阅读说明：本技术 用于处理高蛋白质籽粒以提高其作为食品的使用的方法 (Method for treating high protein kernels to enhance their use as food products ) 是由 G·谢诺 M·吉耶维克 A·热尔曼 H·朱安 M·莱西雷 P·沙普托 P·诺齐埃 C· 于 2018-11-20 设计创作，主要内容包括：本发明涉及用于处理高蛋白质籽粒的方法,所述籽粒选自以下籽粒中的一种：蚕豆、豌豆、白色羽扇豆、蓝色羽扇豆、黄色羽扇豆；其特征在于,所述方法特别包括以下连续步骤：a)使用至少一种上述植物物种的籽粒,条件是这些籽粒具有的蛋白质和/或淀粉和/或脂肪含量的值不低于预定值,并且使用含量低于预定含量的以下群组中的至少一种化合物：抗营养因子(FAN)、粗纤维、中性洗涤纤维(NDF)；b)将步骤a)的籽粒置于至少10巴的压力下,持续超过10秒钟,直至获得高于80℃的温度。(The present invention relates to a method for treating high protein grain selected from one of the following: broad bean, pea, white lupin, blue lupin, yellow lupin; characterized in that said method comprises in particular the following successive steps: a) using kernels of at least one of the above mentioned plant species, provided that they have a value of the protein and/or starch and/or fat content not lower than a predetermined value, and using at least one compound of the following group in an amount lower than the predetermined content: anti-nutritional Factors (FAN), crude fiber, Neutral Detergent Fiber (NDF); b) subjecting the kernel of step a) to a pressure of at least 10 bar for more than 10 seconds until a temperature above 80 ℃ is obtained.)

1. A method for treating protein-rich seeds to increase their value as a foodstuff, in particular as a foodstuff for animals, the seeds being selected from at least one of the following seeds: vicia faba L, Pisum sativum L, Lupinus albus L, Lupinus angusticus L, Lupinus albus L, and Lupinus luteus L,

characterized in that the method comprises the following successive steps:

a) seeds of at least one of the above plant species are used, provided that said seeds have values of protein content and/or starch content and/or fat content greater than or equal to the values indicated in the following table:

and, at least one compound from the following group is used at levels below those shown in the following table: anti-nutritional Factor (FAN), crude fiber, Neutral Detergent Fiber (NDF):

b) pressurizing the seeds in step a) for more than 10 seconds at a pressure of at least 10 bar until a temperature of more than 80 ℃ is reached;

and/or b1) heating the seeds at a temperature exceeding 80 ℃, preferably 90 and 150 ℃ for a time of at least 15 minutes, preferably 30 minutes to 2 hours.

2. The method of claim 1, wherein after performing step a), the seeds are graded.

3. The method according to claim 1, characterized in that after performing said step a), if seeds of different species and/or seeds with different composition in terms of protein, starch, fat, anti-nutritional factors, crude fiber or Neutral Detergent Fiber (NDF) are being treated, said seeds are mixed and fractionated, or fractionated and then mixed.

4. Method according to any one of claims 1 to 3, characterized in that, before step b), a heating step of the prepared seeds is carried out with steam and/or a water-based liquid, for a time greater than 2 minutes, preferably 15 minutes, until a temperature between 30 and 90 ℃ and a humidity greater than 12%, preferably 15%, are obtained.

5. The method of claim 4, wherein the preparatory heating step is carried out in the presence of at least one exogenous enzyme identified in the following families: arabinofuranosidase, beta-glucanase, cellulase, glucoamylase, pectinase, pectin methyl esterase, phytase, protease, xylanase, preferably xylanase, beta-glucanase and pectinase, the exogenous enzyme having been previously added to the seed or mixture.

6. The method according to claim 5, characterized in that in the preliminary heating step carried out in the presence of exogenous enzymes, the humidity is set to more than 15%, preferably 25%, and the preliminary is allowed to last at least 15 minutes, preferably 60 minutes.

7. A method according to any one of claims 4 to 6, wherein the mixture is stirred while the preliminary heating step is being carried out.

8. The method according to any one of claims 3 to 7, characterized in that when mixing is performed and then fractionation, new mixing is performed after the fractionation.

9. The method according to any one of claims 2 to 8, wherein the fractionation is continued until at least 90% of the seeds have a particle size of less than 2000 microns, preferably less than 1500 microns.

10. The method according to any one of claims 3 to 9, characterized in that said mixture is subjected to said step b 1).

11. Method according to any one of the preceding claims, characterized in that the execution of step b) or b1) is interrupted if the content of at least one antinutritional factor in the table below has a value lower than the value also shown below:

12. the method according to any of the preceding claims, wherein after step a) the seeds are dehulled and/or de-coated.

13. The method according to any one of claims 1 to 12, characterized in that after said step a) or after said dehulling and/or decoating step, said seeds are subjected to a specific fractionation and separation according to criteria selected from the group consisting of size, weight, shape, density, aerodynamic, colorimetric or electrostatic parameters.

14. The method according to any one of the preceding claims, characterized in that after or upstream of step a) the seeds are classified according to criteria selected from size, weight, shape, density, aerodynamics, colorimetric or electrostatic parameters.

15. A method according to any of the preceding claims, characterized in that at least one other raw material is mixed with the seed, said other raw material being selected from the group consisting of oilseeds and by-products thereof, oils, protein-rich seed by-products, cereals and by-products thereof, simple and complex carbohydrate sources, oilseed cakes.

16. Method according to the preceding claim, characterized in that the starting material is a lipid source, preferably an oleaginous seed.

17. The method according to any of the preceding claims, characterized in that it comprises a final step of cooling the seeds.

Technical Field

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

Background

Feeding of monogastric livestock, such as broiler chickens and egg-laying poultry, pigs and ruminants is highly dependent on the inclusion of soy flour in the ration. This material is mainly from south america and is not without difficulties in terms of protein autonomy and sustainability due to competitive issues particularly associated with strong social and environmental expectations.

Soybean meal has become a widespread protein source for animal feed due to its high protein content. Today, french uses soybean meal mainly by import and transgenics, and traceability is questioned (Gourdouvelis et al, 2012).

At the same time, new social expectations are emerging in france (food diversity, product origin, environmental impact, product quality), and the use of soybeans in animal production is now threatened by the risk of new social crises (delaneaue et al, 2015).

In animal husbandry, meeting protein requirements is crucial to achieving animal technical performance goals. However, nearly half of the protein-rich raw materials used in animal nutrition in france are imported, most of which correspond to over 300 million tons of soybean meal per year (Bouvarel et al, 2014).

Relative scarcity and fluctuations in soybean prices are affecting farmers' income due to increased asian imports. One of the main challenges of animal production is therefore to reduce the dependence on soybean flour, and therefore to find alternatives to reduce the need to import soybean flour (Bourin and Bouvarel, 2015), while ensuring economic profitability of the farm, in particular by trying not only to optimize the performance of its production tools, but also to make more full use of its products.

Furthermore, for pets such as dogs and cats, their owners are very concerned with the quality of the food they purchase, in particular to confer them vitality and protect them from certain metabolic and digestive diseases or allergies.

Proteins of animal origin in western countries cover two thirds of the protein requirements of humans. However, new vegetarian requirements are emerging and it is becoming increasingly important to introduce protein-rich seeds directly into the human diet. Soybeans account for the majority of this supply, but for the same physiological reasons as animals, humans should be able to obtain a vegetable protein source in the form of seeds with improved nutritional value. Thus, the following description of animals also corresponds to humans. Hereinafter and in the claims, it must be understood that humans belong to monogastric mammals.

In france, the crop lacks diversity, mainly cereals, whose protein yield is guaranteed by the use of nitrogen fertilizers and pesticides (messean et al, 2014). Thus, farmers are looking for high-efficiency, sustainable and feasible farming systems thanks to new crop rotation primary crops that consume less input (nitrogen fertilizer, pesticides) and are cost-effective.

The issues to meet the multiple expectations of consumers and citizens face many challenges, namely:

sustainable diets with nutritional benefits, more naturalness and biodiversity and wide availability;

-local sources of agriculture and food production;

-a non-transgenic mode of production;

protection of the environment by reduction of greenhouse gas emissions, use of phytosanitary products, etc.

Finally, every link in the food chain expresses expectations, which can be summarized as follows:

farmers: crop rotation is expanded, crop rotation is diversified, and production systems of the crop rotation are more sound;

livestock and farmers: local production and consumption of protein in animal diets;

the pet owner: a balanced healthy diet;

the consumer: the nutrient is consumed locally, does not contain transgenic organisms and has higher nutrient density.

But there are other aspects that are far from affecting, presented as:

the mechanism is as follows: limiting the use of soybean imports, nitrogen fertilizers and pesticides, increasing the production of French and European plant proteins;

companies in the whole food chain: seeking differentiation and added value in a stressful economic environment;

quasi-agricultural enterprises: constitute a wide range of service and product networks.

Many approaches may provide partial solutions to meet these many desires, including:

for the consumer, for example:

organic production … but its price is not sufficient to satisfy the maximum number of consumers.

For breeders, for example:

grass or legume feed replaces corn silage, but this will only be a partial replacement in many dairy farms.

By-products such as grains or oil cakes also provide additional protein, but must be limited in order to remain highly efficient;

synthetic amino acids represent another form of contribution and strategy, but only address the primary concerns of farmers, not producers and consumers.

For farmers, for example:

some areas have other crop rotation main crops besides cereals and oilseeds, such as beetroot, potato, fiber flax, etc., but they are not sufficient for individual french farmers.

These examples show that each link can provide some solutions separately, but they are still partial solutions with respect to many of the above expectations, and above all without integrating upstream and downstream, which does not bring added value to the products of our farmers and livestock breeders, especially in terms of confirming consumer and citizen interests.

However, the integrated solution seems to have great potential to "meet" our fields (champs), troughs (auges) and trays (assettes).

Indeed, protein crops or legumes can be a beneficial strategy to diversify crops (either alone or in combination with annual cereals) while meeting protein autonomy requirements for livestock farms and regions.

From an agronomic perspective, the integration of legumes in crop rotation is beneficial in both traditional and organic systems and helps to slow down global warming (Magrini et al, 2016; Schneider et al, 2017).

In fact, they are able to fix atmospheric nitrogen in the soil due to the bacteria contained in the nodules of the roots, thus limiting the use of nitrogen fertilizers (half of the agricultural greenhouse gas emissions are caused by nitrogen fertilizers).

Leguminous plants, on the other hand, also have agronomic and economic value by improving plant traits and reducing input costs (Magrini et al, 2016; Schneider et al, 2017). The insertion of legumes is beneficial for the yield of the following cereals. Thus by just maintaining the "yield" and "reducing nitrogen fertilizer" effects, the gross profit of wheat after planting a protein crop (referred to as protein-wheat) is increased by about +160 @/ha compared to wheat-wheat (i.e. wheat crop followed by wheat crop) (terres univia, 2016; Magrini et al, 2016).

Protein-rich seeds are an important source of protein and energy (in the form of starch in the case of peas and beans). Furthermore, these proteins are rich in amino acids (e.g., lysine) compared to cereals, which enhances their value in balancing animal diets.

The following table shows the nutritional value of the protein-rich pea, broad bean and lupin seeds.

MAT ═ total nitrogen

(sources: Table de composition et de valeur fractional des mate, de fre e re pre e i me aue d e.

However, despite their promising nutritional potential, protein-rich seeds are still not fully used in different species due to low digestibility and the presence of many anti-nutritional factors.

Focusing on the energy uptake of proteins from different seeds by monogastric and ruminant animals, it can be quickly seen that there is an important yet unexplored nutritional potential of about 40 to 50% of energy and 20% of protein (according to INRA, 2002).

These protein-rich seeds have a number of anti-nutritional factors that may limit their use. Their presence leads to limitations and poor technical performance of the incorporated food products, which can be summarized as making practical difficulties due to the competitiveness of protein-rich seeds relative to other protein sources (especially in monogastric species).

The reported major anti-nutritional factors are as follows:

tannic acid:

tannins are thermolabile phenolic compounds located in the seed coat and are known to reduce digestibility of proteins by monogastric animals as they bind to proteins before digestion to form insoluble complexes.

The tannin content is related to the colour of the seeds or even the flowers (Myer et al, 2001). The introduction of beans rich in tannins leads to a lower digestibility in vitro (Bond, 1976) and in vivo in poultry, whereas the tannins-free varieties have a higher digestibility with proteins and amino acids (gateway, 1994; Cr é pon et al, 2010). Tannins cause a decrease in the nitrogen content of the ration of monogastric birds, resulting in a decrease in growth rate and feed efficiency (Carr é and Brillouet 1986; Garrido et al, 1988) and a decrease in egg weight (Martin-Tanguy et al, 1977).

Broad bean varieties without tannins exist. However, their agronomic performance is poor and their input into production has been abandoned today.

The tannins content of seeds of fava beans varies widely, with an average value equal to 0.49g/100g dry matter and a maximum value of 1.70.

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

Fabacebroside and adfabacebroside:

the cotyledons or kernels of the seeds of Vicia faba contain Vicia faba pyrimidine glucoside and Vicia faba pyrimidine nucleoside. They are thermostable glycosides (Muduli et al, 1982) and can cause bean poisoning in people with glucose-6-phosphate dehydrogenase deficiency.

Vicia faba pyrimidine glucoside and companion Vicia faba pyrimidine nucleoside have not been shown to affect digestibility of beans by pigs, but they have been reported to cause a decrease in egg weight in laying hens (Lesser et al, 2005 and Gatta et al, 2013) and a decrease in egg production intensity (Muduli et al, 1981). Early studies established that fabaceine glucoside and fabaceine nucleoside reduced egg production by laying hens (Guillaume et al, 1977; Fru-Nji et al, 2007; Olaboro et al, 1981).

Varieties without faba pyrimidine glucosides and with faba pyrimidine nucleosides exist. They have the advantage over varieties without tannins that a quite satisfactory agronomic performance is obtained (Duc et al, 1999).

Levels found in broad bean seeds were 0.02 to 1.49g/100g (Khamassi et al, 2013).

Antitrypsin factor:

trypsin inhibitors and chymotrypsin inhibitors are inhibitors of proteases (and amylases) that bind to trypsin in the small intestine and prevent protein digestion. In mice, pigs, and in chickens (to a lesser extent), their intake is accompanied by an increased loss of endogenous proteins (rich in sulfur amino acids, excreted) in the form of inhibitor-enzyme complexes (Liener 1979). This phenomenon exacerbates the deficiencies of legume seeds in these amino acids, resulting in slow growth of the animal. Lower enzyme concentrations in the small intestine lead to overactivity and hypertrophy of the pancreas. These molecules, which are proteins in nature, are relatively thermolabile.

For pea, trypsin inhibitor is the major anti-nutritional factor, slightly below the 2% protein content (raw soybean contains 8 times this amount). The varieties of antitrypsin factor vary widely. The trypsin inhibitory activity of 33 European spring pea varieties is 1.69 to 7.56 Trypsin Inhibitory Units (TIU), whereas that of winter pea is 7.34 to 11.24TIU (Leterme et al, 1998). For fava beans, the level of trypsin inhibitors is low, with an average of 2.9TIU (0.88 to 6.25).

Lectin:

the group of compounds is widely distributed in the plant world and is very polymorphic. They are heat-sensitive glycoproteins, whose common feature is the affinity for sugars, which explains their in vitro properties of red blood cell agglutination (binding of lectins to glycosyl residues present on the red blood cell wall), depending to varying degrees on the lectin and animal species considered (Liener 1986). They act in the small intestine by binding and destroying epithelial cells to interfere with absorption of the end products of digestion (Dixon et al, 1992). Their uptake leads to growth retardation, which has not been explained clearly to date.

They account for about 2.5% of pea protein (Perrot, 1995) and 2.0% to 13.0% of broad bean protein.

Alkaloid:

early varieties of lupins were considered bitter. They contain toxic alkaloids (lupetanine) and are not recommended for use in animal feed without prior treatment. Thus, it is known that high levels of certain lupin varieties containing these alkaloids cause adverse effects, manifested by reduced growth performance and feed consumption.

However, with advances in genetics, sweet varieties (with low alkaloid content) have been obtained that no longer constitute a palatability problem.

Oligosaccharide:

these materials are small polymers with carbohydrate properties and are heat stable. Poultry and domestic animals do not have alpha-galactosidase, an enzyme necessary for the hydrolysis of the bond between galactose and glucose and between two galactose molecules. These molecules do not cross the intestinal wall and are therefore intact in the colon (where they are metabolized by the microorganisms present), resulting in reduced growth performance due to reduced uptake. The resulting fermentation results in dyspepsia (flatulence, diarrhea) with the potential to slow food intake (Diaz et al, 2006; Kaysi and Melion, 1992). This growth decline is exacerbated as the amount of lupin incorporation is increased. Experimental results in pigs show that oligosaccharides negatively affect the apparent digestion of protein, fat and some minerals. In addition, the large amount of oligosaccharides increases the empty weight of the small intestine. Since organ tissues such as the intestine are metabolically very active, more energy is required to maintain the animal's basal metabolism, resulting in less energy for growth.

White lupin seeds contain 7 to 14% of alpha-galactosides, the most predominant of 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 oligosaccharides depends not only on the variety but also on the growth and harvesting conditions (Pisarikova et al, 2009). There is a correlation between stachyose and verbascose content and flatulence phenomena, whereas raffinose appears to have less effect. It is also present in broad bean and pea seeds, but to a lesser extent, as shown in the following table.

g/100g MS	Stachyose	Cotton seed candy	Verbascose
				Lupine	2.8-5.3	0.4-1.1	1.4-2.0
Pea (Pisum sativum L.)	2.3–2.6	0.5-0.6	2.2-3.4
				Broad bean	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 major form of phosphorus storage in many plant tissues. Non-ruminant animals cannot obtain phosphorus in this form due to the lack of a phytase enzyme that separates the phosphorus in the phytic acid molecule.

Phytic acid is an important chelator of minerals (e.g., calcium, magnesium, iron, and zinc) and can therefore lead to mineral deficiencies. It also binds to a lesser extent to proteins and starches, resulting in a reduced availability of these nutrients in the digestive tract.

The concentration of phytic acid in fava beans is about 0.2% to 0.7% dry weight.

The introduction of phytase into monogastric feed is now widely used and is the primary means of controlling this anti-nutritional factor.

Fiber:

generally, monogastric animals have limited fiber digestibility. Protein-rich seeds have relatively high fiber: the crude fiber in the pea, broad bean and white lupin seeds was 5.2%, 7.9% and 11.4%, respectively. Thus, especially in monogastric livestock such as chickens, piglets and fish, and young pets such as cats and dogs, this fiber is also a notable class of anti-nutritional factors.

This fiber not only has low digestibility, but also causes less digestibility of proteins and other nutrients because it can act as a "barrier" to animal digestive enzymes by limiting their availability and the occlusion in the field limits nutrient absorption.

In summary, from the above, all the anti-nutritional factors present in protein-rich seeds are to be considered in order to move towards a more limited use of these seeds in monogastric diets and to improve their nutritional and metabolic value.

Technical problem to be solved

The limitations of using protein-rich seeds in animal nutrition are diverse and associated with technical and economic barriers.

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

From an economic point of view, as far as the use of protein-rich seeds alone in monogastric animals is not fully exploited, there is today no technical implementation that can successfully meet the challenges of economic feasibility, both in traditional systems and in organic systems.

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

For example,in the aspect of variety selectionThis approach has been directed to several anti-nutritional factors, with some success in developing these varieties in farmers: this is particularly the case for tannins in peas, faba pyrimidine glucosides and concomitant faba pyrimidine nucleosides in fava beans, and alkaloids in lupins.

However, genetic approaches have not proven their value in the field for many of the anti-nutritional factors in these plant species. In addition, some varieties with very low anti-nutritional factors are low-yielding varieties.

While performing variety selection work on antinutritional factors, it has been testedMany technical methodsTo reduce or eliminate anti-nutritional factors and/or to improve the nutritional and digestive value of the seed.

The different methods tested to date involve mechanical or thermal or thermomechanical or enzymatic methods.

It should be noted that the reference book on technical methods for processing protein-rich seeds is very miscellaneous, incomplete, the information is not very useful and mostly obsolete.

Many published works have attempted to compare one technique to an untreated control, or to compare techniques to each other in a pairwise comparison. Or using generally different in vitro evaluation techniques and/or in vivo studies under different conditions.

Indeed, part of the results of the reference bibliography are outdated, not keeping up with technological changes in the last 30 years; and they only propose comparisons between techniques and do not really consider optimization of these techniques, or let alone combinations of techniques.

For this reason, the current reference book must not give clear conclusions with regard to the technology used and the corresponding parameters, in particular with regard to industrial implementation.

On the other hand, in vivo studies, which are often quite old, are mainly performed on animals with low production genetics and on foods that are less adapted to the current food systems.

In fact, animal genetic selection, the feed consumption index of which increases on average by 2.5% per year, is made for global production, by corn-based feeds and soybean-based feeds traditionally used (Schmidt et al, 2009; Zuidhof et al, 2014).

This does not allow for optimal value-added for more diverse feedstocks in terms of energy and protein sources, such as protein-rich seeds.

Thus, the impact of anti-nutritional Factors (FANs), plant varieties, and technology may be amplified from present day in vivo animal evaluation models. Because of this background, it is increasingly difficult to make current protein-rich seeds perform well.

In this sense, although many technical processes have been tested in the past, some of them should be reexamined in the current technical and economic environment.

The main treatments tested so far for these protein-rich seeds are as follows:

mechanical treatment:

"traditional" mechanical treatment (milling, micronization) destroys the initial structure of the seed by breaking up the cell walls and starch particles. These treatments allow the division into smaller particles by coarse (5 mm particles) or fine (2 to 3mm sieve) grinding, i.e. by crushing, crushing (hammer mill) or shearing (knife mill or knife-toothed drum).

The size of the resulting particles and their destruction determine the degree of exposure of the biochemical components to the digestive agents (rumen microorganisms or intestinal enzymes) and thus their rate of digestion. The tissue structure is preserved to a large extent. However, grinding with a 3mm or even 1mm screen before coagulation can destroy the tissue structure.

Although digestibility (especially in the case of micronisation) can be improved by increasing the digestibility of starch (in pigs and poultry) and protein (in pigs only, which has no effect on poultry), there is no effect on anti-nutritional factors.

Dehulling/de-coating the seed removes hulls or skins from the seed that contain only cellulose, fiber, certain anti-nutritional factors and contaminants.

Dehulling is the mechanical separation of the kernel from the shell, most commonly used for pea, broad bean and soybean seeds. The aim is to reduce the cellulose content to enrich the feedstock and to improve the recovery of the seeds by eliminating certain FAN (e.g. tannins) present in the shell.

Dehulling involves the removal of the film (thin film) present around the kernel, with the same advantages as depacketizing.

Both processes concentrate certain nutrients such as proteins and fats and only separate a fraction of the FAN contained in these membranes.

And (3) heat treatment:

heat treatment includes granulation, baking, exfoliation and autoclaving: the action of heat is combined with external hydration in the form of water or steam under reduced pressure (long term wet steaming at moderate temperature). The superior effects of baking and autoclaving are significant compared to flaking and granulation.

These processes have a more or less important positive effect on energy values in particular, but are not widely used in animal nutrition due to lack of profitability.

Baking at very high temperatures with heat transferred by conduction, convection and radiation, "jet splash", dry short cooking has not proven effective to date. In fact, such processing hardly changes the particle size and therefore does not change the integrity of the seed tissue.

Thermal mechanical treatment:

extrusion cooking is a complex operation which corresponds to several unit operations: mixing, steaming and shaping. By appropriately selecting the machine control parameters, each of these operations can be adjusted according to the material to be processed and the product to be obtained.

Under screw drive, the material is subjected to high temperatures (100 to 200 ℃) and high pressures (50 to 150 bar) and more or less intense shear within a very short time (20 to 60 seconds). Under the action of these physical parameters, the material is subjected to physicochemical changes and homogenization. Through the exit of the die to give it its final shape. The sudden drop in pressure during extrusion causes the instantaneous evaporation of the water present, which can lead to the characteristic expansion of the product.

The first extruder-cooker was a single screw. The second generation of equipment is a twin screw unit (with two parallel, tangential or co-penetrating screws, which rotate in the same or opposite directions); they are more flexible to use, in particular to make it work more regularly.

This cooking-extrusion process causes a destruction of part of the FAN and improves the digestibility of the seeds, but if many of the parameters involved are not controlled (machine type (single/twin), mechanical constraints (screw type, lock, speed, dies), heating constraints (water, steam, duration), appearance constraints (dies for flour, croquettes), the results are variable and not always reproducible.

However, for most protein crops, thermo-mechanical processing is not well known and has yet to be developed.

Enzyme treatment:

all animals secrete enzymes to digest food. However, the digestion process in animals is not 100% efficient. For example, pigs and poultry do not digest 15% to 25% of the food eaten. The ingestion of exogenous enzymes (especially monogastric enzymes) in animal feed improves digestibility of starch, protein, fiber and minerals. This enzymatic contribution makes the growth performance better and reduces waste projected to the environment.

Enzyme supplementation was performed using a single commercial enzyme, which was selected for several enzyme activities. Experiments have shown that this technique is beneficial as a way of improving the nutritional value of protein crops, but it has not proven economically feasible.

Technical treatment and starch gelatinization, protein denaturation and fat utilization rate：

Temperature, pressure and humidity affect the composition of the food product. In many processes, temperature is one of the factors that further disrupt starch structure (Pan et al, 2017; Wang et al, 2016 a; Zhang et al, 2014). However, when the heating temperature is fixed, the water content or pressure plays an equally important role in disrupting the structure and increasing the digestibility of the starch (Wang et al, 2017 b). However, it is important to consider all these parameters, as the same effects, such as protein denaturation, starch gelatinization and starch interaction, can be achieved by different combinations of these parameters. For example, in the extreme, gelatinization may be achieved by treatment under hydrostatic pressure at room temperature. At constant temperature and treatment time, the degree of gelatinization increases with increasing pressure. The higher the temperature, the lower the pressure for complete gelatinization. At constant temperature and pressure, the degree of gelatinization increases with the duration of the treatment.

A good example is the gelatinization of starch, 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). The transition to the different states depends on technical parameters such as pressure, temperature, time and water content. The solid-glass transition temperature may vary depending on humidity and pressure conditions. At this level, the matrix changes from a solid to a mobile rubbery state (Keetels, 1995; Behnke, 2001). Likewise, the melting temperature from glassy to liquid state also depends on humidity and pressure conditions. At this temperature, the matrix becomes liquid (Keetels, 1995). The extent of the transformation depends mainly on the duration and speed of heating (Mariotti et al, 2005; Wang & Copeland, 2013). Maintaining these technical parameters for a long time can lead to a complete transformation of the matrix. It should be noted that gelatinization occurs in those portions of the matrix where the water content is sufficiently high (Hoseney, 1994).

The technical parameters applied during the processing will cause physicochemical changes, such as starch gelatinization or protein denaturation. Finally, nutrients may interact, involving complex changes such as maillard reactions (Sylhus, 2006), or may polymerize with each other to form new structures (Svihus et al, 2005).

Starch gelatinization and protein denaturation alter the structure of the matrix, thereby altering the properties of the matrix. Thus, the viscosity is different between the natural substrate and the treated substrate. These changes positively affect the digestibility of nutrients by animals (Champ and Colonna, 1993). Studies of starch gelatinization under pressure have shown that the treated starch has improved enzymatic digestibility (Hayashi and Hayashida, 1989).

Fat availability (MGD) is an essential parameter to be controlled to increase its value to animals when using lipid-based seeds, and even more so because they are rich in fat. The seed treatment process using mechanical and thermal forces promotes the rupture of cell walls and plasma membranes, resulting in more release of fat contained in lipid vacuoles. Thus, treatment with appropriate techniques can maximize this fat release. The latter benefit to animals is related to improved lipid digestibility (Noblet et al, 2008), to the efficiency of dietary fatty acid deposition in pigs (Chesneau et al, 2009), and to the in vitro biological hydrogenation of fatty acids in cows (Enjalbert et al, 2008).

Treatments adopted by economic participants in animal husbandry:

the observation reports of the breeding industry today are based on the following facts:

1-the resultant varieties have no own characteristics in terms of nutritional composition, other than agronomic characteristics;

2-technical methods for using seeds in rearing are basic methods, since they use only mechanical grinding treatment (or even de-coating in the case of salmon type), heat treatment for granulation and baking, and almost no thermomechanical cooking and extrusion treatment.

In fact, the only uses that exist today are basic and therefore not fully developed, due to the lack of technical and economic feasibility.

In summary, some technical limitations of the use of protein crops in food products, such as in particular certain antinutritional factors such as antitrypsin factor, tannic acid, fabacebrosidase and adfabacebropyrimidine nucleoside, can be lifted by genetics.

The removal of other technical limitations can be accomplished by processing methods such as de-coating to remove tannins contained in the membrane or by thermal processing under conditions that overcome certain heat-sensitive anti-nutritional factors.

Finally, mechanical processing such as grinding helps to improve the nutritional value of the seeds, and thermal processing such as baking and pelleting, or thermal mechanical processing such as cooking extrusion, or enzymatic processing such as addition of enzymes also helps to improve the nutritional value of the seeds.

Thus, the genetic selection routes or technical methods reported in the reference literature each individually set forth a well-known field for improving, on the one hand, the limitation or elimination of antinutritional factors and/or, on the other hand, the digestibility/degradability value of seeds, but none of them can be sufficient to be technically complete and economically beneficial in terms of increasing the technical and economic values, as evidenced by the absence of widespread practice in this field.

In the historical market (the choice of vegetable protein sources in the animal diet is basically based on economic criteria), the position of protein-rich seeds has been reduced to zero with respect to the existing limits, so that soybean meal benefits first, and rapeseed and sunflower oil cakes, and even synthetic amino acids benefit second. .

In new markets (where the trend is not only to produce meat or eggs at competitive prices, but also to meet consumer expectations for greater product traceability and proximity), the challenge is of course to have a competitive protein source, but also a protein source that can be produced and traced locally.

Thus, lupin, fava bean and pea crops have many agronomic and environmental benefits, can provide well-known ecosystem services, and provide benefits recognized by producers and consumers.

In order to successfully reintroduce protein crops into soil and feed troughs in a sustainable manner, there are the following advantageous background requirements: "political" incentives (protein crop assistance program, "ecological plants" program, innovation assistance …), farming assets (below, yield of cereals, reduction of input …), potential countermeasures to many agricultural desires (protein autonomy, economic sustainability …) and social desires (local, non-transgenic, no soybean, biodiversity, environment …).

However, if our animal and plant production system is not technically sound and economically viable, this favorable environment is not sufficient to increase the use of the metro oilseed and protein crops. Therefore, development of new technology, which is technically and economically efficient, must be accompanied to provide the best solution for agriculture and animal husbandry.

The technical treatment adopted by economic participants in animal husbandry is currently only proven. In animal husbandry today, observations are based on non-optimal use of technical processes. As mentioned before, the technical methods for using seeds in farming use only mechanical grinding treatment (or even de-coating for salmon species), only heat treatment (for granulation and roasting), and almost no thermomechanical treatment of cooking and extrusion.

In fact, the only use currently available in monogastric feeding is the basic one and therefore is not fully exploited due to the lack of technical and economic feasibility. Moreover, they cannot be used as a complement to genetic methods.

Therefore, a need not currently addressed is to provide a method for treating protein crops which avoids many of the above-mentioned disadvantages by a combination of different methods which are combined together in a manner which produces a synergistic effect.

In addition, in addition to evaluating these processes for use in animals, it is also beneficial to use methods to evaluate the quality of the technical treatment in terms of potential for use in animals.

In this sense, several methods have been chosen to evaluate the quality of the processing in relation to the main nutritional components of starch, protein and fat, and are shown below:

method for assessing starch gelatinization rate or starch destruction rate

The starch changes its structure from a native structure to a gelatinized structure and then to a new structure during the transition depending on the temperature, pressure, humidity and duration of the treatment. Although the gelatinized structure is attacked by amylase, both the new and native structures are resistant to the action of amylase.

The starch gelatinization rate of the protein-rich seeds was determined according to the method described by Chiang and Johnson (1977). The method is based on an increased enzyme sensitivity, i.e. gelatinized starch granules are easier to hydrolyze than native starch granules. Briefly, total starch is hydrolyzed with amyloglucosidase under specific conditions such that only gelatinized starch is hydrolyzed. To determine the proportion, the total starch was determined according to the standard enzymatic method (AFNOR 2005) as a reference value.

Method for assessing protein solubilization

Technical treatments applied to protein-rich seeds may denature proteins under certain conditions. This denaturation is manifested by the establishment of new linkages (cross-links) between polypeptide chains, which make them aggregated and insoluble. The bonds formed (usually involving lysine and glutamic acid residues) are resistant to enzymatic hydrolysis. This insolubility or lesser degree of solubilization of proteins is beneficial in the field of animal nutrition. For example, it reduces sensitization by disrupting certain epitopes (Toullec et al, 1992) and limits protein degradation in ruminants (Benchamar et al, 1992).

The determination of protein solubility was assessed by dissolving the protein in buffers of different pH values (acidic, neutral, basic). After determining the protein content of the starting material (kjeldahl method) and the proteins solubilized in the different buffers, a material balance was established to determine the solubilized fractions.

Method for fat utilization

This internal method is based on the evaluation of the proportion of fat extracted in the solvent after a predetermined time. The purpose of this test was to simulate the gradual release of fat into the different compartments of the animal's digestive tract.

In short, the test is carried out in four steps:

-preparation of the starting materials: it includes coarse grinding to obtain different particle size distributions, as is the case with grinding performed in the animal feed industry;

-extraction of fat: which comprises contacting a pre-weighed amount of the raw material with an extraction solvent (e.g. petroleum ether) under controlled stirring for a predetermined period of time, in this case 10 minutes. The extraction phase can also be carried out with different durations, thus enabling the release kinetics of the fat.

-solid/liquid separation by filtration: it involves filtering the triturate dissolved in the solvent so as to recover only the liquid phase in a dry tared flask.

-removing the solvent from the extract and weighing the dry residue: which involves evaporating the solvent in which the fat has been dissolved. After evaporation, drying and cooling, the flask was weighed. This cycle is repeated until a constant mass is obtained.

Thus, the seed may be characterized by an MGD, the value of which depends on the set operating conditions.

This released fat (which is called available because it is rapidly available) will be absorbed through the intestinal wall. After such absorption, the animal can use it for its own metabolism.

In a complementary novel manner, we have also selected direct and indirect methods of protein and carbohydrate complexation.

Method for evaluating protein degradability (DE1)

The enzymatic degradability of proteins in protein-rich seeds was measured according to Aufrere et al (1989, 1991) (DE 1). Briefly, samples were hydrolyzed by protease for 1 hour at 40 ℃ in borate-phosphate buffer at pH 8. The determination of degraded nitrogen was performed in the supernatant and correlated to the total amount of nitrogen in the sample.

Method for evaluating N-Carboxymethyllysine (CML) and total lysine

CML and lysine levels were determined using the method of Niquet-L Ridon and Tessier (2011). In general, each sample was reduced and then hydrolyzed in acidic media prior to analysis by High Performance Liquid Chromatography (HPLC) coupled tandem mass spectrometry (MS/MS) detection. Correction of any basis by addition of an internal referenceQualitative influence, the internal standard being CML (CML-D)²) And lysine (lysine-¹⁵N₂) The stable isotope of (1).

Furthermore, heating proteins in the presence of reducing sugars (fructose, lactose, etc.) allows the formation of many complex polymers, including lysine. This reaction, known as non-enzymatic browning or maillard reaction, also helps to reduce the digestibility of proteins.

Method for evaluating acrylamide

Acrylamide is of empirical formula C₃H₅2-acrylamide of NO (acrylamide of acrylic acid). Acrylamide can be formed in particular during cooking at high temperatures of raw materials which are rich in carbohydrates (starch, sugars) and proteins which react with asparagine (maillard reaction). The formation of acrylamide appears to be strongly influenced by the cooking temperature, the water content in the food product, and the "browning" or "carbonation" of the product. Acrylamide is formed during cooking at temperatures of 120 ℃ or higher.

Briefly, acrylamide in samples was extracted in water by stirring, then after centrifugation and recovery of the supernatant, purified on SPE columns, then quantified by UPLC-MS/MS in MRM mode.

The latest technology in the field of the present invention includes the following documents:

JEZIERNY D et al: "The use of grains as a protein source in verification: A review", ANIMAL FEED SCIENCE AND TECHNOLOGY, volume 157, period 3-4, 11 months 2010, page 111-;

PATRICIO SAEZ et al: "Effects of debolling, team-browsing and microwave-irradiation on differential value of white lupin (cervical interbus mykiss) and Atlantic salmon (Salmo salmon)", ARCHIVESOFA ANIMAL TRITION, Vol.69, No. 2, day 3/4 of 2015 (D2);

FR 3 040 588 A1(D3)；

france ESCO MASOERO et al: "Effect of exclusion, expansion and ablation on the nutritional value of peas, fava beans and lupins", ITALIAN JOURNAL OFANIMAL SCIENCE, Vol.4, No. 2, 1/2005 (D4). D1 is like a "catalogue" for independent treatment of protein-rich seeds. This article discusses the possibility of reducing the level of anti-nutritional factors by selection techniques or by techniques known per se, such as physical treatment.

D2 focused on treating only a single seed (white lupin) by de-coating, cooking and microwave treatment.

D3 relates to a method of seed treatment comprising a step of seed germination.

D4 relates to the effect of extrusion, expansion and baking on the nutritional value of certain protein-rich seeds.

Disclosure of Invention

The present invention therefore relates to a method for treating protein-rich seeds selected from at least one of the following seeds to increase their value as a foodstuff, in particular as a foodstuff for animals: vicia faba L, Pisum sativum L, Lupinus albus L, Lupinus angustifolius L, and Lupinus luteus L,

characterized in that the method comprises the following successive steps:

a) seeds of at least one of the above plant species are used, provided that said seeds have values of protein content and/or starch content and/or fat content greater than or equal to the values indicated in the following table:

and, at least one compound from the following group is used at levels below those shown in the following table: anti-nutritional Factor (FAN), crude fiber, Neutral Detergent Fiber (NDF):

b) pressurizing the seeds in step a) for more than 10 seconds at a pressure of at least 10 bar until a temperature of more than 80 ℃ is reached;

and/or b1) heating the seeds at a temperature above 80 ℃, preferably between 90 and 150 ℃, for a time of at least 15 minutes, preferably between 30 minutes and 2 hours.

According to other non-limiting advantageous features of the invention:

-after performing said step a), grading said seeds;

-after carrying out said step a), if seeds of different species and/or seeds with different composition in terms of protein, starch, fat, anti-nutritional factors, crude fiber or Neutral Detergent Fiber (NDF) are involved, said seeds are mixed and fractionated, or fractionated and then mixed;

-before step b), a heating step of the prepared seeds with steam and/or a water-based liquid, for a time greater than 2 minutes, preferably 15 minutes, until a temperature between 30 and 90 ℃ and a humidity greater than 12%, preferably 15%, are obtained;

-the preliminary heating step is carried out in the presence of at least one exogenous enzyme identified in the following families: arabinofuranosidase, beta-glucanase, cellulase, glucoamylase, pectinase, pectin methyl esterase, phytase, protease, xylanase, preferably xylanase, beta-glucanase, and pectinase, the exogenous enzyme having been previously added to a seed or mixture;

-a preliminary heating phase in the presence of exogenous enzymes, setting the humidity to greater than 15%, preferably 25%, and allowing said preliminary to last for at least 15 minutes, preferably 60 minutes;

-stirring the mixture while performing said heating preparation step;

-while mixing and then classifying, a new mixing after classification;

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

-subjecting said mixture to said step b 1);

-interrupting step b) or b1 if the level of at least one antinutritional factor listed in the following table is lower than the level also shown below):

-dehulling and/or de-coating the seeds after step a);

-specific fractionation and separation of the seeds according to criteria selected from size, weight, shape, density, aerodynamics, colorimetry or electrostatic parameters, after said step a) or after said dehulling and/or decoating step;

-after step a) or upstream, classifying said seeds according to criteria selected from size, weight, shape, density, aerodynamics, colorimetric or electrostatic parameters;

-mixing at least one other raw material selected from the group consisting of oilseeds and by-products thereof, oils, protein-rich seed by-products, cereals and by-products thereof, simple and complex carbohydrate sources, oilseed cakes with said seeds;

-the feedstock is a lipid source, preferably an oleaginous seed;

-having a final stage of cooling the seeds.

Detailed Description

In support of the present invention, digestibility tests were performed on the target monogastric species, in which broilers, cocks, pigs and fish were selected.

The evaluation is based on:

energy utilization, which is based on different methods depending on the species: metabolic Energy (EM) of poultry, digestive utilization coefficient of energy (CUD) of pigs, apparent digestive utilization coefficient of energy (CUDa) of fish;

-utilization of proteins based on the determination of the digestive utilization coefficient of the proteins.

Animal technology production performance tests were also performed on the same species (i.e., broiler chicken, layer chicken, pig and fish).

In this case, the aim is to show good growth and/or egg laying performance, or to check whether the growth and/or egg laying performance is the same as the control, provided that the nutritional value of the solution produced by the invention is those values previously determined to be worthwhile by the digestibility test.

The method of the invention consists of a combination of the following steps:

step a): using specific seeds

Use of at least one protein-rich seed considered to be specific due to the fact that: the seeds have a high content of at least one (1) nutrient from protein, starch or fat, preferably two (2), three (3) or even four (4) nutrients, and a low content of at least one heat stable anti-nutritional factor or low value nutrient such as crude fiber or Neutral Detergent Fiber (NDF).

Seeds with high protein, starch and fat content are considered to be high protein, starch and fat seeds if their content is equal to or greater than the following threshold (right column):

the use of the most protein-and/or energy-rich seeds (in particular the starches of the Vicia faba L. or of the pea seeds Pisum sativum L.; lipids of the white lupin seed Lupinus albus L., or of the blue lupin seed Lupinus angustifolius L., or of the yellow lupin seed Lupinus luteus L.) does contribute to making the process of the invention more competitive from a technical and economic point of view; this is not only because of the concentration of the beneficial nutrients but also because of the positive interaction of the biochemical reactions during the technical process (synergistic effect).

Seeds with a low content of thermostable FAN or low value nutrients are considered to be seeds with a low content of thermostable FAN or low value nutrients if their content is below the following threshold (right column):

(MS dry matter.)

There are differences in seed composition within the same species due to factors such as variety, cultural routes, soil climatic conditions, etc. The selection of protein-rich seeds on the basis of their nutritional quality influences the industrial-technical processes to be carried out subsequently. In fact, the presence of proteins, starches and lipids in the matrix is known to affect "processability" and thus the conversion of these nutrients, especially by analysis of starch gelatinization, protein denaturation and fat utilization (which are known to predict improved digestibility). The level of transition from one state to another (solid, glassy, liquid) depends on the proportions of the above-mentioned nutrients.

Thus, under hydrothermal treatment or the like, two seeds of the same species having different compositions will react differently; that is, at the end of the same hydrothermal treatment, the particular seed reserved in the first stage of the invention is characterized by an increase in its denatured protein, gelatinized starch and available fat compared to the seed that was not reserved.

From another point of view, the particular seeds selected in the first stage of the invention require the application of technical processes to them at lower temperatures, pressures, water contents and/or times for the same purposes of protein denaturation, starch gelatinization and availability of fat, i.e. being more efficient processes due to the need for fewer technical constraints.

In addition, the higher the starch and/or protein content in the pea and field seeds and the higher the protein and/or fat content in the lupin seeds, the lower the fibre concentration. Thus, this lower incorporation of low value fiber for animals allows for higher levels of digestion by promoting digestibility of proteins and other nutrients. This fiber serves as a "fort" for the animal digestive enzymes by limiting their availability; and limit intestinal blockage that prevents nutrient absorption.

Step b): pressurized heat treatment step

This step consists in placing the seeds under a pressure of minimum 10 bar, preferably 20 bar, even 30 bar, for a time of greater than 10 seconds, preferably between 10 seconds and 2 minutes, in order to reach a temperature of greater than 80 ℃, preferably greater than 100 ℃, or even comprised between 100 and 150 ℃, even more advantageously between 110 and 140 ℃ and not exceeding 160 ℃; this temperature is advantageously achieved by: self-heating caused by shear forces, friction and compression, possibly also via conduction (heat transfer fluid, electrical resistance, electromagnetic field, etc.) or by the addition of steam.

A non-exhaustive list of pressurized heat treatment apparatuses capable of performing this step is as follows: extruder, cooker-extruder, expander, compressor.

And/or

Step b 1): non-pressure heat treatment step

This step comprises a pressureless heat treatment, the duration of which is extended compared to step b) so that it exceeds 15 minutes, preferably exceeds 30 minutes, or even 30 minutes to 2 hours, at a temperature above 80 ℃, preferably above 90 ℃, or even between 90 and 150 ℃. In the same way, suitable equipment for this pressureless thermal treatment are, for example: a drier, a baking oven and a constant temperature screw.

The purpose of this step (these steps) b) and/or b1) is to reduce heat-sensitive antinutritional factors and inactivate endogenous and/or exogenous enzymes, while improving the digestibility of energy and/or proteins and amino acids, especially in the case of heat treatment under pressure.

Finally, it is made possible (if necessary) to reduce the humidity of the seeds, which should not exceed 14%, preferably 12%, in order to obtain a good preservation of the mixture.

One way of characterizing the effectiveness of the step(s) b) and/or b1) is to evaluate the reduction of at least one heat-sensitive antinutritional factor, the objectives of which are listed in the following table:

(UTI ═ trypsin inhibitory unit)

Another way is to evaluate according to the seeds considered: for peas and beans, the proportion of gelatinized starch is to reach a minimum of 50%, preferably 65%, advantageously 80%; or for pea, broad bean and lupin, the proportion of protein dissolved at alkaline pH is up to a maximum of 55%, preferably 40%, advantageously 30%; or for lupins, a minimum of 40%, preferably 50%, advantageously 60% of fat is available; or for pea and broad bean the 1 hour enzymatic digestibility is to a maximum of 50%, preferably 40%, advantageously 30%, and for lupin the 1 hour enzymatic digestibility is to a maximum of 60%, preferably 50%, advantageously 40%; or in the case of lupins, a minimum of 40% of fat may be utilised; or the content of Maillard compounds such as N-Carboxymethyllysine (CML) or acrylamide. The threshold value not exceeded is N-carboxymethyllysine of 0.020g/kg dry matter, preferably 0.018g/kg dry matter, advantageously 0.015g/kg dry matter for beans and peas, and N-carboxymethyllysine of 0.025g/kg dry matter, preferably 0.020g/kg dry matter, advantageously 0.018g/kg dry matter for lupins; and 110g/kg dry matter, preferably 90g/kg dry matter, advantageously 70g/kg dry matter acrylamide for beans and peas and 300g/kg dry matter, preferably 200g/kg dry matter, advantageously 150g/kg dry matter acrylamide for lupins.

The final step is as follows: cooling down

At the end of the heating step b), the seeds are hot. It is then necessary to cool the seeds of the process to a temperature at which they remain stable for a period of time, to be preserved and stored under good nutritional conditions until consumed.

For example, the temperature should not exceed 30 ℃, preferably 20 ℃ above ambient temperature.

The aforementioned steps of the method according to the invention can also be advantageously implemented by first taking into account the following elements:

mixing and grading of seeds

Although this step is not mandatory, the process of the invention is improved when a fractionation or even a mixing step is performed after step a) of the process of the invention.

This step consists in selecting at least one mechanical mixing technique (when there are at least two raw materials with different properties and/or qualities), and/or a mechanical technique for fractionating the seeds or mixtures, which are arranged in such a way that they are firstly able to obtain a homogeneous mixture of the protein-rich seeds with any other raw material (as described below), and secondly able to break the seed coating and the kernel, so as to bring the digestive enzymes closer to the nutrients and thus improve the digestibility of the seeds.

One preference is to premix the materials prior to classification. It is also possible to first fractionate the materials separately and then mix them, but it is also possible to carry out two mixing operations, one before and one after the fractionation.

Simple and/or combined mechanical stresses for accomplishing these functions may be achieved by impact, cutting, compression, shearing, or friction.

Seed fractionation is characterized by the determination of particle size measurements of the particle size produced by the method of the present invention. Preferably, this mechanical classification produces 90% of the particles having a maximum dimension of less than 2000 μm, preferably less than 1500 μm.

For example, a horizontal hammer mill may be used to achieve this size according to the following parameters:

for a plant with a throughput of 10t/h and a motor of 200kW, the speed of rotation is 2800rpm and the sieve size is 3 mm.

Other common equipment (e.g., hammer and roller mills or crushers, tray mills) can also be used to achieve this size.

Finally, there are other techniques that can accomplish this: a sand wheel mill, a disc mill, a pin mill, a cutting head mill, a bead or ball mill, a blade or crusher, an impactor or impact mill, etc.

Preliminary heat treatment

It is also advantageous to continue the heating step after step a) of the process of the invention, or after the previously described fractionation and/or mixing step.

It is also important to remember that this step can be performed on de-coated seeds and flax cakes (another advantageous step described later).

This step comprises selecting a heating technique that is parameterized in such a way that the following steps and features are followed:

first possibility: hydrothermal preparation step：

This step consists in impregnating the seeds with steam and/or an aqueous liquid so as to bring the previously fractionated seeds to a temperature between 30 and 90 ℃ for a time greater than 2 minutes and to a humidity greater than 12%. In a preferred manner, it is advisable to impregnate the seeds for a duration of more than 5 minutes, or 15 minutes, even 30 minutes, and preferably less than 4 hours, even 8 hours, not more than 24 hours, so as to have a humidity of more than 15%, even 18% and preferably less than 40%, not more than 60%. In a very advantageous manner, it is advisable to impregnate the seeds for a duration of more than 1 hour, even 2 hours, so as to bring the humidity to more than 20%, even 25%. The purpose of this step is to facilitate the subsequent heating step under pressure and/or in the absence of pressure, in particular by improving the heat transfer capacity, causing activation of the endogenous enzymes of the seed and starting the subsequent heat treatment.

In a non-exhaustive manner, the equipment capable of performing this step is: a preparer, a preconditioner and conditioner, a cooking machine, a mixer, a baker, a steam impregnator, a ager.

Or

Second possibility: hydrothermal enzymatic preparation step:

this preliminary step involves applying the same preliminary conditions as described in the first possibility. The simple difference is that at least one exogenous enzyme not present in the protein-rich seed is activated, which exogenous enzyme may be supplied in particular as a processing aid (enzyme extract, etc.) from additives, raw or fermentation raw materials, etc., and added to the process of the invention in one stage before or during the process.

The temperature profile is then selected in such a way that it corresponds to the range of activity of the selected enzyme, but is maintained between 30 and 90 ℃. The time and moisture characteristics required are the same as described in the first possibility, but it is to be taken into account that these exogenous enzymes require more favourable conditions than the endogenous enzymes, since they are not as close to their substrates as possible both spatially and temporally. In this sense, the impregnation conditions should be adjusted so that the impregnation lasts at least 15 minutes, preferably 60 minutes, and preferably less than 4 hours, or even 8 hours, but not more than 24 hours, so that the humidity is greater than 15%, preferably 25%, and preferably less than 40%, but not more than 60%. The enzyme or enzymes to be introduced belong to the family of arabinofuranosidases, beta-glucanases, cellulases, glucoamylases, alpha-amylases, pectinases, pectin methyl esterases, phytases, proteases, xylanases, preferably to the family of xylanases, beta-glucanases and pectinases.

It (or they) are pre-selected for their effectiveness in hydrolyzing specific chemical bonds (either by the animal not completing at all or not completing completely or not quickly enough).

It may also be selected for the following capabilities: the carbohydrates which are not or not sufficiently hydrolysed by the animal are broken down, thereby giving the animal's digestive enzymes better access to other nutrients in the seed.

The equipment capable of performing this step is for example: conditioners, preconditioners and conditioners, cooking machines, mixers, ovens, steam impregnators, agers, reactors, and the like.

It should be noted that a rational addition of water (but also in whole or in part in the seed mixing step) may be carried out in the above-mentioned apparatus, which is necessary for improving the process according to the invention, and that an addition of water vapour (but also in whole or in part in step b) of the process according to the invention, i.e. the step of carrying out the heat treatment under pressure and/or without pressure, may be carried out in the above-mentioned apparatus, which is also necessary for improving the process according to the invention.

With or without the advantageous steps described above, the steps of the method according to the invention can also be carried out, if desired, taking into account the following said basic elements:

classification

The classification step groups the seeds according to criteria of size, weight, shape, density or according to aerodynamic, colorimetric or electrostatic characteristics. The tools used to perform these operations are in particular: screeners, cleaner-separators, screeners, flat screens, density meters, winnowing machines, optical sorters, aeration systems (air columns, air extractors, blowers …), magnetic.

The purpose of this operation may be to separate seeds of different species, to remove impurities, to distribute seeds of the same species, etc. The seeds may be separated from other species, or may be assigned to the same species.

Removing and/or coating layers

The purpose of this step is, on the one hand, to concentrate the protein content and the energy content in the form of starch, in particular in the case of beans and peas, and in the case of lupins; another aspect is to reduce the proportion of fibres and anti-nutritional factors present in the seed coating.

The dehulling and/or decoating step is characterized by a minimum yield evaluated on the basis of the influence of the protein concentration of the seeds concerned, the following table being the expected concentration levels:

	protein concentration
		Broad bean	+ 5%, preferably + 15%, or even + 20%.
Pea (Pisum sativum L.)	+ 5%, preferably + 12%, or even 15%.
		Lupine	+ 5%, preferably + 15%, or even + 20%.

Removing the coating with a very high fibre content (very low protein content) will result in a high protein content in the de-coated seed.

Dehulling and/or decoating is carried out by combining a mechanical stress phase and a separation phase, followed by possible rehydration of the kernel of the seed (if required), followed by a phase of heat pretreatment to facilitate dehulling and/or decoating.

The mechanical stress used to perform a simple and/or combined function of these may be impact, compression or friction. Tools for performing these operations include, but are not limited to: roller and hammer mills or crushers, impact compactors or impact mills, polishers, paddle mills, sand mills, disk mills, pin mills, cutting-head mills, bead mills or ball mills, blade mills or blade crushers.

The separation can be performed according to criteria of size, weight, shape, density or according to aerodynamic, colorimetric or electrostatic characteristics. The tools used for performing these operations are in particular: shaker, cleaner-separator, screener, stonemaser, flat screen, densitometer, winnower, optical classifier, aeration system (air column, air extractor, blower …), magnetic.

Specific fractionation and separation

In order to obtain a more concentrated protein, carbohydrate and/or fat fraction, specific fractionation and separation steps may be added. It should be carried out downstream of or instead of the dehulling/decoating and/or classifying step.

This step is characterized by a minimum yield, expressed as the concentration of protein, carbohydrate or lipid in the obtained fraction or fractions. The protein, carbohydrate or lipid concentrated fraction should preferably contain at least 25%, preferably 35% or even 50% more protein, carbohydrate and/or lipid than the original whole seed.

This separation of the fractions, which can be promoted by a shelling/de-coating step if necessary, will first be carried out by a simple and/or combined mechanical stress step carried out by equipment such as a micronizer, needle, roller, hammer mill or impact mill. Then by means of separation steps according to size, weight, shape, density criteria or according to aerodynamic, colorimetric or electrostatic characteristics, using devices such as screeners, scrubbers, separators, sieves, density meters, turbo separators, selectors, aerators or magnetic systems.

Using other raw materials

It is advantageous to add to the protein-enriched seeds at least one raw material which will be selected according to its technical and/or nutritional and/or economic properties. In fact, depending on the seed or mixture produced by the invention to be used, in particular the goals in terms of animal species and physiological steps, the choice of raw material will be related in particular to the nutritional characteristics and cost price of the raw material.

However, they must also be selected according to their technical advantages, in particular by their mechanical properties and thus their tendency to make the mechanical constraints of the process of the invention more favourable, their rheological and physicochemical properties and therefore their ability to be mixed with protein-rich seeds under wet conditions, their water-absorbing or absorptive capacity, in some cases their ability to bind to proteins, their enzymatic properties and therefore the ability to enhance the enzymatic activity (in particular to improve the digestibility of protein-rich seeds).

Advantageously, when the heating technique involves a heat treatment step under pressure, the protein-rich seeds are combined with a portion of the oilseeds.

For example, to facilitate passage in an extruder, adding fat preferably from oilseeds rather than oil (because of a more evenly dispersed supply of lubricant) allows protein-rich seeds to be treated technically better and increases throughput.

Finally, more generally, the selection of other raw materials is preferably based on the potential for nutritional and economic improvement that can be achieved by applying the process to protein-rich seeds.

Thus, among other raw materials, it would be preferable to first select oilseeds, oils, cereals and their by-products, simple or complex carbohydrate sources, then oilseed cakes, and then all other raw materials commonly used in animal nutrition.

Stirring during preparatory heat treatment

In the aforementioned preliminary heat treatment step, it is an advantage that the mixture is stirred to be subjected to uniform treatment conditions. In fact, stirring:

-homogenising the fractionated seed with water and possibly other additional inputs, in particular with the aim of facilitating the function of bringing the enzyme into contact with the substrate;

-homogenizing the added water and temperature within the seed or mixture;

avoiding the formation of agglomerates, thus favouring the transport conditions of the seeds or of the mixture.

As will be seen below, the method according to the invention makes it possible to achieve advantageous technical results with respect to the prior art.

Indeed, none of the references have generalized the process to achieve the technical and economic improvements obtained by the process of the present invention, particularly with respect to the latest animal production systems characterized by significant genetic improvements, adapting them to feeding systems essentially based on soybean, corn kernel and cereals.

The combination of the above steps allows at least one protein-rich seed to simultaneously have the following characteristics:

the content of at least one (1), preferably two (2) nutrient compounds is higher:

at least one, preferably two, advantageously three or even four thermostable anti-nutritional factors or low-value nutrients The content of the components is reduced:

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

(UTI ═ trypsin inhibitory unit)

Content improvement of at least one evaluation criterion:

(*): hereinafter, the term "solubilized protein" means a protein that is solubilized at an alkaline pH.

Improved levels of energy and/or protein and amino acid digestion: -for monogastric animals:

(EM: metabolic energy; CUD: digestive utilization coefficient)

For ruminants:

the results listed in the table above are compared with those obtained with seeds which have only undergone a granulation step similar to the fractionation step described above and carried out at a temperature lower than 100 ℃. These results are also based on current animal genetics, i.e. the breed selected according to its productivity.

They are the result of digestibility studies on monogastric species by in vivo tests on broiler chickens, fish and pigs, and on ruminants by in vivo tests. These tests are shown below.

Digestibility test

a/Standard broiler digestibility test

On the test field, tests were performed to determine the digestive nitrogen and energy utilization coefficients of standard broiler chickens. The aim was to evaluate the impact of broad bean seed selection and technical treatment provided as described in the present invention. The energy recovery and digestibility of the proteins were calculated from the differences defined by Carr et al, 2013.

Therefore, by combining the best techniques from seed selection to mechanical and thermomechanical processing, we seek to significantly improve the level of digestion: the protein digestibility of broiler chickens increased by + 8%, the energy value increased by + 18%, and broiler chickens are a species that is highly sensitive in the method of digestibility, making it an excellent model for monogastric animals. When broad bean seeds were dehulled in an advantageous manner, a synergistic effect was noted on the utilization values of protein (+ 10%) and energy (+ 24%) after heat treatment. These effects are superior to simple seed selection with addition, dehulling of the seeds and proper heat treatment.

b/Digestibility test in pigs

In the laboratory field, tests were carried out to determine the excrements and the ileal digestibility of growing pigs. The digestibility of each test material was obtained using a differential method by measurement of a basic meal and measurement of a meal containing a portion of the basic meal and one of the test products.

To determine the digestibility of pig excrements, tests were carried out according to the apparatus described by Noblet et al (1989). In short, the principle is to introduce 35% of one of the seeds to be tested into the basic diet (wheat + soybean meal) and to allocate each diet thus prepared to 5 pigs.

This test highlights the advantages of the technical treatment (preparatory and thermal treatment) applied to the whole seeds or to the de-coated seeds, as disclosed in the present invention, the digestibility levels of total nitrogen and energy that can be achieved being statistically equivalent to the control diet based on soy flour. The two heat treatments applied to the seeds produced substantially equivalent results. On the other hand, without technical treatment of the selected seeds of fava beans, their previous dehulling makes it possible to reach the CUD of the total nitrogen and energy of the feed obtained from the soybean meal. In the case of technical treatment of broad bean kernels, the CUD is statistically significantly better than soybean meal, as described herein in the advantageous aspects.

c/Rainbow trout digestibility test

In experimental fish farms, experiments to determine the metabolic utilization of energy and proteins were performed. Energy utilization and protein digestibility were calculated according to the differences defined by Choubert et al, 1982. The following table lists the selected and treated broad bean seeds and their digestibility coefficients.

In the above and below tables, values with the same superscript letter are not significantly different at the 5% threshold.

Processing of selected broad bean seeds by preparation and heat treatment results in improved digestibility of dry matter, energy, protein and starch compared to untreated broad bean seeds. The results produced by the two thermal processes did not differ significantly from each other. However, there are numerical differences that favor the preparatory and further heat treatment (temperature, pressure).

The advantageous use of dehulling of the broad bean seeds prior to heat treatment may further improve digestibility of nutrients (e.g., energy and proteins) to the level of soy flour.

This test highlights the benefits of digestibility of nutrients and advantageous use of dehulling in the treatment of broad bean seeds according to the invention, with the aim of replacing soybean meal.

These results of the intrinsic digestibility of the seeds, although expressing the effects related to the technical method of the invention (synergistic effect of seed selection and technical treatment), should bear in mind that they do not express the synergistic effect of the invention at the level of digestibility of the animals. Some digestibility results were obtained according to the conventional method used in the previous examples. On the other hand, other phenomena are also associated with the digestive consequences of the animal and affect the metabolism of the animal. In fact, if anti-nutritional factors can participate in the digestive value of seeds, they are also responsible for digestive and metabolic disorders, leading to reduced intake and production performance, physiological disorders and various pathologies. The advantage of the method according to the invention is therefore not only that a high level of so-called digestibility of the protein-rich seed is obtained, but also that adverse manifestations and other health problems associated with the high presence of anti-nutritional factors are avoided.

For example, faba bean pyrimidine glucoside and faba bean pyrimidine nucleoside of faba bean have been shown to reduce egg weight and egg strength of laying hens. On the other hand, lectins have the property of red blood cell agglutination, which can lead to growth retardation. For oligosaccharides, they are metabolized by microorganisms in the colon and, due to their fermentation, can cause discomfort at the digestive level (flatulence, diarrhea), which is likely to reduce feed intake, resulting in reduced growth performance.

Because of these advantages, the present invention produces animal technical results that were previously incomparable in animal husbandry.

Several animal technical tests with different species at different farms are shown below, so that the technical advantages obtained by the present invention can be verified under production conditions.

animal technology test of a/egg-laying hens

In the experimental farm, 15% of the mixtures from different processes having 90% broad bean seeds and 10% flax seeds and having predetermined EM and CUD N values were fed to laying hens (Isa-Brown) for about 3 months.

In view of the selected method, which consists in formulating the egg feed with iso-nutrients (metabolic energy, digestible essential amino acids, calcium, phosphorus, etc.), taking into account the differences in the digestibility values previously evaluated, the aim of the present trial was to examine whether the present invention enables a higher level of animal technical performance to be obtained due to the synergistic effect of the reduction of anti-nutritional factors, the effect of which is not introduced into the digestibility values.

Thus, the following table shows:

the nutritional characteristics maintained by broad beans produced by the invention on the one hand and standard broad beans (ground only) on the other hand; these values are predetermined by studying their digestibility according to conventional protocols known to the skilled person;

egg production performance in terms of egg weight, quality of eggs produced (considering egg production number) and consumption index (feed efficiency for producing eggs).

Compared to the control batch with soy flour (well characterized in terms of digestibility values and production performance), it appeared:

the results obtained with standard broad bean seeds (not of the invention) were inferior to the "soy flour" control, which highlights the negative impact of anti-nutritional factors on the egg laying performance of hens. In fact, the main finding is that despite a similar or even slightly higher egg weight, the hens of this batch have a lower quality of eggs produced, which means that their egg production is less; and they must consume more feed to produce the same number of eggs, which is an indication of decreased feed efficiency (increased consumption index).

The result obtained with the technical treatment of the seeds of standard fava beans at a level lower than the present invention is a reduction in productivity. The technical treatments applied to the non-selected seeds are not sufficient to effectively reduce the content of anti-nutritional factors and therefore do not significantly improve the nutritional value of the seeds.

The results obtained with the selected, but not technically treated, broad bean seeds were such that, despite the intrinsic nutritional value being higher than that of the non-selected seeds, the performance was still lower than that of the control batch with soybean flour. The broad bean seeds can not be fully utilized by the laying hens without technical treatment.

The nutritional value of the broad beans produced by the invention (test broad bean seeds) has been significantly higher than that of standard broad beans, while the results obtained with the broad bean seeds of the invention show an improvement in egg weight and quality of production without changing the consumption index.

This is the way to observe the advantages of the selection and the method according to the invention. In the so-called digestibility studies, the protein-rich seeds of the process not only feature a higher nutritional value, but can also inhibit the harmful effects of antinutritional factors while achieving a higher production performance compared to soybean meal, which is a marker of the synergistic effect sought in the step of utilization by the animal.

Animal technology and economic testing of b/labeled broiler poultry

In a tag type chicken reference farm 4400 chickens were raised on two identical buildings according to two feeding programs. The conventional soy flour based procedure was compared to the soy-free and protein-rich seed based procedure of the present invention.

Technical and economic performance was evaluated in terms of consumption, animal weight, growth, feed conversion, mortality and feed cost.

In this test, the nutritional value of fava beans for food formulations is not based on the so-called digestibility value, but on values in the literature. The aim is therefore to check whether the animal-technical performance of the chickens is indeed better than that of the control batches, and if so, to calculate how much the nutritional value of the broad bean seeds should be higher. The following shows the elements:

the technical result obtained thanks to the invention therefore compensates to a large extent for the additional food costs.

Thus, replacing soybean meal with the solution proposed according to the invention up to 8% of the feed allows a significant improvement in technical (weight + 7.5%, GMQ + 7.6%, IC-5.9% and performance index + 13.9%) and economic performance.

According to these animal technology results, the digestibility value of the solution according to the invention alone cannot explain the improved performance. In fact, while the digestibility results were evaluated by calculating the digestibility coefficient at 3313kcal of metabolic energy and 79% of protein, the performance thus obtained allowed an estimate of 11% increase in metabolic energy (i.e. 3675kcal) and 5% increase in protein digestibility (i.e. 83%).

This suggests a synergistic effect associated with better expression at the metabolic level of the animal, which may be linked to a reduction in anti-nutritional factors.

c/Animal technical, environmental and economic testing of standard broiler chickens

In the laboratory, tests to determine the growth performance of broiler chickens were performed. The test was carried out on male chickens fed with RosSPM3 fed with a feed in which the main protein source was soy flour or unselected and technically untreated broad bean seeds (raw broad beans) or the soybean seeds of the invention called "cooked beans" (selected and technically treated). These protein sources were introduced into the growth feed at up to 15% (EM: 2950 kcal/kg; digestible lysine: 11g/kg) and into the fattening feed at up to 20% (EM: 3000 kcal/kg; digestible lysine: 10 g/kg). These feeds have the same nutritional characteristics as soybeans. Animal technical performance was recorded and the impact of these products on the Environment (ECOALIM) and the economy (2018 economic status) was determined.

The growth performance of chickens consuming the iso-nutritive feed in the soy flour, raw beans and cooked bean batches was statistically the same. The consumption index, although not significantly different, shows values numerically favorable for the broad beans of the invention. The consumption index of the latter (1.444) is much lower than that of soybean flour (1.456) and raw fava beans (1.460). When the nutritional value values in the feed of standard broiler chickens are the same, the observed growth performance is the same, which means that the nutritional composition of these feeds is correctly evaluated. These results confirm the digestible EM and LYS content of test faba beans, particularly in the invention. Moreover, to the extent that it is not as good as that of the laying hens, the synergistic effect of the invention is highlighted by the animal performance.

The environmental impact of producing chickens with the broad bean-based feed of the invention on climate change (-41%), phosphorus consumption (-36%), fossil energy consumption (-22%) and acidification (-18%) is positive, neutral to eutrophication (-2%) and negative to land use (+ 13%). The same environmental impact of producing chickens with broad beans not according to the invention has intermediate values.

The economic impact of broiler production was 40.20 euro and 39.45 euro/100 kg live weight, i.e. a drop of-1.9%, assessed by the feed-related part (approximately two thirds of the production cost). The effect is also neutral in terms of french chicken consumption (19.0 kg per year), since it can save 0.14 euro/year. The economic impact of production and consumption of chickens fed with seeds not derived from the present invention is less.

This test shows the dual advantages of the invention in terms of selection of broad bean seeds and appropriate technical treatment. This benefit relates both to the utilization of broad beans produced by the invention by standard broiler chickens and to their environmental weight beneficial to the invention, as well as to the economic weight to french consumers. Thus, the introduction of protein-rich seeds produced by the present invention was validated on standard broiler chickens.

d/Animal husbandry testing of rainbow trout

In the laboratory field, tests to determine the growth performance of rainbow trout were performed. The nutritional value of the broad bean seeds selected according to the invention, which are or are not technically treated and de-coated or not de-coated, were evaluated in advance in rainbow trout parrs raised in fish farms at 17 ℃.

An isoenergetic (23-24kJ/g MS) and an isonitrogenous (43-45% MS) food product comprising 21% fish meal and 25% broad bean seeds or 25% soybean meal to be tested as protein source. The raw materials for forming the fish feed are mixed and ground, and then the fish feed is subjected to hot cooking processing in a double-screw extruder and then is pelletized. These feeds were randomly distributed to trout batches for 84 days.

Compared to the soybean control, there was no difference in animal technical performance, but the technical treatment significantly affected the feed consumption and feed efficiency of the feed containing the seeds derived from the invention: (i) the preparatory and thermal treatments disclosed herein improve feed efficiency and protein efficiency in connection with a reduction in feed consumption. (ii) Dehulling plus heat treatment also improves food efficiency.

In the context of this test, the incorporation of 25% of the broad bean seeds from the invention in the feed allows to achieve the same level of trout growth performance compared to soy flour and iso-nutritional formulas. Even more, certain advantageous technical treatment combinations have a positive effect on the broad bean seeds by increasing the feed efficiency of the broad bean seeds to a level superior to that of the soybean meal. More generally, this illustration demonstrates the synergistic effect of the present invention by the ability of animals to utilize these selected and treated seeds.

Thus, these results indicate that broad bean seeds produced by the present invention are good candidates for replacing soybean meal in trout feed. Furthermore, although aquaculture feeds are made by cooking extrusion, the method according to the invention applied to whole or de-coated broad bean seeds can provide a practical added value by using heat treatment under pressure, thereby improving the nutritional value of these seeds.

e/Animal technical testing of commercial pigs

In a reference farm of fattening pigs, we compared the growth performance of two batches of pigs, whose feed only differs by the contribution of the solution produced by the present invention.

Both the growing and fattening feeds of the two batches had equal nutrient intakes (net energy and digestible amino acids). The solution is provided up to 10% during the growth phase and up to 5% during the fattening phase, replacing the protein cake and the oilseeds.

Since the nutritional value of fava beans used in feed formulations is based on digestibility values, the aim is to check whether the animal technical performance of the animal is equal and, if higher, to see whether there is a possible synergistic effect related to better expression at the metabolic level of the animal.

The following table shows the technical performance data obtained.

The technical performance recorded in this test shows that the average daily gain is increased due to better intake and an equally good gain in weight gain of one kg of feed.

This indicates a synergistic effect between on the one hand a good digestibility of the seeds and on the other hand a positive effect on the intake, which may be associated with a reduction in antinutritional factors, which are often reported to have a negative effect on the intake of food products because they may in particular cause digestive disorders.

As mentioned above, after the animal has ingested the plant material, post-digestion effects of nutrients such as proteins and energy may occur, depending on whether they are derived from the present invention. Previous work reported a decrease in productivity due to altered physiological function and/or digestibility. In these changes to health, allergic reactions may occur. Protein-rich seeds have many documented cases of response after ingestion of these seeds due to the presence of major allergens in the seeds.

f/Dog allergy test

The objective of this work was to study the allergenicity of raw or treated broad bean seeds under different conditions. Sensitization (or reactivity) was assessed by immunoblotting using serum from food-sensitive dogs.

After extracting the proteins from the seeds and selecting the serum from food-sensitive dogs (dogs with immunoglobulin E-type or IgE reactive to protein-rich seeds), their reactivity was investigated by immunoblotting. The extracted protein was cultured in serum containing IgE. When allergenic proteins are present, they bind to IgE in serum and present specific bands on the membrane.

The raw broad bean seeds showed many very strong bands, i.e., strong allergic reactivity. On the other hand, neither the seeds produced by the present invention, i.e. the intact seeds or the de-coated seeds, showed reactivity (no visible bands). It appears that the IgE reactivity initially present in the seed is greatly reduced or even inhibited at the end of the method of the invention.

g/Animal technology testing of cows

On an experimental dairy, experiments were conducted to test the effect of the present invention on the protection of broad bean and lupin seed proteins in the rumen and their digestibility in the intestine.

The test was performed on eight lactating holstein cows fitted with rumen cannulae. The experimental design was a 4 × 4 double latin square matrix. Daily diets, consisting of 60% forage and 40% concentrate (concentres), were fed to the cows twice a day. The forage is a mixture of corn silage (33% of the ration in MS), grass silage (17%), hay (10%), and dry beet pulp (10.75%). The concentrate consisted of corn flour and soy flour for the control diet. The cake was replaced according to the treatment with whole bean seeds, respectively raw white lupin seeds (FEV-ENT-cree and LUP-NT-cree) or white lupin seeds treated according to the INVENTION (FEV-ENT-inventon and LUP-ENT-inventon) or white lupin seeds treated according to the alternative method (treatment temperature outside the proposed range of the INVENTION) (FEV-ENT-ALTER and LUP-ENT-ALTER). The diet was formulated for equal total nitrogen (MAT) (146g/kg MS) and equal net energy (0.99UFL/kg MS), with the concentrate providing 40% of the daily ration MAT.

Rumen NH in the case of seeds of the invention₃Is lower than rumen NH in the case of raw seeds₃Indicating protection of nitrogen from rumen degradation, which increases with increasing temperature, while soy flour yields the lowest values. This protection of nitrogen due to technical treatment was also demonstrated by the reduced 1 hour enzymatic degradability between the raw seeds and the seeds produced according to the invention.

The present invention produces seeds with higher levels of maillard compounds than unprocessed seeds, and in particular has a more pronounced effect on the acrylamide and CML content of lupins.

In addition, the CML content of soy flour is four to seven times higher than that of the seeds produced by the present invention.

In the broad bean latin square matrix, the CML content of excreta in the broad bean diet is lower than that of the control diet containing soy flour. Numerically, more maillard compounds are found in the excreta from seeds from alternative diets than from seeds of the invention. This means that in an alternative approach, the maillard reaction is more likely to reach an irreversible step in the abomasum, leading to over-protection of the nitrogen.

This is confirmed by the reduced apparent digestibility of nitrogen observed in seeds from the alternative method compared to seeds from the present invention. Moreover, the higher content of plasma amino acids from the seeds of the invention compared to the seeds obtained from the substitution process on the one hand and the soy flour on the other hand, confirms the better intestinal absorption of the proteins obtained from the seeds of the invention; thus, it was observed that the utilization of the protein of the seeds produced by the alternative method or the protein produced from the soybean meal was low for animals, since a higher proportion of the protein would escape not only from rumen degradation but also from intestinal digestion and absorption.

These results confirm that after technical treatment of broad bean and lupin seeds, maillard reactions occurred in different ratios. In the case of seeds obtained from the present invention, these reactions protect nitrogen from excessive rumen degradation and are reversible at the acidic pH of the abomasum, thereby optimizing amino acid absorption in the small intestine. On the other hand, at high temperatures (160 ℃), these reactions are no longer reversible in the abomasum, and therefore the "too" protected proteins are no longer absorbed in the intestine.

Finally, from an animal technology point of view, the seeds produced by the present invention are the only seeds that can compete with soybean meal, since they enable the expression of the milk protein production potential of cows. This is not the case for raw seeds and alternative methods of seeds.

h/Study of the economic benefit of the method according to the invention

To perform such an economic analysis, feed formulation software is used, with appropriate information on available raw materials, the nutritional value of the raw materials, the price of these raw materials and the nutritional limitations of the feed for broiler and laying hens at different physiological stages of production.

Thus, after learning the nutritional value and potential price of the best combination of the invention, the economically viable tendencies of the invention were evaluated.

By this same method, the beneficial price of raw materials developed from the best processing combination produced by the present invention can also be assessed. It can thus be seen that the present invention is economically very interesting, especially because of formulation limitations associated with specific specifications (the specifications considered relate to obligations in the feed formulation to be non-transgenic, non-imported or locally sourced).

Three feed formulations for growing broilers are shown below, showing that the solution produced by the invention (mixture of broad bean and soybean seeds, in proportions of 90% and 10%, respectively) is economically more advantageous due to the technical and economic advantages in optimizing the content in non-transgenic formulations, compared to the known raw materials of the prior art (in this case soybean meal and cereals).

A comparative table of three non-transgenic growth feed formulations for broiler chickens: one without the solution created using the present invention and two with the solution created using the present invention.

Through the practice of this formulation, it can be seen that the solution of the invention is optimized in the non-transgenic growth feed formulation for broiler chickens, 13.5% in experimental formulation 1, resulting in the inclusion of 6.3% rapeseed meal and a substantial reduction of 12.7% and 10.3% in soybean meal and corn meal, respectively.

In addition, if it is attempted to remove the non-transgenic soybean flour completely from the test formulation 2, the content of the solution of the invention is 20% and the cost price of the formulation is 1 —/t lower than the original standard formulation.

The formulation study confirms the technical and economic feasibility of the invention.

Finally, as far as the application is concerned, the method which forms the subject of the present invention aims at promoting the inclusion of protein-rich seeds in the diet of monogastric animals in place of other protein sources (such as soya flour or other imported flours), thus satisfying the needs of breeders for greater protein autonomy at the regional level, as well as the needs of consumers for more sustainable livestock products (fed without transgenic organisms and whose food is of local origin).

The field of application of the method of the invention may involve two uses in animal husbandry:

use in the preparation of raw materials

A concentrate based on protein crops is prepared, which becomes a raw material that can be introduced into a complete feed or an auxiliary feed for monogastric animals, intended for industrial and/or farm feed manufacturers. In this case, the minimum incorporation of the selected seed is 20%, preferably 40%.

Other raw materials that make up the concentrate may be subjected to all or part of the steps of the described invention, even more so if the steps make these raw materials profitable.

Thus, in a non-limiting manner, preferred starting materials are seeds or any other amyloid-like product, such as cereals and oilseeds.

Use in the preparation of foodstuffs

Complete or supplemental grain feed is prepared for poultry breeders to feed their monogastric animals. In this other case, the minimum incorporation of the selected seed is 5%, preferably 10%.

Furthermore, the food solutions produced by the present invention vary according to the needs of the food manufacturers and breeders, depending on their location on the one hand in the free zone of france or soybean, locally produced, non-transgenic, and on the other hand in the zone called "Bleu Blanc Coeur" (registered trademark).

Indeed, in the case of french protein requirements, it is preferable to treat broad beans in combination with metro soybeans. It is advantageous to combine it with flax seeds in order to meet the "Bleu-Blanc-Coeur" specification.

In the first case, soy is used to concentrate the protein content of the product. In the second case, flaxseed provides a traceable and guaranteed omega-3. The advantage of this method is that, within the framework of the use of food manufacturers, no additional storage unit is required, but another flax-based product can be substituted, which is usually associated with raw material support without much technical interest (wheat bran, cereals …).

Here is an example of a solution:

1/formulation of non-transgenic/native protein supply chain:

based on 90% broad bean seeds and 10% soybeans;

based on 70% broad bean seeds and 30% soybeans.

Formulation of 2/Bleu-Blanc-Coeur method:

based on 75% broad bean seeds and 25% flaxseed;

based on 50% broad bean seeds and 50% flaxseed;

based on 25% broad bean seeds and 75% flaxseed.

The use of the seeds produced by the present invention may find value in domestic and ruminant animals. Although they were developed for feeding monogastric livestock, the seeds treated according to the invention are fully useful for feeding domestic animals such as dogs, cats and ruminants.

In an advantageous way for ruminant use, the benefits of using carbohydrate degrading enzymes on the one hand and so-called reducing sugar sources on the other hand can be retained. Indeed, in addition to the heat effect, one method of protecting proteins from rumen degradation while improving their intestinal digestibility includes: 1) in the hydrothermal and enzymatic preparation steps of the process, an enzyme is added which is capable of hydrolyzing carbohydrates into glucose units or other simpler reducing sugars (reactive towards proteins); and/or 2) in the make-up step, more or less reducing sugar sources remain based on the use of additional starting materials.

The skilled person knows that the use of protein-rich seeds in ruminants is particularly desirable to reduce the rumen degradability of their proteins, and one of the ways to achieve this reduction is to initiate the first step of the maillard reaction between the amine functions of the proteins and the reducing functions of the sugars.

The present invention proposes to create new conditions for carrying out these reactions, which are reversible, so that the intestinal digestibility of proteins is excellent.

The use of the seeds produced by the present invention is also beneficial in the case of pet breeding. In one aspect, the protein-enriched seeds thus prepared provide a diverse and highly digestible protein and energy source, with reduced antinutritional factors, and with reduced sensitization potential of the protein source. Indeed, the skilled person knows that the risk of allergens is significantly reduced due to the biochemical reaction in one heating step of the method (Franck et al, 2008).

Finally, since this method brings a nutritional added value to plant proteins, the use of this method can also be extended to the human food market, where it is currently expected that the dietary intake of plant proteins by people in developed countries will increase, and more so the risk of sensitization of plant proteins will decrease.

Indeed, the french national food safety agency recommends restoring the protein source balance between animal and vegetable proteins in the human diet, so that the 70/30 ratio becomes 50/50 ratio.

The nutritional limitations of known protein-rich seeds (also known as leguminous food) in monogastric animals are the same as in monogastric humans. This is why we believe that the invention is ultimately directed to the field of animal production (monogastric and ruminant animals), and also to the field of transformation of plant proteins for direct human consumption.

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