likely to be increased by currently existing animal models of in vivo evaluation. Therefore, in such a situation, it is now becoming more and more difficult to demonstrate good results using protein-rich seeds.

It is in this sense that although many processing methods have been tried in the past, some of them deserve to be revisited in the current technical and economic environment.

The main treatments tested to date on these protein-rich seeds are as follows.

Methods of mechanical processing.

The use of classical methods of mechanical processing (grinding, micronization) causes the destruction of the original structure of the seeds as a result of the destruction of cell walls and starch granules. The use of these methods allows separation into smaller particles by coarse grinding (up to 5 mm particles) or fine grinding (mesh size 2-3 mm), i.e. by crushing, tearing (using a hammer mill) or shearing (using a knife mill or roller mill).

The size of the resulting particles and the degree of their destruction determine the degree of influence of digestive agents (rumen microorganisms or intestinal enzymes) on biochemical components and, consequently, on the rate of their breakdown. The tissue structure is largely preserved. However, grinding using a mesh size of 3 mm or even 1 mm followed by agglomeration destroys the tissue structure.

Although digestibility, especially in the case of micronization, can be improved by increasing the digestibility of starch (in pigs and poultry) and protein (in pigs only, no change was found in poultry), no effect on anti-nutritional factors was found.

As a result of shelling / peeling, the husk or peel is separated from the seed, which contains exclusively cellulose, fiber, some anti-nutritional factors and impurities.

Peeling is a method of mechanically separating the grain from the skin, most commonly used for pea seeds, common beans and soybeans. The aim is to reduce the cellulose content in order to enrich the raw material and improve seed recovery by eliminating some of the ANFs present in the husk, such as tannins.

Peeling is the removal of the film (thin skin) around the grain and has the same benefits as skin removal.

As a result of these two methods, the concentration of some nutrients such as protein and fat is increased, and only a part of the ANF contained in these films is separated.

Heat treatment methods.

Thermal processing methods include granulation, frying, delaminating and autoclaving: the effect of heat is combined with the effect of external moisture, used in the form of water or steam, at reduced pressure (long-term moist cooking at moderate temperature). The effect of frying and autoclaving may be superior to that of delamination and granulation.

Such processes can have a more or less important positive effect, in particular on energy value, but are not widely used in the manufacture of animal feed due to lack of profitability.

With regard to baking and blasting, dry and short cooking at very high temperatures using heat transferred by conduction, convection and radiation, no evidence of effectiveness has been demonstrated to date. Indeed, the application of this method practically does not change the particle size distribution and, thus, the integrity of the seed tissues.

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Methods of thermomechanical processing.

Steam extrusion is a complex procedure that is equivalent to several typical processes: kneading, steaming, and shaping. Each of these procedures can be optimized depending on the substance to be treated and the product to be obtained by appropriate selection of plant control parameters.

Driven by screws, this substance is subjected for a very short period of time (20-60 s) to high temperatures (100-200°C), high pressures (50-150 bar (515 MPa)) and more or less intense shear . Under the influence of these physical parameters, the substance undergoes physical and chemical changes and homogenization. As a result of exit through the head, it is given its final shape. The sudden drop in pressure during extrusion causes the water present to evaporate immediately, which can lead to a characteristic expansion of the product.

The first cooking extruders were single screw. The second generation equipment is twin-screw devices (with two screws located in parallel, tangentially or working towards each other, rotating in the same direction or in opposite directions); they are more flexible in application and allow, in particular, to work with greater uniformity.

This steam extrusion method allows the destruction of a part of the ANF and improves the digestibility of the seeds, but the results remain variable and not always reproducible, if many parameters used are not controlled (device type (single screw / twin screw), mechanical limitations (screw type, lock, speed, head etc.), heating-related limitations (water, steam, duration, etc.), yield-related limitations (fine powder head, croquettes, etc.).

However, for most high protein crops, the use of thermomechanical methods is not well known and has yet to be developed.

Enzyme processing methods.

All animals secrete enzymes to digest food. However, the digestive process in animals is not 100% efficient. For example, pigs and poultry do not digest 15 to 25% of the food they eat. The introduction of exogenous enzymes into the composition of animal feed, in particular the enzymes of monogastric animals, improves the digestibility of starches, proteins, fiber and minerals. The participation of such enzymes can improve growth rates and reduce the amount of waste released into the environment.

The addition of enzymes is performed using an isolated commercially available enzyme selected for several enzymatic activities. Studies have shown interest in this method as a way to improve the nutritional value of high protein crops, but this method has not proven to be economically viable.

Processing methods and gelatinization of starch, protein denaturation and the availability of fat for digestion.

Temperature, pressure and humidity affect food components. For many processes, temperature is one of the factors that promotes further degradation of the starch structure (Pan et al., 2017; Wang et al., 2016a; Zhang et al., 2014). However, if the heating temperature remains unchanged, then water content or pressure play an equally important role in the destruction of structures and the increase in digestibility of starch (Wang et al., 2017b). However, it is important to take into account all these parameters, because the same effects, such as protein denaturation, starch gelatinization, and starch interaction reactions, can be obtained with different combinations of these parameters. For example, in the extreme case, gelatinization can be carried out at room temperature by treatment under hydrostatic pressure. At constant values of temperature and duration of treatment, the degree of gelatinization increases with increasing pressure. The higher the temperature, the lower the pressure must be to complete gelatinization. At constant temperatures and pressures, the degree of gelatinization increases with increasing treatment time.

A good example is the process of starch gelatinization, which corresponds to the transition of the matrix from a solid or granular state to a glassy state and then to a liquid state (Cuq et al., 2003). This transition to different states depends on process parameters such as pressure, temperature, time and water content. The glass transition temperature may vary depending on humidity and pressure conditions. At this level, the matrix undergoes a change from a solid state to a mobile rubbery state (Keetels, 1995; Behnke, 2001). Likewise, the melting point at the transition from a glassy to a liquid state also depends on the conditions of humidity and pressure. At this temperature, the matrix becomes liquid (Keetels, 1995). The degree of conversion depends mainly on the duration and rate of heating (Mariotti et al., 2005; Wang & Copeland, 2013). The period of time during which these technological parameters are maintained leads to the complete transformation of the matrix. It should be noted that

- 7 043034 gelatinization occurs in those parts of the matrix, where the water content is high enough (Hoseney, 1994).

Process parameters applied during processing will induce physicochemical changes such as starch gelatinization or protein denaturation. Finally, nutrients can interact with each other, including complex modifications such as Maillard reactions (Svihus, 2006), or polymerize with each other to form a new structure (Svihus et al., 2005).

Starch gelatinization and protein denaturation modify the structures and thus the properties of the matrices. Therefore, the viscosity values of the native matrix and the treated matrix are different. These modifications have a positive effect on animal nutrient uptake (Champ and Colonna, 1993). In a study of pressure gelatinization of starch, an improvement in the digestibility of processed starch by the action of enzymes has been demonstrated (Hayashi and Hayashida, 1989).

The availability of fat for digestion (FA) when using lipid-containing seeds is a parameter that needs to be adjusted in order to increase its value for the animal, and even more so since they (seeds) are enriched in it. Technological methods of seed treatment using mechanical and thermal effects contribute to the rupture of cell walls and plasma membranes, which leads to a higher level of release of fat contained in lipid vacuoles. Thus, the use of adapted processing methods maximizes this release of fat. Interest in the latter for the animal is associated with improved lipid digestibility (Noblet et al., 2008) and the efficiency of dietary fatty acid deposition in pigs (Chesneau et al., 2009), as well as in vitro biohydrogenation of fatty acids for dairy cows (Enjalbert and others, 2008).

Processing methods used by people working in animal husbandry The results of observations obtained in modern breeding organizations are based on the fact that:

- bred varieties do not have their own characteristics in terms of nutritional composition other than agrotechnical characteristics;

- technological methods of using seeds in breeding are basic, since they include only processing methods in the form of mechanical crushing or even peeling in case of use for salmon, heat treatment methods for the purpose of granulation and roasting, and unique processing methods in the form of thermomechanical cooking and extrusion.

Indeed, only these applications presented are currently basic and therefore underdeveloped due to the lack of technical and economic efficiency.

As a result, when using genetic approaches, some regulatory and technical restrictions may be put forward regarding the use of crops with a high protein content in food, for example, in relation to some antinutritional factors, such as antitryptic factors, tannins, vicin and convicin, in particular.

Other limitations may be due to processing methods such as peeling to get rid of the tannins contained in the shell, or thermal processing methods, which, under certain conditions, can eliminate some heat-sensitive anti-nutritional factors.

Finally, mechanical methods such as milling help to improve the nutritional value of seeds as well as thermal processing methods such as roasting and pelletizing, or thermomechanical methods such as steam extrusion, or enzymatic methods such as adding enzymes.

Therefore, individually, each of these pathways: genetic selection or technological methods reported in the literature, offer well-known directions for improvement associated with the limitation or elimination of anti-nutritional factors on the one hand, and / or to improve the digestibility/degradability values of seeds. , on the other hand, but none of them is sufficiently technically complete and economically preferable in terms of technical and economic value, as shown by the lack of widespread practical implementation in this field of technology.

Historically, in a market where the choice of vegetable protein sources present in animal diets is based essentially on economic criteria, the place of protein-rich seeds due to existing restrictions has been negated with the obvious advantage of soybean meal first and, secondly, turn, rapeseed and sunflower meal, and even synthetic amino acids.

In a new market where the trend is not only to produce meat or eggs at a competitive price, but also to meet the wishes of consumers who are inclined to gain more control over production and greater availability of products, the challenge is clearly to have competitive sources of protein, as well as protein sources that are locally produced and can be controlled.

Thus, the cultures of lupine, common beans and peas are characterized by numerous

- 8 043034 agrotechnical and environmental benefits, providing well-known ecosystem services and recognition by producers and consumers as crops of interest.

There are favorable situational factors for successfully re-introducing high protein crops into soil and feeders in an environmentally sound manner: policy incentives (High Protein Crop Assistance Plan, Ecophyio Plan, Innovation Development Assistance, etc.) , agrotechnical advantages (yields of crops sown after crops, use of fewer resources, etc.), possible responses to many agrotechnical (autonomy in the production of feed proteins, economic feasibility, etc.) and social (organization of local production, not using GMOs, use of soy-free raw materials, biodiversity, environment, etc.) wishes.

However, such favorable conditions are not sufficient to expand the scope of oilseeds and high-protein crops within the metropolitan area if the crop and livestock production systems in France are not technically sound and economically viable. Therefore, this expansion must be accompanied by the development of new technically and cost-effective technologies to provide the best solutions for agriculture and livestock.

Evidence of this is only to be found in the processing methods currently used by economic actors in the livestock sector. This remark is based on the sub-optimal application of processing methods in today's livestock sector. As stated earlier, the technological uses of seeds in agriculture include only mechanical crushing or even peeling methods in the case of salmon, only thermal processing methods for granulation and roasting, and unique processing methods in the form of thermomechanical cooking and extrusion.

Indeed, only these presented uses for feeding monogastric animals are currently basic and therefore underdeveloped, due to the lack of technical and economic efficiency. In addition, they do not act as an adjunct to the genetic approach.

Thus, there is currently an unresolved need to develop a method for treating high protein crops that avoids many of the disadvantages detailed above by combining different approaches combined in such a way that they create a synergistic effect.

In addition, in addition to evaluating these methods for their use in animals, there is interest in using these methods to evaluate the quality of processing in terms of their potential for animals.

In this regard, it is clear that for the qualitative evaluation of methods related to the main nutritional components of starch, protein, fat, some methods have been selected, and they are presented below.

A method for assessing the degree of starch gelatinization or starch degradation.

The structure of starches changes from natural structure to gelatinized and then to a new structure in the course of transformation depending on temperature, pressure, humidity and duration of processing. Although the gelatinized structure is susceptible to attack by amylase, both the new structure and the natural structure are resistant to amylase attack.

The degree of starch gelatinization in protein-rich seeds was determined according to the methodology described by Chiang and Johnson (1977). This method is based on an increase in susceptibility to the action of enzymes, i.e. the ability of gelatinized starch grains to hydrolyze more easily than native starch grains. Briefly, total starch is hydrolyzed by amyloglucosidase under specific conditions, whereby only the gelatinized starch is hydrolyzed. To determine its proportion, a determination of total starch is carried out according to the standard method using enzymes (AFNOR (French Association for Standardization; from the French Association Francaise de NORmalization), 2005), and this value serves as a base value.

Method for assessing the solubilization of proteins.

Processing methods applied to protein-rich seeds are likely to result in protein denaturation under certain conditions. Such denaturation is manifested in the formation of new bonds between polypeptide chains (through cross-linking), which leads to their aggregation and insolubility. The bonds formed, usually involving lysine and glutamic acid residues, are resistant to enzymatic hydrolysis. Such insolubility or less solubilization of the protein is of interest in the animal feed industry. For example, allergenicity is reduced by degradation of certain epitopes (Toullec et al., 1992) and protein degradation in ruminants is limited (Benchaar et al., 1992).

Determination of protein solubility is carried out by solubilization of proteins in buffer solutions with different pH values: corresponding to acidic, neutral, alkaline areas. After determining the protein content (Kjeldahl method) in raw materials and proteins in different buffer solutions, evaluate

- 9 043034 material balance to determine the proportion of solubilized protein.

A method for assessing the availability of fat for digestion.

This proprietary method of the inventors is based on an estimate of the proportion of fat extracted into the solvent after a given period of time. The purpose of this assay is to mimic the gradual and staged release of fat in different parts of the animal's digestive tract. Briefly, this analysis is carried out in the following four steps:

preparation of raw materials: the stage consists in coarse grinding in order to obtain a mixture of a non-uniform particle size distribution, as in the case of grinding carried out in the industry in the production of animal feed;

fat extraction: the step consists in bringing the preweighed raw material into contact with the extraction solvent (eg petroleum ether) with controlled stirring for a predetermined period of time, in this case 10 minutes. It is also possible to carry out this extraction phase for different periods of time, which makes it possible to get an idea of the kinetics of fat release;

solid/liquid separation by filtration: this step consists in filtering the particulate matter, dissolved in the solvent, in order to extract only the liquid phase, into a dry and pre-tared flask;

removing the solvent from the extract and weighing the dry residue: the stage consists in evaporating the solvent in which the fat is dissolved. After evaporation, drying and cooling, the flask will be weighed. This cycle is repeated until a constant mass is obtained.

Thus, it is possible to characterize seeds by FA, the values of which depend on the conditions of the technological process.

This released fat, known as digestible because it is rapidly digestible, will be absorbed through the intestinal wall. Absorbed in this way, it can be used by the animal for its own metabolism.

As an additional and innovative evaluation method, the inventors have also chosen direct and indirect methods for the formation of complexes of proteins with carbohydrates.

Protein digestibility assessment method (ED1).

Enzymatic protein digestibility (ED1) in protein-rich seeds was measured according to Aufrere et al. (1989, 1991). Briefly, the sample was subjected to protease hydrolysis in borate phosphate buffer at pH 8 and 40° C. for 1 hour. Determination of the eliminated nitrogen was carried out using the supernatant and referred to the total amount of nitrogen in the sample.

Method for evaluating N-ε-carboxymethyllysine (CMI and total lysine; CML and lysine levels were determined using the method of Niquet-Leridon and Tessier (2011). Finally, each sample is reduced and then hydrolyzed in an acidic medium, and then analyzed by high performance liquid chromatography ( HPLC) combined with tandem mass spectrometry (MS/MS) detection Any influences due to the matrix are corrected by adding an internal standard, a stable CML isotope (CML-D ² ) and lysine (lysine- ¹⁵ N ₂ ).

In addition, heating proteins in the presence of reducing sugars (fructose, lactose, etc.) results in the formation of numerous complex polymers containing lysine. This reaction, called the non-enzymatic browning reaction or the Maillard reaction, also contributes to the reduction in protein digestibility.

Method for assessing the content of acrylamide.

Acrylamide is the generic name for 2-propenamide (acrylic acid amide) with the empirical formula C3H5NO. Acrylamide can be formed, in particular, in the process of steaming at high temperature a raw material rich in carbohydrates (starch, sugars) and protein, which interacts with asparagine (in the Maillard reaction). The formation of acrylamide appears to be strongly influenced by the steaming temperature, the water content of the food, and the browning or carbonization process of the food. Acrylamide is formed during steaming at temperatures of 120°C or higher.

Briefly, the acrylamide in the sample is extracted into water by stirring and then, after centrifugation and recovery of the supernatant, purified on a solid phase extraction (SPE) cartridge and then quantitated using a combination of UPLC (high performance liquid chromatography) and MS/MS in monitoring mode multiple reactions (MRM).

The state of the art in the field of the invention is represented by the following documents:

Jezierny D. et al.: The use of grain legumes as a protein source in pig nutrition: A review, ANIMAL FEED SCIENCE AND TECHNOLOGY, vol. 157, No. 3-4, May 11, 2010, pages 111-128, XP027006418, ISSN: 0377-8401 (D1);

Patricio Saez et al.: Effects of decoating, steam-cooking and microwave-irradiation on digestive value of white lupin (Lupinus albus) seed meal for rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), ARCHIVES OF ANIMAL NUTRITION, vol . 69, No. 2, March 4, 2015 (D2);

FR 3040588 A1 (D3);

Francesco Masoero et al.: Effect of extrusion, expansion and toasting on the nutritional value of peas, fava

- 10 043034 beans and lupins, ITALIAN JOURNAL OF ANIMAL SCIENCE, vol. 4, No. 2, January 1, 2005 (D4). D1 is like a catalog of independent treatments for protein-rich seeds. This article discusses the possibility of reducing the level of anti-nutritional factors using the method of selection or using methods known per se, such as methods of physical processing.

D2 refers to the treatment of only a single seed (Lupinus albus) by peeling, steaming and microwaving.

D3 refers to a seed treatment method including a seed germination step.

D4 is related to the effect of extrusion, expansion, and roasting on the nutritional value of some protein-rich seeds.

Brief description of the essence of the invention

Thus, the present invention relates to a method of treating protein-rich seeds to increase their value as food, in particular for animals, and these seeds are selected from at least one of the following seeds: common bean (Vicia fava L), pea ( Pisum sativum L.), white lupine (Lupinus albus L.), blue lupine (Lupinus angustifolius L.) and yellow lupine (Lupinus luteus L.), characterized in that it includes the following successive stages.

a) The use of seeds from at least one of the aforementioned plant species, provided that they have a value of protein content and/or starch content and/or fat content greater than or equal to the values indicated in the table below.

Content in excess of Nutrient Compounds Protein (g/100 g DM) Starch (g/100 g DM) Fat (g/100g DM) Bob ordinary Vicia fava L. 28 39 Peas Pisum sativum L. 22 45 Lupine white Lupinus albus L. 35 8 Lupine blue Lupinus angustifolius L. 31 5.5 Lupine yellow Lupinus luteus L. 38 5.0

And at least one of the compounds selected from the following group: anti-nutritional factor (ANF), crude cellulose, neutral detergent fiber (NDF), is present at a level below the level indicated in the table below.

View ANF/crude cellulose/NDF Content lower than (g/100 g DM) Bob ordinary Vicia fava L. Tannins 0.3 Vitsin, convicin 0.5 Alpha-galactosides 2.5 crude cellulose 10 NDF 18 Peas Pisum sativum L. Tannins 0.01 Alpha-galactosides 5 crude cellulose 7.5 NDF 18 Lupine white Lupinus albus L. alkaloids 0.1 Alpha-galactosides 10 crude cellulose 16 NDF 25 Lupine blue Lupinus angustifolius L. alkaloids 0.2 Alpha-galactosides 9

- 11 043034

b) Pressurizing the seeds from step a) for more than 10 seconds at a minimum pressure of 10 bar (1000 kPa) until a temperature above 80° C. is reached;

and/or b1) heating the seeds for at least 15 minutes, preferably from 30 minutes to 2 hours, at a temperature in excess of 80°C, preferably in the range of 90 to 150°C.

According to other non-limiting and preferred characteristics of the invention:

after the implementation of the specified stage a) they are subjected to fractionation;

after carrying out said step a) in which seeds of different species and/or different content of protein, starch, fat, anti-nutritional factor, crude cellulose or neutral detergent fiber (NDF) are used, they are mixed and fractionated or fractionated and then mixed;

before step b), the seed preparatory heat treatment step is carried out using steam and/or water-based liquid until temperatures in the range of 30 to 90° C. and a moisture content of more than 12%, preferably 15%, are reached, in over a period of time greater than 2 minutes, preferably 15 minutes;

the pre-cooking step is carried out in the presence of at least one exogenous enzyme identified among the following families: arabinofuranosidases, beta-glucanases, cellulases, glucoamylases, pectinases, pectin-methylesterases, phytases, proteases, xylanases and preferably xylanases, beta-glucanases and pectinases, while the specified exogenous enzyme is added to the seeds or to the mixture in advance;

at the stage of pre-heat treatment in the presence of an exogenous enzyme, the moisture value is set at a level of more than 15%, preferably 25%, and at least 15 minutes for at least 15 minutes, preferably 60 minutes;

when performing the specified stage of preparatory heat treatment, the mixture is stirred;

when mixing is performed and then fractionation, a new mixing is carried out after fractionation;

said fractionation is continued until at least 90% of said seeds have a particle size of less than 2000 microns, preferably less than 1500 microns;

said step b1) is carried out using said mixture;

step b) or b1) will be stopped if the level of at least one of the anti-nutritional factors listed in the table below is less than the level also listed below.

View The nature of anti-nutritional factors Content lower than Bob ordinary Vida fava L. Lectins (g/100 g raw material) 0.10 Antitryptic factors (TIU/mg raw material) 1.5 Peas Pisum sativum L. Lectins (g/100 g raw material) 0.10 Antitryptic factors (TI U/mg raw material) 1.5

After step a) the seeds are subjected to a process of peeling and/or peeling;

after said step a) or after said husking and/or peeling step, said seeds are specifically fractionated and separated according to a criterion selected from size, weight, shape, density, aerodynamic, colorimetric or electrostatic parameter;

after step a) or before said seeds are sorted according to a criterion selected from size, weight, shape, density, aerodynamic, colorimetric or electrostatic parameter;

said seeds are mixed with at least one of other types of raw materials selected from the group consisting of oilseeds and their co-products, oils, co-products of protein-rich seeds, cereals and their co-products, sources of simple and complex carbohydrates and oilseed cake seeds;

said feedstock is a source of lipids, preferably oilseed;

the method includes a final step during which said seeds are cooled.

Detailed description of the invention

To substantiate the present invention, digestibility testing was performed on specified species of monogastric animals, among which chicken, rooster, pig and fish were selected.

The evaluation was carried out according to the following parameters:

energy use, which is based on different approaches depending on the species: metabolizable energy (ME) in the case of poultry, digestibility ratio (DUC) of energy in the case of pigs, true digestibility ratio (DUCa) of energy in the case of fish;

the use of protein, which is based on the determination of the protein digestibility coefficient.

In addition, zootechnical production tests were carried out on the same species, namely chickens, laying hens, pigs and fish.

In this case, the goal is either to show higher growth and/or egg production or to check if growth and/or egg production are identical to those of the control, provided that the nutritional values of the product obtained according to the invention result from values determined in advance by digestibility testing.

The method of the present invention includes a combination of the following steps.

Stage a): use of specific seeds

Consider the case of using at least one protein-rich seed specially prepared for having a high content of at least one (1) nutrient component, preferably two (2), three (3) or even four (4), selected ( s) from protein, starch or fat, and low in at least one thermostable anti-nutritional factor or low value nutrient component such as crude cellulose or neutral detergent fiber (NDF).

Seeds that are high in protein, starch and fat are considered high in protein, starch and fat if their content is equal to or greater than the thresholds below (right column).

View Nutrients connections Change range High Seed (g/kg DM) Minimum Maximum Bob ordinary Vicia fava L. TN 25% 33% > 28% STARCH 32% 49% > 39% Peas Pisum sativum L. TN 19% 27% > 22% STARCH 41% 58% > 45% Lupine white Lupinus albus L. TN 32% 40% > 35% MG 8% 12% > 8% Lupine blue Lupinus angustifolius L. TN 26% 44% > 31% MG 4% 8% > 5.5% Lupine yellow Lupinus luteus L. TN 34% 46% > 38% MG 3% 7% > 5%

In fact, it is preferable to use seeds that are richest in protein and/or energy (in starch, in particular in the case of the seed of the common bean, Vicia fava L.; or the seed of the pea, Pisum sativum L.; and in lipids, in the case of the seed of the white lupine, Lupinus albus L.; or blue lupine seed, Lupinus angustifolius L.; or yellow lupine seed, Lupinus luteus L.) to make this process more competitive from a technical and economic point of view; this is explained not only by the amount of nutrients of interest, but also by the positive interaction at the level of biochemical reactions occurring during the implementation of the technological process (synergistic effect).

Seeds with a low content of thermostable ANF or a low value nutrient are considered as seeds containing low levels of thermostable ANF or a low value nutrient if their content is less than the thresholds below (right column).

- 13 043034

View Nutrients connections Change range (g/100 g DM) Low content seeds (g/100g DM) Minimum Maximum Bob ordinary Vicia fava L. Tannins 0.01 1.25 < 0.30 Vitsin, convicin 0.03 2.00 < 0.50 Alpha-galactosides 1.00 6.20 < 2.50 crude cellulose 7.00 12.00 < 10 NDF 12.40 22.10 < 18 Peas Pisum sativum L. Tannins 0.00 0.74 < 0.01 Alpha-galactosides 2.30 11.30 < 5 crude cellulose 3.70 8.50 < 7.5 NDF 9.00 22.00 < 18 Lupine white Lupinus albus L. alkaloids 0.01 3.00 < 0.1 Alpha-galactosides 6.00 14.00 < 10 crude cellulose 13.60 28.10 < 16 NDF 20.50 35.20 <25 Lupine blue Lupinus angustifolius L. alkaloids 0.01 3.00 < 0.2 Alpha-galactosides 6.00 14.00 < 9.0 crude cellulose 13.60 28.10 < 18 NDF 20.50 35.20 < 29 Lupine yellow Lupinus luteus L. alkaloids 0.01 3.00 < 0.3 Alpha-galactosides 6 16 < 12.0 crude cellulose 13.6 28.1 < 18 NDF 20.50 35.20 < 29

DM means dry matter

Variability in seed composition exists within the same species and is determined by factors such as variety, crop route, pedoclimatic conditions, and so on. The selection of protein-rich seeds based on their nutritional quality has an impact on industrial processes that are carried out downstream. Indeed, the presence of proteins, starch and lipids in matrices influences the processability and therefore the conversion of these nutrients, especially in the assays of starch gelatinization, protein denaturation and fat digestibility, which are known to predict the possibility of improved digestibility. The levels of transition from one state to another (solid, glassy, liquid) depend on the proportion of nutrients mentioned above.

Thus, two seeds of the same species and different compositions will, under the same hydrothermally treatment, react differently; namely, that particular seeds selected in the 1st step of the method of the invention will, after the same hydrothermal treatment, have an increase in protein denaturation, starch gelatinization and digestible fat content compared to unselected seeds.

From another point of view, for the same task associated with protein denaturation, starch gelatinization and fat availability for digestion, in the case of specific seeds selected in the first stage according to the invention, it will be necessary to carry out a technological method with them at lower temperatures, pressures, water content and/or time, i.e. more efficient way because it requires less technological constraints.

In addition, the higher the content of starch and/or protein in the seeds of peas and beans, and

- 14 043034 the content of protein and/or fat in the lupine seed, the lower the amount of fiber. Therefore, this lower inclusion of fiber of low value to the animal results in a higher level of digestibility, promoting the digestibility of protein and other nutrients. Such fiber acts as a shelter for animal digestive enzymes, limiting their availability, and regulates intestinal congestion, which hinders nutrient absorption.

Step b): step of heat treatment under pressure.

This step consists of placing the seeds under conditions of a minimum pressure of 10 bar (1000 kPa), preferably 20 bar (2000 kPa), even 30 bar (3000 kPa), for a period of time greater than 10 s, preferably in the range of 10 s to 2 min. , in order to achieve a temperature above 80°C, preferably above 100°C, or even in the range from 100 to 150°C, and more preferably even in the range from 110 to 140°C and without exceeding 160°C; such a temperature is preferably provided by self-heating due to shear forces, friction and pressing and, in addition, possibly due to the supply of heat from the outside, through convective heat exchange (heat transfer fluid, electrical resistance, electromagnetic fields, etc.) or through the addition of steam.

A non-exhaustive list of heat treatment equipment with which this step can be carried out is given below: extruder, cooking extruder, expander, press.

And/or.

Step b1): non-pressure heat treatment step.

This step consists in heat treatment without application of pressure, the duration of which is increased in comparison with step b) so that it is more than 15 minutes, preferably more than 30 minutes or even in the range from 30 minutes to 2 hours, and the temperature is higher 80°C, preferably above 90°C, or even ranged from 90 to 150°C. In the same way, suitable equipment for such heat treatment without the application of pressure is, for example, a dryer, a fryer, a thermostatically controlled screw.

The purpose of this step(s) b) and/or b1) is to reduce heat sensitive anti-nutritional factors and inactivate endogenous and/or exogenous enzymes while improving energy and/or protein and amino acid availability, especially in the case of heat treatment under pressure.

Finally, this allows, if necessary, to reduce the moisture content of the seeds, which should not exceed 14% moisture, preferably 12%, in order to provide good conditions for the preservation of the mixture.

One way to characterize the effectiveness of this stage (these stages) b) and/or b1) is to evaluate the reduction in the content of at least one heat-sensitive anti-nutritional factor, the objectives of which are presented in the table below.

View Antinutritional factors (ANF) in relation to raw materials Range for seeds with unreduced ANF content ANF Reduced Seeds Minimum Maximum Bob ordinary Vicia fava L. Lectins (g/100 g) 2.00 13.30 < 0.10 Antitryptic factors (TIU/mg) 1.90 10.00 < 1.50 Peas Pisum sativum L. Lectins (g/100 g) 0.30 1.00 < 0.10 Antitryptic factors (TIU/mg) 1.70 15.00 < 1.50

TIU stands for Trypsin Inhibiting Units

Another way is to estimate the proportion of gelatinized starch, taking into account the seeds in question, so that it reaches a minimum value of 50%, preferably 65%, more preferably 80% in the case of peas and common beans; or the proportion of protein solubilized at basic pH values so that it reaches a maximum value of 55%, preferably 40%, more preferably 30% for peas, common beans and lupins; or the proportion of fat available for digestion, so that it reaches a minimum value of 40%, preferably 50%, more preferably 60% for lupine; or that the enzymatic degradability after 1 hour reaches a maximum value of 50%, preferably 40%, more preferably 30% for peas and broad beans and a maximum value of 60%, preferably 50%, more preferably 40% for lupine; or in the case of lupine, the proportion of fat available for digestion, so that it reaches a minimum value of 40%; or evaluate Maillard compounds such as Ne-carboxymethyl-lysine (CML) or acrylamide. Threshold values which must not be exceeded are 0.020 g/kg dry matter, preferably 0.018 g; more preferably 0.015 g Νε-carboxymethyl-lysine for beans and peas, and 0.025 g/kg dry matter, preferably 0.020 g; more preferably 0.018 g Nε-carboxymethyl-lysine for lupine; and 110 g/kg dry matter, preferably 90 g;

more preferably 70 g acrylamide for common beans and peas and 300 g/kg dry matter, preferably 200 g; more preferably 150 g of acrylamide for lupine.

Criteria for evaluation Bob is ordinary. Vicia fava L. Peas Pisum sativum L. Lupine Lupinus spp. Protein solubilized at basic pH (%) < 55% < 55% <55% Gelatinized starch (%) > 50% > 50% - ED1 (%) < 50% < 50% < 60% FA (%) - - >40 CML (g/kg DM) < 0.020 < 0.020 < 0.025 Acrylamide (g/kg DM) <110 <110 <300

Final stage: cooling.

At the end of the heat treatment step b) the seeds are hot. Therefore, it is necessary to refrigerate them in order to reduce the temperature of the seeds after this process to a point where they can remain stable over time and thus be protected and kept in good nutritional status until consumption.

For example, the temperature should not exceed 30°C, be above ambient temperature, preferably should be 20°C.

The preceding steps of the method according to the invention can also be carried out in an advantageous manner, in the first place, taking into account the elements described below.

Mixing and fractionation of seeds.

Although this step is optional, the efficiency of the process is increased if, after step a) of the process, a fractionation or even mixing step is performed.

This stage consists in choosing at least one of the mechanical mixing technologies, where there are at least two starting materials of different nature and/or different quality, and/or one of the mechanical fractionation technologies of the seeds or mixture, fine-tuned in such a way that they allow, firstly, prepare a homogeneous mixture of protein-rich seeds and any additional raw materials and, secondly, break up the seed coat and seed kernels to make nutrients more available to digestive enzymes and thus improve the digestibility of the seeds.

It is preferable to premix the substances before fractionation. It is also possible to first fractionate the substances individually and then mix them, and it is also possible to perform two mixing procedures, one before fractionation and one after it.

Simple and/or combined mechanical actions used to perform these procedures can be performed by impact, cutting, pressing, shearing or abrasion.

To characterize the seed fractionation process, particle size measurement is used, which determines the size of the particles resulting from the application of this method. Preferably, 90% of the particles obtained using such mechanical fractionation have a maximum size of less than 2000 µm and more preferably less than 1500 µm.

This size can be achieved, for example, using a horizontal hammer mill according to the following parameters:

for equipment with a capacity of 10 t / h and an engine power of 200 kW, a rotation speed of 2800 rpm and a grid mesh size of 3 mm.

This size can also be achieved using other conventional equipment such as hammer and roller mills or crushers, paddle mills.

Finally, there are other processing equipment with which this procedure can also be carried out: grinding wheel mill, disc mill, pin mill, cutting head mill, bead or ball mill, blade mill or pendulum mill, hammer mill or impact mill. mill and so on.

Pre-heat treatment.

After step a) of this method or the fractionation and/or mixing steps described

- 16 043034 previously, it is also advantageous to proceed to the heat treatment step.

It is also important to remember that seeds can be used for this stage after removing the skins and flaxseed cake (another preferred stage, described later).

This stage consists in choosing a heat treatment technology with such parameters that they correspond to the following stages and characteristics.

The first option: the stage of preparatory hydrothermal treatment.

This step consists in impregnating the seeds with water vapor and/or liquid water in order to reach a temperature of 30 to 90° C. for a time period of more than 2 minutes and a moisture content of more than 12% for the pre-fractionated seeds. In a preferred method, it is recommended to soak the seeds for a period of time greater than 5 minutes or 15 minutes, even 30 minutes, and preferably less than 4 hours, even 8 hours, without exceeding 24 hours, in order to achieve a moisture content above 15%, even 18%, and preferably below 40%, not exceeding 60%. And in a very preferred method, it is recommended to impregnate the seeds for a period of more than 1 hour, even 2 hours, to achieve a moisture content above 20%, even 25%. This stage solves the problem, in particular, of facilitating the subsequent heat treatment stage, under pressure and/or without pressure, by improving the thermal conductivity, by activating the endogenous seed enzymes and by starting the subsequent heat treatment.

The equipment with which this step can be carried out includes, but is not limited to: pre-treatment device, pre-conditioner and conditioner, steamer, mixer, fryer, steamer, maturation device.

Or.

The second option: the stage of preparatory hydrothermal and enzymatic treatment.

This preparatory stage consists in fulfilling the same preparation conditions that are described for the first option. It differs simply in that at least one exogenous enzyme not present in protein-rich seeds is activated, which can be introduced, in particular, as a processing aid (enzyme extracts, etc.), from auxiliary substances, raw or fermented types of raw materials, etc., and added at one of the preliminary stages of the method or during the implementation of this method.

Temperature characteristics are then chosen so that they correspond to the ranges of activity of the selected enzymes, but remain in the range from 30 to 90°C. The required duration and moisture characteristics are the same as those described for the first option, however, taking into account that these exogenous enzymes require more favorable conditions than endogenous enzymes, since they are not so close to their substrates in spatial and temporal aspects. In this sense, it is necessary to adapt the impregnation conditions in such a way that the duration of the impregnation is at least 15 minutes, preferably 60 minutes and preferably less than 4 hours or even 8 hours, but not exceeding 24 hours, in order to achieve a moisture content of more than 15%, preferably 25% and preferably less than 40% but not exceeding 60%. The enzyme (or enzymes) to be administered belongs (or belong) to the families of arabinofuranosidases, beta-glucanases, cellulases, glucoamylases, α-amylases, pectinases, pectin-methylesterases, phytases, proteases, xylanases, and preferably to the families of xylanases, beta-glucanases and pectinase.

He (or they) will be (are) selected in advance based on his (or their) efficiency in the hydrolysis of specific chemical bonds, hydrolysis, which in the animal body cannot be carried out at all or is carried out incompletely or not fast enough.

It may also be selected for its ability to break down carbohydrates that are not or poorly hydrolyzed in the animal's body, thus providing improved access to other nutrients in such seeds by the action of the animal's digestive enzymes.

The equipment with which this step can be carried out are, for example: pretreatment device, preconditioner and conditioner, steamer, mixer, fryer, steamer, aging device, reactor, and so on.

It should be noted that the possible addition of water, necessary to improve this method, can be carried out in the aforementioned equipment, as well as in whole or in part at the stage of mixing the seeds, and that the addition of steam, also necessary to improve this method, can be carried out in the aforementioned equipment, and also wholly or partly in step b) of this process, i. e. at the stage of heat treatment under pressure and/or without the application of pressure.

The steps of the process according to the invention, including or not including those preferred steps mentioned above, may also be carried out, if necessary, taking into account the points described below.

Sorting.

At this sorting stage, seeds are grouped according to size, weight, shape, density, or according to aerodynamic, colorimetric or electrostatic characteristics. The tools used to carry out these procedures are, in particular: sieve, separator-cleaner, screen, sifter, densimeter table, winnower, optical sorter, active ventilation systems (air box, suction systems, fan

- 17 043034 etc.), magnetic systems.

The purpose of this procedure may be to separate seeds of different species, to remove impurities, to select seeds of identical species, and so on. The seed may be separated from seeds of another species, or it may be selected for seeds of the same species.

Peeling and/or peeling.

The purpose of this stage is, on the one hand, to increase the protein content and store energy in the form of starch, in particular in the case of beans and peas, and protein and energy in the form of lipids in the case of lupine, and, on the other hand, to reduce proportion of fiber and anti-nutritional factors present in the seed coat.

This husking and/or peeling step is characterized by the minimum yield estimated from the effect of increasing the amount of protein in the respective seeds, and a table of expected levels is given below.

Protein content Bean ordinary +5%, preferably +15% or even +20% Peas +5%, preferably +12% or even 15% Lupine +5%, preferably +15% or even +20%

By removing the rind, which is high in fiber (and very low in protein), the seed will achieve a higher protein content when the rind is removed.

Peeling and/or peeling is carried out by combining a mechanical action phase and a separation phase, then, if necessary, carrying out a possible rewetting, before which a pre-heating phase is carried out to facilitate peeling and/or peeling.

The methods of simple and/or combined mechanical action used to perform these procedures may be impact, pressing or abrasion. The tools used to perform these operations include, but are not limited to: roller and hammer mills or crushers, impact compactor or impact mill, grinder, paddle mill, grinding wheel mill, disc mill, pin mill, head mill , bead or ball mill, blade mill or pendulum mill.

Separation can be carried out according to criteria of size, mass, shape, density, or according to aerodynamic, colorimetric or electrostatic characteristics. The tools used to carry out these operations are, in particular: sieve, shaker, separator-cleaner, screen, stone remover, sifter, densimeter table, winnower, optical sorter, active ventilation systems (air box, suction systems, fan, etc. .), magnetic systems.

Special stage of fractionation and separation.

In order to obtain fractions with a higher content of protein, carbohydrates and/or fat, a special fractionation and separation step can be added. It should be performed at a later stage or instead of the husking/removing and/or fractionating stage.

This stage is characterized by the minimum yield, expressed as the content of proteins, carbohydrates or lipids in one or more than one of the resulting fractions. Fractions with a high content of protein, carbohydrates or lipids should preferably contain at least more than 25% protein, carbohydrates and/or lipids compared to the original whole seeds, preferably 35% or even 50%.

Such fractionation, which is facilitated, if necessary, by carrying out the stage of peeling/removing the peel, will be carried out first using a stage of simple and/or combined mechanical action, carried out, for example, using equipment such as colloid mills, pin, roller, hammer or impact mills. And then using a separation step according to the criteria of size, mass, shape, density or according to aerodynamic, colorimetric or electrostatic characteristics using equipment such as screeners, cleaners, separators, sieves, densimeter tables, turbine separators, selectors, systems active ventilation or magnetic systems.

Use of additional raw materials.

An advantage can be obtained if at least one other raw material is added to the protein-rich seeds, which will be selected on the basis of its technological and/or nutritional and/or economic properties. Indeed, depending on the application that is planned for the seeds or mixture obtained according to this method, and on their purpose in terms of animal species and, in particular, the stage of physiological development, the choice (types) of raw materials will be associated especially with nutritional characteristics and the cost of raw materials.

- 18 043034

But they must also be selected on the basis of their technological advantages, in particular their mechanical properties and therefore their propensity to make the mechanical limitations of the process more advantageous, their rheological and physico-chemical properties and hence their ability to be mixed with protein-rich seeds. under conditions of high humidity and their ability to adsorb or absorb water, in some cases their ability to bind to proteins, their enzymatic properties and, therefore, their ability to enhance enzyme activities, in particular, to improve the digestibility of protein-rich seeds.

Preferably, the protein-rich seeds are combined with a portion of the oilseeds when the heat treatment technology includes a pressure heat treatment step.

As an example, the addition of fat, preferably from oilseeds rather than oil, due to a more diffuse and uniform supply of lubricant to facilitate passage through the extruder, allows better processing of protein-rich seeds from a technological point of view and increases production capacity.

Finally, more generally, the choice of additional raw materials is preferably based on the possibility of improving nutritional and economic performance, which can be achieved using this method applied to protein-rich seeds.

Thus, among additional raw materials, preference will be given first to oilseeds, oils, cereals and their co-products, sources of simple or complex carbohydrates, then oilseed cake, then all other raw materials commonly used for animal feed.

Stirring in the preheating step In the previously described preheating step, one of the advantages is that the mixture is stirred in such a way that it is subjected to the same processing conditions. Indeed, when mixing will be carried out:

homogenizing the fractionated seeds, together with water and other possible complementary components, with the aim, in particular, of facilitating the functionality of the enzymes after coming into contact with their substrates;

ensuring uniform distribution of water and equalization of the temperature of the seeds or mixture;

avoiding the formation of agglomerates and thus facilitating the conditions for transporting the seed or mixture.

As will be shown below, the application of the method of the present invention makes it possible to achieve technically advantageous results relative to the prior art.

In fact, none of the methods widely described in the literature achieves such improved technical and economic results as those obtained according to the method of the present invention, in particular, this concerns modern systems for the production of animal products, characterized by significant genetic advances adapted to the system feeding, based mainly on the use of soy, corn and cereals.

The combination of the steps described above results in at least one protein-rich seed that simultaneously has a higher content of at least one (1) nutrient compound, preferably two (2):

Content in excess of Nutrient Compounds Protein (g/100 g DM) Starch (g/100g DM) Fat (g/100g DM) Bob ordinary Vicia fava L. 28 39 Peas Pisum sativum L. 22 45 Lupine white Lupinus albus L. 35 8 Lupine blue Lupinus angustifolius L. 31 5.5 Lupine yellow Lupinus luteus L. 38 5.0

lower content of at least one thermostable anti-nutritional factor or nutritional component having low value, preferably 2, more preferably 3 or even 4:

- 19 043034

View Thermostable anti-nutritional factors Range (g/100g DM) Low content seeds (g/100g DM) Minimum Maximum Bob ordinary Vicia fava L. Peas Pisum sativum L. Tannins 0.01 1.25 < 0.30 Vitsin, konvitsin Alpha-galactosides Crude cellulose NDF Tannins Alpha-galactosides Crude cellulose 0.03 1.00 7.00 12.40 0.00 2.30 3.70 2.00 6.20 12.00 22.10 0.74 11.30 8.50 < 0.50 < 2.50 < 9.00 < 16.00 < 0.01 < 5.00 < 5.50 NDF 9.00 22.00 < 14.00 Lupine white Lupinus albus L. alkaloids 0.01 3.00 < 0.1 Alpha-galactosides 6.00 14.00 < 10 crude cellulose 13.60 28.10 < 16 NDF 20.50 35.20 < 25 Lupine blue Lupinus angustifolius L. alkaloids 0.01 3.00 < 0.2 Alpha-galactosides 6.00 14.00 < 9.0 crude cellulose 13.60 28.10 < 18 NDF 20.50 35.20 < 29 Lupine yellow Lupinus luteus L. alkaloids 0.01 3.00 < 0.3 Alpha-galactosides 6 16 < 12.0 crude cellulose 13.6 28.1 < 18 NDF 20.50 35.20 < 29

lower content of at least one heat-sensitive anti-nutritional factor:

View Antinutritional factors (ANF) in relation to raw materials Range for seeds with unreduced ANF content ANF Reduced Seeds Minimum Maximum Bob ordinary Vicia fava L. Lectins (g/100 g) 2.00 13.30 < 0.10 Antitryptic factors (TIU/mt) 1.90 10.00 < 1.50 Peas Pisum sativum L. Lectin! (g/100 g) 0.30 1.00 < 0.10 Antitryptic factors (TIU/mt) 1.70 15.00 < 1.50

TIU stands for Trypsin Inhibiting Units

- 20 043034

Improved content of at least one evaluation criterion.

Criteria for evaluation Bob usually Vicia fava L. Peas Pisum sativum L. Lupine Lupinus spp. Protein solubilized at basic pH (%)* < 55% < 55% <55% Gelatinized starch (%) > 50% > 50% - ED1 (%) < 50% < 50% < 60% FA (%) - - >40 CML (g/kg DM) < 0.020 < 0.020 < 0.025 Acrylamide (g/kg DM) <110 <110 <300

(*): hereinafter, the term solubilized protein means a protein solubilized at basic pH values;

an improved level of digestibility of energy and / or protein and the amino acids it contains for monogastric animals:

Energy absorption (% increase) Protein/amino acid digestibility from (% increase) Bob usually Peas Lupins Bob usually Peas Lupins Domestic bird ME (kcal) DUC (%) 20% 20% 25% 8% 5% 8% Pigs DUC (%) DUC (%) 20% 10% 15% 8% 5% 8% Fish DUC (%) DUC (%) 25% 15% 18% 12% 8% 12% Other types % increase DUC (%) 20% 10% 15% 8% 4% 6%

(ME: metabolic energy; DUC: digestibility factor); for ruminants:

Theoretical cleavage (TD) (%) Digestibility in the gut (dr) (%) Bob usually Peas Lupins Bob usually Peas Lupins Ruminants < 65 <65 < 65 >98 >98 >98

The results presented in the tables above are comparable to the results for seeds that have only undergone a fractionation step similar to that described above and a granulation step below 100°C. These results are also based on modern research in the field of animal genetics, i.e. on breeds selected on the basis of their productivity.

They are the result of digestibility studies carried out in monogastric species by in vivo testing in chickens, fish and pigs and in ruminants by in vivo testing. The results of these tests are presented below.

digestibility testing.

a) Digestibility testing on standard broilers.

An experimental farm conducted a study to determine the content of digestible nitrogen and the rate of energy use for standard broiler chickens. The objective is to evaluate the impact of the proposed selection and processing steps of the common bean seed as described in this invention. Energy use and protein digestibility are calculated from the difference as defined by Carre et al. (2013).

- 21 043034

Characteristics according to the invention Bob usually And Tseln. Rough Bob usually And Tseln. Proparen. Bob usually A Kernel Proparen. Bob usually In Celn. Rough Bob usually In Celn. Proparen. Bob usually In Kernel Proparen. Seed selection No No No Yes Yes Yes Technological seed treatment No Yes Yes No Yes Yes Characteristics of common bean seeds (in whole seed form or after skin has been removed) Protein, g/100 g DM 29.2 33.3 29.1 32 Starch, g/100 g DM 39.7 46.7 41.4 49.8 Tannins, g/100 g DM Vicin + convicin, g/100 g DM Alpha-galactosides, g/100 g DM Crude fiber, g/100 g DM NDF, g/100 g DM Lectins, g/100 g DM Antitryptic factors, TIU/mg 8.2 7.8 5.2 10.6 15.4 7.7 5.2 Ways to form ( 2.1 9.2 4.6 0 0 8.5 4.6 eotki family. 5.8 1.7 1.1 0.3 3.7 3.1 7.2 0 10.5 0.0 7.0 7.7 3.7 3.1 yang beans Stirring Fractionation Peeling Heat treatment Temperature Duration Pressure No No No No No No Yes Yes Yes Yes Yes Yes No No Yes No No Yes - 140°C 130°C - 140°C 130°C - 20 s 20 s - 20 s 20 s _{op s} 25 bar _{op to} 25 bar 30 bar 30 bar D' (3 MPa) If: . (3 MPa) ^{ν 7} MPa) ^{ν 7} MPa) Characteristics of treated bean seeds ordinary Lectins* Antitryptic factors** 6.7 2.9 0.0 0.8 0.0 0.5 6.1 1.7 0.0 0.9 0.0 0.3 Nutrient absorption DUC of total nitrogen 73 76 76 72 79 80 ME for N, kcal/kg of dry matter (MS (from French, matiere seche)) 2813 3068 3258 2890 3313 3477

DUC of total nitrogen 100 104 104 99 108 110 DUC energy 100 109 116 103 118 124

Thus, by combining the best technologies, from seed selection to mechanical and thermomechanical processing methods, the inventors manage to significantly improve digestibility levels: with an increase of + 8% in the value of protein digestibility, with an increase of + 18% in the value of energy value in chickens, highly sensitive species in terms of digestibility, making them an excellent model for monogastric animals. If the shelling of common bean seeds is carried out in an advantageous way, then after the heat treatment there is a synergistic effect in terms of the consumed value of protein (+10%) and energy (+24%). These effects are superior to those obtained by simply adding the effects of seed selection, seed hulling and adapted heat treatment.

b) Digestibility testing in pigs.

On an experimental farm, a study was conducted to determine the faecal and intestinal digestibility in piglets. Digestibility ratios for each test material were obtained using a difference method, whereby measurements were made using the main feed and measurements were made using feeds containing a fraction of the main feed and one of the test products.

To determine fecal digestibility in pigs, testing was performed according to the scheme described by Noblet et al. (1989). Briefly, the principle is to introduce 35% of one of the seeds to be tested into the main feed (wheat + soy flour) and to distribute each of the feeds thus obtained among 5 pigs.

Characteristics according to the invention Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Seed selection - Yes Yes Yes Yes Yes Yes Technological seed treatment No Yes Yes No Yes Yes Diet characteristics ME in the diet, kcal/kg 5360 kcal/kg MS Digestible protein, g/kg 44g/100g DM

- 23 043034

Characteristics of common bean seeds (in whole seed form or after skin has been removed) Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Protein, g/100 g MS Starch, g/100 g MS Tannins, g/100 g MS Vitsin + convicin, g/100 g MS Alphagalactosides, g/100 g MS Crude fiber, g/100 g MS NDF, g/100 g MS Lectins, g/100 g MS Antitryptic factors, TIU/mg Mixing Fractionation Peeling heat treatment Temperature Humidity Duration Heat treatment Temperature Duration Pressure Lectins, g/100 g DM Antitryptic factors, TIU/mg WITH 31.4 38.0 0.5 0.3 2.6 7.0 13.1 2.0 1.9 seed treatment methods No no no Yes Yes Yes No no no - - 60°C - - 14% - - 15 minutes - 110°С 140°С - 20 s 20 s 20 bar 30 bar - (2 (3 MPa) MPa) Image characteristics 6.7 2.2 0.2 6.6 3.0 2.6 36.1 43.6 0.4 0.3 2.9 1.4 11.1 2.2 2.1 common beans No no no Yes Yes Yes Yes Yes Yes - - 60°C - - 14% - - 15 minutes - 110°С 140°С - 20 s 20 s 20 bar 30 bar - (2 (3 MPa) MPa) botanical seeds 6.7 6.7 1.3 5.6 4.4 2.8 Fecal digestibility ratio (DUC) of products nutrition DUC of total nitrogen 84.9 ^bc 82.1 ^a 84.9 ^sun 84.2 ^b 84.1 ^{b 88.3d} 88.0 Seed faecal digestibility ratio (DUC) DUC of total nitrogen - 80.1 85.9 84.4 84.5 93.5 92.2

This study highlights the advantage of a processing method (preparation and heat treatment) applied to whole or dehusked seed, as disclosed in this invention, in achieving levels of total nitrogen and energy digestibility statistically equivalent to a soy based control diet. flour. These two heat treatment methods applied to the seed lead to essentially equivalent results. On the other hand, the pre-husking step of the selected common bean seed allows, when not processed, to reach the DUC values of total nitrogen and energy for feed made using soybean meal. When the common bean kernel is processed as described for the benefit of this invention, the DUC is clearly and statistically superior to that of soy flour.

- 24 043034

c) Digestibility testing in rainbow trout.

In an experimental fish farm, a study was conducted to determine the use of energy and protein as a result of metabolism. Energy use and protein digestibility are calculated from the difference as determined by Choubert et al., 1982. Data for bred and treated common bean seeds and their digestibility ratio values are presented in the table below.

Characteristics according to the invention Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Seed selection - Yes Yes Yes Yes Yes Yes Technological seed treatment No Yes Yes No Yes Yes Characteristics of common bean seeds (in whole seed form or after peeling) Protein, g/100 g MS 31.4 36.1 Starch, g/100 g MS 38.0 43.6 Tannins, g/100 g MS 0.5 0.4 Vitsin + convicin, g/100 g MS 0.3 0.3 Alphagalactosides, g/100 g MS 2.6 2.9 Crude fiber, g/100 g MS 7.0 1.4 NDF, g/100 g MS 13.1 11.1 Lectins, g/100 g MS 2 2.2 Antitryptic factors, TIU/mg 1.9 2.1 Methods for processing seeds of common beans Mixing - No No No No No No Fractionation - Yes Yes Yes Yes Yes Yes Peeling - No No No Yes Yes Yes Preparatory heat treatment Temperature - - - 60°C - - 60°C Humidity - - - 14% - - 14% Duration - - - 15 minutes - - 15 minutes Heat treatment Temperature - - 110°С 140°С - 110°С 140°C Duration - - 20 s 20 s - 20 s 20 s Pressure - - 20 bar (2 MPa) 30 bar (3 MPa) - 20 bar (2 MPa) 30 bar (3 MPa) Characteristics of Treated Common Bean Seeds Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Lectins, g/100 g DM - 6.7 2.2 0.2 6.7 6.7 1.3

- 25 043034

Antitryptic factors, TIU/mg - 6.6 3.0 2.6 5.6 4.4 2.8 Solubilizers. protein, % - 71 51 23 58 35 24 Gelatinized starch, % - 7 66 83 7 68 85 CML, g/kg DM - 0.007 0.016 0.021 0.008 0.018 0.023 Acrylamide, g/kg DM - < 30 35 62 < 30 38 65 ED1, % - 81 36 35 85 46 39 Digestibility Coefficient (DUC I raw materials DUC of dry matter 83.8 ^cd 53.8 ^{a 68.5th 67.9th} 72.4 ^bc 71.7 ^{ye 78.7d} DUC energy 84.7 ^e 56.9 ^{a 70.8th} 71.9 ^b 74.6 ^s 77.1 ^ye 83.4 ^e protein DUC 97.4 ^e 83.5 ^a 89.3 ^th 92.5 ^{ye 91.1st} 93.5 ^ye 96.7 ^e DUC starch - 74.5 ^a 84.5 ^b 81.4 ^b 75.0 ^a 75.8 ^a 85.0 ^th

Values having the same superscript letters in the above table and in subsequent tables do not differ significantly at the 5% threshold.

Treatment of selected common bean seeds using preparation and heat treatment methods results in improved dry matter, energy, protein and starch digestibility compared to untreated common bean seeds. These two heat treatment methods lead to results that do not differ significantly from each other. However, there is some numerical difference in favor of preparatory and further heat treatment (in terms of temperature, pressure).

The advantageous use of shelling common bean seeds prior to heat treatment further improves the digestibility of nutrients such as energy and protein, approaching that of soybean meal.

This study highlights the benefit of this invention from the processing of common bean seeds in terms of nutrient digestibility, and highlights the advantageous use of shelling to replace soybean meal.

It should be remembered that these internal digestibility results, while expressing the effects associated with the process of the invention (the synergy of seed selection and processing), do not express the synergistic effects of the invention at the level of animal digestion. Some of the digestibility results are obtained in accordance with conventional methods used in the preceding illustrative materials. On the other hand, other phenomena are involved in the process of digestion of food by animals, and they affect the metabolism of the animal. Indeed, if anti-nutritional factors can be involved in changing the digestibility of the seed, then they are also the cause of digestive and metabolic disorders, causing a decrease in consumption and productivity, leading to physiological disorders and various pathologies. Thus, the method of the present invention has the advantage of not only achieving high levels of so-called protein rich seed digestibility, but also avoiding poor performance and other health problems associated with the presence of significant amounts of anti-nutritional factors.

For example, the presence of vicin and convicin in common beans has been shown to reduce egg weight and egg production in laying hens. Lectins, on the other hand, have the ability to cause red blood cells to agglutinate, which can lead to growth retardation. As for oligosaccharides, they are metabolized by colonic microorganisms and cause, due to their fermentation, discomfort at the level of the digestive system (flatulence, diarrhea), which can reduce feed intake, causing poor growth performance.

Due to these advantages, the implementation of the present invention led to zootechnical results that were previously unparalleled in animal husbandry.

Below are the results of several zootechnical studies that were carried out using different species and on different farms, which allows you to test the technical advantages obtained using this invention in production conditions.

a) Zootechnical test on laying hens.

On the experimental farm, for about 3 months, laying hens (Isa Brown breed) were fed with a 15% mixture containing 90% common bean seed and 10% flaxseed, prepared in various ways, with predetermined ME and DUC N values.

Given the chosen methodology used to formulate laying hen feeds, the Isonutrients diet (metabolic energy, digestible essential amino acids, calcium, phosphorus, etc.), and

- 26 043034 taking into account the previously estimated differences in digestibility values, the purpose of this study was to test whether, due to the synergistic effect of reducing the content of anti-nutritional factors (the influence of which is not linked to digestibility indicators), the implementation of this invention contributed to the achievement of a higher level of zootechnical performance.

Therefore, the table below presents:

nutritional characteristics selected for the common bean obtained according to the invention, on the one hand, and for the standard common bean, which was simply subjected to crushing; moreover, these values were determined in advance according to the study of their digestibility in accordance with conventional protocols known to the expert;

Egg laying performance in terms of egg weight, weight of eggs produced (taking into account the number of eggs laid) and consumption rate (feed efficiency per egg).

Characteristics according to the invention soy flour Bob usually A Unprocessed Bob usually And Proparen. 1 Bob usually And Proparen. 2 Bob usually In Raw Bob usually In Proparen. 2 Seed selection - No No No Yes Yes Technological seed treatment No Yes Yes No Yes Diet characteristics ME in the diet, kcal/kg 2800 Digestible lysine, g/kg 6.9 Characteristics of common bean seeds Protein, g/100 g MS 29.2 29.1 Starch, g/100 g MS 39.7 41.4 Tannins, g/100 g MS 0.82 0.58 Vitsin + convicin, g/100 g MS 0.78 0.11 Alpha-galactosides, g/100 g MS 3.0 2.4 Crude cellulose, g/100 g MS 10.6 7.2 NDF, r/100rMS 18.9 15.4

- 27 043034

Lectins, g/100 g MS 5.2 2.0 Antitryptic factors, TIU/mg 5.2 3.7 Methods for processing seeds of common beans soy flour Bob usually A Unprocessed Bob usually And Proparen. 1 Bob usually And Proparen. 2 Bob usually In Neobrab Bob usually In Proparen. 2 Mixing - No No No No No Fractionation - Yes Yes Yes Yes Yes Pre-heat treatment Temperature - - 60°C - 60°C Humidity - - - 14% - 14% Duration - - - 15 minutes - 15 minutes Heat treatment Temperature - - 90°C 140°C - 140°C Duration - - 20 s 20 s - 20 s Pressure - - 15 bar (1.5 MPa) 30 bar (3 MPa) - 30 bar (3 MPa) Characteristics of Treated Common Bean Seeds IU/seed of common beans, kcal/kg 2813 2983 3068 2890 3313 DUC N/seed of common beans, % 73 75 76 72 79 Lectins, g/100 g DM 5.2 5.2 0.0 2.0 0.0 Antitryptic factors, TIU/mg 2.9 2.4 0.8 3.7 0.9 The productivity of laying hens Weight of eggs, g 61.9 ^bc 62.2 ^ab 62.2 ^ai 62.1 ^ai 62.6 ^a 62.8 ^a Mass of eggs issued, g / day 61.0 ^ab 60.5 ^b 59.7 ^e 61.1 ^ab 60.2 ^b 62.0 ^a Consumption rate 1.92 ^e 1.97 ^ab 2.00 ^a 1.94 ^b 1.97 ^ab 1.92 ^e

Compared to a control batch using soy flour, which already performs very well in terms of digestibility and productivity values, it appears that:

the results obtained using the standard bean seed, which is not obtained according to this invention, are inferior to the control soybean meal, which highlights the negative impact of anti-nutritional factors on egg laying performance of chickens. In fact, the main finding is that the hens in this group had lower egg mass despite having the same or slightly higher single egg mass, which means that they laid fewer eggs, and also that they had to consume more feed to produce the same number of eggs, which is a sign of reduced feed efficiency (increased consumption rate);

the results obtained using standard common bean seed, processed but at a lower intensity than according to the present invention, indicate lower productivity. The processing method applied to the unselected seed is not sufficiently effective in terms of reducing the content of anti-nutritional factors and, consequently, in terms of a significant increase in the nutritional value of the seed;

The results obtained with selected but unprocessed common bean seed show performance that, despite having a higher intrinsic nutritional value than unselected seed, remains below that of the soy flour control lot. The seed of common beans cannot be satisfactorily assimilated by laying hens without technological processing;

although the nutritional value of the common bean produced according to the invention (in the common bean seed study) was already significantly higher than that of the standard common bean, the results obtained using the common bean seed of the invention show an improvement in egg weight and weight issued eggs, without changing the indicator for

- 28 043034 consumption.

This is where the advantage of the selection and the method according to the present invention can be seen. Not only does the protein-rich seed treated in this way have a higher nutritional value in so-called digestibility studies, but its use also limits the harmful effects of anti-nutritional factors and at the same time achieves higher productivity values than in the case of soybean. flour, which are signs of a synergistic effect found at the stage of assimilation by animals.

b) Animal and economic testing on Label Rouge broiler chickens.

On a reference chick farm, 4,400 chicks of the Label Rouge type housed in two identical rooms were fed according to 2 feeding programs. A conventional program based on the use of soy flour is compared with a program based on the protein-rich seeds of the invention and does not contain soy.

Technical and economic indicators were evaluated taking into account consumption, animal weight, growth, feed conversion, mortality and feed cost.

In this study, the evaluation of the nutritional value of the common bean used in the composition of the food was not based on the values of the so-called digestibility, but on the basis of literature values. Therefore, the task was to check whether the zootechnical parameters for these chickens actually exceeded those in the control lot, and if this was the case, then to make calculations on how much higher the nutritional values of common bean seeds should be. This information is presented below.

Characteristics according to the invention soy flour Bob ordinary, steamed Seed selection - Yes Technological seed treatment Yes Diet characteristics ME in the diet, kcal/kg 2800 Digestible lysine, g/kg 6.9 Characteristics of common bean seeds Protein, g/100 g MS 51.6 29.2 Starch, g/100 g MS 5.7 41.8 Tannins, g/100 g MS 0.69 0.49 Vitsin + convicin, g/100 g MS - 0.15 Alpha-galactosides, g/100 g MS 4.0 2.2 Crude fiber, g/100 g MS 5.9 7.3 NDF, r/100rMS 12.5 13.0 Lectins, g/100 g MS 2.4 Antitryptic factors, TIU/mg 20.0 3.0 Methods for processing seeds of common beans

- 29 043034

soy flour Bob ordinary, steamed Mixing - No Fractionation - Yes Pre-heat treatment Temperature - 60°C Humidity - 14% Duration - 15 minutes Heat treatment Temperature - 140°C Duration - 20 s Pressure - 30 bar (3 MPa) Characteristics of Treated Common Bean Seeds IU/seed of common beans, kcal/kg - 3305 DUC N/seed of common beans, % - 78 Lectins, g/100 g DM - 0.0 Antitryptic factors, TIU/mg - 0.8 Chick production figures Age of slaughter, days 82 82 Final weight, kg 2.184 2,349*** Average daily weight gain, g/day 26.63 28.65*** Economic indicator of consumption 2.913 2740*** kg products / m ² 23.46 25.10** Water/feed ratio 1.40 1.43* Productivity indicator 89.3 101.5*** Feeding program cost, EUR/t 295.00 300.80 Total feed cost based on 100% 100 102.63 Feed - chick limit based on 100% 100 121.10

Thus, the technical results obtained thanks to this invention have shown a significant compensation for the additional cost of feed.

Thus, the replacement of soy flour with a product proposed according to this invention, in an amount up to 8% of the feed inclusive, led to a significant improvement in technical (weight + 7.5%, average daily weight gain (GMQ; from the French Gain Moyen Quotidien) +7, 6%, technical consumption index (CI; from the English Consumption Index) -5.9% and productivity index + 13.9%) and economic indicators.

According to the results of these zootechnical tests, it is not possible to explain the improvement in performance only by the digestibility values of the product according to the invention. In fact, despite the fact that the calculated digestibility results were 3313 kcal for metabolic energy and 79% for the protein digestibility coefficient, the figures obtained in this way allow us to estimate an increase in metabolic energy of 11% (i.e. 3675 kcal), and the coefficient protein digestibility by 5% (i.e. 83%).

This is indicative of a synergistic effect associated with improved metabolic performance in the animal, and which may be associated with a reduction in anti-nutritional factors.

c) Testing of zootechnical, ecological and economic indicators on a standard broiler chicken.

On an experimental farm, a study was conducted to determine the growth rates of broiler chickens. This study was performed on cockerels (ROSS PM3) fed a diet in which the main protein sources were soybean meal or unselected and unprocessed common bean seeds (referred to as unprocessed common bean) or bean seeds of the invention (selected and processed processing), labeled as steamed bean.

These protein sources were incorporated up to and including 15% in growth feed formulation (ME: 2950 kcal/kg; digestible lysine: 11 g/kg) and 20% in finish feed formulation (ME: 3000 kcal/kg; digestible lysine: 10 g/kg). These feeds had the same nutritional characteristics as SOYA. Specified zootechnical indicators and determined the impact of these products on the environment

- 30 043034 (ECOALIM) and the economy (economic situation in 2018).

Characteristics according to the invention soy flour Bob ordinary, unprocessed. Bob ordinary, steamed Seed selection No Yes Technological seed treatment No Yes Diet characteristics ME in diet, kcal/kg For growth: 2950 | finish fattening: 3000 Digestible lysine, g/kg For growth: 11 | finish fattening: 10 Characteristics of common bean seeds soy flour Bob ordinary, unprocessed. Bob ordinary, steamed Protein, g/100 g MS 29.2 Yes Starch, g/100 g MS 39.7 43.2 Tannins, g/100 g MS 0.82 0.54 Vitsin + convicin, g/100 g MS 0.78 0.29 Alpha-galactosides, g/100 g MS 3.0 2.4 Crude fiber, g/100 g MS 10.6 8.8 NDF, g/100 g MS 18.9 18.7 Lectins, g/100 g MS 5.2 3.0 Antitryptic factors, TIU/mt 5.2 2.5 Methods for processing seeds of common beans Mixing - No No Fractionation - Yes Yes Pre-heat treatment Temperature - - 60°C Humidity - - 14% Duration - - 15 minutes Heat treatment Temperature - - 140°C Duration - 20 s Pressure - 30 bar (3 MPa) Characteristics of Treated Common Bean Seeds IU/seed of common beans, kcal/kg 2813 3313 DUC N/seed of common beans, % 73 79 Lectins, g/100 g DM 5.2 0 Antitryptic factors, TIU/mt 2.9 0.9 Chick rearing productivity Consumption rate 1.456 1,460 1.444 Environmental indicators Phosphorus intake 100 79 64 Energy consumption from organic residues 100 84 79 Changing of the climate 100 72 59 Acidification 100 82 83 Eutrophication 100 102 99 land use 100 119 114 Economic indicators Feed costs for 100 kg live weight 40.20 euros 39.03 euros 39.45 euros Annual cost of chick consumption 7.64 euros 7.42 euros 7.50 euros

The growth rates of chicks in the soybean meal, raw bean, and parboiled bean groups fed the same nutritional diet were statistically identical. Consumption figures, although not significantly different, show values that, in numerical terms, give

- 31 043034 preference for the common bean according to the invention. The latter has a lower consumption rate (1,444) than soy flour (1,456) and whole bean (1,460) batches. Obtaining identical growth rates observed when standard broilers are fed the same nutritional value in their feeders means that the formulations of these feeds have been evaluated correctly for their nutritional content. These results confirm the content of digestible ME and digestible LYS in the studied common beans, and especially in the beans according to the invention. The authors of the invention emphasize that the synergistic effect of this invention is manifested in relation to the indicators of productivity and the condition of the animals, although to a lesser extent than in hens.

The environmental impact of producing chicks using common bean feed according to the invention is positive in terms of climate change (-41%), phosphorus intake (-36%), energy intake from organic residues (-22%) and acidification (18 %); eutrophication neutral (-2%) and land use negative (+13%). The same environmental impact in the production of chickens using common beans, not related to this invention, is characterized by intermediate values.

The economic effect in broiler production, estimated in terms of the feed-related part, which is about two thirds of the cost of production, is equal to the difference between 40.20 euros and 39.45 euros / 100 kg of live weight, i.e. there is a decrease of 1.9%. Based on the French consumption of chickens, which is 19.0 kg/year, this effect is objectively neutral, since the savings is 0.14 euros/year. The economic effect in the production and consumption of chickens fed with seeds that are not related to this invention is less important.

This study demonstrates the dual benefit of this invention in the selection of common bean seeds and the use of adapted processing. This advantage is due both to the standard broiler chicken's use of the common beans produced according to the invention, and to the favorable environmental and economic impact of the invention for the French consumer. Thus, the introduction of protein-rich seeds obtained according to the invention is confirmed on a standard broiler chicken.

d) Testing of livestock technology on rainbow trout.

In the experimental farm, a study was conducted to determine the growth rates of rainbow trout. The nutritional value of common bean seeds, selected as described in the invention, processed or not processed as described in the invention, and with or without peeling, was previously evaluated on juvenile rainbow trout bred at 17°C in fish farm.

Feeds with the same energy (23-24 kJ/g DM) and nitrogen content (43-45% DM) contained 21% fishmeal and 25% bean seeds to be tested or 25% soybean meal as a protein source. After mixing and pulverizing the fish food raw materials, the fish food was subjected to a thermal steaming process in a twin screw extruder and then subjected to granulation. These feeds were distributed among the groups of trout, without limitation, for 84 days.

Characteristics according to the invention Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Seed selection - Yes Yes Yes Yes Yes Yes Technological seed treatment - No Yes Yes No Yes Yes Diet characteristics ME in the diet, kcal/kg 5360 kcal/kg MS Digestible protein, g/kg 44g/100g DM

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Characteristics of common bean seeds (in whole seed form or after peeling) Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Protein, g/100 g MS 31.4 36.1 Starch, g/100 g MS 38.0 43.6 Tannins, g/100 g MS 0.5 0.4 Vitsin + convicin, g/100 g MS 0.3 0.3 Alphagalactosides, g/100 g MS 2.6 2.9 Crude cellulose, g/100 g MS 7.0 1.4 NDF, g/100 g MS 13.1 5.5 Lectins, g/100 g MS 2.0 2.2 Antitryptic factors, TIU/mg 1.9 2.1 Methods for processing seeds of common beans Mixing No No No No No No Fractionation - Yes Yes Yes Yes Yes Yes Peeling - No No No Yes Yes Yes Preparatory heat treatment Temperature - - - 60°С - - 60°С Humidity - - - 14% - - 14% Duration - - - 15 minutes - - 15 minutes Heat treatment Temperature - - 110°C 140°C - 110°C 140°С Duration - - 20 s 20 s - 20 s 20 s Pressure - - 20 bar (2 MPa) 30 bar (3 MPa) - 20 bar (2 MPa) 30 bar (3 MPa) Characteristics of Treated Common Bean Seeds Protein DUC, % 97.4 83.5 89.3 92.5 91.1 93.5 96.7 DUG energy, % 84.7 56.9 70.8 71.9 74.6 77.1 83.4 Lectins, g/100 g DM - 6.7 2.2 0.2 7.3 6.7 1.3 Antitryptic factors, TIU/mg - 6.6 3.0 2.6 7.2 4.4 2.8 35 24

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Prod) trout farming activity Soev. flour Bob usually Whole. Rough Bob usually Whole. Proparen. 1 Bob usually Whole. Proparen. 2 Bob usually Core Raw Bob usually Core Proparen. 1 Bob usually Core Proparen. 2 Consumption rate 3.17 3.18 3.17 3.18 3.49 3.14 3.20 Relative daily consumption 1.24ab 1.33 ^b 1.26 ^b 1.28 ^ab 1.36 ^ab 1.20 ^bc 1.20 ^a Feed efficiency 1.13ab 1.05 ^a 1.11 ^ab 1.11 ^ab 1.11 ^ab 1.14 ^ab 1.19 ^b Protein Utilization Efficiency 2.65ab 2.52 ^ab 2.69 ^a 2.67 ^ab 2.58 ^ab 2.73 ^ab 2.88 ^b

No difference in zootechnical performance compared to the SOY control was found, however, processing methods have a significant impact on feed intake and feed efficiency in the case of feed containing seeds according to the invention: (1) pre-treatment and heat treatment, which described in this invention improve feed efficiency and protein efficiency associated with reduced feed intake; (2) peeling combined with heat treatment also improves feed efficiency.

In the context of this study, feeding 25% of common bean seeds prepared according to the invention, as compared to soybean meal and in compositions with the same nutritional value, achieved the same levels of trout growth performance. And it is even more likely that certain preferred combinations of processing methods have had a positive effect on common bean seeds by improving their feed efficiency to the extent that they are superior to soybean meal. More generally, this example shows the synergistic effect of the invention in terms of digestibility that the animal was able to demonstrate in the case of such selected and treated seeds.

Thus, these results indicate that the common bean seeds produced according to the invention are good candidates for replacing soybean meal in trout feeds. In addition, although aquafeeds are prepared by steam extrusion using heat treatment under pressure, it appears that the method of the invention applied to whole seeds of common beans or after removal of their skins provides real added value in order to improve the nutritional value of these seeds.

f) Study of zootechnical indicators on slaughter pigs.

On a model farm for fattening pigs, the inventors compared the growth rates of two groups of pigs whose diet differed only by the addition of the product obtained according to this invention.

Growth and finisher foods in both groups had intake values with the same nutritional value (in terms of net energy and digestible amino acids). The specified product was presented 10% inclusive in the growth phase and 5% in the final phase instead of protein-containing cakes and oilseeds.

Since the nutritional values of the common bean used in the feed formulation were based on digestibility values, the aim was to test whether the animals' zootechnical performance was the same and, if higher, to see if a synergistic effect was possible associated with improvement reflected in the metabolic rate of the animal.

The table below shows the received data related to technical indicators.

Characteristics according to the invention soy flour Bob ordinary, steamed Seed selection - Yes Technological seed treatment Yes Diet characteristics soy flour Bob ordinary, steamed

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ME in the diet, kcal/kg For growth: 2300 | finish fattening: 2150 Digestible lysine, g/kg For growth: 7.5 | finish fattening: 6.5 Characteristics of the tested raw materials Protein, g/100 g MS 29.2 Starch, g/100 g MS 41.8 Tannins, g/100 g MS 0.49 Vitsin + convicin, g/100 g MS 0.15 Alpha-galactosides, g/100 g MS 2.2 Crude cellulose, g/100 g MS 7.3 NDF, r/100rMS 13.0 Lectins, g/100 g MS 2.4 Antitryptic factors, TIU/mg 3.0 Methods for processing seeds of common beans Mixing - No Fractionation - Yes Pre-heat treatment Temperature - 60°C Humidity - 14% Duration - 15 minutes Heat treatment Temperature - 140°C Duration - 20 s Pressure - 30 bar (3 MPa) Characteristics of Treated Common Bean Seeds IU/seed of common beans, kcal/kg 3305 DUC N/seed of common beans, % 78 Lectins, g/100 g DM 0.0 Antitryptic factors, TIU/mg 0.8 Pig rearing efficiency Average daily weight gain, g/day 773 804** Economic indicator of consumption 2.78 2.80*

The technical data reported in this study show an increase in average daily weight gain due to a more satisfactory intake and an equally satisfactory additional effect in terms of weight gain per kilogram of feed.

This indicates a synergistic effect between the good digestibility of the seeds on the one hand and the positive effect on consumption on the other hand, the latter effect being possibly related to the reduction of anti-nutritional factors that are regularly reported to negatively affect food intake, in particular due to the digestive disorders they cause.

As mentioned above, the consequences of the digestion of nutrients such as protein and energy sources can be manifested in animals after their consumption of vegetable raw materials, depending on whether it is or is not a raw material according to this invention. Previously published work reported a decrease in performance as a result of changes in physiological functions and/or digestibility. Among these health-related changes, allergic reactions may occur. For protein-rich seeds, many cases of such reactions have been documented after consumption of these seeds due to the presence of major allergens in these seeds.

f) Allergenicity testing in dogs.

The aim of this work is to study the allergenicity of untreated or treated common bean seeds under different conditions. Allergenicity (or reactivity) was assessed by immunoblotting using serum samples from dogs with food sensitivities.

After extracting the protein from the seeds and collecting sera from food-sensitive dogs, those that react with the formation of immunoglobulin type E, or IgE, to protein-rich seeds, the reactivity is studied by immunoblotting. The extracted proteins are incubated in the presence of serum samples containing IgE. If an allergenic protein is present, then it binds to IgE in

- 35 043034 serum, and a specific zone appears on the membrane, as shown in the drawing.

Characteristics according to the invention soy flour Bob is ordinary. Solid Rough Bob is ordinary. Whole Steamed Bob is ordinary. Core Steamed Seed selection - Yes Yes Yes Technological seed treatment No Yes Yes Characteristics of common bean seeds (in whole seed form or after peeling) Protein, g/100 g MS 29.2 36.1 Starch, g/100 g MS 41.8 43.6 Tannins, g/100 g MS 0.49 0.4 Vitsin + convicin, g/100 g MS 0.15 0.3 Alpha-galactosides, g/100 g MS 2.2 2.9 Crude cellulose, g/100 g MS 7.3 1.4 NDF, g/100 g MS 13.0 5.5 Lectins, g/100 g MS 2.4 2.2 Antitryptic factors, TIU/mg 3.0 2.1 Methods for processing seeds of common beans Mixing - No No No Fractionation - Yes Yes Yes Preparatory heat treatment Temperature - - 60°С 60°С Humidity - - 14% 14% Duration - - 15 minutes 15 minutes Heat treatment Temperature - - 140°С 130°С 20 s Duration - - 20 s Pressure - - 30 bar (3 MPa) 25 bar (2.5 MPa) Characteristics of Treated Common Bean Seeds soy flour Bob is ordinary. Solid Rough Bob is ordinary. Whole Steamed Bob is ordinary. Core Steamed Lectins, g/100 g DM 6.7 0.2 1.3 Antitryptic factors, TIU/mg 6.6 0.8 2.8

Untreated common bean seeds show many very intense zones, i.e. strong reactivity to allergens. On the other hand, the seeds obtained according to the invention from whole seeds or from seeds after peeling, do not show any reactivity (no visible zones). Apparently, the IgE reactivity inherent in these seeds is significantly reduced or even suppressed after the end of the application of the method according to the invention.

g) Testing of zootechnical parameters in dairy cows.

A study was conducted on an experimental dairy farm to investigate the effect of the present invention on the protection of common bean and lupine seed proteins in the rumen and on their intestinal digestibility.

This study was performed on eight Holstein dairy cows with a cannula inserted into the rumen. The plan of the experiment was a double Latin square 4x4. The cows were fed twice a day with a diet consisting of 60% fodder plants and 40% concentrates. Forage plants were a mixture of corn silage (33% ration DM), cereal silage (17%), hay (10%) and dry beet pulp (10.75%). The composition of the concentrate included ground corn and soy flour for control diets. The cake was replaced in accordance with the indicated treatments with whole bean seeds, respectively, white lupine seeds, untreated (BOB

- 36 043034

WHOLE - RAW. and LUPIN-Whole. - RAW.) or processed according to the invention (BOB-Whole - INVENTION and LUPIN-Whole - INVENTION) or processed according to an alternative method in which the processing temperature is outside the recommendations of this invention (BOB-Whole - ALTERN. and LUPIN- WHOLE - ALTERN.). Diets were prepared equal in terms of total nitrogen (TN) (146 g/kg DM) and net energy source (0.99 milk feed units (from French Unite Fourragere Lait; UFL)/kg DM) and the concentrate provided 40% TN from diet.

Characteristics according to the invention SOYA BOBZELNNEPROCESSED. BOBLI ELNALTERN. BOBGOALINVENTION LUPINZELLN UNPROCESSED. LUPINZELNALTERN. LUPINZELINVENTION Seed selection Soev. flour Yes Yes Yes Yes Yes Yes Technological seed treatment No No Yes No No Yes Diet characteristics TN, g/kg MS 146 Net energy, U FL/kg MS 0.99 Intestinal Digestible Protein (PDI), g/kg 92 80 99 91 80 100 92 Rumen protein balance (BalProRU), g/kg 3.47 14.82 -2.33 5.5 15.54 -2.61 5.71 Digestible protein from food (PDIF), g/kg 44 thirty 51 42 thirty 52 42 Rumen-degradable starch, g/kg 155 162 153 153 123 123 123 Digestible lysine, % of PDI 6.82 7.18 7.09 7.1 6.85 6.51 6.44 Digestible methionine, % of PDI 2.53 2.62 2.29 2.38 2.67 2.31 2.42 Characteristics of common bean or lupine seeds SOYA BOBZELNNEPROCESSED. BOBTSELNALTERN. BOBGOALINVENTION LUPINZELLN UNPROCESSED. LUPINZELNALTERN. LUPINZELINVENTION Protein, g/100 g DM 51.6 29.3 33.3 Starch, g/100 g DM 5.7 39.1 - Fat, g/100 g DM 1.6 7.0 13.3 Tannins, g/100 g DM 0.69 0.8 - Vitsin + convicin, g/100 g DM - 0.10 - Alkaloids, g/100 g DM - - 0.02

- 37 043034

Alphagalactosides, g/100 g DM 4.0 2.3 0.93 Crude fiber, g/100 g DM 5.9 10 12.9 NDF, r/100rDM 12.5 18.7 18.3 Lectins, g/100 g DM - 66.7 - Antitryptic factors, TIU/mg 20.0 2.5 - Seed Treatment Methods Mixing - No No No No No No Fractionation Pre-heat treatment Yes Yes Yes Yes Yes Yes Temperature - - 60°C 60°С - 60°С 60°С Humidity - - 14% 14% - 14% 14% Duration Heat treatment - - 15 minutes 15 minutes - 15 minutes 15 minutes Temperature - - 160°C 140°C - 160°С 140°С Duration - - 20 s 20 s - 20 s 20 s Pressure - - 40 bar (4 MPa) 30 bar (3 MPa) - 30 bar (3 MPa) 20 bar (2 MPa) Characteristics of the treated seeds Lectins, g/100 g DM - 66.7 28.6 13.3 - - - Antitryptic factors, TIU/mg - 2.5 1.0 0.9 - - - thirty 82 35 35 84 53 47 Acrylamide, g/100 g DM 34 35 39 69 34 507 175 CML, g/100 g DM 0.123 0.008 0.023 0.017 0.009 0.029 0.023 Indicators for treated bean seeds ordinary BEAN- BOBTSELNAL- BEAN- Lumpmi LU- LU- P 1/11—1 SOYA WHOLE- WHOLE- I 1i1pTSELNNE- PINZELN- 1 1IP- WHOLE- PROCESSED. TERN. SHAVING OBR- ALTERN. BRE- LO I AN. SHADOW 0.63 True (bean) 0.64 0.63 0.66 0.68 0.64 0.68 N digestibility, g/g /0.66 (loop) Total amino acid content, µM (in plasma) 2662 (bob) / 2642 (loop) 2401 2858 2985 2608 2411 2921 Irreplaceable 1034 (bob) / 956 (loop) amino acids, μM (in plasma) 898 1125 1164 774 914 1024 119 _NH3 , mg/l (bean) 145 130 133 140 125 140 (in rumen juice) /120 (loop) Milk protein yield: detected - expected, g/day 11.3 (bob) / -2.7 (loop) -65.0 -56.9 5.1 -68.6 -67.9 -29.7

Lower levels of NH ₃ in the rumen with seeds according to the invention than with untreated seeds demonstrate protection of nitrogen compounds from ruminal degradation increasing with increasing treatment temperature, while soybean meal results in the lowest values. This protection of nitrogen-containing compounds due to technological treatment is also confirmed by the fact that when treated for 1 hour with enzymes, their degradability in seeds obtained according to this invention is reduced compared to untreated seeds.

The content of Maillard compounds in seeds obtained according to this invention is higher than in untreated seeds, in particular with a more pronounced effect on the content of acrylamide and

CML in the case of lupine.

In addition, the content of CML in soy flour is four to seven times higher than that in the seeds obtained according to the invention.

In the bean experiment using the Latin square technique, fecal CML was found to be lower in the bean diet than in the soy flour control diet. Quantitatively, a higher content of Maillard compounds was found in the faeces when using seeds from alternative diets than seeds according to the invention. This means that in alternative processes Maillard reactions were more likely to reach an irreversible step in the abomasum, resulting in overprotection of the nitrogen compounds.

This is evidenced by the decrease in true nitrogen uptake observed when using seeds from alternative methods compared to seeds according to the invention. In addition, the higher plasma amino acid content when using seeds obtained according to the invention, compared with seeds from alternative methods on the one hand, and soy flour on the other hand, demonstrates better digestion in the intestine of proteins from seeds obtained according to the invention. ; thus, there is a lower availability to the animal of proteins from seeds produced according to alternative methods or proteins from soybean meal, since a higher proportion of the latter will avoid not only degradation in the rumen, but also degradation and absorption in the intestine.

These results confirm that the Maillard reactions proceeded after the implementation of the methods of technological processing of seeds of common beans and lupine in various ratios. In the case of the seeds obtained according to the invention, these reactions provide protection of nitrogen-containing compounds from excessive digestion in the rumen and are reversible under acidic pH values of the abomasum, thus allowing optimal absorption of amino acids in the small intestine. On the other hand, after treatment at high temperature (160° C.), these reactions are no longer reversible in the abomasum, and the proteins, thus overprotected, are no longer absorbed in the intestine.

Finally, in zootechnical terms, the seeds obtained according to the invention are the only ones that can compete with soybean meal, since they provided an opportunity to express the potential for the amount of milk protein produced by cows. This does not apply to untreated seeds and seeds from alternative methods.

h) Study of the economic advantages of the method of the present invention.

In order to conduct this economic analysis, a feed composition software was used with relevant information on digestible raw materials, nutritional value of raw materials, prices for these raw materials, and nutritional restrictions on feeds for broilers and laying hens at different stages. physiological development.

Thus, after collecting information regarding the nutritional value and possible prices of the best combinations according to the invention, this invention was evaluated for economic feasibility.

Using the same approach, it was also possible to estimate prices for raw materials of interest, developed on the basis of the best combination of methods implemented according to this invention. And based on this, it was found that the invention was quite economically relevant, especially considering the formulation of compositional restrictions associated with specific specifications (specifications related to the obligation to have raw materials in the composition of feed compositions that do not contain GMOs, are not imported or are raw materials of local production).

Below are the data on three feed compositions for broiler rearing, demonstrating the economic benefits, taking into account the product obtained according to the invention (mixtures of seeds of ordinary beans and soybeans in the ratio of 90% and 10%, respectively), due to its technical and economic priority in terms of its inclusion in terms of optimization in the composition of non-GMO compositions, in comparison with known types of raw materials of the prior art (in this case, soy flour and cereals).

Data comparison table for three non-GMO broiler feed compositions, one prepared without the product of the invention and two prepared with the base prepared according to the invention.

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Standard composition, WITHOUT used, of the proposed product according to the invention Study composition 1, C used, of the proposed product according to the invention Study composition 2, C used, of the proposed product according to the invention The composition of the compositions Non-GMO soy flour 21.0% 8.3% Product according to the invention (90% common bean seed + 10% soybeans) 13.5% 20.0% Rapeseed cake 6.3% 8.0% Wheat 41.9% 45.0% 45.0% Barley 11.1% Corn 25.0% 14.3% 1.3% Gluten corn 5.2% 5.7% 7.6% Soybean oil 4.0% 4.0% 4.0% Minerals and vitamins 2.3% 2.3% 2.3% Amino acids 0.6% 0.6% 0.7% Nutritional characteristics exchange energy 3100 kcal 3100 kcal 3100 kcal Protein 19.5% 19.5% 19.5% Digestible lysine 10.3 g/kg 10.3 g/kg 10.3 g/kg Calcium 0.79% 0.79% 0.79% Phosphorus 0.40% 0.40% 0.40% Cost price production 289.5 euro/ton 287.1 euro/ton 288.5 euro/ton

Through this composition experiment, it can be seen that the amount of the product according to the invention in the non-GMO broiler chicken feed composition is optimized at 13.5% in test composition 1, resulting in a rapeseed meal content of 6.3% and a significant reduction in soy flour and corn flour by 12.7% and 10.3%, respectively.

In addition, if we strive for the complete removal of non-GMO soy flour from the studied composition 2, then the amount of the product according to the invention will be 20%, while the cost of this composition, compared to the original standard composition, will be lower by 1 euro per one ton.

This study of the compositions demonstrates the technical and economic feasibility of the invention.

Finally, in terms of applications, the method of the present invention aims to promote the inclusion of protein-rich seeds in the diet of monogastric animals as a substitute for other protein sources such as soy flour or other imported flour, and therefore to satisfy the needs of livestock breeders to gain more autonomy in the production of feed proteins at the territorial level, as well as the needs of consumers in relation to the provision of more environmentally friendly animal products, while animal feed should not contain GMOs, and food products should be locally produced.

The scope of the method of the present invention may relate to two types of application for animal husbandry.

Application for the preparation of raw materials.

Preparation of a concentrate based on high protein cultures as a raw material for inclusion in the composition of a complete or supplementary feed for monogastric animals and intended for industrial and / or agricultural producers. In this case, the minimum application rate of said selected seeds is 20%, preferably 40%.

Other raw materials included in this concentrate may be subjected to all stages or parts of the stages of the specified method according to the invention, especially if this gives an advantage to these types of raw materials.

Thus, it is preferred that the feedstock be seeds or any other starchy product such as cereals and oilseeds, in a non-limiting manner.

Application for the preparation of a feed product.

Preparation of complete or supplementary cereal-based feed for livestock breeders to feed their monogastric animals. In this different case, the minimum application rate of said selected seeds is 5%, preferably 10%.

In addition, the food produced according to the invention differs according to the needs of food producers and livestock producers, depending on whether they are positioned as non-GMO, whether they are products of local production, French sectors or sectors for the production of soy-free products, on the one hand, or for the sector called Bleu Blanc Coeur (registered trademark) on the other.

Indeed, with regard to the protein requirements of the French, it is preferable to process common beans along with soybeans grown within the metropolis. Taking into account compliance with the requirements of Bleu-Blanc-Coeur, the inventors are interested in combining them with flax seeds.

In the first case, soy is used to increase the protein content in the product. In the second case, the presence of omega-3 fatty acids, which are monitored and guaranteed, is provided by flaxseed. This approach has the advantage, within the scope of food producers, that there is no need for additional storage, but a replacement with another flax-based product is necessary, which often involves the purchase of raw materials without great technical advantages (wheat bran, cereals, etc.). .d.).

Here is an example of compositions.

1) Formulations for non-GMO/protein-containing local food chains:

at the rate of 90% seeds of common beans and 10% soybeans; at the rate of 70% common bean seeds and 30% soybeans.

2) Compositions for the Bleu-Blanc-Coeur approach:

at the rate of 75% common bean seeds and 25% linseeds;

at the rate of 50% common bean seeds and 50% flaxseeds;

at the rate of 25% common bean seeds and 75% flaxseeds.

The seeds obtained according to the invention may also be useful for pets and ruminants. Although they have been developed for feeding monogastric farm animals, seeds treated according to the invention are fully suitable for feeding domestic animals such as dogs and cats and ruminants.

In a preferred method of use for ruminants, the interest in using, on the one hand, carbohydrate-degrading enzymes, and, on the other hand, sources of so-called reducing sugars can be retained. Indeed, in addition to the thermal effect, one of the ways to protect the protein from its breakdown in the rumen while improving its digestibility in the intestine is: 1) adding, at the stage of the method consisting in hydrothermal and enzymatic pre-treatment, enzymes capable of hydrolyzing carbohydrates to glucose units or other simpler reducing sugars that are protein reactive; and/or 2) in the selection at an additional stage based on the use of additional raw materials, sources of reducing sugars to some extent.

It is known to the skilled person that in order to assimilate protein-rich seeds in ruminants, it is necessary, in particular, to reduce the degree of cleavage of their protein in the rumen, and that one way to achieve this is to stimulate the first stages of the Maillard reaction between the amino functional groups of proteins and the reducing functional groups of sugars.

The purpose of this invention is to create new conditions for the implementation of these reactions, which are reversible, which allows for excellent digestibility of proteins in the intestine.

Also of interest is the use of the seeds obtained according to the invention in the context of pet food. On the one hand, the protein-rich seeds obtained in this way provide a source of varied and highly digestible protein and energy, characterized by a lower content of anti-nutritional factors, and a protein source with reduced allergic potential, on the other. Indeed, the skilled person knows that due to the biochemical reactions taking place in one of the heat treatment steps of the method, the risk of developing allergic reactions is significantly reduced (Franck et al., 2008).

- 41 043034

Finally, the application of this method could also be extended to human food markets due to the added nutritional value that vegetable proteins provide, and their consumption by humans in the diets of the population of developed countries is now expected to increase, especially since the risk of developing allergic reactions from the use of these vegetable proteins is reduced.

Indeed, the French National Agency for Food Safety ANSES (from the French Agence nationale de securite sanitaire, de I'alimentation, de I'environnement et du travail) recommended that the balance between animal and vegetable protein sources in the human diet be restored and in this way to move from 70/30 ratio to 50/50 ratio.

The nutritional limitations known to monogastric animals for protein-rich seeds, otherwise known as legumes, are the same as for humans, since they too have a monogastric stomach. That is why the inventors believe that this invention is ultimately intended for both the animal industries (for raising monogastric and ruminant animals) and for industries related to the conversion of vegetable proteins for direct human consumption.

Claims (17)

1. A method of treating protein-rich seeds to increase their food value, said seeds being selected from at least one of the following seeds: common bean (Vicia fava L.), pea (Pisum sativum L.), white lupine (Lupinus albus L.), blue lupine (Lupinus angustifolius L.) and yellow lupine (Lupinus luteus L.), characterized in that it includes the following successive stages: a) the use of seeds from at least one of the above plant species, provided that they have a protein content and/or starch content and/or fat content greater than or equal to the values indicated below, g/100 g dry matter (DM) : common bean (Vicia fava L.): protein - 28, starch - 39; peas (Pisum sativum L.): protein - 22, starch - 45; white lupine (Lupinus albus L.): protein - 35, fat - 8; blue lupine (Lupinus angustifolius L): protein - 31, fat - 5.5; yellow lupine (Lupinus luteus L.): protein - 38, fat - 5.0; and contain at least one of the compounds selected from the following group: anti-nutritional factor (ANF), crude cellulose, neutral detergent fiber (NDF), in an amount that is at a lower level than the values below, g/100 g DM: common bean (Vicia fava L.): tannins - 0.3, vicin, convicin - 0.5, alpha-galactosides - 2.5, - 44 043034 crude cellulose - 10, NDF - 18; peas (Pisum sativum L.): tannins - 0.01, alpha-galactosides - 5, crude cellulose - 7.5, NDF - 18; white lupine (Lupinus albus L.): alkaloids - 0.1, alpha-galactosides - 10, crude cellulose 16, NDF - 25; blue lupine (Lupinus angustifolius L.): alkaloids - 0.2, alpha-galactosides - 9, crude cellulose - 18, NDF - 29; yellow lupine (Lupinus luteus L.): alkaloids - 0.3, alpha-galactosides - 12, crude cellulose 18, NDF - 29; b) subjecting the seeds to pressure from step a) for more than 10 seconds at a minimum pressure of 10 bar (1000 kPa) until a temperature above 80° C. is reached; and/or b1) heating the seeds for a minimum of 15 minutes at a temperature in excess of 80°C. 2. The method according to claim 1, characterized in that after the implementation of the specified stage a) they are subjected to fractionation. 3. Method according to claim 1, characterized in that after said step a) and if seeds of different species and/or with different content of protein, starch, fat, anti-nutritional factor, crude cellulose or neutral detergent fiber (NDF) are used , they are mixed and fractionated or fractionated and then mixed. 4. The method according to one of claims 1 to 3, characterized in that before step b) the step of preparatory heat treatment of the seeds is carried out using steam and/or water-based liquid until temperatures in the range of 30 up to 90°C and a humidity of more than 12%, preferably 15%, for a period of more than 2 minutes, preferably 15 minutes. 5. The method according to claim 4, characterized in that the stage of preparatory heat treatment is carried out in the presence of at least one exogenous enzyme identified from the following families: arabinofuranosidases, beta-glucanases, cellulases, glucoamylases, pectinases, pectinmethylesterases, phytases, proteases, xylanases , and preferably xylanases, beta-glucanases and pectinases, wherein said exogenous enzyme is added to the seeds or to the mixture in advance. 6. The method according to claim 5, characterized in that in the stage of pre-heat treatment in the presence of an exogenous enzyme, the moisture value is set at a level of more than 15%, preferably 25%, and the pre-heat treatment is carried out for at least 15 minutes, preferably 60 minutes . 7. Process according to one of claims 4 to 6, characterized in that the mixture is stirred during said preparatory heat treatment step. 8. Method according to one of claims 3 to 7, characterized in that when mixing is carried out and then fractionation, a new mixing is carried out after said fractionation. 9. Method according to one of claims 2 to 8, characterized in that said fractionation is continued until at least 90% of said seeds have a particle size of less than 2000 µm, preferably less than 1500 µm. 10. Process according to one of claims 3 to 9, characterized in that said step b1) is carried out on said mixture. 11. The method according to one of claims 1 to 10, characterized in that the implementation of step b) or b1) is stopped if the content of at least one of the anti-nutritional factors listed below is lower than the value also listed below : common bean (Vicia fava L.): lectins - 0.10 g/100 g of raw material, antitriptic factors 1.50 TIU/mg of raw material; pea (Pisum sativum L.): lectins - 0.10 g/100 g of raw material, antitriptic factors - 1.50 TIU/mg of raw material. 12. A method according to one of claims 1 to 11, characterized in that after step a), said seeds are shelled and/or peeled. 13. The method according to one of claims 1 to 12, characterized in that after said step a) or after said shelling and/or peeling step, said seeds are specially fractionated and separated according to a criterion selected from size, weight, shape , density, aerodynamic, colorimetric or electrostatic parameter. 14. A method according to one of claims 1 to 13, characterized in that after or before step a) said seeds are sorted according to a criterion selected from size, weight, shape, density, aerodynamic, colorimetric or electrostatic parameter. 15. The method according to one of claims 1 to 14, characterized in that said seeds are mixed with at least one of the other types of raw materials selected from the group consisting of oilseeds and their co-products, oils, co-products of protein-rich seeds, cereals and their co-products, sources of simple and complex carbohydrates and oilseed cake. 16. The method according to one of claims 1 to 15, characterized in that said raw material is a source of lipids, preferably oilseed. 17. The method according to one of claims 1 to 16, characterized in that it includes a final step during which said seeds are cooled.