Process for utilising secondary products from the agri-food industry

Abstract

The present invention relates to a process for the utilisation of a secondary product of edible flour processing, in which a purified solution comprising monosaccharides is obtained by hydrolysis of the starch component of said waste product, and a solid residue, rich in insoluble fibres and proteins and / or oligopeptides, and a liquid fraction, rich in soluble proteins and fibres, are separated out.

Description

[0001] PROCESS FOR UTILISING SECONDARY PRODUCTS FROM THE AGRI-FOOD INDUSTRY

[0002] The present invention relates to a process that, by using secondary products from the agri-food industry, such as waste biomass from the processing of edible flours, as raw material, makes it possible to obtain a variety of organic compounds with high added value that can be utilised through reuse in the development of industrial applications.

[0003] In fact, this process makes it possible both to extract, separate and recover second-generation sugars suitable for use in chemical and fermentation processes from the structural carbohydrates present in these biomasses, and to optimise the extraction of fibres and proteins that can be widely used in the animal feed or food industries.

[0004] Sugars referred to as 'second-generation' sugars are sugars that can be obtained from biomass waste from the agri-food industry, thus without taking food resources away from humans.

[0005] Through various chemical, physico / chemical and / or enzyme treatments, these biomasses can in fact be broken down into their main components (starch, cellulose, hemicellulose, lignin and proteins), obtaining simple sugars from the polysaccharide-rich components for use as raw materials for the production of chemical compounds for the development of final applications (such as fuels like ethanol, polymers like PHA, monomers like 1,4 BDO, etc.).

[0006] The quality and composition of second-generation sugars govern how possible it is to use these sugars as a carbon source in a culture medium for the growth and production of different microorganisms such as bacteria, fungi or yeasts (possibly genetically modified) in fermentation processes.

[0007] In fermentation processes, the culture medium acts as a medium for the growth of microorganisms, providing the essential elements for proper growth and the production of metabolites. For example, carbon is the main element in the composition of cells and is also used in compounds as a source of energy. However, the ability of microorganisms to utilise certain carbon compounds varies depending on the species and strain. Some microorganisms preferentially consume simple saccharides with six carbon atoms such as glucose and fructose; this is the case, for example, of the microorganisms used to produce bio-butanediol. Disaccharides are metabolised by a smaller number of microorganisms; see, for example, patent application WO 2015 / 158716, which describes a process for the production of 1,4-butanediol comprising fermentation by a microorganism with at least one metabolic pathway for the synthesis of 1,4-butanediol in a culture medium comprising a mixture of glucose and sucrose. By contrast, complex pentoses and saccharides are only used by a minority of microorganisms. Furthermore, in the particular case of cereals (including pseudocereals) and leguminous crops, the milling industry, which as is well known is directed towards the production of edible flours as a primary product, produces intermediate products and secondary products that 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, from the stages in processing. These intermediates and secondary products, which are rich in protein and / or fibre, are therefore suitable for use as ingredients and / or semi-finished products in food and nutraceutical products. They do, however, require further processing steps to remove unwanted elements such as residual starch, salts, oils and fatty acids, which are present in varying quantities depending on the starting product (cereal, seed, legume) and the degree and type of milling.

[0008] On the other hand, the application of second-generation saccharides in fermentation also requires a degree of purity high enough not to inhibit the growth of microorganisms and not to interfere with the processes of fermentation and purification of the compounds produced. It is well known that impurities resulting from the production processes of second- generation sugars, such as organic acids, high molecular weight molecules and high concentrations of salts, adversely affect the metabolism of microorganisms and impair their use in fermentation processes.

[0009] On an industrial level, fermentation processes typically require working with substrates that have a limited amount of impurities at the appropriate concentrations so that they can be used without interfering with the production and / or purification process.

[0010] For example, commercially available monosaccharide syrups, obtained directly from maize or potato starch, typically have a nitrogen and ash content of less than 0.05% by weight.

[0011] The present invention overcomes the aforementioned drawbacks by allowing the secondary products of edible flour processing to be completely utilised. Indeed, the process according to the invention allows for complete conversion of the starch present into a highly purified and concentrated monosaccharide syrup, which is therefore suitable for use in fermentation processes and available in high yields due to the possibility that other structural carbohydrates present (cellulose and hemicellulose) can also be converted into monosaccharides. At the same time, the process according to the present invention makes it possible to maximise the recovery of fibres and organic nitrogen compounds (for example amino acids, oligopeptides, proteins, etc.) with improved characteristics and composition due to the reduction in salt and soluble components. All process products are therefore suitable for new high value-added applications in both the chemical and food industries. In particular, the object of the present invention is a process for the utilisation of a secondary product of edible flour processing comprising starch, comprising the steps of:

[0012] (a) subjecting this secondary product to hydrolysis of the starch in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibres, proteins and / or oligopeptides and optionally salts, oils and fatty acids;

[0013] (b) subjecting said aqueous mixture to solid / liquid separation, resulting in a solid fraction comprising insoluble fibres and insoluble proteins and / or oligopeptides and a liquid fraction comprising soluble monosaccharides, fibres and soluble proteins and / or oligopeptides and optionally salts;

[0014] (c) subjecting said liquid fraction from step b) to a purification treatment including:

[0015] (cl) at least one membrane separation operation, removing a soluble protein fraction and obtaining an aqueous solution comprising soluble monosaccharides; and subsequently

[0016] (c2) at least one passage of said aqueous solution through a bed comprising one or more ion exchange resins, resulting in a purified solution comprising monosaccharides.

[0017] The term 'secondary product of edible flour processing' within the meaning of the present invention means a biomass obtained as a secondary product of the milling of cereals, pseudocereals, legumes or mixtures thereof, comprising starch and other structural carbohydrates such as cellulose and / or hemicellulose.

[0018] Said solid fraction obtained in step b) is substantially starch-free and enriched in fibres and organic nitrogen (in the form of proteins and / or oligopeptides), compared to the starting biomass. Said solid fraction can advantageously be subjected to subsequent separation of the protein component from the fibre-rich component.

[0019] According to one aspect of the invention, the process optionally includes a step of drying the solid fraction separated in step b) to facilitate subsequent separation of the protein component from the fibre-rich component.

[0020] According to one aspect of the invention, step c) of the process optionally comprises a step c3) of concentrating the purified solution comprising monosaccharides obtained in step c2).

[0021] Finally, the process according to the invention comprises an optional step of growth of a strain of microorganism capable of producing chemical intermediates and / or polyhydroxyalkanoates in the presence of a carbon source comprising the monosaccharides of the purified aqueous solution obtained from step c2). This growth step is preferably preceded by the concentration step c3) described above. Advantageously, process steps a)-c) are carried out at temperatures not exceeding 100°C, preferably below 80°C and preferably below 60°C, thus preserving the structure and function of the components present.

[0022] Monosaccharides produced by the process according to the present invention are, for example, glucose, fructose, arabinose, rhamnose, galactose, mannose, xylose. The term 'polysaccharides' is understood to include, for example, starch, cellulose, heteropolysaccharides such as hemicellulose (which includes xylans, glucuronoxylans, arabinoxylans, glucomannans, xyloglucans).

[0023] The term 'oligosaccharides' is used in this application to refer to all molecules of a carbohydrate nature consisting of two to ten monosaccharide units. Examples of oligosaccharides thus include disaccharides such as sucrose, maltose and cellobiose, and some maltodextrins.

[0024] The process will be described in more detail below with reference to the block diagram in Figure 1.

[0025] This secondary product of the processing of edible flours comprising starch, fed at step a) of the process, is derived from one or more cereals, selected for example from grain or wheat, rice, barley, oats, spelt, rye, millet, maize; from one or more pseudo-cereals, selected for example from amaranth, buckwheat, quinoa and chia; from one or more legumes, selected for example from peas, chickpeas, lentils, beans, broad beans; or from mixtures thereof.

[0026] The biomass obtained as a secondary product from milling of the plant species listed above mainly consists of fibres (cellulose, hemicellulose and lignin), vitamins, proteins, enzymes, mineral salts, lipids, and includes residual amounts of starch that vary depending on the type of processing and the process step from which they originate.

[0027] 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 by dry weight in the case of cereals and pseudocereals and preferably less than 40%, more preferably less than 30% by weight by dry weight in the case of legumes. The starch content of said biomass advantageously is greater than 5%, 10%, 15% or 20% by weight relative to the dry weight of the biomass.

[0028] The total fibre content (soluble and insoluble, excluding starch) of said biomass is more than 20 % by weight; a fibre content of 25 % or more, 30 % or more, 40 % or more by dry weight of said biomass is particularly preferred.

[0029] The protein content of this biomass is preferably 15% or more by weight, more preferably 20% or more,

[0030] These biomasses have a lipid content of advantageously less than 5%, preferably less than 2% and more preferably less than 1.5% by weight by dry weight; according to a particularly advantageous aspect, the lipid content is less than or equal to 1% or 0.5% by weight relative to the dry weight.

[0031] The use of cereal biomass, preferably wheat, is particularly advantageous.

[0032] As is well known, the process of milling cereals has the purpose of separating the endosperm, which constitutes the starchy and preponderant part of the caryopsis, from the bran and the germ, to obtain flours (flour or semolina, depending on grain size) from the endosperm. This process typically involves alternating stages of milling (involving 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 (that is bran, possibly with the germ).

[0033] Milling may be preceded by one or more stages of dehulling, which consists of removing the outermost layers of the caryopsis, by means of an abrasive action that may be more or less intense, generally without significantly damaging the endosperm-rich core intended for milling. The bran and / or germ fractions separated during the milling process thus constitute secondary products of edible flour processing suitable for feeding to process step a) according to the present invention.

[0034] According to a preferred aspect, these secondary products of edible flour processing consist of the bran (possibly in its finer variant called middlings) optionally including the germ, that is from a fraction of the residual caryopsis after removal of the endosperm.

[0035] According to another preferred aspect, these secondary products of edible flour processing include middlings, finer middlings and groats, that is products richer in starch than bran but still not usable in breadmaking processes. These can be used as such or mixed with bran and / or middlings.

[0036] Outer and inner bran, for example, have a starch content of between 15 and 30% by weight relative to the dry weight of the biomass, protein and oligopeptides of between 15 and 30%, hemicellulose of between 20 and 30%, cellulose of between 10 and 15%, lignin of less than 10%, and ash of less than 10%.

[0037] The amount of protein and oligopeptides present can be derived by multiplying the organic nitrogen present by an appropriate correction factor.

[0038] On the other hand, fine middlings, middlings and groats typically have a starch content of between 50 and 80% by weight, a protein content of between 10 and 30%, and a hemicelluloses content of between 3 and 10% by weight relative to the dry weight of the biomass.

[0039] The process according to the invention may optionally comprise a preliminary step of treating said biomass, whether obtained as a secondary product of 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, this preliminary stage or pre-treatment advantageously includes heat 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 aimed at reducing the lipid content of the biomass or reducing the fibre content, for example through hydrolysis mediated by enzymes such as cellulases and hemicellulases.

[0040] According to a preferred embodiment, the process of the present invention therefore comprises, before step a), a step of defatting the said secondary product from the processing of edible flours comprising starch, for example by extraction of the lipid component in one or more organic solvents or supercritical fluids (such as CO2) or by physical extraction.

[0041] Suitable organic solvents are for example acetone, ethanol, butanol, hexane, tetrahydrofuran, 2-methyltetrahydrofuran (2-MeTHF), cyclopentyl methyl ether, cyclopentanone. Among them, ethanol and hexane are preferred; ethanol is even more preferred.

[0042] Physical extraction is advantageously performed e.g. by cold pressure, instantaneous controlled pressure drop (DIC), pulsed electric fields (PEF), and microwave irradiation (MW)).

[0043] A solvent-free-extraction or a green-solvent extraction are preferred.

[0044] According to a preferred aspect, the biomass subjected to step a) is in the form of pellets and comprises an amount of dust lower than 50% by weight, preferably between 42% and 22% by weight. It advantageously has a water content of approximately 10% by weight.

[0045] During step a) of the process according to the invention, said biomass is subjected to a reaction of hydrolysis (or saccharification) of the polysaccharide chains of the starch to obtain monosaccharides, by means of one or more enzymes belonging to the family of hydrolases and suitable for hydrolysing starch such as amylases. Other hydrolytic enzymes such as cellulases and hemicellulases can be used, before step a), during step a) or during a subsequent treatment of the solid residue separated in step b), in order to maximise the conversion to monosaccharides by also hydrolysing the polysaccharide chains of cellulose and hemicellulose. Amylase and cellulase are preferred.

[0046] These enzymes can be used individually or in mixtures and can be added to the starting biomass at different times depending on the desired hydrolysis product, with the appropriate operating conditions being adopted for each enzyme.

[0047] 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.). Enzymes that make it possible to obtain mixtures of monosaccharides comprising mainly glucose are particularly suitable for use in step a) of the process. These enzymes belong advantageously to the amylase class and are preferably selected from alpha-amylase, glucoamylase (or amyloglucosidase or gamma- amylase) and their mixtures. Examples of suitable commercial products are 'Spezyme Alpha PF' and 'Optidex L-400', supplied by IFF.

[0048] The use of at least one alpha-amylase and at least one glucoamylase or a mixture thereof is preferred.

[0049] The hydrolysis operation in said step a) is carried out in the presence of water, preferably keeping the biomass (that is the secondary product of edible flour processing including starch) in an aqueous suspension.

[0050] According to a preferred aspect, this is carried out by feeding said biomass with an initial dry weight of at least 5%, preferably at least 10%, more preferably at least 20% by weight relative to the volume of the aqueous suspension.

[0051] According to another preferred aspect, this 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) of, for example, between 5% and 50% in the hydrolysis reactor, preferably 40% by weight with respect to the volume of the aqueous suspension, advantageously between 7% and 20% by weight with respect to the volume of the aqueous suspension.

[0052] During the hydrolysis reaction, the pH value is chosen according to the enzymes used, for example by adding mineral acids such as sulfuric acid, as well as temperature. The pH and temperature conditions can be kept constant or modulated during the process, depending on specific requirements.

[0053] For example, according to a preferred embodiment, the reaction is carried out in the presence of amylase and the pH is advantageously kept between 3.5 and 7 (more preferably between 4.5 and 5.5), while the temperature during the reaction is preferably kept at 30° to 130°C, more preferably between 40° and 70°C, more preferably between 50° and 60°C.

[0054] The hydrolysis reaction has a variable duration depending on the conditions used, in particular the type and concentration of enzymes. Advantageously it is between 0.1 and 120 hours, for example between 0.5 and 48 hours, more advantageously between 1 and 6 hours.

[0055] The hydrolysis step is optionally carried out in the presence of bacteriostatic and / or bactericidal agents capable of containing the growth of contaminating microorganisms. Examples are shortchain fatty acids such as nonanoic acid, hydroxy acids (for example lactic acid, citric acid), parabens, benzalkonium chloride, quaternary ammonium salts. Preferred examples are pelargonic acid, lactic acid, citric acid. Alternatively, the hydrolysis in step a) can be achieved chemically and / or physically, for example using mineral acids such as HC1 and H2SO4, or solid acids such as sulfonated organic resins, even in the absence of enzymes.

[0056] The hydrolysis in step a) can be carried out through a continuous or semi-continuous process or, alternatively, in batch mode.

[0057] During step b) of the process, the mixture obtained at the end of the hydrolysis step is subjected to at least one solid / liquid separation operation during which a liquid fraction containing the sugars and soluble components is removed from a solid fraction containing the insoluble components, including fibres, proteins and / or oligopeptides. Advantageously, one or more separation operations are performed in series.

[0058] This separation operation in step b) is carried out according to techniques known to those skilled in the art, and for example includes one or more operations selected from pressing, settling, sedimentation, centrifuging, filtration, or any other suitable technique for solid-liquid separation, and combinations thereof.

[0059] The separation operation in step b) can therefore be performed by one or more devices capable of separating, optionally by compression, a solid fraction and a liquid fraction. Examples of suitable devices are a decanter, a settler, a filter press, a hydrocyclone, a belt filter, a rotary filter, a centrifuge.

[0060] In addition, activated carbon and diatomaceous earth are optionally used to aid separation.

[0061] According to a preferred aspect of the invention, separation of the liquid fraction takes place via a belt filter, advantageously equipped with a backwash system.

[0062] According to another aspect, separation of the liquid fraction takes place via a decanter.

[0063] One or more aliquots of the liquid fraction separated by the solid / liquid separation operation in step b) can be conveniently recycled to step a), with the dual advantage of recovering an important fraction of still-active enzymes and increasing the concentration of saccharides in the liquid phase, with considerable benefits in terms of both water consumption and equipment size.

[0064] In a preferred form of the process according to the invention, in step b) the mixture undergoes a first separation of an insoluble solid fraction and a liquid fraction. The liquid fraction thus obtained can be treated again by an additional solid-liquid separation operation to recover further solid fraction which can be combined with the former.

[0065] The separation operation described in step b) may optionally be followed by a step of washing the solid fraction of the hydrolysed mixture with one or more solvents to facilitate the recovery of soluble monosaccharides and at the same time mainly remove impurities (for example salts, soluble proteins or oligopeptides, etc.). This washing step can advantageously be performed using water, more advantageously by performing several successive washes in series, even more advantageously by counter current washing and / or by backwashing. The wash waters can be collected, optionally concentrated and combined with the previously separated liquid fraction.

[0066] The solid fraction obtained at the end of step b) mainly consists of insoluble material including insoluble fibres, proteins and / or oligopeptides, may further contain for example oils, fatty acids and preferably has a water content of less than 85%, more preferably less than 80%, more preferably less than 75%. The extremely low starch and ash content (starch, where present, preferably less than 5%, more preferably less than or equal to 3%, less than or equal to 2%, even more preferably less than or equal to 1% by weight in relation to the dry weight; ash preferably less than 5%, more preferably less than or equal to 3% by weight in relation to the dry weight), make said solid fraction particularly suitable for applications in the food industry. Starch content can be determined using the method in "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).

[0067] Ash (for example silicates, chlorides, bromides, nitrates, sulfates, phosphates, sodium, potassium) are determined for example by means of the method "Determination of Ash 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. and Templeton, D.; Technical Report NREL / TP-510-42622, 2008) by changing the holding time for the temperature of 575°C to at least 5 hours.

[0068] Where, for example, secondary products of the processing of cereals such as wheat are subjected to the process according to the invention, this solid fraction advantageously comprises at least 45% by weight, preferably at least 50% by weight of polysaccharides (consisting essentially of hemicellulose and cellulose) and at least 18% by weight, preferably at least 20% by weight of proteins (including oligopeptides and free amino acids) relative to its dry weight. At least 50% by weight of the polysaccharides in the said solid fraction are advantageously hemicelluloses and even more advantageously consist of arabinoxylans.

[0069] The said solid fraction advantageously comprises at least 30% by weight of hemicelluloses, wherein said hemicelluloses preferably consist of arabinoxylans. Cellulose and hemicellulose can be quantified, for example, by means of 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.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D: Technical Report NREL / TP-510-42618, 2012). In particular, the amount of cellulose is determined by subtracting the amount of starch from the total amount of glucans measured.

[0070] The total protein content in the solid fraction is determined by analysing the total nitrogen according to the Kjeldahl method and then multiplying the value obtained by a conversion factor defined according to the nature of the biomass analysed.

[0071] The invention thus also relates to a composition prepared from a secondary product of the processing of edible flours from cereals, pseudocereals and / or legumes, essentially free of starch and comprising, with respect to the dry weight of said composition, at least 45% by weight of polysaccharides (of which at least 50% by weight are hemicelluloses and preferably consists of arabinoxylans), at least 18% by weight of proteins (including oligopeptides and free amino acids) and preferably less than 5% ash, more preferably less than or equal to 3% by weight.

[0072] According to a preferred aspect of the process, this solid fraction is recovered and advantageously subjected to one or more subsequent concentration and / or drying steps.

[0073] Such concentration and / or drying may involve one or more stages of liquid removal, for example using presses, such as screw presses, with the dual advantage of increasing the recovery of the liquid phase and reducing the volume of the solid fraction subjected to subsequent treatment.

[0074] Such drying is advantageously carried out using techniques known in the industry. The operation can be conducted in batches 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 disposable flow or recirculation of the drying gases.

[0075] Conveniently, the operation is conducted by choosing a drying system that does not damage the solids while preserving in particular the structure of the protein component. Especially advantageous are systems that lead to drying by minimising the time spent at high temperature and possibly maintaining relatively low temperatures (for example ring dryers, flash dryers). Such drying is advantageously followed by milling and separation operations (for example sieving operations, air classification) in order to facilitate separation of the components. A particularly advantageous aspect of the present invention is the possibility of further valorise the solid fraction obtained in step b) of the process. For example, since it is substantially devoid of starch, it is particularly easy to separate said solid fraction into a composition rich in protein and a composition rich in fibre, both of which have high nutritional value and can be used as food additives, for both human and animal nutrition. The protein component can also find application in agriculture, for example, after hydrolysis, as a biostimulant.

[0076] The polysaccharide fibres present in the solid fraction obtained from step b) of the process (for example cellulose, hemicellulose) have the advantage that they can be easily separated by physical or chemical treatments or combinations thereof known to those skilled in the art. For example, they can be advantageously separated by extraction, for example in an alkaline environment and / or in the presence of organic solvents (for example DMSO, imidazole, alcohols such as ethanol, propanol, t-butyl alcohol), or by hydrothermal treatments, also assisted by, for example, ultrasound or microwaves.

[0077] Such processes are typically conducted under conditions that result in degradation of the sugars present. Thus, one of the unexpected advantages of the present invention lies in the possibility of easily recovering high value plant fibres such as arabinoxylans from the composition constituting the solid fraction recovered from step b), while minimising the loss of soluble sugars, which are previously separated in the liquid fraction. For example, according to a preferred embodiment, the process according to the invention comprises the further steps of: i. subjecting the solid fraction separated in step b) to 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 ii. hydrolysing one or more of the polysaccharide chains thus separated to reduce their molecular weight to oligosaccharides or to break them down completely into monosaccharides.

[0078] Hydrolysates rich in arabinoxylans in particular may be obtained from hemicellulose; these have many positive health-promoting effects and can therefore be used in the food and nutraceutical industry for the production of food and beverages. Hydrolysis is advantageously carried out, for example, by means of enzymes, with commercial enzymes (such as xylanase and arabinase) and under conditions known to those skilled in the art.

[0079] According to an alternative aspect of the process, said solid fraction is directly subjected to an additional step of hydrolysis of the polysaccharide chains of cellulose and / or hemicellulose, as mentioned above for step a) of the process, adapting the mode of hydrolysis to the desired product. Such hydrolysis also favours the release of any proteins from the solid fraction. By subjecting the hydrolysis product thus obtained to a subsequent solid / liquid separation operation, for example according to methods chosen from those described above for step b), it is possible to increase the recovery yield of monosaccharides and possible soluble proteins more or less selectively.

[0080] Consequently, another object of the present invention is a process for the utilisation of a secondary product of edible flour processing comprising starch, the said process comprising the steps of:

[0081] (a) subjecting this secondary product to hydrolysis of the starch in the presence of water and preferably one or more enzymes, resulting in an aqueous mixture comprising monosaccharides, fibres, proteins and / or oligopeptides and optionally salts, oils and fatty acids;

[0082] (b) subjecting said aqueous mixture to solid / liquid separation, resulting in a solid fraction comprising insoluble fibres and insoluble proteins and / or oligopeptides and a liquid fraction comprising soluble monosaccharides, fibres and soluble proteins and / or oligopeptides and optionally salts; and wherein the said solid fraction is further subjected to at least a partial hydrolysis of the polysaccharidic chains of cellulose and / or hemicellulose contained therein.

[0083] The said further hydrolysis can be performed on one or more of the polysaccharide chains of cellulose and / or hemicellulose and can be advantageously preceded by a selective separation of the polysaccharide chains of hemicellulose from those of cellulose, e.g. as described above according to steps i. and ii.. According to a preferred aspect, the said further hydrolysis it is preferably performed by means of xylanases.

[0084] In process step c), the liquid fraction from step b) undergoes a purification treatment comprising at least one membrane separation operation (cl).

[0085] This membrane separation is , for example, selected from filtration (for example microfiltration and / or ultrafiltration and / or nanofiltration), reverse osmosis and electrodialysis.

[0086] Preferably this separation in step cl) includes at least one filtration operation selected from ultrafiltration and nanofiltration.

[0087] Depending on the characteristics of the liquid fraction subjected to separation operations in step cl) of the process, those skilled in the art are able to select the type of membrane used, taking into consideration the material it is made of, its electrochemical properties and its porosity.

[0088] The type of membrane chosen will determine the pressures and other optimum operating conditions. Those skilled in the art will be able to assess the plant conditions to ensure product yield and quality, defining whether the operation should be conducted in batch or continuous mode, whether and how much diafiltration is required (that is dilution of the retentate by adding water and repeating the separation operation), achievable concentration, and species rejection. For example, the separation in step cl) is effectively performed using both organic membranes, either of natural origin (for example rubbers, polysaccharides) or synthetic origin (for example polymer membranes), and inorganic membranes, such as ceramic, metal or glass membranes. Of the organic membranes, those preferred are polyamides, polyimides, poly alkylenes, polyether imide, polyarene ether, poly(ether ketone), polycarbonates, cellulose acetate and derivatives.

[0089] Specific examples of suitable organic membranes are polysulfones, polyamides, polypyperazine amides, polyethylene, polytetrafluoroethylene (PTFE), polypropylene, polyvinyl alcohol, polystyrene, polybenzimidazoles (PBI), polyphenylenes, polyphosphazenes, polyvinylidene fluoride (PVDF), polyether sulfones (PES), polyacrylonitrile (PAN), polyvinyl chloride (PVC).

[0090] Both isotropic (or symmetrical) and anisotropic (or asymmetrical) membranes and composite membranes are suitable.

[0091] Preferably, anisotropic membranes are used.

[0092] Membranes can be formed in different configurations, for example in 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 ratio of surface area / volume occupied.

[0093] Membrane separation operations according to the invention may be carried out in batch or continuous mode; depending on the case, a normal (perpendicular) or tangential flow filtration method respectively is preferably used.

[0094] Membrane separation operations under tangential flow are preferred.

[0095] Nanofiltration operations are preferably carried out according to the invention using membranes made of material selected from the group consisting of: polysulfones, polypiperazine amide, polyamide, polyimide.

[0096] The main purpose of step cl) is to obtain a purified sugar solution (that is an aqueous solution of soluble monosaccharides) and, secondarily, a soluble protein fraction.

[0097] Several separation operations with membranes having various cut-offs can be conveniently combined to produce one or more of the flows described above.

[0098] A particularly advantageous set-up involves the use of a first nanofiltration membrane with a 600-800 Da cut-off. The resulting permeate is a purified sugar solution, while the retentate undergoes a second ultrafiltration treatment (for example, a 2-10 kDa cut-off). This treatment separates a permeate containing salts and other low molecular weight compounds from the retentate (comprising a concentrated solution of proteins and soluble fibres).

[0099] The soluble protein fraction obtained from step cl) has a high organic nitrogen content in the form of proteins, oligopeptides and / or simple amino acids and a low ash content. It is therefore suitable for use in the food sector. It advantageously comprises at least 10% protein by weight of its dry weight, preferably at least 15% by weight, more preferably at least 20% by weight and even more preferably 50% or more by weight of protein. Examples of proteins are albumins and globulins. The soluble protein content may be determined, for example, by the determination of total nitrogen, obtained according to the Kjeldahl method as mentioned above or by means of special analysers. The free amino acids content is preferably of 5% or less by weight relative to its dry weight.

[0100] It also advantageously contains soluble oligosaccharides, such as xylooligosaccharides and arabinooligosaccharides, preferably in amounts of at least 10% by weight relative to dry weight, which characteristically have prebiotic activity and can therefore be used in the food and nutraceutical industry. According to a preferred aspect, the said 10% by weigh consists of arabinoxylans.

[0101] A further advantage of the present invention is therefore the possibility of further utilising the soluble protein fraction obtained as a filtration retentate, in particular as a nanofiltration retentate, in step cl).

[0102] For instance, it may be subjected to further processes of hydrolysis of the saccharide components that may be present; it may be subjected to membrane processes, to separate a fraction rich in soluble arabinoxylans and / or further concentrate the protein content; it may also be conveniently dried by conventional methods, for example by pre-concentration followed by spray drying.

[0103] The membrane separation operations described above allow a soluble protein fraction to be removed during step cl), resulting in an aqueous solution containing sugars.

[0104] Subsequently, the resulting aqueous solution preferably undergoes at least one passage through a bed comprising one or more ion exchange resins (step c2), aimed at removing residual impurities, including organic molecules, salts and / or amino acids still present.

[0105] Depending on the characteristics of the liquid solution entering step c2) of the process, those skilled in the art will be able to select the type of resin to be used, taking into consideration the material it is made of, its electrochemical properties, its porosity, its selectivity, stability and its adsorption capacity.

[0106] These ion exchange resins may be of the anionic or cationic type. Strong cationic and anionic resins are preferred. Typical commercial products are Dowex 88 and Dowex 77. A favoured arrangement is a sequential pass over cationic-anionic resins, possibly followed by a subsequent sequential pass over cationic-anionic resins conducted in series, so as to maximise resin utilisation yields.

[0107] Treatment with resins can be conducted in batch operations, or more conveniently with continuous processes, for example with a carousel or simulated moving bed.

[0108] This purification step c), in particular separation cl), allows a number of compounds and impurities to be removed from the aqueous solution comprising soluble monosaccharides, resulting in a purified sugar solution (that is, comprising monosaccharides).

[0109] Once concentrated, this purified solution comprising monosaccharides has a level of purity suitable for use in fermentation processes to produce biochemicals such as 1,4-BDO.

[0110] Each process step according to the present invention may advantageously be carried out independently in batch mode, in continuous mode or in semi-continuous mode. According to a preferred embodiment, each of the steps a)-c) is carried out in continuous mode.

[0111] According to a preferred embodiment, the process according to the invention comprises an optional step c3) of concentrating said purified solution comprising monosaccharides.

[0112] This concentration step is preferably carried out by one or more operations known in the industry, selected for example from adsorption, reverse osmosis, crystallisation, evaporation, distillation. Multi-effect evaporators, potentially with mechanical or thermal recompression, and low residence time devices such as falling or scraped films are particularly suitable.

[0113] In a particularly advantageous aspect, a syrup of monosaccharides, preferably monosaccharides with 6 carbon atoms, is obtained, and is suitable for use as such in fermentation processes as a carbon source for the growth of microorganisms.

[0114] The purified solution obtained at the end of step c2) or step c3) advantageously has an ash content of less than 0.1% by weight, preferably 0.005% to 0.05% by weight in relation to the dry weight, and a total nitrogen content of preferably less than 0.1% by weight, more preferably 0.005% to 0.05% by weight in relation to the dry weight. The conductibility is advantageously below 25 microS / cm a 25°C, it is measured for example on a purified solution with an average monosaccharide concentration of 35 g / L using a probe suitable for reading conductivity in the range 0.001-500 pS / cm, such as Inlab 741-ISM (Mettler Toledo).

[0115] Ash content can be quantified for example using the NREL method mentioned above, by changing the holding time for the temperature of 575 °C to at least 5 hours. Total nitrogen in the liquid samples may be determined, for example, by using an analyser such as the TOC-L (Shimadzu) instrument, diluting the sample appropriately to comply with calibration and adjustment limits.

[0116] A further object of the present invention is therefore a monosaccharide composition, preferably C6, obtained as a secondary product of the milling of cereals, pseudocereals and / or legumes, preferably having an overall monosaccharide content of 80% or more, more preferably greater than or equal to 85% by weight in relation to the dry weight of the composition, an ash content of less than 0.1% by weight, preferably from 0.005% to 0.05% by weight in relation to the dry weight, and a total nitrogen content of preferably less than 0.1% by weight, more preferably from 0.005, more preferably from 0.01, to 0.05% by weight in relation to the dry weight.

[0117] This composition preferably comprises up to 19%, more preferably up to 15%, even more preferably up to 10% by weight relative to the dry content of oligosaccharides, advantageously in the form of disaccharides or trisaccharides. More advantageously, this composition preferably comprises up to 3.0%, more preferably up to 2.5%, even more preferably up to 2.0% by weight of disaccharides or trisaccharides, relative to the dry content.

[0118] The said composition has a kinematic viscosity, measured at a monosaccharide concentration of 700 g / L, of 30 cSt or less, preferably 26 cSt or less and advantageously from 10 to 25 cSt.This composition can be obtained through the process according to the present application, for example by feeding the by-product of milling cereals, pseudocereals and / or legumes.

[0119] The glucose : fructose ratio in this composition is preferably of above 4 : 1, more preferably of 5 : 1 or above, even more preferably of 6 : 1 or above.

[0120] According to a preferred aspect, said monosaccharide composition has a glucose content of more than 70% and a fructose content of between 10-15% by weight relative to the dry weight of the composition; arabinose and / or xylose are preferably present. Said composition can be obtained by the process according to the present application, using the secondary product of milling cereals, for example wheat, as the starting biomass.

[0121] The fructose in the syrup obtained may optionally be converted into glucose, using techniques known to those skilled in the art, according to the needs of the final application.

[0122] The invention also relates to use of the monosaccharide composition obtained from the process and use of the monosaccharide composition described above as a carbon source for the growth of a strain of microorganism capable of producing chemical intermediates and / or polyhydroxy alkanoates .

[0123] This composition may be used as such or after one or more further treatments aimed, for example, at concentrating the monosaccharides. It may also be used as a sole carbon source or in mixtures with other first- generation sugars. In the case of mixtures, these may comprise from 1 to 99% by weight, preferably from 10 to 80%, more preferably from 15 to 65% by weight of monosaccharides from the monosaccharide composition described above.

[0124] A preferred example is the use as a carbon source for the growth of a strain of microorganism capable of producing 1,4-butanediol (1,4-BDO). According to a preferred form of use, the purified monosaccharide composition described above is used as a component of a culture medium in a fermentation process carried out in the presence of one or more microorganisms with at least one metabolic pathway for the synthesis of 1,4-BDO.

[0125] This fermentation may for example be carried out as a further process step according to the invention using the purified solution obtained in step c2) or optional step c3).

[0126] The sugars supplied to said microorganisms for the production of 1,4-BDO may all be second generation sugars resulting from the saccharification of structural carbohydrates present in the secondary products of edible flour processing or mixtures of these with first generation sugars characterised by a high level of purity. In the case of mixtures, these may comprise from 1 to 99% by weight, preferably from 15 to 65% by weight with respect to total sugars of sugars derived from saccharification of the secondary products of edible flour processing.

[0127] The culture medium may include other substances necessary for growth and sustenance of the microorganism during the fermentation stage, 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, mineral salts, yeast extracts, and trace elements such as Cobalt, Calcium and Copper. Cobalt, Calcium and Copper can be dosed into the culture medium, for example, as salts such as cobalt chloride, calcium chloride and copper chloride. Generally, the culture medium includes at least one sugar, usually glucose and optionally one or more sugars other than glucose, in concentrations between 10 and 100 g / L. As the microorganism consumes one or more sugars during the fermentation phase, it may be necessary to reintroduce these sugars into a fermentation reactor. This reintroduction may be carried out continuously or discontinuously, in a manner known to those skilled in the art.

[0128] To limit the content of unused sugars and thus optimise the economy of the process, the supply of one or more sugars is advantageously interrupted or gradually decreased before the end of fermentation. With regard to other components of the culture medium, the culture medium generally contains salts, essential minerals, and antifoaming agents. The culture medium may be prepared in any way known to those skilled in the art, for example by mixing all its components together or pre-mixing some of them; for example, sugars may be added later. It is also possible to use a commercially available culture medium as a starting point and suitably modify its composition at a later stage, for example when bringing the culture medium into contact with the microorganism having at least one metabolic pathway for the synthesis of 1,4-BDO from a renewable source. During fermentation, the assembly consisting of the microorganism and the culture medium including the sugars is maintained under conditions suitable for exploiting the metabolic pathway for the synthesis of 1,4-BDO from renewable sources. Those skilled in the art will also able to check the progress of the process during fermentation, for example by checking one or more parameters and possibly acting on them to bring the process back to conditions suitable for the production of 1,4-BDO.

[0129] The process according to the invention will now be described according to a non-limiting example.

[0130] EXAMPLES

[0131] Determination of cellulose and hemicellulose content

[0132] 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). In particular, the amount of cellulose is determined by subtracting the amount of starch from the total amount of glucans measured.

[0133] Determination of starch content

[0134] Starch was quantified using 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.

[0135] Determination of ash content

[0136] 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) by changing the holding time for the temperature of 575°C to at least 5 hours.

[0137] Determination of protein content The soluble proteins in liquid samples were determined using the TOC-L instrument (Shimadzu) by diluting appropriately to comply with calibration and adjustment limits and multiplying the value obtained by the grain conversion factor, which is specifically 5.7.

[0138] Total protein in solid samples was determined by analysing total nitrogen content using the Kjeldahl method and multiplying the value obtained by the same conversion factor defined according to the nature of the biomass analysed.

[0139] Insoluble proteins were then determined by the difference between total and soluble proteins.

[0140] Example 1

[0141] A secondary product of the edible wheat flour process underwent process step a) according to the diagram depicted in Figure 1.

[0142] This secondary product, with a water content of 9,7 ± 0.4% by weight, had the composition shown in Table 1:

[0143] Batch enzyme hydrolysis step a) for conversion of the starch present into glucose was carried out by adding water to the biomass to a dry biomass content of 16,5% w / w. The alpha-amylase enzyme 'Spezyme Alpha PF' (5 p E / g dry biomass) and the glucoamylase enzyme 'Optidex E- 400' (5 pL / g dry biomass), both from the supplier IFF, were added to the mixture. Pelargonic acid was added at 0,45 g / g dry biomass resulting in a final slurry of 17,9 Kg total weight.

[0144] The pH was maintained at 5.0 by adding sulfuric acid and the temperature was held at 57 °C for 6 hours.

[0145] Solid / liquid separation step b) was carried out using a centrifuge equipped with fixed angle rotor at 7500 rpm for 10 minutes at 20°C. The liquid filtration fraction of the hydrolysate was recovered and filtered by using 25 m metallic sieve.

[0146] Subsequently, the wet solid was resuspended using water with wash water / dry hydrolysate weight ratio of 4 and finally centrifuged using the same condition reported above. The liquid fraction from the washing was filtered as described above and all the liquid fractions were pooled to pass to step c).

[0147] The solid fraction, consisting mainly of insoluble fibres and proteins, was recovered and dried by oven at 50 °C.

[0148] The recovered solid fraction, dried to a moisture content of 8,6 ± 0.3%, had the composition in Table 2.

[0149] The liquid fraction containing glucose and other soluble substances was purified (step cl)) by nanofiltration with single module 2,79 m2polyamide spiral membrane with a cut-off of 600- 800 Da. During the filtration, the retentate was recirculated and mixed with feeding stream while the permeate was recovered separately (batch configuration). Furthermore, two steps of diafiltration with overall water weight / retentate filtration weight ratio of 2 were performed in order to maximise glucose recovery.

[0150] The diafiltrate retentate, representing the soluble protein fraction, with a water content of 94.3+ 0.1% by weight, was characterised and showed a high content of organic nitrogen in the form of amino acids and / or proteins and / or oligopeptides (24.8+ 0.5% by weight in relation to the dry weight, a glucose content of 27.9 + 0.2% and a fructose content of 3,6 + 0.2% by weight in relation to the dry weight).

[0151] Glucose and fructose content was determined by means of high-pressure liquid chromatography (HPLC) with an RID detector and Biorad Aminex HPX-87H 300mm x 7.8mm column and pre- column (Flow: 0.6 mL / minute; oven temperature: 50°C; detector temperature: 35°C; eluent: 5mM H2SO4).

[0152] Example 2

[0153] A secondary product of the edible wheat flour process underwent process step a) according to the diagram depicted in Figure 1.

[0154] This secondary product, with a water content of 11.3 ± 0.5% by weight, had the composition shown in Table 3:

[0155] Batch enzyme hydrolysis step a) for conversion of the starch present into glucose was carried out by adding water to the biomass to a dry biomass content of 16,5% w / w. The alpha-amylase enzyme 'Spezyme Alpha PF' (5 pL / g dry biomass) and the glucoamylase enzyme 'Optidex L- 400' (5 pL / g dry biomass), both from the supplier IFF, were added to the mixture. The resulting final slurry was of 350,8 Kg total weight.

[0156] The pH was maintained at 5.0 by adding sulfuric acid and the temperature was held at 57 °C for 3 hours.

[0157] Solid / liquid separation step b) was carried out using a belt filter equipped with a washing system to facilitate sugar recovery. This belt filter is a horizontal vacuum system with polypropylene filter cloth with an air permeability of 1850 L / sec / m2. The liquid filtration fraction of the hydrolysate was separated out in the first zone of the belt filter. Subsequently, the wet solid was washed in 3 zones in a counter-current arrangement and a wash water / dry hydrolysate weight ratio of 4 at the inlet to the solid / liquid separation system. Finally, the wet solid underwent a drying process in the last zone. The liquid fraction from the washing and drying zones was pooled with the liquid fraction from the first zone to pass to step c). The solid fraction consisting mainly of insoluble fibres and proteins, was recovered and dried by flash drying.

[0158] The recovered solid fraction, dried to a moisture content of 7.5+ 0.4%, had the composition in Table 4.

[0159] The liquid fraction containing glucose and other soluble substances was purified (step cl)) by nanofiltration with two 8.1 m2modules of polyamide spiral membranes with a cut-off of 600- 800 Da connected in series (total filtration area of 16.2 m2). The filtration was performed in batch configuration, thus recirculating and mixing the retentate with feeding stream, and recovering the permeate.

[0160] The filtration step was followed by four stages of diafiltration of the retentate obtained, arriving at a diafiltration water weight / retentate filtration weight ratio of 4, in order to maximise glucose recovery.

[0161] The diafiltrate retentate, representing the soluble protein fraction, with a water content of 98.4 + 0.1% by weight, was characterised and showed a high content of organic nitrogen in the form of amino acids and / or proteins and / or oligopeptides (73.5 + 0.1% by weight in relation to the dry weight, a glucose content of 10.3 + 0.2% and a fructose content of 2.1 + 0.2% by weight in relation to the dry weight).

[0162] Glucose and fructose content was determined by means of high-pressure liquid chromatography (HPLC) with an RID detector and Biorad Aminex HPX-87H 300mm x 7.8mm column and precolumn (Flow: 0.6 mL / minute; oven temperature: 50°C; detector temperature: 35°C; eluent: 5mM H2SO4).

[0163] Example 3 A secondary product of the edible wheat flour process underwent process step a) according to the diagram depicted in Figure 1.

[0164] This secondary product, with a water content of 10.0 ± 0.9% by weight, had the composition shown in Table 5:

[0165] Batch enzyme hydrolysis step a) for conversion of the starch present into glucose was carried out by adding water to the biomass to a dry biomass content of 16.5% w / w. The alpha-amylase enzyme 'Spezyme Alpha PF' (5 pL / g dry biomass) and the glucoamylase enzyme 'Optidex L- 400' (5 pL / g dry biomass), both from the supplier IFF, were added to the mixture.

[0166] Pelargonic acid was added at 0,45 g / g dry biomass resulting in a final slurry of 1072.9 Kg total weight.

[0167] The pH was maintained at 5.0 by adding sulfuric acid and the temperature was held at 57°C for 6 hours.

[0168] Solid / liquid separation step b) was carried out using a belt filter equipped with a washing system to facilitate sugar recovery. This belt filter is a horizontal vacuum system with polypropylene filter cloth with an air permeability of 1850 L / sec / m2. The liquid filtration fraction of the hydrolysate was separated out in the first zone of the belt filter. Subsequently, the wet solid was washed in 3 zones in a counter-current arrangement and a wash water / dry hydrolysate weight ratio of 4 at the inlet to the solid / liquid separation system. Finally, the wet solid underwent a drying process in the last zone. The liquid fraction from the washing and drying zones was pooled with the liquid fraction from the first zone to pass to step c).

[0169] The solid fraction, consisting mainly of insoluble fibres and proteins, was recovered and dried by flash drying. The recovered solid fraction, dried to a moisture content of 6.9 ± 0.3%, had the composition in Table 6.

[0170] The liquid fraction containing glucose and other soluble substances was purified (step cl)) by nanofiltration with two 8.1 m2modules of polyamide spiral membranes with a cut-off of 600- 800 Da connected in series (total filtration area of 16.2 m2). The filtration was performed in continuous configuration, thus directly recovering the retentate and the permeate in different storage tanks. The retentate, representing the soluble protein fraction, with a water content of 90.8 ± 0.1% by weight, was characterised and showed a high content of organic nitrogen in the form of amino acids and / or proteins and / or oligopeptides (24.9 ± 0.1% by weight in relation to the dry weight, a glucose content of 44.8 ± 0.2% and a fructose content of 3.8 ± 0.2% by weight in relation to the dry weight).

[0171] Glucose and fructose content was determined by means of high-pressure liquid chromatography (HPLC) with an RID detector and Biorad Aminex HPX-87H 300mm x 7.8mm column and precolumn (Flow: 0.6 mL / minute; oven temperature: 50°C; detector temperature: 35°C; eluent: 5mM H2SO4).

[0172] The membrane permeate was then treated on cationic ion-exchange resin dowex 88 in a single step followed by a single step on dowex 77 anionic ion-exchange resin (step c2)).

[0173] The aqueous solution eluted from the anion resin was then concentrated in a forced circulation evaporator while maintaining a vacuum level such that the concentration was performed at a temperature below 80°C.

[0174] Following concentration of the liquid phase, a 63.8% w / w syrup comprising glucose and fructose in a total quantity of more than 85% w / w and having a nitrogen content of below 0.02% w / w and an ash content of 0.038% w / w by dry weight was obtained. The ratio of glucose to fructose was 6.4.

[0175] The monosaccharide syrup obtained from secondary products by the process according to the invention has therefore proven to be equivalent to commercially available syrups made from starches, in terms of both nitrogen and ash content.

[0176] This syrup, in a mixture with technical grade (first generation) glucose at 32% w / w (weight of second generation glucose and fructose / weight of total sugars), was used as a carbon source in a fermentation process for the production of 1,4-BDO. The resulting sugar mixture had a glucose concentration of 669.3 g / L and a fructose concentration of 32.24 g / L.

[0177] An Escherichia coli strain equipped with a metabolic pathway for the synthesis of 1,4-BDO was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of Luria Bertani medium fortified with 15 g / L first- generation glucose. The Erlenmeyer flask was then agitated at 275 rpm at a temperature of 35°C, overnight, resulting in a preinoculum.

[0178] Subsequently, 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; 10 g / L first generation glucose; 1 mL / L MgSCU IM; 1 mL / L CaCh 0.1 M; 1.25 mL / L Trace Elements; 0.5 mL / L Streptomycin 100 mg / mL).

[0179] The flask was incubated at 35°C, agitating the contents at 275 rpm for approximately 8 hours. After this incubation period the optical density reached an OD value (optical density measured at 600nm) of approximately 3-4 OD and the culture was used to inoculate a 0.016 OD seeding fermenter.

[0180] After approximately 18 hours, an aliquot of the seed fermentation was used to inoculate a production fermenter containing 1 litre of medium of OD 4 (KH2PO4 1.73 g / L; (NH4)2SO4 0.83 g / L; NH4H2PO4 0.96 g / L; Na2SO40.30 g / L; Ca Citrate.4H20 0.038 g / L; Citric Acid CeHxO? 0.20 g / L; MgSO4 IM (4 mL / L); Trace Elements 2.5 mL / L; Antifoam 0.1 mL / L) and 20 g / L of first generation glucose.

[0181] In the course of fermentation, the mixture of first and second generation sugars described above (prepared by mixing the second generation sugar syrup obtained according to the procedure according to the invention with technical grade glucose) was progressively fed into the fermenter by a fed batch process so as to keep the overall concentration of glucose and fructose in the culture medium constant in the range of 30-60 g / L, for about 30 hours from the start of fermentation, then progressively reducing it until a glucose concentration of about 0 g / L was obtained at the end of fermentation (about 36 hours from inoculation). The production fermenter was maintained under the following conditions: stirring speed 700- 900 rpm, air flow 0.4 vvm (L air / L medium / minute), pH 6.75, temperature 35 °C.

[0182] Samples of the fermentation broth were taken at different times to assess the production of 1,4-BDO by high-pressure liquid chromatography (HPLC) analysis.

[0183] The 1,4-BDO 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.

[0184] Based on the data collected, Titre and Productivity were determined, where:

[0185] "Titre" (g / L): concentration of 1,4-BDO in the reaction medium at the end of fermentation time;

[0186] "Productivity" (g / L / h): average synthesis rate of 1,4-BDO, calculated as titre / hour of fermentation.

[0187] The results obtained are shown in Table 7 in comparison with the reference results for a fermentation conducted under the same operating conditions, but fed exclusively with technical grade sugar.

[0188] The comparison of process performance for the production of 1,4-BDO by fermentation clearly demonstrates how the use of a mixture containing the monosaccharide composition produced according to the invention as a carbon source yields results that are quite comparable to those achieved with technical grade glucose. Such sugars do not interfere with normal microorganism growth and are efficiently converted by this into 1 ,4-butanediol, demonstrating the high degree of purity achieved by the monosaccharide composition produced according to the invention. At the same time, the substitution of first-generation sugars by sugars derived from the secondary products of edible flour processing improves the economy of the process as well as its sustainability.

Claims

CLAIMS1. Process for the utilisation of a secondary product from the processing of edible flours comprising starch, comprising the steps of: a) subjecting said secondary product to hydrolysis of the starch in the presence of water, resulting in an aqueous mixture comprising monosaccharides, fibres, proteins and / or oligopeptides; b) subjecting said aqueous mixture to a solid / liquid separation, resulting in a solid fraction comprising insoluble fibres and insoluble proteins and / or oligopeptides, and a liquid fraction comprising soluble monosaccharides, soluble fibres and proteins and / or oligopeptides, and optionally salts; c) subjecting said liquid fraction resulting from step b) to a purification treatment comprising cl) at least one membrane separation operation, removing a soluble protein fraction and obtaining an aqueous solution comprising soluble monosaccharides; and subsequently c2) at least one passage of said aqueous solution through a bed comprising one or more ion exchange resins, resulting in a purified solution comprising mono s accharides .

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

3. Process according to claim 1 in which said product is a biomass comprising more than 20% by weight of total fibre, excluding starch, 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 wherein said hydrolysis in step a) is carried out in the presence of a content of said secondary product of at least 5%, preferably at least 10%, more preferably at least 20%, and preferably less than 40% by weight relative to the volume of the aqueous mixture.

5. Process according to one or more of claims 1- 4 wherein said step a) is carried out in the presence of at least one amylase enzyme, preferably selected from alpha-amylase, glucoamylase (or amyloglucosidase or gamma-amylase) and mixtures thereof.

6. Process according to one or more of claims 1 - 5 wherein one or more aliquots of the liquid fraction separated in step b) are recycled to step a).

7. Process according to one or more of claims 1 - 6 comprising an additional step of drying the solid fraction separated in step b).

8. Process according to any of claims 1-7 wherein said solid fraction separated in step b) is subjected to separation of the protein component from the fibre-rich component.

9. Process according to one or more of claims 1 - 8 comprising a further step of hydrolysis of the cellulose and / or hemicellulose polysaccharide chains of the solid fraction separated in step b), in the presence of water and one or more enzymes.

10. Process according to one or more of claims 1 - 9 wherein the separation in step cl) comprises at least one ultrafiltration and / or nanofiltration operation.

11. Process according to one or more of claims 1 - 10 comprising a step c3) of concentrating the purified solution comprising monosaccharides obtained in step c2).

12. Process according to claim 11 in which said concentration is carried out by one or more operations selected from adsorption, dialysis, reverse osmosis, crystallisation, evaporation and distillation.

13. Monosaccharide composition, preferably C6, having an overall monosaccharide content of 80% or more, wherein the glucose : fructose ratio is of above 4 : 1, an ash content of less than 0.1% by weight, preferably 0.005% to 0.05% by weight, and a total nitrogen content of less than 0.1% by weight, preferably 0.005%, more preferably from 0.01% to 0.05% by weight relative to the dry weight of the composition.

14. Monosaccharide composition according to claim 13 obtained at the end of the process according to any one of claims 1-12.

15. Use of a monosaccharide composition obtained by the process according to one or more of claims 1 - 12, or the monosaccharide composition according to claim 13, as a carbon source for the growth of a microorganism strain capable of producing chemical intermediates and / or polyhydroxy alkanoates.

16. Use according to the preceding claim for the growth of a microorganism strain capable of producing 1,4-butanediol.

17. Composition, obtainable as a solid fraction in step b) of the process in claim 1, essentially free of starch and comprising at least 45% by weight of polysaccharides, at least 18% by weight of protein (including oligopeptides and free amino acids) and ash preferably less than 5%, more preferably no more than 3% by weight of the dry weight of said composition.

18. Composition, obtainable as a soluble protein fraction in step cl) of the process in claim 1, comprising one or more oligosaccharides, preferably selected from xylooligosaccharides and / or arabinooligosaccharides, and at least 10% protein,preferably at least 15% by weight, more preferably at least 20% by weight of protein, relative to its dry weight.

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