Process for the production of chemical intermediates and protein hydrolysates from agribusiness by-products

Abstract

The present invention relates to a process that, by using as raw materials by-products from the agribusiness industry, such as waste biomass from the processing of food flours, enables chemical intermediates and protein hydrolysates yielding amino acids with biostimulant action for plants to be produced from the polysaccharides and proteins present in such biomass. The process comprises sugar release by enzymes, fermentation, ethanol separation and protein hydrolysis by enzymes including solid-liquid separations at different points of the procedure and the optional release of arabinoxylans.

Description

[0001] PROCESS FOR THE PRODUCTION OF CHEMICAL INTERMEDIATES AND PROTEIN HYDROLYSATES FROM AGRIBUSINESS BY-PRODUCTS

[0002] The present invention relates to a process that makes it possible to produce organic compounds of high added value, which can be utilised in the development of various applications, through the use, as raw materials, of by-products from the agribusiness industry, such as waste biomass from the processing of food flours, preferably the defatted waste obtained after flour production. Through this process it is in fact possible to produce chemical intermediates (e.g. bioethanol) and soluble fibre having applications, for example, in the food sector, and protein hydrolysates for use respectively as an ingredient in animal feed or human food products, or as a plant biostimulant, from the polysaccharides and proteins present in these biomasses. By chemical intermediates are meant molecules resulting from specific chemical and / or biochemical reactions that may be used as such or used in reactions to synthesise complex products. Bioethanol is one example of a chemical intermediate produced from biomass.

[0003] Bioethanol is a renewable energy source produced by a fermentation process from biomass of different kinds.

[0004] First- generation bioethanol, for example, is obtained from the fermentation of biomasses such as starchy cereals (e.g. corn and wheat) and sugar crops (such as beet and sugarcane).

[0005] Processes using biomass derived from starchy cereals typically involve an initial step of separating and sorting the seeds, which are then milled to produce a flour. Water is then added to the flour, and the resulting mixture is subjected to a short cooking process at high temperatures ("gelatinisation") to promote release of the starch. After gelatinisation, the starch undergoes hydrolysis, for example with enzymes, resulting in the release of sugars into solution. These sugars are then converted to ethanol and carbon dioxide during the subsequent or simultaneous fermentation step. The ethanol in the fermentation broth is finally separated by distillation from a residue containing unconverted substrate, yeast, and any fermentation byproducts.

[0006] Distillers' Dried Grains (DDGs) and a soluble portion, which can be concentrated and added to the dried distilled grains are typically separated from this residue, resulting in what are known as Distillers' Dried Grains with Solubles (DDGS). Such DDGS are generally used as fertilizers or in animal feed, where they are a source of organic phosphorus, digestible protein, fibre and fat.

[0007] The characteristics and composition of DDGS vary and are, for example, influenced by the initial biomass (type of cereal, variety and environmental growing conditions) and the process conditions used in production (temperature, cooking time, distillation, dehydration, pelletisation, etc.).

[0008] On average, DDGS contain 23-36% protein, 3-9% fat and less than 14% fibre (source: Distillers Grains Technology Council, https: / / distillersgrains.org / distillers-grains / ).

[0009] In WO 2022 / 157226, the DDGS from a process to produce bioethanol from corn undergo extraction with water and / or organic solvents. The extracts obtained are used directly as pesticides and / or as plant biostimulants.

[0010] However the process for the production of first-generation bioethanol has the major disadvantage of taking feedstock away from human and animal nutrition, thus creating conflicts between their use for food and energy.

[0011] An alternative to first-generation bioethanol is second-generation bioethanol, which is produced from lignocellulosic biomass comprising cellulose, hemicellulose and lignin. Such biomasses are non-food raw materials and include, for example, crop residues, wood residues, dedicated energy crops and industrial wastes.

[0012] However the process for second-generation ethanol production is subject to technical problems such as recalcitrance of the biomass to hydrolyse, requiring pretreatment that often results in the formation of compounds that inhibit fermentation, and difficulty in finding enzymes that are efficient at hydrolysing cellulose and hemicellulose, even at a cost that is competitive with the first-generation enzymes that hydrolyse starch.

[0013] An alternative is the production of bioethanol from by-products of the agribusiness, such as waste biomass from food flour processing.

[0014] In the particular case of the processing of cereals (including pseudocereals) and legumes in the milling industry, which as is well known is directed towards producing food flours as the primary product, intermediate products and by-products such as bran, middlings, shreds and germs are obtained from the processing steps. These contain starch, salts, soluble fibres (glucans, inulin, some hemicelluloses, etc.), insoluble fibres (cellulose, hemicellulose and lignin), oils and fatty acids, and organic nitrogen in the form of proteins, oligopeptides or simple amino acids. Through various chemical, physical-chemical and / or enzyme treatments these intermediates and by-products can be broken down into their main components (starch, cellulose, hemicellulose, lignin and proteins), resulting in compositions rich in simple sugars for use as substrates for bioethanol production, and proteins for conversion into protein hydrolysates with, for example, a biostimulant action for plants.

[0015] The papers by Cripwell et al. (Applied Energy, 2015, Vol. 160, pages 610-617) and Favaro et al. (Applied Energy, 2013, Vol. 102, pages 170-178) describe the use of wheat bran for bioethanol production. In Cripwell et al.'s process, the saccharification and fermentation steps occur simultaneously using amylolytic strains of Saccharomyces cerevisiae and an enzyme cocktail of cellulosolytic enzymes. In Favaro et al. the biomass undergoes acid treatment prior to the hydrolysis step.

[0016] Siepmann et al. (Semina: Ciencias Agrarias, Londrina, v. 41, no. 6, Supplement 2, p. 2951- 2966, 2020) describe a process for the production of bioethanol from deoiled rice bran, in which the starting biomass is pretreated with proteases before being hydrolysed to promote starch hydrolysis and increase bioethanol production. The use of proteases for the same purpose is also described in US 2011 / 014671.

[0017] In these documents protein hydrolysis produces oligopeptides and free amino acids that can be metabolised by yeast during fermentation, resulting in a decrease in the protein content of the residual biomass, which is of little value.

[0018] The document WO 2023 / 148756 Al describes a process in which a milled grain is saccharified and subjected to ethanol fermentation. The stillage after ethanol distillation is treated with cellulase enzymes before protein separation. Thus, the obtained proteins comprise monosaccharides deriving from cellulose hydrolysis which require further steps to be removed. In contrast to the above documents, the Applicant has now identified a process comprising a protein hydrolysis step downstream of fermentation, preferably after separation of the chemical intermediate (e.g. ethanol) from a residue comprising fibre, protein, and / or oligopeptides.

[0019] Instead of being a waste by-product, in the process according to the invention said residue is advantageously subjected to protein hydrolysis, thus enriching it into free oligopeptides and amino acids. These oligopeptides and free amino acids have an advantageous bio stimulatory effect on plants of various kinds. A protein hydrolysate of this kind is therefore particularly suitable for use as a plant biostimulant. For farmers, biostimulants are a possible alternative or supplement to the use of conventional chemical fertilisers. For example, by applying protein hydrolysate-based biostimulants to plants of various kinds it is possible to improve nutrient uptake and assimilation (e.g. nitrate nitrogen and iron), stimulate endogenous plant defence responses to biotic and abiotic stresses (such as salinity, drought, and temperature extremes), and influence product quality (for example by promoting higher antioxidant content, higher protein level, and lower nitrate content). In addition, the presence in such a protein hydrolysate of phytates such as phytic acid and its salts, which are particularly abundant in cereals, pseudocereals, and legumes, makes it an advantageous source of phosphorus for the plants to which it is applied. Another possible use of protein hydrolysates comprising oligopeptides and free amino acids obtained from by-products of the agribusiness industry according to the present invention is as an ingredient for animal and / or human nutrition. These oligopeptides and free amino acids in fact have an advantageous amino acid composition that makes them particularly suitable for use in the food industry.

[0020] Thus, the present invention makes it possible to achieve full utilisation of the by-products of food flour processing: the polysaccharides present are converted with high efficiency into chemical intermediates (e.g. bioethanol) and / or soluble fibre, while the proteins and / or oligopeptides are converted into protein hydrolysates that are suitable, for example, for use as plant bio stimulants. Soluble fibres such as xylo-oligosaccharides, arabino-oligosaccharides, and / or arabinoxylo-oligosaccharides may also be separated from the residue obtained after the removal of ethanol, and because of their prebiotic activity they can find use in the food and nutraceutical industries.

[0021] Dietary intake of soluble fibre comprising arabinoxylans results in benefits for the human body such as antitumour and immunomodulatory activities or reduces the risk of the occurrence of certain chronic diseases such as type II diabetes, obesity, or coronary artery disease. In particular, arabinoxylans bring about positive effects on what is known as the intestinal microbiota by promoting the production of Short Chain Fatty Acids (SCFAs), such as butyric acid, thus improving the metabolism of intestinal epithelial cells. The rheological, gelling, and emulsifying properties of arabinoxylans make them particularly suitable for use as a food ingredient in the preparation of, for example, pasta, bread, biscuits, and beverages with improved technological characteristics and benefits to human health. In addition, the presence of ferulic acid esterified with arabinoxylans (feruloylated arabinoxylans) gives the soluble fibres an additional beneficial effect as an antioxidant.

[0022] Increasing market demand for products with a high content of arabinoxylans has given rise to the need to develop new technologies to derive these compounds in a sustainable manner. The present invention enables soluble fibre compositions comprising arabinoxylans to be obtained from by-products of food flour processing.

[0023] All products of the process are therefore suitable for new high value-added applications in the chemical, food or agrichemical industries.

[0024] The Applicant has surprisingly found a process that, as an alternative to or in conjunction with the production of ethanol, also allows other chemical intermediates and / or polyhydroxyalkanoates to be produced. Through this process protein hydrolysates and chemical intermediates and / or polyhydroxyalkanoates can be produced from a by-product of food flour processing including starch, fibre, protein and / or oligopeptides, said process comprising the steps of: a) hydrolysing the starch in said by-product in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibre, protein and / or oligopeptides; b) fermenting said aqueous mixture obtained from step a) in the presence of at least one microorganism capable of producing chemical intermediates and / or polyhydroxy alkanoates, resulting in a fermentation broth including chemical intermediates and / or polyhydroxy alkanoates, fibre, proteins and / or oligopeptides; c) separating said chemical intermediates and / or polyhydroxyalkanoates from said fermentation broth, obtaining a first residue comprising fibre, proteins and / or oligopeptides; d) subjecting at least one part of the proteins and / or oligopeptides present in said first residue to protein and / or oligopeptide hydrolysis, resulting in a second residue comprising hydrolysed proteins and / or oligopeptides and fibre; e) subjecting said second residue comprising hydrolysed proteins and / or oligopeptides to solid / liquid separation, resulting in: e)i. a solid fraction, including fibre, and e)ii. a liquid fraction comprising a protein hydrolysate comprising oligopeptides and free amino acids.

[0025] Said chemical intermediates are preferably selected from the group that includes, or consists of: diols, monoalcohols, hydroxy acids, monoacids and diacids, amino acids and amides.

[0026] Examples of diols are ethanediol, propanediol, butanediol (preferably 1,4-butanediol) and hexanediol. Examples of monoalcohols are butanol and ethanol. One example of hydroxy acids is lactic acid. Examples of monoacids are acetic acid and formic acid. Examples of diacids are succinic acid and fumaric acid.

[0027] As regards the polyhydroxyalkanoates (PHAs) which can be produced according to the present invention, these are generally polymers containing repeating units having the general formula (I):

[0028] -O-CHRi-(CH2)n-CO- (I) wherein Ri represents a hydrogen atom, or is selected from C1-C12 alkyl groups, C4-C16 cycloalkyl groups, C2-C12 alkenyl groups, optionally substituted with at least one group selected from halogens such as, for example, fluorine, chlorine, bromine, -CN, -OH, -COOH, -OR3, - COOR3 wherein R3 represents a C1-C4 alkyl group or a benzyl group, n is an integer comprised between 1 and 6, preferably 1 or 2. Preferably Ri is methyl or ethyl and n is 1 or 2.

[0029] Polyhydroxyalkanoates (PHAs) can be both homopolymers and copolymers or terpolymers. In the case of copolymers or terpolymers, they can consist of different repeating units having general formula (I) in combination with at least one repeating unit deriving from comonomers which are capable of copolymerizing with hydroxyalkanoates, for example lactones or lactams. In the latter case, the repeating units having general formula (I) are present in an amount equal to at least 10% by moles with respect to the total moles of repeating units.

[0030] Examples of repeating units having general formula (I) derive from: 3-hydroxybutyrate, 3- hydroxyvalerate, 3-hydroxyhexanoate, 3 -hydroxyoctanoate, 3-hydroxyundec-10-enoate, 4- hydroxy valerate .

[0031] Preferred polyhydroxyalkanoates (PHAs) are selected from the group consisting of: poly-3- hydroxybutyrate (PHB), poly- 3 -hydroxy valerate (PHV), poly-3-hydroxyhexanoate (PHH), poly-3-hydroxyoctanoate (PHO), poly(3-hydroxybutyrate-co-3-hydroxy-valerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), poly(3-hydroxybutyrate-co-4- hydroxybutyrate) (PHBB), poly(3-hydroxyoctanoate-co-3-hydroxy-undecen- 10-enoate) (PHOU), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-c-4-hydroxyvalerate) (PHBVV), or mixtures thereof. Poly-3-hydroxybutyrate, poly(3-hydroxybutyrate-co-3-hydroxy-valerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) are particularly preferred.

[0032] S. .

[0033] In particular, this process has been found to be particularly effective for ethanol production.

[0034] Thus, a preferred object of the present invention is a process for the production of ethanol and protein hydrolysates from a by-product of food flour processing comprising starch, fibre, protein and / or oligopeptides, said process comprising the steps of: a) hydrolysing the starch into said by-product in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibre, protein and / or oligopeptides; b) fermenting said aqueous mixture obtained from step a) in the presence of at least one microorganism capable of producing ethanol, resulting in a fermentation broth comprising ethanol, fibre, protein and / or oligopeptides; c) separating ethanol from said fermentation broth, resulting in a first residue comprising fibre, protein and / or oligopeptides; d) subjecting at least one part of the proteins and / or oligopeptides present in said first residue to protein and / or oligopeptide hydrolysis, resulting in a second residue comprising hydrolysed proteins and / or oligopeptides and fibre; e) subjecting said second residue comprising hydrolysed proteins and / or oligopeptides to solid / liquid separation, resulting in: e)i. a solid fraction, including fibres, and e)ii. a liquid fraction comprising a protein hydrolysate comprising oligopeptides and free amino acids.

[0035] According to one aspect of the invention, steps a)-e) of the process are advantageously carried out at temperatures not exceeding 150°C, preferably not exceeding 130°C, more preferably below 100°C, even more preferably below 80°C or below 60°C, thus allowing the structure and function of the components present to be preserved. The above operations can be carried out once in each step or repeated several times under the same or different conditions.

[0036] According to one aspect of the invention, steps a)-e) of the process are advantageously carried out at pH values below 7, preferably below 6, more preferably of 5 or below, thus reducing undesired microbial proliferation. These pH values are preferably achieved by the addition of organic acids, for example selected from lactic acid, malic acid, citric acid and tartaric acid. Within the meaning of the present invention, by the term "by-product of food flour processing" is meant the intermediate products and by-products of the milling industry, that is a biomass obtained as a by-product of milling cereals, pseudocereals, legumes or their mixtures, which includes starch, fibres such as cellulose and / or hemicellulose, and protein and / or oligopeptides. The monosaccharides produced through the process according to the present invention are sugars such as glucose, fructose, arabinose, rhamnose, galactose, mannose, and xylose.

[0037] The term "polysaccharides" is to be understood to include, for example, starch, cellulose, heteropolysaccharides such as hemicellulose (which includes xylans, glucuronoxylans, arabinoxylans, glucomannans, xyloglucans).

[0038] The term "oligosaccharides" is used in this application to refer to the set of molecules of a carbohydrate nature consisting of two to ten monosaccharide units. Examples of oligosaccharides therefore include disaccharides such as sucrose, maltose and cellobiose, and some maltodextrins. Oligosaccharides substituted e.g. by acetylation and / or containing organic acid residues (e.g. ferulic acid) are included. The term "oligopeptides" is used in this application to mean the set of molecules of a peptide nature consisting of two to twenty amino acids and may include dipeptides, tripeptides, tetrapeptides and pentapeptides. Where not otherwise indicated, the term "fibre" is intended to include both insoluble fibres such as cellulose, lignin, and some hemicelluloses and soluble fibres such as some hemicelluloses, glucans, and inulin. This term does not include starch. The term "soluble fibre" refers to any fibre that is soluble in an aqueous environment. Examples of soluble fibre therefore include disaccharides such as xylobiose, cellobiose and some arabinoxylans and some beta-glucans.

[0039] The term "phytates" in this application refers to the set of molecules consisting of inositol to which phosphate groups are covalently bonded, such as phytic acid, inositol-pentaphosphoric acid, inositol-tetraphosphoric acid, inositol-triphosphoric acid, inositol-diphosphoric acid, or inositol-monophosphoric acid, or their salts.

[0040] The process will be described in more detail below with reference to the block diagram in Figure 1, which illustrates the embodiment in Example 1.

[0041] Said by-product from food flour processing comprising starch, fibre, protein, and / or oligopeptides fed to step a) of the process is derived from one or more cereals chosen, for example, from wheat or rice, barley, oats, spelt, rye, millet and corn or from one or more pseudo-cereals chosen, for example, from amaranth, buckwheat, quinoa, and chia or from one or more legumes chosen, for example, from peas, chickpeas, lentils, beans, broad beans or is derived from mixtures thereof.

[0042] The biomass obtained as a by-product of the milling of the plant species listed above results in mainly fibre (cellulose, hemicellulose, and lignin), vitamins, proteins, enzymes, mineral salts, phytates and lipids, and includes varying amounts of residual starch depending on the type and stage of processing from which these originate.

[0043] The starch content of said biomass, in particular, is preferably less than 70%, more preferably less than 60%, even more preferably less than 50% by weight relative to dry weight of the biomass in the case of cereals and pseudocereals and preferably less than 40%, more preferably less than 30%, by weight relative to dry weight of the biomass in the case of legumes. The starch content of said biomass is advantageously higher than 5, 10, 15, 20% by weight relative to the dry weight of the biomass.

[0044] The total fibre content (soluble and insoluble) of said biomass is more than 20% by weight; a fibre content of 25% or more, 30% or more, 40% or more relative to the dry weight of said biomass is especially preferred.

[0045] The protein content of said biomass is preferably 15% by weight or more, more preferably 20% or more.

[0046] Said biomass advantageously has a lipid content of less than 5%, preferably less than 2% and more preferably less than 1.5% by weight relative to dry weight of the biomass; according to a particularly advantageous aspect, the lipid content is less than or equal to 1% or 0.5% by weight relative to dry weight of the biomass.

[0047] Said biomass has a phytate content advantageously from 0.5% to 5.0%, preferably from 1% to 4.5%, and more preferably from 2% to 3.5% by weight relative to dry weight of the biomass. Preferably, said by-products of food flour processing are in the form of cylindrical pellets having a diameter of 0.2 to 2.0 cm, more preferably 0.4 to 1.5 cm, even more preferably 0.5 to 1.0 cm, and comprise a dust content of less than 50% by weight, preferably between 42% and 22% by weight relative to the total weight of biomass.

[0048] Such by-products of food flour processing preferably have a moisture content of 0.5 to 20.0%, more preferably 5 to 15%, even more preferably 2 to 10% relative to the total weight of biomass. Particularly advantageous is the use of cereal biomass having a starch content of 10% to 40% by weight, preferably 15% to 35%, more preferably 20% to 30% by weight relative to the dry weight of said cereal biomass.

[0049] The total fibre content (soluble and insoluble) of said preferred cereal biomass is more than 20% by weight; a total fibre content of 25% or more, 30% or more, 40% or more by weight relative to the dry weight of said cereal biomass is especially preferred. Its arabinoxylans content is between 15% and 30% by weight, preferably between 15% and 25%, more preferably between 20% and 25% by weight relative to the dry weight of said cereal biomass. Such arabinoxylans include ferulic acid and / or its esters.

[0050] Said preferred cereal biomass contains fructose as monosaccharide and / or in the form of oligosaccharide and / or polysaccharide.

[0051] The protein content of said preferred cereal biomass is preferably greater than or equal to 15% by weight, more preferably greater than or equal to 20% by weight relative to dry weight of the biomass. The protein content of said preferred cereal biomass is advantageously less than 40, 30, 25% by weight relative to the dry weight of the cereal biomass.

[0052] Said preferred cereal biomass advantageously has a lipid content of less than 5%, preferably less than 2% and more preferably less than 1.5% by weight relative to dry weight of the biomass; according to a particularly advantageous aspect, the lipid content is less than or equal to 1% or 0.5% by weight relative to dry weight of the biomass.

[0053] Advantageously said preferred cereal biomass has a phytate content of 2.0% to 4.0%, preferably 3.0% to 4.0% and more preferably 3.0% to 3.5% by weight relative to dry weight of the biomass. Its lignin content is between 1% and 10% by weight, preferably between 2% and 5% by weight relative to dry weight of the biomass. Said preferred cereal biomass has an ash content that is advantageously below 10% by weight, preferably from 3% to 8%, more preferably from 5% to 7% by weight relative to dry weight of the biomass.

[0054] The above described preferred cereal biomass is advantageously a cereal defatted biomass, for example deriving from wheat, preferably durum wheat.

[0055] As is well known, the process of milling cereals is intended to separate the endosperm, which constitutes the starchy and preponderant part of the caryopsis, from the bran and germ, in order to obtain flours (flour or semolina, depending on grain size) from the endosperm. Said process typically involves alternating stages of milling (involving the opening or breaking of the caryopsis, with reduction of the grain size of the endosperm) and separation of the flours from their respective bran fractions (i.e. bran, possibly with the germ).

[0056] Milling may be preceded by one or more stages of dehulling, which involves removing the outermost layers of the caryopsis by an abrasive action of greater or lesser intensity, generally without significantly damaging the endosperm-rich core intended for milling.

[0057] The bran and / or germ fractions separated during the milling process therefore constitute byproducts of food flour processing suitable for feeding to step a) of the process according to the present invention.

[0058] According to a preferred aspect, said by-products of food flour processing consist of the bran (possibly in its finer variant called middlings) optionally including the germ, i.e. from a fraction of the residual caryopsis after removal of the endosperm. Preferably, said by-products of food flour processing are defatted.

[0059] According to another preferred aspect, said by-products of food flour processing include middlings of various grades, i.e. products richer in starch than bran but still not suitable for use in baking processes. These can be used as such or in a mixture with bran and / or middlings.

[0060] Bran and middlings, for example, have a starch content between 15 and 30% by weight relative to the dry weight of the biomass, protein and oligopeptides between 15 and 30%, hemicellulose between 20 and 30%, cellulose between 10 and 15%, lignin less than 10%, and ash less than 10%.

[0061] The amount of protein and oligopeptides present can be derived by multiplying the organic nitrogen present by an appropriate correction factor.

[0062] By contrast, flour, middlings, and fine bran typically have starch contents between 50 and 80% by weight, protein between 10 and 30%, and hemicelluloses between 3 and 10% by weight relative to the dry weight of the biomass. The process according to the invention may optionally include a preliminary step of treating said biomass, whether obtained as a by-product from the cereal, pseudocereal and / or legume milling industry, in order to make it more accessible to the hydrolysis carried out in the first step of the process, and to stabilise it biologically. For example, such a preliminary step or pretreatment advantageously includes heat and / or ozone treatment and / or washing with water, carried out under conditions of time and temperature known to those skilled in the art. Other useful preliminary operations may be intended to reduce the lipid content of the biomass or reduce the fibre content, such as by hydrolysis mediated by enzymes such as cellulase and hemicellulase. Other pretreatments may also be physical (e.g. mechanical reduction to powder, irradiation), chemical-physical (e.g. steam explosion) or chemical (e.g. ozonolysis, dilute acid hydrolysis, alkaline hydrolysis, processes using organic solvents).

[0063] During step a) of the process according to the invention, said by-products undergo a hydrolysis (or saccharification) reaction of the polysaccharide chains of the starch to yield monosaccharides. Said hydrolysis may be performed with one or more enzymes belonging to the hydrolase family, preferably those suitable for hydrolysing starch, such as, for example, amylases. Preferably, said enzymes are added in step a) of the process.

[0064] Other hydrolytic enzymes such as cellulase and hemicellulase may be used before step a), during step a) or during subsequent treatment of the solid fraction separated in step e) to maximise conversion to monosaccharides by also hydrolysing the polysaccharide chains of cellulose and hemicellulose.

[0065] Said enzymes may be used singly or in mixtures and may be added to the starting biomass at different times depending on the desired hydrolysis product, adopting the appropriate operating conditions for each enzyme.

[0066] Depending on the enzymes selected, those skilled in the art will be able to adopt the necessary hydrolysis or saccharification conditions (reaction medium, pH, temperature, duration, etc.).

[0067] Enzymes that make it possible to obtain mixtures of monosaccharides comprising mainly glucose are particularly suitable for use in step a) of the process.

[0068] Said enzymes advantageously belong to the amylase class and are preferably chosen from alpha-amylase, glucoamylase (or amyloglucosidase or gamma-amylase) and mixtures thereof. Examples of suitable commercial products are "Spezyme® Alpha PF" and "Optidex® L-400," supplied by IFF.

[0069] The use of at least one alpha-amylase and at least one glucoamylase or a mixture thereof is preferred. According to one embodiment, the hydrolysis in step a) may be preceded or followed by an additional hydrolysis step a2), preferably in the presence of at least one enzyme capable of hydrolysing cellulose or hemicellulose fibres. Said enzyme is chosen from cellulase, hemicellulase and combinations thereof. Said additional hydrolysis step a2) improves the monosaccharide content of aqueous mixture to be fed in the subsequent fermentation step b). If step a) is followed by step a2), the product of step a2) is still referred to hereafter as the product of step a).

[0070] According to a further embodiment, the hydrolysis in step a) may be performed in the presence of at least one enzyme capable of hydrolysing cellulose and / or hemicellulose fibres.

[0071] Said cellulases and hemicellulases may include any enzyme that has cellulosolytic or hemicellulosolytic activity, respectively. The cellulases and hemicellulases may be part of an enzyme cocktail comprising one or more cellulases, one or more hemicellulases, or a mixture thereof.

[0072] Said hemicellulases are preferably chosen from xylanases, mannanases, arabinases and combinations thereof. Xylanases are preferred.

[0073] According to a preferred embodiment, the hydrolysis in step a) is carried out without adding enzymes capable of hydrolysing cellulose or hemicellulose fibres.

[0074] The hydrolysis operation in said step a) is carried out in the presence of water, preferably keeping the biomass (i.e. the by-product of food flour processing including starch) in aqueous suspension.

[0075] According to a preferred aspect, said operation is carried out by feeding said biomass with an initial dry weight of at least 2%, preferably at least 10%, more preferably at least 20% by weight, even more preferably at least 30% by weight, in relation to the weight of the aqueous suspension. Said aqueous suspension includes all the components added during the hydrolysis step, such as biomass, water, enzymes and acid / base solution for pH correction.

[0076] According to another preferred aspect, said operation is carried out by gradually feeding said biomass (for example semi-continuously or continuously) so as to maintain a solids content (in terms of dry weight) in the hydrolysis reactor of between, for example, 2% and 50%, preferably between 2% and 40% by weight relative to the weight of the aqueous suspension, advantageously between 7% and 25% by weight relative to the weight of the aqueous suspension.

[0077] During the hydrolysis reaction, the pH value is adjusted according to the enzymes used, such as through the addition of mineral acids such as sulfuric acid, or organic acids such as lactic acid, as is the temperature. The pH and temperature conditions may be kept constant or modulated throughout the process, depending on specific requirements.

[0078] For example, according to a preferred embodiment, the reaction is carried out in the presence of amylase and advantageously maintaining a pH 3 or more and 7 or less, preferably between 3.5 and 7 (more preferably between 4.5 and 5.5), while the temperature is preferably maintained at values from 30° to 130°C during the reaction, more preferably between 40° and 70°C, more preferably between 50° and 60°C.

[0079] The duration of the hydrolysis reaction will vary depending on the conditions adopted, particularly the type and concentration of enzymes, and the degree of hydrolysis desired. Advantageously it will be between 0.1 and 120 hours, for example between 0.5 and 48 hours, more advantageously between 10 and 30 hours, even more advantageously between 5 and 24 hours.

[0080] The hydrolysis step is optionally carried out in the presence of antimicrobial agents capable of containing the growth of any microbial contamination. Said antimicrobial agents preferentially include bactericidal and / or bacteriostatic agents. Examples are antibiotics, C2-C10 chain fatty acids such as nonanoic acid, hydroxy acids (e.g. lactic acid, citric acid), parabens, triazides, benzalkonium chloride, quaternary ammonium salts. Preferred examples are lactic acid, citric acid.

[0081] As an alternative to the use of enzymes, the hydrolysis in step a) can be performed chemically and / or physically, such as by the use of mineral acids, such as HC1 and H2SO4, or solid acids, such as sulfonated organic resins even in the absence of enzymes.

[0082] The hydrolysis in step a) may be carried out through a continuous or semi-continuous process or, alternatively, in batch mode.

[0083] Preferably step a) is carried out until conversion of 70% or more, more preferably 90% or more, even more preferably 95% or more of the starch initially present is achieved. This conversion is, for example, determined on the basis of the residual starch ((i.e. initial starch - residual starch) / initial starch).

[0084] At the end of step a), an aqueous mixture including monosaccharides, fibre, proteins and / or oligopeptides, and optionally salts, oils and fatty acids is obtained.

[0085] According to one embodiment of the present invention, the aqueous mixture obtained at the end of step a) can optionally be subjected to a solid-liquid separation step a3) to obtain: a3)i. a liquid fraction, including monosaccharides, and a3)ii. a solid fraction, including fibre, proteins, and / or oligopeptides; The separation operation in step a3) may include one or more operations chosen from settling, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis, and combinations thereof. Centrifuging, settling, and / or filtration operations are preferred, for example filtration on gauze and / or microfiltration and / or ultrafiltration.

[0086] Such separation operation in step a3) may be performed using devices chosen from, for example, a decanter, a sedimenter, a filter press, a hydrocyclone, a belt filter, a rotary filter, and a centrifuge.

[0087] For example, step a3) includes an initial centrifuging operation followed by washing of the solid fraction and a further centrifuging operation to separate the wash waters from the washed solid fraction.

[0088] Liquid fraction a3)i comprising monosaccharides obtained as a result of step a3) may be wholly or partially fed into fermentation in step b) of the process according to the invention for the purpose of producing chemical intermediates and / or polyhydroxyalkanoates. According to one aspect, such liquid fraction is concentrated by techniques known to those skilled in the art before being fed to fermentation step b). One example of a known concentration technique, but not limiting for the purposes of the present invention, is evaporation by the distillation of water using rotary or thin-film evaporators, for example.

[0089] Thus, the object of the present invention is a process for the production of chemical intermediates and / or polyhydroxyalkanoates and protein hydrolysates from a by-product of food flour processing comprising starch, fibre, protein and / or oligopeptides, said process comprising the steps of: a) hydrolysing the starch in said by-product in the presence of water and preferably one or more enzymes, yielding an aqueous mixture comprising monosaccharides, fibre, protein and / or oligopeptides; a3) optionally separating the aqueous mixture obtained at the end of step a) obtaining: a3)i. a liquid fraction, including monosaccharides, and a3)ii. a solid fraction, including fibre, proteins, and / or oligopeptides; b) fermenting said aqueous mixture obtained from step a) or said liquid fraction a3)i in the presence of at least one microorganism capable of producing chemical intermediates and / or polyhydroxyalkanoates, resulting in a fermentation broth comprising chemical intermediates and / or polyhydroxyalkanoates, fibre, proteins and / or oligopeptides; c) separating said chemical intermediates and / or polyhydroxyalkanoates from said fermentation broth, obtaining a first residue comprising fibre, proteins and / or oligopeptides; d) subjecting at least one part of the proteins and / or oligopeptides present in said first residue and / or, if separation a3) is performed, subjecting said solid fraction a3)ii to protein and / or oligopeptide hydrolysis, yielding a second residue comprising hydrolysed proteins and / or oligopeptides and fibre; e) subjecting said second residue comprising hydrolysed proteins and / or oligopeptides to solid / liquid separation, yielding: e)i. a solid fraction, including fibre, and e)ii. a liquid fraction comprising a protein hydrolysate including oligopeptides and free amino acids.

[0090] Said aqueous mixture obtained from step a) or said liquid fraction a3)i are subjected to fermentation step b) in the presence of at least one microorganism capable of producing ethanol or other chemical intermediates and / or polyhydroxy alkanoates.

[0091] Said microorganism capable of producing ethanol may be chosen from microorganisms belonging to the genera: Saccharomyces, Zygosaccharomyces, Candida, Hansenula, Kluyveromyces, Debaromyces, Nadsonias, Lipomyces, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Cryptococcus, Trichosporon, Aureobasidium, Lipomyces, Phaffia, Rhodotorula, Yarrowia, Schwanniomyces . Preferably said microorganism belongs to the genus Saccharomyces. Even more preferably said microorganism is Saccharomyces cerevisiae.

[0092] Butanediol, for example, may be produced by microorganisms of the genus Klebsiella, Enterobacter, Escherichia', a microorganism of the genus Escherichia genetically modified with at least one metabolic pathway for 1,4-butanediol synthesis is preferred.

[0093] Examples of microorganisms suitable for the production of polyhydroxyalkanoates are bacteria belonging to the genera Bacillus, Rhodococcus, Pseudomonas, Ralstonia, Cupriavidus, Protomonas, Alcaligenes, Escherichia and Leuconostoc. Archea belonging to the genus Haloferax are similarly suitable for the production of polyhydroxy alkanoates.

[0094] For the purposes of the present invention, said microorganism in step b) may be wild-type, obtained by essentially biological procedures or genetically modified, and capable of metabolising glucose and / or xylose.

[0095] According to one embodiment of the present invention, said microorganism can be fed directly to the fermentation step b) (known as "direct pitching"). According to an alternative embodiment, before being fed to fermentation said microorganism is propagated, resulting in an inoculum. Advantageously, said inoculum is prepared with a portion of the aqueous mixture comprising monosaccharides, fibre, proteins and / or oligopeptides obtained during step a) or at the end of step a), with the optional addition of urea as a nitrogen source.

[0096] In the case where a microorganism of the genus Saccharomyces is used, to obtain said inoculum the microorganism is fed to a fermentation device in the presence of a culture medium including sugars and preferably urea as a source of nitrogen, and when the microorganism reaches a cell concentration (dry weight) of 1 g / L or more, preferably between 3 g / L and 6 g / L, more preferably between 2 and 5 g / L, the inoculum is fed to the fermentation. To obtain said inoculum it may be propagated at a temperature of between 20°C and 40°C, preferably between 25°C and 35°C, and / or for a time of between 1 hour and 30 hours, preferably between 4 hours and 24 hours, and / or at an automatically fed air flow rate of between 1 L / Lh and 60 L / Lh, preferably between 10 L / Lh and 30 L / Lh, and / or at a pH of between 3 and 7, preferably between 4 and 7, more preferably between 4.5 and 6.5. To maintain the pH in the above ranges, an aqueous solution of at least one inorganic base such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or mixtures thereof, preferably potassium hydroxide, or at least one organic or inorganic acid such as, for example, lactic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or mixtures thereof, preferably sulfuric acid, may be added. In accordance with a preferred embodiment of the present invention said microorganism of the genus Saccharomyces is inoculated at an initial cell concentration (dry weight) of between 0.1 g / L and 2 g / L, preferably between 0.2 g / L and 1 g / L. Where a genetically modified microorganism of the genus Escherichia having at least one metabolic pathway for the synthesis of 1,4-butanediol is used, fermentation is preferably preceded by step a3) and is performed by feeding liquid fraction a3)i comprising monosaccharides to step b).

[0097] According to one aspect, liquid fraction a3)i comprising possibly concentrated monosaccharides is mixed wholly or partly with other carbon sources such as other sugars to obtain a mixture to be fed in the subsequent fermentation step b).

[0098] Solid fraction a3)ii including fibres, proteins and / or oligopeptides obtained as a result of step a3) may undergo protein and / or oligopeptide hydrolysis in step d).

[0099] According to a further aspect, the invention therefore relates to a process for the production of 1,4-butanediol and protein hydrolysates from a by-product of food flour processing comprising starch, fibre, protein and / or oligopeptides, said process comprising the steps of: a) hydrolysing the starch in said by-product in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibre, protein and / or oligopeptides; a3) separating the aqueous mixture obtained at the end of step a), obtaining: a3)i. a liquid fraction, including monosaccharides, and a3)ii. a solid fraction, including fibre, proteins, and / or oligopeptides, b) fermenting liquid fraction a3)i comprising monosaccharides in the presence of at least one microorganism having at least one metabolic pathway for 1,4-butanediol synthesis, resulting in a fermentation broth comprising 1,4-butanediol; c) separating the 1,4-butanediol from said fermentation broth; d) subjecting solid fraction a3)ii comprising fibre, proteins and / or oligopeptides to protein and / or oligopeptide hydrolysis, resulting in a residue comprising hydrolysed proteins and / or oligopeptides and fibre; e) subjecting said residue from step d) comprising hydrolysed proteins and / or oligopeptides to solid / liquid separation, to yield: e)i. a solid fraction, including fibre, and e)ii. a liquid fraction comprising a protein hydrolysate including oligopeptides and free amino acids.

[0100] According to this aspect, step a) is preferably accompanied, preceded or followed by hydrolysis of the cellulose or hemicellulose fibres present in said by-product, preferably in the presence of at least one enzyme chosen from cellulase, hemicellulase and combinations thereof, thereby reducing the fibre content in the subsequent process steps in favour of the monosaccharide content in the liquid fraction fed to fermentation step b). According to this aspect, step c) is carried out according to known techniques. Preferably it is carried out through at least one operation chosen from filtration, distillation, centrifuging, extraction and evaporation.

[0101] Also according to this aspect, the residual fermentation broth obtained at the end of step c) after the separation of 1,4-butanediol may also undergo hydrolysis during step d).

[0102] In step b) of the process according to the invention, fermentation is performed at a temperature of 20°C to 40°C, preferably 25°C to 35°C.

[0103] Such fermentation is preferably performed for a time of from 5 to 100 hours, preferably from 5 to 72 hours, more preferably from 6 to 30 hours, even more preferably from 8 to 24 hours. Such a time is suitable, for example, if a microorganism of the genus Saccharomyces is used.

[0104] Said fermentation is preferably performed at a pH of between 3 and 7, preferably between 4 and 7, more preferably between 4.5 and 6.5. Such a pH is, for example, suitable when using a microorganism of the genus Saccharomyces. To maintain the pH within the desired ranges an aqueous solution of at least one base such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonium hydroxide, or mixtures thereof, preferably potassium hydroxide, or at least one organic or inorganic acid may be added. Examples of organic acids are: lactic, malic, citric and tartaric acids. Examples of inorganic acids are: phosphoric acid, sulfuric acid, hydrochloric acid, or mixtures thereof. Phosphoric, sulfuric, lactic and citric acid are preferred.

[0105] According to one aspect, when the fermentation in step b) is carried out in the presence of at least one microorganism able to produce polyhydroxyalkanoates or at least one microorganism able to produce 1,4-butanediol, the separation step a3) is advantageously performed. According to this aspect, the liquid fraction a3)i., obtained by said separation, is mixed with another source of monosaccharides, such as for example glucose syrup, before to be fed in fermentation step b). To this end, preferably a weight ratio of monosaccharides from the liquid fraction a3)i to first generation monosaccharides of 1:1, more preferably of 1:2, even more preferably of 1:5 is used.

[0106] In cases where a microorganism of the genus Escherichia endowed with at least one metabolic pathway for the synthesis of 1,4-butanediol is used and separation step a3) is carried out, it is advantageous to feed liquid fraction a3)i comprising possibly concentrated monosaccharides to a culture used as a base for the growth of that microorganism.

[0107] Such culture medium will comprise the substances necessary for growth and sustenance of the microorganism during the fermentation step, such as elements like C, H, O, N, K, S, P, Fe, Ca, Co, Mn, Mg. Typically, the culture medium may include one or more components selected from the group consisting of sugars other than glucose, protein hydrolysates, proteins, amino acids, organic acids, vitamins, minerals, yeast extracts, and trace elements such as Cobalt, Calcium, and Copper. Cobalt, Calcium, and Copper may be dosed into the culture medium, for example, as salts such as Cobalt chloride, Calcium chloride, and Copper chloride. The culture medium may also contain antifoaming agents.

[0108] Generally, the culture medium will include at least one sugar, usually glucose and optionally one or more sugars other than glucose, in concentrations of between 10 and 100 g / L.

[0109] The culture medium may be prepared in any manner known to those skilled in the art, such as by mixing all its components together or pre-mixing some of them; for example, sugars may be added later. Said sugars may be commercial syrups, technical grade powders, or consist wholly or partly of the monosaccharides of liquid fraction a3)i. A commercially available culture medium may also be used as a starting point and its composition suitably modified at a later stage, for example, when bringing the culture medium into contact with the microorganism of the genus Escherichia endowed with at least one metabolic pathway for the synthesis of 1,4-butanediol.

[0110] The fermentation in step b) may be carried out in one or more steps, in batch mode, in fed-batch mode, or in continuous mode. Batch mode is preferred.

[0111] Preferably, fermentation step b) is continued until the sugars in the culture medium are depleted, that is until a residual sugar concentration of less than 50 g / L, preferably less than 10 g / L, more preferably less than 1 g / L is reached.

[0112] The fermentation in step b) is optionally carried out in the presence of antimicrobial agents capable of containing the growth of any microbial contamination. Said antimicrobial agents can be selected by the skilled in the art in a way that they do not interfere with the microorganisms used to perform the fermentation. Preferentially, said antimicrobial agents include bactericidal and / or bacteriostatic agents. Examples are antibiotics, C2-C10 chain fatty acids such as nonanoic acid, hydroxy acids (e.g. lactic acid, citric acid), parabens, triazotides, benzalkonium chloride, quaternary ammonium salts. Preferred examples are lactic acid and citric acid.

[0113] During step b), as an alternative or concurrently with ethanol production, one or more microorganisms capable of producing polyhydroxyalkanoates and / or chemical intermediates other than ethanol, such as, for example, succinic acid and / or 1,4-butanediol, may advantageously be present.

[0114] At the end of fermentation step b), a fermentation broth comprising chemical intermediates and / or polyhydroxyalkanoates, fibre, proteins and / or oligopeptides, and optionally salts, oils and fatty acids is obtained. Such broth is subjected to one or more separation operations in step c) to recover said chemical intermediates and / or polyhydroxyalkanoates produced, for example to remove ethanol from a first residue comprising fibre, proteins and / or oligopeptides and optionally salts, oils and fatty acids. According to one aspect of the invention, said fermentation broth comprises the microorganism described above.

[0115] This separation operation in step c) is carried out according to techniques known to those skilled in the art, for example it comprises one or more operations chosen from distillation, centrifuging, extraction and evaporation. Distillation is preferred.

[0116] The distillation in step c) can be carried out according to methods known to those skilled in the art, for example as described in "Ethanol distillation: the fundamentals" (1999), Chapter 18, p. 269-288, Katzen R., Madson P.W. and Moon G.D. Jr, KATZEN International, Inc., Cincinnati, Ohio, USA. If there is no step a3), the first residue obtained after removal of the chemical intermediate by distillation in step c) contains 16 to 60% by weight, preferably 22 to 35%, of proteins and / or oligopeptides and / or 20 to 80% by weight, preferably 30 to 50%, of fibres such as arabinans, glucans, xylans with respect to the dry weight of the residue. According to a particularly advantageous aspect, said first residue contains cells or cellular residues of the microorganism(s) employed during step b) of the process.

[0117] Such first residue can be used as a plant biostimulant and / or fertilizer and / or biopesticide. The presence of phytates provides a source of phosphorus that makes the agricultural use of such a first residue particularly advantageous.

[0118] In addition, the ash that may be present in the first residue may find use as a component of fibres.

[0119] Total protein and / or oligopeptide content is understood to be determined by total nitrogen analysis according to the Kjeldahl method and subsequent multiplication of the value obtained by a conversion factor defined according to the nature of the biomass analysed.

[0120] Fibre content is understood to be determined by the NREL / TP-510-42618 method.

[0121] According to the process according to the invention, at least one part of the proteins and / or oligopeptides present in the first residue obtained in step c) undergoes protein and / or oligopeptide hydrolysis in subsequent step d), resulting in a second residue comprising hydrolysed proteins and / or oligopeptides. Said hydrolysed proteins and / or oligopeptides are hereinafter also referred to as "protein hydrolysate."

[0122] If the fermentation broth obtained at the end of step b) and subjected to separation in step c) also includes the microorganism in step b), the proteins contained in said microorganism are also hydrolysed in step d) of the process according to the invention.

[0123] According to a preferred embodiment, the solid-liquid separation step a3) is not performed and the first residue including fibre, proteins and / or oligopeptides obtained at the end of step c) may undergo an optional step c2) including one or more further separation operations chosen from centrifuging, filtration or settling to obtain c2)i. a liquid fraction including salts, and c2)ii. a solid fraction including fibre, proteins, and / or oligopeptides;

[0124] According to one aspect, said liquid fraction c2)i advantageously comprises soluble fibre and soluble proteins and can be further enhanced by, for example, separating said soluble fibre and soluble proteins by one or more operations, selected from, for example, adsorption, ion exchange, reverse osmosis, microfiltration, ultrafiltration and nanofiltration. Ultrafiltration and / or nanofiltration, preferably in tangential flow, are preferred. The soluble fibre and soluble proteins thus obtained find use, for example, in food and nutraceuticals, either as such, or when concentrated or after undergoing hydrolysis of the protein and / or polysaccharide component.

[0125] According to one aspect, they are sent to step d) and / or step f) of the process according to the present invention.

[0126] Said solid fraction c2)ii advantageously undergoes step d) of protein and / or oligopeptide hydrolysis in the process according to the invention.

[0127] When the solid-liquid separation step a3) is performed, said optional step c2) is not done.

[0128] The said optional step c2) is preferred for example when the chemical intermediate is separated by distillation.

[0129] The proteins and / or oligopeptides in step d) can be hydrolysed out by any known method suitable for breaking down proteins into free amino acids and / or low molecular weight peptides. The hydrolysis may, for example, be chemical, enzyme, or a combination thereof. Enzyme hydrolysis is preferred.

[0130] Enzyme hydrolysis may be performed with any enzyme or mixture of enzymes capable of hydrolysing proteins into free amino acids and / or lower molecular weight peptides.

[0131] Enzyme hydrolysis occurs through proteolytic enzymes, known as proteases, which can cleave the peptide chain of proteins at specific points and hydrolyse them to free amino acids and / or low molecular weight oligopeptides. Proteases may be endoproteinases and / or exoproteinases. In a preferred embodiment, enzyme hydrolysis is carried out using an acid protease. In a particularly preferred embodiment, said protease is an aspartate endopeptidase.

[0132] In one alternative embodiment, enzyme hydrolysis is carried out using an alkaline protease.

[0133] In one alternative embodiment, enzyme hydrolysis is carried out using a neutral protease. Examples of suitable commercial products are "Prolyve PAC 30L PF" supplied by Soufflet Biotechnologies and "Sunson® PRA100," supplied by Sunson Industry Group Co., Ltd.

[0134] The enzyme hydrolysis is performed by selecting appropriate amounts of reactants, reaction conditions, and reaction sequences to obtain an appropriate level of enzyme activity. For example, the enzyme hydrolysis is performed by placing the first residue obtained in step c) in water at a ratio of 1:2 to 1:5 between the dry weight of the residue and water (expressed as w / w). When the separation step c2) is performed, the enzyme hydrolysis is preferably performed on the solid fraction c2)ii at a concentration of 10% by weight in water, preferably of 8%, more preferably of 6% by weight in water. Proteases, in liquid or solid formulation, are added to the resulting suspension in a weight ratio of 0.01 to 30 mg, preferably 0.5 to 10 mg, more preferably 0.1 to 3 mg of enzyme per 1 g of protein in the residue. The resulting suspension can be incubated at a pH and temperature appropriate to the specific protease used, and for an adequate period of time to obtain the desired amount of proteolysis.

[0135] When using a neutral protease, the suspension may be incubated at pH 5.5 to 8.5, preferably 7.5 to 8.5.

[0136] If an alkaline protease is used, the suspension may be incubated at pH 10 to 14, preferably 11 to 13.

[0137] In the preferred case of using an acid protease, the suspension may be incubated at pH 2 to 6, preferably 3 to 5.

[0138] The temperature is advantageously kept between 40 and 70°C, preferably 50 and 60°C.

[0139] The stirring is advantageously set at between 100 and 300 rpm, preferably 150 and 250 rpm. Hydrolysis is advantageously continued for a time of at least 2 hours, for example 24 to 168 hours, preferably 48 to 96 hours, more preferably 60 to 72 hours.

[0140] The protein hydrolysis in step d) is optionally carried out in the presence of antimicrobial agents capable of containing the growth of any microbial contamination. Said antimicrobial agents preferentially include bactericidal and / or bacteriostatic agents. Preferred examples of antimicrobial agents are lactic acid and citric acid.

[0141] Enzyme hydrolysis may be carried out in any suitable device for the purpose known to those skilled in the art, such as a reactor with temperature control and stirring. The second residue comprising hydrolysed proteins and / or oligopeptides obtained at the end of step d) is subjected to at least one solid / liquid separation operation in step e) during which a solid fraction comprising fibre is removed from a liquid fraction comprising a protein hydrolysate comprising oligopeptides and free amino acids.

[0142] Advantageously, one or more separation operations are carried out in series.

[0143] The separation operation in step e) may include one or more operations chosen from settling, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis, and combinations thereof. Centrifuging, settling and / or filtration operations are preferred, such as filtration on gauze and / or microfiltration and / or ultrafiltration.

[0144] According to one aspect, solid / liquid separation step e) includes one or more microfiltration operations.

[0145] For example, the microfiltration may be performed using PES polymer membranes or ceramic filters.

[0146] According to another aspect, solid / liquid separation step e) comprises centrifuging operations. For example, step e) includes an initial centrifuging operation followed by washing of the solid fraction and a further centrifuging operation to separate the wash waters from the washed solid fraction.

[0147] This separation operation in step e) may be performed using devices such as a decanter, settler, filter press, hydrocyclone, belt filter, rotary filter, and centrifuge.

[0148] According to one aspect, in step e) the residue including hydrolysed proteins and / or oligopeptides undergoes initial separation of an insoluble solid fraction and a liquid fraction. The resulting liquid fraction may optionally be treated again by an additional solid-liquid separation operation to recover additional solid fraction that can be combined with the former. Alternatively, all or some of the liquid fraction obtained from the first separation may advantageously be reintroduced into protein and / or oligopeptide hydrolysis step d) to utilise any still active enzyme present.

[0149] Solid fraction e)i obtained at the end of process step e) consists mainly of fibre and may contain proteins, oils, fatty acids, phytates and lignin. Such solid fraction preferably has a water content of less than 70% and a starch content of preferably less than 1% by weight, relative to dry weight of the solid fraction. Said solid fraction preferably has a polysaccharide and oligosaccharide content (cellulose and hemicellulose) of more than 40%, more preferably more than 50%, more preferably more than 60% by weight, relative to dry weight of the solid fraction. Such solid fraction preferably has a lignin content of less than 15%, preferably less than 10% by weight and / or a lipid content of less than 5%, preferably less than 2% by weight, relative to dry weight of the solid fraction. Said solid fraction has a phytate content that is advantageously lower than that of the by-product of food flour processing fed to step a) of the process according to the invention.

[0150] If said solid fraction e)i contains residual starch, it may be subjected to further hydrolysis with amylase and glucoamylase to obtain sugars for reintroduction into fermentation step b).

[0151] Starch content may be determined using the method "Determination of Cellulosic Glucan Content in Starch Containing Feedstocks" developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Michel, K.; Sluiter, J.; Payne C.; Ness, R.; Thornton, B.; Reed, M.; Schwartz, A.; and Wolfrum, E.; Technical Report NREL / TP-2800-76724, 2021).

[0152] Cellulose, hemicellulose and lignin content may be determined by the method "Determination of Structural Carbohydrates and Lignin in Biomass" developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D; Technical Report NREL / TP-510-42618, 2012). Ash (for example silicates, chlorides, bromides, nitrates, sulfates, phosphates, sodium, potassium) is determined, for example, by the "Determination of Ash in Biomass" method developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J. and Templeton, D.; Technical Report NREL / TP-510-42622, 2008), changing the holding time at temperature 575°C to at least 5 hours.

[0153] Where, for example, by-products of the processing of cereal grains such as wheat undergo the process according to the invention, said solid fraction e)i advantageously comprises at least 50% by weight, preferably at least 60% by weight, relative to its dry weight, of polysaccharides and oligosaccharides consisting essentially of hemicellulose and cellulose. Such solid fraction e)i advantageously comprises more than 50%, more advantageously more than 60% by weight of hemicellulose relative to the weight of polysaccharides and oligosaccharides (cellulose and hemicellulose); such hemicellulose is mainly composed of arabinoxylans. The arabinoxylans in such solid fraction advantageously contain ferulic acid or its esters and have a ratio of arabinose to xylose by weight of between 0.4 and 0.9, preferably between 0.5 and 0.8, even more preferably between 0.6 and 0.7. Such a solid fraction possesses a phytate content of preferably less than 4%, more preferably less than 3%, even more preferably less than 2% by weight in relation to its dry weight of the solid fraction.

[0154] Thus the present invention also relates to a composition, obtainable as a solid fraction in step e) of the process according to the invention, preferably performed from by-products of wheat processing, that is essentially free of starch (i.e. less than 5%, preferably less than or equal to 2% by weight on a dry weight basis) and comprising at least 50% by weight of cellulose and hemicellulose and having a lignin content of less than 15% by weight and a lipid content of less than 5% by weight, on a dry weight basis.

[0155] Cellulose and hemicellulose may be quantified, for example, by the method "Determination of Structural Carbohydrates and Lignin in Biomass" developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D; Technical Report NREL / TP-510-42618, 2012). Specifically, the amount of cellulose is determined by subtracting the amount of starch from the total amount of glucans measured.

[0156] A particularly advantageous aspect of the present invention is the possibility of also making use of solid fraction e)i obtained in step e) of the process.

[0157] According to a first embodiment, said solid fraction e)i undergoes anaerobic digestion with the production of biogas. According to a second embodiment, said solid fraction e)i undergoes treatments to recover soluble fibres and / or separate cellulose fibres from hemicellulose fibres. Said solid fraction does in fact contain fibres such as cellulose and hemicellulose, which can advantageously be easily separated by physical, chemical, biochemical treatments or combinations thereof known to those skilled in the art.

[0158] According to a first aspect of said second embodiment, soluble fibres are recovered from said solid fraction e)i, preferably by subjecting it to the following steps: f) subjecting said solid fraction e)i to one or more physical, chemical and / or biochemical treatments to obtain a third residue comprising fibres, including arabinoxylans. g) separating the third residue obtained in step f) to obtain: g)i. A liquid fraction comprising soluble fibres, including arabinoxylans and g)ii. a solid fraction.

[0159] Said chemical, biochemical and / or physical treatments in step f) may be the same or different from each other and carried out in any order.

[0160] Examples of physical treatments are cavitation and extrusion.

[0161] Said treatment in step f) preferably includes one or more operations chosen from enzyme hydrolysis, extraction and hydrothermal treatment.

[0162] Said enzyme hydrolysis in step f) occurs advantageously with enzymes suitable for the selective hydrolysis of the polysaccharide chains of fibres (such as cellulase, xylanase and arabinase) and under conditions known to those skilled in the art.

[0163] Said enzymes may be used individually or in mixtures and may be added to the starting biomass at different times depending on the desired hydrolysis product, adopting the appropriate operating conditions for each enzyme.

[0164] Depending on the enzymes selected, those skilled in the art will be able to adopt the necessary hydrolysis or saccharification conditions (reaction medium, pH, temperature, time, etc.).

[0165] Enzymes that can be used to obtain soluble fibre mixtures comprising arabinoxylans are particularly suitable for use in step f) of the process.

[0166] Said enzymes advantageously belong to the class of hemicellulases, of bacterial or fungal origin, and are preferably chosen from xylanases, arabinases and mixtures thereof. The use of at least one endo-xylanase is preferred. Examples of suitable commercial products are "Rohalase SEP Visco" supplied by AB Enzymes, and "Viscamyl Pro," supplied by IFF.

[0167] Enzyme hydrolysis may optionally be performed in the presence of one or more accessory enzymes that promote separation between polysaccharides and lignin, such as laccase, feruloyl esterase, glucuronidase, acetylxylanesterase, beta-glucosidase, and / or lytic polysaccharide monooxygenases (LPMOs).

[0168] The hydrolysis operation in said step f) is carried out in the presence of water, preferably keeping solid fraction e)i of the process in aqueous suspension.

[0169] According to a preferred aspect, said operation is carried out by feeding said solid fraction e)i with an initial dry weight of at least 2%, preferably at least 5%, more preferably at least 8% by weight relative to the weight of the aqueous suspension. Said aqueous suspension further optionally comprises acids and / or / bases added for pH correction, such as mineral acids (e.g. sulfuric acid) and / or organic acids (e.g. lactic acid).

[0170] During the hydrolysis reaction, the pH value and temperature are chosen according to the enzymes used. The pH and temperature conditions may be kept constant or modulated throughout the process, depending on specific requirements.

[0171] For example, according to a preferred embodiment, the reaction is carried out in the presence of xylanase while advantageously maintaining the pH between 4.0 and 7.5 (more preferably between 4.5 and 6.5), while the temperature during the reaction is preferably maintained at values from 30° to 80°C, more preferably between 40° and 70°C, more preferably between 50° and 60°C.

[0172] The hydrolysis reaction has a variable duration depending on the conditions adopted, particularly the type and concentration of enzymes, and the degree of hydrolysis desired. Advantageously the time is between 0.1 and 72 hours, for example between 0.5 and 48 hours, more advantageously between 1 and 30 hours, even more advantageously between 2 and 24 hours.

[0173] The hydrolysis step is optionally carried out in the presence of antimicrobial agents that can contain the development of any microbial contamination, as described above for step a).

[0174] Said extraction in step f) takes place at, for example, basic pH. The basic pH extraction operation is carried out in the presence of water, preferably keeping the solid fraction obtained from step e) of the process in aqueous suspension, and is advantageously performed with stirring and in the presence of one or more basifying agents. Examples of basifying agents are alkali metal hydroxides, such as sodium hydroxide or potassium hydroxide, or alkaline earth metal hydroxides such as, for example, calcium hydroxide or magnesium hydroxide, or mixtures thereof. Said extraction may be carried out at pH of between 7 and 14, more preferably between 10 and 14, even more preferably between 12 and 14, at a temperature of between 25 and 80°C, more preferably between 30 and 70°C, even more preferably between 40 and 60°C. The extraction reaction has a variable duration depending on the conditions adopted, such as the pH and temperature employed. Advantageously it runs for between 0.1 and 48 hours, more preferably between 0.5 and 24 hours, even more preferably between 1 and 20 hours.

[0175] Said hydrothermal treatment in step f) is carried out in the presence of water, preferably keeping the solid fraction obtained from step e) of the process in aqueous suspension. According to a preferred aspect, said operation is carried out by feeding in said solid fraction with an initial dry weight of at least 2%, preferably at least 5%, more preferably at least 8% by weight relative to the weight of the aqueous suspension, optionally adding one or more acids or one or more bases for pH correction. The operation is preferably performed at a pH of between 1 and 7, more preferably between 2 and 6, even more preferably between 3 and 5, at a temperature of over 100°C, more preferably over 120°C, even more preferably over 150°C. Advantageously, the operation lasts between 1 and 60 minutes, more preferably between 5 and 30 minutes, even more preferably between 10 and 20 minutes.

[0176] The third residue comprising fibre, comprising arabinoxylans obtained at the end of step f), is subjected to at least one solid / liquid separation operation in step g) during which a liquid fraction comprising soluble fibre, comprising arabinoxylans is removed from a solid fraction. Advantageously, one or more separation operations are carried out in series, optionally bringing together the solid fractions obtained. Liquid fraction g)i obtained from a first separation, or part of it, may advantageously be reintroduced into fibre hydrolysis step f) in order to reuse any enzymes still active in it.

[0177] The separation operation in step g) may include one or more operations chosen from settling, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis, and combinations thereof. Centrifuging, settling and / or filtration operations such as filtration on gauze and / or microfiltration and / or ultrafiltration are preferred; centrifuging operations are more preferred.

[0178] This separation operation in step g) may be performed using one or more of devices chosen from decanters, settlers, filter presses, hydrocyclones, belt filters, rotary filters and centrifuges. Solid fraction g)ii from the process consists mainly of fibre and may contain proteins, oils, fatty acids and lignin. It may be advantageously used for the production of biogas.

[0179] Where the process according to the invention is started from wheat processing by-products, liquid fraction g)i advantageously comprises more than 50% by weight of carbohydrates relative to the dry weight of the liquid fraction.

[0180] The soluble fibre content, comprising arabinoxylans, in said liquid fraction g)i is advantageously more than 20%, more preferably more than 40%, even more preferably more than 60% of the total dry weight of the liquid fraction. With respect to the total of said soluble fibres, arabinoxylans constitute at least 30%, more preferably at least 50%, even more preferably at least 80% by weight. Such arabinoxylans advantageously comprise ferulic acid and / or its esters and have a ratio of arabinose to xylose by weight of between 0.3 and 0.6, preferably between 0.3 and 0.5, more preferably between 0.3 and 0.4.

[0181] The content of soluble proteins (and / or oligopeptides and / or amino acids) is preferably less than 30%, more preferably less than 20%, even more preferably less than 10% of the total dry weight of the liquid fraction.

[0182] Therefore, object of the present invention is also a composition of soluble fibre, comprising arabinoxylans, containing more than 50% by weight of carbohydrates and having a soluble fibre content of more than 20% of the total dry weight relative to the dry weight of said composition, wherein said arabinoxylans constitute at least 30% by weight with respect to the total soluble fibre.

[0183] Said liquid fraction g)i may be further purified and / or concentrated by known techniques. The soluble fibres comprising arabinoxylans in it may be used as food or nutraceutical ingredients on account of their rheological, functional and beneficial properties for human health.

[0184] According to a second aspect of said second embodiment, solid fraction e)i undergoes separation of cellulose fibres from hemicellulose fibres by extraction, for example in a basic environment and / or in the presence of organic solvents (e.g. DMSO, imidazole, alcohols such as ethanol, propanol, tert-butyl alcohol), or by hydrothermal treatments, also assisted by, for example, ultrasound or microwaves.

[0185] Such processes are typically performed under conditions that allow high-value fibres such as arabinoxylans to be recovered from that solid fraction.

[0186] According to such an embodiment, said solid fraction e)i is therefore preferably subjected to:

[0187] 1) selective separation of the polysaccharide chains of hemicellulose from those of cellulose by extraction in basic aqueous solution (possibly in the presence of polar organic solvents such as ethanol) and subsequent acidification and

[0188] 2) hydrolysis of one or more of the polysaccharide chains thus separated to reduce their molecular weight to obtain oligosaccharides or to completely break them down into mono s accharides .

[0189] Said hydrolysis advantageously takes place by, for example, enzyme means, using commercial enzymes (such as cellulase, xylanase and arabinase) and under conditions known to those skilled in the art.

[0190] In particular, hydrolysates rich in five-carbon-atom monosaccharides such as xylose and arabinose and / or oligoxylans such as arabinoxylans can be obtained from hemicellulose. Said arabinoxylans, as indicated above, are endowed with numerous positive effects in promoting health and can thus be used in the food and nutraceutical industry for food and beverage production.

[0191] Said five-carbon- atom monosaccharides such as xylose and arabinose may advantageously be recycled in fermentation step b) as a substrate for bioethanol production.

[0192] Glucose may be obtained from the hydrolysis of cellulose, and this may advantageously be recycled in fermentation step b) as a substrate for ethanol production or used as a substrate in fermentations for the production of chemical intermediates, such as 1,4-butanediol or succinic acid, and / or polyhydroxyalkanoates.

[0193] Before undergoing said treatments for the recovery of soluble fibre and / or for the separation of cellulose fibres from hemicellulose fibres, solid fraction e)i obtained at the end of step e) may advantageously be subjected to one or more concentration and / or drying operations, resulting in a concentrated / dried solid fraction.

[0194] Said concentration and / or drying may involve one or more steps of liquid removal, such as by means of presses, such as screw presses, with the dual advantages of increasing recovery of the liquid phase and reducing the volume of the solid fraction subjected to subsequent treatment. Said drying is advantageously carried out by techniques known in the industry. The operation may be performed in batch or continuously, for example using plate dryers, drum dryers, fixed- bed dryers, tunnel dryers, belt dryers, fluidised bed dryers, flash dryers, ring dryers, pan dryers, spouted-bed dryers, heated directly or indirectly, with a one-way flow or the recycling of the drying gases.

[0195] Especially advantageous are systems that lead to drying by minimising the time spent at high temperature and possibly maintaining relatively low temperatures (e.g. ring dryers, flash dryers). Said drying is advantageously followed by milling and sieving operations to promote separation of the components.

[0196] According to a third embodiment, said solid fraction e)i directly undergoes an additional step of hydrolysis of the cellulose and / or hemicellulose polysaccharide chains, as mentioned above for step a) of the process, adapting the manner of hydrolysis to the product desired. Optionally, said additional hydrolysis step may be preceded by pretreatment aimed at making the polysaccharide chains more accessible to the enzymes used for the aforementioned hydrolysis. Said additional hydrolysis step allows monosaccharides that can be recycled in fermentation step b) to be recovered.

[0197] Thus, in the process according to the invention, the hydrolysis of the cellulose and / or hemicellulose polysaccharide chains, when performed, is advantageously conducted before step b) of fermentation (e.g. in step a2)) or after the separation of protein hydrolysate (e.g. hydrolysis of solid fraction e)i). In this way, the protein hydrolysate obtained is essentially free of monosaccharides. By "essentially free of monosaccharides " is meant the presence of monosaccharides in amount less than 5% by weight, preferably less than 4% by weight or more preferably less than 3% by weight relative to dry weight of the protein hydrolysate.

[0198] Liquid fraction e)ii obtained at the end of step e) comprises a protein hydrolysate including oligopeptides and free amino acids.

[0199] Said protein hydrolysate may optionally undergo one or more filtration operations, which allow further purification of the hydrolysate, removing any residual impurities from the oligopeptides and free amino acids.

[0200] The process according to the invention may therefore comprise an optional step h) of subjecting said liquid fraction e)ii comprising a protein hydrolysate to purification, resulting in an additional liquid fraction comprising a purified protein hydrolysate.

[0201] Said purification includes, or advantageously consists of, one or more filtration operations, such as filtration on gauze and / or microfiltration and / or ultrafiltration. Preferably said filtration operations include ultrafiltration.

[0202] Depending on the characteristics of the protein hydrolysate subjected to said filtration operations in optional step h) of the process, those skilled in the art will be able to select the type of membrane to be used, taking into consideration the material it is made of, its electrochemical properties and its porosity.

[0203] The type of membrane chosen will determine the pressures and other optimal operating conditions. Those skilled in the art will be able to assess the plant conditions such that yield and product quality are guaranteed, defining whether the operation should be performed in batch or continuous mode, whether and how much diafiltration is needed (i.e. dilution of the retentate by the addition of water and repeating the separation operation), the concentration achievable, and the reinjection of materials.

[0204] For example, filtration of step h) may be effectively carried out by employing either organic membranes, of natural origin (e.g. rubbers, polysaccharides) or synthetic origin (e.g. polymer membranes), or inorganic membranes, such as ceramic, metal or glass membranes.

[0205] Of the organic membranes, polyamides, polyimides, poly alkylenes, polyether imide, polyarene ether, poly(ether ketone), polycarbonates, cellulose acetate and derivatives are preferred.

[0206] Specific examples of suitable organic membranes are polysulfones, polyamides, polypyperazine amide, polyethylene, polytetrafluoroethylene (PTFE), polypropylene, polyvinyl alcohol, polystyrene, polybenzimidazoles (PBI), polyphenylenes, polyphosphazenes, polyvinylidene fluoride (PVDF), polyether sulfones (PES), polyacrylonitrile (PAN), and polyvinyl chloride (PVC).

[0207] Both isotropic (or symmetrical) and anisotropic (or asymmetrical) membranes and composite membranes are suitable.

[0208] Preferably, anisotropic membranes are used.

[0209] The membranes may be formed in different configurations, such as flat, tubular, capillary or hollow fibre form. Flat membranes may be used as such in filter-press type systems, in rotary systems, or wound in spiral modules to increase the surface area / occupied volume ratio.

[0210] Separation operations using a membrane according to the invention may be carried out in batch or continuous mode; depending on the case, a normal (perpendicular) or tangential flow regime filtration method respectively is preferably used.

[0211] Separation operations through membrane in tangential flow regime are preferred.

[0212] The protein content of said purified protein hydrolysate according to the present invention is advantageously more than 40% by weight, more than 50% by weight, preferably more than 60% by weight, and more preferably more than 70% relative to the dry weight of the protein hydrolysate. The soluble fibers content of said purified protein hydrolysate is advantageously below 35% by weight, preferably below 25% by weight relative to the dry weight of the protein hydrolysate. The monosaccharide content of said purified protein hydrolysate is less than 10% by weight, advantageously from 0.1% to 9.9% by weight, from 0.5% to 8.0% by weight, from 1.0 to 5.0% by weight relative to the dry weight of the protein hydrolysate. Ashes content of said purified protein hydrolysate is less than or equal to 10% by weight, advantageously from 0.1% to 7.9% by weight, from 0.5% to 6.0% by weight, from 2.0 to 4.0% by weight relative to the dry weight of the protein hydrolysate.

[0213] The liquid fraction comprising a protein hydrolysate obtained in step e) or step h) may optionally be concentrated by known techniques, for example by distillation, evaporation, or reverse osmosis, until the desired concentration of protein substance is achieved.

[0214] The process according to the invention may therefore comprise an optional step j) of subjecting said liquid fraction comprising a protein hydrolysate obtained as a result of step e) or step h) to concentration, resulting in a concentrated protein hydrolysate.

[0215] According to one aspect, such liquid fractions comprising the protein hydrolysate (or purified protein hydrolysate) and / or concentrated protein hydrolysate are partly recycled in fermentation step b) for the purpose of providing nitrogenous components (such as oligopeptides and free amino acids) or a source of phosphorus (such as phytates or phosphates) useful for the growth and metabolism of the microorganism(s). Those skilled in the art will know the amount of said liquid fraction or concentrated protein hydrolysate that can be appropriately recycled in step b), based on the concentration of the nitrogen components and phosphorus sources present in said liquid fraction or concentrated protein hydrolysate, and based on the needs of the microorganism during fermentation step b).

[0216] Both the second residue comprising hydrolysed proteins and / or oligopeptides obtained in step d), liquid fractions comprising protein hydrolysate (obtained in step e)ii) or purified protein hydrolysate (obtained in optional step h)), and concentrated protein hydrolysate (obtained in optional step j)) are particularly suitable for use as plant biostimulants and / or fertilizers and / or biopesticides. The protein hydrolysate advantageously contains phytates that may be a source of phosphorus for plants, reducing or eliminating the need to add other phosphorus-based molecules in the formulation of plant biostimulants and / or fertilizers and / or biopesticides.

[0217] A second aspect of the present invention is therefore the use of said protein hydrolysate as a plant biostimulant and / or fertilizer and / or biopesticide. Use as a plant biostimulant is preferred. A third aspect of the present invention is a plant biostimulant comprising said protein hydrolysate.

[0218] A further aspect of the present invention is a plant fertilizer comprising said protein hydrolysate. A further aspect of the present invention is a plant biopesticide comprising said protein hydrolysate.

[0219] The protein hydrolysate comprises 0.1 to 30 g / L, preferably 1 to 25 g / L, more preferably 5 to 20 g / L, of total amino acids.

[0220] The concentrated protein hydrolysate includes 3 to 900 g / L, preferably 30 to 750 g / L, more preferably 150 to 600 g / L, even more preferably 200 to 400 g / L, advantageously 300 to 400 g / L, of total amino acids.

[0221] The protein hydrolysate according to the present invention advantageously comprises 5, preferably 10 to 40% by weight, preferably 15 to 25% by weight of free amino acids, relative to the total amino acids. The presence of a high amount of free amino acids promotes rapid and efficient foliar as well as root uptake of the protein hydrolysate by plants.

[0222] According to a preferred aspect, the protein hydrolysate contains phytates.

[0223] The type of action protein hydrolysates exert on plants depends on their composition. Specific amino acids can in fact be associated with a certain effect on plants. Glutamic acid, for example, promotes plants’ resistance to various environmental stresses, being active in resistance mechanisms in adverse situations and promoting the assimilation of inorganic nitrogen. Proline, for example, accelerates recovery time from abiotic stress and reduces its effect by strengthening cell walls (Hayat et al., 2012, Plant Signal Behav. 7(11): 1456-1466), and increases pollen fertility (Mattioli et al., 2018, BMC Plant Biol. 18( 1 ):356) . Glycine and alanine stimulate plant growth by supporting chlorophyll synthesis, chelating micronutrients, improving pollen fertility or resistance to low temperatures (Mikula K. et al., 2022, Environ Sci Pollut Res). Histidine is known to induce resistance to some pathogens through activation of the ethylene pathway in plants (Seo S. et al., 2016, Plant Cell Physiol. 57(9): 1932-42). Cysteine is important because it is involved in detoxification in mitochondria and in the response to pathogens (Romero L.C. et al., 2014, Molecular Plant, Volume 7, Issue 2, pages 264-276). Threonine is an important metabolite involved in defence against abiotic stresses, such as cold, salinity and aridity (Muthuramalingam P. et al., 2018, Scientific Reports, 8: 9270).

[0224] The composition of protein hydrolysates greatly depends on the origin of the raw material from which they are derived and the process used to produce them.

[0225] Advantageously, the protein hydrolysate of the invention has an ash content of less than or equal to 10%, preferably of less than 8%, advantageously from 0.1% to 7.9% by weight, from 0.5% to 6.0% by weight, from 2.0 to 4.0% by weight relative to the dry weight of the hydrolysate. According to an aspect, the said ash content advantageously derives directly from the process of the invention when for example the above described separation step c2) is performed. According to another aspect, the said ash content is obtained when one or more of the starch hydrolysis reaction of step a), the fermentation of step b) and the protein hydrolysis of step d) is performed using an organic acid to control the pH, e.g. selected from lactic acid, malic acid, citric acid and tartaric acid; lactic acid is preferred.

[0226] The protein hydrolysate according to the present invention advantageously comprises one or more amino acids chosen from glutamic acid, proline, serine, glycine, alanine, threonine, cysteine, and histidine.

[0227] The protein hydrolysate according to the present invention comprises at least 10%, preferably 12 to 30%, more preferably 14 to 22% of glutamic acid relative to the % of total amino acids.

[0228] The protein hydrolysate according to the present invention comprises at least 3%, preferably at least 5%, more preferably 5 to 15%, more preferably 7 to 10% of proline relative to the % of total amino acids.

[0229] The protein hydrolysate according to the present invention comprises at least 3.5%, preferably 4 to 10%, even more preferably 4 to 8% of serine relative to the % of total amino acids.

[0230] The protein hydrolysate according to the present invention comprises at least 4%, preferably at least 5%, more preferably 6 to 15%, even more preferably 8 to 11% of glycine relative to the % of total amino acids. The protein hydrolysate according to the present invention comprises at least 4%, preferably 5 to 15%, more preferably 6 to 10% of alanine relative to the % of total amino acids.

[0231] The protein hydrolysate according to the present invention includes at least 2%, preferably 2.5 to 5% of threonine relative to the % of total amino acids.

[0232] The protein hydrolysate according to the present invention comprises at least 1.5%, preferably 2 to 5% of cysteine relative to the % of total amino acids.

[0233] The protein hydrolysate according to the present invention comprises at least 1%, preferably 1 to 5%, even more preferably 2 to 4% of histidine relative to the % of total amino acids.

[0234] The concentrated protein hydrolysate according to the present invention comprises at least 10% by weight of dry matter, preferably 20 to 80% by weight, more preferably 30 to 60% by weight. The dry matter of the protein hydrolysate is understood as the weight of the residual portion of the protein hydrolysate after removal of the water it contains and may be determined, for example, according to ASTM El 756 - 08.

[0235] With regard to macronutrient content, the concentrated protein hydrolysate includes 1 to 45% by weight, preferably 2 to 25% by weight, of organic nitrogen, as measured by the Kjeldahl method, relative to the dry weight of the hydrolysate.

[0236] According to the preferred aspect in which the fermentation broth obtained at the end of step b) and subjected to ethanol separation in step c) also includes the previously described microorganism, the protein hydrolysate according to the present invention also includes oligopeptides, free amino acids and cell residues (such as polysaccharides, lipids and nucleic acids) derived from that microorganism.

[0237] The protein hydrolysate according to the present invention is substantially constituted by oligopeptides having a molecular weight below IKDa and / or free amino acids, thereby advantageously favouring the plant up-take.

[0238] A further aspect of the present invention is therefore a plant biostimulant comprising said protein hydrolysate and said cell residues derived from the microorganism used in step b) of fermentation. Said biostimulant therefore advantageously comprises a combination of plant- derived and microorganism-derived components.

[0239] Such a biostimulant may optionally include one or more additives and / or adjuvants that make a product suitable for field applications and enhance its properties in both fertigation and spray application. Such additives and adjuvants may be selected from: carriers, adhesives, filmforming agents, humectants, penetrating agents, cold protectants, emulsifying agents, wetting agents, dispersants, stabilisers, preservatives, diluents, micronutrients, macronutrients, and combinations thereof. The carrier is selected from the group including but not limited to: lignite, bentonite, peat, vermiculite, charcoal, soil mixtures, manure, or combinations thereof.

[0240] The cold protector is selected from the group including but not limited to: polyethylene glycol (PEG), glycerol, DMSO, polyvinyl alcohol, sodium alginate, gelatin, gellan, or combinations thereof.

[0241] Adhesive adjuvants with film-forming and wetting power, used to allow the biostimulant active ingredient to remain on the leaf as long as possible and penetrate inside the cuticle, are selected from the group including but not limited to: xanthan gum, carboxymethylcellulose (CMC), gum arabic, polyvinylpyrrolidone (PVP), chitosan, alginate, modified or complexed starches or combinations thereof.

[0242] Wetting additives are selected from the group including but not limited to: polyol alcohol esters, ethoxylated and nonethoxylated sorbitans, alkylpolyglucosides or combinations thereof. The function of these additives is to keep biostimulant products from drying and thus crystallizing on the leaf, leaving them moist and thus able to act.

[0243] Penetrating agents as well as emulsifiers and wetting agents serve to promote entry of the biostimulant into the leaf cuticle and are selected from the group including but not limited to: cationic surfactants, anionic surfactants, nonionic surfactants, silicon-based surfactants and their combinations. These surfactants may be natural, semisynthetic, synthetic or combinations thereof.

[0244] Examples of surfactants are: ethoxylated and ethoxylated-propoxylated sorbitans, ethoxylated alcohols, block or star copolymers, partially hydrolysed and ethoxylated polyol esters, ethoxylated and / or propoxylated vegetable oils.

[0245] Dispersant additives, used to stabilise the formulation over time, are selected from the group including but not limited to: lignin sulfonates and block copolymers, ethoxylated tristyrylphenols, dioctyl sulfosuccinates and the like, naphthalene sulfonates and EO / PO block copolymers, star copolymers, xanthan gum or combinations thereof.

[0246] The stabiliser and preservative are selected from the group including but not limited to: potassium sorbate, sorbic acid, lactic acid, trehalose, sugars, mannitol, citric acid, polyglutamic acid, benzoic acid or sodium benzoate, propylene glycol, propionic acid, zinc sulfate, iron sulfate, copper sulfate, l,2-benzisothiazolin-3-one (BIT) and the like, 2, 2', 2"- (hexahydro- 1,3,5- triazine 1,3,5 triyl) triethanol and the like, pentane- 1,2-diol, 3-phenylpropane-l-ol and the like or combinations thereof.

[0247] The diluent is selected from the group including but not limited to: ionic buffer diluent solution, salt solution, water or combinations thereof. The biostimulant according to the invention may be administered to plants together with solutions containing micro- and / or macronutrients or may be formulated together with them. Examples of micronutrients are: iron, nickel, copper, zinc, boron, manganese, calcium, sodium, molybdenum, and magnesium, variously complexed such as with EDTA and other organic complexing agents.

[0248] Examples of macronutrients are: nitrogen, potassium, and phosphorus. The presence of phytates reduces or eliminates the need for the addition of other sources of phosphorus.

[0249] The biostimulant according to the invention may be in solid or liquid form. In the liquid form, which is the preferred form, the biostimulant (having a dry matter of about 50% by weight) is placed in contact with or applied to plants at a concentration ranging from about 1 g / L to about 20 g / L (i.e. diluted with a final dry matter ranging from about 0.5 g / L to 10 g / L).

[0250] Said biostimulant may be applied to the soil, rhizosphere, aerial or nonaerial parts of plants, selected from the group including roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits, seeds and combinations thereof.

[0251] In some embodiments the biostimulant is applied as a foliar spray solution in a dose of 1 to 20 g per litre of water.

[0252] In other embodiments the biostimulant is applied directly to the soil by fertigation in a dose of 1 to 20 g per litre of water.

[0253] The biostimulant according to the invention may be administered to plants such as agricultural crops, horticultural crops, floricultural crops, fruit crops or any combination thereof.

[0254] Among agricultural crops, the biostimulant is preferably applied to tomato, eggplant, squash, cucumber, zucchini, lettuce, cauliflower, strawberry, and tobacco crops.

[0255] The application of biostimulant results in improved performance of the plants to which it is administered. Such improved performance includes one or more of: an increase in the number, size, weight, or quality of hypogeal or aerial biomass, for example roots, shoots, leaves, flowers, anthers, stigmas, stamens, fruits, and / or seeds, an increase in photosynthetic activity or chlorophyll content, an increase in protein, dietary fibre, P-carotene, essential oils and / or specific plant metabolites, improved absorption or utilisation of nutrients or minerals, increased resistance to biotic and / or abiotic stresses.

[0256] According to one aspect, the application of biostimulant by fertigation results in improved growth delta (difference between leaf length measured before administration of the protein hydrolysate and that measured 3 weeks after administration) of the first true leaf of plants treated. According to another aspect, the application of biostimulant by fertigation results in improved dry weight of plants treated. Plant dry weight can be measured, for example, drying plants in an oven at 105°C±l.

[0257] According to another aspect, the application of biostimulant by fertigation results in improved SPAD index of plants treated. SPAD index is a measure of chlorophyll content of the leaves and plant health.

[0258] According to another aspect, the administration of biostimulant results in an improved germination of seeds.

[0259] Each process step according to the present invention may advantageously be carried out independently in batch mode, continuous mode or semi-continuous mode. According to a preferred embodiment, each of steps a)-e) is carried out in continuous mode.

[0260] Advantageously, each step in the process according to the invention is performed using conditions conducive to the performance of the next step, without adversely affecting the products obtained.

[0261] The process according to the invention will now be described according to a non-limiting example.

[0262] METHODS USED IN THE EXAMPLES

[0263] Determination of cellulose and hemicellulose content

[0264] Cellulose and hemicellulose were determined using the method developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Sluiter, A.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D: "Determination of Structural Carbohydrates and Lignin in Biomass." Technical Report NREL / TP-510-42618, 2012). Specifically, the amount of cellulose is determined by subtracting the amount of starch from the total amount of glucans measured.

[0265] Determination of starch content

[0266] Starch was quantified by the method developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Katie Michel, Justin Sluiter, Courtney Payne, Ryan Ness, Brittany Thornton, Michelle Reed, Alexa Schwartz, and Ed Wolfrum): "Determination of Cellulosic Glucan Content in Starch Containing Feedstocks" Technical Report NREL / TP-2800-76724, February 2021.

[0267] Determination of ash content

[0268] Ash content was quantified using the method developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory (Determination of Ash in Biomass Laboratory Analytical Procedure (LAP) Issue Date: 7 / 17 / 2005 A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, and D. Templeton), modifying the holding time of the temperature of 575°C to at least 5 hours.

[0269] Determination of protein content

[0270] The soluble protein in liquid samples was determined using the TOC-L instrument (Shimadzu), appropriately diluting to meet calibration limits and multiplying the value obtained by the grain conversion factor, which is specifically 6.25.

[0271] The total protein in solid samples was determined by analysing the total nitrogen content by the Kjeldahl method and multiplying the value obtained by the same conversion factor defined according to the nature of the biomass analysed.

[0272] Insoluble proteins were then determined by the difference between total and soluble proteins.

[0273] EXAMPLE 1,

[0274] A by-product of the process of processing food flours from wheat was subjected to the process according to the invention, according to the scheme depicted in Figure 1.

[0275] Said by-product, with a water content of 10% by weight, had the composition shown in Table 1 :

[0276] Table 1: Composition of the wheat processing by-product fed to step a)

[0277] Step a)

[0278] The above by-product underwent enzyme hydrolysis in step a) to transform the starch present into glucose.

[0279] Two hydrolysis were carried out using two 3.6-litre reactors with a configuration for solid substrates, each filled to 30% volume. The first hydrolysis was performed for the yeast propagation and the second one for fermentation step b); the second hydrolysis started with 16h of delay with respect to the first hydrolysis. 811 g of demineralised water was added to each reactor, which were then heated to a reaction temperature of 57°C. Once this temperature was reached, biomass was added a little at a time (189 g for each reactor, considering 10% moisture in the biomass).

[0280] Next, 6 mg of antibiotic (Lactrol) was added, and the pH was raised to 5 by the addition of 10% vol / vol phosphoric acid.

[0281] Next, a-amylase enzyme (IFF, SPEZYME® ALPHA PF, Alpha-Amylase 13775-15225 AAU / g) and glucoamylase enzyme (IFF, OPTIDEX L-400, Glucoamylase 350 - 390 GAU / g Transglucosidase 0 - 10 TGU / 100GAU) were added. The dosage for both enzymes was 1 mL / kg dry solid material (0.17 mL each).

[0282] Hydrolysis was continued for 24 hours at a temperature of 57°C with stirring at 100 rpm. During the test the pH was maintained in the range 4.8-5.3 by the addition of 10% vol / vol phosphoric acid.

[0283] At the end of the test the released sugars (glucose and fructose) were quantified and found to be 50-60 g / L.

[0284] At the end of hydrolysis the reactor temperature was lowered to 32°C to allow the next fermentation step.

[0285] Step b)

[0286] Nutrients (Urea -1 g / L and Corn Steep Liquor 5 g / L) and 0.25 g / L of rehydrated Saccharomyces cerevisiae yeast (LeSaffre's Ethanol Red ® - Leaf yeast, rehydrated according to the manufacturer's instructions) were added to the same reactor as in step a).

[0287] The yeast was propagated in batch mode, at 32°C, with stirring fixed at 200 rpm, with pH monitored in the range of 4.5 to 5.3 and with aeration of 10 vvh (volumes of gas / volume culture / hour).

[0288] After 16 hours propagation, when cell growth of at least 14 to 16 times the initial concentration was reached, a portion of the propagate, amounting to about 180 g, was transferred to the fermentation reactor (i.e. the reactor where the second hydrolysis was performed) to achieve a yeast concentration of at least 0.4-0.6 g / L.

[0289] Fermentation was performed in batch mode, at 32°C, with stirring fixed at 100 rpm, pH monitored in the range 4.5-5.3 and in the absence of aeration.

[0290] Fermentation was performed in the presence of nutrients such as urea (1 g / L) and Com Steep Liquor (2.5 g / L) and lasted 24 hours.

[0291] The fermentation yield was 0.42 grams of ethanol compared to the grams of fermentable sugars obtained after the hydrolysis step. The final ethanol titre was found to be 24 g / L.

[0292] Step c) The fermentation broth was transferred to a rotavapor flask in order to remove the ethanol produced. The oil bath was maintained at 80-83°C, the condenser was cooled with running water to a temperature of 10-15°C, and the vacuum was maintained at 400 mbar and then decreased to 200 mbar. The flask was rotated at about 100 rpm for 4 hours.

[0293] Step d)

[0294] 1 kg of residue obtained from the separation of ethanol from the fermentation broth in step c), having the composition given in Table 2, was resuspended in 2 litres of water and the pH was raised to 7.9 by the addition of 10N KOH. The resulting suspension was hydrolysed using BS- L enzyme (neutral protease supplied by Enzyme Supplies) and employing an enzyme loading of about 1:350 w / w, expressed as protein content of enzyme and total protein present in the biomass, respectively. Enzyme hydrolysis was performed at 55°C for 5 hours with stirring at 200 rpm.

[0295] Table 2: Composition of the residue obtained from the separation of ethanol from the fermentation broth in step c).

[0296] Step e)

[0297] After protein hydrolysis step d) ended, a liquid phase was separated from a solid phase by centrifuging at 8000 rpm for 30 min. The liquid fraction was characterised in terms of total amino acids, which amounted to 7.25 g / L, and free amino acids, which amounted to 21.6% of the total amino acids. The amino acid content of the liquid fraction is shown in Table 3 below:

[0298] Table 3. Amino acid content of the liquid fraction separated in step e).

[0299] EXAMPLE 2,

[0300] Step a

[0301] A by-product of the process of processing food flours from wheat was subjected to step a) of starch hydrolysis under the same conditions as indicated in Example 1. At the end of the enzyme reaction the liberated monosaccharides (glucose and fructose) were quantified and found to be about 75 g / L.

[0302] Step a3)

[0303] The aqueous mixture obtained as a result of step a) was centrifuged at 7000 rpm for 30 min to separate liquid fraction a3)i containing the monosaccharides from solid fraction a3)ii. Solid fraction a3)ii was washed with demineralised water to maximise monosaccharide recovery and centrifuged again to separate the wash waters from the washed solid. The wash waters were combined with liquid fraction a3)i, and the washed solid fraction was subjected to protein hydrolysis step d) under the conditions described below.

[0304] The monosaccharide solution obtained by combining liquid fraction a3)i and the wash waters was transferred to a rotavapor flask and concentrated more than 10 times by evaporating some of the water present. Concentration was performed at a temperature of 70°C while maintaining a vacuum of about 250 mbar for the first few hours and gradually decreasing it to 120 mbar for the last few hours of evaporation. The flask was kept rotating at about 40 rpm for a total of 4 hours. The monosaccharide syrup obtained from the concentration of the monosaccharide solution was mixed with commercial glucose syrup in a ratio of 1:4 by weight. The resulting mixture was used in the subsequent fermentation step.

[0305] Step b)

[0306] A strain of Escherichia coli having with a metabolic pathway for the synthesis of 1,4-butanediol was inoculated into a 250-mL Erlenmeyer flask containing 25 mL of Luria Bertani medium fortified with 10 g / L technical grade glucose. The flask was then stirred at 275 rpm at a temperature of 35°C for 16 hours, yielding a preinoculum.

[0307] Next, an aliquot of the preinoculum was transferred to a 1000 mL Erlenmeyer flask containing 200 mL of a second culture medium (12.78 g / L M9 Minimal Salt; 15 g / L technical grade glucose; 1 mL / L IM MgSCL; 1 mL / L 0.1 M CaCh; 1.0 mL / L 1000X Trace Elements; 0.5 mL / L Streptomycin 100 mg / mL).

[0308] The flask was incubated at 35°C, stirring the contents at 275 rpm for about 8 hours. After this incubation period the optical density reached an OD value (optical density measured at 600 nm) of about 2-5 OD, and the culture was used to inoculate a seeding fermenter at OD 0.5.

[0309] After about 18 hours, an aliquot of the seeded fermentation was used to inoculate a production fermenter containing 1 litre of medium (K2HPO41.76 g / L; H3PO41.16 g / L; H2SO4O.68 g / L; Ca Citrate.4H2O 0.038 g / L; MgSO4 0.48 g / L; 1000X Trace Elements 2.0 mL / L; Antifoam 0.1 mL / L and glucose syrup 50 g / L) at OD 4.

[0310] During fermentation, the sugar mixture (prepared by mixing liquid fraction a3)i concentrate with a commercial glucose syrup) was progressively fed into the production fermenter (fed batch process), so that the overall concentration of glucose and fructose in the culture medium was kept constant in the range of 30-60 g / L, for about 30 hours from the start of fermentation, and then progressively reduced until the glucose concentration at the end of fermentation (34 hours from inoculation) was about 0 g / L.

[0311] The productive fermenter was maintained under the following conditions: stirring speed 700-900 rpm, air flow 0.4 vvm (L air / L medium / minute), pH 6.2, temperature 35 °C.

[0312] Samples of the fermentation broth were taken at different times to assess the output of 1,4-butanediol using high-pressure liquid chromatography (HPLC) analysis, and a concentration of 130 g / L was measured at the end of the fermentation stage.

[0313] 1,4-butanediol content was analysed by HPLC equipped with an RID detector and a Biorad Aminex HPX-87H 300mm x 7.8mm column and corresponding pre-column, using the following operating conditions: flow rate 0.6 mL / minute, oven temperature 50°C, detector temperature 35 °C, eluent 5mM H2SO4.

[0314] Step c)

[0315] The fermentation broth obtained at the end of step b) was processed to obtain a liquid fraction comprising the 1,4-butanediol produced, which can be subsequently purified as shown in Example 1 of patent WO 2019 / 102030.

[0316] Step d)

[0317] 125 g of solid fraction a3)ii, having a protein content of 34.1% with respect to dry weight of solid fraction, was resuspended in demineralised water at a total solids concentration of 7.57%. The pH of the suspension was raised to 3.75 by the addition of H3PO4. The resulting suspension was hydrolysed using Prolyve PAC 30L PF enzyme (acid protease supplied by Soufflet Biotechnologies) and employing an enzyme loading of about 1:1000 w / w, expressed as protein content of enzyme and total protein present in the biomass, respectively. Enzyme hydrolysis was performed at 55 °C for 22 hours with stirring at 200 rpm.

[0318] Step e)

[0319] After protein hydrolysis step d) ended, a liquid phase was separated from a solid phase by centrifuging at 7000 rpm for 30 minutes.

[0320] The liquid fraction was characterised in terms of total amino acids, amounting to 159 mmol / L (about 17.5 g / L), and free amino acids, amounting to 61 mmol / L (about 6.7 g / L).

[0321] The solid fraction was washed with demineralised water and centrifuged again to separate the wash waters. The composition of the resulting washed solid fraction is shown in Table 4.

[0322] Table 4: Composition of the washed solid fraction obtained from separation of the protein hydrolysate produced in step d). The hemicellulose present in the solid fraction was found to consist mainly of arabinoxylans, from which an arabinose / xylose ratio of 0.62 was determined (by the procedure described above for the determination of cellulose and hemicellulose).

Claims

CLAIMS1. Process for the production of chemical intermediates and / or polyhydroxyalkanoates and protein hydrolysates from a by-product of food flour processing including starch, fibre, protein and / or oligopeptides, said process including the steps of: a) hydrolysing the starch into said by-product in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibre, protein and / or oligopeptides; a3) optionally separating the aqueous mixture obtained at the end of step a) obtaining: a3)i. a liquid fraction, including monosaccharides, and a3)ii. a solid fraction, including fibres, proteins, and / or oligopeptides; b) fermenting said aqueous mixture obtained from step a) or said liquid fraction a3)i in the presence of at least one microorganism capable of producing chemical intermediates and / or polyhydroxyalkanoates, resulting in a fermentation broth comprising chemical intermediates and / or poly hydroxy alkanoates, fibre, proteins and / or oligopeptides; c) separating said chemical intermediates and / or polyhydroxyalkanoates from said fermentation broth, obtaining a first residue comprising fibre, proteins and / or oligopeptides; d) subjecting at least one part of the proteins and / or oligopeptides present in said first residue and / or, if separation a3) is performed, subjecting said solid fraction a3)ii to protein and / or oligopeptide hydrolysis, resulting in a second residue comprising hydrolysed proteins and / or oligopeptides and fibre; e) subjecting said second residue comprising hydrolysed proteins and / or oligopeptides to solid / liquid separation, resulting in: e)i. a solid fraction, including fibres, and e)ii. a liquid fraction comprising a protein hydrolysate including oligopeptides and free amino acids.

2. Process according to claim 1, in which said by-product of food flour processing is a biomass obtained from milling cereals, pseudocereals and / or legumes.

3. Process according to claim 2, in which said by-product is a biomass comprising more than 20% by weight of total fibre and at least 15% by weight of protein relative to the dry weight of said biomass.

4. Process according to one or more of claims 1-3, in which said chemical intermediates are chosen from ethanediol, propanediol, butanediol, hexanediol, butanol, ethanol, lactic acid, acetic acid, formic acid, succinic acid and fumaric acid.

5. Process according to claim 4, in which said chemical intermediates are chosen from 1,4- butanediol and ethanol.

6. Process according to one or more of claims 1-5, including the steps of: a) hydrolysing the starch into said by-product in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibre, protein and / or oligopeptides; b) fermenting said aqueous mixture obtained from step a) in the presence of at least one microorganism capable of producing ethanol, resulting in a fermentation broth comprising ethanol, fibre, protein and / or oligopeptides; c) separating ethanol from said fermentation broth, resulting in a first residue comprising fibre, protein and / or oligopeptides; d) subjecting at least one part of the proteins and / or oligopeptides present in said first residue to protein and / or oligopeptide hydrolysis, resulting in a second residue comprising hydrolysed proteins and / or oligopeptides and fibre; e) subjecting said second residue comprising hydrolysed proteins and / or oligopeptides to solid / liquid separation, resulting in: e)i. a solid fraction, including fibre, and e)ii. a liquid fraction comprising a protein hydrolysate including oligopeptides and free amino acids.

7. Process according to one or more of claims 1-6, in which said hydrolysis in step a) is carried out in the presence of a content of from 5 to 50% dry weight of said by-product relative to the weight of the aqueous suspension.

8. Process according to one or more of claims 1-7, in which said step a) is carried out in the presence of at least one amylase enzyme, preferably chosen from alpha-amylase, glucoamylase (or amyloglucosidase or gamma-amylase) and mixtures thereof.

9. Process according to one or more of claims 1-8, in which said hydrolysis step a) is preceded or followed by an additional hydrolysis step a2) in the presence of at least one enzyme capable of hydrolysing cellulose or hemicellulose fibres.

10. Process according to one or more of claims 1-9, in which said hydrolysis step a), and / or said fermentation in step b) and / or said protein and / or oligopeptide hydrolysis are carried out in the presence of antimicrobial agents chosen from: antibiotics, C2-C10 chain fattyacids, hydroxy acids, parabens, triazides, benzalkonium chloride, and quaternary ammonium salts.

11. Process according to claim 10, in which said antimicrobial agent is lactic acid.

12. Process according to one or more of claims 1-11, in which the microorganism in step b) is chosen from microorganisms belonging to the genera: Saccharomyces, Zygosaccharomyces, Candida, Hansenula, Kluyveromyces, Debaromyces, Nadsonias, Lipomyces, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, Trigonopsis, Brettanomyces, Cryptococcus, Trichosporon, Aureobasidium, Lipomyces, Phaffia, Rhodotorula, Yarrowia, Schwanniomyces.

13. Process according to claim 12, in which the microorganism in step b) is Saccharomyces cerevisiae.

14. Process according to one or more of claims 1-13, in which said step c) comprises one or more operations chosen from distillation, centrifuging, extraction and evaporation.

15. Process according to one or more of claims 1-14, in which the solid-liquid separation step a3) is not performed and the first residue comprising fibre, proteins and / or oligopeptides obtained at the end of step c) is subjected to a step c2) comprising one or more separation operations selected from centrifuging, filtration and settling, yielding c2)i. a liquid fraction including salts, and c2)ii. a solid fraction including fibre, proteins, and / or oligopeptides; in which said solid fraction c2)ii comprising fibre, proteins and / or oligopeptides is fed to step d) of protein and / or oligopeptide hydrolysis.

16. Process according to claim 15, in which said liquid fraction c2)i comprises soluble fibre and soluble proteins that are separated by one or more operations, chosen from adsorption, ion exchange, reverse osmosis, microfiltration, ultrafiltration or nanofiltration.

17. Process according to one or more of claims 1-16, in which hydrolysis of the proteins and / or oligopeptides in step d) is carried out in the presence of at least one protease, preferably an acid protease.

18. Process according to one or more of claims 1-17, in which solid / liquid separation step e) comprises one or more operations chosen from settling, centrifuging, filtration, microfiltration, nanofiltration, ultrafiltration, ion exchange, osmosis, and combinations thereof.

19. Process according to one or more of claims 1-18, comprising the steps of:f) subjecting said solid fraction e)i to one or more physical, chemical and / or biochemical treatments obtaining a third residue comprising fibre, including arabinoxylans. g) separating the third residue obtained in step f), obtaining: g)i. a liquid fraction comprising soluble fibre, including arabinoxylans and g)ii. a solid fraction.

20. Process according to claim 19 in which the treatment in said step f) comprises one or more operations chosen from enzyme hydrolysis, extraction and hydrothermal treatment.

21. Process according to claim 20 in which said enzyme hydrolysis is carried out using enzymes belonging to the class of hemicellulases, of bacterial or fungal origin, chosen from xylanase, arabinase, and mixtures thereof.

22. Process according to one or more of claims 1-21, further comprising a step h) of subjecting said liquid fraction e)ii comprising a protein hydrolysate to purification, in which a liquid fraction comprising a purified protein hydrolysate is obtained.

23. Process according to any one or more of claims 1-22, further comprising a step j) of subjecting said liquid fraction comprising protein hydrolysate or said purified protein hydrolysate to concentration, resulting in a concentrated protein hydrolysate.

24. Protein hydrolysate, obtainable as a component of liquid fraction e)ii according to one or more of claims 1-21 or as purified protein hydrolysate according to claim 22 or as concentrated protein hydrolysate according to claim 23, comprising more than 40% by weight of proteins, below 35% by weight of soluble fibres, less than 10% by weight of monosaccharide, less than or equal to 10% by weight, advantageously from 0.1% to 7.9% by weight of ashes, relative to the dry weight of the protein hydrolysate.

25. Use of protein hydrolysate of claim 24 or obtained according to one or more of claims 1- 23 as a plant biostimulant and / or fertilizer and / or biopesticide.

26. Plant biostimulant comprising protein hydrolysate according to claim 24 or obtained according to one or more of claims 1-23.

27. Composition, obtainable as a solid fraction e)i in step e) of the process according to the invention, essentially starch-free and comprising at least 40% by weight of cellulose and hemicellulose and having a lignin content of less than 15% by weight and / or a lipid content of less than 5% by weight relative to dry weight of the composition.

28. Soluble fibre composition, obtainable as a liquid fraction g)i according to one or more of claims 19-21, comprising arabinoxylans, containing more than 50% by weight of carbohydrates and having a soluble fibre content of more than 20% of the total dry weightrelative to the dry weight of said composition, wherein said arabinoxylans constitute at least 30% by weight with respect to the total soluble fibre.

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