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WO2012069889A1 - Integrated process for extracting proteins and arabinoxylans from brewer's spent grain - Google Patents

Integrated process for extracting proteins and arabinoxylans from brewer's spent grain Download PDF

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Publication number
WO2012069889A1
WO2012069889A1 PCT/IB2010/055699 IB2010055699W WO2012069889A1 WO 2012069889 A1 WO2012069889 A1 WO 2012069889A1 IB 2010055699 W IB2010055699 W IB 2010055699W WO 2012069889 A1 WO2012069889 A1 WO 2012069889A1
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WO
WIPO (PCT)
Prior art keywords
arabinoxylans
extraction
proteins
ethanol
citric acid
Prior art date
Application number
PCT/IB2010/055699
Other languages
French (fr)
Portuguese (pt)
Inventor
Manuel António COIMBRA RODRIGUES DA SILVA
Isabel Maria Pinto Leite Viegas Oliveira Ferreira
Maria Angélica MARTINS ROCHA
Elisa Marisa Ferreira Vieira
Jorge Manuel Alexandre Saraiva
Olívia Maria DE CASTRO PINHO
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Universidade De Aveiro
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Publication of WO2012069889A1 publication Critical patent/WO2012069889A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12F—RECOVERY OF BY-PRODUCTS OF FERMENTED SOLUTIONS; DENATURED ALCOHOL; PREPARATION THEREOF
    • C12F3/00—Recovery of by-products
    • C12F3/06—Recovery of by-products from beer and wine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
    • A23J1/005—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from vegetable waste materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention has application in the areas of utilization / recovery of one of the brewery by-products, the dreche, with a view to obtaining products that can be used as ingredients in the food, dietetic and pharmaceutical industry.
  • the present invention describes an integrated process for extracting protein and arabinoxylans from the brewery without the need for any pretreatment by the use of alkaline reagents and their separation by changing pH and adding ethanol:
  • This integrated brewing protein and arabinoxylan extraction process is based on the principle that proteins and arabinoxylans are soluble in alkaline reagents and therefore extractable in these solvents.
  • the alkaline extract separated from the fraction A solid solution of citric acid is added.
  • the acidification of the medium leads to the precipitation of proteins, allowing their removal, remaining the arabinoxylans in solution.
  • the solution containing the arabinoxylans is acidified with concentrated hydrochloric acid to a pH of less than 2, which are then collected by precipitation after the addition of ethanol to a concentration preferably of 70% (v / v). Citric acid remains soluble in the ethanol solution.
  • Extraction may use alkaline reagents of increasing concentrations, thereby sequentially extracting the proteins and arabinoxylans with different structural characteristics and properties.
  • Potassium hydroxide is the alkaline reagent used in the integrated protein and arabinoxylan extraction process of the proposed brewery.
  • the use of this reagent leads to the formation of potassium chloride (KCl) after acidification with hydrochloric acid (HCl).
  • the solubility of KCl in aqueous solutions containing 70% ethanol is 1,874 g per 100 g solution at 25 ° C and 2,834 g per 100 g solution at 40 ° C.
  • the solubility of KCl in aqueous solutions containing 60% ethanol is 3,759 g per 100 g solution at 25 ° C and 5,444 g per 100 g solution at 40 ° C.
  • Sodium hydroxide may be used as a substitute for potassium hydroxide as an alkaline reagent to be used.
  • the extraction process can be carried out in batch or continuous.
  • the invention is used for the simultaneous extraction of proteins and arabinoxylans by a process to obtain proteins and arabinoxylans as value added products in an integrated and sequential manner.
  • the extraction process allows the recycling of used citric acid and ethanol as well as the recovery of potassium chloride (or sodium chloride), with substantial economic gains.
  • the process is environmentally clean and allows the most abundant by-product of the brewing industry to be used to obtain protein-rich and arabinoxylan extracts that can be used as food ingredients. Examples of possible applications of arabinoxylans for use as dietary fiber and prebiotics (US Patents 2008/012162 Al and WO 2010/020639 Al).
  • the protein rich material may be used to obtain supplemental protein hydrolyzates in different types of food, dietary products and pharmaceuticals.
  • This integrated brewing protein and arabinoxylans extraction process allows 65-70% of the brewery's arabinoxylans and 86-96% of the protein to be extracted, in a process where the dreche does not need to undergo any pre-treatment such as drying.
  • WO 2008/047081 Al and / or grinding
  • the process uses as an alkaline reagent neutralizing agent a saturated citric acid solution.
  • This innovative use has the advantage of using a food grade acid (E 330) with a higher acidity than most organic acids. and with high solubility in water (61.8% m / m at 25 ° C) and in ethanol (38.3% m / m at 25 ° C).
  • the high solubility of citric acid in water allows selective precipitation of proteins after neutralization and acidification of the medium, preferably up to pH 3, a value that can be reached due to the acidity of this acid.
  • the high solubility of citric acid in ethanol allows it not to co-precipitate along with arabinoxylans.
  • citric acid Since citric acid is used to neutralize alkaline extracts, it will exist in the form of citrate. For its conversion to citric acid, For the reasons given above, a strong acid must be added, which is why the process uses a concentrated hydrochloric acid solution (food additive 507). Thus, the citrate is converted to citric acid, with formation of potassium chloride or sodium chloride, depending on the alkaline reagent used. These salts, at the concentrations at which they are formed, remain soluble in ethanol solutions, which allows the recovery of arabinoxylans without co-precipitation of these salts. Ethanol is a GRAS - 184.1293 (Generally Recognized as Safe) solvent, generally used for precipitation purification of polysaccharides.
  • the brewery corresponds to the insoluble part of the must, consisting essentially of the fibrous parts (the husks) of malted barley grains and residues of insoluble unmalted cereals sometimes added to the recipe ( barley, wheat, maize, among others), used as the main raw material in beer production.
  • the dreche is separated from the wort by filtration after the "brewing" phase.
  • Beer production yields 15-20 kg of dreche per hectoliter of beer produced.
  • the amount of dreche may vary depending on the type of beer produced and the associated moisture content, typically between 70 and 80%. It is a byproduct of high interest given its nutritional and functional characteristics. In Portugal beer breweries have been used almost exclusively for animal feed, in particular for cattle. However, the biotechnology, food and pharmaceutical sectors may offer alternatives for their recovery.
  • Dreche is a lignocellulosic material containing protein (25-30%), lignin (-28%), hemicelluloses (-25%, mostly arabinoxylans) and cellulose (-17%) in dry waste (Celus, I; Brijs, Delcour, JA (2006) "The effects of malting and mashing on barley protein extractability" Journal of Cereal Science, 44, 203-211; Teimo, J.; Westereng, B.; Horn, SJ; Forssell, P .; Robertson, JA; Faulds, CB; Waldron KW; Buchert, J .; Eijsink, VGH (2009), "Enzymatic Solubilization of Brewers' Spent Grain by Combined Action of Carbohydrases and Peptidases", Journal of Agricultural and Food Chemistry, 57, 3316-3324).
  • Dreche's main proteins are hordeins (A, B and C) constituting more than 50% of the total, followed by gluten
  • the separation of the dreche into two fractions: protein (high protein and fat and low fiber) and fibrous (protein low and arabinoxylan) by pressing and sieving can be performed wet, as described in European Patent 0443813A1 and the process developed by Heineken (Schwencke, KV (2006), "Sustainable, cost-effective, and feasible solutions for the treatment of brewers' spent grains," Master Brewers Association of the Americas Technical Quartely, 43, 199-202).
  • a dreche suspension is prepared in hot water (80 ° C) and then passed through a sieve.
  • Extractions of the dreche protein fraction in alkaline media are described, inter alia, in Japanese patent 51-129776 which describes the alkaline extraction of the dreche at pH 11-12 at 104 ⁇ ° C-121 ⁇ ° C, with isoelectric precipitation of proteins to obtaining the protein fraction.
  • this process involves protein decomposition due to the drastic conditions of high extraction temperature, resulting in low protein yield and deterioration of product quality.
  • the process requires a large amount of energy for high temperature extraction.
  • the product "MNG-3 / Biobran” has an active ingredient developed from arabinoxylans extracted from rice bran (Ghoneum, M. & Gollapudi, S. (2003), "Modified arabinoxylan rice bran (MGN-3 / biobran) sensitizers. human T cell leukemia cells to death receptor (CD95) -induced apoptosis. ", Cancer Letters, 201, 41-49), structurally similar to the arabinoxylans of the dreche.
  • the brewery Prior to sequential extraction, the brewery was pretreated with an 80% (v / v) ethanol solution. ) at reflux to give the ethanol-insoluble residue which was subjected to two washes for two hours, first with cold water and then with hot water. The hot water-insoluble residue was further subjected to enzymatic treatment with pronase, a proteolytic enzyme. Only after this pretreatment was the dreche treated with alkaline reagents. The obtained alkaline extracts were separated from the residue by centrifugation and filtration, neutralized with acetic acid and thoroughly dialyzed against deionized water and finally lyophilized. Based on the above studies, the present invention proposes an integrated protein and arabinoxylan extraction process from the brewery.
  • This process has the advantage of simultaneously extracting the proteins and arabinoxylans, which are separated by changing the pH of the alkaline extract together with ethanol precipitation.
  • a saturated citric acid solution as a neutralizing agent allows, after precipitation of proteins by pH change and removal by filtration or centrifugation, the solution containing the arabinoxylans can be treated with ethanol for selective precipitation of polysaccharides without co-precipitation of citric acid.
  • a strong acid preferably concentrated hydrochloric acid, which allows the protonation of citrate (insoluble in ethanol solutions) with formation of citric acid (soluble in ethanol solutions) is proposed.
  • Co-precipitation of the formed salts does not occur due to their solubility in the proposed ethanol solutions.
  • This process allows the collection of arabinoxylans without the need for a dialysis step, which significantly shortens the process time.
  • a dialysis process there is a need for a minimum of 3 water changes, each dialysis water being in contact for the same 6 hours, which totals 18 hours.
  • the fractions are diluted by contact with dialysis water and a time consuming and costly concentration step is required due to the need for water evaporation.
  • Another advantage of this process is the reuse of used citric acid and ethanol, in addition to the collection of potassium chloride. or sodium chloride however formed.
  • the present invention proposes an integrated extraction process of proteins and arabinoxylans (AX) from the brewery through the use of alkaline reagents. Proteins and arabinoxylans are soluble in alkaline reagents and therefore extractable in these solvents.
  • the alkaline extract is separated from the solid fraction and further acidification with a concentrated citric acid solution leads to the precipitation of the proteins, allowing them to separate from the remaining soluble arabinoxylans.
  • the extracted protein fraction is washed with ethanol to remove trace trace citric acid, also facilitating its drying.
  • the arabinoxylans are recovered by acidification to pH below 2 with a concentrated hydrochloric acid solution, followed by the addition of ethanol to form a solution, preferably up to 70% (v / v) in ethanol, which leads to the insolubilization of the arabinoxylans. and consequently to its precipitation.
  • Arabinoxylans are recovered by decantation, filtration and / or centrifugation.
  • the extracted polysaccharide fraction is washed with ethanol for removal of trace trace citric acid and drying.
  • Acidification at pH below 2 allows complete protonation of citric acid, which is thus soluble in aqueous ethanol solutions, allowing the separation of arabinoxylans without their co-precipitation.
  • the KCl or NaCl salt meanwhile formed by the acidification of citrate to citric acid is soluble in ethanol solutions and does not co-precipitate with arabinoxylans.
  • Dreche does not require any treatment prior to the extraction of proteins and arabinoxylans and as the simultaneous extraction of these two sets of compounds occurs, the process is faster and easier to apply than if separate processes for the extraction of proteins and arabinoxylans were used. .
  • the citric acid and ethanol used in the process are recycled and the potassium chloride or sodium chloride formed depending on the alkaline reagent used in the integrated protein and arabinoxylan extraction process is recovered.
  • Extracted proteins and arabinoxylans have added value in relation to the dreche, and are obtained by an environmentally clean process, not involving the use of toxic components and the reagents used are food grade and can therefore be used as ingredients in the industry. food, dietetic and pharmaceutical products.
  • the extraction process is characterized in that the alkaline reagent used in step a) is potassium hydroxide or sodium hydroxide.
  • the extraction process is characterized in that the alkaline reagent is used at a ratio of 1: 2 (w / v) (drought weight per volume of alkaline reagent solution).
  • the extraction process is characterized in that the alkaline reagent is used with increasing concentrations between 0.1 M and 4 M, preferably 0.1 M, 0.5 M and 4 M.
  • the extraction process is characterized in that the extraction takes place for 24 hours at room temperature from a dreche sample without any pretreatment.
  • the extraction process is characterized in that it can be carried out in batch or continuously.
  • step b) occurs by decantation, filtration and / or centrifugation.
  • the proteins and / or arabinoxylans and / or their derivatives are characterized by being obtained by the processes described above.
  • the use of the residue obtained after the extraction of proteins and / or arabinoxylans and / or their derivatives is characterized as a source of insoluble and / or combustible dietary cellulose and / or fiber and / or raw material for the paper industry.
  • the process of the present invention is based on the sequential and integrated extraction of the protein fraction and AX from the dreg with KOH solutions with increasing concentrations in the ratio of 1: 2 (m / v) (drab mass per volume of the KOH solutions). ) at room temperature for 24 h.
  • the flowchart outlines the integrated process for the extraction of proteins and arabinoxylans from the dreche. After alkaline extraction (1) with increasing concentrations of KOH [0.1 M (1) a; 0.5 M (l) b and 4 M (l) c] and acid precipitation with a concentrated citric acid solution to pH less than or equal to 4, preferably pH 3 (2), are obtained three protein fractions designated respectively by FP 1, FP 2 and FP 3.
  • the arabinoxylans are precipitated by acidification with concentrated HCl to pH less than or equal to 2 (3) and addition of ethanol to a 70% (v / v) solution, yielding three arabinoxylan fractions designated respectively as AX 1, AX 2 and AX 3. Finally, the residue is washed dreche with water for 24 h at room temperature (4) to maximize the removal of arabinoxylans; the last arabinoxylan fraction, designated AX 4, being obtained.
  • Arabinoxylans and proteins are extracted by a sequential process with increasing solutions of potassium hydroxide (or sodium hydroxide) at concentrations between 0.1 M and 4 M, preferably 0.1 M, 0.5 M and 4 M. M from a sample brewery without any pretreatment.
  • potassium hydroxide or sodium hydroxide
  • the flowchart outlines the integrated process for the extraction of proteins and arabinoxylans from the brewery.
  • Extractions with potassium hydroxide or sodium hydroxide may be carried out at room temperature for 24 hours with occasional stirring. 5 mM sodium metabisulphite may be added as an antioxidant agent. Alternatively, extractions with potassium hydroxide or sodium hydroxide may be performed for shorter periods if they are performed above room temperature.
  • a ratio of 1: 2 (w / v) (drought mass per volume of KOH solutions) is used, an aqueous solution of 0,1 M KOH + 5 mM Na 2 S 2 0s, (la) of the flowchart.
  • Arabinoxylans are recovered by precipitation in 70% (v / v) solutions in ethanol, and separated by decantation, filtration and / or centrifugation and washed with ethanol (3) from the flow chart. In this step, three arabinoxylan fractions designated respectively as AX 1, AX 2 and AX 3 are obtained.
  • residue 4 is finally washed for 24 h at room temperature (or alternatively for shorter periods using higher temperatures) (4) from the flowchart.
  • the supernatant obtained after precipitation of the arabinoxylans with ethanol is distilled, which allows the recovery of ethanol and the KCl (or NaCl) containing citric acid solution.
  • This aqueous solution is treated with ethanol for salt precipitation, which is collected by decantation and / or filtration and / or centrifugation.
  • the citric acid solution is evaporated to collect the ethanol and the citric acid remaining in the distillation aqueous solution.
  • This citric acid solution will be further used (5) from the flowchart. In this step the reuse of proteins and arabinoxylans which have not precipitated in phases (2) and (3) and which return to the circuit may occur.
  • Residue 1 was extracted with 278 mL of 0.5 M potassium hydroxide solution and 5 mM sodium metabisulfite under the same time, temperature and stirring conditions. The residue was separated from the alkaline extract by decantation and filtration, respectively designated Residue 2 and Extract 2.
  • Residue 2 was extracted with 278 mL of 4 M potassium hydroxide solution and 5 mM sodium metabisulfite, with 24 hour contact and occasional stirring.
  • the residue from the alkaline extract was separated by decantation and filtration, which were designated Residue 3 and Extract 3, respectively.
  • Extract 4 was acidified with a concentrated citric acid solution to pH around 4 and then with concentrated hydrochloric acid to a pH below 2. Then the arabinoxylans were precipitated. with the addition of ethanol to 70% (v / v) ethanol concentration, the soluble citric acid remaining as it is fully protonated (in acid form) at a pH below 2.
  • a prior evaluation of the protein content of the dreche samples was carried out, obtaining concentrations ranging from 8 to 10 g per 100 g of dreche as such (32 and 40 g of dry dreche).
  • concentrations ranging from 8 to 10 g per 100 g of dreche as such (32 and 40 g of dry dreche).
  • (1) a between 29-38% of the total proteins present in the dreche are separated.
  • (1) b between 26-46% of the total proteins present in the nursery are separated.
  • (1) c are separated between 10- 19% of the total proteins present in the dreche
  • the final residue has low amounts of protein in the order of 3-10%.
  • FP 2 and FP 3 are not pure, contain 60% protein, arabinoxylans, fat, ashes and other constituents of dreche. Studying the composition of these protein fractions revealed that they mostly include hordeins A, B and C.
  • the final residue consists mainly of cellulose, some lignin and the remaining arabinoxylans, while proteins exist in a trace amount.
  • the results obtained with this integrated protein and arabinoxylan extraction process when extrapolated to one ton of dreche as such indicate that between 86-96% of the protein and 66% of the total arabinoxylan can be extracted from the brewery.
  • 59% which corresponds to 40 kg of arabinoxylans of the initial 68 kg, are water soluble, with the remaining 7% remaining in the protein fraction corresponding to 4.6 kg of arabinoxylans of the initial 68 kg.
  • Figure 1 Sequential extraction of protein fraction and AX from the dreche with KOH solutions with increasing concentrations to 1: 2 ratio for 24 at room temperature.

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Abstract

The present invention proposes an integrated process for extracting proteins and arabinoxylans from brewer' s spent grain, without the need to subject the spent grains to any pre-treatment, through the use of alkaline reagents followed by selective precipitation by acidification of the medium and addition of ethanol. The present invention is applicable in the areas of reuse or valorization of brewer' s spent grain, with the aim to obtain products that can be used as ingredients in the food industry, and in the production of dietetic and pharmaceutical products. The final residue obtained after extraction of the proteins and arabinoxylans can be used as a source of cellulose, as an insoluble dietetic fiber or, possibly, as a fuel or raw material for the paper-making industry.

Description

DESCRIÇÃO  DESCRIPTION
"PROCESSO INTEGRADO DE EXTRACÇÃO DE PROTEÍNAS E  "INTEGRATED PROTEIN EXTRACTION PROCESS AND
ARABINOXILANAS DA DRECHE CERVEJEIRA"  ARABINOXILANAS OF THE DRECHE BEER "
Domínio Técnico da Invenção : Technical Field of the Invention:
A presente invenção tem aplicação nas áreas de aproveitamento/valorização de um dos subprodutos da indústria cervejeira, a dreche, com vista à obtenção de produtos passíveis de serem usados como ingredientes na indústria alimentar, de produtos dietéticos e farmacêuticos .  The present invention has application in the areas of utilization / recovery of one of the brewery by-products, the dreche, with a view to obtaining products that can be used as ingredients in the food, dietetic and pharmaceutical industry.
Mais concretamente, a presente invenção descreve um processo integrado de extracção de proteínas e arabinoxilanas da dreche cervejeira, sem que esta necessite de qualquer tratamento prévio, através da utilização de reagentes alcalinos e a sua separação por alteração de pH e adição de etanol:  More specifically, the present invention describes an integrated process for extracting protein and arabinoxylans from the brewery without the need for any pretreatment by the use of alkaline reagents and their separation by changing pH and adding ethanol:
(1) extracção de proteínas e arabinoxilanas com reagentes alcalinos num processo sequencial com soluções de concentração crescente entre 0,1 e 4 M de hidróxido de potássio ou hidróxido de sódio, preferencialmente, 0,1 M, 0,5 M e 4,0 M, a partir de uma amostra de dreche cervejeira sem qualquer tipo de pré-tratamento ;  (1) extraction of proteins and arabinoxylans with alkaline reagents in a sequential process with solutions of increasing concentration between 0.1 and 4 M potassium hydroxide or sodium hydroxide, preferably 0.1 M, 0.5 M and 4.0 M, from a sample of brewery without any pretreatment;
(2) separação dos extractos alcalinos por decantação, filtração e/ou centrifugação;  (2) separating the alkaline extracts by decantation, filtration and / or centrifugation;
(3) precipitação das proteínas e sua separação das arabinoxilanas por acidificação do meio com uma solução de ácido cítrico, preferencialmente, uma solução saturada;  (3) protein precipitation and its separation from arabinoxylans by acidifying the medium with a citric acid solution, preferably a saturated solution;
(4) recolha das proteínas por filtração ou centrifugação, ficando as arabinoxilanas na solução aquosa;  (4) protein collection by filtration or centrifugation, leaving the arabinoxylans in the aqueous solution;
(5) lavagem das proteínas precipitadas com etanol, para remoção de vestígios de ácido cítrico; (6) acidificação da solução aquosa remanescente com ácido clorídrico, preferencialmente com uma solução concentrada de deste ácido; (5) washing the precipitated proteins with ethanol to remove traces of citric acid; (6) acidifying the remaining aqueous solution with hydrochloric acid, preferably with a concentrated solution of this acid;
(7) adição de etanol, preferencialmente até perfazer 70% (v/v), para precipitação das arabinoxilanas ;  (7) adding ethanol, preferably to 70% (v / v), for precipitation of arabinoxylans;
(8) recolha das arabinoxilanas por decantação, filtração e/ou centrifugação;  (8) collection of arabinoxylans by decantation, filtration and / or centrifugation;
(9) lavagem das arabinoxilanas precipitadas com etanol, para remoção de vestígios de ácido cítrico;  (9) washing ethanol precipitated arabinoxylans to remove traces of citric acid;
(10) repetição dos passos 2 a 9 para cada solução alcalina usada ;  (10) repeating steps 2 through 9 for each alkaline solution used;
(11) neutralização do resíduo final, resultante da precipitação das proteínas e arabinoxilanas, com solução de ácido cítrico até o resíduo ficar a pH ácido, preferencialmente a pH 3, seguido de filtração e/ou centrifugação, permitindo obter uma fracção aquosa rica em arabinoxilanas que pode ser recuperada seguindo os passos 6 a 9, e a obtenção de um resíduo rico em celulose;  (11) neutralization of the final residue resulting from the precipitation of the proteins and arabinoxylans with citric acid solution until the residue is at acidic pH, preferably at pH 3, followed by filtration and / or centrifugation, yielding an arabinoxylan-rich aqueous fraction which may be recovered by following steps 6 to 9 and obtaining a cellulose rich residue;
(12) reciclagem do ácido cítrico e etanol usados no processo integrado e recuperação do sal de cloreto de potássio ou cloreto de sódio entretanto formados, pela destilação do etanol, preferencialmente utilizando pressão reduzida. O ácido cítrico e o sal que permanecem na solução aquosa não destilada são separados por adição de etanol, que precipita selectivamente o sal e dissolve o ácido cítrico. O etanol é removido por destilação, podendo a solução saturada de ácido cítrico formada ser usada nos passos seguintes de neutralização.  (12) recycling the citric acid and ethanol used in the integrated process and recovering the meanwhile formed potassium chloride or sodium chloride salt by distilling ethanol, preferably using reduced pressure. The citric acid and salt remaining in the undistilled aqueous solution are separated by the addition of ethanol, which selectively precipitates the salt and dissolves the citric acid. Ethanol is distilled off, and the saturated citric acid solution formed can be used in the next steps of neutralization.
Este processo integrado de extracção de proteínas e arabinoxilanas da dreche cervejeira tem como base o princípio de que as proteínas e as arabinoxilanas são solúveis em reagentes alcalinos e, por isso, extractáveis nestes solventes. Ao extracto alcalino separado da fracção sólida é adicionada uma solução concentrada de ácido cítrico. A acidificação do meio leva à precipitação das proteínas, permitindo a sua remoção, permanecendo as arabinoxilanas em solução. A solução contendo as arabinoxilanas é acidificada com ácido clorídrico concentrado até pH inferior a 2, sendo estas posteriormente recolhidas por precipitação após adição de etanol até uma concentração, preferencialmente, de 70% (v/v). O ácido cítrico permanece solúvel na solução de etanol. This integrated brewing protein and arabinoxylan extraction process is based on the principle that proteins and arabinoxylans are soluble in alkaline reagents and therefore extractable in these solvents. The alkaline extract separated from the fraction A solid solution of citric acid is added. The acidification of the medium leads to the precipitation of proteins, allowing their removal, remaining the arabinoxylans in solution. The solution containing the arabinoxylans is acidified with concentrated hydrochloric acid to a pH of less than 2, which are then collected by precipitation after the addition of ethanol to a concentration preferably of 70% (v / v). Citric acid remains soluble in the ethanol solution.
A extracção pode utilizar reagentes alcalinos de concentrações crescentes, procedendo-se deste modo a uma extracção sequencial das proteínas e das arabinoxilanas com características estruturais e propriedades diferentes. Extraction may use alkaline reagents of increasing concentrations, thereby sequentially extracting the proteins and arabinoxylans with different structural characteristics and properties.
O hidróxido de potássio é o reagente alcalino utilizado no processo integrado de extracção de proteínas e de arabinoxilanas da dreche cervejeira que se propõe. A utilização deste reagente leva à formação de cloreto de potássio (KC1) após a acidificação com ácido clorídrico (HC1) . A solubilidade do KC1 em soluções aquosas contendo 70% de etanol é de 1, 874 g por 100 g de solução, à temperatura de 25°C e de 2, 834 g por 100 g de solução, à temperatura de 40°C. A solubilidade do KC1 em soluções aquosas contendo 60% de etanol é de 3, 759 g por 100 g de solução, à temperatura de 25°C e de 5, 444 g por 100 g de solução, à temperatura de 40°C. (Pinho, S.P. e Macedo, E.A. (2005), "Solubility of NaCl, NaBr, and KC1 in Water, Methanol, Ethanol, and Their Mixed Solvents", Journal of Chemical and Engineering Data, 50, 29-32) . Nestas condições, é possível separar as arabinoxilanas por precipitação nestas soluções, permanecendo o KC1 em solução. Dada a solubilidade do cloreto de sódio (NaCl) nas soluções de etanol ser comparável ou até superior à solubilidade do KC1 (Li, M.Y.; Constantinescu, D.; Wang, L.S.; Mohs, A.; Gmehling, J. (2010), "Solubilities of NaCl, KC1, LiCl, and LiBr in Methanol, Ethanol, Acetone, and Mixed Solvents and Correlation Using the LIQUAC Model", Industrial & Engineering Chemistry Research, 49, 4981- 4988), o hidróxido de sódio pode ser usado como substituto do hidróxido de potássio como reagente alcalino a utilizar. O processo de extracção pode ser realizado em batelada ("batch") ou em continuo. Potassium hydroxide is the alkaline reagent used in the integrated protein and arabinoxylan extraction process of the proposed brewery. The use of this reagent leads to the formation of potassium chloride (KCl) after acidification with hydrochloric acid (HCl). The solubility of KCl in aqueous solutions containing 70% ethanol is 1,874 g per 100 g solution at 25 ° C and 2,834 g per 100 g solution at 40 ° C. The solubility of KCl in aqueous solutions containing 60% ethanol is 3,759 g per 100 g solution at 25 ° C and 5,444 g per 100 g solution at 40 ° C. (Pine, SP and Macedo, EA (2005), "Solubility of NaCl, NaBr, and KCl in Water, Methanol, Ethanol, and Their Mixed Solvents", Journal of Chemical and Engineering Data, 50, 29-32). Under these conditions, it is possible to separate the arabinoxylans by precipitation in these solutions, leaving the KCl in solution. Because the solubility of sodium chloride (NaCl) in ethanol solutions is comparable to or even greater than the solubility of KCl (Li, MY; Constantinescu, D .; Wang, LS; Mohs, A .; Gmehling, J. (2010), "Solubilities of NaCl, KCl, LiCl, and LiBr in Methanol, Ethanol, Acetone, and Mixed Solvents and Correlation Using the LIQUAC Model", Industrial & Engineering Chemistry Research, 49, 4981-4988), Sodium hydroxide may be used as a substitute for potassium hydroxide as an alkaline reagent to be used. The extraction process can be carried out in batch or continuous.
O invento é utilizado para a extracção simultânea de proteínas e arabinoxilanas por um processo que permite obter as proteínas e arabinoxilanas como produtos com valor acrescentado, de um modo integrado e sequencial. O processo de extracção, nos moldes em que é aplicado, permite a reciclagem do ácido cítrico e do etanol usados, assim como a recuperação de cloreto de potássio (ou cloreto de sódio) , com ganhos substanciais em termos económicos. O processo é ambientalmente limpo e permite o aproveitamento do subproduto mais abundante da indústria cervejeira para obter, de forma integrada, extractos ricos em proteínas e arabinoxilanas que podem ser utilizados como ingredientes alimentares. São exemplos de possíveis aplicações das arabinoxilanas a sua utilização como fibra dietética e pré- bióticos (Patentes US 2008/012162 Al e WO 2010/020639 Al) . O material rico em proteínas poderá ser utilizado para obtenção de hidrolizados proteicos passíveis de suplementação em diferentes tipos de alimentos, produtos dietéticos e produtos farmacêuticos.  The invention is used for the simultaneous extraction of proteins and arabinoxylans by a process to obtain proteins and arabinoxylans as value added products in an integrated and sequential manner. The extraction process, as it is applied, allows the recycling of used citric acid and ethanol as well as the recovery of potassium chloride (or sodium chloride), with substantial economic gains. The process is environmentally clean and allows the most abundant by-product of the brewing industry to be used to obtain protein-rich and arabinoxylan extracts that can be used as food ingredients. Examples of possible applications of arabinoxylans for use as dietary fiber and prebiotics (US Patents 2008/012162 Al and WO 2010/020639 Al). The protein rich material may be used to obtain supplemental protein hydrolyzates in different types of food, dietary products and pharmaceuticals.
Este processo integrado de extracção de proteínas e arabinoxilanas da dreche cervejeira, permite extrair 65-70% das arabinoxilanas da dreche e 86-96% da proteína, num processo em que a dreche não necessita de ser submetida a nenhum pré-tratamento como a secagem (WO 2008/047081 Al) e/ou a moagem (US 2010/7709033 B2), integra todas as extracções e permite reciclar o ácido cítrico e etanol, recuperando também o sal formado (KC1 ou NaCl) . This integrated brewing protein and arabinoxylans extraction process allows 65-70% of the brewery's arabinoxylans and 86-96% of the protein to be extracted, in a process where the dreche does not need to undergo any pre-treatment such as drying. (WO 2008/047081 Al) and / or grinding (US 2010/7709033 B2), incorporates all extractions and allows recycling of citric acid and ethanol, also recovering the salt formed (KCl or NaCl).
A utilização da dreche cervejeira sem tratamento prévio, através da utilização directa de reagentes alcalinos, de modo sequencial e com concentração crescente constitui a novidade do processo proposto. Outra inovação a realçar é a precipitação selectiva e sequencial das proteínas por acidificação do meio com uma solução de ácido cítrico e das arabinoxilanas por adição de HC1 seguida de etanol. Dadas as características químicas e de solubilidade dos reagentes envolvidos, nomeadamente, as suas características de solubilidade em água e em soluções de água e etanol, é possível a separação por precipitação diferenciada das proteínas e arabinoxilanas da dreche sem a co-precipitação de sais. Por fim, este processo inovador, permite a reciclagem do ácido cítrico e etanol, que poderão ser usados nos passos seguintes, e a recuperação do sal (cloreto de potássio ou cloreto de sódio) . The use of brewery without previous treatment through the direct use of alkaline reagents, sequentially and with increasing concentration is the novelty of the proposed process. Another innovation to highlight is the selective and sequential precipitation of proteins by acidifying the medium with a solution of citric acid and arabinoxylans by the addition of HCl followed by ethanol. Given the chemical and solubility characteristics of the reagents involved, namely their solubility characteristics in water and in water and ethanol solutions, it is possible to separate by precipitation of the proteins and arabinoxylans of the dreche without the co-precipitation of salts. Finally, this innovative process allows the recycling of citric acid and ethanol, which can be used in the following steps, and the recovery of salt (potassium chloride or sodium chloride).
0 processo usa como agente de neutralização do reagente alcalino uma solução saturada de ácido cítrico. Esta utilização inovadora tem a vantagem de utilizar um ácido de grau alimentar (E 330), com uma acidez mais elevada do que a maioria dos ácidos orgânicos
Figure imgf000006_0001
e, com elevada solubilidade em água (61,8% m/m a 25°C) e em etanol (38,3% m/m a 25°C) . A elevada solubilidade do ácido cítrico em água permite a precipitação selectiva das proteínas após a neutralização e acidificação do meio, preferencialmente até pH 3, valor possível de atingir devido à acidez deste ácido. A elevada solubilidade do ácido cítrico em etanol permite que este não co-precipite juntamente com as arabinoxilanas. Dado que o ácido cítrico é utilizado para neutralização dos extractos alcalinos, este vai existir na forma de citrato. Para a sua conversão a ácido cítrico, pelas razões acima indicadas, é necessário adicionar um ácido forte, razão pela qual o processo usa uma solução concentrada de ácido clorídrico (aditivo alimentar 507). Deste modo, o citrato é convertido em ácido cítrico, com formação de cloreto de potássio ou cloreto de sódio, dependendo do reagente alcalino utilizado. Estes sais, nas concentrações em que são formados, permanecem solúveis nas soluções de etanol, o que permite a recuperação das arabinoxilanas sem co-precipitação destes sais. O etanol é um solvente GRAS - 184.1293 (Generally Recognized as Safe), geralmente utilizado na purificação de polissacarídeos por precipitação . The process uses as an alkaline reagent neutralizing agent a saturated citric acid solution. This innovative use has the advantage of using a food grade acid (E 330) with a higher acidity than most organic acids.
Figure imgf000006_0001
and with high solubility in water (61.8% m / m at 25 ° C) and in ethanol (38.3% m / m at 25 ° C). The high solubility of citric acid in water allows selective precipitation of proteins after neutralization and acidification of the medium, preferably up to pH 3, a value that can be reached due to the acidity of this acid. The high solubility of citric acid in ethanol allows it not to co-precipitate along with arabinoxylans. Since citric acid is used to neutralize alkaline extracts, it will exist in the form of citrate. For its conversion to citric acid, For the reasons given above, a strong acid must be added, which is why the process uses a concentrated hydrochloric acid solution (food additive 507). Thus, the citrate is converted to citric acid, with formation of potassium chloride or sodium chloride, depending on the alkaline reagent used. These salts, at the concentrations at which they are formed, remain soluble in ethanol solutions, which allows the recovery of arabinoxylans without co-precipitation of these salts. Ethanol is a GRAS - 184.1293 (Generally Recognized as Safe) solvent, generally used for precipitation purification of polysaccharides.
Antecedentes da Invenção: Background of the Invention:
A dreche cervejeira, muitas vezes também designada como "resíduos de malte" ou "bagaço de malte", constitui o principal subproduto gerado durante o processo de produção de cerveja.  Brewery, often also referred to as "malt waste" or "malt bagasse", is the main by-product generated during the brewing process.
Em termos genéricos, pode-se dizer que a dreche cervejeira corresponde à fracção insolúvel do mosto, sendo essencialmente constituída pelas partes fibrosas (as cascas) dos grãos da cevada maltada e de resíduos de fracções insolúveis de cereais não maltados por vezes adicionados à receita (cevada, trigo, milho, entre outros), utilizados como principal matéria-prima na produção de cerveja. A dreche é separada do mosto por filtração, após a fase de "brassagem".  In general terms, it can be said that the brewery corresponds to the insoluble part of the must, consisting essentially of the fibrous parts (the husks) of malted barley grains and residues of insoluble unmalted cereals sometimes added to the recipe ( barley, wheat, maize, among others), used as the main raw material in beer production. The dreche is separated from the wort by filtration after the "brewing" phase.
A produção de cervejas origina 15-20 kg de dreche por cada hectolitro de cerveja produzida. A quantidade de dreche pode variar em função do tipo de cerveja produzida e do teor em humidade associado, sendo tipicamente entre 70 a 80%. Trata-se de um subproduto com elevado interesse, dadas as suas características em termos nutricionais e funcionais. Em Portugal, as dreches cervejeiras têm sido destinadas, praticamente exclusivamente, à alimentação animal, em particular de gado bovino. Contudo, os sectores da biotecnologia, da alimentação humana e da indústria farmacêutica podem constituir alternativas para a sua valorização . Beer production yields 15-20 kg of dreche per hectoliter of beer produced. The amount of dreche may vary depending on the type of beer produced and the associated moisture content, typically between 70 and 80%. It is a byproduct of high interest given its nutritional and functional characteristics. In Portugal beer breweries have been used almost exclusively for animal feed, in particular for cattle. However, the biotechnology, food and pharmaceutical sectors may offer alternatives for their recovery.
A dreche é um material lenhocelulósico que contém proteína (25-30%), lenhina (-28%), hemiceluloses (-25%, maioritariamente, arabinoxilanas ) e celulose (-17%) em resíduo seco (Celus,I; Brijs, K.; Delcour, J.A. (2006) "The effects of malting and mashing on barley protein extractability" Journal of Cereal Science, 44, 203-211; Teimo, J.; Westereng, B; Horn, S.J.; Forssell, P . ; Robertson, J.A.; Faulds, C.B.; Waldron K.W.; Buchert, J.; Eijsink, V.G.H. ( 2009 ) , "Enzymatic solubilization of Brewer 's Spent Grain by Combined action of carbohydrases and peptidases", Journal of Agricultural and Food Chemistry, 57, 3316-3324). As principais proteínas da dreche são as hordeínas (A, B e C) constituindo mais de 50% do total, seguidas das gluteninas . A fracção menos abundante inclui as albuminas (cerca de 2%) .  Dreche is a lignocellulosic material containing protein (25-30%), lignin (-28%), hemicelluloses (-25%, mostly arabinoxylans) and cellulose (-17%) in dry waste (Celus, I; Brijs, Delcour, JA (2006) "The effects of malting and mashing on barley protein extractability" Journal of Cereal Science, 44, 203-211; Teimo, J.; Westereng, B.; Horn, SJ; Forssell, P .; Robertson, JA; Faulds, CB; Waldron KW; Buchert, J .; Eijsink, VGH (2009), "Enzymatic Solubilization of Brewers' Spent Grain by Combined Action of Carbohydrases and Peptidases", Journal of Agricultural and Food Chemistry, 57, 3316-3324). Dreche's main proteins are hordeins (A, B and C) constituting more than 50% of the total, followed by glutenins. The least abundant fraction includes albumin (about 2%).
Diversas tentativas têm sido realizadas para obter produtos enriquecidos em proteínas da dreche, nomeadamente, após moagem e crivagem da dreche seca (Patentes americanas nos 4377601 e 4547382). No entanto, neste processo as cascas ficam extremamente finas após a moagem, pelo que a separação da fracção rica em proteínas se torna difícil e, por isso, o produto resultante ainda apresenta alto teor de proteínas (cerca de 40%) . Adicionalmente, como a dreche cervejeira contém 70-80% de humidade, é necessária uma grande quantidade de energia para a sua secagem. Alternativamente, a separação da dreche em duas fracções: proteica (rica em proteínas e gordura e pobre em fibras) e fibrosa (pobre em proteínas e rica em arabinoxilanas ) por prensagem e crivagem pode ser realizada a húmido, como descrevem a patente europeia 0443813A1 e o processo desenvolvido pela Heineken (Schwencke, K.V. (2006), "Sustainable, cost-effective, and feasible solutions for the treatment of brewers' spent grains," Master Brewers Association of the Américas Technical Quartely, 43, 199- 202) . No último caso, é preparada uma suspensão de dreche em água quente (80°C) e posteriormente esta é passada através de um crivo. Several attempts have been made to obtain products enriched in the drêche proteins, particularly after grinding and sieving the dried drêche (US Patent Nos 4,377,601 and 4,547,382). However, in this process the shells become extremely thin after grinding, so the separation of the protein rich fraction becomes difficult and therefore the resulting product still has a high protein content (about 40%). In addition, as the brewery contains 70-80% humidity, a large amount of energy is required for drying. Alternatively, the separation of the dreche into two fractions: protein (high protein and fat and low fiber) and fibrous (protein low and arabinoxylan) by pressing and sieving can be performed wet, as described in European Patent 0443813A1 and the process developed by Heineken (Schwencke, KV (2006), "Sustainable, cost-effective, and feasible solutions for the treatment of brewers' spent grains," Master Brewers Association of the Americas Technical Quartely, 43, 199-202). In the latter case, a dreche suspension is prepared in hot water (80 ° C) and then passed through a sieve.
Extracções da fracção proteica da dreche em meio alcalino estão descritas, nomeadamente, na patente Japonesa 51- 129776 que descreve a extracção alcalina da dreche a pH 11- 12, à temperatura de 104°C-121°C, com precipitação isoeléctrica das proteínas para obtenção da fracção proteica. Contudo, este processo envolve decomposição das proteínas, devido às condições drásticas de elevada temperatura da extracção, resultando num rendimento baixo das proteínas e deterioração da qualidade do produto. Além disso, o processo requer grande quantidade de energia para a extracção a elevada temperatura.  Extractions of the dreche protein fraction in alkaline media are described, inter alia, in Japanese patent 51-129776 which describes the alkaline extraction of the dreche at pH 11-12 at 104Â ° C-121Â ° C, with isoelectric precipitation of proteins to obtaining the protein fraction. However, this process involves protein decomposition due to the drastic conditions of high extraction temperature, resulting in low protein yield and deterioration of product quality. In addition, the process requires a large amount of energy for high temperature extraction.
Mais recentemente foi realizada na Universidade de Leuven na Bélgica (Celus, I.; Brijs, K.; Delcour, J.A. ( 2007 ) , "Enzymatic Hydrolysis of Brewers' spent grain proteins and technofuncional properties of the resulting hydrolysates", Journal of Agricultural and Food Chemistry, 55, 8703-8710) a preparação de concentrados de proteína da dreche após extracção alcalina (17% m/v) com hidróxido de sódio (NaOH) 0,1 M a 60°C. Após 60 minutos de extracção, as amostras foram filtradas e as proteínas do filtrado foram precipitadas por acidificação a pH 4 usando ácido cítrico 2 M. Most recently it was held at the University of Leuven in Belgium (Celus, I.; Brijs, K.; Delcour, JA (2007), "Enzymatic Hydrolysis of Brewers' spent grain proteins and technofunctional properties of the resulting hydrolysates", Journal of Agricultural and Food Chemistry, 55, 8703-8710) the preparation of protein concentrates from the table after alkaline extraction (17% w / v) with 0.1 M sodium hydroxide (NaOH) at 60 ° C. After 60 minutes of extraction, the samples were filtered and the filtrate proteins were precipitated by acidification to pH 4 using 2M citric acid.
O produto "MNG-3 /Biobran" tem um princípio activo desenvolvido a partir de arabinoxilanas extraídas do farelo do arroz (Ghoneum, M. & Gollapudi, S. (2003), "Modified arabinoxylan rice bran (MGN-3/biobran) sensitizes human T cell leukemia cells to death receptor (CD95) -induced apoptosis.", Câncer Letters, 201, 41-49), estruturalmente semelhantes às arabinoxilanas da dreche.  The product "MNG-3 / Biobran" has an active ingredient developed from arabinoxylans extracted from rice bran (Ghoneum, M. & Gollapudi, S. (2003), "Modified arabinoxylan rice bran (MGN-3 / biobran) sensitizers. human T cell leukemia cells to death receptor (CD95) -induced apoptosis. ", Cancer Letters, 201, 41-49), structurally similar to the arabinoxylans of the dreche.
Com o objectivo de extrair e caracterizar as arabinoxilanas da dreche cervejeira, foi realizado no Institute of Food Research (IFR), no Reino Unido (Mandalari, G.; Faulds, C. B.; Sancho, A. I.; Saija, A.; Bisignano, G.; LoCurto, R.; Waldron, K. W. (2005), "Fractionation and characterization of arabinoxylans from brewers' spent grain and wheat bran.", Journal of Cereal Science, 42, 205-212), um estudo em que as arabinoxilanas da dreche cervejeira foram extraídas com reagentes alcalinos (Mandalari et al . , 2005). Estas foram maioritariamente extraídas sequencialmente com soluções de hidróxido de potássio em concentração crescente de 0,5 M, 1 M e 4 M. Antes da extracção sequencial, a dreche cervejeira foi pré-tratada com uma solução de etanol a 80% (v/v) em refluxo, obtendo-se o resíduo insolúvel em etanol que foi submetido a duas lavagens durante duas horas, em primeiro lugar com água fria e depois com água quente. O resíduo insolúvel em água quente foi ainda submetido a um tratamento enzimático com pronase, uma enzima proteolítica . Só depois deste pré-tratamento é que a dreche foi submetida a tratamento com reagentes alcalinos. Os extractos alcalinos obtidos foram separados do resíduo por centrifugação e filtração, tendo sido neutralizados com ácido acético e dialisados exaustivamente contra água desionizada e, por fim, liofilizados . Com base nos estudos referidos, a presente invenção propõe um processo de extracção integrado de proteínas e arabinoxilanas da dreche cervejeira. Este processo tem a vantagem de extrair as proteínas e arabinoxilanas em simultâneo, que são separadas por alteração do pH do extracto alcalino em conjunto com a precipitação em etanol. A utilização de uma solução saturada de ácido cítrico como agente de neutralização permite que, após a precipitação das proteínas por alteração do pH e a sua remoção por filtração ou centrifugação, a solução contendo as arabinoxilanas possa ser tratada com etanol para precipitação selectiva dos polissacarídeos sem que haja co- precipitação do ácido cítrico. Para isso, é proposta uma solução inovadora de acidificação do extracto com um ácido forte, preferencialmente, o ácido clorídrico concentrado, que permite a protonação do citrato (insolúvel em soluções de etanol) com formação de ácido cítrico (solúvel em soluções de etanol) . Também não ocorre a co-precipitação dos sais formados (cloreto de potássio ou o cloreto de sódio) devido à sua solubilidade nas soluções de etanol propostas. Este processo permite a recolha das arabinoxilanas sem ser necessário recorrer a um passo de diálise, o que diminui significativamente o tempo de duração do processo. Num processo de diálise, há a necessidade de proceder a um mínimo de 3 mudanças de água, devendo cada água de diálise estar em contacto pelo mesmo 6 horas, o que totaliza 18 horas. A esta operação há a acrescentar o facto de as fracções ficarem diluídas pelo contacto com a água de diálise, tendo que se realizar um passo de concentração, moroso e com custos energéticos devido à necessidade de evaporação da água. Outra vantagem deste processo é a reutilização do ácido cítrico e do etanol usados, para além da recolha do cloreto de potássio ou cloreto de sódio entretanto formados. Até ao momento não foi descrito nenhum processo integrado de extracção das proteínas e arabinoxilanas da dreche, da sua recolha após utilização de ácido cítrico nem da reciclagem total dos reagentes utilizados, tal como se atesta pela consulta ao artigo de revisão recentemente publicado sobre dreche cervejeira e onde se referem várias possibilidades de utilização deste subproduto (Gupta, M. ; Abu-Ghannam, N.; Gallaghar, E. (2010), "Barley for brewing: characterist ic changes during malting, brewing and applications of its by- products.", Comprehensive Reviews in Food Science and Technology, 9, 318-328.) e o mesmo se verifica para a patente internacional WO 2008/047081 Al. In order to extract and characterize the arabinoxylans from the brewery, it was carried out at the Institute of Food Research (IFR) in the United Kingdom (Mandalari, G.; Faulds, CB; Sancho, AI; Saija, A.; Bisignano, G. ; LoCurto, R.; Waldron, KW (2005), "Fractionation and Characterization of Arabinoxylans from Brewers' Waste Grain and Wheat Bran.", Journal of Cereal Science, 42, 205-212), a study in which dreche arabinoxylans Brewers were extracted with alkaline reagents (Mandalari et al., 2005). These were mostly extracted sequentially with increasing 0.5 M, 1 M and 4 M potassium hydroxide solutions. Prior to sequential extraction, the brewery was pretreated with an 80% (v / v) ethanol solution. ) at reflux to give the ethanol-insoluble residue which was subjected to two washes for two hours, first with cold water and then with hot water. The hot water-insoluble residue was further subjected to enzymatic treatment with pronase, a proteolytic enzyme. Only after this pretreatment was the dreche treated with alkaline reagents. The obtained alkaline extracts were separated from the residue by centrifugation and filtration, neutralized with acetic acid and thoroughly dialyzed against deionized water and finally lyophilized. Based on the above studies, the present invention proposes an integrated protein and arabinoxylan extraction process from the brewery. This process has the advantage of simultaneously extracting the proteins and arabinoxylans, which are separated by changing the pH of the alkaline extract together with ethanol precipitation. The use of a saturated citric acid solution as a neutralizing agent allows, after precipitation of proteins by pH change and removal by filtration or centrifugation, the solution containing the arabinoxylans can be treated with ethanol for selective precipitation of polysaccharides without co-precipitation of citric acid. For this purpose, an innovative solution of acidification of the extract with a strong acid, preferably concentrated hydrochloric acid, which allows the protonation of citrate (insoluble in ethanol solutions) with formation of citric acid (soluble in ethanol solutions) is proposed. Co-precipitation of the formed salts (potassium chloride or sodium chloride) does not occur due to their solubility in the proposed ethanol solutions. This process allows the collection of arabinoxylans without the need for a dialysis step, which significantly shortens the process time. In a dialysis process, there is a need for a minimum of 3 water changes, each dialysis water being in contact for the same 6 hours, which totals 18 hours. In addition, the fractions are diluted by contact with dialysis water and a time consuming and costly concentration step is required due to the need for water evaporation. Another advantage of this process is the reuse of used citric acid and ethanol, in addition to the collection of potassium chloride. or sodium chloride however formed. So far, no integrated process has been described for the extraction of dreche proteins and arabinoxylans, their collection after use of citric acid or the total recycling of the reagents used, as evidenced by reference to the recently published review article on brewery and where various uses of this by-product are mentioned (Gupta, M.; Abu-Ghannam, N.; Gallaghar, E. (2010), "Barley for brewing: characterist ic changes during malting, brewing and applications of its by-products. ", Comprehensive Reviews in Food Science and Technology, 9, 318-328.) And the same is true for international patent WO 2008/047081 A1.
Sumário da Invenção : Summary of the Invention:
O presente invento propõe um processo de extracção integrado das proteínas e das arabinoxilanas (AX) da dreche cervejeira através da utilização de reagentes alcalinos. As proteínas e as arabinoxilanas são solúveis em reagentes alcalinos e, por isso, extractáveis nestes solventes. O extracto alcalino é separado da fracção sólida e a posterior acidificação com uma solução concentrada de ácido cítrico leva à precipitação das proteínas, permitindo a sua separação das arabinoxilanas, que permanecem solúveis. A fracção proteica extraída é lavada com etanol para a remoção do ácido cítrico vestigial, facilitando também a sua secagem. As arabinoxilanas são recuperadas por acidificação até pH inferior a 2 com uma solução concentrada de ácido clorídrico, seguido de adição de etanol até formar uma solução, de preferência, até 70% (v/v) em etanol, que leva à insolubili zação das arabinoxilanas e, consequentemente, à sua precipitação. As arabinoxilanas são recuperadas por decantação, filtração e/ou centrifugação. A fracção polissacarídica extraída é lavada com etanol para a remoção do ácido cítrico vestigial e secagem. A acidificação a pH inferior a 2 permite a protonação completa do ácido cítrico, sendo este assim solúvel nas soluções aquosas de etanol, permitindo a separação das arabinoxilanas sem a sua co-precipitação . 0 sal de KC1 ou NaCl entretanto formado pela acidificação do citrato a ácido cítrico é solúvel nas soluções de etanol, pelo que não co-precipita com as arabinoxilanas. The present invention proposes an integrated extraction process of proteins and arabinoxylans (AX) from the brewery through the use of alkaline reagents. Proteins and arabinoxylans are soluble in alkaline reagents and therefore extractable in these solvents. The alkaline extract is separated from the solid fraction and further acidification with a concentrated citric acid solution leads to the precipitation of the proteins, allowing them to separate from the remaining soluble arabinoxylans. The extracted protein fraction is washed with ethanol to remove trace trace citric acid, also facilitating its drying. The arabinoxylans are recovered by acidification to pH below 2 with a concentrated hydrochloric acid solution, followed by the addition of ethanol to form a solution, preferably up to 70% (v / v) in ethanol, which leads to the insolubilization of the arabinoxylans. and consequently to its precipitation. Arabinoxylans are recovered by decantation, filtration and / or centrifugation. The extracted polysaccharide fraction is washed with ethanol for removal of trace trace citric acid and drying. Acidification at pH below 2 allows complete protonation of citric acid, which is thus soluble in aqueous ethanol solutions, allowing the separation of arabinoxylans without their co-precipitation. The KCl or NaCl salt meanwhile formed by the acidification of citrate to citric acid is soluble in ethanol solutions and does not co-precipitate with arabinoxylans.
A dreche não necessita de qualquer tratamento prévio à extracção das proteínas e arabinoxilanas e como ocorre a extracção simultânea destes dois conjuntos de compostos, o processo é mais rápido e de mais fácil aplicação do que se fossem utilizados processos separados para a extracção das proteínas e arabinoxilanas. 0 ácido cítrico e o etanol usados no processo são reciclados e o cloreto de potássio ou cloreto de sódio formados dependendo do reagente alcalino utilizado no processo integrado da extracção das proteínas e das arabinoxilanas, são recuperados. As proteínas e as arabinoxilanas extraídas possuem valor acrescentado em relação à dreche, e são obtidos por um processo ambientalmente limpo, não envolvendo a utilização de componentes problemáticos ao nível da toxicidade e os reagentes utilizados têm grau alimentar pelo que podem ser usados como ingredientes na indústria alimentar, de produtos dietéticos e farmacêuticos. Dreche does not require any treatment prior to the extraction of proteins and arabinoxylans and as the simultaneous extraction of these two sets of compounds occurs, the process is faster and easier to apply than if separate processes for the extraction of proteins and arabinoxylans were used. . The citric acid and ethanol used in the process are recycled and the potassium chloride or sodium chloride formed depending on the alkaline reagent used in the integrated protein and arabinoxylan extraction process is recovered. Extracted proteins and arabinoxylans have added value in relation to the dreche, and are obtained by an environmentally clean process, not involving the use of toxic components and the reagents used are food grade and can therefore be used as ingredients in the industry. food, dietetic and pharmaceutical products.
0 processo de extracção das proteínas e das arabinoxilanas da dreche cervejeira é, assim, caracterizado por ser integrado e compreender as seguintes etapas:  The process of extracting proteins and arabinoxylans from the brewery is thus characterized by being integrated and comprising the following steps:
a) extracção da dreche com reagentes alcalinos;  (a) extraction of the laboratory with alkaline reagents;
b) separação do extracto alcalino; c) neutralização até o pH ser igual ou inferior a 4, preferencialmente pH 3, com solução concentrada de ácido cítrico; b) separation of the alkaline extract; c) neutralizing until the pH is 4 or less, preferably pH 3, with concentrated citric acid solution;
d) acidificação até o pH ser igual ou inferior a 2 com ácido clorídrico concentrado;  d) acidifying until the pH is 2 or less with concentrated hydrochloric acid;
e) adição de etanol, preferencialmente até perfazer 70% (v/v) ;  e) adding ethanol, preferably to 70% (v / v);
f) lavagem das fracções proteicas e das fracções de arabinoxilanas com etanol para eliminar eventuais vestígios de ácido cítrico e secagem das fracções.  (f) washing the protein fractions and arabinoxylan fractions with ethanol to remove any traces of citric acid and drying the fractions.
g) reciclagem do etanol e do ácido cítrico concentrado por destilação;  (g) recycling of ethanol and concentrated citric acid by distillation;
h) recuperação do cloreto de potássio ou cloreto de sódio ;  h) recovery of potassium chloride or sodium chloride;
i) repetição das etapas anteriores.  i) repetition of the previous steps.
O processo de extracção é caracterizado por o reagente alcalino utilizado na etapa a) ser o hidróxido de potássio ou o hidróxido de sódio. The extraction process is characterized in that the alkaline reagent used in step a) is potassium hydroxide or sodium hydroxide.
O processo de extracção é caracterizado por o reagente alcalino ser utilizado com um rácio de 1:2 (m/v) (massa de dreche por volume de solução do reagente alcalino) . The extraction process is characterized in that the alkaline reagent is used at a ratio of 1: 2 (w / v) (drought weight per volume of alkaline reagent solution).
O processo de extracção é caracterizado por o reagente alcalino ser utilizado com concentrações crescentes entre 0,1 M e 4 M, preferencialmente, 0,1 M, 0,5 M e 4 M. The extraction process is characterized in that the alkaline reagent is used with increasing concentrations between 0.1 M and 4 M, preferably 0.1 M, 0.5 M and 4 M.
O processo de extracção é caracterizado por a extracção decorrer durante 24 horas à temperatura ambiente a partir de uma amostra de dreche sem qualquer tipo de pré- tratamento 0 processo de extracção é caracterizado por poder ser realizado em batelada ou em continuo. The extraction process is characterized in that the extraction takes place for 24 hours at room temperature from a dreche sample without any pretreatment. The extraction process is characterized in that it can be carried out in batch or continuously.
0 processo de extracção é caracterizado por a etapa b) ocorrer por decantação, filtração e/ou centrifugação. The extraction process is characterized in that step b) occurs by decantation, filtration and / or centrifugation.
As proteínas e/ou as arabinoxilanas e/ou os seus derivados são caracterizados por serem obtidos pelo processos descritos anteriormente. The proteins and / or arabinoxylans and / or their derivatives are characterized by being obtained by the processes described above.
A utilização do resíduo obtido depois da extracção das proteínas e/ou das arabinoxilanas e/ou seus derivados é caracterizada por ser uma fonte de celulose e/ou fibra dietética insolúvel e/ou combustível e/ou matéria-prima para a indústria do papel. The use of the residue obtained after the extraction of proteins and / or arabinoxylans and / or their derivatives is characterized as a source of insoluble and / or combustible dietary cellulose and / or fiber and / or raw material for the paper industry.
Descrição Geral da Invenção: General Description of the Invention:
0 processo da presente invenção tem como base a extracção integrada e sequencial da fracção proteica e das AX da dreche com soluções de KOH com concentrações crescentes para o rácio de 1:2 (m/v) (massa de dreche por volume das soluções de KOH), à temperatura ambiente, durante 24 h. 0 fluxograma esquematiza o processo integrado para a extracção de proteínas e arabinoxilanas da dreche. Após extracção alcalina (1) com concentrações crescentes de KOH [0,1 M (l)a; 0,5 M (l)b e 4 M (l)c] e precipitação ácida com uma solução concentrada de ácido cítrico até pH inferior ou igual a 4, preferencialmente pH 3 (2), são obtidas três fracções proteicas designadas, respectivamente, por FP 1, FP 2 e FP 3. Na fracção solúvel em ácido efectua-se a precipitação das arabinoxilanas por acidificação com HC1 concentrado até pH inferior ou igual a 2 (3) e adição de etanol até formar uma solução a 70% (v/v), sendo obtidas três fracções de arabinoxilanas designadas, respectivamente, por AX 1, AX 2 e AX 3. Finalmente, efectua-se uma lavagem do resíduo da dreche com água, durante 24 h à temperatura ambiente (4) para maximização da remoção de arabinoxilanas; sendo obtida a última fracção de arabinoxilanas, designada por AX 4. The process of the present invention is based on the sequential and integrated extraction of the protein fraction and AX from the dreg with KOH solutions with increasing concentrations in the ratio of 1: 2 (m / v) (drab mass per volume of the KOH solutions). ) at room temperature for 24 h. The flowchart outlines the integrated process for the extraction of proteins and arabinoxylans from the dreche. After alkaline extraction (1) with increasing concentrations of KOH [0.1 M (1) a; 0.5 M (l) b and 4 M (l) c] and acid precipitation with a concentrated citric acid solution to pH less than or equal to 4, preferably pH 3 (2), are obtained three protein fractions designated respectively by FP 1, FP 2 and FP 3. In the acid-soluble fraction the arabinoxylans are precipitated by acidification with concentrated HCl to pH less than or equal to 2 (3) and addition of ethanol to a 70% (v / v) solution, yielding three arabinoxylan fractions designated respectively as AX 1, AX 2 and AX 3. Finally, the residue is washed dreche with water for 24 h at room temperature (4) to maximize the removal of arabinoxylans; the last arabinoxylan fraction, designated AX 4, being obtained.
Por último ocorre a reciclagem do ácido cítrico e etanol usados no processo integrado e recuperação do cloreto de potássio ou cloreto de sódio se, em vez do hidróxido de potássio for usado o hidróxido de sódio (5) . Finally there is recycling of citric acid and ethanol used in the integrated process and recovery of potassium chloride or sodium chloride if sodium hydroxide is used instead of potassium hydroxide (5).
Descrição Detalhada da Invenção: Detailed Description of the Invention:
1. Extracção com reagentes alcalinos  1. Extraction with alkaline reagents
As arabinoxilanas e as proteínas são extraídas por um processo sequencial com soluções crescentes de hidróxido de potássio (ou hidróxido de sódio) com as concentrações entre 0,1 M e 4 M, preferencialmente, de 0,1 M, 0,5 M e 4 M a partir de uma amostra de dreche cervejeira sem qualquer tipo de pré-tratamento . O fluxograma esquematiza o processo integrado para a extracção de proteínas e arabinoxilanas da dreche cervejeira.  Arabinoxylans and proteins are extracted by a sequential process with increasing solutions of potassium hydroxide (or sodium hydroxide) at concentrations between 0.1 M and 4 M, preferably 0.1 M, 0.5 M and 4 M. M from a sample brewery without any pretreatment. The flowchart outlines the integrated process for the extraction of proteins and arabinoxylans from the brewery.
As extracções com hidróxido de potássio ou hidróxido de sódio podem ser efectuadas à temperatura ambiente, durante 24 horas, com agitação ocasional. Pode ser adicionado metabissulfito de sódio 5 mM como agente antioxidante. Em alternativa, as extracções com hidróxido de potássio ou hidróxido de sódio podem ser realizadas por períodos mais curtos, se forem realizadas acima da temperatura ambiente. Na primeira extracção alcalina da dreche utiliza-se, na proporção 1:2 (m/v) (massa de dreche por volume das soluções de KOH) , uma solução aquosa de KOH 0,1 M + Na2S20s 5 mM, (la) do fluxograma. Na segunda extracção alcalina (extracção do resíduo 1) utiliza-se uma solução aquosa de KOH 0,5 M + Na2S205 5 mM, (lb) do fluxograma. A terceira extracção alcalina (extracção do resíduo 2) efectua-se com KOH 4 M + Na2S205 5 mM (lc) do fluxograma. Os extractos alcalinos obtidos (extracto 1, extracto 2 e extracto 3) são separados dos resíduos (resíduo 1, resíduo 2 e resíduo 3) por decantação, filtração e/ou centrifugação. Extractions with potassium hydroxide or sodium hydroxide may be carried out at room temperature for 24 hours with occasional stirring. 5 mM sodium metabisulphite may be added as an antioxidant agent. Alternatively, extractions with potassium hydroxide or sodium hydroxide may be performed for shorter periods if they are performed above room temperature. For the first alkaline extraction of the dreche, a ratio of 1: 2 (w / v) (drought mass per volume of KOH solutions) is used, an aqueous solution of 0,1 M KOH + 5 mM Na 2 S 2 0s, (la) of the flowchart. In the second alkaline extraction (extraction of the residue 1) is used an aqueous solution of KOH + 0.5 M Na 2 S 2 0 5 5 mM (lb) of the flowchart. The third alkaline extraction (residue extraction 2) is carried out with 4 M KOH + 5 mM Na 2 S 2 0 5 (1c) from the flow chart. The alkaline extracts obtained (extract 1, extract 2 and extract 3) are separated from the residues (residue 1, residue 2 and residue 3) by decantation, filtration and / or centrifugation.
2. Tratamento dos extractos 2. Treatment of extracts
Todos os extractos alcalinos obtidos após decantação, filtração e/ou centrifugação (Extracto 1, Extracto 2 e Extracto 3) são acidificados com uma solução de ácido cítrico concentrada até pH inferior ou igual a 4, preferencialmente pH 3, (2) do fluxograma. Este valor de pH permite a precipitação das proteínas da dreche cervejeira (maioritariamente, hordeínas e gluteninas) que são separadas por decantação e filtração e lavadas com etanol. Neste passo são obtidas três fracções proteicas designadas, respectivamente, por FP 1, FP 2 e FP 3. As fracções solúveis em ácido 1, 2 e 3, que contêm as arabinoxilanas , são, por sua vez, acidificadas com ácido clorídrico concentrado até um pH inferior a 2 para que o ácido cítrico seja solúvel em etanol. As arabinoxilanas são recuperadas por precipitação em soluções a 70 % (v/v) em etanol, e separadas por decantação, filtração e/ou centrifugação e lavadas com etanol, (3) do fluxograma. Neste passo, são obtidas três fracções de arabinoxilanas designadas, respectivamente, por AX 1, AX 2 e AX 3.  All alkaline extracts obtained after decantation, filtration and / or centrifugation (Extract 1, Extract 2 and Extract 3) are acidified with a concentrated citric acid solution to pH less than or equal to 4, preferably pH 3, (2) of the flowchart. This pH value allows the precipitation of the brewery proteins (mostly hordeins and glutenins) which are separated by decantation and filtration and washed with ethanol. Three protein fractions designated respectively as FP 1, FP 2 and FP 3 are obtained in this step. The acid soluble fractions 1, 2 and 3 containing the arabinoxylans are in turn acidified with concentrated hydrochloric acid to pH less than 2 so that citric acid is soluble in ethanol. Arabinoxylans are recovered by precipitation in 70% (v / v) solutions in ethanol, and separated by decantation, filtration and / or centrifugation and washed with ethanol (3) from the flow chart. In this step, three arabinoxylan fractions designated respectively as AX 1, AX 2 and AX 3 are obtained.
Com o intuito de maximizar a remoção de arabinoxilanas efectua-se uma lavagem final do resíduo 4, durante 24 h à temperatura ambiente (ou em alternativa por períodos mais curtos, usando temperaturas mais elevadas), (4) do fluxograma . 0 sobrenadante obtido após precipitação das arabinoxilanas com etanol é destilado, o que permite a recuperação do etanol e da solução de ácido cítrico contendo KC1 (ou NaCl) . Esta solução aquosa é tratada com etanol para precipitação do sal, que é recolhido por decantação e/ou filtração e/ou centrifugação. A solução de ácido cítrico é evaporada para recolha do etanol e do ácido cítrico que fica na solução aquosa resultante da destilação. Esta solução de ácido cítrico será posteriormente utilizada, (5) do fluxograma. Neste passo pode ocorrer o reaproveitamento de proteínas e de arabinoxilanas que não tenham precipitado nas fases (2) e (3) e que voltam a entrar no circuito. In order to maximize arabinoxylan removal, residue 4 is finally washed for 24 h at room temperature (or alternatively for shorter periods using higher temperatures) (4) from the flowchart. The supernatant obtained after precipitation of the arabinoxylans with ethanol is distilled, which allows the recovery of ethanol and the KCl (or NaCl) containing citric acid solution. This aqueous solution is treated with ethanol for salt precipitation, which is collected by decantation and / or filtration and / or centrifugation. The citric acid solution is evaporated to collect the ethanol and the citric acid remaining in the distillation aqueous solution. This citric acid solution will be further used (5) from the flowchart. In this step the reuse of proteins and arabinoxylans which have not precipitated in phases (2) and (3) and which return to the circuit may occur.
Exemplo : Example:
Para uma mais fácil compreensão da invenção descrevem-se de seguida exemplos de realizações preferenciais do invento, as quais, contudo, não pretendem, limitar o objecto da presente invenção.  For an easier understanding of the invention, the following are examples of preferred embodiments of the invention which, however, are not intended to limit the scope of the present invention.
A presente invenção é agora descrita com mais detalhe no exemplo seguinte, em que foram utilizadas amostras de 135 g de dreche cervejeira sem qualquer pré-tratamento e com um teor de humidade entre 72% e 76%. A extracção foi realizada à temperatura ambiente. The present invention is now described in more detail in the following example, where 135 g brewery samples were used without any pretreatment and with a moisture content between 72% and 76%. Extraction was performed at room temperature.
Na primeira extracção alcalina, à amostra de dreche foram adicionados 278 mL de uma solução contendo hidróxido de potássio 0,1 M e metabissulfito de sódio 5 mM. O contacto da amostra com estes reagentes realizou-se durante 24 horas, procedendo-se a agitação ocasional. Por decantação e filtração do extracto alcalino, obtiveram-se o Resíduo 1 e o Extracto 1. 0 Resíduo 1 foi extraído com 278 mL de solução de hidróxido de potássio 0,5 M e metabissulfito de sódio 5 mM, nas mesmas condições de tempo, temperatura e agitação. Procedeu-se à separação do resíduo do extracto alcalino por decantação e filtração, sendo designados, respectivamente, por Resíduo 2 e Extracto 2. In the first alkaline extraction, 278 ml of a solution containing 0.1 M potassium hydroxide and 5 mM sodium metabisulfite were added to the dreche sample. The sample was contacted with these reagents for 24 hours with occasional stirring. Decantation and filtration of the alkaline extract gave Residue 1 and Extract 1. Residue 1 was extracted with 278 mL of 0.5 M potassium hydroxide solution and 5 mM sodium metabisulfite under the same time, temperature and stirring conditions. The residue was separated from the alkaline extract by decantation and filtration, respectively designated Residue 2 and Extract 2.
O Resíduo 2 foi extraído com 278 mL de solução de hidróxido de potássio 4 M e metabissulfito de sódio 5 mM, com contacto durante 24 horas e agitação ocasional. Separou-se o resíduo do extracto alcalino por decantação e filtração, que foram designados por Resíduo 3 e Extracto 3, respectivamente . Residue 2 was extracted with 278 mL of 4 M potassium hydroxide solution and 5 mM sodium metabisulfite, with 24 hour contact and occasional stirring. The residue from the alkaline extract was separated by decantation and filtration, which were designated Residue 3 and Extract 3, respectively.
Ao Resíduo 3 adicionaram-se 200 mL de água, deixando-se em contacto durante 24 horas, com agitação ocasional. Separou- se o resíduo final do extracto 4 por decantação e filtração O extracto 4 foi acidificado com uma solução concentrada de ácido cítrico até pH próximo de 4 e depois com ácido clorídrico concentrado até um pH inferior a 2. De seguida, as arabinoxilanas foram precipitadas com adição de etanol até perfazer uma concentração em etanol de 70% (v/v), permanecendo o ácido cítrico solúvel, por estar totalmente protonado (na forma ácida) ao pH inferior a 2. To Residue 3 was added 200 mL of water and left in contact for 24 hours with occasional stirring. The final residue of extract 4 was separated by decantation and filtration. Extract 4 was acidified with a concentrated citric acid solution to pH around 4 and then with concentrated hydrochloric acid to a pH below 2. Then the arabinoxylans were precipitated. with the addition of ethanol to 70% (v / v) ethanol concentration, the soluble citric acid remaining as it is fully protonated (in acid form) at a pH below 2.
Efectuou-se uma avaliação prévia do teor proteico das amostras de dreche, obtendo-se concentrações que variaram entre 8 e 10 g por 100 g de dreche tal e qual (32 e 40 g de dreche seca) . Na primeira extracção alcalina, (l)a, são separadas entre 29-38% do total de proteínas presentes na dreche. Na segunda extracção alcalina, (l)b, são separadas entre 26-46% do total de proteínas presentes na dreche. Na terceira extracção alcalina, (l)c, são separadas entre 10- 19 % do total de proteínas presentes na dreche 0 resíduo final apresenta quantidades baixas de proteína na ordem dos 3-10%. A prior evaluation of the protein content of the dreche samples was carried out, obtaining concentrations ranging from 8 to 10 g per 100 g of dreche as such (32 and 40 g of dry dreche). In the first alkaline extraction, (1) a, between 29-38% of the total proteins present in the dreche are separated. In the second alkaline extraction, (1) b, between 26-46% of the total proteins present in the nursery are separated. In the third alkaline extraction, (1) c, are separated between 10- 19% of the total proteins present in the dreche The final residue has low amounts of protein in the order of 3-10%.
As fracções proteicas resultantes do processo extractivo FP The protein fractions resulting from the extractive process FP
1, FP 2 e FP 3 não são puras, contêm 60% de proteína, arabinoxilanas , gordura, cinzas e outros constituintes da dreche. O estudo da composição destas fracções proteicas revelou que incluem maioritariamente hordeínas A, B e C. 1, FP 2 and FP 3 are not pure, contain 60% protein, arabinoxylans, fat, ashes and other constituents of dreche. Studying the composition of these protein fractions revealed that they mostly include hordeins A, B and C.
Efectuou-se uma avaliação prévia do teor de arabinoxilanas em amostras de dreche cervejeira que variaram entre 23,3%- 35,3% determinado relativamente à massa seca da dreche. A prior assessment of the arabinoxylan content was carried out in brewery samples ranging from 23.3% - 35.3% determined relative to the dry mass of the dreche.
Na primeira extracção alcalina (l)a são extraídas 11% do total das arabinoxilanas presentes na dreche cervejeira, em que 3% estão presentes na fracção FP 1 e as restantes 7% constituem a fracção AX 1. Na segunda extracção alcalina (l)b são extraídas 18% do total das arabinoxilanas presentes na dreche cervejeira, em que 4% estão presentes na fracção FP 2 e as restantes 14% constituem a fracção AXIn the first alkaline extraction (l) a 11% of the total arabinoxylans present in the brewery are extracted, where 3% are present in fraction FP 1 and the remaining 7% constitute fraction AX 1. In the second alkaline extraction (l) b 18% of the total arabinoxylans present in the brewery are extracted, with 4% being present in fraction FP 2 and the remaining 14% constituting fraction AX.
2. Na terceira extracção alcalina (l)c são extraídas 27% do total das arabinoxilanas presentes na dreche cervejeira constituem a fracção AX 3. Com o intuito de maximizar a remoção das arabinoxilanas da dreche cervejeira efectuou-se uma lavagem final do resíduo 4 em que se consegue extrair 9% de arabinoxilanas que constitui a fracção AX 4. O resíduo final apresenta quantidades baixas de arabinoxilanas na ordem dos 15%. 2. In the third alkaline extraction (l) and 27% of the total arabinoxylans present in the brewery are extracted, they constitute fraction AX 3. In order to maximize the removal of the arabinoxylans from the brewery, a final wash of residue 4 was carried out. 9% arabinoxylans can be extracted from the AX 4 fraction. The final residue has low amounts of arabinoxylans in the order of 15%.
Perante estes resultados, pode-se afirmar que o resíduo final é constituído maioritariamente por celulose, alguma lenhina e as arabinoxilanas remanescentes, enquanto as proteínas existem numa quantidade vestigial. Os resultados obtidos com este processo de extracção integrado de proteínas e arabinoxilanas quando extrapolados para uma tonelada de dreche tal e qual, indicam que podem ser extraídas, entre 86-96% das proteínas e 66% das arabinoxilanas totais da dreche cervejeira. Do total de arabinoxilanas extraídas por este processo, 59%, que corresponde a 40 kg de arabinoxilanas das 68 kg iniciais são solúveis em água, permanecendo as restantes 7% na fracção proteica que correspondem a 4,6 kg de arabinoxilanas das 68 kg iniciais. Estes valores são meramente indicativos, dependendo do tipo de dreche cervejeira usada. In view of these results, it can be said that the final residue consists mainly of cellulose, some lignin and the remaining arabinoxylans, while proteins exist in a trace amount. The results obtained with this integrated protein and arabinoxylan extraction process when extrapolated to one ton of dreche as such indicate that between 86-96% of the protein and 66% of the total arabinoxylan can be extracted from the brewery. Of the total arabinoxylans extracted by this process, 59%, which corresponds to 40 kg of arabinoxylans of the initial 68 kg, are water soluble, with the remaining 7% remaining in the protein fraction corresponding to 4.6 kg of arabinoxylans of the initial 68 kg. These values are purely indicative, depending on the type of brewery used.
Descrição das Figuras Description of the Figures
Para uma mais fácil compreensão da invenção juntam-se em anexo as figuras, as quais, representam realizações preferenciais do invento que, contudo, não pretendem, limitar o objecto da presente invenção.  For an easier understanding of the invention, attached are figures which represent preferred embodiments of the invention which, however, are not intended to limit the scope of the present invention.
Figura 1 - Extracção sequencial da fracção proteica e de AX da dreche com soluções de KOH com concentrações crescentes para o rácio de 1:2, durante 24 à temperatura ambiente. Figure 1 - Sequential extraction of protein fraction and AX from the dreche with KOH solutions with increasing concentrations to 1: 2 ratio for 24 at room temperature.
Referências : References :
Artigos científicos:  Scientific articles:
Celus, I; Brijs, K.; Delcour, J.A. (2006) "The effects of malting and mashing on barley protein extractability" Journal of Cereal Science, 44, 203-211.  Celus, I; Brijs, K .; Delcour, J.A. (2006) "The effects of malting and mashing on barley protein extractability" Journal of Cereal Science, 44, 203-211.
Celus, I.; Brijs, K.; Delcour, J.A. (2007), "Enzymatic Hydrolysis of Brewers' spent grain proteins and technofuncional properties of the resulting hydrolysates", Journal of Agricultural and Food Chemistry, 55, 8703-8710. Ghoneum, M. & Gollapudi, S. (2003), "Modified arabinoxylan rice bran (MGN-3/biobran) sensitizes human T cell leukemia cells to death receptor (CD95 ) -induced apoptosis.", Câncer Letters, 201, 41-49.  Celus, I .; Brijs, K .; Delcour, J.A. (2007), "Enzymatic Hydrolysis of Brewers' spent grain proteins and technofunctional properties of the resulting hydrolysates", Journal of Agricultural and Food Chemistry, 55, 8703-8710. Ghoneum, M. & Gollapudi, S. (2003), "Modified Arabinoxylan rice bran (MGN-3 / biobran) sensitizing human T cell leukemia cells to death receptor (CD95) -induced apoptosis.", Cancer Letters, 201, 41- 49
Gupta, M.; Abu-Ghannam, . ; Gallaghar, E. (2010), "Barley for brewing: characteristic changes during malting, brewing and applications of its by-products . ", Comprehensive Reviews in Food Science and Technology, 9, 318-328.  Gupta, M .; Abu-Ghannam,. ; Gallaghar, E. (2010), "Barley for brewing: characteristic changes during malting, brewing and applications of its by-products.", Comprehensive Reviews in Food Science and Technology, 9, 318-328.
Mandalari, G.; Faulds, C. B . ; Sancho, A. I.; Sai a, A.; Bisignano, G.; LoCurto, R.; Waldron, K. W. (2005), "Fractionation and characterization of arabinoxylans from brewers' spent grain and wheat bran.", Journal of Cereal Science, 42, 205-212 Mandalari, G .; Faulds, C. B. ; Sancho, A. I .; Sai a, A .; Bisignano, G .; LoCurto, R .; Waldron, K. W. (2005), "Fractionation and characterization of arabinoxylans from spent grain brewers and wheat bran.", Journal of Cereal Science, 42, 205-212
Pinho, S.P. e Macedo, E.A. (2005), "Solubility of NaCl, NaBr, and KC1 in Water, Methanol, Ethanol, and Their Mixed Solvents", Journal of Chemical and Engineering Data, 50, 29-32.  Pinho, S.P. and Macedo, E.A. (2005), "Solubility of NaCl, NaBr, and KCl in Water, Methanol, Ethanol, and Their Mixed Solvents", Journal of Chemical and Engineering Data, 50, 29-32.
Schwencke, K.V. (2006), "Sustainable, cost-effective, and feasible solutions for the treatment of brewers' spent grains" Master Brewers Association of the Américas Tecnhical Quartely, 43, 199-202.  Schwencke, K.V. (2006), "Sustainable, Cost-Effective, and Feasible Solutions for the Treatment of Brewers' Spent Grains" Master Brewers Association of the Americas Technological Quartely, 43, 199-202.
Teimo, J.; Westereng, B; Horn, S.J.; Forssell, P . ; Robertson, J.A.; Faulds, C.B.; Waldron K.W.; Buchert, J.; Eijsink, V.G.H. (2009) "Enzymatic solubilization of Brewer ' s Spent Grain by Combined action of carbohydrases and peptidases" Journal of Agricultural and Food Chemistry, 57, 3316-3324. Teimo, J .; Westereng, B; Horn, SJ; Forssell, P. ; Robertson, JA; Faulds, CB; Waldron KW; Buchert, J .; Eijsink, VGH (2009) "Enzymatic solubilization of Brewer's Spent Grain by Combined Action of Carbohydrases and Peptidases "Journal of Agricultural and Food Chemistry, 57, 3316-3324.
Li, M.Y.; Constantinescu, D.; Wang, L.S.; Mohs, A.; Gmehling, J. (2010), "Solubilities of NaCl, KC1, LiCl, and Li Br in Methanol, Ethanol, Acetone, and Mixed Solvents and Correlation Using the LIQUAC Model", Industrial & Engineering Chemistry Research, 49, 4981-4988.  Li, M.Y .; Constantinescu, D .; Wang, L.S .; Mohs, A .; Gmehling, J. (2010), "Solubilities of NaCl, KCl, LiCl, and Li Br in Methanol, Ethanol, Acetone, and Mixed Solvents and Correlation Using the LIQUAC Model", Industrial & Engineering Chemistry Research, 49, 4981-4988.
Patentes : Patents:
U.S. Pat. N°4377601 (Dreese, P.C.; Hoseney, R.C.), 1983. U.S. Pat. No. 4,377,601 (Dreese, P.C .; Hoseney, R.C.), 1983.
"Method of Removing Hulls from Brewer ' s spent grain". "Method of Removing Hulls from Brewer's Spent Grain".
U.S. Pat. N° 4547382 (Gannon, J.J.; Hawley P.), 1985. U.S. Pat. No. 4547382 (Gannon, J.J.; Hawley P.), 1985.
"Process of separating Husks from dried spent grains". "Process of separating husks from dried spent grains".
E.P. 0 443 813 Al (Kishi, S . ; Kimura, T . ; Minami, T . ; E.P. 0 443 813 Al (Kishi, S.; Kimura, T.; Minami, T.;
Kobayashi, H.) 1991. Protein-rich Products of brewer ' s spent grain origin. Kobayashi, H.) 1991. Protein-rich Products of brewers' spent grain origin.
Japanese Laid-Open Patent Publication No. Sho 51-129776 (U.S. Pat. N° 2008/012162 Al (Delcour, J.; Courtin, C . ; Broekaert, W.; Swennen, K.; Verbeke, K.; Rutgeers, P.), 2008. "Prebiotic preparation" .  Japanese Laid-Open Patent Publication No. Sho 51-129776 (US Patent No. 2008/012162 A1 (Delcour, J .; Courtin, C.; Broekaert, W .; Swennen, K .; Verbeke, K .; Rutgeers, P.), 2008. "Prebiotic preparation".
W.O. Pat. N° 2008/047081 Al (Waldron, K.W.; Waldron (husband) , F . ; Faulds, C.B.; Wilde, P.J.), 2008. W.O. Pat. No. 2008/047081 Al (Waldron, K.W .; Waldron (husband), F.; Faulds, C.B .; Wilde, P.J.), 2008.
"Production surfactants" . Production surfactants.
W.O. Pat. N° 2010/020639 Al (Ekhart, P.F.; Van Der Saag, H.; Pos-Semiers, S . ; Van Den Abbeele, P . ; Van De Wiele, T.), 2010. "Arabinoxylans for modulating the barrier function of the intestinal surface".  W.O. Pat. No. 2010/020639 Al (Ekhart, PF; Van Der Saag, H.; Pos-Semiers, S.; Van Den Abbeele, P.; Van De Wiele, T.), 2010. "Arabinoxylans for modulating the barrier function of the intestinal surface ".
U.S. Pat. N° 7709033 B2 (Kivst, S . ; Carlsson, T . ; Lawther, J.M.; DeCastro, F.B.), 2010. "Process for the fract ionation of cereal brans". As reivindicações seguintes representam adicionalmente realizações preferenciais da presente invenção. US Pat. No. 7709033 B2 (Kivst, S.; Carlsson, T.; Lawther, JM; DeCastro, FB), 2010. "Process for the fracture of cereal brans". The following claims further represent preferred embodiments of the present invention.

Claims

REIVINDICAÇÕES
1. Processo de extracção das proteínas e das arabinoxilanas da dreche cervejeira, caracterizado por ser integrado e compreender as seguintes etapas: 1. Process for the extraction of proteins and arabinoxylans from the brewery, characterized by being integrated and comprising the following steps:
a) extracção das proteínas e arabinoxilanas da dreche com reagentes alcalinos;  (a) extraction of the proteins and arabinoxylans from the dreche with alkaline reagents;
b) separação do extracto alcalino;  b) separation of the alkaline extract;
c) neutralização até o pH ser igual ou inferior a 4, preferencialmente pH 3, com solução concentrada de ácido cítrico;  c) neutralizing until the pH is 4 or less, preferably pH 3, with concentrated citric acid solution;
d) acidificação até o pH ser igual ou inferior a 2 com ácido clorídrico concentrado;  d) acidifying until the pH is 2 or less with concentrated hydrochloric acid;
e) adição de etanol, preferencialmente até perfazer 70% (v/v) ;  e) adding ethanol, preferably to 70% (v / v);
f) lavagem das fracções proteicas e das fracções de arabinoxilanas com etanol para eliminar eventuais vestígios de ácido cítrico e secagem das fracções.  (f) washing the protein fractions and arabinoxylan fractions with ethanol to remove any traces of citric acid and drying the fractions.
g) reciclagem do etanol e do ácido cítrico concentrado por destilação;  (g) recycling of ethanol and concentrated citric acid by distillation;
h) recuperação do cloreto de potássio ou cloreto de sódio ;  h) recovery of potassium chloride or sodium chloride;
i) repetição das etapas anteriores.  i) repetition of the previous steps.
2. Processo de extracção, de acordo com a reivindicação anterior, caracterizado por o reagente alcalino utilizado na etapa a) ser o hidróxido de potássio ou o hidróxido de sódio . Extraction process according to the preceding claim, characterized in that the alkaline reagent used in step a) is potassium hydroxide or sodium hydroxide.
3. Processo de extracção de acordo com a reivindicação 2, caracterizado por o hidróxido de potássio ou hidróxido de sódio ser utilizado com um rácio de massa de dreche por volume de solução do reagente alcalino de 1:2 (m/v) . Extraction process according to claim 2, characterized in that the potassium hydroxide or sodium hydroxide is used with a ratio of dreic mass per volume of alkaline reagent solution of 1: 2 (m / v).
4. Processo de extracção de acordo com as reivindicações 2- 3 caracterizado por o reagente alcalino a ser utilizado com concentrações crescentes entre 0,1 M e 4 M, preferencialmente 0,1 M, 0,5 M e 4 M. Extraction process according to claims 2-3, characterized in that the alkaline reagent is to be used with increasing concentrations between 0.1 M and 4 M, preferably 0.1 M, 0.5 M and 4 M.
5. Processo de extracção de acordo com qualquer das reivindicações anteriores caracterizado por a extracção decorrer durante 24 horas à temperatura ambiente a partir de uma amostra de dreche sem qualquer tipo de pré- tratamento . Extraction process according to one of the preceding claims, characterized in that the extraction takes place for 24 hours at room temperature from a dreche sample without any pretreatment.
6. Processo de extracção, de acordo com as reivindicações anteriores, caracterizado por o processo de extracção ser realizado em batelada ou em continuo. Extraction process according to the preceding claims, characterized in that the extraction process is carried out in batch or continuously.
7. Processo de extracção de acordo com a reivindicação 1 caracterizado por a etapa b) ocorrer por decantação, filtração e/ou centrifugação. Extraction process according to claim 1, characterized in that step b) takes place by decantation, filtration and / or centrifugation.
8. Proteínas e/ou arabinoxilanas e/ou seus derivados são caracterizados por serem obtidos pelo processos descrito nas reivindicações anteriores. Proteins and / or arabinoxylans and / or derivatives thereof are characterized by being obtained by the processes described in the preceding claims.
9. Utilização do resíduo obtido depois da extracção das proteínas e/ou das arabinoxilanas e/ou seus derivados pelo processo descrito nas reivindicações 1-7 caracterizada por ser uma fonte de celulose e/ou fibra dietética insolúvel e/ou combustível e/ou matéria-prima para a indústria do papel . Use of the residue obtained after extraction of the proteins and / or arabinoxylans and / or their derivatives by the process described in claims 1-7 characterized in that it is a source of insoluble and / or combustible dietary cellulose and / or fiber and / or matter. for the paper industry.
PCT/IB2010/055699 2010-11-24 2010-12-09 Integrated process for extracting proteins and arabinoxylans from brewer's spent grain WO2012069889A1 (en)

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EP4427599A1 (en) 2023-03-10 2024-09-11 Proteinrise Spólka z ograniczona odpowiedzialnoscia Method of protein extraction

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021237363A1 (en) * 2020-05-28 2021-12-02 Terra Bioplastics Inc. System and method for producing byproducts from spent grains
EP4427599A1 (en) 2023-03-10 2024-09-11 Proteinrise Spólka z ograniczona odpowiedzialnoscia Method of protein extraction
PL444063A1 (en) * 2023-03-10 2024-09-16 Proteinrise Spółka Z Ograniczoną Odpowiedzialnością Protein extraction method

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