Preparation method of low-solubility gelatin-carboxymethyl chitosan composite food packaging film

Abstract

The present invention discloses a method for preparing a low-solubility gelatin-carboxymethyl chitosan composite food packaging film, comprising the following steps: 1) adding carboxymethyl chitosan to distilled water to obtain a carboxymethyl chitosan solution; 2) adding gelatin to distilled water to obtain a gelatin solution; 3) mixing the gelatin solution with the carboxymethyl chitosan solution, adding glycerol, and stirring to obtain a gelatin-carboxymethyl chitosan film solution; 4) adding a reducing sugar to the gelatin-carboxymethyl chitosan film solution, stirring, and removing impurities to obtain a film-forming liquid, adjusting the pH value of the film-forming liquid and pouring it into a plastic dish, drying to form a film, and after removing the film, performing an MR reaction to obtain a low-solubility gelatin-carboxymethyl chitosan composite food packaging film. The present invention achieves modification of the gelatin-carboxymethyl chitosan composite film by adding a reducing sugar to the gelatin-carboxymethyl chitosan composite film solution, and further, by screening the type and concentration of the reducing sugar, the modified composite film has low water solubility, elongation at break, and tensile strength.

Description

Technical Field

[0001] The invention belongs to the technical field of food packaging, and particularly relates to a method for preparing a low-solubility gelatin-carboxymethyl chitosan composite food packaging film. Background Art

[0002] With the development of society, packaging has gradually become an essential component of commodities and a crucial means of maintaining food freshness and extending its shelf life. Plastic is one of the most commonly used materials for food packaging, offering advantages such as waterproofness, durability, mature production technology, and low cost. However, the extensive use of plastic products can lead to serious environmental problems, causing significant "white pollution" to the natural environment. Furthermore, when heated, plastic food packaging is prone to producing harmful gases and odors, which have certain toxic side effects on the human body and affect the flavor of the food. With both ecological and food safety issues urgently needing to be addressed, the development of new, green and safe food packaging has become a research priority in the field of food packaging.

[0003] Bio-based membranes refer to novel, biodegradable, and safe food packaging materials made from natural biomaterials such as polysaccharides, proteins, and lipids through blending, grafting, and stabilized molding techniques. Current research on bio-based membranes primarily focuses on polysaccharide, protein, lipid, and composite bio-based membranes.

[0004] Gelatin is a water-soluble protein produced by partial or complete hydrolysis of collagen. It offers advantages such as low cost and high availability. Furthermore, gelatin exhibits excellent biocompatibility and film-forming properties, making it widely used in food packaging and other industries. Carboxymethyl chitosan is a water-soluble chitosan derivative obtained by reacting chitosan with monochloroacetic acid under alkaline conditions. Carboxymethyl chitosan exhibits excellent film-forming properties, biocompatibility, and biodegradability, making it widely used in food packaging and other fields.

[0005] Bio-based films for food packaging require excellent mechanical properties, transparency, barrier properties, and water resistance. Films made from a single film-forming matrix often struggle to meet these requirements, necessitating the creation of composite films to improve their performance. While gelatin-carboxymethyl chitosan composite films exhibit promising mechanical properties, their high water solubility due to the presence of numerous hydrophilic groups limits their application in high-moisture food packaging. Therefore, it is necessary to employ appropriate methods to reduce the water solubility of gelatin-carboxymethyl chitosan films. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention aims to provide a method for preparing a low-solubility gelatin-carboxymethyl chitosan composite food packaging film.

[0007] The present invention is achieved through the following technical solutions:

[0008] The method for preparing a low-solubility gelatin-carboxymethyl chitosan composite food packaging film is characterized in that the method comprises the following steps:

[0009] 1) dissolving carboxymethyl chitosan in distilled water with stirring to prepare a carboxymethyl chitosan solution;

[0010] 2) Dissolve gelatin in distilled water in a water bath and cool to room temperature to prepare a gelatin solution.

[0011] 3) Mixing the gelatin solution and the carboxymethyl chitosan solution, adding glycerol, and stirring at room temperature for 30 minutes to prepare a gelatin-carboxymethyl chitosan membrane solution;

[0012] 4) adding reducing sugar to the gelatin-carboxymethyl chitosan film liquid prepared in step 3), stirring at room temperature for 30 minutes, filtering to remove impurities, and preparing a film-forming liquid. The film-forming liquid is adjusted to a pH value and poured into a plastic dish. The film is dried at 25° C. for 48 hours to form a film. After removing the film, the film is placed in a forced air drying oven for MR reaction to prepare a low-solubility gelatin-carboxymethyl chitosan composite food packaging film.

[0013] Furthermore, the solid-liquid ratio of carboxymethyl chitosan to distilled water g:mL is 0.1:10, the solid-liquid ratio of gelatin to distilled water g:mL is 0.2:5, and the mass ratio of carboxymethyl chitosan to gelatin in the gelatin-carboxymethyl chitosan film liquid is 1:2.

[0014] Furthermore, in step 2), the water bath temperature is 60° C., and the water bath time is 30 min.

[0015] Furthermore, in step 3), the amount of glycerol added accounts for 25% of the total mass of carboxymethyl chitosan and gelatin.

[0016] Furthermore, in step 4), the reducing sugar is xylose, and the concentration of xylose is 5%-10%, preferably 6.25%.

[0017] Furthermore, the pH value of the membrane-forming solution in step 4) is 7-10, preferably 8.7.

[0018] Furthermore, in step 4), the temperature of the MR reaction is 70-90° C., and the reaction time is 24 h.

[0019] The present invention achieves modification of the gelatin-carboxymethyl chitosan composite membrane by adding reducing sugar into the gelatin-carboxymethyl chitosan composite membrane liquid, and further by screening the type and concentration of reducing sugar, the modified composite membrane has lower water solubility, elongation at break and tensile strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Effects of different types of reducing sugars on the water solubility of gelatin-carboxymethyl chitosan films after heating (different English letters indicate significant differences between data (p<0.05));

[0021] Figure 2 Effects of heating and xylose concentration on the water solubility of gelatin-carboxymethyl chitosan film (different English letters indicate significant differences between the data (p<0.05)).

[0022] Figure 3 The effect of the initial pH value of the membrane-forming solution on the water solubility of MR / G-CMCS (different English letters indicate significant differences between the data (p<0.05));

[0023] Figure 4 The effect of reaction temperature on the water solubility of MR / G-CMCS (Note: different English letters indicate significant differences between the data (p<0.05));

[0024] Figure 5 The effect of reaction time on the water solubility of MR / G-CMCS (Note: different English letters indicate significant differences between the data (p<0.05));

[0025] Figure 6 The following is the appearance comparison between G-CMCS and G-CMCS-X;

[0026] Figure 7 Comparison of water resistance of G-CMCS and G-CMCS-X;

[0027] Figure 8 UV-visible absorption spectra of (A) G-CMCS and (B) G-CMCS-X, A is G-CMCS, B is G-CMCS-X;

[0028] Figure 9 Fourier transform infrared spectra of (A) G-CMCS and (B) G-CMCS-X;

[0029] Figure 10 X-ray diffraction spectra of (A) G-CMCS and (B) G-CMCS-X;

[0030] Figure 11 The micromorphology of G-CMCS and G-CMCS-X. DETAILED DESCRIPTION

[0031] The present invention is described in further detail below in conjunction with specific implementation methods to facilitate a better understanding of the present technical solution.

[0032] Example: Preparation of membrane

[0033] Gelatin-carboxymethyl chitosan film: Weigh 0.2 g of carboxymethyl chitosan into 20 mL of distilled water and stir to dissolve. Weigh 0.4 g of gelatin into 10 mL of distilled water and incubate in a 60°C waterbath for 30 min. Cool to room temperature and mix with the carboxymethyl chitosan solution. Add 25% (w / w, glycerol / gelatin and carboxymethyl chitosan) glycerol and stir at room temperature for 30 min to obtain a gelatin-carboxymethyl chitosan film solution. Filter to remove impurities and pour the film solution into a plastic dish (100 mm × 100 mm). Dry at 25°C for 48 h to form a film. Remove the film and label it as G-CMCS.

[0034] MR-modified gelatin-carboxymethyl chitosan membrane: Reducing sugar was added to the gelatin-carboxymethyl chitosan membrane solution, stirred at room temperature for 30 minutes, and filtered to remove impurities. This solution had an initial pH of 8.7. The solution was poured into a plastic dish and dried at 25°C for 48 hours to form a membrane. After removal, the membrane was placed in a forced air drying oven for MR. This membrane was designated MR / G-CMCS. Membranes prepared under the same conditions without the addition of reducing sugar were designated H / G-CMCS.

[0035] The membrane was equilibrated in a constant temperature and humidity chamber at 25° C. and 50% relative humidity for 24 hours, and then its water solubility was measured. The mechanical properties of the membrane were measured after equilibration for 48 hours under the same conditions.

[0036] Experimental example

[0037] Screening of reducing sugar types: The preparation of MR / G-CMCS was the same as in the example, wherein the reducing sugars were xylose, arabinose, glucose, galactose, fructose, maltose, and lactose, with a concentration of 7.50% (w / w, reducing sugar / gelatin and carboxymethyl chitosan). After film formation, the reaction was carried out at 60°C for 6 hours. Using water solubility as an indicator, the reducing sugar with the best effect of reducing the water solubility of the gelatin-carboxymethyl chitosan film was screened.

[0038] like Figure 1As shown, the water solubility of both G-CMCS (control 1) and H / G-CMCS (control 2) was 100%, indicating that heating had no significant effect on the water solubility of gelatin-carboxymethyl chitosan films (p>0.05). The water solubility of MR / G-CMCS (all 100%) with the addition of glucose, lactose, and maltose remained unchanged (p>0.05). However, the water solubility of MR / G-CMCS prepared with the addition of the other four reducing sugars decreased significantly (p<0.05), with the MR / G-CMCS prepared with xylose exhibiting the lowest water solubility. This may be due to the tight cross-linking of gelatin, carboxymethyl chitosan, and reducing sugar molecules during MR, which exposes the hydrophobic groups of gelatin and promotes interactions between macromolecules, resulting in a more compact MR / G-CMCS network and reduced film water solubility. Monosaccharides (pentoses and hexoses) are more susceptible to MR than disaccharides, and pentoses are more susceptible than hexoses. Lactose and maltose are disaccharides, glucose, fructose, and galactose are hexoses, and arabinose and xylose are pentoses. MR / G-CMCS with xylose has the lowest water solubility, likely due to its highest degree of cross-linking. Wang et al., studying the antioxidant activity of the reaction products of whey protein isolate with xylose, fructose, glucose, lactose, and maltose, found that xylose is more likely to undergo MR, resulting in the strongest antioxidant activity. Therefore, xylose was selected as the reducing sugar to reduce the water solubility of MR / G-CMCS.

[0039] Optimization of reducing sugar concentration: The preparation of MR / G-CMCS was the same as in Example 1, wherein the xylose concentrations were 5.00%, 6.25%, 7.50%, 8.75% and 10.00%, respectively. After film formation, the reaction was carried out at 60°C for 6 hours. The reducing sugar concentration was optimized based on water solubility.

[0040] like Figure 2As shown, heating had no significant effect on the water solubility of gelatin-carboxymethyl chitosan films (p>0.05). However, the addition of xylose reduced the water solubility of MR / G-CMCS. Increasing the xylose concentration from 5.00% to 6.25% significantly decreased the water solubility of MR / G-CMCS (p<0.05). This may be because increasing xylose concentration increases the degree of MR, which increases the cross-linking of MR / G-CMCS and enhances the hydrophobic interaction of gelatin, leading to a decrease in the water solubility of the film. As the xylose concentration increased from 6.25% to 10.00%, the water solubility of MR / G-CMCS showed a decreasing trend, but the difference was not significant (p>0.05). The water solubility ranged from 38.98% to 39.68%. This may be because at a xylose concentration of 6.25%, most of the free amino groups in the film-forming matrix bonded to the carbonyl groups of xylose, resulting in a high degree of MR under these reaction conditions. Further increases in xylose concentration only slightly deepened the MR, resulting in no significant change in water solubility (p>0.05). When studying the effect of xylose concentration on the water solubility of peanut protein films, Lin Weijing found that as the xylose concentration increased from 1% to 2%, 5%, and 10%, the water solubility of the protein films decreased. However, when the concentration increased from 10% to 20%, the water solubility of the protein films did not change significantly (p>0.05). Therefore, 6.25% xylose concentration was selected as the concentration to reduce the water solubility of MR / G-CMCS.

[0041] Effects of initial pH value of membrane-forming solution, reaction temperature and time on the properties of MR / G-CMCS

[0042] (1) Initial pH value of the film-forming solution

[0043] The initial pH values of the membrane-forming solution were adjusted to 7, 8, 9, 10, and 11 using 2 mol / L HCl or 7.5 mol / L NaOH, respectively. After membrane formation, the solution was reacted at 60 °C for 6 h, and the water solubility and mechanical properties of MR / G-CMCS were measured.

[0044] (2) Reaction temperature

[0045] The initial pH value of the membrane-forming solution was 8.7. After the membrane was formed, the reaction was carried out at 50℃, 60℃, 70℃, 80℃, and 90℃ for 6 h, and the water solubility and mechanical properties of MR / G-CMCS were measured.

[0046] (3) Reaction time

[0047] The initial pH value of the membrane-forming solution was 8.7. After the membrane was formed, the reaction was carried out at 60 °C for 1.5 h, 3 h, 6 h, 12 h, and 24 h, respectively, and the water solubility and mechanical properties of MR / G-CMCS were measured.

[0048] Determination method

[0049] Moisture content: Following the method of Kchaou et al., cut the membrane into small pieces with a mass of approximately 0.1 g, place them in a constant-weight weighing bottle, and weigh them, recorded as m0 (g). Dry the membrane in a 105°C oven until the mass is constant, recorded as m1 (g). Moisture content is calculated as follows:

[0050]

[0051] Water solubility: Following the method of Kchaou et al., the membrane was cut into small pieces of approximately 0.1 g and weighed, denoted as m0. The membrane was placed in 30 mL of distilled water, allowed to stand at room temperature for 24 h, then removed and dried in a 105°C oven to a constant mass, denoted as m1 (g). Water solubility was calculated as follows:

[0052]

[0053] Where WC is the moisture content of the membrane.

[0054] Mechanical properties: The thickness of the film was measured using a thickness gauge. Random points were taken from each film and the average value was used to calculate its tensile strength.

[0055] Following the method of Nurual et al., the membrane was cut into 10 mm × 60 mm strips and placed between clamps with an initial distance of 40 mm. The tensile strength (TS) and elongation at break (EAB) of the membrane were tested using an electric tensile testing machine at a stretching speed of 30 mm / min. TS and EAB were calculated as shown in Equations 1 and 2:

[0056]

[0057] Where F is the maximum tensile force (N) that the membrane withstands during the stretching process; S is the cross-sectional area of the membrane sample (m 2 ).

[0058]

[0059] Where L is the length added when the membrane breaks (mm); L0 is the initial length of the membrane (mm).

[0060] Data Processing: All experimental points were replicated in at least three times, with six replicates for film thickness and mechanical properties. Data are presented as mean ± standard deviation. Microsoft Excel was used for data processing, and SPSS 22.0 software was used for statistical analysis. Tukey's test was used for data analysis (p < 0.05 indicated a significant difference). Graphs were generated using OriginPro 2018 software.

[0061] Effect of initial pH value of membrane-forming solution on water solubility and mechanical properties of MR / G-CMCS

[0062] Water solubility: Figure 3 As shown, the water solubility of the MR-treated gelatin-carboxymethyl chitosan film was significantly reduced compared to control 1 (p < 0.05). When the initial pH of the membrane-forming solution was between 7 and 10, the water solubility of the MR / G-CMCS film did not differ significantly from its natural pH (8.7) (p > 0.05), ranging from 44.45% to 55.56%. When the pH of the membrane-forming solution reached 11, the water solubility of the film increased significantly to 58.77 ± 1.06% (p < 0.05). Literature reports suggest that alkaline conditions favor MR, with the water solubility of the film decreasing with increasing MR. This is inconsistent with the results of this study. This may be because the membrane-forming solution environment is relatively mild at pH values between 7 and 9, resulting in little difference in the degree of gelatin structural unfolding. However, as the pH of the membrane-forming solution increases (10 and 11), gelatin denaturation increases under strong alkaline conditions, exposing more hydrophilic groups and leading to increased water solubility of the MR / G-CMCS.

[0063] Mechanical properties: As shown in Table 1, compared with control 1, the TS of MR / G-CMCS prepared with different initial pH values of the membrane-forming solution were all greater, with the maximum at the natural pH value of 8.7; there was no significant difference in EAB (p>0.05). When the initial pH value of the membrane-forming solution was 7-9, there was no significant difference in the TS and EAB of MR / G-CMCS (p>0.05). This may be because the increase in the pH value of the membrane-forming solution from 7 to 9 did not significantly affect the MR degree and had no significant effect on the tertiary structure of gelatin. When the pH value of the membrane-forming solution was 10, the TS and EAB of the membrane increased significantly (p<0.05). This may be because the intermolecular interaction of gelatin was strengthened at this pH value of the membrane-forming solution, resulting in a denser structure of the membrane. When the pH value of the membrane-forming solution increased to 11, the TS and EAB of the membrane decreased. This may be because the pH value of the membrane-forming solution was too high, resulting in excessive denaturation of gelatin, and the loose structure of the membrane, resulting in poor mechanical properties.

[0064] Table 1: Effect of initial pH value of the membrane-forming solution on the mechanical properties of MR / G-CMCS

[0065]

[0066]

[0067] Note: Different English letters in the same column indicate significant differences between the data (p<0.05).

[0068] Effect of reaction temperature on water solubility and mechanical properties of MR / G-CMCS

[0069] Water solubility: Figure 4As shown, the addition of xylose had no significant effect on the gelatin-carboxymethyl chitosan film (p>0.05). The water solubility of MR / G-CMCS decreased with increasing reaction temperature. When the temperature was between 50°C and 70°C, the water solubility of the film decreased significantly with increasing temperature (p<0.05). This may be because the degree of MR increased with increasing temperature, which enhanced the cross-linking between gelatin, carboxymethyl chitosan, and xylose molecules, exposed the hydrophobic groups in the gelatin, and made the membrane structure more compact, leading to a decrease in the water solubility of the film. Although the water solubility of the film decreased at temperatures between 70°C and 90°C, the difference was not significant (p>0.05). This may be because the degree of MR of MR / G-CMCS was high at 70°C, which reduced the water solubility to a certain level. Further increases in temperature did not significantly affect the exposure of hydrophobic groups and the membrane structure, resulting in no significant change in water solubility (p>0.05). Kchaou et al. also observed a similar phenomenon when studying the effect of temperature on the water solubility of gelatin-based films.

[0070] Mechanical Properties: Table 2 shows that the addition of xylose had no significant effect on the mechanical properties of the gelatin-carboxymethyl chitosan film (p>0.05). Reaction temperature had no significant effect on the TS of MR / G-CMCS (p>0.05), but heating significantly reduced the EAB of MR / G-CMCS (p<0.05). Heating increased the TS and decreased the EAB of MR / G-CMCS. This may be because increasing temperature increases the MR of the film, enhancing the interaction between the gelatin, carboxymethyl chitosan, and xylose molecules and making the film structure denser, leading to an increase in TS. However, excessive cross-linking of the gelatin, carboxymethyl chitosan, and xylose molecules limits the mobility of the film-forming molecules, resulting in a decrease in the EAB of the film. Hazaveh et al., studying the effects of ribose and fructose on the mechanical properties of gelatin films, also found that as the TS of the films increased, the EAB decreased.

[0071] Table 2: Effect of reaction temperature on mechanical properties of MR / G-CMCS

[0072]

[0073] Note: Different English letters in the same column indicate significant differences between the data (p<0.05).

[0074] Effect of reaction time on water solubility and mechanical properties of MR / G-CMCS

[0075] Water solubility: Figure 5As shown in the results, the addition of xylose had no significant effect on the water solubility of gelatin-carboxymethyl chitosan films (p>0.05); however, the water solubility of the films decreased significantly with increasing reaction time (p<0.05). This may be because the MR degree of the films increased, the cross-linking between gelatin, carboxymethyl chitosan, and xylose molecules was enhanced, the hydrophobic groups in the gelatin were exposed, and the membrane structure became more compact, resulting in a decrease in the water solubility of the films. Stevenson et al., when studying the effect of heating time on the water solubility of films, also found that the water solubility of gelatin-ribose films decreased with increasing heating time.

[0076] Mechanical Properties: Table 3 shows that the addition of xylose had no significant effect on the mechanical properties of the gelatin-carboxymethyl chitosan film (p>0.05). However, with increasing heating time, the TS of the MR / G-CMCS significantly increased (p<0.05), while the EAB decreased. This is likely due to the increased MR level of the MR / G-CMCS, which enhanced the interaction between the gelatin, carboxymethyl chitosan, and xylose molecules and made the film structure denser, leading to increased TS. However, excessive cross-linking reduced the mobility of gelatin and other film-forming molecules, resulting in a decrease in the film's EAB. Lin Weijing also observed a similar phenomenon when studying the effect of heating time on the mechanical properties of peanut protein films.

[0077] Table 3: Effect of reaction time on mechanical properties of MR / G-CMCS

[0078]

[0079] Note: Different English letters in the same column indicate significant differences between the data (p<0.05).

[0080] Taking water solubility as the evaluation index, when 6.25% xylose was added, the water solubility of the prepared MR / G-CMCS was the lowest, at 39.07±0.04%, which was significantly lower than that of unmodified G-CMCS (water solubility 100%). Therefore, 6.25% xylose was the most appropriate choice to reduce the water solubility of MR / G-CMCS.

[0081] The effects of the initial pH value of the film-forming solution, reaction temperature, and reaction time on MR / G-CMCS were investigated, using water solubility and mechanical properties as evaluation indicators. The results showed that adjusting the initial pH value of the film-forming solution increased the water solubility of MR / G-CMCS, while increasing the reaction temperature and time decreased it. The water solubility was lowest at a reaction temperature of 90°C, reaching 25.35±0.31%. Changing the initial pH value of the film-forming solution and increasing the reaction temperature and time enhanced the mechanical properties of MR / G-CMCS. When the initial pH value of the film-forming solution was 10, the elongation at break of MR / G-CMCS reached a maximum of 63.28±0.95%. The tensile strength of MR / G-CMCS reached a maximum of 9.29±0.19 MPa when the reaction time was 24 h.

[0082] Example 2

[0083] According to the method of Example 1, a series of gelatin-carboxymethyl chitosan films were prepared under different conditions, as shown in Table 4.

[0084] Table 4: Experimental groups

[0085]

[0086] Except that the membranes used for Fourier transform infrared spectroscopy and X-ray diffraction spectroscopy were equilibrated in a desiccator, the equilibration conditions for the other membranes were the same as those in Example 1.

[0087] Membrane properties: According to the CIELAB color scale, the color is expressed as L * (100=white; 0=black), a * (+, red; -, green) and b * (+, yellow; -, blue). Use a colorimeter to compare the white plate (L0 * =94.52,a0 * =-0.23,b0 * =3.47) as the background, and the color parameter L of the film was measured. * 、a * and b * , and use color parameters to calculate color difference (ΔE * ), as follows:

[0088]

[0089] Calculate BI using the measured color parameters, as shown in Equations 3 and 4

[0090]

[0091]

[0092] Where, L * 、a * 、b * is the color parameter of the sample, L1 * 、a1 * 、b1 * is the color parameter of the control film.

[0093] The appearance was photographed according to the method of Etxabide et al. The film was cut into strips of 15 mm × 60 mm and attached to the surface of white A4 paper printed with the word "Appearance". The appearance of the film was photographed using a digital camera.

[0094] Moisture content: The determination method is the same as above.

[0095] Swelling ratio: Cut the membrane into small pieces of approximately 0.1 g according to the method of Rui et al., weigh them, and record them as m0 (g). Place the membrane in water for 30 minutes, then quickly remove it, place it on filter paper, absorb the surface water, and weigh it, and record it as m1 (g). The swelling ratio is calculated as follows:

[0096]

[0097] Water solubility: The water solubility was determined as above, with additional photographic presentation. Referring to the method of Park et al., the membrane was cut into 250 mm × 250 mm blocks, immersed in 30 mL of distilled water, and the state of the membrane was recorded using a digital camera.

[0098] Water contact angle: Following the method of Ahammed et al., cut the membrane into 10 mm × 20 mm pieces and attach them to a clean glass slide, ensuring that there are minimal air bubbles between the membrane and the slide. Turn the knob to drip approximately 3 μL of distilled water from a syringe onto the membrane surface. Immediately measure the water contact angle using a contact angle meter.

[0099] Water vapor permeability coefficient: Following the method of Chen et al., the membrane was cut into 50 mm × 50 mm pieces. The membrane thickness was measured and sealed with paraffin wax in the mouth of a 50 mL centrifuge tube (filled with 20 mL of distilled water). After weighing, the membrane was placed in a desiccator and the weight change was recorded every 24 hours until the mass change was less than 5%. The water vapor permeability coefficient (g / (m·s·Pa)) was calculated as follows:

[0100]

[0101] Where w is the weight difference of the centrifuge tube within 24 hours after the water vapor permeation rate stabilizes (g), d is the sample thickness (m), and s is the effective area of the sample (m 2 ), p is the water vapor partial pressure on the sample (Pa), and t is the interval time (s).

[0102] Thickness and mechanical properties: The measurement method is the same as above.

[0103] The determination of thickness and mechanical properties is the same as 2.3.3.3.

[0104] Membrane characterization

[0105] UV-visible absorption spectroscopy: Referring to the method of Zhong et al., the membrane was cut into strips of 10 mm × 30 mm and attached to the inside of a quartz cuvette. The absorbance of the sample at 200–800 nm was measured using a UV-visible spectrophotometer, with air as a control.

[0106] Fourier transform infrared spectroscopy: According to the method of Liu et al., the film was placed in the middle of the sample stage and measured using a Fourier transform infrared spectrometer with an attenuated total reflectance accessory. The number of scans was 32 and the resolution was 4 cm -1 , measuring range 650-4000cm -1 .

[0107] X-ray diffraction spectroscopy: Following the method of Kchaou et al., the membrane was spread flat on a test glass slide and measured using an X-ray diffractometer. The measurement range was 5-50°, the scanning speed was 10° / min, the voltage was 40 kV, and the current was 15 mA.

[0108] Micromorphology: Referring to the method of Li et al., the film samples were treated by sputtering gold, and the microscopic images of the films were obtained in a suitable field of view using a scanning electron microscope.

[0109] Data processing: The data processing method is the same as above.

[0110] Experimental results

[0111] Properties of G-CMCS and G-CMCS-X

[0112] Color, BI value and appearance: The changes in color, BI value and appearance are related to MR. Table 5 shows the color parameters and BI value of gelatin-carboxymethyl chitosan film.

[0113] As shown in Table 5, the L * The values were 93.57-93.64, with no significant difference (p>0.05). * The value was 93.56±0.06. With the increase of reaction temperature and time, the L * The value shows a decreasing trend, especially at 90℃, L * The value was significantly decreased (p<0.05), and the L * The value is the smallest, which is 80.48±0.42. This may be because the amino groups in gelatin and carboxymethyl chitosan and the carbonyl groups of xylose undergo MR to generate brown melanoidins, which causes the gelatin-carboxymethyl chitosan film to darken.

[0114] Table 5: Comparison of color and BI values between G-CMCS and G-CMCS-X

[0115]

[0116]

[0117] Note: Different English letters in the same column indicate significant differences between the data (p<0.05).

[0118] As shown in Table 5, the a of F1* The value is -0.61±0.01. With the increase of reaction temperature and time, the a * The value shows a decreasing trend, * The smallest value is F1-90-2, which is -0.92±0.02. * The value is -0.74±0.02. With the increase of reaction temperature and time, the a * The values showed a trend of first decreasing and then increasing, decreasing from -1.28±0.10 (F2-50-1) to -12.43±0.05 (F2-90-1), and then increasing to 1.81±0.38 (F2-90-2).

[0119] As shown in Table 5, the b of F1 * The value is 2.16±0.05, and heating leads to the b * The value increased significantly (p < 0.05), which may be due to the weak MR between gelatin and carboxymethyl chitosan caused by heating, which can be proved by the changes in the UV-visible absorption spectrum of 3.4.2.1 (A). With the increase of reaction temperature and time, the b value of the film * There was no significant difference in the values (p>0.05), ranging from 5.36 to 6.08. * The value is 2.50±0.08. With the increase of reaction temperature and time, the b * The value increased significantly (p<0.05), and the b * The maximum value is 87.37±0.52, at which point the membrane is yellow-brown. Figure 6 This may be because MR generates melanin in the later stage, which causes the gelatin-carboxymethyl chitosan film to change from colorless to yellow-brown. Kchaou et al. also found that as the heating temperature increases, the b * The value showed an increasing trend. When glucose was added, the gelatin-glucose membrane b * The increase in the value was more significant (p<0.05).

[0120] As shown in Table 5 and Figure 6 As shown, the ΔE of G-CMCS * There was no significant difference between the values (p>0.05), ΔE * The values are 3.20-3.92. With the increase of reaction temperature and time, the ΔE * The value showed a significant increasing trend (p<0.05), which may be due to the deepening of MR and the increase of late-stage melanin products, which led to the deepening of G-CMCS-X color. Stevenson et al. studied the ΔE of gelatin-ribose membranes at different temperatures and time. *When the value changes, it is found that with the increase of heating temperature and time, ΔE * The value increased significantly (p<0.05).

[0121] The BI value is an important parameter for measuring the degree of MR, and it increases with increasing MR. As shown in Table 5, the BI value of F1 was 1.78 ± 0.05. Heating significantly increased the BI value of G-CMCS (p < 0.05), likely due to the MR between gelatin and carboxymethyl chitosan. However, there were no significant differences in the BI values across different reaction temperatures and times (p > 0.05), ranging from 5.21 to 5.76, indicating that MR in G-CMCS under these experimental conditions was relatively weak. The BI value of F2 was 2.04 ± 0.07. The BI value of G-CMCS-X increased significantly with increasing reaction temperature and time (p < 0.05), reaching the highest value of 249.55 ± 3.49 for F2-90-2, indicating that the degree of MR in G-CMCS-X increased significantly with increasing reaction temperature and time (p < 0.05). When Stevenson et al. studied the BI values of gelatin-ribose membranes at different temperatures and durations, they found that with the increase of heating temperature and time, the difference in BI values of the membranes became more significant (p<0.05).

[0122] Moisture content: Moisture content is a critical parameter for packaging materials. As shown in Table 6, the moisture content of F1 was 16.68 ± 0.44%, while the moisture content of heated G-CMCS ranged from 16.38% to 16.78%, showing no significant difference from F1 (p > 0.05). The moisture content of F2 was 16.71 ± 0.45%, while the moisture content of heated G-CMCS-X ranged from 15.96% to 17.39%, showing no significant difference from F2 (p > 0.05). With the increase of reaction temperature and time, the moisture content of G-CMCS-X showed a trend of first increasing and then decreasing. The moisture content of F2-50-2 was the highest, which was 17.61±0.46%, and the moisture content of F2-90-2 was the lowest, which was 15.96±0.58%. This may be because the MR degree of F2-50-2 was low, and there was still xylose that had not been completely reacted in the membrane. The xylose containing a large number of hydroxyl groups combined with water, resulting in an increase in its moisture content; compared with F2-50-2, the moisture content of F2-90-2 was significantly reduced (p<0.05). This may be because the amino groups in gelatin and / or carboxymethyl chitosan and the carbonyl groups in xylose were tightly cross-linked through MR, making it impossible for water molecules to combine with gelatin, carboxymethyl chitosan and xylose molecules, resulting in a decrease in the moisture content of the membrane.

[0123] Table 6: Comparison of properties of G-CMCS and G-CMCS-X

[0124]

[0125] Note: / represents unmeasurable; different English letters in the same column indicate significant differences between the data (p<0.05).

[0126] Swelling ratio: The swelling ratio is an important indicator of membrane water resistance. As shown in Table 6, the swelling ratio of G-CMCS could not be determined because it dissolved in water after immersion for 30 minutes. The swelling ratios of F2 and F2-50-1 were 9.22±2.47 and 9.41±2.03, respectively, showing no significant difference (p>0.05). F2 and F2-50-1 swelled with water and exhibited severe deformation. With increasing reaction time and temperature, the swelling ratio of G-CMCS-X decreased significantly (p<0.05), with F2-90-2 exhibiting the lowest swelling ratio of 0.70±0.03. This may be because MR forms a dense network structure between gelatin, carboxymethyl chitosan and xylose, and the strong interaction of intramolecular and intermolecular hydrogen bonds prevents water molecules from binding to them. In addition, MR will generate melanin-like substances that are insoluble in water in the later stage, and the electrostatic repulsion between gelatin molecules decreases. Therefore, the swelling ratio of G-CMCS-X tends to decrease with the increase of reaction temperature and time.

[0127] Water solubility: The present invention uses water solubility as the core indicator to characterize the water resistance of the membrane. As shown in Table 6, G-CMCS, F2 and F2-50-1 are all dissolved. With the increase of reaction time and temperature, the water solubility of F2-50-2, F2-90-1 and F2-90-2 significantly decreases (p<0.05), and the water solubility of F2-90-2 is the lowest, which is 26.95±2.96%. This may be because heating causes MR between gelatin, carboxymethyl chitosan and xylose. The Schiff base produced by the reaction is subsequently cyclized to produce Amadori compounds, and then water-insoluble melanoidins are formed. At the same time, the hydrophobic groups of the gelatin molecules are exposed, and the hydrophobic interactions are strengthened, resulting in a decrease in the water solubility of G-CMCS-X.

[0128] In order to more intuitively demonstrate the water resistance of gelatin-carboxymethyl chitosan film, it was immersed in water. Figure 7 As shown, G-CMCS completely dissolved within 30 minutes, while G-CMCS-X had not yet dissolved. After 15 hours, F2 and F2-50-1 dissolved. This may be because F2 and F2-50-1 did not undergo MR or did so to a low degree, resulting in insufficient crosslinking between the gelatin, carboxymethyl chitosan, and xylose molecules and the presence of numerous hydrophilic hydroxyl groups within the membrane, leading to poor water resistance. After 5 days, F2-50-2 dissolved. At this time, F2-90-1 absorbed water and swelled, exhibiting severe deformation, while F2-90-2 only expanded slightly. After 30 days, F2-90-1 had not completely dissolved, forming small, colorless fragments. At this point, F2-90-2 still retained its intact membrane shape, demonstrating excellent water resistance.

[0129] Water contact angle: The water contact angle is an important indicator of membrane surface hydrophobicity. Generally, a larger water contact angle indicates a more hydrophobic membrane surface. As shown in Table 6, the water contact angle of F1 was 71.32±3.52°. With increasing reaction temperature and time, the water contact angle of G-CMCS decreased significantly (p<0.05). F1-90-2 had the smallest water contact angle, at 62.61±3.52°. This may be due to the exposure of hydrophilic groups within the membrane surface during heating, resulting in a decrease in the water contact angle. The water contact angle of F2 was significantly lower than that of F1, at 57.03±2.60° (p<0.05). This may be because the added xylose contains a large number of hydroxyl groups, which readily form hydrogen bonds with water molecules, thereby enhancing the membrane's surface hydrophilicity. With increasing reaction temperature and time, the water contact angle of G-CMCS-X gradually decreased, with F2-90-2 having the smallest water contact angle, at 50.47±0.96°. Generally, cross-linking polymers reduces their water solubility but enhances their hydrophilicity. Kwak et al., when cross-linking gelatin with sucrose, glucose, and fructose to prepare electrospun nanofibers, found that the water contact angles of the three sugars cross-linked with gelatin nanofibers were in the order of sucrose > glucose > fructose, while their water absorption capacities showed opposite trends. This is similar to the findings of the present study on swelling ratio and water contact angle.

[0130] Water vapor permeability coefficient: Water vapor can penetrate into the interior of the package or escape from the interior, causing continuous changes in food quality. Therefore, the water vapor permeability coefficient of food packaging film should be as small as possible to prevent moisture exchange between food and the external environment.

[0131] As shown in Table 6, the water vapor permeability coefficient of F1 is 7.85±0.46 (×10 -11 g / m·s·Pa). With the increase of reaction temperature and time, the water vapor permeability coefficient of G-CMCS did not change significantly (p>0.05), ranging from 7.39 to 8.15 (×10 - 11 g / m·s·Pa). The water vapor permeability coefficient of F2 is 8.57±0.96(×10 -11 g / m·s·Pa), which is higher than the water vapor permeability coefficient of G-CMCS. This may be because xylose contains hydrophilic hydroxyl groups, which are easy to combine with water molecules. With the increase of reaction temperature and time, the water vapor permeability coefficient of G-CMCS-X showed an overall decreasing trend. The water vapor permeability coefficient of F2-90-2 was the smallest, which was 7.40±0.16 (×10 -11 g / m·s·Pa), which may be because MR promotes the close binding between protein and sugar molecules, making the membrane structure more compact, thereby reducing the water vapor permeability coefficient of the membrane.

[0132] Thickness and Mechanical Properties: As shown in Table 7, the thickness of F2-90-2 was 59.17±1.47μm, significantly greater than that of the other groups (p<0.05). The thicknesses of the other groups showed no significant differences (p>0.05), ranging from 53.50 to 56.50μm. This may be because G-CMCS-X underwent MR, and the MR-modified membrane F2-90-2 had the highest degree of MR and the most MRPs, resulting in increased thickness. Li et al., studying the effect of adding MRPs on chitosan membrane thickness, also found that the membrane thickness increased with the increase in MRPs.

[0133] TS and EAB are important performance indicators for packaging materials. Food packaging materials require high mechanical strength to maintain food integrity. As shown in Table 7, heat treatment had no significant effect on the mechanical properties of G-CMCS (p > 0.05). With increasing reaction temperature and time, the mechanical properties of G-CMCS-X initially increased and then decreased. This may be due to the enhanced MR and intermolecular covalent bonding, leading to enhanced mechanical properties. However, the TS and EAB of F2-90-2 decreased significantly, possibly due to excessive cross-linking of gelatin, carboxymethyl chitosan, and xylose, which reduced the macromolecular fluidity and decreased mechanical properties.

[0134] Table 7: Comparison of thickness and mechanical properties of G-CMCS and G-CMCS-X

[0135]

[0136] Note: Different English letters in the same column indicate significant differences between the data (p<0.05).

[0137] Characterization of G-CMCS and G-CMCS-X

[0138] UV-visible absorption spectrum: UV-visible absorption spectrum can characterize the degree of film MR and correlate it with the changes in color and BI value. 294 ) is associated with the formation of colorless compounds in the early stages of MR, which may be precursors to the formation of melanoidins (brown and water-insoluble compounds) in the final stages of MR. Figure 8 As shown in (A), the characteristic absorption peak of Schiff base (one of the early products of MR) is near 280nm. After heating, the absorption value of G-CMCS at 280nm increases but is generally small. This is because gelatin and carboxymethyl chitosan have a weak MR. Heating increases the A of G-CMCS. 294 , but A under different heating temperature and time 294 The difference is small, indicating that heating has little effect on the UV-visible absorption spectrum of G-CMCS. Figure 8As shown in (B), with the increase of reaction temperature and time, the A 294 The A 294 A above 50℃ 294 , indicating that the membrane enters the stage of producing colorless compounds earlier at 90 °C than at 50 °C.

[0139] Absorbance at 420 nm (abbreviated as A 420 ) is related to the formation of melanin, the late product of MR. Figure 8 As shown in (A) and (B), the A of G-CMCS 420 Almost zero, while the A of F2-50-1, F2-50-2 and F2-90-1 294 There is an increasing trend, but A 420 The A of F2-90-2 is lower, indicating that MR has reached the stage of colorless compound formation, but slightly extended to the final stage of melanoidin formation. 294 and A 420 The sharp increase in indicates that MR has reached its final stage, with a higher degree of MR and a darker color of the membrane.

[0140] In summary, the UV-visible absorption spectrum of G-CMCS changed little, and the heat treatment caused a weak MR between gelatin and carboxymethyl chitosan. With the increase of reaction temperature and time, the A of G-CMCS-X 294 and A 420 The mid-stage and late-stage products increased, and the MR degree increased, which is related to Figure 6 The results of the membrane color change were consistent with that of the MR of G-CMCS-X. The increased MR of G-CMCS-X generated more water-insoluble melanoidins, resulting in a significant decrease in water solubility and swelling ratio (p < 0.05).

[0141] Fourier transform infrared spectroscopy: FTIR is an effective technique for characterizing interactions between polymer molecules and a crucial method for studying molecular structure. It effectively reveals crosslinking between substances. When strong interactions occur between substances, generating new functional groups, corresponding new characteristic peaks appear in the spectrum. Analyzing the spectrum reveals the strength of these interactions and the effects of reaction temperature and time on the structure of the substance. Therefore, the present invention utilizes FTIR spectroscopy to study changes in the molecular structure within the membrane.

[0142] like Figure 9As shown in the figure, the infrared spectra of G-CMCS and G-CMCS-X show similar shapes and positions. The addition of xylose will introduce hydroxyl groups. After heating, the carbonyl and amino groups in G-CMCS-X react to form CN bonds. However, gelatin and carboxymethyl chitosan will also form CN bonds when heated, so no new characteristic absorption peaks appear in the infrared spectrum of G-CMCS-X. G-CMCS and G-CMCS-X have 3298 cm -1 and 3294cm -1 A broad characteristic peak appears at 2930 cm, which belongs to the amide A band. This is the stretching vibration peak of the hydroxyl and amino groups. The characteristic absorption peak of G-CMCS-X here moves to the low wave number direction, indicating that the hydrogen bond force within or between the protein-sugar polymer molecules is enhanced; -1 and 2933cm -1 The stretching vibration absorption peaks of C=C and C=O of primary and secondary amines are 1635, 1554 and 1238 cm-1, respectively. -1 and 1635, 1551, 1238cm -1 Characteristic absorption peaks of amide I (C=O bond stretching vibration), II (NH bond bending vibration), and III (CN bond and NH bond stretching vibration) bands appeared at the 400 nm wavelength. MR occurred between the carbonyl group of xylose and the amino groups of gelatin and carboxymethyl chitosan, and conjugation induced N-substituted glycosylamines. These glycosylamines were arranged into 1-amino-1-deoxy-2-ketose, known as Amadori rearrangement products, and then furfural Schiff bases were formed, which participated in the formation of aldols, aldimines, and melanoidins. The characteristic absorption peak of Schiff base (C=N bond) is about 1640 cm -1 The absorption peak of the amide I band is located nearby, resulting in the overlap of the characteristic peaks of the Schiff base, or the amount of Schiff base generated is too small, so it is difficult to observe the spectral changes caused by MR from the infrared spectrum. Yuan et al. studied the effect of adding dialdehyde glucomannan to gelatin on the properties and structure of the membrane and found that although the significant change in membrane color and the increase in the peak area of the photoelectron spectrum proved that the membrane did undergo MR and generated Schiff base, the infrared spectrum did not show the characteristic absorption peak of Schiff base. 1554cm -1 The absorption peak near 1551 cm -1 The conformation of gelatin changed from helix to random coil and the molecular order decreased. In addition, the G-CMCS and G-CMCS-X showed a strong affinity at 1035 cm -1 An absorption peak appears at , which is the characteristic peak of hydroxyl group in glycerol.

[0143] In summary, although the UV-visible absorption spectrum of A 294 、A 420The significant increase in the BI value (p < 0.05) proves that MR occurs in G-CMCS-X after heating, but the infrared spectrum of the film does not change significantly. It may be that the characteristic absorption peaks of Schiff base are overlapped, or the amount generated is too small to be detected.

[0144] X-ray diffraction spectrum: X-ray diffraction is a method to study the atomic arrangement and crystal structure of substances, and is widely used in the characterization and analysis of substances such as crystal powders and film materials. Figure 10 As shown in Figure 2, all spectra show a sharp peak at 2θ = 7.8° and a broad peak at 2θ = 21.4°. The sharp peak at 2θ = 7.8° is due to the triple helical structure of gelatin, and its intensity corresponds to the triple helical content; the broad amorphous peak at 2θ = 21.4° is related to the distance between amino acid residues along the helix. Figure 10 As shown in (A), heating slightly decreases the intensity of the two diffraction peaks, indicating that heating has little effect on the crystallinity of G-CMCS. Figure 10 As shown in (B), with the increase of reaction temperature and time, the intensities of the two diffraction peaks of G-CMCS-X decreased significantly and shifted to the right, indicating that the heating treatment reduced the crystallinity of the film. This may be because MR formed strong intermolecular interactions between gelatin, carboxymethyl chitosan and xylose, resulting in changes in the polymer structure.

[0145] Micromorphology: Scanning electron microscopy is one of the effective means to observe membrane morphology at the microscopic level. Figure 11 Shown are the surfaces and cross-sections of G-CMCS and G-CMCS-X. Surface observation reveals that the surfaces of F1 and F1-90-1 are similar, with larger and more abundant patches of material on the surface of F2. The surface of F2-90-1 is rougher, resulting in higher water vapor permeability coefficients for F2 and F2-90-1 than for F1 and F1-90-1. Cross-sections reveal no obvious delamination in any of the membranes, indicating good compatibility between the gelatin and carboxymethyl chitosan molecules, as well as between the gelatin, carboxymethyl chitosan, and xylose molecules. Compared to the cross-section of F1, the cross-section of F2 is rougher, likely due to the addition of xylose. The cross-section of F1-90-1 is more uniform than that of F1, but exhibits fine cracks on the surface. The cross-section of F2-90-1 is denser and more compact than that of F1. This is likely due to the tight cross-linking between gelatin, carboxymethyl chitosan, and xylose molecules caused by MR, forming an ordered, dense structure. Consequently, the water solubility and swelling ratio of F2-90-1 are lower.

[0146] The color, moisture content, water solubility, swelling ratio, water vapor permeability coefficient, thickness and mechanical properties of G-CMCS heated by the present invention were not significantly affected (p>0.05), and the effect on the 294 and A 420The effects of Fourier transform infrared spectroscopy and X-ray diffraction spectroscopy were minor. Increasing the reaction temperature and time significantly reduced the water contact angle of G-CMCS (p<0.05), and the membrane cross-section became denser, but cracks existed.

[0147] With increasing heating temperature and time, the color of G-CMCS-X deepened, its water solubility, swelling ratio, and water contact angle decreased significantly (p < 0.05), and its mechanical properties improved. UV-visible absorption spectroscopy indicated that G-CMCS-X underwent magnetic resonance (MR), with the degree of MR increasing with increasing heating temperature and time. Fourier transform infrared spectroscopy showed minimal changes, while X-ray diffraction spectroscopy revealed a decrease in the crystallinity of G-CMCS-X. Microscopic morphology revealed a more uniform and dense membrane structure.

[0148] The mechanism of MR reducing the water solubility of gelatin-carboxymethyl chitosan film: MR occurs between the amino groups in gelatin and carboxymethyl chitosan and the carbonyl groups in xylose, generating water-insoluble melanoidins and promoting cross-linking between gelatin, carboxymethyl chitosan and xylose, resulting in a denser membrane structure and enhanced water resistance of the membrane.

Claims

1. A method for preparing a low-solubility gelatin-carboxymethyl chitosan composite food packaging film, characterized in that: The preparation method comprises the following steps: 1) Dissolve carboxymethyl chitosan in distilled water and stir to prepare a carboxymethyl chitosan solution; 2) Dissolve gelatin in distilled water in a water bath and cool to room temperature to prepare a gelatin solution. 3) Mix the gelatin solution and carboxymethyl chitosan solution, add glycerol, and stir at room temperature for 30 minutes to prepare a gelatin-carboxymethyl chitosan membrane solution; 4) Reducing sugar was added to the gelatin-carboxymethyl chitosan film liquid prepared in step 3), and the mixture was stirred at room temperature for 30 minutes. Impurities were removed by filtration to obtain a film-forming liquid. The pH of the film-forming liquid was adjusted to 8.7 and poured into a plastic dish. The film was dried at 25°C for 48 hours to form a film. After removing the film, the film was placed in a forced air drying oven for MR reaction to obtain a low-solubility gelatin-carboxymethyl chitosan composite food packaging film. The reducing sugar was xylose, and the xylose concentration was 6.25%.

2. The method for preparing a solubility gelatin-carboxymethyl chitosan composite food packaging film according to claim 1, characterized in that The solid-liquid ratio of carboxymethyl chitosan to distilled water (g:mL) was 0.1:10, the solid-liquid ratio of gelatin to distilled water (g:mL) was 0.2:5, and the mass ratio of carboxymethyl chitosan to gelatin in the gelatin-carboxymethyl chitosan membrane solution was 1:

2.

3. The method for preparing a solubility gelatin-carboxymethyl chitosan composite food packaging film according to claim 1, characterized in that In step 2), the water bath temperature is 60°C and the water bath time is 30 min.

4. The method for preparing a solubility gelatin-carboxymethyl chitosan composite food packaging film according to claim 1, characterized in that In step 3), the amount of glycerol added accounts for 25% of the total mass of carboxymethyl chitosan and gelatin.

5. The method for preparing a solubility gelatin-carboxymethyl chitosan composite food packaging film according to claim 1, characterized in that The temperature of the MR reaction in step 4) is 70-90°C, and the reaction time is 24 hours.

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