Title

PROCESS FOR TREATING OF BREWING INDUSTRY BY-PRODUCTS.

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Technical field

[0001] The present invention belongs to the technical field of food industry by-product treatment and recovery, more specifically it belongs to the field of treatment and recovery of brewing industry by-products, specifically the residual spent grains from brewing process, i.e., the mash- ing residue consisting of malt husks and other insoluble parts remaining from the mashing process.

Present status of the art

[0002] Brewer' s spent grains constitute the main by-prod- uct of the brewing industry, accounting for about 85% of the total waste from the brewing process. On average, the annual production of brewer's spent grains is about 40 million tons, of which about 8 million are produced in Europe alone and 240, 000 t/year in Italy. Only 30% of brewer's spent grains are currently reused, mainly as feed in the livestock sector, while most are disposed of in landfills.

[0003] Given the large quantities of brewer' s spent grains that are produced annually, their very early degra- dation, their low market value, their low degree of reuse, and the consequent issues related to their environmental sustainability, the problem of reusing these wastes by giv- ing them a new and more profitable use has long been felt.

[0004] Methods have been proposed for using the spent grains to produce ethanol (Rojas-Chamorro et al., Ethanol production from brewers' spent grain pretreated by dilute phosphoric acid, Energy & Fuels, 2018) , activated carbon (Mussatto et al., Production, characterization and application of activated carbon from brewer' s spent grain lignin, Bioresource Technolog, 2010; Osman et al., Upcycling brewer's spent grain waste into activated carbon and carbon nanotubes for energy and other applications via two-stage activation, J Chem Technol Biotechnol, 2020) , lactic acid (Mussatto et al., Brewer' s spent grain as raw material for lactic acid production by Lactobacillus delbrueckii , Bio- technology Letters, 2007; Mussatto et al., Effects of medium supplementation and pH control on lactic acid production from brewer' s spent grain, Biochemical Engineering Journal, 2008) , xylitol (Mussatto et al., Establishment of the optimum initial xylose concentration and nutritional supplementation of brewer' s spent grain hydrolysate for xylitol production by Candida guilliermondii , Process Biochemistry, 2008) . However, these processes are economically very expen- sive and characterized by rather low production yields.

[0005] Thus, the need is felt to valorize brewer's spent grains by recovering their main components, namely protein (-30%) , hemicellulose (-25%) , lignin (-28%) , and cellulose (-17%) .

[0006] This is both with a view to reducing the volume of by-products of the brewing process to be disposed of in landfills, and for economic reasons related to the profits that can be derived from the sale of the extracted compo- nents, the market value of which is significantly higher than that of the brewer's spent grains.

[0007] Brewer' s spent grains thus possess great potential for reuse because of the added value of their components.

[0008] Numerous studies have been conducted to develop techniques for deconstructing, separating and recovering the components (protein, cellulose, hemicellulose and lignin) of brewers' spent grains, however, most of them are focused on recovering only the protein fraction, while little attention has been paid to recovering both the fibrous or lignocellulosic part, which is the main constituent of the spent grains, and the sub-components of the latter (hemicel- lulose, cellulose, lignin) .

[0009] In addition, none of the currently known tech- niques have been implemented at an industrial level, partly because of the high cost of drying the spent grains, which result from the production process with a high moisture con- tent (-80%) .

[0010] Separation and recovery of protein from brewer' s spent grains has received increasing attention in recent years due to the increased demand for protein foods for humans and animals. Numerous studies have been conducted for recovering proteins from spent grains by using traditional alkaline extraction methods followed by acid-induced precip- itation (Connolly et al., Characterisation of protein-rich isolates and antioxidative phenolic extracts from pale and black brewers' spent grain, Int . J. Food Sci. Technol., 2013; Viera et al., Valuation of brewer' s spent grain using a fully recyclable integrated process for extraction of proteins and arabinoxylans , Ind. Crops Prod. 2014; Connolly et al., Isolation of peptides from a novel brewers spent grain protein isolate with potential to modulate glycaemic response, Int. J. Food Sci. Technol., 2017; Musatto, Brewer' s spent grain: a valuable feedstock for industrial applications, J. Sci. Food Agric., 2014; Piritta, Enzymatic fractionation of brewer' s spent grain and bioconversion of lignin-rich fractions in a colon model in vitro, Doctoral Dissertations Aalto University publication, 2016) ; methods employing enzymes have also been developed, leading to the isolation of protein hydrolysates (Treimo et al., Enzymatic solubilization of proteins in brewer' s spent grain, J. Agric. FoodChem, 2008; Bi et al, Proteomic profiling of barley spent grains guides enzymatic solubilization of the remaining proteins, Appl . Microbiol. Biotechnol., 2018) .

[0011] However, none of the methods known so far have been implemented on an industrial scale because of the high costs of preliminary drying operations of the spent grains, as well as the high costs of the enzyme species required and the low efficiency of the protein separation process.

[0012] On the other hand, recovery of the fibrous frac- tion of the spent grains, constituting about 60% of this by- product, is a largely unexplored field; in particular, cel- lulose, hemicellulose, and lignin can be obtained by frac- tionation of the lignocellulosic component of brewer's spent grains .

[0013] Cellulose is used in a variety of sectors, for example, in the production of paper and cardboard, or as an additive in polymeric materials, or even in the production of bioethanol; cellulose can also be converted into deriva- tives such as cellophane, rayon, cellulose acetate, typi- cally used in textiles, for the production of plastics, var- ious consumer products, etc.

[0014] Hemicellulose and lignin are typically regarded as low-quality and low-value by-products, yet they can be used as substrates in bio-refineries for the production of a very wide range of bio-products of industrial interest, such as raw materials for chemicals, additives, biofuels and even energy .

[0015] For example, hemicellulose conversion processes have been developed for the production of ethanol, xylitol, butanediol, polyhydroxyalkanoates, organic acids (succinic acid, butyric acid, etc. ) , furfural (Chandel et al, Bioconversion of hemicellulose into ethanol and value-added products, 2018) ; in this case, the extraction of hemicellulose in the form of monomeric sugars is required. Also known are uses of hemicellulose in macromolecule form as a source of new materials, with special reference to composite materi- als .

[0016] Possible uses of lignin include: the use of depol- ymerized lignin in polyf unct ional aromatic monomers for use as a raw material in the polymer industry; the use in mac- romolecule form as an additive, or in polymer mixtures, or composites in the synthesis of copolymers; the use of lignin as a source of carbon-based materials such as carbon fibers. [0017] There is, however, a particular need to obtain high-quality lignin and hemicellulose, i.e., with a high degree of purity, which can be directly reused for other purposes in a wide variety of production sectors, without the need for further processing.

[0018] A method of fractionating wet spent grains by tra- ditional alkaline, or enzymatic, or sodium bisulf ite-medi- ated extraction has been proposed in the literature to re- cover a high-protein product and a high-fiber product (Yanhong He et al., Wet fractionation process to produce high protein and high fiber products from brewer's spent grain, Food and Bioproducts Processing, 2019) .

[0019] Patent application WO 2020/214502 Al discloses a process for extracting the spent grains from which a protein fraction and a fiber-rich fraction are isolated; separation is done using a classifier mill. The fibrous fraction is then processed into paper material for packaging; in one possible form of implementing the method, preliminary drying of the spent grains to moisture values less than or equal to 70% is provided.

[0020] Methods are also known for recovering the protein component and cellulose, in the form of nanofibers, by al- kaline treatment of dried brewer's spent grains; other known methods allow the recovery of hemicellulose alone in hydro- lyzed form (Mussatto et al., Chemical characterization and liberation of pentose sugars from brewer' s spent grain, Jour- nal of Chemical Technology and Biotechnology, 2006) , of cel- lulose alone (hydrolyzed and non-hydrolyzed) (Mussatto et al., Optimum operating conditions for brewer' s spent grain soda pulping, Carbohydrate Polymers, 2006; Mussatto et al., Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer' s spent grain, Enzyme and Microbial Technology, 2008) and lignin alone (Mussatto et al. , Lignin recovery from brewer' s spent grain black liquor, Carbohy- drate Polymers, 2007) .

[0021] Patent application WO 2020/234761 Al describes a process for recovering only the components of the lignocel- lulosic structure (lignin, hemicellulose, cellulose) from biomass, including brewer's spent grains. This process in- volves the use of deep eutectic extraction (DES) solvents, but these are very expensive, especially in view of an in- dustrial-scale application. In particular, for brewer's spent grains, the use of choline acetate in combination with glycolic acid is contemplated; disadvantageously, both gly- colic acid and the direct precursor of choline acetate, cho- line hydroxide, have toxic properties for humans and the environment. In addition, the process illustrated in WO 2020/234761 Al involves preliminary drying of the spent grains, and their components are selectively isolated using additional organic solvents (ethanol) , which increases the costs associated with the process.

[0022] Another currently known process for recovering cellulose, hemicellulose, and lignin from brewer's spent grains involves the use of ionic liquids as the lignin ex- traction solvent (Outeirino et al., A novel approach to the biorefinery of brewery spent grain, Process Biochemistry; Biorefining brewery spent grain polysaccharides through biotuning of ionic liquids , Carbohydrate Polymers, 2019) . The use of such ionic solvents entails economic disadvantages due to their high cost, all the more so in view of indus- trial-scale applications; moreover, this method is limited to obtaining only lignin in macromolecule form and cellulose and hemicellulose in hydrolyzed form.

[0023] Patent US 10240006 B2 discloses a method for iso- lating functionalized lignin from lignocellulosic biomass by hydrolysis under supercritical conditions combined with re- active extraction. The method, however, is limited to ob- taining only lignin in macromolecule form, and cellulose and hemicellulose in hydrolyzed form.

[0024] Patent application US 5430142 A relates to a pro- cess for the isolation of functionalized xylans from ligno- cellulosic biomass by reactive extraction. However, this process involves preliminary delignification of the biomass by an organosolv process and/or treatment by hypochlorites, which can have significant negative environmental impacts.

[0025] None of the aforementioned methods that involve reactive extraction to isolate components of lignocellulosic matrices, however, relate to brewer's spent grains.

[0026] At the current state of the art, there are no known processes for integral fractionation of components of wet brewer' s spent grains that include at least one reactive extraction step.

[0027] It should be noted that, in the present patent text, the term fractionation refers to the subdivision of a heterogeneous product, such as indeed brewer's spent grains, into the fractions of homogeneous component elements, namely, in the present case, protein, cellulose, hemicellu- lose and lignin.

Objects and summary of the invention

[0028] Thus, the object of the present invention is to provide a technique for deconstructing, separating, and integrally recovering the components of brewer's spent grains, thus enhancing the value of these waste by-products, making it possible to reuse them in other processes, and reintroducing them into a production cycle.

[0029] A second object of the present invention is to provide a process for recovering the components of brewer' s spent grains that does not require prior dewatering, drying, or desiccation of the wet spent grains resulting from the brewing process, so as to reduce costs, both from an energy and economic point of view.

[0030] Further object of the present invention is to pro- vide a process for the fractionation of the essential com- ponents constituting the brewer's spent grains, also func- tionalized, with a high degree of purity.

[0031] Not least object of the present invention is to provide a process for the separation of the protein component of brewer' s spent grains and for the separation of the lig- nocellulosic component into cellulose, functionalized hemi- cellulose, and lignin, possibly functionalized.

[0032] These and other objectives that will become clear to the expert in the field have been achieved by implementing a sequential extraction process of said components.

[0033] It should be pointed out that although specific reference is made in this patent text to brewer' s spent grains, the process object of the present application can also be usefully applied to other agro-food residues con- taining lignocellulose, such as olive tree foliage, residual olive pomace from the oil extraction process, plant residues from cereal threshing, and other similar plant matrices.

[0034] In the case of its application to brewer' s spent grains, the process which is the subject of the present patent application makes it possible to achieve, in a repro- ducible manner, the separation of the protein component of brewer' s spent grains and also the separation of cellulose, functionalized hemicellulose and lignin, possibly function- alized, with a high degree of purity.

[0035] More specifically, the present invention comprises a reactive extraction step i.e., with simultaneous function- alization and extraction (by precipitation) of hemicellu- lose, thus enabling hemicellulose to be obtained in the form of a high-quality (pure) and functionalized macromolecule, increasing its value for biorefineries.

[0036] Functionalization takes place by means of organic compounds capable of selectively giving rise to esterifica- tion reaction (containing groups -XC(=O)R; X=OC(=O)R, OH, etc. ; where R can be an alkyl, fluoroalkyl, alkenyl, aryl, fluoroaryl) , or to etherification, amidation. Esterification or etherification, amidation of hemicellulose can result in superior properties of the modified hemicellulose itself in applications such as polymer mixtures and composites for the (bio) plastic packaging industry. Indeed, the added groups improve miscibility and compatibility between polymers. In a possible variant of the present invention, the process includes a combination of the functionalization and extrac- tion by precipitation of lignin, for example, by means of alkyl-type organic compounds (preferably hydroxyalkyls) .

[0037] Most advantageously, the treatment process object of the present invention can be implemented semi-continu- ously or continuously, so as to improve efficiency and ensure absolute benefits from the point of view of the safety of operators .

[0038] It should be emphasized that the fractions that are isolated by means of the present process exhibit a high degree of purity, being basically composed of a homogeneous component (protein, cellulose, hemicellulose or lignin) without a significant presence of other foreign elements. Brief description of the drawings

[0039] Fig. 1 shows a flow chart of a form of implemen- tation of the process for treating brewer' s spent grains according to the present invention.

[0040] Entering step (A) are the spent grains (1) , water and an inorganic base (NaOH) , and exiting this step are the proteins (2) and fibers (3) , which then enter step (B) where they are combined with water and an inorganic base (NaOH) , following this step, cellulose (4) , and hemicellulose (5) and lignin (6) are obtained solubilized. Hemicellulose (5) and lignin (6) supply step (C) , where an organic compound is added that selectively reacts with hemicellulose (5) , and further downstream, functionalized hemicellulose (7) and solubilized lignin (6) are obtained, the latter feeds step (D) , together with a strong acid (11) and produces a solid fraction containing lignin (8) , finally obtaining water and salts (9) . Particularly in step (D) , functionalized lignin can be obtained if an organic compound is substituted in place of the strong acid.

[0041] Fig. 2 shows an FTIR-ATR spectrum of a sample of functionalized hemicellulose with a -OC(=O)Ph group obtained with the method subject of the present invention.

Detailed description of two embodiments of the invention

[0042] In its most essential embodiment, the present pro- cess of fractionation of the components of wet brewers' spent grains takes place in a single reactor and includes the following main steps :

(A) extraction of the protein phase from the lignocellulo- sic phase (itself composed of cellulose, hemicellulose, and lignin) of the brewer' s spent grains and separation of said protein phase;

(B) extraction of hemicellulose and lignin and separation of cellulose from said lignocellulosic fraction; (C) extraction by precipitation and separation of function- alized hemicellulose;

(D) precipitation and separation of lignin, possibly func- tionalized .

[0043] In step (A) , wet brewer' s spent grains resulting from a brewing process are fed into a discontinuous, semi- continuous or continuous reactor operating at atmospheric pressure and are mixed with a fluid consisting of water and an inorganic base, preferably sodium hydroxide (NaOH) .

[0044] The volume of water is such that the concentration in water of the inorganic base, preferably sodium hydroxide, is between 2, 5% and 6% m/V, considering a quantity of inor- ganic base between 2, 5% and 6% by weight relative to the dry matter of the spent grains; optimum results are obtained with a quantity of inorganic base between 4, 5% and 5, 5% by weight. The process temperature is maintained between 30 °C and 90°C, preferably between 50° and 70°C. The mixture is maintained under intermittent or continuous stirring for be- tween 0.5 and 5 hours, preferably at least 2 hours.

[0045] In a preferred embodiment, step (A) is preceded by the grinding of wet brewer's spent grains. Grinding facili- tates the mixing of the spent grains themselves with the liquid extracting medium during step (A) and, consequently, subsequent separation processes.

[0046] Inside the reactor, the brewer' s spent grains treated with sodium hydroxide in an aqueous solution result in a mixture consisting essentially of a first liquid frac- tion comprising solubilized proteins, and a first solid frac- tion, the latter comprising the lignocellulosic fibrous fraction of the brewer's spent grains, which in turn consists of hemicellulose, cellulose and lignin.

[0047] In step (A) the protein component is solubilized, generating a first liquid fraction comprising protein and a first solid fraction comprising hemicellulose, cellulose and lignin .

[0048] Preferably, in step (A) , the separation between the insoluble lignocellulosic residue and the liquid portion is refined by conventional methods such as filtration or centrifugation .

[0049] In a particularly complete embodiment of the pro- cess, water washing of the insoluble lignocellulosic resi- due, repeated one to three or more times, is included to maximize the recovery of the protein fraction. The solid lignocellulosic residue is used in the next step (B) of the process .

[0050] In the second step (B) , the solid residue result- ing from the previous step (A) , i.e., the fiber, is mixed inside a reactor with a fluid consisting of water and an inorganic base, preferably sodium hydroxide (NaOH) ; the vol- ume of water is such that the concentration in water of the inorganic base, preferably sodium hydroxide, is between 2, 5% and 6% m/V, considering a quantity of inorganic base between 30% and 55% by weight with respect to the dry matter of the fiber. The process temperature is preferably maintained be- tween 30°C and 90°C.

[0051] Excellent results have been obtained using a NaOH solution with a concentration between 4, 5% to 5, 5% m/V and maintaining the process temperature between 30°C and 50°C.

[0052] The mixture can be kept under intermittent or con- tinuous stirring for between 0.5 and 24 hours, preferably at least 6 hours .

[0053] Inside the reactor, treatment of the lignocellu- losic fraction of the spent grains with the aqueous sodium hydroxide solution produces a mixture consisting essentially of a second liquid fraction comprising solubilized hemicel- lulose and lignin and a second solid fraction comprising cellulose .

[0054] Preferably, in step (B) , the separation of the cellulosic solid residue from the liquid fraction is refined by conventional methods such as filtration or centrifuga- tion. The liquid fraction is used in the next step (C) .

[0055] In the third step (C) of the process, the liquid fraction from the previous step (B) is mixed inside the reactor with an organic compound. In the embodiment described here, the concentration of the organic compound in the liquid fraction is between 1% and 9% m/V, preferably between 4% and 6% m/V.

[0056] The organic compound can be any suitable organic compound that selectively reacts with hemicellulose at a process temperature below 100°C, preferably forming ester, ether, or amide bonds with it. The mixture can be kept under intermittent or continuous stirring for between 0.5 and 2 hours, preferably at least 1.5 hours.

[0057] The resulting mixture consists essentially of a third liquid fraction containing solubilized lignin and a third solid fraction comprises functionalized hemicellulose. [0058] In the preferred embodiment, described here, dur- ing step (C) a reactive separation of the hemicellulose oc- curs by means of the organic compound: the hemicellulose contained in the liquid fraction is functionalized by ester- ification, etherification, and amidation, and as the reac- tion progresses, precipitation of the functionalized hemi- cellulose occurs due to the progressive decrease in the sol- ubility characteristics of the derivative. In this example, the organic compound used is benzoyl chloride, which func- tionalizes hemicellulose by esterification.

[0059] In step (C) , the separation between the function- alized hemicellulose and the liquid fraction is refined by conventional methods such as filtration or centrifugation. Preferably, the precipitate obtained is washed with water, or ethanol, or both, to remove possible residual organic compound and other impurities. The liquid fraction is used in the next step (D) .

[0060] In step (D) of the present process, the liquid fraction resulting from step (C) is acidified to pH between 2 and 3 with a strong acid such as, for example, a mineral acid such as hydrochloric acid or sulfuric acid. The result- ing suspension consists essentially of lignin, and the lat- ter is separated from the liquid fraction, consisting essen- tially of water and salts, by centrifugation.

[0061] In a variation of the process that is the object of the present invention, instead of step (D) described above, step (C) is followed by the step (E) of extraction of functionalized lignin.

[0062] In step (E) , the liquid fraction from step (C) is additioned with an alkyl-type organic compound, preferably alkylthiols, with which it reacts to form a solid fraction comprising the functionalized lignin and the supernatant. Said organic compound can be any organic compound having characteristics such that it reacts with lignin, preferably with the phenolic -OH functional groups of lignin, at a temperature below 100°C.

[0063] Experimental tests conducted on a predetermined amount of brewer' s spent grains with a moisture content of 78% (determined according to the official Analytica EBC 12.2 method) , revealed excellent results both in terms of the efficiency of separation of the protein fraction and the extraction efficiency of the cellulosic component, hemicel- lulose and lignin.

[0064] The protein content in the liquid portion result- ing from the first step (A) of the process, was measured by total nitrogen determination. The test was carried out according to standard AOAC 945.18-B and AOAC 920.53 methods. The operation was repeated on the fibrous residue. The pro- tein separation efficiency, given by the ratio: [Protein in liquid fraction (g) /total protein in liquid and fiber frac- tion (g) ]x 100, was greater than or equal to 40 %.

[0065] The separation efficiency of cellulose was found to be greater than 80%. The amount of cellulose separated from the fibrous residue in the second step (B) of the pro- cess, was determined gravimetrically after drying the cel- lulose sample at 60°C for 24 hours.

[0066] The yield of functionalized hemicellulose was found to be more than 85%.

[0067] As an example, results are reported for the reac- tive extraction of hemicellulose present in the liquid frac- tion from the second step (B) of the process using a prede- termined amount of benzoyl chloride (step C) , carried out at a temperature preferably between 30°C and 50°C for between 1 and 2 hours, in the presence of NaOH (concentration: 2, 5% to 6% m/V) .

[0068] Successful functionalization of hemicellulose was confirmed by Fourier transform, attenuated total reflectance infrared spectroscopy (FTIR-ATR) . Figure 2 shows an analysis conducted on a sample of functionalized hemicellulose ob- tained by the process that is the object of the present invention. Proper, typical and characteristic signals of the bonds of an ester functional group of the OC(=O)Ph type are evidently recognizable.

[0069] Finally, the amount of lignin recovered by precip- itation with mineral acid from the liquid portion extracted in the fourth step (D) of the process was determined gravimet rically after drying at 60 °C for about 24 hours. A lignin recovery efficiency greater than 78% was measured.