Method for upgrading and reconstructing waste coffee grounds into antioxidant dietary fibers

Abstract

A method for upgrading waste coffee grounds into antioxidant dietary fibers comprises the following steps: a) performing microwave-assisted extraction (MAE) on a waste coffee grounds (SCG) material in water at a temperature of at most 200 DEG C for a time of at least 5 minutes; and then b) subjecting the waste coffee grounds material extracted according to step a) to enzyme-assisted extraction (EAE) in an aqueous solution comprising an enzyme having endoglucanase and / or cellulase activity, and recovering soluble substances. In a preferred embodiment, the SCG is pre-treated prior to the MAE extraction step, the pre-treatment being selected from pulsed electric field treatment or high voltage extraction, or both.

Description

Technical Field

[0001] The present invention relates to an improved process for upcycling spent coffee grounds (SCG) into antioxidant dietary fiber. Background Art

[0002] Coffee is a widely consumed, high-value product worldwide, and its consumption continues to grow, reaching approximately 10.1 billion kilograms in 2020, an increase of 1 billion kilograms from 2015 (International Coffee Organization, 2021). All steps in coffee processing generate significant amounts of waste annually. The production of instant coffee and brewing coffee at the home / retail level generate approximately 6 million tons of spent coffee grounds (SCG) annually.

[0003] In line with circular economy principles, recent research efforts have focused on investigating the composition of SCG to explore its potential for reuse. Polysaccharides, oligosaccharides, lipids, fatty acids, amino acids, proteins, alkaloids (e.g., caffeine, trigonellin), phenols, minerals, lignin, melanoidins, and volatile compounds are all valuable components of SCG.

[0004] SCG has functional qualities due to the high content of fiber, tannins and phenolics and therefore has a high antioxidant potential. SCG fiber is largely insoluble and consists mainly of cellulose and hemicellulose, with a high amount of lignin also present.

[0005] In addition, SCG also contains a large amount of lipids and proteins, which have high biological value. It has been suggested that SCG can be used as a bulk material for a variety of purposes, including adsorbents, fillers, polymer production additives, animal feed supplements and soil fertilizers. However, SCG is also a source of valuable bioactive compounds. The most important of these are diterpenoid esters and phenolic substances, while condensed tannins and hydrolyzable tannins are other active polyphenols present in SCG. Melanoidins are the end products of the Maillard reaction, which have high molecular weight and variable composition, and usually contain SCG phenolic substances. Melanoidins have different biological activities, including prebiotics, antioxidants and antibacterials. Dietary melanoidins avoid the digestive process in the gastrointestinal tract and become substrates for the production of short-chain fatty acids (SCFAs) by the intestinal flora, and then regulate the microbiota.

[0006] In addition to melanoidins, manno-oligosaccharides (MOS) are also defined as prebiotics due to their ability to influence the intestinal microbiota by promoting the proliferation of specific beneficial species. Therefore, given the high concentrations of fiber and melanoidins, components from SCG can be used as prebiotics, or they can also be used as commensals to support the growth of probiotics.

[0007] Extraction of these compounds from food by-products typically employs polar or intermediate-polarity solvents. Unfortunately, most of the valuable coffee molecules are extracted during brewing, while the remaining molecules in SCG are trapped within a rich fibrous network, requiring extensive hydrolysis to extract and utilize them as a source of functional ingredients. Enzyme-assisted extraction (EAE) holds great promise for achieving efficient and economical hydrolysis of plant matrices. Due to the high content of lignin, cellulose, and hemicellulose, different enzymes, including β-glucanases, cellulases, and hemicellulases, can be used to solubilize the insoluble components of SCG.

[0008] In addition to enzymes, physical methods can also be used to hydrolyze SCG, and in particular microwave treatment (microwave-assisted extraction, MAE) is very effective for extracting oligosaccharides and phenolic fractions (Passos et al., 2013). Whether enzymatic or physical extraction, particle size is an important factor affecting the extraction of phenolic compounds.

[0009] The object of the present invention is to develop a new method to increase the amount of soluble matter extracted from SCG, thereby obtaining antioxidant dietary fiber suitable for human consumption.

[0010] Another object of the present invention is to provide a method which allows obtaining prebiotic functional ingredients comprising oligosaccharides, preferably in combination with polyphenols, with an improved yield, which have a positive effect on the intestinal microbiota and can trigger anti-inflammatory pathways at the level of intestinal epithelial cells.

[0011] Another object of the present invention is to provide a process which allows obtaining prebiotic functional ingredients with improved antioxidant properties. Summary of the Invention

[0012] The subject matter of the invention is defined by the appended claims.

[0013] The method of the present invention combines a microwave assisted extraction (MAE) step performed on an unprocessed spent coffee grounds material with an enzyme assisted extraction (EAE) step performed on a spent coffee grounds material that is the extracted residue of the MAE step.

[0014] According to the present invention, it has been found that by combining the two steps, the total amount of valuable soluble substances is increased, since this total amount is higher than that obtained by subjecting the same raw SCG to MAE and EAE separately, under the same process conditions as those adopted for each step of the combined process. Furthermore, it has been found that by carrying out the extraction process in the order of MAE followed by EAE, the amount of soluble substances obtained is significantly increased compared to the amount obtained using the reverse order.

[0015] The method of the present invention is further described by the following detailed description and working examples with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the following examples and figures:

[0017] SCG-C represents the amount of soluble matter (SM) released from SCG in water after cryogenic grinding;

[0018] SCG-M represents the amount of soluble matter (SM) released from SCG in water after MAE;

[0019] SCG-E represents the amount of soluble substances (SM) released from SCG in water after EAE;

[0020] SCG-CE represents the amount of soluble matter (SM) released from SCG in water after cryogenic grinding and EAE;

[0021] SCG-ME represents the amount of soluble matter (SM) released from SCG in water after MAE and EAE.

[0022] In the attached figure:

[0023] Figure 1 A step-by-step approach for upcycling SCG into a prebiotic functional ingredient is described. Figure 1 The grinding and degreasing steps are optional;

[0024] Figure 2 is a pie chart showing the composition of raw SCG used in the following examples;

[0025] Figure 3 is a schematic flow chart showing the material balance of the method of the present invention;

[0026] Figure 4 is a bar graph showing the quantification of galactose, fucose, arabinose, rhamnose, glucose, mannose, and fructose in raw SCG extracted from an industrial coffee brewing process and in SCG after combined microwave extraction and enzyme treatment (SCG-ME);

[0027] Figure 5 are bar graphs showing the quantification of galactose, fucose, arabinose, glucose, mannose, rhamnose, and xylose in SCG from café au lait before any treatment (SCG), after cryogenic grinding (SCG-C), after MAE (SCG-M), after EAE (SCG-E), after the combination of cryogenic grinding and EAE (SCG-CE), and after the combination of MAE and EAE (SCG-ME).

[0028] Figure 6 Shown are the composition and properties of the functional ingredients obtained after the spray drying step.

[0029] Figure 7 are bar graphs showing the amount of soluble fraction released in water of SCG before any treatment (SCG), after cryogenic grinding (SCG-C), after MAE (SCG-M), after EAE (SCG-E), after cryogenic grinding and enzyme treatment combination (SCG-CE), and after MAE and EAE combination (SCG-ME);

[0030] Figure 8 are bar graphs showing the antioxidant properties of SCG from café au lait before any treatment (SCG), after cryogenic grinding (SCG-C), after MAE (SCG-M), after EAE (SCG-E), after the combination of cryogenic grinding and EAE (SCG-CE), and after the combination of MAE and EAE (SCG-ME) as evaluated by DPPH, ABTS, and FRAP assays. DETAILED DESCRIPTION

[0031] Analytical methods

[0032] The raw SCG used in the process of the invention may come from different sources; it may be waste from coffee machines (from domestic appliances or from coffee shops) or from industrial coffee brewing plants.

[0033] Figure 2 Shown is the approximate composition of raw SCG after heat stabilization from an industrial coffee brewing process.

[0034] Table 1 shows the approximate composition of raw SCG after heat stabilization from coffee house production.

[0035] Table 1. Approximate composition of spent coffee grounds after thermal stabilization.

[0036]

[0037] A proximate analysis was performed to determine the percentages of moisture, ash, protein, fat, soluble and insoluble fiber, and carbohydrates. Details are as follows:

[0038] - moisture is determined according to AACC method n. 44-15.02;

[0039] - ash content determined according to AACC method n.08-01;

[0040] - Protein content was assessed by the Dumas method using a Flash EA 1112NC analyzer (ThermoFisher Scientific Inc, Waltman, USA) following the manufacturer's protocol using a conversion factor of 6.25 commonly applied to SCG ( Massaya et al., 2019 );

[0041] - Fat content is determined by Soxhlet extraction. Weigh 3-5 grams of sample into an extraction thimble. Add 200 mL of petroleum ether at 40-60°C to a flat-bottom flask containing the zeolite. Carefully record all weights for fat determination. Connect the flat-bottom flask to an extractor equipped with a sample thimble and a cooler and place it on a heating block. Turn on the heating and extract for 5 hours. After cooling, evaporate the flat-bottom flask using a Büchi rotary evaporator. Leave the thimble and evaporated flat-bottom flask in a fume hood overnight to allow any residual petroleum ether to evaporate. Weigh the dried flat-bottom flask and use the amount of fat extracted to determine the fat content by comparing it with the amount of sample used.

[0042] - Soluble and insoluble fiber were determined by using the K-TDFR-200A Megazyme kit (Megazyme, Ireland) according to AACC method n. 32-07.01.

[0043] Specifically, to determine the soluble dietary fiber (SDF) content, 4 volumes of 60°C preheated EtOH (95%) were added to the sample in a 50 mL Greiner tube. The precipitate was allowed to form at room temperature for 60 minutes. The sample was centrifuged and the supernatant discarded. The precipitate was transferred to a 15 mL Greiner tube of known weight. The tube was sealed with tissue paper and the precipitate was freeze-dried.

[0044] The analysis was done in duplicate and the SDF was determined using Equation 2, taking into account the dilution used during extraction.

[0045] SDF:

[0046] - Carbohydrates were determined by the difference method (Nitisewojo, 1995).

[0047] - Determine the total amount of dry matter present in the soluble fraction (SDM) by taking ±2 mL of the soluble fraction (supernatant after centrifugation) and transferring it to an aluminum pan. Carefully record the weight of the sample and the aluminum pan. Dry the pan in an oven at 100°C overnight until the weight is constant. Determine the dry weight and soluble solids content of the sample, taking into account the dilution used during the extraction process, and use equal weights.

[0048] Formula 1 is expressed as mg SDM / g SCG.

[0049]

[0050] Total phenolic content (TPC) is determined based on the Folin-Ciocalteu method of Singleton and Rossi (1965). Folin-Ciocalteu is a bright yellow reagent. By measuring the absorbance at a wavelength of 760 nm, the number of hydroxyl groups present in the sample can be determined. These hydroxyl groups are directly correlated with the presence of phenols.

[0051] 50g of NaCO3 is dissolved in 250mL of MiliQ water to make 20% NaOH. A gallic acid calibration curve is drawn using the following concentrations: 0.025, 0.05, 0.1, 0.15, and 0.25mg / mL. 0.2mL of sample (standard or blank) is mixed with 1.88mL of MiliQ water, 0.11mL of Folin-phenol reagent, and 0.31mL of NaCO3. The tube is carefully shaken and placed in a dark environment at room temperature for 60 minutes. Then, absorbance is measured using a spectrophotometer (Cary 60 spectrophotometer (Agilant Technologies Inc.)), and gallic acid equivalents (GAE) are calculated by comparing the calibration value with the sample absorbance.

[0052] Among the SCGs analyzed from industrial coffee brewing processes ( Figure 2 ) had the highest concentration of insoluble fiber (50.83 g / 100 g SCG dry matter) and the lowest concentration of soluble fiber (4.00 g / 100 g SCG dry matter). The fat content of the analyzed raw materials was approximately 20% by weight (dry matter), while the fat content of the café au lait was only about 15% (Table 1).

[0053] Antioxidant activity

[0054] The antioxidant activity of SCG extract was evaluated by DPPH, ABTS and FRAP assays ( Figure 8 ).

[0055] For DPPH analysis, the method described by Brand-Williams et al. (1995) was used with some modifications. A 0.1 mM solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol was prepared. 50 μL of sample (standard or blank) was added to 1.95 mL of DPPH solution. The sample was vortexed and incubated at room temperature in the dark for 60 minutes. After incubation, the absorbance was measured at a wavelength of 515 nm.

[0056] Process Description

[0057] In a preferred optional embodiment, the raw SCG is ground prior to the extraction step to reduce its particle size, preferably to a particle size with D[4,3] of less than 1 mm, preferably less than 100 microns.

[0058] Since raw SCG typically contains water (up to 70% by weight), it is preferred to dry the raw SCG to a moisture content of about 10% or less before grinding.

[0059] For the purpose of grinding, any grinding method known in the art can be used as long as the method ensures a particle size of less than 1000-900 microns; however, grinding using a ball mill or more preferably using cryogenic grinding is particularly preferred.

[0060] As mentioned above, the amount of fat can vary over a relatively large range depending on the source of SCG. Although the fat content has little effect on the yield of soluble substances in the process, in a preferred embodiment of the present invention, in order to improve the sensory properties of the target probiotic functional ingredients and reduce oxidation phenomena, it is recommended to apply a defatting step before extraction to reduce the fat content to less than 5%, preferably less than 2%.

[0061] Defatting can be accomplished by methods known in the art, but preferably Soxhlet extraction, preferably using petroleum ether. The extraction time is 2 hours to 16 hours.

[0062] Alternatively, supercritical carbon dioxide extraction can be used.

[0063] According to the present invention, microwave-assisted extraction (MAE) is used to increase the soluble fraction. Microwave-assisted extraction is a process that uses microwave energy to heat the solvent in contact with the sample to separate the analytes in the sample matrix into the solvent. The ability to rapidly heat the sample-solvent mixture is inherent to MAE and is a major advantage of this technique. By using a closed container, extraction can be performed at elevated temperatures, which accelerates the mass transfer of the target compound from the sample matrix. Single-wave extraction is preferred.

[0064] Water is the preferred solvent for MAE, preferably in a mass ratio (SCG / water) of 1 :3 to 1 :4. MAE may be applied at a temperature of about 200°C for a time ranging from 2 to 10 min, preferably 5 to 10 min.

[0065] According to the method of the present invention, the MAE extracted SCG slurry is then directly subjected to enzyme assisted extraction (EAE). Alternatively, the soluble material can be separated and recovered from the solid extracted SCG phase before being subjected to EAE.

[0066] The EAE step b) is performed in an aqueous solution comprising an enzyme having endo-beta-glucanase and / or cellulase activity.

[0067] In addition, an enzyme having mannanase, xylanase, and / or hemicellulase activity or side activity may also be used or added to the enzyme having endo-β-glucanase and / or cellulase activity. The enzyme concentrations are preferably 33 to 66 μL / g SCG for an enzyme having 300 amyloglucosidase units / mL of endo-β-glucanase activity and 11 to 40 μL / g SCG for an enzyme having 700 endo-glucanase units / g of cellulase activity.

[0068] The extraction is preferably carried out at a temperature ranging from 45 to 65°C, at a pH of 4.5 to 6, for a period of 3 to 15 hours.

[0069] Preferred commercial enzymes that may be used include Viscozyme TM L and Celluclast TM 1.5L.

[0070] Viscozyme L is a widely used food-grade enzyme that hydrolyzes plant tissue (Novozymes, 2023); Viscozyme's primary enzymatic activity is endo-β-glucanase, but it also has secondary activities as xylanase, cellulase, and hemicellulase. Celluclast 1.5L is a food-grade enzyme with cellulase activity that breaks down plant fibrous tissue and improves the extraction yield of vegetable products (Novozymes, 2023).

[0071] The enzyme reaction can be stopped by raising the temperature (eg, to 90°C) and maintaining that temperature (eg, for 10 minutes).

[0072] After EAE, the supernatant containing soluble material was collected after centrifugation.

[0073] The soluble matter can be dried by methods known in the art (including freeze drying and drum drying) to finally obtain the antioxidant dietary fiber. Preferably, the antioxidant dietary fiber is obtained by spray drying.

[0074] In other embodiments of the present invention, the raw SCG may be subjected to a pretreatment selected from pulsed electric field treatment (PEF) or high pressure extraction (HPE) or both prior to MAE extraction. PEF and HPE may preferably be performed as pretreatments after grinding and defatting, between or after these steps, but must be performed before MAE extraction.

[0075] PEF is a non-thermal method that improves mass transfer by electroporating cell membranes using brief (nanosecond to millisecond) and moderately intense electrical pulses. This method, with low energy consumption and minimal environmental impact, accelerates the release of intracellular chemicals and improves the extraction rate and yield of various components from plant matrices. PEF is preferably used under the following parameters: field strength of 1.5 kV / cm; 20 Hz; pulse width of 2 μs; conductivity of 0.5 mS / cm; and test temperature of 10-12°C. The number of pulses can range from 1,000 to 10,000, with a solid / liquid ratio of 30-42.5%.

[0076] HPE operates at low temperatures (typically up to 60°C) and high pressures (typically 100-600 MPa) to rapidly extract compounds, requiring low amounts of organic solvents while providing similar recoveries to other extraction techniques. As mentioned above, PEF is a good alternative to traditional thermal treatments because it reduces extraction time and solvent consumption, and increases extraction yields.

[0077] To achieve the goals of this patent, HPE can also be used as a pretreatment. In this case, the SCG sample can be suspended in deionized water at a ratio of 2 / 3 v / v and then packed into a plastic bag under vacuum. The pressure and time conditions can range from 200 MPa to 600 MPa and 1 to 5 minutes, respectively.

[0078] Example 1

[0079] Cryogenic grinding (optional) and particle size assessment

[0080] By using 6875D SCG was finely ground using a cryogenic grinder (Spex Sample Prep, USA). Specifically, 20 g of SCG was ground three times, each lasting 5 minutes, with a setting of 10 cycles per second (cps).

[0081] Particle size distribution was analyzed using a Mastersizer 3000 (Malvern Instruments, Worcestershire, UK) equipped with an Aero S dispersion accessory. A particle refractive index of 1.530 was optimized for coffee grounds. D[3,2] (surface weight mean diameter), D[4,3] (volume weight mean diameter), and D(50) (volume median diameter) values were obtained. In addition, the parameters D(10) and D(90) were calculated and represent the proportion of particles with a diameter less than the respective parameter value (10% and 90%), respectively.

[0082] The particle size distribution of dried SCG was characterized by the following parameters: D[3,2] of 102.8 ± 2.6 μm, D[4,3] of 372.4 ± 7.2 μm, D(10) of 34.2 ± 0.5 μm, D(50) of 292.3 ± 4.9 μm, and D(90) of 638 ± 11.7 μm. The cryogenic grinding process significantly reduced the particle size of SCG. In fact, the D[3,2] of SCG-C was 33.7 ± 0.6 μm, D[4,3] of 93.6 ± 3.3 μm, D(10) of 15.0 ± 0.2 μm, D(50) of 44.4 ± 0.9 μm, and D(90) of 243.1 ± 8.5 μm. Thus, the reduction in D(50) was the greatest, resulting in an 85% reduction in the particle size of 50% of the SCG-C particles.

[0083] In a previous study of SCG (from an Iberital IB7 coffee machine), cryogenic grinding resulted in a total extraction of 40% of soluble matter.

[0084] Example 2: Degreasing step d) (optional)

[0085] The defatting process is preferably performed when the fat content in the raw SCG extracted by Soxhlet extraction is high (eg >15%).

[0086] 3 grams of SCG were weighed in a thimble and placed in an extractor connected to a flat bottom flask filled with 200 mL of petroleum ether and placed on a heater. After 5 hours of extraction, the thimble containing defatted SCG was left overnight to dry.

[0087] Example 3: Microwave-assisted extraction step a)

[0088] SCG was processed by using Monowave 400 (Anton Paar, Austria).

[0089] The Anton Paar Monowave product line is a series of high-performance single-mode microwave reactors designed for small to medium-sized microwave syntheses (installed microwave power: 850 W (single magnetron)).

[0090] In a G30 wide neck glass vial were added 4 g of SCG and 12 g of demineralized water.

[0091] The treatment was carried out using a Monowave 400, with a stirrer speed of 300 rpm and a maximum power of 850 W to reach 200° C., maintaining the temperature at 200° C. for 10 minutes, and then reducing the temperature to 70° C. The time required to reach the set temperature was 145 seconds, and a maximum pressure of 30 bar was reached. Including cooling to 55° C., the total treatment time was approximately 27 minutes.

[0092] To optimize extraction time, we studied the soluble matter in SCG extracted using only a single wave, with extraction times ranging from 10 minutes to 2 minutes. There was no significant difference between 10 minutes and 5 minutes, but there was a difference between 10 minutes and 2 minutes. Therefore, the preferred extraction time is 5 to 10 minutes, with 5 minutes being the most preferred.

[0093] Example 4: Enzyme-assisted extraction step b)

[0094] Using commercial enzyme Viscozyme TM and Celluclast TM The slurry produced by the MAE step of Example 3 or the unprocessed cryogenically ground SCG of Example 1 were subjected to enzyme-assisted extraction using the following process conditions:

[0095] μL enzyme / g SCG: 66 μL / g Viscozyme

[0096] 22.6μL / g Celluclast

[0097] and 33 μL / g Viscozyme

[0098] 11.2μL / g Celluclast

[0099] Temperature 55℃

[0100] pH: 5.95

[0101] The duration is 4 to 14 hours.

[0102] In terms of soluble matter, no significant differences were observed between the two enzyme amounts and treatment times: the soluble matter was approximately 30-33%.

[0103] Example 5: Spray Drying Step

[0104] A Büchi mini spray dryer (Büchi Laboratoriums-Technik, Switzerland) was used. At least 100 mL of supernatant extract was used in the spray drying. A peristaltic pump pumped the SCG supernatant to the atomizer. A fluid nozzle was used for atomization. Internally supplied compressed air was used. The compressed air was regulated by a flow meter, and cooling water was circulated through the jack around the nozzle. The inlet dry air was set to 120°C, the aspiration rate was 95%, and the pump was 20%. The inlet dry air flowed through the main chamber simultaneously with the spray after passing through an electric heater. The dry powder sample was collected from the bottom of the cyclone separator.

[0105] The supernatant obtained after centrifugation of the MAE and EAE extracts of Example 4 was spray-dried under the following conditions:

[0106] Inlet temperature: 120-160℃

[0107] Suction rate: 95%

[0108] Pump: 20%

[0109] Example 6

[0110] Figure 3 The layout of the method of the present invention according to a specific working example is shown.

[0111] 3 g of dried SCG from an industrial coffee brewing process was diluted in water (12 mL of water) at a 1:4 dilution and subjected to MAE and EAE sequentially according to the preferred conditions determined in the previous examples. The enzyme reaction was then stopped by raising the temperature to 90°C for 10 minutes; 24 mL of water was added.

[0112] The dry matter in 36 mL was 0.95 g, with a solubility of 31.7%. The extracted slurry was centrifuged to obtain approximately 23.5 mL of supernatant from 36 mL (yield 65.4%). The supernatant was spray-dried to obtain 0.43 g of the desired functional ingredient; the overall yield of the process was 14.3%.

[0113] Example 7

[0114] Unprocessed SCG from industrial coffee brewing plants was used, which had a fat content of approximately 20%.

[0115] Grinding process: step c)

[0116] The grinding process was performed to reduce the particle size of the sample, as this should facilitate the following steps. The SCG was processed by using a Bosch TSM6A013B household grinder, where 30 g of SCG was ground for 5 minutes using a rated power of 180 W.

[0117] Degreasing process: step d)

[0118] Degreasing was performed to account for the high amount of fat previously detected on the SCG.Degreasing was performed in a beaker where 50 g of SCG was stirred in 200 mL of petroleum ether at 300 rpm for 16 hours.

[0119] Microwave-assisted extraction: step a)

[0120] Defatted SCG was processed according to the method described in Example 3. All samples were frozen at -20°C until subsequent processing or analysis.

[0121] Enzyme extraction: step b)

[0122] Enzyme extraction was performed sequentially at a ratio of 1:4 (SCG:water). L is mainly composed of endo-β-glucanase and The 1.5L incubation system consists of a 1000 FBG / g Viscozyme enzyme to hydrolyze (1,3)- or (1,4)-linkages in β-D glucans, and a 700 EGU / g cellulase enzyme to hydrolyze (1,4)-β-D-glucosidic bonds in cellulose and other β-D glucans. Both were supplied by Novozymes (Denmark). The recommended dosages are 200-400 mL and 100-200 mL per ton of vegetables, respectively. The optimal pH ranges are 3.3-5.5 and 4.0-6.0, respectively, and the optimal temperatures are 40-50°C and 50-60°C, respectively. Based on previous experiments, the concentrations used were 33 μL / g Viscozyme and 11.2 μL / g Celluclast, respectively. The incubation time was 4 hours. The temperature was set at 55°C and the pH at 5.95. The enzyme reaction was stopped at 90°C for 10 minutes. The supernatant was collected after centrifugation at 4500 rpm for 10 minutes.

[0123] Spray drying step

[0124] A Büchi mini spray dryer (Büchi Laboratoriums-Technik, Switzerland) was used. At least 100 mL of supernatant extract was used for spray drying. A peristaltic pump pumped the SCG supernatant to the nebulizer. A fluid nozzle was used for atomization. Internally supplied compressed air was used. The compressed air was regulated by a flow meter, and cooling water was circulated through the jack around the nozzle. The inlet dry air was set to 120°C, the suction rate was 95%, and the pump was 20%. This inlet dry air flowed through the main chamber simultaneously with the spray after passing through an electric heater. The dry powder sample was collected from the bottom of the cyclone separator.

[0125] Example 8

[0126] Unprocessed SCG from an industrial coffee brewing process was used, which had a fat content of approximately 20%.

[0127] Grinding step c)

[0128] The grinding process was performed to reduce the particle size of the sample, as this should facilitate the subsequent steps. SCG was processed by using: a) a 6875D Freezer / Mill cryogenic grinder, for example, grinding 20 g of SCG three times for 5 minutes each time, set at 10 cycles per second (cps); b) a Bosch TSM6A013B household grinder, grinding 30 g of SCG for 5 minutes, rated at 180 W; c) a ball mill, grinding 30 g of SCG at 100 rpm for 30 minutes.

[0129] Degreasing step d)

[0130] Degreasing was performed to address the high levels of fat previously detected. Various methods were employed, including a) Soxhlet extraction: 3 g of SCG was weighed in a cannula and placed in an extractor connected to a flat-bottomed flask filled with 200 mL of petroleum ether, which was then placed on a heater. After 5 hours of extraction, the cannula containing the defatted SCG was left overnight to dry. b) and c) degreasing was performed without heat treatment, but rather by simply stirring the sample with a solvent such as petroleum ether. In b), 10 g of SCG was mixed with 225 mL of petroleum ether and stirred at 300 rpm for 30 minutes; in c), 50 g of SCG was mixed with 200 mL of petroleum ether and stirred at 300 rpm for 16 hours.

[0131] Microwave-assisted extraction step a)

[0132] Defatted SCG were processed using a Monowave 400 (Anton Paar, Austria) according to Example 3. All samples were frozen at -20°C before subsequent processing or analysis.

[0133] Enzyme extraction step b)

[0134] Enzyme extraction was performed using a 1:4 ratio (SCG:water) as described in Example 7. L and The concentrations in 1.5 L were a) 66 μL / g Viscozyme and 22.6 μL / g Celluclast; and b) 33 μL / g Viscozyme and 11.2 μL / g Celluclast. The incubation times were a) 14 hours and b) 4 hours. The temperature was set to 55°C and the pH to 5.95. The enzyme reaction was stopped at 90°C for 10 minutes. The supernatant was collected after centrifugation at 4500 rpm for 10 minutes.

[0135] Spray drying step

[0136] A Büchi mini spray dryer (Büchi Laboratoriums-Technik, Switzerland) was used. At least 100 mL of supernatant extract was used for spray drying. The effect of adding additives was studied by the following process: 5% maltodextrin was added to the supernatant extract and the solution was stirred until the maltodextrin was dissolved before spray drying. A peristaltic pump pumped the SCG supernatant to the atomizer. A fluid nozzle was used for atomization. An internally supplied compressed air was used. The compressed air was regulated by a flow meter and cooling water was circulated through the jack around the nozzle. The inlet dry air was set to 120°C, the suction rate was 95%, and the pump was 20%. This inlet dry air flowed through the main chamber simultaneously with the spray after passing through an electric heater. The dry powder sample was collected from the bottom of the cyclone separator.

[0137] result

[0138] The antioxidant activities measured with DPPH and ABTS were 33 mM Trolox eq / g and 14 mg Trolox eq / g, respectively. The total polyphenol content was 28.46 mg gallic acid eq / g.

[0139] Example 9

[0140] Unprocessed SCG from an industrial coffee brewing process was used, which had a fat content of approximately 20%.

[0141] Pulsed Electric Field (PEF)

[0142] Spent coffee grounds were pretreated by pulsed electric field (PEF). The treatment was performed by mixing the sample with 40.5% MS water, with a voltage of 1.5 kV and a pulse number of 10,000.

[0143] Grinding step c)

[0144] A grinding process has been performed to reduce the particle size of the sample, as this should facilitate the following steps. SCG was ground using a Bosch TSM6A013B household grinder for 5 minutes with a rated power of 180W.

[0145] Degreasing step d)

[0146] Soxhlet extraction In this step, 3 grams of SCG were weighed into a thimble and placed into an extraction beaker filled with petroleum ether. The extraction beaker was placed into the instrument. The temperature was set to 135°C. After 3 hours of degreasing, the thimble containing the defatted SCG was left to dry overnight. The sample was pre-treated before analysis.

[0147] Microwave-assisted extraction step a)

[0148] Defatted SCG were processed by using Monowave 400 (Anton Paar, Austria) according to Example 3. All samples were frozen at -20°C before subsequent processing or analysis.

[0149] Enzyme-assisted extraction step b)

[0150] Enzyme extraction was performed in the order described in Example 7, step b).

[0151] result

[0152] Antioxidant activity was measured by DPPH and dry matter.

[0153] The HPE-pretreated sample had a dry matter content of 44.17 mM Trolox eq / g, with a dry matter content of 7.6%. Enzymatic treatment of the HPE-pretreated spent coffee grounds sample increased the yield to 12.96%, and the antioxidant activity, measured by DPPH, was 54.26 mM Trolox eq / g. A combination of enzyme treatment and single-wavelength-assisted extraction of the HPE-pretreated sample demonstrated a dry matter yield of 38%.

[0154] Example 10

[0155] High Pressure Method (HPE)

[0156] High-pressure extraction (HPE) operates at low temperatures (typically up to 60°C) and high pressures (typically 100-600 MPa) to rapidly extract compounds with low organic solvent requirements while providing similar recoveries to other extraction techniques. It is a good alternative to traditional thermal treatments because it reduces extraction time and solvent consumption, while increasing extraction yields. It is set up using a 1 / 1.5 ratio of water at 600 MPa for 5 minutes.

[0157] Grinding step c)

[0158] The SCG was ground using a Bosch TSM6A013B household grinder for 5 minutes, with a rated power of 180W.

[0159] Degreasing step d)

[0160] According to the defatting step in Example 9, Soxhlet extraction was used Degrease.

[0161] Microwave-assisted extraction step a)

[0162] SCG was processed as described in Example 3 by using Monowave 400 (Anton Paar, Austria).

[0163] Enzyme-assisted extraction step b)

[0164] Enzyme extraction was performed as described in Example 9 using a 1:4 ratio (SCG:water).

[0165] result

[0166] Antioxidant activity was measured by DPPH and dry matter.

[0167] The HPE-pretreated sample had a dry matter content of 50.59 mM Trolox eq / g, with a dry matter content of 5.98%. Enzymatic treatment of the HPE-pretreated spent coffee grounds increased the yield to 11.44%, and the antioxidant activity, measured by DPPH, was 52.86 mM Trolox eq / g. A combination of enzyme treatment and single-wave assisted extraction of the HPE-pretreated sample showed a dry matter yield of 38%.

[0168] chemical properties

[0169] The soluble matter in all extracts was evaluated. Overall, the soluble matter ranged from 3.92 ± 0.24 to 33.53 ± 0.48 g / 100 g of SCG ( Figure 7 ).

[0170] Cryogenic grinding increased the soluble matter extracted from SCG-C by 40% compared to SCG (3.92 ± 0.24 g / 100 g of SCG) and from SCG-CE by 30% compared to SCG-E (12.96 ± 0.22 g / 100 g of SCG).

[0171] Characteristics of antioxidant dietary fiber

[0172] Table 2 below shows the properties of the spray dried extracts using MAE followed by EAE.

[0173] Table 2

[0174]

[0175] Figure 4 Shown are the quantification of galactose, fucose, arabinose, rhamnose, glucose, mannose and fructose in SCG after a combination of microwave extraction and enzymatic treatment (SCG-ME from an industrial coffee brewery).

[0176] Mannose (41.35 ± 0.79 mg / g SCG dm), glucose (35.5 ± 3.28 mg / g SCG dm), and galactose (23.81 ± 0.08 mg / g SCG dm) were the most abundant sugars detected in SCG-ME. As observed in previous studies (not reported here), enzymatic treatment had a greater impact on sugar concentrations than microwave treatment. The high concentrations of mannose and galactose in SCG-ME confirm the breakdown of galactomannan polymers from insoluble fibers. SCG can be considered a good source of galactomannans, which are composed of linked 1,4-mannan chains and galactose derivatives with varying degrees of branching.

[0177] like Figure 8 As shown in the bar graphs, SCG-ME exhibited the highest free radical scavenging activity, with values of 89.21 ± 0.70 and 30.31 ± 0.18 μmol Trolox / g SCG in the ABTS and DPPH assays, respectively, and 126.97 ± 4.14 μmol FeSO₄ in the FRAP assay, demonstrating how both microwave and enzyme treatments enhanced the antioxidant properties of SCG. Indeed, SCG-M and SCG-E were other samples with high antioxidant properties, with SCG-M exhibiting higher values in all three assays, but with no statistical difference in the DPPH assay. Cryogenic grinding did not enhance the antioxidant properties of SCG, and no statistical differences were found between SCG and SCG-C, or between SCG-E and SCG-CE.

[0178] According to the present invention, it has been found that the benefits of the present invention in terms of the amount of soluble matter extracted are significantly increased if the EAE step is performed after the MAE step, rather than in the reverse order; by comparison, the following examples show the results of performing the MAE step after the EAE step.

[0179] Example 11 (Comparative): Enzyme-assisted extraction followed by microwave-assisted extraction

[0180] The SCG used was obtained from an industrial coffee brewing process, dried to a moisture content of approximately 5%, and ground as described in Example 7. grind:

[0181] 1. Degrease SCG with petroleum ether at a ratio of 1:4 (SCG / petroleum ether) for 16 hours at room temperature with stirring at 300 rpm.

[0182] 2. Enzyme extraction was performed on SCG diluted with water at a ratio of 1:4. Viscozyme and Celluclast were added at concentrations of 33 uL / g and 11.3 uL / g, respectively, at pH 5.9 and 55°C.

[0183] 3. SCG was pretreated with enzymes, and the supernatant was further extracted by using Monowave 400. The process was performed by reaching 200°C using a stirrer speed of 300 rpm and a maximum power of 850 W, then keeping the temperature constant for 10 minutes, and then cooling to 70°C.

[0184] Dry matter analysis

[0185] The supernatants from both treatments were separated by centrifugation at 4700 x g for 10 minutes. Approximately 1 mL of the supernatant was first transferred to an aluminum pan. The weight of the sample and pan was carefully recorded. The pan was oven-dried at 100°C overnight until the weight was constant. The dry weight of the sample was determined, taking into account the dilution used during the extraction process, and expressed as mg SDM / g SCG. The SCG extraction was repeated three times.

[0186]

[0187] Example 12: Microwave-assisted extraction followed by enzyme-assisted extraction

[0188] The same SCG as in Example 11 was used.

[0189] 1. Degrease SCG according to the same procedure and conditions as in step 1 of Example 11.

[0190] 2. Following step 3 of Example 11, defatted SCG was extracted using Monowave 400, and the SCG was diluted with water at a ratio of 1:4.

[0191] 3. The SCG and supernatant in step 2 were further extracted by enzyme-assisted extraction by adding Viscozyme and Celluclast, where the concentrations and conditions were the same as those in step 2 of Example 11.

[0192] Dry matter analysis

[0193] The supernatants from both treatments were separated by centrifugation at 4700 x g for 10 minutes. Approximately 1 mL of the supernatant was first transferred to an aluminum pan. The weight of the sample and pan was carefully recorded. The pan was oven-dried at 100°C overnight until the weight was constant. The dry weight of the sample was determined, taking into account the dilution used during the extraction process, and expressed as mg SDM / g SCG. The SCG extraction was performed in triplicate.

[0194]

Claims

1. A method for upcycling spent coffee grounds (SCG) into antioxidant dietary fiber, comprising the following steps: a) subjecting the spent coffee grounds material to microwave assisted extraction (MAE) in water at a temperature of up to 200° C. and for a period of at least 5 minutes, after which: - b) subjecting the spent coffee grounds material extracted according to step a) to enzyme assisted extraction (EAE) in an aqueous solution comprising an enzyme having endo-β-glucanase and / or an enzyme having cellulase activity and recovering the soluble material.

2. The method according to claim 1, further comprising the steps of: c) Grinding said spent coffee grounds material to be subjected to microwave assisted extraction to a particle size having a D[4,3] of less than 100 microns, preferably by cryogenic grinding.

3. The method according to claim 1 or 2, further comprising the steps of: d) prior to step a), defatting the spent coffee grounds to reduce the fat content to less than 5% by weight.

4. The method according to claim 3, wherein the defatting step d) is performed by Soxhlet extraction using petroleum ether for a period of at least 2 hours.

5. The method according to any one of claims 1 to 4, comprising the steps of: The soluble matter from steps a) and b) is spray-dried to obtain the antioxidant dietary fiber in a spray-dried form.

6. The method according to any one of claims 1 to 5, wherein in step b), the amount of enzyme having endo-β-glucanase activity equivalent to 300 amyloglucosidase units / mL is 33 to 66 μL / g SCG.

7. The process according to any one of claims 1 to 6, wherein in step b) the amount of enzyme having a cellulase activity equivalent to 700 endo-glucanase units / g SCG is 11 to 40 μL / g SCG.

8. The method according to any one of claims 1 to 7, wherein the enzyme-assisted extraction step is carried out at a temperature of 45 to 65°C, at a pH of 4.5 to 6, for a period of 3 to 15 hours.

9. The method according to any one of the preceding claims, wherein the MAE step a) is performed by applying microwave assisted extraction to a slurry of SGC in water, wherein the slurry has a SGC / water ratio of 1:3 to 1:

4.

10. The method according to any one of claims 1 to 9, wherein prior to the MAE extraction step, the SCG is subjected to a pretreatment selected from pulsed electric field treatment or high voltage extraction or both.

11. The method according to claim 10, wherein the pulsed electric field treatment and / or the high-pressure extraction are performed before the grinding step c) and the defatting step d).

12. The method according to claim 10 or claim 11, wherein the pulsed electric field treatment is performed by mixing SCG with water at a solid / liquid ratio of 30% to 42.5% and with a pulse number of 1,000 to 10,000.

13. The method of claim 10 or claim 11, wherein the high-pressure extraction is carried out at a temperature below 60°C and a pressure of 100 to 600 MPa.

14. A dry antioxidant dietary fiber obtainable by the method according to any one of claims 1 to 12.

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