METHOD FOR PREPARING AN UMAMI FOOD INGREDIENT FROM A HEMP-BASED PLANT SUBSTRATE

Abstract

The invention relates to the field of plant-based protein foods. More particularly, the invention concerns a process for preparing an umami food ingredient from a hemp seed-based substrate. This process is based on the fermentation of the substrate by at least one strain of Bacillus subtilis. This process may include a second modification, in particular a second fermentation, to modify the organoleptic properties of the product resulting from the first fermentation. This process allows the preparation of a high-quality plant-based protein ingredient with desirable organoleptic properties. The resulting food product can be used as an organoleptic substitute for animal proteins.

Description

Title of the invention: PROCESS FOR PREPARING AN UMAMI FOOD INGREDIENT FROM A HEMP-BASED PLANT SUBSTRATE

[0001] The invention relates to the field of plant protein-based foods. More particularly, the invention relates to a method for preparing an umami food ingredient obtained from a hemp seed-based substrate. This method is based on the fermentation of the substrate by at least one strain of Bacillus subtilis. This method may comprise a second modification, in particular a second fermentation, in order to modify the organoleptic properties of the product resulting from the first fermentation. This method allows the preparation of a plant protein ingredient of high nutritional quality having interesting organoleptic properties. The food product obtained can be substituted for animal proteins. Field of the invention

[0002] It is now accepted that excessive consumption of animal products has a significant environmental cost for the planet. According to ADEME, the average French diet emits four times more greenhouse gases (GHG) than a vegan diet, or nearly a ton more carbon. Faced with this growing awareness, consumers are increasingly interested in alternatives to meat. This change in perspective has led manufacturers to develop protein and textured products in order to get closer to the uses of meat from plants whose environmental impact is much lower. Thus, in addition to traditional products such as tofu, tempeh or seitan, products have appeared in recent years aimed at imitating meat in the form of, for example, minced meat, steaks or vegetable bacon.The choice of plant raw material and its processing result in products that vary in terms of texture, taste, smell, appearance and nutritional quality. The challenges in this sector are significant and consumer expectations are high.

[0003] Very often, the grassy or bitter taste and texture of these processed foods is a barrier to adoption by the majority. One of the development axes of these new protein products of plant origin is based on the fermentation of plant protein isolates which makes it possible to improve the organoleptic properties of the food. But this technique requires particular know-how regarding the choice of microbial strains involved in the fermentation and the conditions of this transformation which must be adapted to each plant material.

[0004] Natto, a traditional Japanese product obtained by Fermentation of soybeans with a strain of Bacillus subtilis natto. The resulting product is not textured.

[0005] Document JP2008072917 describes a method for preparing a barley and soybean-based food obtained by fermentation with a Bacillus subtilis natto strain. Soybean is used as a fermentation aid, since fermentation of barley alone by the Bacillus subtilis natto strain is not effective.

[0006] Document CN115404227 describes a method for using hemp seed residues to produce nattokinase by solid-state fermentation. This fermentation is obtained by inoculating at least three different strains of Bacillus. The fermented product is centrifuged to extract the enzyme.

[0007] While the development of alternative products to meat is booming, diversification of the offer is desirable in order to satisfy consumers, an essential criterion for achieving a more environmentally friendly food transition. Statement of the invention

[0008] The present invention relates to a process for preparing an umami food ingredient which can replace animal proteins from an organoleptic point of view, from a plant substrate comprising at least 50% by weight of hemp seeds, said process comprising the following steps: - Pretreatment of said seeds to allow the opening of their seed coat - Aqueous impregnation of said substrate at a temperature between 25°C and 100°C - Fermentation in solid medium of said substrate by contact with at least one strain of Bacillus subtilis for a period of between 12h and 72h, at a temperature of between 37°C and 53°C.

[0009] The hemp-based food ingredient obtained by fermentation is rich in nutrients and has interesting organoleptic properties. Advantages of the invention

[0010] The inventors have developed an innovative way of using hemp for human consumption. This involves fermenting a substrate based on hemp seeds with a strain of Bacillus subtilis. The resulting fermented product is nutritionally rich and made attractive for human consumption thanks to satisfactory organoleptic properties.

[0011] The method according to the invention allows the preparation of a vegetable culinary ingredient rich in textured proteins from hemp seeds or a substrate rich in hemp seeds. The hemp proteins thus treated are more assimilable than untreated vegetable proteins. In particular, the quantity of amino acids in the ingredient - such as glutamate, responsible for the imami taste, but also aspartate, glutamine and glycine - is increased compared to that present in the initial substrate, as well as that of many metabolites.

[0012] The method according to the invention comprises the fermentation of a hemp-based substrate using a Bacillus subtilis bacterium. The product obtained has an umami taste appreciated by consumers. It can be offered as a substitute for animal proteins as part of a vegetarian diet or more broadly for anyone wishing to eat a balanced diet while limiting their consumption of animal protein.

[0013] The new fermented ingredient can be processed from a taste point of view either by culinary transformation (cooking, mixing, seasoning, etc.), or by carrying out a second modification such as another fermentation. These transformation methods make it possible to modify the organoleptic properties of the ingredient, in particular its taste and texture, so as to obtain substitute products for meat, fish, cheese, etc.

[0014] Compared to soy, the most widely used plant protein source today, the fermented ingredient from hemp seeds is not likely to cause allergies and is free from antinutritional factors. Likewise, compared to pea proteins, which are known to cause digestive problems and flatulence.

[0015] The present invention therefore completes the offer of culinary raw materials and food products of 100% plant origin by proposing a process for preparing products with high quality nutritional properties and organoleptic properties of interest to the consumer. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to a process for preparing an “umami” type food ingredient from a plant substrate comprising at least 50% by weight of hemp seeds, said process comprising the following steps: - Pretreatment of said seeds to allow the opening of their integument - Aqueous impregnation of said substrate at a temperature between 25°C and 100°C. - Fermentation in solid medium of said substrate by contact with at least one strain of Bacillus subtilis for a period of between 12h and 72h, at a temperature of between 37°C and 53°C.

[0017] In the context of the present invention, the hemp seeds used have a THC content in accordance with current regulations.

[0018] The seed pretreatment step aims to open the seed coat to allow moistening of the protein-rich material under the seed coat. Opening can be achieved by different methods such as decortication, grinding or germination... The pretreatment step must at least allow the seed coat to open but can consist of a deeper transformation of the seed. This pretreatment can be carried out just before the impregnation step or upstream, or even at a distance from the implementation of the process; it is indeed possible to implement this process from ground, crushed, mixed seeds, in particular from hemp co-products, such as okara. Okara corresponds to the oilseed residues of hemp seeds obtained during the manufacture of vegetable drinks.

[0019] The seeds thus pretreated (at least open) are then moistened or hydrated by impregnation in an aqueous solution.

[0020] In a preferred embodiment, the seeds are moistened by soaking in a bath heated between 25°C and 100°C, preferably between 25° and 65°C, even more preferably between 35°C and 45°C until saturation. The bath consists of an aqueous solution, preferably water. The seeds are then drained to remove excess water.

[0021] The seeds thus impregnated are fermented in a solid medium by adding at least one strain of Bacillus subtilis in the form of an inoculum applied to the seeds. Fermentation in a solid medium consists of ensuring a sufficient humidity level to promote fermentation but without the substrate and bacteria being in a liquid medium. This fermentation can take place in an oven or a fermenter so as to maintain a constant humidity level and promote bacterial culture and the fermentation process. The temperature and duration of the fermentation can be modulated according to the desired level of fermentation which influences the appearance and consistency of the fermented “hemp paste”; the adjustment of these parameters is well known to those skilled in the art. Generally speaking, fermentation will take place for a period of between 12 and 72 hours, at a temperature of between 37°C and 53°C.

[0022] The fermentation of the hemp seed-based substrate is carried out using at least one strain of Bacillus subtilis. Among the strains of Bacillus subtilis that can be used for this fermentation, mention may be made of: Bacillus subtilis var. natto, Bacillus subtilis GB03, Bacillus subtilis QST 713.... The inoculum may comprise one or more strains of Bacillus. In a preferred embodiment, the inoculum comprises at least one strain of Bacillus subtilis var. natto.

[0023] A fermentation rate allowing the transformation of hemp seeds into a textured ingredient is obtained by adding at least 3g of inoculum in 1.5 to 4.5 kg of hemp-based substrate.

[0024] The hemp-based substrate comprises at least 50% hemp seeds. It may also comprise rice grains, oat flakes, fabaceae (such as chickpeas, yellow peas), vegetables, etc. The substrate may also consist of 100% hemp seeds. According to embodiments in accordance with the invention, the hemp-based substrate comprises at least 60%, preferably 70%, and even more preferably at least 80%, or even at least 90% hemp seeds.

[0025] As previously indicated, by "hemp seeds" is meant hemp seeds as such and any derivative product resulting from the processing of hemp seeds, such as the co-products of such processing.

[0026] This produces an "umami" type ingredient. Umami is associated with glutamic acid and its derivatives such as monosodium glutamate. It constitutes one of the five basic tastes, in addition to sweet, sour, bitter and salty. For example, meats and fish such as pork, cold cuts, steak and salmon have a basic umami taste. This unique flavor is also found in vegetables such as tomatoes, cabbage, mushrooms, cheeses and green tea. The umami taste is characterized by its depth and length in the mouth, but also by its roundness.

[0027] The product obtained at the end of fermentation using at least one Bacillus subtilis strain can be used as a ready-to-cook food ingredient. It is defined by an odor, taste and consistency that differentiate it from the initial substrate and depend on the fermentation conditions. It has a neutral to basic pH between 7 and 10. The modifications induced by fermentation are identifiable by quantification of amino acids and metabolites. Such analyses have highlighted an increase in glutamate, responsible for the umami taste, but also other amino acids such as aspartate, glutamine and glycine.

[0028] The organoleptic properties of the food ingredient can be modulated to suit by carrying out other modification steps, either on the substrate pretreated before fermentation with Bacillus subtilis, or the substrate fermented with Bacillus subtilis. These additional modifications allow adjustment of the taste, smell and / or consistency according to the desired characteristics, and the production of food products such as meat, fish, cheese or others.

[0029] In a first embodiment, this second modification step consists of a second fermentation using at least one ferment chosen from a lactic acid bacterium, a yeast or a fungus. It can be carried out on the substrate pretreated before fermentation with Bacillus subtilis, or on the substrate fermented with Bacillus subtilis.

[0030] When this ferment is a lactic acid bacterium, the fermentation will take place between 20 and 30 °C for a period of between 12 and 72 hours. It is not necessary to adjust the pH of the fermented substrate, as fermentation by lactic acid bacteria can be initiated on the substrate fermented with Bacillus. Examples of bacteria that may be suitable for this fermentation are lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus grasseri, Lactobacillus sakei etc.

[0031] When this ferment is a yeast, the fermentation will take place between 18 and 28 °C for a period of between 12 and 72 hours. Examples of yeasts that may be suitable for this fermentation are Saccharomyces cerevisiae, Candida utilis etc.

[0032] When this ferment is a fungus, the fermentation will take place between 20 and 37 °C for a period of between 12 and 72 h. Examples of fungi that may be suitable for this fermentation are Rhizopus spp. and Aspergillus spp.

[0033] Depending on the microorganism used, different types of modification of the product fermented by Bacillus can be observed, such as a drop in pH to a minimum of 7, a change in the odor (mainly with yeasts), an increase in the fermentability (mainly with fungi), a modification of the taste (mainly with bacteria) of the final product.

[0034] In addition, the pH of the fermented product can be modified, for example to bring it back to around 7 for reasons of preservation or stabilization. pH adjustments are known to those skilled in the art.

[0035] The inventors have shown that products fermented with a Bacillus subtilis strain exhibit an increase in the content of certain amino acids. Experimental data show that the contents of aspartate, glutamine, glutamate and glycine are increased by at least a factor of 2 and up to 10 times more after a first fermentation compared to the contents present in the hulled seeds before fermentation. A second fermentation makes it possible to further increase these contents.

[0036] Thus the invention also relates to a food ingredient of the “umami” type obtained from a hemp-based plant substrate in which the level of certain amino acids is increased compared to the level present in the hemp seeds before fermentation with a Bacillus subtilis strain.

[0037] Among the amino acids whose level is increased in the fermented product, we can cite aspartate, glutamine, glutamate and glycine.

[0038] The increase in the amino acid level is at least a factor of 2 but can be increased by at least a factor of 3, 4 or 5 after a simple fermentation with a Bacillus subtilis strain. This level is improved by a second transformation, as indicated previously, and can reach up to a factor of 5, or even 10 and even 15. The experimental conditions can also be modulated to optimize this factor.

[0039] In particular, an “umami” type food ingredient obtained by fermentation with a Bacillus subtilis strain from a hemp-based plant substrate according to the invention presents: - a glutamate content at least 2 times higher than that of unfermented pretreated hemp seeds, preferably at least 5 times higher, and even more preferably at least 10 or 20 times higher.

[0040] The present invention will be better understood from reading the following examples, provided for illustration purposes and in no way to be considered as limiting the scope of the present invention. EXAMPLES

[0041] The hemp seeds used in the process according to the invention may be, for example, whole germinated seeds, crushed hulled hemp seeds or hulled hemp seeds, mixed with water, mixed and then filtered (recovery of the solid phase).

[0042] The hemp-based substrate may comprise, in addition to hemp seeds, for example, cooked rice grains, oat flakes such as groats, cooked or canned chickpeas, cooked or canned red beans, defrosted spinach, etc.

[0043] EXAMPLE 1: Simple fermentation of hulled hemp seeds and analysis of changes in amino acid content by HPLC

[0044] Step 1: Preparation of the substrate

[0045] Preheat the induction hob, and boil water in a beaker (-100°C).

[0046] Pour the hulled hemp seeds into boiling water.

[0047] Cook the seeds for 20 minutes at -100°C.

[0048] Keep the water boiling during cooking.

[0049] Step 2: Preparation of the inoculation solution

[0050] Preparation of an inoculum under sterile conditions of bacillus subtilis in sterile physiological water.

[0051] Store at 37°C in a rotating incubator for approximately 1 hour.

[0052] Step 3: inoculation of the substrate

[0053] After cooking, filter the seeds and return them to the beaker.

[0054] / !\ handle in sterile area (psm).

[0055] Inoculate the seeds with the ferment (suspension of bacillus subtilis natto).

[0056] Mix the seeds.

[0057] Step 4: fermentation

[0058] Spread the seeds in layers in a ventilated container.

[0059] Store in an oven at 37°C for 48 to 72 hours under saturating humidity. Analysis of the fermented product

[0060] Table 1 describes the content of certain amino acids in hulled seeds before fermentation, then in the product fermented with a strain of Bacillus subtilis. The analyses were carried out using high-performance liquid chromatography (HPLC).

[0061] [Tables 1] Hulled seeds 1st fermentation with Bacillus vr natto N=3 Mean standard deviation Mean standard deviation Aspartate 20.2 13.9 31.5 13.2 Glutamine 13.9 14.9 23.3 12.6 Glutamate 64.2 73.3 128.4 94.0 Glycine 24.3 17.6 30.7 16.4

[0062] Table 1: Aspartate, glutamine, glutamate and glycine contents after a first fermentation stage from hulled hemp seeds analyzed by HPLC. The results are expressed in nmol.mg DW1.

[0063] These results show that the fermentation conditions with a strain of Bacillus subtilis used allow a transformation of hemp seeds associating an increase in the amino acid content which makes them better assimilated by the body.

[0064] In particular, an increase in glutamate, a molecule associated with the umami taste, will be noted. They also confirm the fermentation activity of Bacillus subtilis bacteria on the hemp substrate.

[0065] EXAMPLE 2: Double fermentation of hulled hemp seeds and analysis of changes in amino acid content by HPLC

[0066] Step 1: Preparation of the substrate

[0067] Preheat the induction hob, and heat water in a beaker (~50°C).

[0068] Pour the hulled hemp seeds into the hot water.

[0069] Soak the seeds at 50°C for 1 hour while stirring. Drain the seeds lightly.

[0070] Step 2: Preparation of the Bacillus inoculation solution

[0071] Preparation of an inoculum of Bacillus subtilis var. natto from an isolate. Carrying out a DO measurement. Dilution. Inoculation with 1 mL per 300g of pre-treated hemp seeds. Mix the inoculated seeds and place them in a ventilated container.

[0072] Step 3: Bacillus fermentation

[0073] Distribute the seeds in a ventilated container.

[0074] Store in an oven at 43°C for 36 to 48 hours under saturating humidity.

[0075] Step 4: preparation of the lactic acid bacteria inoculation solution

[0076] Preparation of a Lactobacillusplantarum inoculum from an industrial sachet as indicated by the supplier. Inoculation with 1 mL per 300g of once-fermented hemp seeds.

[0077] Mix the inoculated seeds with the inoculum.

[0078] Step 5: fermentation of Lactobacillus

[0079] Distribute the seeds in a sealed container.

[0080] Store in an oven at 37°C for 36 to 48 hours under saturating humidity. Analysis of the fermented product

[0081] Table 2 describes the content of certain amino acids in hulled seeds before fermentation, then in the product fermented with a strain of Bacillus subtilis. The analyses were carried out using high-performance liquid chromatography (HPLC).

[0082] [Tables2] Hulled seeds 1st fermentation with Bacillus vr natto 2nd fermentation with Lactobacillus plantarum N=3 Mean standard deviation Mean standard deviation Mean standard deviation Aspartate 20.2 13.9 31.5 13.2 31.9 32.6 Glutamine 13.9 14.9 23.3 12.6 60.6 53.8 Glutamate 64.2 73.3 128.4 94.0 276.7 235.1 Glycine 24.3 17.6 30.7 16.4 48.3 37.7

[0083] Table 2: Aspartate, glutamine, glutamate and glycine contents after double fermentation from hulled hemp seeds analyzed by HPLC. The results are expressed in nmol.mg DW1.

[0084] These results show that the organoleptic properties, here measured by evaluating the increase in amino acid content, are improved by carrying out a double fermentation. It will be noted that the second fermentation with Lactobacillus plantarum further increases the amino acid content compared to that present in the product fermented with Bacillus var. natto. In particular, the quantity of glutamate is increased by more than a factor of 4 compared to the pretreated but unfermented seeds.

[0085] EXAMPLE 3: Fermentation of hulled hemp seeds and analysis of changes in amino acid content by GC-MS

[0086] The experimental conditions of Example 3 are the same as those implemented in Examples 1 and 2.

[0087] The analyses were carried out by gas chromatography coupled with mass spectrometry (GC-MS); the results are given in GC-MS units, i.e. the metabolite peaks are normalized to Ribitol (control) and dry weight. The metabolite contents are expressed in arbitrary units (semi-quantitative determination).

[0088] The results are presented in Table 3.

[0089] [Tables3] Pretreated seeds 1st fermentation with a Bacillus Vr Natto strain 2nd fermentation with a Lacto-bacillus plantarum strain N=3 Mean standard deviation Mean standard deviation Mean standard deviation Aspartate 0.087 0.095 0.283 0.155 0.412 0261 Aspartic acid 1 0.055 0.058 0.184 0.098 0.315 0.196 Aspartic acid 2 0.031 0.037 0.098 0.057 0.097 0.065 Glutamine 0.176 0250 0.352 0310 1.024 0.987 Glutamine 3 0.176 0.250 0.352 0.310 1.024 0.987 Glutamate 0.342 0282 2.197 1.692 6.784 2.805 L-glutamic acid 2 0.060 0.072 0.915 0.769 2.736 1.446 L-pyroglutami c acid 0.282 0.210 1.282 0.926 4.048 1.359 N-carbamyl-L-Glutamic acid 1 Trace amounts Glycine 0.072 0.032 0.157 0.083 0.214 0.153 Glycine 0.072 0.032 0.157 0.083 0.214 0.153

[0090] Table 3: GC-MS results measuring the contents of Aspartate, Glutamine, Glutamate and Glycine.

[0091] These results confirm the variations of Aspartate, Glutamine, Glutamate and Glycine measured by HPLC from the pretreated seeds then for the first fermentation with a strain of B. subtilis vr natto and after a second fermentation with Lactobacillus plantarum (N=3).

[0092] Conclusion of Examples 1 to 3:

[0093] Fermentation of hemp seeds using a strain of B. subtilis allows the content of certain amino acids to be modified, resulting in an improvement in the organoleptic properties of the fermented products, with single or double fermentation. A significant increase in glutamate can be noted, which confirms the possibility of increasing the umami taste via fermentation, as well as a significant increase in glycine, which provides a sweet taste, and in aspartate and glutamine, also associated with taste.

[0094] EXAMPLE 4: Analysis of metabolites in fermented products with a strain of Bacillus subtilis

[0095] The metabolite content in the pretreated seeds, simply fermented with Bacillus subtilis, was studied by GC-MS. The conditions for preparing the fermented products are those described in Example 1.

[0096] The results are presented in Table 4.

[0097] [Tables4] Metabolites Pretreated seeds Standard deviation Simple fermentation Standard deviation Factor 3-(4-hydroxyphenyl)propionic acid 0.004 0.001 0.148 0.079 40 2,6-Diaminopimelic acid 2 0.001 0.000 0.025 0.008 36 Citrulline 2 0.002 0.001 0.049 0.013 32 Arabinose 0.001 0.000 0.031 0.007 32 Av-cide L-glutamic 2 0.120 0.072 2.744 0.769 23 5-hydroxy-L-tryptophan 0.001 0.000 0.017 0.005 22 Hydrocinnamic acid 0.003 0.002 0.071 0.038 22 Linoelaidic acid 0.003 0.002 0.065 0.019 21 arabitol 0.004 0.000 0.068 0.019 18 glutamine 3 0.066 0.046 1.055 0.310 16 phenethylamine 0.006 0.023 0.085 0.020 14 4-hydroxyphenylacetic acid 0.001 0.000 0.016 0.004 14 cystine 3 0.010 0.007 0.126 0.035 13 omithin 2 0.172 0.438 1.844 0.466 11 Mucic acid 0.001 0.000 0.010 0.003 10 3-indolelactic acid 2 0.002 0.001 0.021 0.010 10 D-Ala-D-Ala2 0.016 0.008 0.130 0.030 8 histidine 3 0.123 0.079 1.004 0.308 8 L-pyroglutamic acid 0.563 0.210 3.846 0.926 7 Malonic acid 1 0.001 0.000 0.009 0.002 7 5-aminovaleric acid 1 0.887 0.616 5.332 2.263 6 Lactic acid 0.058 0.018 0.336 0.156 6 glycerol 1-phosphate 0.004 0.002 0.023 0.006 6 cysteine ​​2 0.001 0.000 0.004 0.001 5 . Guanidinopropionic acid 1 0.003 0.002 0.014 0.004 5 asparagine 2 0.011 0.006 0.061 0.017 5 2-hydroxybutyric acid 0.008 0.003 0.043 0.015 5 Aspartic acid 1 0.111 0.058 0.552 0.098 5 3-phenyllactic acid 0.017 0.002 0.085 0.028 5 Aspartic acid 2 0.063 0.037 0.295 0.057 5 Glycolic acid 0.002 0.000 0.010 0.002 5 4-hydroxyphenylglycine 2 0.015 0.006 0.063 0.013 4 3-ureidopropionate 2 0.001 0.000 0.005 0.002 4

[0098] Table 4: GC-MS results representing the quantity of metabolites in pretreated seeds vs. after simple fermentation using a B. subtilis natto strain (N=3). The last column represents the increase factor.

[0099] EXAMPLE 5: Analysis of metabolites in double-fermented products with a strain of Bacillus subtilis and a strain of lactic acid bacteria

[0100] The metabolite content in the pretreated seeds, doubly fermented with Bacillus subtilis and a strain of Lactobacillus plantarum were studied by GC-MS. The conditions for preparing the fermented products are those described in Example 2.

[0101] The results are presented in Table 5.

[0102] [Tables5] Metabolites Pretreated seeds Standard deviation Double fermentation Standard deviation Factor 3-(4-hydroxyphenyl)propionic acid 0.004 0.001 3.377 1.130 902 Hydrocinnamic acid 0.003 0.002 0.371 0.134 115 L-glutamic acid 2 0.120 0.072 5.704 1.770 48 glutamine 3 0.066 0.046 3.072 0.987 46 histidine 3 0.123 0.079 3.861 1.057 31 3-indolelactic acid 2 0.002 0.001 0.061 0.016 29 4-hydroxyphenylacetic 0.001 0.000 0.028 0.006 25 5-aminovaleric acid 1 0.887 0.616 20.021 4.541 23 L-pyroglutamic acid 0.563 0.210 8.786 2.165 16 thymine 0.004 0.002 0.058 0.012 15 5-hydroxy-L-tryptophan 0.001 0.000 0.011 0.003 15 2-hydroxybutyric acid 0.008 0.003 0.085 0.021 10 Guanidinopropionic acid 1 0.003 0.002 0.023 0.005 9 3 -pho sphogly cerate 0.000 0.000 0.002 0.000 9 3-ureidopropionate 2 0.001 0.000 0.009 0.002 8 asparagine 2 0.011 0.006 0.087 0.034 8 xanthine 0.021 0.003 0.157 0.059 7 Mucic acid 0.001 0.000 0.007 0.001 7 Linoelaidic acid 0.003 0.002 0.021 0.005 7 2,6-diaminopimelic acid 2 0.001 0.000 0.004 0.001 6 Aspartic acid 1 0.111 0.058 0.643 0.223 6 arabinose 0.001 0.000 0.006 0.001 6 Lactic acid 0.058 0.018 0.313 0.134 5 lysine 2 0.158 0.054 0.805 0.347 5 . threitol 0.003 0.002 0.012 0.001 5 Saccharic acid 0.003 0.000 0.012 0.001 5 Malonic acid 1 0.001 0.000 0.006 0.001 5 4-hydroxyphenylglycine 2 0.015 0.006 0.066 0.008 5 glycine 0.145 0.032 0.643 0.153 4

[0103] Table 5: GC-MS results representing the quantity of metabolites in pretreated seeds vs. after double fermentation using first a strain of B. subtilis natto and then a strain of Lactobacillus plantarum (N=3). The last column represents the increase factor.

[0104] Conclusion on Examples 4 and 5:

[0105] It can be noted that the fermentation of hemp seeds with at least one strain of Bacillus subtilis induces a significant increase in a large number of metabolites associated with health claims and / or taste notes.

[0106] If we consider those which are increased by at least a factor of 10, we find in particular: - 3-(4-hydroxyphenyl)propionic acid often used as an additive, as a preservative. - Citrulline, known for its ability to reduce muscle breakdown - Arabinose, a natural sugar inhibitor, which has pre- biotics. - L-glutamic acid is associated with maintaining the integrity of the intestinal walls and has pre- and probiotic effects. It is used in industry in the form of salts and is the basis of umami taste. - 5-hydroxy-L-tryptophan which contributes to mental concentration. - Hydrocinnamic acid known for its anti-inflammatory, antioxidant and anti-tumor properties. - Linoleic acid, derived from linoleic acid, omega 3, positive for the cardiovascular system. - Arabitol which provides a sweet flavor. - Glutamine involved in immune protection, maintenance of intestinal wall integrity and acid-base balance. - Phenylethylamine, an essential amino acid, plays a role in stimulating the central nervous system.

[0107] The increased presence of these compounds demonstrates the beneficial effect of fermentation of hemp seeds with Bacillus subtilis, possibly combined with a lactic acid bacterium to increase the effects.

[0108] EXAMPLE 6: Simple fermentation of a substrate derived from hemp seeds

[0109] Step 1: Preparation of hemp seeds

[0110] Use hulled hemp seeds and soak them in 3 volumes of water at room temperature. Roughly mix the preparation. Filter and keep the solid part.

[0111] Step 2: Preparation of the inoculation solution

[0112] Preparation of an inoculum under sterile conditions of Bacillus subtilis in sterile physiological water.

[0113] Store at 37°C in a rotating incubator for approximately 1 hour.

[0114] Step 3: inoculation of the substrate

[0115] Take the solid preparation obtained from the hemp seeds.

[0116] / !\ Handle in sterile area (PSM).

[0117] Inoculate the preparation with the ferment (suspension of Bacillus subtilis natto).

[0118] Mix.

[0119] Step 4: fermentation

[0120] Spread the inoculated preparation in a layer in a ventilated container.

[0121] Store in an oven at 37°C for 48 to 72 hours under saturating humidity.

[0122] EXAMPLE 7: Simple fermentation of 70% hemp seeds with 30% cooked rice

[0123] Step 1: Preparation of hemp seeds

[0124] Preheat the induction hob, and heat water in a beaker (~50°C).

[0125] Pour the hulled hemp seeds into the hot water.

[0126] Soak the seeds at 50°C for 1 hour while stirring. Drain the seeds lightly.

[0127] Step 2: preparing the rice grains

[0128] Wash the rice grains several times in cold water then bring a pan of water to a boil. Boil and cook the seeds according to the supplier's instructions. Drain the cooked seeds and wait for them to cool to room temperature.

[0129] Step 3: Preparation of the Bacillus inoculation solution

[0130] Preparation of an inoculum of Bacillus subtilis var. natto from an isolate. Carrying out a OD measurement. Dilution. Inoculation with 1 mL per 200g of pre-treated hemp seeds mixed with 100g of cooled cooked rice. Mix the inoculated seeds and place them in a ventilated container.

[0131] Step 4: Bacillus fermentation

[0132] Spread the seeds in a ventilated container.

[0133] Store in an oven at 43°C for 36 to 48 hours under saturating humidity.

Claims

Claims

1. Process for the preparation of an “umami” type food ingredient which can replace animal proteins from an organoleptic point of view, from a plant substrate comprising at least 50% by weight of hemp seeds, said process comprising the following steps: - Pretreatment of said seeds to allow the opening of their integument - Aqueous impregnation of said substrate at a temperature between 25°C and 100°C - Solid medium fermentation of said substrate by contacting with at least one strain of Bacillus subtilis for a period of between 12h and 72h, at a temperature between 37°C and 53°C.

2. A method according to claim 1 wherein said pretreatment consists of decortication, grinding or germination.

3. A method according to any preceding claim wherein said strain of Bacillus is added to the substrate, the amount added being at least 3g of bacterial inoculum in 1.5 to 4.5 kg of hemp-based substrate.

4. A method according to any preceding claim wherein said strain of Bacillus is a strain of Bacillus var. natto.

5. A method according to any preceding claim wherein said substrate further comprises rice grains, oat flakes, fabaceae or vegetables.

6. Method according to one of the preceding claims further comprising a second fermentation carried out either on the substrate pretreated before fermentation with said at least one strain of Bacillus subtilis, or on the fermented substrate obtained at the end of the fermentation with said at least one strain of Bacillus subtilis, this second fermentation being carried out using at least one ferment chosen from a lactic acid bacterium, a yeast or a fungus.