A quercetin bactericide, its preparation and application

Abstract

This invention discloses a quercetin bactericide, its preparation, and its application, belonging to the field of fresh fruit and vegetable preservation technology. The preparation method includes the following steps: adding quercetin and a catalyst to water to obtain mixture I; adding p-anisaldehyde to an ethanol aqueous solution to obtain mixture II; mixing mixture I and mixture II, adding acid to adjust the pH to acidic, heating the reaction, filtering, washing, and drying to obtain the quercetin bactericide. The preparation method of this invention uses molecular engineering technology to introduce p-anisaldehyde into the quercetin molecule, forming a two-dimensional stacked structure. The quercetin bactericide prepared by the method of this invention has stronger antibacterial activity and photosensitizing properties. When used in conjunction with visible light, it can significantly extend the preservation effect while minimizing nutrient loss in fruits and vegetables.

Description

Technical Field

[0001] This invention relates to the field of fresh fruit and vegetable preservation technology, and particularly to a quercetin bactericide, its preparation and application, and a composite packaging film. Background Technology

[0002] Microbial contamination is one of the most difficult challenges in maintaining the safety of fruits and vegetables. It can occur at different stages of food processing, posing a significant threat to various foods and increasing the risk of human infection. Traditional sterilization methods (including physical and chemical sterilization) cannot fully meet the requirements of the food industry due to their instability, uncontrollability, and complexity. For example, fresh-cut fruits are popular due to their nutritional value and convenience; however, the risk of spoilage increases further during the cutting and processing of fresh fruits and vegetables, and the cut surfaces are conducive to bacterial survival, thus further limiting the product's shelf life. Traditional high-temperature, high-pressure sterilization or chemical additive sterilization methods can cause irreversible damage to fruit quality, making them unsuitable for market use.

[0003] Quercetin is widely distributed, primarily derived from edible plants, and exhibits diverse biological functions. Studies have shown that quercetin demonstrates antibacterial potential through mechanisms such as cell wall damage, inhibition of protein / nucleic acid synthesis, and its role as a metabolic antagonist, providing new insights into addressing multidrug resistance in clinical settings. Furthermore, the application of natural polyphenols in food packaging offers a novel solution for preserving fresh fruits and vegetables, meeting consumer demand for safe food while mitigating the environmental impact of traditional food packaging. However, current research also indicates that quercetin has a relatively weak inhibitory effect on Gram-positive bacteria, limiting its application scope. Therefore, developing effective and sustainable antibacterial technologies using quercetin is crucial for the future development of fresh agricultural production. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first aspect of this invention provides a method for preparing a quercetin bactericide. The second aspect of this invention provides a quercetin bactericide that exhibits excellent inhibitory effects against Gram-positive bacteria. The third aspect of this invention provides applications of the aforementioned quercetin bactericide. These are specifically achieved through the following techniques.

[0005] The first aspect of this invention provides a method for preparing a quercetin bactericide, comprising the following steps:

[0006] Quercetin and the catalyst were added to water to obtain mixture I; anisaldehyde was added to an ethanol aqueous solution to obtain mixture II.

[0007] Mix the mixture I and the mixture II, add acid to adjust the pH to acidic, heat to react, filter, wash, and dry to obtain quercetin bactericide.

[0008] The preparation method of this invention promotes the dissolution of quercetin by adding a catalyst, resulting in a relatively uniform and stable first mixture. The catalyst also promotes the reaction. An acidic environment is conducive to the nucleophilic substitution reaction between anisaldehyde and quercetin. Furthermore, the preparation method of this invention triggers the supramolecular assembly of quercetin and anisaldehyde through acid-catalyzed nucleophilic substitution and π-π interactions, yielding a quercetin bactericide with a two-dimensional stacked structure, achieving excellent optical properties and preservation stability.

[0009] Preferably, the mass ratio of quercetin to p-anisaldehyde is (0.5-1):(0.05-0.1).

[0010] Preferably, the catalyst is at least one of sodium carbonate or sodium bicarbonate.

[0011] Preferably, the heating reaction conditions are: temperature 55-65℃, time 20-30h.

[0012] Preferably, the mass ratio of quercetin to catalyst is (0.5-1):(0.01-0.1).

[0013] Preferably, the pH value is 3-5.

[0014] A second aspect of the present invention provides a quercetin bactericide prepared by the above preparation method, wherein the quercetin bactericide has a two-dimensional stacked structure.

[0015] The third aspect of this invention provides the application of the above-mentioned quercetin bactericide in the preservation of fruits and vegetables, the application method comprising the following steps:

[0016] The film-forming material is dispersed in an organic solvent and stirred at room temperature to prepare a solution; the above-mentioned quercetin bactericide is added to the solution and stirred to obtain a uniform mixed solution; then the mixed solution is poured onto a glass film-forming substrate and dried to obtain a composite packaging film;

[0017] After the composite packaging film is applied to the surface of fresh fruits and vegetables, they are treated with visible light.

[0018] Preferably, the mass ratio of quercetin bactericide to polylactic acid is (0.0005-0.005):(0.01-0.25).

[0019] Preferably, the conditions for light treatment are: irradiance of 50-150 mW / cm². 2 The time should be no less than 20 minutes.

[0020] Preferably, the film-forming material includes at least one of polylactic acid, polypropylene, or polyethylene.

[0021] This invention proposes a novel natural supramolecular assembly-mediated food packaging film based on quercetin and p-anisaldehyde to address the problem of biocontamination in fresh food safety. It achieves excellent optical properties and preservation stability not only through acid-catalyzed nucleophilic substitution and π-π interactions triggering supramolecular assembly, but also by adding the assembled photosensitive quercetin bactericide to a polylactic acid solution to form a flexible nanocomposite packaging film with highly efficient photo-triggered antibacterial properties, effectively inhibiting bacterial growth on fruit surfaces. The quercetin of this invention incorporates p-anisaldehyde through molecular engineering, giving the quercetin molecule stronger light absorption, a narrower band gap, higher charge separation efficiency, and stronger electron-rich capacity. These properties facilitate photoactivation of quercetin to release reactive oxygen species, resulting in effective bacterial killing.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] 1. The quercetin bactericide of the present invention has strong photoactivity and bactericidal properties;

[0024] 2. The composite packaging film prepared using the quercetin bactericide of the present invention can play a good role in preserving fresh fruits and vegetables with cuts; and it has photosensitive properties. When used in combination with visible light treatment, it can significantly extend the preservation effect, reduce the loss of nutrients in fruits and vegetables, and extend the shelf life of cut cherry tomatoes to 10 days. Attached Figure Description

[0025] Figure 1 middle: Figure 1 (a) shows the preparation process of the quercetin bactericide in Example 1; Figure 1 (b) is a SEM image of the quercetin bactericide of Example 1; Figure 1 (c) is a schematic diagram of the intermolecular interactions of the quercetin bactericide in Example 1; Figure 1 (d) is a kinetic simulation diagram of the molecular structure stacking of the quercetin bactericide in Example 1.

[0026] Figure 2 middle: Figure 2 (a) is a schematic diagram of the band gap of the quercetin bactericide in Example 1; Figure 2 (b) is a schematic diagram of the light absorption capacity of the quercetin bactericide (QC-MB) in Example 1; Figure 2 (c) is a schematic diagram of the charge separation efficiency of the quercetin bactericide in Example 1; Figure 2 (d) is the photocurrent diagram of the quercetin bactericide in Example 1;

[0027] Figure 3 This is a schematic diagram illustrating the bactericidal effect of the quercetin bactericide of the present invention;

[0028] Figure 4This is a schematic diagram illustrating the principle of quercetin bactericide combined with light sterilization according to the present invention. Detailed Implementation

[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] This invention provides a method for preparing a quercetin bactericide, comprising the following steps:

[0031] S1. Add quercetin and sodium carbonate to water to obtain mixture I; add p-anisaldehyde to an aqueous ethanol solution to obtain mixture II;

[0032] S2. Mix the mixture I and mixture II from step S1, add dilute nitric acid to adjust the pH value, and heat the mixture in a water bath to obtain a solid product. Wash and dry the solid product to obtain the quercetin bactericide.

[0033] Optionally, the mass ratio of quercetin, catalyst, and p-anisaldehyde is (0.5-1):(0.01-0.1):(0.05-0.1).

[0034] Optionally, the pH can be set to 3-5.

[0035] Optionally, the water bath heating reaction conditions are: temperature 55-65℃, time 20-30h.

[0036] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] In the following examples and comparative examples, quercetin and p-anisaldehyde were both plant-derived and purchased from Maclean's, with quercetin purity ≥95% and p-anisaldehyde purity ≥98%. Sodium carbonate was purchased from Aladdin, with a relative molecular weight of 105. Polylactic acid particles were purchased from Aladdin, with a molecular weight Mw of approximately 60,000.

[0038] Example 1

[0039] This embodiment provides a method for preparing a quercetin bactericide, comprising the following steps:

[0040] S1. Weigh quercetin, sodium carbonate, and p-anisaldehyde in a mass ratio of 0.8:0.02:0.8; then add quercetin and sodium carbonate to water to obtain mixture I; add p-anisaldehyde to an ethanol aqueous solution to obtain mixture II.

[0041] S2. Mix the mixture I and mixture II from step S1, add dilute nitric acid to adjust the pH to 4, and then stir and react for 24 hours under hydrothermal conditions at 60°C to obtain a solid product; wash and dry the solid product to obtain quercetin bactericide.

[0042] The quercetin bactericide prepared in Example 1 was characterized and tested. The test results are as follows: Figure 1 As shown. Figure 1 (a) shows the preparation process of quercetin bactericide. The figure shows that a nano-framework with good photocatalytic activity was prepared using a simple hydrothermal synthesis method. Under acidic conditions, the aldehyde group forms C through protonation. + The ions, including resorcinol-containing quercetin, undergo nucleophilic attack, forming conjugated chains through electrophilic substitution reactions. The spontaneous self-assembly from monomers to nanoaggregates is triggered by intermolecular forces, including strong π-π interactions and some hydrogen bonds. Figure 1 (b) shows the SEM test of the quercetin bactericide. The figure shows that the synthesized material exhibits a two-dimensional nanosheet stacked morphology with an average diameter of about 2 μm. Figure 1 (c) The independent gradient model is used to intuitively present the intermolecular interactions. As can be seen from the figure, the degree of intermolecular interaction can be represented by the volume of the interaction region between conjugates. Figure 1 (d) shows the kinetic simulation of the molecular structure of the quercetin bactericide. The figure reveals that the composite bactericide molecules assemble into typical H-type aggregates, with a slip angle of approximately 83.4°(θ) between the two π-stacked groups and an average interaction energy of 59.48 kcal / mol. -1 .

[0043] The quercetin bactericide of Example 1 was optically characterized, and the results are as follows: Figure 2 As shown. Figure 2 (a) It can be seen that the quercetin bactericide in this embodiment has a narrower band gap; Figure 2 (b) It can be seen that, compared with quercetin, the quercetin bactericide (QC-MB) of this embodiment has a stronger light absorption capacity; Figure 2 (c) It can be seen that the quercetin bactericide in this embodiment has a higher charge separation efficiency. The band gap energies of quercetin and quercetin bactericide (QC-MB) are 3.21 eV and 2.41 eV, respectively. Figure 2 (d) represents the photocurrent of quercetin and quercetin bactericide (QC-MB). As can be seen from the figure, compared with quercetin, the photocurrent of quercetin bactericide (QC-MB) in this embodiment has a greater increase, indicating that the separation efficiency of electron-hole pairs is higher. Figure 2 This demonstrates that the engineered self-assembled and reinforced quercetin prepared in this embodiment possesses excellent molecular structure and morphological characteristics, resulting in improved optical properties. Analysis indicates that this is due to the optimization effect of molecular assembly on intersystem crossing (ISC).

[0044] Example 2

[0045] This embodiment provides a method for preparing a quercetin bactericide, comprising the following steps:

[0046] S1. Weigh quercetin, sodium carbonate, and p-anisaldehyde in a mass ratio of 0.5:0.01:0.05; then add quercetin and sodium carbonate to water to obtain mixture I; add p-anisaldehyde to an ethanol aqueous solution to obtain mixture II.

[0047] S2. Mix the mixture I and mixture II from step S1, add dilute nitric acid to adjust the pH to 3, and then stir and react for 20 hours under hydrothermal conditions at 65°C to obtain a solid product; wash and dry the solid product to obtain quercetin bactericide.

[0048] Example 3

[0049] This embodiment provides a method for preparing a quercetin bactericide, comprising the following steps:

[0050] S1. Weigh quercetin, sodium carbonate, and p-anisaldehyde in a mass ratio of 1:0.1:0.1; then add quercetin and sodium carbonate to water to obtain mixture I; add p-anisaldehyde to an ethanol aqueous solution to obtain mixture II.

[0051] S2. Mix the mixture I and mixture II from step S1, add dilute nitric acid to adjust the pH to 5, and then stir and react for 30 hours under hydrothermal conditions at 55°C to obtain a solid product; wash and dry the solid product to obtain quercetin bactericide.

[0052] Comparative Example 1

[0053] The preparation method of the quercetin bactericide in this comparative example is basically the same as that in Example 1, except that in step S1, anisaldehyde is replaced with an equal mass of quercetin.

[0054] Comparative Example 2

[0055] The preparation method of the quercetin bactericide in this comparative example is basically the same as that in Example 1, except that in step S1, quercetin is replaced with an equal mass of p-anisaldehyde.

[0056] Comparative Example 3

[0057] The preparation method of the quercetin bactericide in this comparative example is basically the same as that in Example 1, except that dilute nitric acid was not added in step S2.

[0058] Application examples

[0059] The applicant used quercetin bactericide and polylactic acid (PLA) to prepare composite packaging film, with a mass ratio of quercetin bactericide to PLA of (0.0005-0.005):(0.01-0.25). The applicant conducted numerous experiments, confirming that the composite packaging film of this application could be obtained when the mass ratio of quercetin bactericide to PLA was within the above range. For ease of comparison, in the following examples and comparative methods for preparing composite packaging film, the mass ratio of quercetin bactericide to PLA is 0.003:0.2.

[0060] The quercetin bactericides from Examples 1-3 and Comparative Examples 1-3 were used to prepare composite packaging films, and the procedures were as follows:

[0061] P1. Weigh quercetin bactericide and polylactic acid according to the mass ratio; then dissolve polylactic acid in dichloromethane and stir at room temperature for 1 hour to obtain a packaging film solution;

[0062] P2. Add quercetin bactericide to the packaging film solution in step P1, stir and mix well to obtain a mixture;

[0063] P3. Pour the mixture from step P2 onto a glass film-forming substrate and allow it to dry naturally at room temperature for 48 hours to obtain a composite packaging film.

[0064] The Staphylococcus aureus (ATCC25923) used in this invention was provided by the College of Food Science and Technology, Northwest A&F University. The method for culturing Staphylococcus aureus is as follows:

[0065] (1) The bacteria were streaked onto LB agar plates using the streak plate technique and incubated overnight at 37°C. Then, single colonies of Staphylococcus aureus were picked from the incubated plates and inoculated into 50 mL of LB broth and incubated at 37°C with shaking for 12 h.

[0066] (2) The shaker was set to 180 rpm at 37℃ for 24 h and 48 h respectively; then the bacterial suspension was centrifuged at 4℃ for 5 min, washed three times with physiological saline, resuspended with physiological saline and the OD of Staphylococcus aureus was adjusted. 600nm =0.8, yielding approximately 10 8 The bacterial concentration was determined by CFU / mL; the bacterial suspension was then diluted 100-fold to obtain 10... 6 Working concentration of CFU / mL.

[0067] The composite packaging films obtained in Examples 1-3 and Comparative Examples 1-3 were used to conduct preservation tests on freshly cut fruits. Fresh cherry tomatoes were selected as the experimental subjects. The selection method for cherry tomatoes was as follows: cherry tomatoes of similar size and maturity without any mechanical damage were selected from farmers' markets, washed with tap water, and their surfaces were disinfected by spraying with a 75% ethanol solution; after drying in a sterile environment, they were cut to form a 1×1 cm incision on the surface of the cherry tomatoes to simulate the freshly cut surface of the fruit. The incision was treated with a solution of 10% ethanol. 6 Cover the cherry tomatoes with gauze containing CFU / mL Staphylococcus aureus to simulate a bacterial infection environment and incubate for 1 hour. Divide the cherry tomatoes into 8 groups for later use.

[0068] Experimental setup: The composite packaging film of Example 1 was applied to the surface of cherry tomatoes with cuts, and the coated cherry tomatoes were then exposed to AM 1.5G illumination (irradiance 100mW / cm²). 2 Under these conditions, the light was applied for 20 minutes using a commercial 300W xenon lamp (Perfectlight Technology, Beijing). Examples 2-3 and Comparative Examples 1-3 all used the same treatment method as Example 1. The applicant conducted extensive experiments to confirm that when the visible light irradiance is 50-150 mW / cm², the light is suitable for the application of xenon lamps. 2 When the light exposure time is not less than 20 minutes, the bactericidal activity of the quercetin bactericide of the present invention can be activated. The light conditions here simulate the irradiation conditions of fluorescent lamps used in daily life.

[0069] Control group setup: blank control group and Light(-) group (covered only with the composite packaging film of Example 1, without light exposure). The cherry tomatoes in the blank control group were not treated in any way. The cherry tomatoes in the Light(-) group were only covered with the packaging film obtained in Example 1 and were not subjected to light treatment.

[0070] The eight groups of cherry tomatoes were placed in PP plastic boxes and kept at room temperature to simulate the actual storage conditions during food processing.

[0071] The cherry tomatoes in each group were placed for 0, 2, 4, and 10 days, respectively, and then the pH changes, soluble solids (TSS) content, and total phenolic content (TPC) of each group were measured and evaluated. The corresponding detection and evaluation methods were all conventional techniques in this field.

[0072] pH changes were monitored using a precise pH meter: 10g of cherry tomato sample was taken from each of the above 8 groups, ground evenly, and a pH electrode was inserted into it. After the reading stabilized, the data was recorded as the pH value of each group of samples. The results are shown in Table 1 below.

[0073] Table 1. pH change data for each group

[0074]

[0075]

[0076] As shown in Table 1, the pH values ​​of Examples 1-3 changed very little, while the pH values ​​of the other groups changed significantly. This indicates that the quercetin bactericide of the present invention can effectively prevent cherry tomatoes from going rancid under light conditions and has excellent bactericidal properties.

[0077] Evaluation of soluble solids (TSS) content: The sugar content of cherry tomatoes in each group was monitored using a handheld refractometer. From each of the eight groups, 5g of cherry tomatoes were ground, filtered, and the filtrate was measured using a handheld refractometer. The measurement was repeated three times, and the average value was taken. The results are shown in Table 2 below.

[0078] Table 2. Soluble solids (TSS) content data for each group.

[0079]

[0080] As shown in Table 2, none of the groups had a significant effect on the soluble solids (TSS) content of cherry tomatoes, which also confirms that the quercetin bactericide used in this invention will not have an adverse effect on the quality of cherry tomatoes.

[0081] Given the high activity of reactive oxygen species during photodynamic sterilization, antioxidant properties are of great importance. This invention investigated the concentration of total phenolic compounds (TPC) in cherry tomatoes before and after photodynamic sterilization; the TPC was tested using the Folin-Ciocalteu method, which included the following three steps:

[0082] (1) Preparation of standard solution: Weigh 0.1g gallic acid and add 50mL of distilled water to make up to 100mL to obtain a 1000mg / L gallic acid standard solution. Prepare a series of standard solutions with concentrations of 0mg / L, 12.5mg / L, 25mg / L, 50mg / L, 100mg / L, 200mg / L and 400mg / L respectively.

[0083] (2) Sample preparation: Take 5g of sample from each of the above 8 groups, grind it, add 20mL of distilled water and filter it. After processing at 3000 rpm offline for 10 minutes, take the supernatant and dilute it 50 times.

[0084] (3) Test and calculation: Take 1 mL of sample from the supernatant diluted in step (2) of each group, add 1 mL of Folin-Ciocalteu colorimetric reagent and 3 mL of 20% sodium carbonate solution, mix well, react at 50℃ for 30 minutes, and measure the absorbance at 765 nm. Plot a standard curve and calculate the concentration of total phenolic compounds (TPC) in each group. The results are shown in Table 3 below.

[0085] Table 3 Concentrations of total phenolic compounds (TPC) in each group

[0086]

[0087] As shown in Table 3, none of the groups had a significant effect on the total phenolic compound concentration of cherry tomatoes, which also confirms that the quercetin bactericide used in this invention will not have an adverse effect on the quality of cherry tomatoes.

[0088] Appearance evaluation: Primarily based on the visible growth of microbial colonies. Four groups were set up for appearance evaluation. Experimental group setup: The composite packaging film of Example 1 (QC-MB Light(+)) was used to cover Staphylococcus aureus colonies, and then placed under AM 1.5G illumination (irradiance 100mW / cm²). 2 Under these conditions, the light was applied for 20 minutes using a commercial 300W xenon lamp (Perfectlight Technology, Beijing).

[0089] Control group setup: Blank control group (Control Light(+)), Light(-) group (QC-MB Light(-)), and Comparative Example 1 (Qu Light(+)). The Staphylococcus aureus colonies in the blank control group underwent no treatment. The Staphylococcus aureus colonies in the Light(-) group were only covered with the packaging film obtained in Example 1 and were not subjected to light treatment. The Staphylococcus aureus colonies in Comparative Example 1 were covered with the composite packaging film of Comparative Example 1 and then subjected to light treatment under the same conditions as in Example 1.

[0090] The growth of microbial colonies in each group is as follows: Figure 3 As shown. Figure 3 The dots in the image represent the number of bacterial colonies. (From...) Figure 3 It is evident that the composite packaging film of Example 1 exhibits excellent bactericidal effects under light irradiation. This is because the composite packaging film of the present invention possesses strong photoactivity; under visible light catalysis, it can be activated to release more reactive oxygen species, thereby exerting a superior bactericidal effect.

[0091] The bactericidal and preservative principle of the quercetin bactericide of this invention is as follows: Figure 4 As shown. By Figure 4 It is known that under visible light catalysis, the composite packaging film of the present invention can be activated to release more reactive oxygen species, thereby exerting an excellent bactericidal effect.

[0092] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing a quercetin bactericide, characterized by, The method comprises the following steps: adding quercetin and a catalyst into water to obtain a mixed solution I; adding p-anisaldehyde into an aqueous ethanol solution to obtain a mixed solution II; mixing the mixed solution I and the mixed solution II, adding an acid to adjust the pH value to be acidic, heating and reacting, filtering, cleaning, and drying to obtain the quercetin fungicide; the pH value is 3-5.

2. The production method according to claim 1, characterized by, The mass ratio of the quercetin and the p-anisaldehyde is (0.5-1):(0.05-0.1).

3. The preparation method according to claim 1, characterized in that, The catalyst comprises at least one of sodium carbonate or sodium bicarbonate.

4. The method of claim 1, wherein, The mass ratio of the quercetin and the catalyst is (0.5-1):(0.01-0.1).

5. The preparation method according to claim 1, characterized in that, The heating and reacting condition is that the temperature is 55-65℃ and the time is 20-30h.

6. The quercetin fungicide prepared by the preparation method in any one of claims 1-5.

7. The use of the quercetin bactericide of claim 6 in the preservation of fruits and vegetables, characterized in that, The application method comprises the following steps: dispersing a film-forming material in an organic solvent, stirring at room temperature, and preparing a solution; adding the quercetin fungicide into the solution, stirring, and obtaining a uniform mixed solution; pouring the mixed solution onto a film-forming substrate, drying, and obtaining a composite packaging film; covering the composite packaging film on the surface of fresh fruits and vegetables, and performing visible light illumination treatment.

8. Use according to claim 7, characterized in that, The mass ratio of the quercetin fungicide and the film-forming material is (0.0005-0.005):(0.01-0.25).

9. Use according to claim 7, characterized in that, The conditions of the visible light irradiation treatment are: irradiance 50-150 mW / cm 2 ; time not less than 20 min.

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