Chopstick surface mould detection method

Abstract

The invention relates to the technical field of hygiene detection, and discloses a chopstick surface mold detection method which comprises the following steps: S1, selecting to-be-detected chopsticks; s2, placing the chopsticks in an environment with humidity and temperature conditions for standing so as to induce mould breeding; s3, performing disinfection treatment on the chopsticks to be detected, wherein the disinfection mode is one of high-pressure steam sterilization, boiling water boiling, microwave heating, ozone disinfection cabinet treatment or alcohol wiping; s4, using a sterilized swab to collect surface samples of the head end and the splicing seam of the chopstick, and transferring the surface samples into the extracting solution; s5, reacting the extracting solution with a detection composition containing a color change indicator and a protease substrate; s6, performing visual judgment according to the color change, and judging whether active mould exists on the surfaces of the chopsticks or not. The method provided by the invention realizes rapid visual determination of the chopstick disinfection effect, is simple and convenient in detection and high in sensitivity, and is suitable for mold residue evaluation of chopsticks made of various materials.

Description

Technical Field

[0001] The invention relates to the technical field of sanitation detection, in particular to a method for detecting mold on the surface of chopsticks. Background Art

[0002] Chopsticks are widely used in Asian households and restaurants, and their surface hygiene is closely linked to public health. Especially in high-humidity, frequently used environments, chopstick surfaces are a vulnerable carrier for mold spores and microbial residues. If not thoroughly cleaned, mold can form biofilms on the chopstick surface, releasing metabolic products that pose a potential threat to human health.

[0003] In the existing technology, most of the research on chopstick hygiene focuses on the selection of materials or the safety assessment of surface coatings. There are few systematic studies on the response patterns of mold residues between different disinfection methods and chopsticks of different materials. In some existing methods, in order to verify the disinfection effect, they often rely on microscopic observation, microbial culture or molecular biology methods (such as PCR detection, colony counting, etc.). Although the above technologies have a certain degree of accuracy, they generally have the problems of long detection cycles, strong dependence on experimental conditions, and poor adaptability to actual application scenarios, making it difficult to adapt to the rapid evaluation needs of daily households or catering.

[0004] In addition, different chopstick materials respond significantly differently to disinfection treatments. Wooden and bamboo chopsticks are highly absorbent and easily retain moisture and contaminants, while stainless steel and alloy chopsticks exhibit higher resistance to contamination due to their dense surfaces. However, traditional disinfection processes usually do not make detailed assessments of the differences between materials and still use a unified treatment process, which may cause some materials (such as melamine chopsticks) to deform or pose safety risks under specific disinfection conditions. For example, the surface of melamine chopsticks may release harmful substances during microwave heating, and the existing testing scheme does not jointly evaluate this usage risk with the actual disinfection effect, and there are certain blind spots.

[0005] In terms of mold detection methods, some color development methods have been applied in food residue detection or environmental monitoring. However, these methods are mostly based on colorimetric systems involving specific metal ions or acid-base reactions, and are not directly related to the response of microbial enzyme activity. The lack of a real-time visual detection mechanism that reflects the metabolic activity of mold enzymes has disconnected current verification methods for the sterilization efficacy of chopsticks from the actual mold removal results, making it difficult to form an effective feedback mechanism to guide the adjustment of disinfection strategies.

[0006] Therefore, the present invention proposes a method for detecting mold on the surface of chopsticks to overcome the shortcomings of the prior art. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides a method for detecting mold on the surface of chopsticks, which solves the problem in the existing technology of lacking rapid, visual, and enzyme activity-related detection of the disinfection effect of chopsticks made of different materials.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions: a method for detecting mold on the surface of chopsticks, comprising the following steps: S1. Select chopsticks to be tested; S2. Place the chopsticks in an environment with appropriate humidity and temperature to induce mold growth; S3. Disinfecting the chopsticks to be tested by one of high-pressure steam sterilization, boiling water, microwave heating, ozone disinfection cabinet treatment, or alcohol wiping; S4. Use a sterile swab to collect surface samples from the tip and seam of the chopsticks and transfer them to the extraction solution. S5, reacting the extract with a detection composition containing a color change indicator and a protease substrate; S6. Visually inspect the chopsticks based on color changes to determine whether active mold is present on their surface.

[0009] Preferably, the chopsticks include wooden chopsticks, bamboo chopsticks, stainless steel chopsticks, alloy chopsticks or melamine chopsticks.

[0010] Preferably, the steps of step S2 include: The chopsticks are placed in a sealed container, and the relative humidity of the environment is controlled at 80-95% and the temperature is controlled at 20-30°C through a humidifier. The container is left standing for 72 to 120 hours.

[0011] Preferably, step S4 includes: Use a sterile cotton swab moistened with 1-3 ml of saline to wipe the tip and seam of the chopsticks 5-10 times each. Place the swab in 5-10 ml of extract and oscillate at 200 rpm for 1-3 minutes to release surface microorganisms and metabolites.

[0012] Preferably, the extract is physiological saline with a pH value of 6.5-7.5.

[0013] Preferably, step S5 includes: Take 100-500 μl of the extract sample and mix it with a detection composition containing a color-changing indicator and a protease substrate in a volume ratio of 1:1, then place it in a reaction container and react for 30-90 seconds under static conditions to allow the color-changing indicator and the protease substrate to undergo a visible color change.

[0014] Preferably, the detection composition containing a color-changing indicator and a protease substrate comprises the following components in parts by mass: Bromocresol green: 2-10 parts; Protease substrate dye: 4-12 parts; Polyethylene glycol solubilizer: 20-60 parts; Boric acid buffer stabilizer: 10-30 parts; Hydroxypropyl methylcellulose adsorbent: 1-10 parts; Deionized water: 25-35 parts.

[0015] Preferably, step S6 includes: Observe the color of the reaction solution. If it is blue or green, it is judged as negative. If it is yellow or orange, it is judged as weakly positive. If it is red or reddish brown, it is judged as positive, indicating obvious fungal enzyme activity and the presence of a large amount of residue.

[0016] The present invention provides a method for detecting mold on the surface of chopsticks. It has the following beneficial effects: 1. This invention utilizes a combination of multi-material chopsticks, constant temperature and humidity induction, and on-site colorimetric detection to rapidly determine residual mold activity on chopstick surfaces in a realistic simulated environment. Compared to traditional methods that rely on culture in petri dishes or microscopic observation, this approach overcomes the technical bottlenecks of long processing times, complex procedures, and low sensitivity.

[0017] 2. This invention establishes a quantitative comparison system between different chopstick materials and disinfection methods, effectively identifying the most suitable sterilization treatment method for each material. Existing techniques generally fail to distinguish between the differences in the sterilization response of chopstick materials, making precise disinfection selection difficult. This solution solves the problem of incomplete sterilization caused by a "one-size-fits-all" disinfection method.

[0018] 3. By optimizing the formulation of a detection composition based on the principle of color development through enzyme activity, this invention achieves color development within one minute, visual identification, and the elimination of sophisticated equipment. Unlike conventional laboratory methods such as enzyme-linked immunosorbent assays and PCR, this solution enables the expansion of testing beyond the laboratory into the home and restaurant sector.

[0019] 4. This invention incorporates microwave treatment restriction analysis and melamine material safety boundary specifications to clearly integrate microwave prohibition information with detection reaction results. Existing technologies often overlook the safety hazards posed by the thermal properties of materials, which can easily cause chopstick deformation or harmful releases. This solution significantly improves the scientific and safe matching of materials and disinfection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of the detection method of the present invention. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Unless otherwise stated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The color change indicator used was bromocresol green, an analytically pure product produced by Sinopharm Chemical Reagent Co., Ltd., model BR; The protease substrate dye used was N-benzyloxycarbonyl-L-phenylalanine p-nitrophenyl ester, a high-purity protease substrate provided by MacLean Biochemical Technology Co., Ltd., model number B827340; The polyethylene glycol solubilizer used was PEG-400 provided by Aladdin Reagent Company, model number P110041; The borate buffer stabilizer used was the standard buffer provided by Shanghai Yuanye Biotechnology Co., Ltd., model number YS10050; the hydroxypropyl methylcellulose adsorbent used was pharmaceutical grade HPMC provided by Tianjin Kailiying Pharmaceutical Technology Co., Ltd., model number HPMC-606; Deionized water used was ultrapure water from Shanghai Jingan Biotechnology Co., Ltd., model DW1001.

[0023] See also Figure 1 The present invention provides a method for detecting mold on the surface of chopsticks. S1. Select chopsticks to be tested; S2. Place the chopsticks in an environment with appropriate humidity and temperature to induce mold growth; S3. Disinfecting the chopsticks to be tested by one of high-pressure steam sterilization, boiling water, microwave heating, ozone disinfection cabinet treatment, or alcohol wiping; S4. Use a sterile swab to collect surface samples from the tip and seam of the chopsticks and transfer them to the extraction solution. S5, reacting the extract with a detection composition containing a color change indicator and a protease substrate; S6. Visually inspect the chopsticks based on color changes to determine whether active mold is present on their surface.

[0024] For step S1, ensure that the selected chopstick samples are suitable for mold testing, representative, and practical. Select samples from five common chopstick materials: bamboo, wooden, stainless steel, alloy, and melamine. When selecting, check that the chopsticks are clean, free of visible cracks, oil stains, or other contaminants. When selecting samples, ensure that the chopsticks are representative in material and meet the research requirements.

[0025] The cleanliness of the chopstick surface and the integrity of its material directly impact mold growth and detection results. Especially in high-humidity environments, different chopstick materials have varying susceptibility to mold. Bamboo and wooden chopsticks, due to their natural fiber structure, are more hygroscopic, while stainless steel and alloy chopsticks are less susceptible to mold. Therefore, comparing chopsticks of different materials can help verify differences in mold growth under different environmental conditions.

[0026] For step S2, provide a suitable environment for mold to promote its growth on the surface of chopsticks and produce metabolites. Place the sterilized chopsticks in an environment with a humidity control of 80%-95% and a temperature control of 20℃-30℃. This environmental condition can be achieved through a humidity chamber, a constant temperature device or a humidifier. Let the chopsticks stand for 72 hours to 120 hours. At this time, mold will gradually grow on the surface of the chopsticks and produce metabolites. Note that the chopsticks need to be placed in a dark environment because mold growth is more active under conditions with less light.

[0027] Mold growth requires moisture and a suitable temperature. When humidity is above 80%, mold easily grows on the surface of chopsticks. When the temperature is controlled between 20°C and 30°C, mold reproduces faster and accelerates the accumulation of metabolic products. This process simulates the environmental conditions of the rainy season, which is the environment where mold is most likely to grow under natural conditions.

[0028] Regarding step S3, the five disinfection methods used in the present method for detecting mold on the surface of chopsticks vary in their treatment and scope of application. In practice, these methods retain varying degrees of mold metabolites, potentially impacting the color development and interpretation accuracy of subsequent testing. Therefore, the choice of disinfection method is not only influenced by the chopstick material but also closely linked to the stability and reliability of the test results.

[0029] Taking high-pressure steam sterilization as an example, this method achieves a strong penetrating sterilization effect by releasing high-temperature, high-pressure steam under closed conditions. This method is suitable for heat-resistant materials such as wood, bamboo, or metal, and can effectively remove residual microorganisms on the surface and in the crevices of chopsticks. However, high temperature conditions may affect certain sensitive metabolites, and if the processing parameters are not properly controlled, it may affect the detection sensitivity to a certain extent.

[0030] Boiling water, as a thermal treatment method, operates under relatively mild conditions and is suitable for most common household chopstick materials, especially absorbent wood and bamboo. During the treatment process, boiling water not only physically sterilizes the chopsticks but also prevents significant damage to metabolic products. It also helps maintain a certain level of moisture on the chopstick surface, making it highly adaptable to post-mold induction testing.

[0031] Microwave heating utilizes the energy of electromagnetic waves to stimulate water molecules within chopsticks, creating a thermal effect, making it suitable for rapid processing. This method is somewhat effective in removing surface microorganisms, and with a short processing time, it also has relatively minimal interference with mold metabolites. Under specific conditions, it can balance efficiency with pre-treatment integrity. However, it is not suitable for metal chopsticks or some heat-sensitive plastics, presenting certain limitations.

[0032] Ozone disinfection is a low-temperature oxidation treatment method that works by disrupting microbial cell membranes through the oxidative properties of ozone molecules. This method sterilizes without introducing additional heat, making it suitable for treating temperature-sensitive materials. Its dry operation reduces the impact of sample moisture fluctuations on subsequent reaction systems, offering advantages for preserving the state of metabolites.

[0033] Alcohol wiping is a common and easy-to-use surface disinfection method. It primarily inactivates surface microorganisms through protein denaturation, making it suitable for rapid treatment. However, due to its limited penetration and range of action, it may not be as effective as other methods for treating residual mold in caulking joints or micropores of materials. Despite this, alcohol disinfection has good versatility and is suitable for routine treatment in non-specialized settings.

[0034] In summary, by setting up multiple disinfection methods and then subjecting chopsticks to subsequent testing after each treatment, the test results can reflect the effectiveness of different disinfection methods. Because the detection method is based on the presence of active mold, under the same mold induction conditions, if the test result after disinfection is negative or the color development is weak, it can be considered that the treatment method has better mold removal ability in actual use.

[0035] In step S4, mold spores and metabolites are collected from the surface of the chopsticks to provide samples for subsequent testing. Use a sterile cotton swab to gently wipe the tip and seams of the chopsticks. These areas are often high-risk for mold growth. The sample from the swab is transferred to an extraction solution, typically saline or another suitable buffer, to dissolve the mold metabolites and facilitate subsequent analysis.

[0036] Cotton swabs are effective in collecting mold spores and their metabolites from the chopstick surface. Using saline as an extraction fluid gently preserves mold activity while extracting metabolites from the chopstick surface into the liquid, providing a suitable sample for subsequent mold testing.

[0037] In step S5, the protease in the mold metabolites reacts with the protease substrate in the detection composition to produce a visible color change. The intensity and nature of the color change can reflect the activity level of the mold, thereby helping to determine whether the mold is present and its activity level.

[0038] Prepare the reaction solution: Take an appropriate amount of extract (usually mold metabolites extracted with physiological saline or other buffer solution) and the required amount of detection composition.

[0039] Mix the extract and the test composition: The extract is mixed with the detection composition in a specified ratio.

[0040] Reaction time: The reaction is carried out at room temperature for 30-90 seconds. During this time, the protease in the mold metabolites will react with the protease substrate in the detection composition.

[0041] Reaction vessel: Select appropriate reaction vessels (e.g., test tubes, microplates, etc.) to ensure that the liquids can be fully mixed and contact the reactants.

[0042] Observe the reaction: After the reaction is complete, observe the color change with the naked eye or an optical instrument. The type and intensity of the color change can indicate the degree of mold activity.

[0043] According to the requirements of this step, the detection composition includes the following main components, and the functions and proportions of each component are as follows: Color change indicator (2-10 parts): Function: A color-changing indicator is a chemical substance that changes color under acidic or alkaline conditions. In this method, the color-changing indicator changes color in response to the acidic groups in mold metabolites, creating a visual effect. The color change is proportional to the intensity of mold activity.

[0044] The preferred ratio is 2-10 parts, ensuring that the color change of the reaction solution is sufficiently obvious and stable.

[0045] Protease substrate dye (4-12 parts): Function: Protease substrate dyes react with proteases secreted by molds. Proteases hydrolyze the substrate dye, releasing acidic groups. This reaction causes a color change, indicating the metabolic activity of the mold.

[0046] Optimal ratio: 4-12 parts, to ensure sufficient substrate for the protease reaction in the mold metabolites to achieve a significant color change.

[0047] Polyethylene glycol solubilizer (20-60 parts): Function: Polyethylene glycol is a commonly used solubilizer that effectively increases the solubility of various components in a solution, making the reactants more uniform. Its presence can promote the mixing of substrate and indicator, improving the efficiency of the reaction.

[0048] The preferred ratio is 20-60 parts to ensure the solubility of each component in the reaction solution and avoid inconsistent reaction results due to uneven components.

[0049] Boric acid buffer stabilizer (10-30 parts): Function: Boric acid buffer can maintain a stable pH value in the reaction system. Proteases in mold metabolites are typically more active within a certain pH range. Therefore, using boric acid buffer can ensure that reaction conditions remain within the optimal range, thus preventing pH fluctuations from affecting the reaction results.

[0050] The preferred ratio is 10-30 parts, which can effectively maintain the pH stability of the reaction system and ensure the smooth hydrolysis reaction of the protease substrate.

[0051] Hydroxypropyl methylcellulose adsorbent (1-10 parts): Function: Hydroxypropyl methylcellulose is a substance with excellent adsorption properties. It can help fix key components of mold metabolites on the reaction interface, improving the sensitivity of the reaction. It also helps remove impurities in the reaction system, ensuring the accuracy of the test results.

[0052] The preferred ratio is 1-10 parts, which can effectively enhance the reaction sensitivity and reduce the influence of interfering substances.

[0053] Deionized water (25-35 parts): Function: Deionized water is the main solvent of the reaction solution. It can provide a suitable solution environment to facilitate the full dissolution and reaction of other reactants.

[0054] The preferred ratio is 25-35 parts, ensuring the appropriate concentration of the solution and providing sufficient reaction medium for other ingredients.

[0055] When the extract contains mold metabolites, these products often contain proteases. Proteases have the ability to hydrolyze proteins or substrates. In this method, proteases hydrolyze the protease substrate dye, releasing acidic groups, which promote a color change. As the protease substrate hydrolyzes, the released acidic groups react with a color-changing indicator. The color-changing indicator changes color in an acidic environment. The intensity of this change is proportional to the concentration and activity level of the mold metabolite. By observing the degree of color change, the level of mold activity can be determined. A significant and rapid color change indicates strong mold activity; a weaker color change may indicate low mold activity or the absence of active mold.

[0056] In step S6, the mold activity is quickly judged by the color change, thereby determining whether there is mold on the surface of the chopsticks.

[0057] Observe the color change of the reaction solution and determine the fungal activity according to the following standards: Blue or green: judged as negative, no mold activity was detected; Yellow or orange: judged as weak positive, the mold activity is weak; Red or reddish brown: judged as positive, obvious fungal enzyme activity, and the presence of a large amount of residues.

[0058] The color change of the color-changing indicator is directly related to the activity of the mold. By observing the color change, the presence and activity of the mold can be intuitively determined. This method is simple and efficient, and does not require complex equipment.

[0059] Example 1: S1. Select chopsticks to be tested Five pairs of unpainted wooden chopsticks used daily were selected. The chopsticks were inspected for surface integrity, showing no visible cracks, oil stains, or other visible dirt. Wooden chopsticks have a natural fiber structure and are highly absorbent, making them representative and suitable for mold growth experiments.

[0060] S2, induce mold growth The chopsticks were placed in a sealed humidity-controlled chamber (constant temperature and humidity chamber). The humidity was maintained at 85-90% and the temperature was set at 20-30°C. The chamber was then left to stand for 96 hours under light-shielding conditions to simulate the humid environment of the rainy season, promoting mold growth and the accumulation of metabolites on the chopstick surfaces.

[0061] S3. Disinfection After the mold induction was completed, the five pairs of wooden chopsticks were disinfected in the following five different ways: Wooden chopsticks 1: sterilize in an autoclave (121°C, 15 minutes). Wooden chopsticks 2: Boil in boiling water for 10 minutes; Wooden chopsticks 3: Microwave (700W) for 2 minutes; Wooden chopsticks 4: Place in ozone sterilizer for 30 minutes; Wooden chopsticks 5: Use a cotton cloth soaked in 75% alcohol to wipe the surface and gaps, and dry at room temperature.

[0062] After the disinfection process is completed, each set of wooden chopsticks will no longer be exposed to external sources of pollution and will immediately enter the next step of the testing process.

[0063] S4: Collect surface samples Using a sterile cotton swab that has been sterilized at high temperature and high pressure, wipe the tip and seam of each pair of chopsticks evenly five times to ensure coverage of high-risk areas. Immediately after collection, the cotton swab is inserted into an EP tube containing 5 mL of normal saline (pH 6.5-7.5), rotated at 200 rpm for 2 minutes, and allowed to stand for 5 minutes to extract the mold spores and their metabolites remaining on the surface of the chopsticks to form a sample extract for testing.

[0064] S5: Reacting with the detection composition The extract (100 μL) was mixed with the prepared detection composition in a 1:1 ratio, added dropwise to a transparent reaction well plate, reacted at room temperature for 60 seconds, and the color change was observed.

[0065] The detection composition comprises the following components in parts by mass: Color change indicator: 2 parts; Protease substrate dye: 4 parts; Polyethylene glycol solubilizer: 20 parts; Boric acid buffer stabilizer: 10 parts; Hydroxypropyl methylcellulose adsorbent: 1 part; Deionized water: 30 parts.

[0066] Reaction Principle: If proteases are present in the sample, they hydrolyze the substrate, releasing acidic groups that trigger a color change in the color indicator. The intensity of the color change reflects the level of fungal activity.

[0067] S6: Color determination and result analysis Visual judgment is performed based on the color change results of the reaction wells. The standards are as follows: Blue or green: judged as negative, no obvious fungal enzyme activity; Yellow or orange: judged as weak positive, there are residual active molds but the number is limited; Red or reddish brown: judged as positive, obvious fungal enzyme activity, and a large amount of residues.

[0068] Color determination and result analysis are shown in Table 1: Table 1: Chopstick number Disinfection method Color rendering Judgment results Evaluation of sterilization effect Wooden chopsticks 1 High-pressure steam sterilization blue-green Negative Very strong Wooden chopsticks 2 Boil water light yellow weak positive Strong Wooden chopsticks 3 Microwave heating orange-yellow weak positive Strong Wooden chopsticks 4 Ozone treatment red Positive generally Wooden chopsticks 5 alcohol wipes Reddish brown Positive Weaker High-pressure steam sterilization is the most effective method to completely inactivate mold; Microwaves and boiling water have some inhibitory effect on mold, but not completely; Under these conditions, ozone and alcohol treatments did not sufficiently remove mold spores, and significant enzyme activity remained.

[0069] Example 2: S1. Select chopsticks to be tested Five pairs of unpainted bamboo chopsticks for daily use were selected. The surfaces of the chopsticks were observed to have no obvious cracks, oil stains or other visible dirt, indicating good surface integrity.

[0070] S2, induce mold growth The bamboo chopsticks were placed in a sealed humidity control chamber (constant temperature and humidity chamber). The humidity was maintained at 85-90% and the temperature was set at 20-30°C. The chamber was then left to stand for 72 hours under light-shielding conditions to simulate the humid environment of the rainy season, promoting mold growth and the accumulation of metabolites on the chopstick surfaces.

[0071] S3. Disinfection After the mold induction was completed, the five pairs of bamboo chopsticks were disinfected in the following five different ways: Bamboo chopsticks 1: Sterilize in an autoclave (121°C, 15 minutes). Bamboo chopsticks 2: Boil in boiling water for 10 minutes; Bamboo chopsticks 3: Microwave (700W) for 2 minutes; Bamboo chopsticks 4: Place in ozone sterilizer for 30 minutes; Bamboo chopsticks 5: Use a cotton cloth soaked in 75% alcohol to wipe the surface and gaps, and dry at room temperature.

[0072] After the disinfection process is completed, each set of bamboo chopsticks will no longer be exposed to external sources of pollution and will immediately enter the next step of the testing process.

[0073] S4: Collect surface samples Using a sterile cotton swab that has been sterilized at high temperature and high pressure, wipe the tip and seam of each pair of chopsticks evenly five times to ensure coverage of high-risk areas. Immediately after collection, the cotton swab is inserted into an EP tube containing 5 mL of normal saline (pH 6.5-7.5), rotated at 200 rpm for 2 minutes, and allowed to stand for 5 minutes to extract the mold spores and their metabolites remaining on the surface of the chopsticks to form a sample extract for testing.

[0074] S5: Detection of composition reaction The extract (100 μL) was mixed with the prepared detection composition in a 1:1 ratio, added dropwise to a transparent reaction well plate, reacted at room temperature for 60 seconds, and the color change was observed.

[0075] Test composition formula (by mass): Color change indicator: 10 parts; Enzyme substrate: 12 parts; Polyethylene glycol: 60 parts; Boric acid buffer: 30 parts; Hydroxypropyl methylcellulose: 10 parts; Deionized water: 35 parts.

[0076] Reaction Principle: If proteases are present in the sample, they hydrolyze the substrate, releasing acidic groups that trigger a color change in the color indicator. The intensity of the color change reflects the level of fungal activity.

[0077] S6: Color determination and result analysis Visual judgment is performed based on the color change results of the reaction wells. The standards are as follows: Blue or green: judged as negative, no obvious fungal enzyme activity; Yellow or orange: judged as weak positive, there are residual active molds but the number is limited; Red or reddish brown: judged as positive, obvious fungal enzyme activity, and a large amount of residues.

[0078] Color determination and result analysis are shown in Table 2: Table 2: High-pressure steam sterilization: High-pressure steam sterilization has the best effect. The mold on the surface of bamboo chopsticks is completely removed, and the test result is blue-green, indicating that the mold enzyme activity is completely inhibited and the bactericidal effect is extremely strong.

[0079] Boiling water: Boiling water disinfection has a good removal effect on bamboo chopstick mold, but it cannot completely remove it. The color is light yellow and it is judged as weak positive, indicating that there is still a small amount of mold remaining and the bactericidal effect is strong.

[0080] Microwave heating: Microwave disinfection has a certain effect on mold on bamboo chopsticks. The color turns orange-yellow, indicating that there is some mold activity, and the effect is average.

[0081] Ozone disinfection: The effect of ozone disinfection is poor, and the color is red, which means that the metabolic activity of mold still exists and the bactericidal effect is weak.

[0082] Alcohol wiping: Alcohol wiping has the worst disinfection effect, and its color is reddish-brown, indicating that the mold activity is strong and the bactericidal effect is limited.

[0083] Example 3: S1. Select chopsticks to be tested Five pairs of stainless steel chopsticks for daily use were selected. The surfaces of the chopsticks were observed to have no obvious cracks, oil stains or other visible dirt, indicating good surface integrity.

[0084] S2, induce mold growth The stainless steel chopsticks were placed in a sealed humidity control device (constant temperature and humidity chamber). The ambient humidity was controlled at 85-90% and the temperature was set at 20-30°C. The chamber was then left to stand for 120 hours under light-shielding conditions to simulate the humid environment of the rainy season, promoting mold growth and the accumulation of metabolites on the surface of the chopsticks.

[0085] S3. Disinfection After the mold induction was completed, five pairs of stainless steel chopsticks were disinfected in the following five different ways: Stainless steel chopsticks 1: Sterilize in a high-pressure steam sterilizer (121°C, 15 minutes); Stainless steel chopsticks 2: Boil in boiling water for 10 minutes; Stainless steel chopsticks 3: (not applicable); Stainless steel chopsticks 4: Place in ozone disinfection cabinet for 30 minutes; Stainless steel chopsticks 5: Use a cotton cloth soaked in 75% alcohol to wipe the surface and gaps, and dry at room temperature.

[0086] After each set of stainless steel chopsticks is disinfected, they will no longer be exposed to external sources of contamination and will immediately enter the next testing process.

[0087] S4: Collect surface samples Using a sterile cotton swab that has been sterilized at high temperature and high pressure, wipe the tip and seam of each pair of chopsticks evenly 10 times to ensure coverage of high-risk areas. Immediately after collection, the cotton swab is inserted into an EP tube containing 5 mL of normal saline (pH 6.5-7.5), rotated at 200 rpm for 2 minutes, and allowed to stand for 5 minutes to extract the mold spores and their metabolites remaining on the surface of the chopsticks to form a sample extract for testing.

[0088] S5: Detection of composition reaction The extract (100 μL) was mixed with the prepared detection composition in a 1:1 ratio, added dropwise to a transparent reaction well plate, reacted at room temperature for 60 seconds, and the color change was observed.

[0089] Test composition formula (by mass): Color change indicator: 3 parts; Enzyme substrate: 5 parts; Polyethylene glycol: 40 parts; Boric acid buffer: 15 parts; Hydroxypropyl methylcellulose: 5 parts; Deionized water: 30 parts.

[0090] Reaction Principle: If proteases are present in the sample, they hydrolyze the substrate, releasing acidic groups that trigger a color change in the color indicator. The intensity of the color change reflects the level of fungal activity.

[0091] S6: Color determination and result analysis Visual judgment is performed based on the color change results of the reaction wells. The standards are as follows: Blue or green: judged as negative, no obvious fungal enzyme activity; Yellow or orange: judged as weak positive, there are residual active molds but the number is limited; Red or reddish brown: judged as positive, obvious fungal enzyme activity, and a large amount of residues.

[0092] Color determination and result analysis are shown in Table 3: Table 3: Chopstick number Disinfection method Color rendering Judgment results Evaluation of sterilization effect Stainless steel chopsticks 1 High-pressure steam sterilization blue-green Negative Very strong Stainless steel chopsticks 2 Boil water light green Negative powerful Stainless steel chopsticks 3 (Unsterilized) yellow weak positive No disinfection Stainless steel chopsticks 4 Ozone treatment light yellow weak positive generally Stainless steel chopsticks 5 alcohol wipes orange-yellow weak positive Generally weak Since the surface of stainless steel chopsticks does not absorb water, the degree of mold growth is much lower than that of wooden or bamboo chopsticks; Both high-pressure steam sterilization and boiling water sterilization can completely remove the mold activity on the surface, and the test results are negative, indicating that the sterilization effect on metal chopsticks is stable; Weak fungal enzyme activity can still be detected on the metal surface after oxygen and alcohol disinfection, originating from the chopstick end joints or food residue attachment areas, indicating that the cleaning is not thorough; Although the untreated control group was made of stainless steel, it still showed a weak positive result due to the high humidity environment and trace contamination, verifying that even materials that are not easily mold-attached still have residual risks in a high humidity environment.

[0093] Example 4: S1. Select chopsticks to be tested Five pairs of alloy chopsticks for daily use were selected. The surfaces of the chopsticks were observed to have no obvious cracks, oil stains or other visible dirt, indicating good surface integrity.

[0094] S2, induce mold growth The alloy chopsticks were placed in a sealed humidity control device (constant temperature and humidity chamber). The ambient humidity was controlled at 85-90% and the temperature was set at 20-30°C. The temperature and humidity were maintained constant. The chopsticks were left to stand for 120 hours under light-shielding conditions to simulate the humid environment of the rainy season, promoting mold growth and the accumulation of metabolites on the chopsticks' surfaces.

[0095] S3. Disinfection After the mold induction was completed, the five pairs of alloy chopsticks were disinfected in the following five different ways: Alloy chopsticks 1: sterilize in a high-pressure steam sterilizer (121°C, 15 minutes); Alloy chopsticks 2: Boil in boiling water for 10 minutes; Alloy chopsticks 3: Microwave (700W) for 2 minutes; Alloy chopsticks 4: Place in ozone disinfection cabinet for 30 minutes; Alloy chopsticks 5: Use a cotton cloth soaked in 75% alcohol to wipe the surface and gaps, and dry at room temperature.

[0096] After the disinfection process is completed, each combination of gold chopsticks will no longer be exposed to external pollution sources and will immediately enter the next step of the testing process.

[0097] S4: Collect surface samples Using a sterile cotton swab sterilized by high temperature and high pressure, wipe the tip and seam of each pair of chopsticks evenly six times to ensure coverage of high-risk areas. Immediately after collection, the cotton swab is inserted into an EP tube containing 5 mL of normal saline (pH 6.5-7.5), rotated at 200 rpm for 2 minutes, and allowed to stand for 5 minutes to extract the mold spores and their metabolites remaining on the surface of the chopsticks to form a sample extract for testing.

[0098] S5: Detection of composition reaction The extract (500 μL) was mixed with the prepared detection composition in a 1:1 ratio, added dropwise to a transparent reaction well plate, reacted at room temperature for 60 seconds, and the color change was observed.

[0099] Test composition formula (by mass): Color change indicator: 5 parts; Enzyme substrate: 6 parts; Polyethylene glycol: 50 parts; Boric acid buffer: 20 parts; Hydroxypropyl methylcellulose: 6 parts; Deionized water: 31 parts.

[0100] Reaction Principle: If proteases are present in the sample, they hydrolyze the substrate, releasing acidic groups that trigger a color change in the color indicator. The intensity of the color change reflects the level of fungal activity.

[0101] S6: Color determination and result analysis Visual judgment is performed based on the color change results of the reaction wells. The standards are as follows: Blue or green: judged as negative, no obvious fungal enzyme activity; Yellow or orange: judged as weak positive, there are residual active molds but the number is limited; Red or reddish brown: judged as positive, obvious fungal enzyme activity, and a large amount of residues.

[0102] Color determination and result analysis are shown in Table 4: Table 4: Chopstick number Disinfection method Color rendering Judgment results Evaluation of sterilization effect Alloy chopsticks 1 High-pressure steam sterilization blue-green Negative Very strong Alloy chopsticks 2 Boil water light yellow weak positive powerful Alloy chopsticks 3 Microwave heating yellow weak positive Strong Alloy chopsticks 4 Ozone disinfection cabinet treatment light orange weak positive generally Alloy chopsticks 5 alcohol wipes orange-yellow weak positive Generally weak High-pressure steam sterilization has the most significant effect on removing mold from alloy chopsticks. The test result is blue-green, indicating that the mold has been completely removed and the effect is extremely strong. Boiling water and microwave heating have better inhibitory effects on mold. The test results are light yellow and yellow, indicating that there are a small amount of mold residues, which is weak positive and has a strong bactericidal effect. Ozone disinfection and alcohol wiping are less effective, with the color appearing light orange and orange-yellow, indicating that mold activity still exists and the bactericidal effect is limited; Microwave disinfection is particularly suitable for alloy chopsticks, with short disinfection time and good effect. However, for chopsticks made of other materials, microwaves will cause deformation and make them difficult to use.

[0103] Example 5: S1. Select chopsticks to be tested Five pairs of melamine chopsticks for daily use were selected. The surfaces of the chopsticks were observed to have no obvious cracks, oil stains or other visible dirt, indicating good surface integrity.

[0104] S2, induce mold growth The melamine chopsticks were placed in a sealed humidity control device (constant temperature and humidity chamber). The ambient humidity was controlled at 85-90% and the temperature was set at 20-30°C. The temperature and humidity were maintained constant. The chopsticks were left to stand for 120 hours under light-shielding conditions to simulate the humid environment of the natural rainy season, promoting mold growth and the accumulation of metabolites on the chopsticks' surfaces.

[0105] S3. Disinfection After the mold induction was completed, the five pairs of melamine chopsticks were disinfected in the following five different ways: Melamine chopsticks 1: Use high pressure steam sterilizer (121℃, 15 minutes); Melamine chopsticks 2: Boil in boiling water for 10 minutes; Melamine chopsticks 3: Microwave (700W) for 2 minutes; Melamine chopsticks 4: Place in ozone disinfection cabinet for 30 minutes; Melamine chopsticks 5: Use a cotton cloth soaked in 75% alcohol to wipe the surface and gaps, and dry at room temperature.

[0106] After the disinfection process is completed, each set of melamine chopsticks will no longer be exposed to external pollution sources and will immediately enter the next step of the testing process.

[0107] S4: Collect surface samples Using a sterile cotton swab that has been sterilized at high temperature and high pressure, the tip and joint of each pair of chopsticks were evenly wiped seven times to ensure coverage of high-risk areas. The cotton swab was immediately inserted into an EP tube containing 5 mL of normal saline (pH 6.5-7.5), and the tube was rotated at 200 rpm for 2 minutes and allowed to stand for 5 minutes to extract the mold spores and their metabolites remaining on the surface of the chopsticks to form a sample extract for testing.

[0108] S5: Detection of composition reaction The extract (200 μl) was mixed with the prepared detection composition in a ratio of 1:1, added dropwise to a transparent reaction well plate, reacted at room temperature for 60 seconds, and the color change was observed.

[0109] Test composition formula (by mass): Color change indicator: 5 parts; Enzyme substrate: 6 parts; Polyethylene glycol: 50 parts; Boric acid buffer: 20 parts; Hydroxypropyl methylcellulose: 6 parts; Deionized water: 31 parts.

[0110] Reaction Principle: If proteases are present in the sample, they hydrolyze the substrate, releasing acidic groups that trigger a color change in the color indicator. The intensity of the color change reflects the level of fungal activity.

[0111] S6: Color determination and result analysis Visual judgment is performed based on the color change results of the reaction wells. The standards are as follows: Blue or green: judged as negative, no obvious fungal enzyme activity; Yellow or orange: judged as weak positive, there are residual active molds but the number is limited; Red or reddish brown: judged as positive, obvious fungal enzyme activity, and a large amount of residues.

[0112] Color determination and result analysis are shown in Table 5: Table 5: Chopstick number Disinfection method Color rendering Judgment results Evaluation of sterilization effect Melamine chopsticks 1 High-pressure steam sterilization blue-green Negative Very strong Melamine chopsticks 2 Boil water light yellow weak positive powerful Melamine chopsticks 3 Unsterile red Positive No disinfection Melamine chopsticks 4 Ozone disinfection cabinet treatment orange color weak positive generally Melamine chopsticks 5 alcohol wipes orange-yellow weak positive Generally weak High-pressure steam sterilization showed the best effect, with complete elimination of mold and negative color development; Boiling water can effectively inhibit mold, but there is still a small amount of residue, which is a weak positive; The unsterilized sample of melamine chopsticks 3 showed red, which was positive, indicating that mold was highly active in a humid environment; Although ozone treatment and alcohol wiping can reduce activity, the bactericidal effect is relatively limited and is only weakly positive; Microwave heating should not be used on melamine materials to avoid deformation of the material or release of harmful substances. This is not implemented.

[0113] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting mold on the surface of chopsticks, characterized in that: The following steps are involved: S1. Select chopsticks to be tested; S2. Place the chopsticks in an environment with appropriate humidity and temperature to induce mold growth; S3. Disinfecting the chopsticks to be tested by one of high-pressure steam sterilization, boiling water, microwave heating, ozone disinfection cabinet treatment, or alcohol wiping; S4. Use a sterile swab to collect surface samples from the tip and seam of the chopsticks and transfer them to the extraction solution. S5, reacting the extract with a detection composition containing a color change indicator and a protease substrate; S6. Visually inspect the chopsticks based on color changes to determine whether active mold is present on their surface.

2. The method for detecting mold on the surface of chopsticks according to claim 1, wherein: The chopsticks include wooden chopsticks, bamboo chopsticks, stainless steel chopsticks, alloy chopsticks or melamine chopsticks.

3. The method for detecting mold on the surface of chopsticks according to claim 1, characterized in that: The steps of step S2 include: Place the chopsticks in a sealed container, control the relative humidity of the environment at 80-95% through a humidifier, control the temperature at 20-30°C, and let it stand for 72 to 120 hours.

4. The method for detecting mold on the surface of chopsticks according to claim 1, wherein: The step S4 comprises: Use a sterile cotton swab moistened with 1-3 ml of saline to wipe the tip and seam of the chopsticks 5-10 times each. Place the swab in 5-10 ml of extract and oscillate at 200 rpm for 1-3 minutes to release surface microorganisms and metabolites.

5. The method for detecting mold on the surface of chopsticks according to claim 4, characterized in that: The extract is physiological saline with a pH value of 6.5-7.

5.

6. The method for detecting mold on the surface of chopsticks according to claim 1, characterized in that: The step S5 comprises: Take 100-500 μl of the extract sample and mix it with a detection composition containing a color-changing indicator and a protease substrate in a volume ratio of 1:1, then place it in a reaction container and react for 30-90 seconds under static conditions to allow the color-changing indicator and the protease substrate to undergo a visible color change.

7. The method for detecting mold on the surface of chopsticks according to claim 6, characterized in that: The detection composition containing a color change indicator and a protease substrate comprises the following components in parts by mass: Color change indicator: 2-10 parts; Protease substrate dye: 4-12 parts; Polyethylene glycol solubilizer: 20-60 parts; Boric acid buffer stabilizer: 10-30 parts; Hydroxypropyl methylcellulose adsorbent: 1-10 parts; Deionized water: 25-35 parts.

8. The method for detecting mold on the surface of chopsticks according to claim 1, characterized in that: The step S6 comprises: Observe the color of the reaction solution. If it is blue or green, it is judged as negative. If it is yellow or orange, it is judged as weakly positive. If it is red or reddish brown, it is judged as positive, indicating obvious fungal enzyme activity and the presence of a large amount of residue.

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