Process for upcycling spent coffee grounds into antioxidant dietary fiber

Abstract

To develop a novel process for increasing the amount of soluble matter extracted from SCG and obtaining an antioxidant dietary fiber suitable for human consumption.SOLUTION: A process for upcycling spent coffee grounds (SCG) into an antioxidant dietary fiber comprises the steps of: (a) performing a microwave assisted extraction (MAE) of the spent coffee grounds in water at a temperature up to 200°C for a time of at least 5 min, followed by (b) performing an enzymatic assisted extraction (EAE) of the spent coffee ground material extracted according to step (a) in an aqueous solution comprising an enzyme having endo-β-glucanase activity and / or an enzyme having cellulase activity, and recovering soluble matter. In a preferred embodiment, prior to MAE step, the SCG is subjected to a pretreatment selected from a pulsed electric field treatment or a high-pressure extraction or both.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an improved method for upcycling coffee brew residue (or spent coffee ground (SCG)) into antioxidant dietary fiber. [Background technology]

[0002] Coffee is a widely consumed and highly appreciated product worldwide, and its consumption is continuously increasing, reaching approximately 10.1 billion kg in 2020, 1 billion kg more than in 2015 (International Coffee Organization, 2021). The entire coffee processing process generates a significant amount of waste annually. Instant coffee production and coffee extraction at the domestic / retail level generate approximately 6 million tonnes of coffee brew residues (SCG) annually.

[0003] Recent research activities have focused on the study of the composition of SCG to explore the possibility of reusing coffee brew residues, as suggested by the principles of circular economy. Polysaccharides, oligosaccharides, lipids, aliphatic acids, amino acids, proteins, alkaloids (caffeine, trigonelline, etc.), phenols, minerals, lignin, melanoidins, and volatile compounds are valuable components of SCG.

[0004] SCG has functional qualities due to its high fiber, tannin, and phenolic content, which results in high antioxidant capacity. The fiber in SCG is mostly insoluble and consists mostly of cellulose and hemicellulose, with a high content of lignin.

[0005] Furthermore, SCG contains significant amounts of lipids and proteins of high biological value. SCG has been proposed as a bulk material for several applications, including adsorbents, fillers, additives for polymer production, animal feed supplements, and soil fertilizers. However, SCG is also a valuable source of bioactive compounds. Diterpene alcohol esters and phenols are the most prominent, while condensed tannins and hydrolyzable tannins are other active polyphenols found in SCG. Melanoidins are end products of the Maillard reaction, have high molecular weights, vary in composition, and typically contain phenols in SCG. Melanoidins have been associated with various biological activities, including prebiotic, antioxidant, and antimicrobial activities. Dietary melanoidins bypass digestion and serve as substrates for the production of short-chain fatty acids (SCFAs) by gut bacteria, thereby modulating the microbiota.

[0006] In addition to melanoidins, mannooligosaccharides (MOS) have been defined as prebiotics due to their ability to influence the intestinal microflora by promoting the growth of certain beneficial species. Therefore, SCG-derived ingredients containing high concentrations of fiber and melanoidins can be used as prebiotics or as symbiotic substances to support the growth of probiotics.

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

[0008] Besides enzymes, physical methods can also be used for hydrolysis of SCG, and microwave treatment (Microwave-Assisted Extraction; MAE) was particularly effective in extracting oligosaccharides and phenolic fractions (Passos et al., 2013). In both enzymatic and physical extraction methods, particle size is an important factor influencing the extraction of phenolic compounds. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Claudia P. Passos et al., Microwave superheated water extraction of polysaccharides from spent coffee grounds, Carbohydrate Polymers 94 (2013) 626-633 Summary of the Invention [Problem to be solved by the invention]

[0010] The aim of the present invention is to develop a new method to increase the amount of soluble material extracted from SCG and obtain antioxidant dietary fibre suitable for human consumption.

[0011] A further object of the present invention is to provide a method which makes it possible to obtain, in improved yield, prebiotic functional ingredients comprising oligosaccharides, preferably linked to polyphenols, which have a positive effect on enterobacteriaceae and are capable of inducing anti-inflammatory pathways at the level of the intestinal epithelium. [Means for solving the problem]

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

[0013] The method of the present invention combines a microwave-assisted extraction (MAE) step of raw coffee extract material with an enzyme-assisted extraction (EAE) step carried out on the coffee extract material that is the extraction residue of the MAE step. [Effects of the Invention]

[0014] It has been found in accordance with the present invention that by combining the two steps, the overall amount of valuable soluble material is increased, as it is higher than the overall amount obtained by subjecting the same raw SCG to MAE and then to EAE separately, under the same process conditions employed for each step in the combined process. Furthermore, it has been found that by performing the extraction steps in the order of MAE followed by EAE, the amount of soluble material obtained is significantly increased compared to the amount obtained in the reverse order.

[0015] The method of the present invention is further illustrated by the following detailed description and examples, which refer to the accompanying drawings. [Brief explanation of the drawings]

[0016] In the following examples and figures: SCG‐C indicates the amount of soluble material (SM) released into water by SCG after cryogenic grinding; SCG‐M indicates the amount of soluble material (SM) released into water by SCG after MAE; SCG‐E indicates the amount of soluble substances (SM) released into the water by SCG after EAE; SCG‐CE indicates the amount of soluble material (SM) released into water by SCG after cryo-grinding and EAE; SCG-ME indicates the amount of soluble material (SM) released into water by SCG after MAE and EAE. [Figure 1] The stepwise method adopted for upcycling SCG into a prebiotic functional ingredient is shown, with the grinding and degreasing steps in Figure 1 being optional. [Figure 2] 1 is a pie chart showing the composition of raw SCG used in the following examples. [Figure 3] 1 is a schematic flow chart showing the mass balance of the process of the present invention. [Figure 4] Histogram showing the quantification of galactose, fucose, arabinose, galactose, glucose, mannose, and fructose in SCG after combined microwave extraction and enzymatic treatment (SCG-ME) and extraction of raw SCG from an industrial coffee extraction method. [Figure 5] Histograms showing the quantification of galactose, fucose, arabinose, glucose, mannose, rhamnose, and xylose in SCG from coffee houses before treatment (SCG), after cryo-grinding (SCG-(C), MAE (SCG-M), EAE (SCG-E), a combination of cryo-grinding and EAE (SCG-CE), and a combination of MAE and EAE (SCG-ME). [Figure 6] Together with Table 2, the composition and properties of the functional ingredients obtained after the spray drying process are shown. [Figure 7] Histograms showing the amount of soluble fraction released into water before treatment (SCG), after cryo-grinding (SCG-(C), after MAE (SCG-M), after EAE (SCG-E), combined cryo-grinding and enzyme treatment (SCG-CE), and combined MAE and EAE (SCG-ME). [Figure 8] Histograms showing the antioxidant properties of SCG from coffee houses before treatment (SCG), after cryo-grinding (SCG-(C), MAE (SCG-M), EAE (SCG-E), a combination of cryo-grinding and EAE (SCG-CE), and a combination of MAE and EAE (SCG-ME), as assessed by DPPH, ABTS, and FRAP assays. DETAILED DESCRIPTION OF THE INVENTION

[0017] (Analysis method) The raw SCG used in the method of the invention may come from different sources, it may be waste from coffee machines (from domestic appliances or coffee houses) or from industrial coffee extraction plants.

[0018] FIG. 2 shows the approximate composition of raw SCG from an industrial coffee extraction process after heat stabilization.

[0019] Table 1 shows the approximate composition of raw SCG after heat stabilization from the coffee house. JPEG2025124616000001.jpg93160

[0020] Proximate analysis was performed to determine the percentages of moisture, ash, protein, lipid, soluble dietary fiber, insoluble dietary fiber, and carbohydrates, as detailed below. Moisture was measured according to AACC method n. 44‐15.02; Ash content was measured according to AACC method no. 08-01. Protein content was assessed by the Dumas method using a Flash EA 1112 NC analyzer (Thermo Fisher Scientific Inc., Waltman, USA) according to the manufacturer's protocol, with a conversion factor of 6.25 typically applied to SCG (Massaya et al.). Lipid content was determined by Soxhlet extraction. 3–5 g of sample was weighed into an extraction thimble. 200 mL of petroleum ether (40–60°C) was added to a flat-bottom flask containing boiling chips. All weights were carefully recorded for lipid determination. The flat-bottom flask was connected to the extractor and condenser containing the sample thimble and placed on a heating block. Heating was initiated and extraction was performed for 5 hours. After cooling, the flat-bottom flask was evaporated using a Büchi rotary evaporator. The thimble and evaporated flat-bottom flask were left in a fume hood (or fume hood) overnight to evaporate the remaining petroleum ether. The dried flat-bottom flask was weighed, and the amount of extracted lipid compared to the amount of sample used was used to determine the lipid content. Soluble and insoluble dietary fiber were measured using the K-TDFR-200A Megazyme kit (Megazyme, Ireland) according to AACC Method n. 32-07.01.

[0021] Specifically, to measure the amount of soluble dietary fiber (or water-soluble dietary fiber; SDF), 4% by volume (vol) of preheated 60°C EtOH (95%) was added to the sample in a 50 mL Greiner tube. A precipitate was allowed to form at room temperature for 60 minutes. The sample was centrifuged, and the supernatant was discarded. The pellet was transferred to a 15 mL Greiner tube of known weight. The tube was closed with cotton paper, and the pellet was freeze-dried.

[0022] The analysis was performed in duplicate and the SDF was determined using Equation 2, taking into account the dilution used during extraction. SDF: (Dry sample weight) / (Initial sample weight) x 100% Equation 2 Carbohydrates were measured by differential (Nitisewojo, 1995). The total dry matter present in the soluble fraction (SDM) was determined by transferring ±2 mL of the soluble fraction (supernatant after centrifugation) to an aluminum dish. The weight of the sample and aluminum dish was carefully recorded. The dish was dried overnight in an oven at 100 °C until a constant weight was reached. The dry weight of the sample was determined to determine the soluble solids content, which, taking into account the dilution used in the extraction process, was expressed as mg SDM / g SCG using Equation 1. SDM = (dry sample weight) / (wet sample weight) Equation 1 Total phenolic content (TPC) was determined based on the Folin-Ciocalteu method by Singleton and Rossi (Singleton and Rossi, 1965). Folin-Ciocalteu is a bright yellow reagent. By measuring the absorbance at 760 nm, the number of hydroxyl groups present in the sample can be determined. These hydroxyl groups are directly related to the presence of phenols.

[0023] 20% NaOH was prepared by solubilizing 50 g of NaCO3 in 250 mL of MiliQ water. Gallic acid calibration curves were prepared at the following concentrations: 0.025, 0.05, 0.1, 0.15, and 0.25 mg / mL. 0.2 mL of sample, standard, or blank was mixed with 1.88 mL of MiliQ water, 0.11 mL of Folin-Ciocalteu reagent, and 0.31 mL of NaCO3. The tubes were carefully shaken and left at room temperature in the dark for 60 minutes. The absorbance was then measured using a spectrophotometer (Cary 60 spectrophotometer (Agilant Technologies Inc.)). Gallic acid equivalents (GAE) were calculated by comparing the absorbance of the sample with that of the calibration curve.

[0024] The analyzed SCG from the industrial coffee extraction process (Figure 2) showed the highest concentration of insoluble dietary fiber (50.83 g / 100 g dry matter of SCG) and the lowest concentration of soluble dietary fiber (4.00 g / 100 g dry matter of SCG). The lipid content of the analyzed raw material was about 20% (dry matter), while the lipid content from the coffee house (Table 1) was only about 15%.

[0025] (Antioxidant activity) The antioxidant activity of the SCG extract was evaluated using the DPPH, ABTS, and FRAP methods (Figure 8).

[0026] For DPPH analysis, a 0.1 mM solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol was prepared using the method described by Brand-Williams et al. (1995) with minor modifications. 50 μL of sample, standard, or blank was added to 1.95 mL of DPPH solution. The sample was vortexed and allowed to stand at room temperature in the dark for 60 minutes. After incubation, absorbance was measured at 515 nm.

[0027] (Method description) In a preferred optional embodiment, prior to the extraction step, the raw SCG is milled to reduce the particle size, preferably to a particle size with a D[4,3] of less than 1 mm, more preferably less than 100 μm.

[0028] Raw SCG typically contains moisture (up to 70% by weight), so it is preferred to dry the raw SCG to a moisture content of about 10% by weight or less before grinding.

[0029] For the purpose of milling, any milling method known in the art that can ensure a particle size of 1000 to less than 900 μm can be used, but particularly preferred is milling by ball milling, or more preferably cryogenic milling.

[0030] As mentioned above, depending on the source of SCG, the amount of lipids can vary within a relatively wide range. Although it has been found that the lipid content does not have a strong influence on the yield of soluble matter in this process, in a preferred embodiment of the present invention, it is also recommended to apply a defatting step before extraction to reduce the lipid content to less than 5%, preferably less than 2%, in order to improve the organic character of the desired prebiotic functional components and reduce oxidation phenomena.

[0031] Defatting may be accomplished by methods known in the art, preferably Soxhlet-type extraction using petroleum ether, with extraction times ranging from 2 to 16 hours.

[0032] Supercritical carbon dioxide extraction can also be used.

[0033] According to the present invention, microwave-assisted extraction (MAE) is used to increase the soluble fraction. Microwave-assisted extraction is a method that uses microwave energy to heat a solvent in contact with a sample to partition analytes from the sample matrix into the solvent. The ability to rapidly heat the sample-solvent mixture is inherent in MAE and is a major advantage of this technique. The use of a closed vessel allows extraction to be performed at elevated temperatures, facilitating mass transfer of target compounds from the sample matrix. Preferably, monowave extraction is used.

[0034] The solvent for MAE is preferably water, preferably in a mass ratio (SCG / water) of 1: 3 to 1: 4. MAE can be applied at a temperature of about 200°C for 2 to 10 minutes, preferably 5 to 10 minutes.

[0035] According to the method of the present invention, the MAE extracted SCG slurry is then subjected directly to enzyme-assisted extraction (EAE). Alternatively, soluble materials may be separated and recovered from the solid extracted SCG phase which is then subjected to EAE.

[0036] The EAE step (b) is carried out in an aqueous solution containing an enzyme having endo-β-glucanase activity and / or cellulase activity.

[0037] Enzymes with mannanase, xylanase and / or hemicellulase activity or secondary activities may also be used or added to the enzymes with endo-β-glucanase and / or cellulase activity. The enzyme concentrations are preferably: 33-66 μL / g SCG for an enzyme with endo-β-glucanase activity of 300 amyloglucosidase units / mL, and 11-40 μL / g SCG for an enzyme with cellulase activity of 700 endo-glucanase units / g.

[0038] The extraction is preferably carried out at a temperature ranging from 45 to 65°C, at a pH of 4.5 to 6, and for a time period ranging from 3 to 15 hours.

[0039] Preferred commercially available enzymes that can be used include Viscozyme® L and Celluclast® 1.5L.

[0040] Viscozyme L is a broad-spectrum food-grade enzyme that hydrolyzes plant tissues (Novozymes, 2023). Viscozyme's primary enzymatic activity is endo-β-glucanase, but it also has secondary xylanase, cellulase, and hemicellulase activities. Celluclast 1.5L is a food-grade enzyme with cellulase activity that breaks down fibrous plant tissues, increasing the extraction yield of vegetable products (Novozymes, 2023).

[0041] The enzymatic reaction may be stopped by raising the temperature to, for example, 90° C. and maintaining this temperature for, for example, 10 minutes.

[0042] After EAE, the supernatant containing soluble material is collected by centrifugation.

[0043] Drying of the soluble materials to ultimately obtain the antioxidant dietary fiber may be accomplished by methods known in the art, including freeze drying and drum drying. Preferably, the antioxidant dietary fiber is obtained by spray drying.

[0044] In a further embodiment of the invention, the raw SCG prior to MAE extraction may be subjected to a pretreatment selected from pulsed electric field treatment (PEF) or high pressure extraction (HPE), or both, which may be carried out during or after grinding and degreasing, preferably as a pretreatment following these steps, but not necessarily prior to MAE extraction.

[0045] PEF is a non-thermal approach to improve mass transfer by permeabilizing cell membranes through electroporation (or electroporation) under the influence of short (nanosecond to millisecond) moderate-intensity electric pulses. This method, which consumes little energy and has minimal environmental impact, can accelerate the release of intracellular chemicals and increase the production rate and yield of various components from plant matrices. Preferably, PEF can be used with the following parameters: electric field strength 1.5 kV / cm, 20 Hz, pulse width 2 μs, conductivity 0.5 mS / cm, and a resting temperature of 10–12°C. The number of pulses is 1,000–10,000, and the solid-liquid ratio is 30–42.5%.

[0046] HPE is performed at low temperatures (typically below 60°C) and high pressures (typically 100-600 MPa) to rapidly extract compounds, requiring less organic solvent and achieving similar recovery rates to other extraction techniques. Like PEF, it offers a good alternative to traditional thermal processing because it reduces extraction time, solvent consumption, and increases extraction yield.

[0047] For the purposes of this patent, HPE may be used as a pretreatment. In this scenario, the SCG sample is suspended in deionized water at a 2 / 3 v / v ratio and then packed into a plastic bag under vacuum. The pressure and time conditions can vary from 200 MPa to 600 MPa and 1 minute to 5 minutes, respectively. [Example]

[0048] Example 1 Cryogenic grinding (optional) and particle size evaluation SCG was pulverized using a Cryo-miller 6875D Freezer / Mill® (Spex Sample Prep, USA). Specifically, 20 g of SCG was milled three times for 5 minutes at a setting of 10 cycles per second (cps). 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 and used for coffee powder. Values ​​for D[3,2] (area-based mean diameter), D[4,3] (volume-based mean diameter, or volume-weighted mean diameter), and D(50) (volume median diameter, or volume median diameter) were obtained. Additionally, the parameters D(10) and D(90) were calculated, indicating the fraction of particles with diameters less than the respective parameter values ​​(10% and 90%, respectively).

[0049] The particle size distribution of dried SCG was characterized by the following parameters: D[3,2] = 102.8 ± 2.6 μm, D[4,3] = 372.4 ± 7.2 μm, D(10) = 34.2 ± 0.5 μm, D(50) = 292.3 ± 4.9 μm, and D(90) = 638 ± 11.7 μm. Cryomilling significantly reduced the particle size of SCG. Indeed, SCG-C had a D[3,2] = 33.7 ± 0.6 μm, D[4,3] = 93.6 ± 3.3 μm, D(10) = 15.0 ± 0.2 μm, D(50) = 44.4 ± 0.9 μm, and D(90) = 243.1 ± 8.5 μm. Therefore, the decrease in D(50) was the largest, resulting in an 85% particle size reduction in 50% of the particles in SCG-C.

[0050] In a previous study using SCG (obtained from the Iberital IB7 coffee maker), the total amount of soluble matter extracted by cryogenic grinding was 40%.

[0051] Example 2 Degreasing step (d) (optional) ) A delipidation step is preferably performed when the amount of lipid in the raw SCG extracted with Soxhlet is high (eg, greater than 15%).

[0052] Weigh 3 g of SCG into a filter thimble and place it in an extractor connected to a flat-bottom flask containing 200 mL of petroleum ether placed on a heater. After 5 hours of extraction, leave the filter thimble containing the defatted SCG overnight to dry.

[0053] Example 3 Microwave-assisted extraction step (a) ) SCG was processed using Monowave400 (Anton Paar, Austria). Anton Paar's Monowave product line is a series of high-performance monomode microwave reactors designed for small- to medium-scale microwave synthesis (installed microwave power: 850 W (single magnetron)).

[0054] 4 g of SCG and 12 g of demineralized water were added to a wide-mouth glass bottle G30.

[0055] The Monowave400 treatment was carried out at a stirrer speed of 300 rpm and a maximum power of 850 W to reach 200°C, maintain the temperature at 200°C for 10 minutes, and then reduce 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. The total treatment time, including cooling to 55°C, was approximately 27 minutes.

[0056] To optimize the extraction time, we examined the soluble matter in SCG extracted using only a monowave oven by varying the extraction time from 10 minutes to 2 minutes. There was no significant difference between 10 minutes and 5 minutes, but there was a significant difference between 10 minutes and 2 minutes. Therefore, the preferred extraction time is 5 to 10 minutes, with 5 minutes being the most preferred.

[0057] Example 4 Enzyme-assisted extraction process (b) ) Enzyme-assisted extraction was carried out sequentially on the slurry obtained from the MAE process of Example 3, or independently on the raw cryogenically ground SCG of Example 1, using the commercially available enzymes Viscozyme® and Celluclast® under the following process conditions: TIFF2025124616000002.tif36135

[0058] No significant difference was observed in terms of soluble matter between the two enzyme doses and treatment times: soluble matter about 30-33%.

[0059] Example 5 Spray drying process ) A Büchi mini spray dryer (Büchi Laboratoriums‐Technik, Switzerland) was used. A minimum of 100 mL of supernatant extract was used for spray drying. A peristaltic pump delivered the SCG supernatant to the atomizer. Atomization was performed using a fluid nozzle. Compressed air supplied by the company was used. The compressed air was regulated by a flow meter, and cooling water was circulated through a jack installed around the nozzle. The inlet drying air was set at 120°C, with suction at 95% and pump at 20%. This inlet drying air passed through an electric heater and then flowed simultaneously with the spray liquid through the main chamber. Dry powder samples were collected from the bottom of the cyclone.

[0060] The supernatant obtained after centrifugation of the MAE and EAE extraction products from Example 4 was spray dried under the following conditions: Inlet temperature: 120~160℃ Suction speed: 95% Pump: 20% So I went.

[0061] Example 6 FIG. 3 shows a layout of the method of the present invention according to a specific embodiment.

[0062] 3 g of dried SCG from an industrial coffee extraction process was diluted in water at a 1:4 dilution (12 mL water) and subjected sequentially to MAE and EAE according to the preferred conditions defined in the previous example, after which the enzymatic reaction was stopped by raising the temperature to 90°C for 10 min and adding 24 mL water.

[0063] The dry matter in 36 mL was 0.95 g, giving a solubilization yield 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 component (overall yield: 14.3%).

[0064] Example 7 Raw SCG from an industrial coffee extraction plant was used and had a lipid content of approximately 20%.

[0065] Grinding process: Process (c) The grinding process was carried out to reduce the particle size of the sample to facilitate the next step. 30 g of SCG was ground using a Bosch TSM6A013B household grinder at a rated power of 180 W for 5 minutes.

[0066] Degreasing process: Step (d) Defatting was performed to address the high amount of lipids previously detected in SCG. Defatting was performed by placing 50 g of SCG in 200 mL of petroleum ether in a beaker stirred at 300 rpm for 16 hours.

[0067] Microwave-assisted extraction: step (a) Defatted SCG was processed according to the method described in Example 3. All samples were frozen at −20° C. before further processing or analysis.

[0068] Enzyme extraction: Step (b) Enzyme extraction was performed sequentially with SCG and water in a ratio of 1:4. Viscozyme® L consists of endo-β-glucanase (100 FBG / g), which primarily hydrolyzes (1,3)- or (1,4)-linkages in β-D-glucans. Celluclast® 1.5L consists of cellulase (700 EGU / g), which hydrolyzes (1,4)-β-D-glucosidic linkages in cellulose and other β-D-glucans. These products are supplied by Novozymes (Denmark). The recommended dosages were 200–400 mL and 100–200 mL per ton of vegetables, respectively. The optimal pH ranges were 3.3–5.5 and 4.0–6.0, respectively, and the optimal temperatures were 40–50°C and 50–60°C, respectively. Based on previous experiments, the concentrations used were 33 μL / g for Viscozyme and 11.2 μL / g for Celluclast. 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. After centrifugation at 4500 rpm for 10 minutes, the supernatant was collected.

[0069] Spray drying process A Büchi Mini Spray Dryer (Büchi Laboratoriums‐Technik, Switzerland) was used. A minimum of 100 mL of supernatant extract was used for spray drying. A peristaltic pump delivered the SCG supernatant to the atomizer. Atomization was performed using a fluid nozzle. Compressed air supplied by the company was used. The compressed air was regulated by a flow meter, and cooling water was circulated through a jack installed around the nozzle. The inlet drying air was set at 120°C, with suction at 95% and pump at 20%. This inlet drying air passed through an electric heater and then flowed simultaneously with the spray liquid through the main chamber. Dry powder samples were collected from the bottom of the cyclone.

[0070] Example 8 Raw SCG from an industrial coffee extraction process was used, with a lipid content of approximately 20%.

[0071] Grinding process (c) The milling process was performed to reduce the particle size of the sample to facilitate the following steps: (a) using a Cryo-miller 6875D freezer / mill, for example, by milling 20 g of SCG three times for 5 minutes at a setting of 10 cycles per second (cps), (b) using a Bosh TSM6A013B domestic mill, by milling 30 g of SCG for 5 minutes at a rated power of 180 W, and (c) using a ball mill, by milling 30 g of SCG for 30 minutes at a speed of 100 rpm.

[0072] Degreasing process (d) Delipidation was performed on the previously detected high amounts of lipids. Different methods were used, such as (a), (b), and (c). (a) In the Soxhlet method, 3 g of SCG was weighed into a filter thimble and placed in an extractor connected to a flat-bottom flask containing 200 mL of petroleum ether placed on a heater. After 5 hours of extraction, the filter thimble containing the delipidated SCG was left to dry overnight. In (b) and (c), delipidation was performed without heat treatment; the sample was simply stirred 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 stirred in 200 mL of petroleum ether and stirred at 300 rpm for 16 hours.

[0073] Microwave-assisted extraction step (a) Defatted SCG was processed using Monowave400 (Anton Paar, Austria) according to Example 3. All samples were frozen at −20° C. before further processing or analysis.

[0074] Enzyme extraction process (b) Enzyme extraction was performed using 1.5 L of Viscozyme® L and Celluclast® in a 1:4 SCG:water ratio as described in Example 7. The concentrations used 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 at 55°C and the pH at 5.95. The enzyme reaction was stopped at 90°C for 10 minutes. After centrifugation at 4500 rpm for 10 minutes, the supernatant was collected.

[0075] Spray drying process A Büchi Mini Spray Dryer (Büchi Laboratoriums‐Technik, Switzerland) was used. A minimum of 100 mL of supernatant extract was used for spray drying. The effect of additives was investigated by adding 5% maltodextrin to the supernatant extract and stirring the solution until the maltodextrin dissolved before spray drying. A peristaltic pump delivered the SCG supernatant to the atomizer. Spraying was performed using a fluid nozzle. Compressed air supplied by the company was used. The compressed air was regulated by a flow meter, and cooling water was circulated through a jack installed around the nozzle. The inlet drying air was set at 120°C, with suction at 95% and pump at 20%. This inlet drying air passed through an electric heater and then flowed simultaneously with the spray liquid through the main chamber. Dry powder samples were collected from the bottom of the cyclone.

[0076] result The antioxidant activities measured with DPPH and ABTS were 33 mM Trolox equivalents / g and 14 mg Trolox equivalents / g, respectively. The total polyphenol content was 28.46 mg gallic acid equivalents / g.

[0077] Example 9 Raw SCG with a lipid content of approximately 20% obtained from an industrial coffee extraction process was used.

[0078] Pulsed Electric Field (PEF) Coffee extract residue was pretreated with pulsed electric fields (PEF) by mixing the sample with 40.5% MS water at 1.5 kV and 10,000 pulses.

[0079] Grinding process (c) The grinding process was carried out to reduce the particle size of the sample to facilitate the next step. The SCG was ground for 5 minutes using a Bosch TSM6A013B household grinder at a rated power of 180 W.

[0080] Degreasing process (d) Soxhterm was used for degreasing. In this process, 3 g of SCG was weighed into a filter thimble and placed in an extraction beaker filled with petroleum ether. The extraction beaker was then placed in the apparatus. The set temperature was 135°C. After 3 hours of degreasing, the filter thimble containing the degreasing SCG was left to dry overnight. The sample was analyzed as a pretreatment.

[0081] Microwave-assisted extraction step (a) Defatted SCG was processed using Monowave400 (Anton Paar, Austria) according to Example 3. All samples were frozen at −20° C. before further processing or analysis.

[0082] Enzyme-assisted extraction process (b) The enzymatic extraction was carried out sequentially as described in Example 7, step (b).

[0083] result Antioxidant activity was measured in terms of DPPH and dry matter.

[0084] The dry matter content of the HPP-pretreated sample was 44.17 mM Trolox equivalents / g, representing a dry matter yield of 7.6%. Enzymatic treatment of the HPP-pretreated coffee extract sample increased the yield to 12.96%, with an antioxidant activity of 54.26 mM Trolox equivalents / g, measured as DPPH. Combining enzyme treatment and monowave-assisted extraction of the pretreated HPP sample resulted in a dry matter yield of 38%.

[0085] Example 10 High Pressure Processing (HPP) High-pressure processing (HPP) operates at low temperatures (usually below 60°C) and high pressures (typically 100-600 MPa) to rapidly extract compounds, requiring less organic solvent while achieving similar recovery rates to other extraction techniques. It is a good alternative to traditional thermal processing because it reduces extraction time and solvent consumption and increases extraction yield. It is set at 600 MPa for 5 minutes in a 1:1.5 ratio with water.

[0086] Grinding process (c) The SCG was ground for 5 minutes using a Bosch TSM6A013B household grinder at a rated power of 180 W.

[0087] Degreasing process (d) Degreasing was carried out using Soxhterm according to the degreasing process of Example 9.

[0088] Microwave-assisted extraction step (a) SCG was processed as described in Example 3 using a Monowave400 (Anton Paar, Austria).

[0089] Enzyme-assisted extraction process (b) Enzyme extraction was performed as described in Example 9 with SCG:water in a ratio of 1:4.

[0090] result Antioxidant activity was measured in DPPH and dry matter. The dry matter content of the HPP-pretreated sample was 50.59 mM Trolox equivalents / g, representing a dry matter yield of 5.98%. Enzymatic treatment of the HPP-pretreated coffee extract sample increased the yield to 11.44%, with an antioxidant activity of 52.86 mM Trolox equivalents / g, measured as DPPH. The pretreated HPP sample, combined with enzyme treatment and monowave-assisted extraction, showed a dry matter yield of 38%.

[0091] Chemical characterization Soluble matter was assessed for all extracts. Overall, soluble matter ranged from 3.92 ± 0.24 to 33.53 ± 0.48 g / 100 g (Figure 7).

[0092] Cryogenic grinding increased the extractable soluble matter by 40% in SCG-C compared to SCG (3.92 ± 0.24 g / 100 g SCG) and by 30% in SCG-CE compared to SCG-E (12.96 ± 0.22 g / 100 g SCG).

[0093] Antioxidant dietary fiber properties Table 3 below shows the properties of the extracts spray dried using MAE followed by EAE. JPEG2025124616000003.jpg123164

[0094] Figure 4 shows the quantification of galactose, fucose, arabinose, galactose, glucose, mannose, and fructose in SCG (SCG-ME) after combined microwave extraction and enzymatic treatment.

[0095] Mannose (41.35 ± 0.79 mg / g‐SCG dry matter (dm)), glucose (35.5 ± 3.28 mg / g‐SCG dry matter), and galactose (23.81 ± 0.08 mg / g‐SCG dry matter) were the most abundant sugars detected in SCG‐ME. As observed in previous studies (not reported here), enzymatic treatment had a greater effect on sugar concentrations than microwave treatment. The presence of high concentrations of mannose and galactose in SCG‐ME confirmed the disruption of galactomannan polymers from insoluble dietary fiber. SCG is considered a good source of galactomannan, consisting of linked 1,4 mannan chains with galactose branches of different degrees of branching.

[0096] As shown in the histograms in Figure 8, SCG-ME exhibited the highest radical scavenging activity, with 89.21 ± 0.70 and 30.31 ± 0.18 μmol of Trolox / g-SCG using the ABTS and DPPH methods, respectively, and 126.97 ± 4.14 μmol of FeSO4 using the FRAP method. This confirms how both microwave and enzymatic treatments improved the antioxidant properties of SCG. Indeed, SCG-M and SCG-E were other samples with high antioxidant properties. SCG-M showed high values ​​in all three test methods, but no statistical difference was observed using the DPPH method. Cryogenic grinding did not increase the antioxidant properties of SCG, and no statistical difference was observed between SCG and SCG-C, or between SCG-E and SCG-CE.

[0097] In accordance with the present invention, it has been found that the benefits of the present invention, in terms of the amount of soluble material extracted, are significantly greater when the EAE step is performed following the MAE step, rather than when the steps are performed in reverse order. For comparison, the following examples show results when the MAE step is performed following the EAE step.

[0098] Example 11 (Comparative Example): Enzyme-assisted extraction followed by microwave-assisted extraction ) SCG from an industrial coffee extraction process was used, dried to a moisture content of approximately 5% and ground, as described in Example 7. : 1. SCG was defatted with petroleum ether at a ratio of 1:4 (SCG / petroleum ether) for 16 h at room temperature and stirred at 300 rpm. 2. Enzyme extraction was performed with SCG diluted 1:4 with water. Viscozyme and Celluclast were added at concentrations of 33 μL / g and 11.3 μL / g, respectively, at pH 5.9 and 55°C. 3. The enzyme-pretreated SCG and the supernatant were further extracted using Monowave 400. The temperature was raised to 200°C with a stirrer speed of 300 rpm and a maximum power of 850 W, and the temperature was kept constant for 10 minutes, after which it was lowered to 70°C.

[0099] Dry matter analysis The supernatant obtained after both treatments was separated by centrifugation at 4700 × g for 10 minutes. Approximately 1 mL of the supernatant was transferred to an aluminum dish. The weights of the sample and aluminum dish were carefully recorded. The aluminum dish was dried overnight in an oven at 100 °C. The dry weight of the sample was measured and expressed as mg SDM / g SCG, taking into account the dilution used in the extraction method. SCG extractions were performed in triplicate. JPEG2025124616000004.jpg45164

[0100] Example 12 Microwave-assisted extraction followed by enzyme-assisted extraction ) The same SCG as in Example 11 was used. 1. SCG was delipidated according to the same procedure and conditions as in step 1 of Example 11. 2. Defatted SCG diluted 1:4 with water was extracted using Monowave400 according to the procedure in step 3 of Example 11. 3. The SCG and supernatant from step 2 were further extracted by enzyme-assisted extraction with the addition of Viscozyme and Celluclast at the same concentrations and under the same conditions as in step 2 of Example 11.

[0101] dry matter analysis The supernatant obtained after both treatments was separated by centrifugation at 4700 × g for 10 minutes. Approximately 1 mL of the supernatant was transferred to an aluminum dish. The weight of the sample and aluminum dish was carefully recorded. The aluminum dish was dried overnight in an oven at 100 °C. The dry weight of the sample was measured and expressed as mg SDM / g SCG, taking into account the dilution used in the extraction method. SCG extraction was performed in triplicate. JPEG2025124616000005.jpg48164

Claims

1. (a) subjecting a coffee extraction residue material to microwave-assisted extraction (MAE) in water at a temperature of not more than 200°C for at least 5 minutes; (b) subjecting the coffee extract residue material extracted according to step (a) to enzyme-assisted extraction (EAE) in an aqueous solution containing an enzyme having endo-β-glucanase activity and / or an enzyme having cellulase activity, and recovering the soluble substances. A method for upcycling coffee grounds (SCG) into antioxidant dietary fiber, comprising:

2. (c) grinding the coffee extraction residue material to be subjected to microwave-assisted extraction, preferably by cryogenic grinding, to a particle size having a D[4,3] of less than 100 μm. The method of claim 1 further comprising:

3. 3. The method of claim 1 or 2, further comprising the step of: (d) prior to step (a), defatting the coffee extract residue material to reduce the lipid content to less than 5% by weight.

4. 4. The method of claim 3, wherein the defatting step (d) is carried out by Soxhlet extraction with petroleum ether for at least 2 hours.

5. 5. The method of any one of claims 1 to 4, comprising spray drying the soluble material from steps (a) and (b) to obtain the antioxidant dietary fiber in spray dried form.

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

7. The method according to any one of claims 1 to 6, wherein in step (b), the amount of the enzyme having cellulase activity equivalent to 700 endoglucanase units / g is 11 to 40 µL per 1 g of SCG.

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

9. 9. The method according to any one of claims 1 to 8, wherein the MAE step (a) is carried out by applying microwave-assisted extraction to a slurry of SGC in water at a SCG / 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 step, the SCG is subjected to a pre-treatment selected from pulsed electric field treatment or high pressure extraction, or both.

11. 11. The method of claim 10, wherein the pulsed electric field treatment and / or high pressure extraction is carried out before the grinding step (c) and the degreasing step (d).

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

13. 12. The method according to 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 dried antioxidant dietary fiber obtained by the method according to any one of claims 1 to 12.

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