Method for preparing an umami food ingredient from a hemp-based plant substrate

Abstract

The invention relates to the field of plant protein-based food products. More particularly, the invention relates to a method for preparing an umami food ingredient obtained from a hempseed-based substrate. The 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 makes it possible to prepare a plant-based protein ingredient having a high nutritional value and advantageous organoleptic properties. The obtained food product can be used as a substitute for animal proteins from an organoleptic perspective.

Description

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 a strain of Bacillus subtilisnatto. 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 invention

[0002] It is now widely 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 shift in perspective has led manufacturers to develop protein and textured products to approximate the uses of meat from plants, which have a much lower environmental impact. Thus, in addition to traditional products such as tofu, tempeh, or seitan, products have emerged in recent years that aim to imitate meat, such as minced meat, steaks, or vegetable bacon.The choice of plant-based raw materials and their processing results 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] Often, the grassy or bitter taste and texture of these processed foods are a barrier to widespread adoption. One of the areas of development for these new plant-based protein products is based on the fermentation of plant protein isolates, which improves the food's organoleptic properties. However, this technique requires specific expertise in the selection of microbial strains involved in fermentation and the processing conditions, which must be adapted to each plant material.

[0004] Natto, a traditional Japanese product made by fermenting soybeans with a strain of Bacillus subtilis natto, has long been known. 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 strain of Bacillus subtilis natto. Soybean is used as a fermentation aid, since fermentation of barley alone by the strain of Bacillus subtilis natto is not effective.

[0006] Document CN115404227 describes a solid-state fermentation process using a hemp by-product. This fermentation is carried out using hemp seed meal, which results from the extraction of hemp oil. The objective of this fermentation is to produce an enzyme, nattokinase. The fermentation is carried out in the presence of 3 strains of Bacillus: Bacillus subtilis CGMCC No. 13932, Bacillus natto BNCC 194961 and Bacillus licheniformis BNCC 132622.

[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.

[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, said pretreated seeds containing at least 8% of lipidsAqueous impregnation of said substrate at a temperature between 25°C and 100°CSolid medium fermentation of said substrate by contacting with one or more strains of Bacillus subtilisnattofor a period of between 12h and 72h, at a temperature 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 to use hemp for human consumption. This involves fermenting a hemp seed-based substrate with a strain of Bacillus subtilisnatto. The resulting fermented product is nutritionally rich and attractive for human consumption thanks to its satisfactory organoleptic properties.

[0011] The process 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. Hemp proteins thus treated are more assimilable than untreated vegetable proteins.

[0012] The resulting food ingredient is particularly interesting from a nutritional point of view: When the substrate is made of 100% hemp seeds, the total protein content approaches 25% in the fermented product, which is high compared to plant substrates available on the market. This contributes to the nutritional value of the product. The soluble protein content is significantly increased compared to unfermented hemp seeds, which significantly improves the digestibility of the product. This addresses a current problem in the use of hemp seeds in human nutrition. The high level of fatty acids in the fermented substrate (100% hemp seeds) is between 8% and 30%, which means that the product is an interesting source of lipids, particularly omega 3 and omega 6.Fermentation of the hemp-based substrate by a strain of Bacillus natto allows the release of a large number of molecules that are both beneficial to health and contribute to the organoleptic properties of the product.

[0013] In particular, the amount 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.

[0014] The resulting product has an umami taste that consumers appreciate. It can be offered as a substitute for animal protein as part of a vegetarian diet or, more broadly, for anyone wishing to eat a balanced diet while limiting their consumption of animal protein.

[0015] 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 processing 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.

[0016] Compared to soy, the most widely used plant-based protein source today, fermented hemp seed ingredients are unlikely to cause allergies and are free of antinutrients. Similarly, compared to pea protein, which is known to cause digestive problems and flatulence.

[0017] 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

[0018] 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 seed coat, said pretreated seeds containing at least 8% by weight of lipidsAqueous impregnation of said substrate at a temperature between 25°C and 100°C.Solid medium fermentation of said substrate by contacting with one or more strains of Bacillus subtilisnattofor a period of between 12h and 72h, at a temperature between 37°C and 53°C.

[0019] In the context of the present invention, the hemp seeds used have a THC content in accordance with current regulations.

[0020] The seed pretreatment step aims to open the seed coat to allow moistening of the protein-rich material under the coat. Opening can be achieved by different methods such as decortication, grinding or germination... The pretreatment step must at least allow the opening of the seed coat 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 by-products, such as okara. Okara corresponds to the residues of hemp seeds obtained during the manufacture of vegetable drinks.

[0021] The pretreatment applied to open the seed coat is not intended to deoil the seed. Thus, the methods of treating hemp seeds used to prepare hemp flours, commonly called "hemp seed meal" are not part of the scope of the invention; similarly, hemp flours resulting from processes aimed at extracting the oil are not part of the substrates used in the fermentation processes employed in the present invention.

[0022] On the contrary, for the food ingredient to be nutritionally rich, it is desirable that the lipids are preserved. Thus, in particular embodiments of the invention:the pretreatment must not aim to deoil the seedsthe pretreatment must not induce the loss of more than 50% by weight of the quantity of lipids contained in the native seedsthe quantity of lipids must be greater than or equal to 8% by weight of the pretreated seedsthe quantity of lipids in the pretreated seeds is preferably greater than 10%, even more preferably greater than 15%, or even 20%, 25% and even more 30% by weight.Knowing that the quantity of lipids present in unhulled hemp seeds is between 25% and 35% by weight and reaches approximately 50% when the seeds are hulled, and that these lipids are interesting from a nutritional point of view due to their balanced content of omega 3, 6 and 9 fatty acids, it is desirable that pre-treated hemp seeds be as rich as possible in lipids.

[0023] The seeds thus pretreated (at least open) are then moistened or hydrated by impregnation in an aqueous solution.

[0024] 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.

[0025] The seeds thus impregnated are fermented in a solid medium by adding one or more strains of Bacillus subtilisnatto 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, fermentation takes place for a period of between 12 and 72 hours, at a temperature of between 37°C and 53°C.Preferably, the humidity level of the substrate before fermentation with the strain(s) of Bacillus subtilisnatto is between 45% and 68%.

[0026] The fermentation of the hemp seed substrate is carried out using at least one strain of Bacillus subtilis natto. The inoculum may include one or more strains of Bacillus subtilis natto. Various strains of Bacillus natto are described in the literature, such as Bacillus natto Miyagino, natto starterstrain BEST195, Natto Starter Powder Naruse Strain, Natto Starter Spores (Nattomoto), or the strains cited in WO2014 / 162919 or WO2015 / 163120.

[0027] A fermentation rate allowing the transformation of hemp seeds into a textured ingredient is obtained by adding to the hemp-based substrate a strain of Bacillus natto (i) either in solid form by adding at least 3g of bacterial ferment in 1.5 to 4.5 kg of substrate, (ii) or in liquid form by adding an inoculum having an optical density OD = 1.

[0028] The “plant substrate” used to obtain the food ingredient according to the invention is based on hemp seeds and 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.

[0029] For example, particular plant substrates may contain:

[0030] - 70% hemp seeds and 30% cooked rice

[0031] - 80% hemp seeds and 20% chickpeas.

[0032] As previously stated, "hemp seeds" means hemp seeds as such and any product resulting from the processing of hemp seeds, namely co-products such as okara. The co-products that can be used have, like pre-treated seeds, a quantity of lipids greater than or equal to 8% by weight.

[0033] Fermentation produces an umami-like ingredient. Umami is associated with glutamic acid and its derivatives, such as monosodium glutamate. It is one of the five basic tastes, along with 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, cheese, and green tea. The umami taste is characterized by its depth and length in the mouth, but also by its roundness.

[0034] The product obtained after at least one fermentation using a strain of Bacillus subtilisnatto 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 changes induced by fermentation can be identified by quantifying 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.

[0035] The organoleptic properties of the food ingredient can be modulated by carrying out further modification steps, either on the substrate pretreated before fermentation with the strain of Bacillus subtilisnatto, or on the substrate fermented with the strain of Bacillus subtilisnatto. 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.

[0036] In a preferred embodiment of the invention, this second modification step consists of a second fermentation carried out using at least one ferment chosen from a lactic acid bacterium, a yeast or a fungus. This second fermentation can be carried out on the substrate fermented with Bacillus subtilis natto. This second fermentation can also be carried out on the substrate pretreated before fermentation with Bacillus subtilis natto.

[0037] 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.

[0038] 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, Candidautilise etc.

[0039] When this ferment is a fungus, fermentation will take place between 20 and 37 °C for a period of between 12 and 72 h. Examples of fungi suitable for this fermentation are Rhizopus spp. and Aspergillus spp.

[0040] 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 smell (mainly with yeasts), an increase in the fermentability (mainly with fungi), a change in the taste (mainly with bacteria) of the final product.

[0041] In addition, the pH of the fermented product can be modified, for example to bring it back to around 7 for preservation or stabilization reasons. pH adjustments are known to those skilled in the art.

[0042] The inventors have shown that products fermented with a strain of Bacillus subtilisnatto exhibit an increase in both total protein and soluble protein levels.

[0043] Experimental data show that the content of certain amino acids is increased, in particular 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 hulled seeds before fermentation. A second fermentation allows these contents to be increased further.

[0044] 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 subtilisnatto strain.

[0045] Among the amino acids whose levels are increased in the fermented product are aspartate, glutamine, glutamate and glycine.

[0046] The increase in amino acid levels 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 rate is improved by a second transformation, as indicated above, 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.

[0047] In particular, an "umami" type food ingredient obtained by fermentation with a Bacillus subtilisnatto strain from a hemp-based plant substrate according to the invention has 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.

[0048] The present invention will be better understood from the following examples, provided for illustration purposes and in no way to be considered as limiting the scope of the present invention. EXAMPLES

[0049] The hemp seeds used in the process according to the invention may be, for example, whole sprouted seeds, crushed hulled hemp seeds or hulled hemp seeds, mixed with water, mixed and then filtered (recovery of the solid phase).

[0050] The hemp-based substrate may include, in addition to hemp seeds, for example, cooked rice grains, oat flakes such as groats, cooked or canned chickpeas, cooked or canned kidney beans, defrosted spinach, etc.

[0051] EXAMPLE 1: Simple fermentation of hulled hemp seeds and analysis of changes in amino acid content by HPLC

[0052] Step 1: Substrate preparation

[0053] Preheat the induction hob, and boil water in a beaker (~100°C).

[0054] Pour the hulled hemp seeds into boiling water.

[0055] Cook the seeds for 20 minutes at ~100°C.

[0056] Keep the water boiling while cooking.

[0057] Step 2: Preparation of the inoculation solution

[0058] Preparation of an inoculum under sterile conditions of Bacillus subtilis in sterile physiological water.

[0059] Store at 37°C in a rotating incubator for approximately 1 hour.

[0060] Step 3: inoculation of the substrate

[0061] After cooking, strain the seeds and return them to the beaker.

[0062] Handle in a sterile area (psm).

[0063] Inoculate the seeds with the ferment (suspension of Bacillus subtilis natto).

[0064] Mix the seeds.

[0065] Step 4: fermentation

[0066] Spread the seeds in a layer in a ventilated container.

[0067] Store in an oven at 37°C for 48 to 72 hours under saturating humidity. Analysis of the fermented product

[0068] Table 1 describes the content of certain amino acids in hulled seeds before fermentation, and then in the product fermented with a strain of Bacillus subtilis. The analyses were carried out using high-performance liquid chromatography (HPLC).

[0069] Hulled seeds1st fermentationwithBacillusvrnattoN=3MeanStandard deviationMeanStandard deviationAspartate20.213.931.513.2Glutamine13.914.923.312.6Glutamate64.273.3128.494.0Glycine24.317.630.716.4

[0070] 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 DM -1 .

[0071] 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.

[0072] In particular, there was an increase in glutamate, a molecule associated with umami taste. They also confirmed the fermentation activity of Bacillus subtilis bacteria on the hemp substrate.

[0073] EXAMPLE 2: Double fermentation of hulled hemp seeds and analysis of changes in amino acid content by HPLC

[0074] Step 1: Preparation of the substrate

[0075] Preheat the induction hob, and heat water in a beaker (~50°C).

[0076] Pour the hulled hemp seeds into the hot water.

[0077] Soak the seeds at 50°C for 1 hour while stirring. Drain the seeds lightly.

[0078] Step 2: Preparation of the Bacillus inoculation solution

[0079] Preparation of an inoculum of Bacillus subtilis var. natto from an isolate. Carrying out an OD measurement. Dilution. Inoculation with 1 mL per 300g of pre-treated hemp seeds. Mix the inoculated seeds and place them in a ventilated container.

[0080] Step 3: Bacillus fermentation

[0081] Spread the seeds in a ventilated container.

[0082] Store in an oven at 43°C for 36 to 48 hours under saturating humidity.

[0083] Step 4: Preparation of the lactic acid bacteria inoculation solution

[0084] Preparation of a Lactobacillus plantarum inoculum from an industrial sachet as indicated by the supplier. Inoculation with 1 mL per 300g of once-fermented hemp seeds.

[0085] Mix the inoculated seeds with the inoculum.

[0086] Step 5: fermentation of Lactobacillus

[0087] Spread the seeds in a sealed container.

[0088] Store in an oven at 37°C for 36 to 48 hours under saturating humidity. Analysis of the fermented product

[0089] Table 2 describes the content of certain amino acids in hulled seeds before fermentation, and then in the product fermented with a strain of Bacillus subtilis. The analyses were carried out using high-performance liquid chromatography (HPLC).

[0090] Hulled seeds1st fermentation with Bacillus vr natto2nd fermentation with Lactobacillus plantarumN=3MeanStandard deviationMeanStandard deviationMeanStandard deviationAspartate20.213.931.513.231.932.6Glutamine13.914.923.312.660.653.8Glutamate64.273.3128.494.0276.7235.1Glycine24.317.630.716.448.337.7

[0091] 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 DM -1 .

[0092] 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 should 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 amount of glutamate is increased by more than a factor of 4 compared to the pretreated but unfermented seeds.

[0093] EXAMPLE 3: Fermentation of hulled hemp seeds and analysis of changes in amino acid content using GC-MS

[0094] The experimental conditions of Example 3 are the same as those implemented in Examples 1 and 2.

[0095] Analyses were performed using gas chromatography-mass spectrometry (GC-MS); results are given in GC-MS units, i.e., metabolite peaks are normalized to Ribitol (control) and dry weight. Metabolite contents are expressed in arbitrary units (semi-quantitative determination).

[0096] The results are presented in Table 3.

[0097] Pretreated seeds1st fermentation with a strainBacillus Vr Natto2nd fermentation with a strainLactobacillus plantarumN=3MeanStandard DeviationMeanStandard DeviationMeanStandard DeviationAspartate0.0870.0950.2830.1550.4120.261Aspartic acid 10.0550.0580.1840.0980.3150.196Aspartic acid 20.0310.0370.0980.0570.0970.065Glutamine0.1760.2500.3520.3101.0240.987Glutamine 30.1760.2500.3520.3101.0240.987Glutamate0.3420.2822.1971.6926.7842.805L-glutamic acid 20.0600.0720.9150.7692.7361.446L-pyroglutamic acid0.2820.2101.2820.9264.0481.359N-carbamyl-L-Glutamic acid 1Trace quantitiesGlycine0.0720.0320.1570.0830.2140.153Glycine0.0720.0320.1570.0830.2140.153

[0098] Table 3: GC-MS results measuring Aspartate, Glutamine, Glutamate and Glycine contents.

[0099] These results confirm the variations of Aspartate, Glutamine, Glutamate and Glycine measured by HPLC from pretreated seeds then for the first fermentation with a strain of B. subtilisvrnatto and after a second fermentation with Lactobacillus plantarum (N=3).

[0100] Conclusion of Examples 1 to 3:

[0101] Fermentation of hemp seeds using a strain of B. subtilis allows for modification of the content of certain amino acids, resulting in an improvement in the organoleptic properties of fermented products, whether through single or double fermentation. A significant increase in glutamate can be noted, confirming the possibility of increasing umami taste through fermentation, as well as a significant increase in glycine, which provides a sweet taste, and in aspartate and glutamine, also associated with taste.

[0102] EXAMPLE 4: Analysis of metabolites in fermented products with a strain of Bacillus subtilis

[0103] The metabolite content in pretreated seeds, simply fermented with Bacillus subtilis, was studied by GC-MS. The conditions for preparing the fermented products were those described in Example 1.

[0104] The results are presented in Table 4.

[0105] MétabolitesGrainesprétraitéesEcartTypeSimple fermentationEcartTypeFacteurAcide 3-(4-hydroxyphenyl)propionic0.0040.0010.1480.07940Acide 2,6-diaminopimelic 20.0010.0000.0250.00836citrulline 20.0020.0010.0490.01332arabinose0.0010.0000.0310.00732Av-cide L-glutamic 20.1200.0722.7440.769235-hydroxy-L-tryptophan0.0010.0000.0170.00522Acide hydrocinnamic0.0030.0020.0710.03822Acide linoelaidic0.0030.0020.0650.01921arabitol0.0040.0000.0680.01918glutamine 30.0660.0461.0550.31016phenethylamine0.0060.0230.0850.02014Acide 4-hydroxyphenylacetic0.0010.0000.0160.00414cystine 30.0100.0070.1260.03513ornithine 20.1720.4381.8440.46611Acide mucic0.0010.0000.0100.00310Acide 3-indolelactic 20.0020.0010.0210.01010D-Ala-D-Ala20.0160.0080.1300.0308histidine 30.1230.0791.0040.3088Acide L-pyroglutamic0.5630.2103.8460.9267Acide malonic 10.0010.0000.0090.0027Acide 5-aminovaleric 10.8870.6165.3322.2636Acide lactic0.0580.0180.3360.1566glycerol 1-phosphate0.0040.0020.0230.0066cysteine 20.0010.0000.0040.0015Guanidinopropionic acid 10.0030.0020.0140.0045asparagine 20.0110.0060.0610.01752-hydroxybutyric acid0.0080.0030.0430.0155Aspartic acid 10.1110.0580.5520.09853-phenyllactic acid0.0170.0020.0850.0285Aspartic acid 20.0630.0370.2950.0575Glycolic acid0.0020.0000.0100.00254-hydroxyphenylglycine 20.0150.0060.0630.01343-ureidopropionate 20.0010.0000.0050.0024.

[0106] Table 4: GC-MS results representing the amount of metabolites in pretreated seeds vs. after simple fermentation using a strain of B. subtilis natto (N=3). The last column represents the increase factor.

[0107] EXAMPLE 5: Analysis of metabolites in double-fermented products with a strain of Bacillus subtilis and a strain of lactic acid bacteria

[0108] The metabolite content in 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.

[0109] The results are presented in Table 5.

[0110] MétabolitesGrainesprétraitéesEcartTypeDouble fermentationEcartTypeFacteurAcide 3-(4-hydroxyphenyl)propionic0.0040.0013.3771.130902Acide hydrocinnamic0.0030.0020.3710.134115Acide L-glutamic 20.1200.0725.7041.77048glutamine 30.0660.0463.0720.98746histidine 30.1230.0793.8611.05731Acide 3-indolelactic 20.0020.0010.0610.01629Acide 4-hydroxyphenylacetic0.0010.0000.0280.00625Acide 5-aminovaleric 10.8870.61620.0214.54123Acide L-pyroglutamic0.5630.2108.7862.16516thymine0.0040.0020.0580.012155-hydroxy-L-tryptophan0.0010.0000.0110.00315Acide 2-hydroxybutyric0.0080.0030.0850.02110Acide guanidinopropionic 10.0030.0020.0230.00593-phosphoglycerate0.0000.0000.0020.00093-ureidopropionate 20.0010.0000.0090.0028asparagine 20.0110.0060.0870.0348xanthine0.0210.0030.1570.0597Acide mucic0.0010.0000.0070.0017Acide linoelaidic0.0030.0020.0210.0057Acide 2,6-diaminopimelic 20.0010.0000.0040.0016Acide aspartic 10.1110.0580.6430.2236arabinose0.0010.0000.0060.0016Acide lactic0.0580.0180.3130.1345lysine 20.1580.0540.8050.3475threitol0.0030.0020.0120.0015Saccharic acid0.0030.0000.0120.0015Malonic acid 10.0010.0000.0060.00154-hydroxyphenylglycine 20.0150.0060.0660.0085glycine0.1450.0320.6430.1534.

[0111] 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.

[0112] Conclusion on Examples 4 and 5:

[0113] It can be noted that the fermentation of hemp seeds with at least one strain of Bacillus subtilisnatto induces a significant increase in a large number of metabolites associated with health claims and / or taste notes.

[0114] If we consider those that 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 prebiotic properties. L-glutamic acid associated with maintaining the integrity of the intestinal walls, which has pre- and probiotic effects. It is used in industry in the form of salts and is the basis of the umami taste. 5-hydroxy-L-tryptophan which contributes to mental concentration. Hydrocinnamic acid known for its anti-inflammatory, anti-oxidant and anti-tumor properties. Linoleic acid, derived from linoleic acid, omega 3, positive for the cardiovascular system. Arabitol which provides a sweet flavor.Glutamine is involved in immune protection, maintaining intestinal wall integrity and acid-base balance. Phenylethylamine, an essential amino acid, plays a role in stimulating the central nervous system.

[0115] The increased presence of these compounds demonstrates the beneficial effect of fermenting hemp seeds with Bacillus subtilisnatto, possibly combined with a lactic acid bacterium to increase the effects.

[0116] EXAMPLE 6: Simple fermentation of a substrate derived from hemp seeds

[0117] Step 1: Preparing the hemp seeds

[0118] Use hulled hemp seeds and soak them in 3 parts water at room temperature. Roughly mix the mixture. Strain and keep the solid part.

[0119] Step 2: Preparation of the inoculation solution

[0120] Preparation of an inoculum under sterile conditions of Bacillus subtilis in sterile physiological water.

[0121] Store at 37°C in a rotating incubator for approximately 1 hour.

[0122] Step 3: inoculation of the substrate

[0123] Take the solid preparation obtained from the hemp seeds.

[0124] Handle in a sterile area (PSM).

[0125] Inoculate the preparation with the ferment (suspension of Bacillus subtilis natto).

[0126] Mix.

[0127] Step 4: Fermentation

[0128] Spread the inoculated preparation in a layer in a ventilated container.

[0129] Store in an oven at 37°C for 48 to 72 hours under saturating humidity.

[0130] EXAMPLE 7: Simple fermentation of 70% hemp seeds with 30% cooked rice

[0131] Step 1: Preparing the hemp seeds

[0132] Preheat the induction hob, and heat water in a beaker (~50°C).

[0133] Pour the hulled hemp seeds into the hot water.

[0134] Soak the seeds at 50°C for 1 hour while stirring. Drain the seeds lightly.

[0135] Step 2: Preparing the rice grains

[0136] Wash the rice grains several times in cold water, then bring a pot of water to a boil and cook the grains according to the supplier's instructions. Drain the cooked grains and wait for them to cool to room temperature.

[0137] Step 3: Preparation of the Bacillus inoculation solution

[0138] Preparation of an inoculum of Bacillus subtilis var. natto from an isolate. Carrying out an 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.

[0139] Step 4: Bacillus fermentation

[0140] Spread the seeds in a ventilated container.

[0141] Store in an oven at 43°C for 36 to 48 hours under saturating humidity.

[0142] EXAMPLE 8: Increase in the level of soluble proteins in a hemp seed substrate by fermentation with a strain Bacillus subtilis natto

[0143] Hulled hemp seeds were autoclaved at 121°C for 20 min. They were then inoculated with DO=1 and fermented with a B. natto strain. Total protein was measured using the Kjeldahl method (Lynch JM, Barbano DM. Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products. J AOAC Int. 1999 Nov-Dec;82(6):1389-98. PMID: 10589493) and soluble protein was measured using the Lowry method (Waterborg JH, Matthews HR. The Lowry method for protein quantitation. Methods Mol Biol. 1994;32:1-4. doi: 10.1385 / 0-89603-268-X:1. PMID: 7951715).

[0144] It is observed that after 48 hours of fermentation under temperature conditions ranging from 28 to 40°C, for a humidity of the substrate before fermentation between 55 and 68%, the concentration of soluble proteins increases between 0.5 and 1.5 times. After 48 hours, the increase is not very significant.

[0145] Under these conditions, the total protein level is not modified.

[0146] The inoculum volume has no significant influence on this parameter under the conditions tested where the OD was set at 1.

[0147] EXAMPLE 9: Increase in the total protein level in a substrate based on hemp seeds, alone or mixed with chickpeas, by fermentation with a strain of Bacillus subtilis natto

[0148] Three types of substrate were prepared: Hulled hemp seeds autoclaved at 121°C for 20 min. Pre-cooked chickpeas. A mixture consisting of 80% hemp seeds as described above and 20% chickpeas as described above.

[0149] These substrates were then fermented with a B. natto strain by varying the incubation temperature. The total protein level was measured after 48 h using the Kjeldahl method. The results are presented in Table 6.

[0150]

[0151] Table 6: Total protein levels depending on the nature of the substrate and the fermentation temperature. On 100% hemp seed substrate

[0152] It is observed that the concentration of total proteins increases as a function of the incubation temperature to reach a maximum at 45°C with a maximum of approximately 23.7 g of proteins per 100 g of fresh product for a substrate made of 100% hemp seeds. This represents an increase in total proteins of approximately 16% on fresh product over 48 hours and corresponds to approximately 39.5 g of proteins per 100 g of dry product.

[0153] Observation of fermented seeds shows the presence of threads characteristic of PGA (poly-glutamic acid) which is correlated with an increase in glutamic acids during fermentation.

[0154] On the substrate consisting of a mixture of hemp / chickpeas (80 / 20)

[0155] Fermentation was carried out using autoclaved hulled hemp seeds mixed with canned (or cooked) chickpeas. The ratio is 80% hemp seeds and 20% chickpeas.

[0156] It is observed that the total protein concentration in the starting substrate decreases slightly due to the presence of chickpeas. However, a progressive increase in total protein concentration is observed at the fermentation temperature. The 45°C condition allows a concentration of 19.6 g of total protein to be obtained per 100 g of fresh product. This represents an increase in total protein of 14% on fresh product over 48 hours.

[0157] Observation of fermented seeds shows the presence of threads characteristic of PGA (poly-glutamic acid) which is correlated with an increase in glutamic acids during fermentation.

[0158] These results show the interest of fermenting a substrate based on hemp seeds to increase the protein level.

[0159] Conclusion of Examples 8 and 9 relating to protein levels: The results show that the levels of soluble and total proteins can be modulated by changing the fermentation temperature. If one wishes to simultaneously increase the level of soluble and total proteins with the aim of simultaneously optimizing these two parameters, fermentation at a temperature between 38-40°C appears to be a good compromise.

[0160] EXAMPLE 10: Aromatic profile of the food ingredient obtained by fermentation of a hemp-based substrate with a Bacillus subtilis strain and cooked in the form of a steak in comparison with a beef steak Sample preparation

[0161] Hemp steak: it was prepared from a substrate consisting of 100% hulled hemp seeds soaked in boiling water for 10 min then inoculated with an OD = 1 with a strain of Bacillus natto. Fermentation was carried out from a substrate with a humidity level of 45% at a temperature of 45 ° C for 24 h or 48 h. The fermented products constitute the hemp steaks. In 20 mL vials, 2 g of hemp steak were weighed. About 0.5 g of cooking oil was added and the whole was centrifuged for 3 min at 2000 rpm. The steaks were cooked in an incubator at 150 ° C for 10 min.

[0162] Beef steak: beef steak that has been cooked for 2 minutes at 110°C.

[0163] Instrumental analysis: Samples were analyzed using the solid phase micro extraction (SPME)-GC-MS-SCD method following the supplier's recommendations on a polar column.

[0164] Data processing: The obtained spectra were analyzed with Agilent MassHunter Unknowns analysis software.

[0165] Validation of the results and identification of the compounds were carried out by comparison of spectra with the NIST 23 database and retention indices. A compound is putatively identified when its similarity (the match factor) is greater than 70% and / or its retention index is between + / - 30 compared to the literature. Statistical analyses, PCA, Heatmap, dendrogram were carried out with MetaboAnalyst software.

[0166] Analytical strategy: the objective of these analyses is first of all to develop an analysis method by carrying out a first profile by GC-ToF-MS, then to carry out another profile of the sulfur compounds by GC-MS-SCD.

[0167] The aroma profiles of the three products are described in Table 7.

[0168] Compounds (odors) Beef Steak (%) 24h Steak (%) 48h Steak (%) Thresholds (ug / L) 2,5(6)-Dimethylpyrazine (roasted, grilled) 1002647128500.02 2-Methylpyrazine (green, grilled) 100198397334100 Dimethyl Disulfide (onion, cabbage, truffle) 10013599193245 Methanethiol (sulfurous) 100622134780.08 Butanal, 2-methyl- (malty, green) 10057985516145 Carbonyl Sulfide (unknown) 10049403467 N / A 3-Methyl Butanal (malty, chocolate, green)100418932921002,3,5-Trimethylpyrazine (roast, potato)100296945039Hydrogen sulfide (feces, rotten eggs, sulfur)10023149608Benzaldehyde (almond, hazelnut, wood)1001950249350Pentane (alkane)10015663388N / AStyrene (sweet)100141712541402-Thiophenecarboxaldehyde (wine, unknown)10012711059N / AAcetic acid (vinegar)10012321560282792-Octanone (soapy, green apple)100993986N / AFormic acid (acrid)1008833870N / A3-Methylpyridazine (unknown)100823DIV / 0N / A2-Acetylfuran (sweet)10068232810000Acetone100548319300000Toluene100503410N / A3-Furaldehyde / furfural (sulfurous,butterscotch)100503778N / ABenzonitrile (almond, rancid, volatile)100388DIV / 0N / AIsoamyl Alcohol (alcoholic, malty)1003709919250Ethyl Octanoate (fruity, sweet, floral)10030688746,42-Pentylfuran1002837842903Pyridine (burnt)100222335N / A2-Butanone (acetone, etheric)10020922655133Ethyl Acetate (fruit, pineapple, sweet)1001706079169b-Myrcene (spicy)10058102223-Octanol (mushroom, melon, lemon, mint)1004567181-Octen-3-ol (mushroom, earthy, rubber)1004115425.85Heptane (sweet-ethereal)10041149N / A1-Pentanol (fruity, green)100284532440.8Hexanal (green, grassy, ​​fruit)1001435398Unknown RI=140510062N / AUnknown RI=1476100%0DIV / 0N / A,

[0169] Table 7: Relative surfaces of the different volatile compounds between the 2 hemp steaks, the beef steak is considered as reference (100%).

[0170] Beef is a meat known for being a source of a strong Umami taste, as is the case with pork, fish, shellfish and mushrooms.

[0171] Using cooked beef as a standard shows that hemp steaks after 24 and 48 hours of fermentation develop aromatic qualities similar to those of beef. However, some of these qualities are greatly enhanced, particularly sulfur notes, as is the case for Dimethyl Disulfide or methanethiol. Grilled and roasted notes are probably exacerbated by the extensive cooking of hemp steaks.

[0172] On the other hand, if we compare the results of fermented hemp steaks with what is known about the taste characteristics of hemp seeds alone (e.g.: Sensory Wheel and Lexicon for the Description of Cold-Pressed Hemp Seed Oil, February 2023, Foods 12(3):661), we observe that we find the notes of "hazelnuts" and "almonds" characteristic of the starting hemp seed.

[0173] On the other hand, fermentation brings many aromatic notes with a dominance around the “mushroom”, the “truffle” which are known and described to have an Umami taste (Dimethyl Disulfide, 3-Octanol and 1-Octene-3-ol).

[0174] Finally, the duration of fermentation has an importance in the development of the aroma since we observe that between 24h and 48h the grilled and sulfurous notes are particularly present while they are almost divided by 10 in the hemp steaks fermented for 48h (2,5(6)-dimethylpyrazine, 2-methylpyrazine, Dimethyl Disulfide).

[0175] This analysis attests to the taste interest of hemp steaks prepared from a fermented substrate.

Claims

Process for preparing 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 seed coat, said pretreated seeds containing at least 8% lipidsAqueous impregnation of said substrate at a temperature between 25°C and 100°CSolid medium fermentation of said substrate by contacting with one or more strains of Bacillus subtilisnattofor a period of between 12h and 72h, at a temperature between 37°C and 53°C. The method of claim 1 wherein said pretreatment consists of decortication, grinding or germination. Method according to one of claims 1 or 2, in which said fermentation is carried out at a temperature between 38 and 40°C. Method according to one of the preceding claims, in which the humidity level of the substrate is between 45% and 68%. Method according to one of the preceding claims in which said strain of Bacillus is added to said substrate, (i) either in solid form by adding at least 3g of bacterial ferment in 1.5 to 4.5 kg of substrate, (ii) or in liquid form, by adding an inoculum of Bacillus subtilis natto having an optical density OD = 1. A method according to any preceding claim wherein said pretreated hemp seeds are hemp okara. A method according to any preceding claim wherein said substrate further comprises rice grains, oat flakes, fabaceae or vegetables. Method according to one of the preceding claims further comprising a second fermentation carried out using at least one ferment chosen from a lactic bacterium, a yeast or a fungus. The method of claim 8 wherein said second fermentation is carried out on the substrate fermented with said strain of Bacillus subtilis natto. The method of claim 8 wherein said second fermentation is carried out on the substrate pretreated before fermentation with said strain of Bacillus subtilis natto.

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