Method for producing chitosan-based antioxidant film

Abstract

The present invention concerns a method for producing a chitosan-based antioxidant film, the method comprising the steps of autoclaving spent ground coffee with distilled water to obtain a spent ground coffee extract; filtering the spent ground coffee extract; homogenizing the filtered spent ground coffee extract; mixing the homogenized spent ground coffee extract with a chitosan solution comprising chitosan, glycerol, and acetic acid, so as to obtain a chitosan-spent ground coffee mixture comprising 1-10% v / v spent ground coffee extract; filtering and homogenizing the chitosan-spent ground coffee mixture; casting the homogenized chitosan-spent ground coffee mixture into a mold; and drying the casted chitosan- spent ground coffee mixture to obtain the chitosan-based antioxidant film. The chitosan-based antioxidant film obtained from the process of the present invention could be applied to foods and other commodities that are sensitive to lipid and photo oxidation and presents an environmentally friendly alternative to single-use plastics.

Description

[0001]METHOD FOR PRODUCING CHITOSAN-BASED ANTIOXIDANT FILM Technical Field of the Invention The present invention generally relates to active packaging materials, and more particularly to an antioxidant chitosan- based film with spent ground coffee extract. Background of the Invention Biodegradable active packaging film has been studied extensively in recent years due to public concerns about food quality and safety. Active packaging is an innovative development that provides some salient functions in food preservation other than providing a barrier, protection, and containment. Incorporating different kinds of active compounds and substances from natural resources, e.g., plant extracts, into packaging materials is the main method to develop active packaging. It may contain antimicrobial, antifungal, and antioxidant properties and agents and nutrients that can enhance the shelf-life of the products being protected. Recent discoveries in antioxidant active packaging have focused on the use of extracts from natural sources such as green tea, rosemary essential oil, tea polyphenols, Thymus moroderi or Thymus piperella essential oils, and Lycium barbarum or goji berry fruit. Apart from the mentioned sources of antioxidants, coffee is also known for its antioxidant properties. It is widely consumed locally. Waste from coffee shops or spent / used ground coffee can still be extracted to maximize its antioxidant potential. In the United States, tons of residues after brewing ground coffee at restaurants, cafeterias, and in households are produced annually but all of them are disposed of. Antioxidants can still be recovered from the spent ground coffee. Extracts can be incorporated into the chitosan films or other biodegradable matrix to enhance its antioxidant properties. Chitosan is a natural carbohydrate polymer [β-(1-4)-2- acetamido-£-glucose units] and is yielded from deacetylation of chitin [poly-β-(1-4)-N-acetyl-2-glucosamine]. Although chitosan has its antimicrobial and antioxidant properties, there is a limitation in being a practical antioxidant source. Hence, it can be improved further by adding extract from spent ground coffee as a potential source of polyphenols. In the meat industry, synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert- butylhydroquinone (TBHQ), and propyl gallate (PG) are commonly used to prevent oxidative changes. These antioxidants contain carcinogenic effects that could be harmful to consumers. Chitosan-based films comprising coffee and its byproducts and methods for their production have been reported in the art. For example, CN116496525A provides a preparation method of coffee silver skin cellulose / chitosan-based natural pigment film comprising the following steps: (1) drying coffee silver skin, crushing, and sequentially performing water immersion, alkaline hydrolysis, bleaching, washing and drying to obtain coffee silver skin cellulose; (2) mixing the coffee silver skin cellulose with water to obtain a cellulose solution, and mixing a natural pigment with a chitosan solution to obtain a pigment chitosan solution; and (3) mixing a cellulose solution with the pigment chitosan solution, adding glycerol, standing, degassing, pouring into a mold, and drying to obtain the coffee silver skin cellulose / chitosan-based natural pigment film. CN116413260A discloses an intelligent indicator film capable of real-time monitoring of the pickle fermentation process, characterized in that: the Robusta coffee bark extract with anthocyanin content not less than 20g / 100g is added to the chitosan- glycerin matrix to obtain The film- forming liquid is obtained by cutting the film-forming liquid after drying the film; when in use, the intelligent indicator film is pasted on the cover of the kimchi jar and fully contacted with the gas in the kimchi jar. However, none of these disclose a method for producing chitosan-based antioxidant film as shown by the present invention. Summary of the Invention The object of the present invention is to provide a process for producing an antioxidant chitosan-based film mixed with spent ground coffee extract (SGCE) through casting method. It is also an object of the present invention to provide a process for producing an antioxidant and biodegradable chitosan-based film that could prolong the shelf-life of products that are sensitive to lipid and photooxidation. Therefore, the present invention provides a method for producing a chitosan-based antioxidant film, the method comprising the steps of: a) autoclaving spent ground coffee with distilled water to obtain a spent ground coffee extract; b) filtering the spent ground coffee extract; c) homogenizing the filtered spent ground coffee extract; d) mixing the homogenized spent ground coffee extract with a chitosan solution comprising chitosan, glycerol, and acetic acid, so as to obtain a chitosan- spent ground coffee mixture comprising 1-10% v / v spent ground coffee extract; e) filtering and homogenizing the chitosan-spent ground coffee mixture; f) casting the homogenized chitosan-spent ground coffee mixture into a mold; and g) drying the casted chitosan-spent ground coffee mixture to obtain the chitosan-based antioxidant film. Brief Description of the Figures FIG. 1 shows light transmission of chitosan-based film without and with spent ground coffee extract. Results marked with different letters are statistically different (p < 0.05). FIG. 2 shows DPPH scavenging activity of the chitosan-based film without and with different amounts of SGCE. Results marked with different letters are statistically different (p < 0.05). FIG. 3 shows FTIR pattern of the chitosan-based film without and with different amounts of SGCE. FIG. 4 shows change in weight percentage of the antioxidant chitosan-based film without and with SCGE vs storage time, days. Detailed Description of the Invention The present invention relates to a method for producing a chitosan-based antioxidant film. The chitosan-based antioxidant film is a biodegradable film made from chitosan, a natural polymer derived from chitin, which is found in the shells of crustaceans like shrimp and crabs. These films are designed to provide packaging solutions with improved properties such as antimicrobial activity, barrier properties, and antioxidant capacity. The inventors found that spent coffee ground extract obtained by autoclaving spent ground coffee in distilled water provides the antioxidant properties to the chitosan-based antioxidant film, making it suitable as packaging material for foods and other commodities that are sensitive to lipid and photo oxidation and presents an environmentally friendly alternative to single-use plastics. “Spent ground coffee” refers to the coffee grounds left over after brewing coffee. “Spent ground coffee extract” refers to a water extract obtained from autoclaving spent ground coffee in distilled water. Spent ground coffee is autoclaved in distilled water to obtain a spent ground coffee extract. Preferably, the autoclaving step is performed at 110 °C at 5 psi for 20 to 30 minutes. Preferably, the mass volume ratio of the spent ground coffee to the distilled water is 25-35g:400mL. The spent ground coffee extract is then filtered. Filtration helps to clarify the spent ground coffee extract and remove impurities such as suspended solids, fine particles, and other impurities from the spent coffee grounds. The filtration of the spent ground coffee extract may be performed using, for example, a cheesecloth. The filtered spent ground coffee extract is then homogenized. Homogenization of the spent ground coffee extract is performed to achieve a uniform dispersion of the components within the extract. Homogenization can be achieved using various types of equipment, including high-pressure homogenizers, colloid mills, or ultrasonic homogenizers. Preferably, the homogenizing step is performed for 15 minutes at 300 pm. The homogenized spent ground coffee extract is then mixed with a chitosan solution comprising chitosan, glycerol, and acetic acid, so as to obtain a chitosan-spent ground coffee mixture comprising 1-10% v / v spent ground coffee extract. The chitosan-spent ground coffee mixture is then filtered and homogenized. Preferably, the homogenizing of the chitosan- spent ground coffee mixture is performed for 1 hour. The homogenized chitosan-spent ground coffee mixture is then casted into a mold. Finally, the casted chitosan-spent ground coffee mixture is dried to obtain the chitosan-based antioxidant film. Preferably, the drying step is performed at 30-34 °C for 10-14 days. Other advantages and features of the present invention will be apparent from the examples. Examples Example 1: Collection, extraction of spent ground coffee, and characterization of the coffee extract 1.1 Collection of spent ground coffee Samples were collected from a local coffee shop in Metro Manila. Moisture content was measured using HR 73 Halogen Moisture Analyzer. The collected raw materials have an average moisture content of 67%. Samples were packed in foil, sealed, then kept frozen until use. 1.2 Extraction of spent ground coffee The SGCE was obtained using mild hydrothermal pretreatment extraction based on the procedure of Conde and Mussatto, Isolation of polyphenols from spent coffee grounds and silverskin by mild hydrothermal pretreatment, Preparative Biochemistry and Biotechnology, 2016, 46(4): 406-409. Six treatment combinations were considered for the extraction experiment (Table 1). Table 1. Treatments with various amounts of SGC and extraction time. Treatments Amount of spent Extraction time, ground coffee, g minutes n autoclaved at 110 °C at 5 psi. The mixture was filtered using cheesecloth and homogenized for 15 minutes. The aqueous extracts were collected and pooled in a reagent bottle, wrapped with foil, and stored at 2 °C until use. The aqueous extracts obtained from the six treatments have dark brown color. The effect of extraction time on the yield of SGCE is shown in Table 2. Based on the findings, longer extraction time resulted in an increase in TPC value of the coffee extract. 1.3 Characterization of spent ground coffee extract – Total Phenolic assays The total phenolic content of the extracts was determined according to Folin-Ciocalteau method described by Slinkard and Singleton (1997) and Siripatrawan and Harte (2010). SCGE (10 mL) was diluted with deionized water and adjusted to 100 mL. Sample solutions (5 mL) with appropriate dilution was added to 200 µL of the freshly prepared Folin-Ciocalteau reagent. After 8 minutes, sodium carbonate (15% w / v) was added to the solvent. The reaction mixture was allowed to react in the dark for 30 minutes at ambient temperature. The absorbance at 760 nm was measured by an ultraviolet spectrophotometer (UV-VIS model 1800). The control blank was done in the same way with deionized water. Gallic acid was used as the standard, and results were expressed as gallic acid equivalents per gram of sample. The results gathered confirmed that the residue of the brewed coffee still contains a significant amount of phenolic compound that could be used as antioxidant. The results could be attributed to the extraction process, roasting, and variety of coffee products. The variety of spent ground coffee used was not identified. Table 2. Total phenolic content values of SGCE residue samples. Treatment Amount of Ratio (amount of TPC (ml GAE L i i i ml re significantly different between the amount of SCG and extraction time (p<0.05) Antioxidant activity of the coffee extract The antioxidant activity of the film samples was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay according to Siripatrawan and Harte (2010). A 3 mL of film extract solution was mixed with 1 mL of 1 mM methanolic solution of commercially available DPPH. The mixture was vortexed and incubated in the dark at ambient temperature for 30 minutes. The absorbance was measured at 517 nm. The percentage of DPPH free radical quenching activity was calculated using the following Equation 1: DPPH scavenging effect (%) = (AbsDPPH – AbsEXTRACT) / (AbsDPPH) Where, AbsDPPHis the absorbance value at 517 nm of the methanolic solution of DPPH and AbsEXTRACTis the absorbance value at 517 nm for the sample extracts. Each sample was assayed three times. Table 3. DPPH radical scavenging activity of SGCE with different treatments. Treatment Amount of Ratio (amount of DPPH Means with different letters (a-d) in the last column are significantly different between the amount of SCG and extraction time (p<0.05) Treatment 1, which has the lowest amount of SGCE and shortest autoclave time, showed the lowest DPPH radical scavenging activity of 6.29±1.16. Treatment 5 (35g:20 min) showed the highest DPPH radical scavenging activity (32.47±3.93) and is significantly different (p<0.05) among the treatments. At longer extraction time (30 minutes), the DPPH scavenging effect decreased significantly, when T6 is compared with T5. This could be due to the decomposition of the antioxidant associated with total phenolic content. All the treatments have considerable DPPH radical scavenging activity. Example 2: Development and characterization of antioxidant chitosan-based packaging film mixed with spent ground coffee extract 2.1 Development of antioxidant film Chitosan film-forming solution was prepared according to Siripatrawan et al., 2010, with a slight modification. A series of preliminary experiments was conducted to optimize the right amount of acetic acid, glycerol, and chitosan in the mixture. 1 g of chitosan powder was dissolved into a 250 mL acetic acid solution. Glycerol (0.5 g) was added as plasticizer. The solution was stirred for 12 hours. Subsequently, the chitosan-based solution was filtered using a cheesecloth. Aqueous SCGE at 0, 1, 3, 5, 7, and 10% (v / v) were added and then centrifuged for another 1 hour. The solutions with different amounts of SCGE (230 grams / tray) were casted on a PP rectangular shaped tray (16 cm x 32 cm) and dried at ambient conditions (30-34 °C) for 10-14 days. All samples were prepared in three replicates and recovered after drying. The increasing amount of SGCE added to the chitosan solution corresponds to an increasing darker color of the packaging film when compared to the control. 2.2 Evaluation of the properties of antioxidant chitosan-based film Moisture content (%) The moisture content (MC) of films was determined according to the procedure of Rachtanapun et al. (2021) with slight modification. Samples were cut into 6 cm x 6 cm size then dried in a 105 °C oven for 24 h. The final mass before and after drying were recorded. The moisture content was calculated using the following Equation 2: Moisture content (%) = (Mi-Mf) / (Mi) X 100 Where Mi is the mass of initial samples (g) and Mf is the mass of dried samples (g). Table 4. Moisture content of chitosan-based film without and with different amounts of SGCE. Amount of SGCE, % Moisture content (%) Results marked with different letters (a-c) show a statistically significant difference among the samples (p<0.05) The addition of increasing amounts of SGCE (3 - 10%) showed a significant decrease in moisture content when compared to control. The addition of 1% SGCE to chitosan film resulted in a non-significant increase in moisture content. Water-soluble matter The water-soluble matter (SM) of the films was determined in triplicate as described by Rachtanapun et al. (2021) with slight modifications. Film samples (6 cm x 6 cm) were dried at 105 °C for 24 h, kept in desiccators for 24 h, weighed initial dry weight (Wi), and then immersed in 50 mL of distilled water for 24 h at 25 °C and manually stirred occasionally. Subsequently, the samples were dried in a 105 °C oven for 24 h to obtain the final dry weight (Wf). The SM was calculated according to the following Equation 3: SM (%) = (Wi – Wf) / (Wi) X 100 Where Wi is the initial dry mass of the sample (g) and Wf is the final dry mass of the sample (g) after incubation in distilled water. Table 5. Water-soluble matter of chitosan-based film without and with different amounts of SGCE. Amount of SGCE, % Water soluble matter (%) 5 21.24±0.34b7 19.09±0.37ca s (p<0.05) The water-soluble matter of the films with the highest SGCE (10%) had a slight difference while the samples with 3 – 7% extract showed a significant difference from the control. Film thickness of the developed film Film thickness was measured using a digital micrometer (Mitutoyo Absolute, Tester Sangyo Ltd., Tokyo, Japan). Five replicates were done for each treatment. Five measurements were taken at random position of the film samples and mean values were calculated. Table 6. Thickness of chitosan-based film without and with different amounts of SGCE. Amount of SGCE, % Thickness, microns The developed films resulted in very slight differences in thickness. The thickness obtained after drying ranged from 39.72±3.95 to 41.47±5.11 microns. Statistically, there was no significant differences (p > 0.05) among the samples. Light transmission rate A spectrophotometer (UV1800) was used to measure the light barrier and ultraviolet barrier properties of the films (Fang et al., 2002). Samples (3.0 cm in length and 1.0 cm in width) were fixed in place in a cuvette for the light beam to pass through the films. Light transmission rate was determined at a wavelength of 525 nm. As shown in Fig. 1, increasing the amount of spent ground coffee extract resulted in an increasing UV / visible barrier property of the chitosan-based film. The percentage of light transmission in films with extract significantly decreased (p<0.05) when compared to the control films. Films mixed with 7% and 10% SGCE resulted in a lower transmission rate percentage of 49.24 and 46.53, respectively. Lower transmission rate values correspond to a higher light or UV / visible barrier property. The results gathered could be related to the phenolic compounds present in SGCE, giving a natural darker color to the developed films. Color parameter Hunter color (L*, a*, b*) values were measured using the Minolta Chromameter (CR-300, Minolta Camera Co., Osaka, Japan). Six readings at different positions on each film were measured. Table 7. Color parameters (L*, a*, b*) of chitosan-based film without and with different amounts of SGCE. Amount of SGCE, % L* a* b* 0 87.79±1.02f1.93±0.44f0.99±1.20fn The lightness (L*) of the film significantly decreased while a* and b* (yellowness) values greatly increased (p<0.05) with increasing amount if SGCE added. Depending on the amount of SGCE added, a light to dark brownish color was observed after drying the film. The values obtained in Table 7 support the appearance of the film when visually inspected. Water vapor transmission rate and oxygen transmission rate The water vapor transmission rate (WVTR) and oxygen transmission rate (OTR) of the antioxidant films were measured using the Permatran-W (Model 3 / 33, MOCON) and OX-Tran (Model 2 / 21, MOCON), respectively. The WVTR and OTR of the developed film were not detected during the initial and validation tests. This could be due to the high permeability of the developed films which is beyond the detection limit of the testing machine. Based on the result, the films developed could be considered to have high water and oxygen permeability. Mechanical properties Tensile strength (TS) and percentage of elongation at break (%E) was measured using the Universal Testing Machine (UTM) (Model AGS-J Series, Shimadzu, Japan) following the ASTM Standard Test Method D 882-91 (ASTM 2003). Table 8. Tensile strength and elongation at break (%) of chitosan-based film without and with different amounts of SGCE. Amount of SGCE, % Tensile strength Elongation at break (%) 0 24 73±6 27 4 06±0 75 g g ly increased with increasing amount of SGCE added. It is also higher compared to control. Film with 3% SGCE had the highest tensile strength (31.65±2.69) among the group whereas control was slightly higher (4.06±0.75%) in elongation at break. Antioxidant activity The antioxidant activity of the film samples was evaluated using DPPH free radical scavenging assay following the procedure of Siripatrawan (2010). Film extract solution, 3 mL, was mixed with 1 mL of 1 mM methanolic solution of commercially available DPPH. The mixture was vortexed and incubated in the dark at ambient temperature for 30 minutes. Absorbance was then measured at 517 nm in the UV VIS machine (model 1800). The percentage of DPPH free radical quenching activity was computed using Equation 1. Results showed that DPPH scavenging activity of the films significantly increased (p < 0.05) as SGCE concentration added increased (Fig. 2). Fourier Transform Infrared (FTIR) Analysis Fourier Transform Infrared (FTIR, Model IR Prestige 21, Shimadzu) spectrometry was carried out to observe the structural interactions of chitosan films incorporated with SCGE. The FTIR values of the different samples are shown in Fig. 3. All samples followed a similar wavelength with the spectral bands. A very slight displacement was observed in samples with added SGCE (3290.56 – 3352.28 cm-1) when compared to control (3354.21 cm-1) in the O-H stretching. Films with 7% SGCE showed the highest displacement (3290.56 cm-1). Meanwhile, films with 3% and 7% SGCE in the C-H group showed a slight shift also at 1028.06 cm-1and 1026.13 cm-1, respectively. This may indicate the interaction between the chitosan and the phenolic compound in the SGCE. Field Emission Scanning Electron Microscopy (FESEM) FESEM imaging was conducted using the Dual Beam Helios Nanolab 600i with accelerating volume of 2.0 kV and beam current of 86 Pa. Samples were sputter-coated with gold (Au) prior to analysis. The test was done by the Advanced Device and Materials Testing Laboratory (ADMATEL), DOST Compound, Bicutan, Taguig City. Compared to the control, pigments were detected on samples with added SGCE. Films with higher concentrations of SGCE (5, 7, and 10%) presented a more emphasized pigment. Control showed a smooth and pigment-free microstructure surface. These observations could validate the presence of active compounds in the films. Example 3: Biodegradability study on the developed chitosan- based films 3.1 Collection and preparation of garden soil Garden soil was collected from the Department of Environment and Natural Resources (DENR) nursery station in Bicutan, Taguig City. The specific content of the soil was not identified, and no fertilizers and compost were added during the experiment. Preparation of samples Biodegradability test was conducted according to the procedure of Bonilla and Sobral (2019). Samples were cut to 6 cm x 6 cm dimension, placed in a net, and buried in soil at least 7 cm deep. The soil was loosely loaded in a 10 L capacity recycled, cut out plastic water container. Samples were prepared in three replicates and stored in a chamber at 58 °C and 58% RH. Occasional watering was done to keep the soil wet. Chitosan- based antioxidant films were weighed and appearance was visually observed and documented until the end of storage study. 3.2 Evaluation of the packaging film Change in weight The change in weight was measured and calculated throughout the storage using Equation 2 as described in Example 2. Fig. 4 shows the change in weight of the films with and without SGCE. High and increasing trend on the change in weight percentage was observed in all samples at the end of the experiment. On day 10, samples with SGCE were higher than that of the control. Slight differences were observed on both samples from day 17 to day 44 although when visually inspected, samples with SGCE, disintegration was more apparent after 30 days. The material can be considered completely disintegrable when 90% of the sample mass shall be lost within 90 days of test, and sample residues are below 2 mm based on Standard ISO 20200:2015. Change in visual appearance The visual change in appearance of the films was documented to determine the degree of biodegradability during storage. All samples were prepared with homogeneous structure, with no visible holes and transparent and brownish color for control and samples with SGCE, respectively. Creases and rough surfaces were observed on both samples after 10 days. No disintegration was observed until day 22 for samples with SGCE and Day 30 for the control. Disintegration started to manifest on samples with SGCE after 30 days whereas at day 37, control samples begun to break down also. Samples with SGCE seemed to disintegrate faster than that of the control. Both samples continued to disintegrate until the end of the composting condition at laboratory scale (Day 44). This could mean that the developed film is biodegradable and compostable. 5 10

Claims

CLAIMS 1. A method for producing a chitosan-based antioxidant film, the method comprising the steps of: a) autoclaving spent ground coffee with distilled water to obtain a spent ground coffee extract; b) filtering the spent ground coffee extract; c) homogenizing the filtered spent ground coffee extract; d) mixing the homogenized spent ground coffee extract with a chitosan solution comprising chitosan, glycerol, and acetic acid, so as to obtain a chitosan- spent ground coffee mixture comprising 1-10% v / v spent ground coffee extract; e) filtering and homogenizing the chitosan-spent ground coffee mixture; f) casting the homogenized chitosan-spent ground coffee mixture into a mold; and g) drying the casted chitosan-spent ground coffee mixture to obtain the chitosan-based antioxidant film.

2. The method of claim 1, wherein the autoclaving step a) is performed at 110 °C at 5 psi for 20 to 30 minutes.

3. The method of claim 1, wherein the mass volume ratio of the spent ground coffee to the distilled water is 25- 35g:400mL.

4. The method of claim 1, wherein the homogenizing step c) is performed for 15 minutes at 300 pm.

5. The method of claim 1, wherein the homogenizing in step e) is performed for 1 hour.

6. The method of claim 1, wherein the drying step g) is performed at 30-34 °C for 10-14 days.

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