Preparation method of surface cleaning disinfectant for fresh fruits and vegetables and application of surface cleaning disinfectant in fruit and vegetable cleaning

Abstract

The invention discloses a preparation method of a fresh fruit and vegetable surface cleaning disinfectant and application of the fresh fruit and vegetable surface cleaning disinfectant in fruit and vegetable cleaning.The preparation method comprises the steps that firstly, the antibacterial effect of citric acid, tartaric acid and sodium hypochlorite is evaluated, the synergistic antibacterial effect of different compounding modes is optimized, and meanwhile the optimal compounding formula is screened out; and the antibacterial activity is verified by using a time-sterilization curve. The compound disinfectant has an obvious inhibition effect on typical food-borne pathogenic bacteria such as salmonella typhimurium and escherichia coli O157: H7, and meanwhile, in fruit and vegetable cleaning application, the effectiveness of the synergistic antibacterial effect of the method is evaluated under a cherry tomato and lettuce system; and the molecular mechanism of sterilization is explored from the aspects of cell membrane morphology and property change at the cellular level, and the synergistic effect mechanism of citric acid and sodium hypochlorite is discussed. The method can be applied to fresh fruit and vegetable surface cleaning and disinfection, and an important theoretical basis is provided for guaranteeing the safety of fresh fruit and vegetable microorganisms.

Description

Technical Field

[0001] The present invention relates to the field of microbial safety of fresh fruits and vegetables, specifically to the synergistic bactericidal effect of the compound of citric acid and sodium hypochlorite in organic acids on foodborne pathogenic bacteria and the application effect in an actual food system. The technical research analyzes the synergistic bactericidal mechanism, aiming to develop a method applicable to the surface cleaning and disinfection of fresh fruits and vegetables. Background Art

[0002] Foodborne diseases are one of the major public health problems globally. Approximately 600 million people suffer from foodborne diseases every year, resulting in about 420,000 deaths. Among them, the microbial contamination of fresh fruits and vegetables is an important cause of foodborne diseases. Therefore, the surface cleaning and disinfection of fresh fruits and vegetables have become the key to technical research. Among fresh fruit and vegetable products, leafy vegetables (such as lettuce, spinach, etc.) and vine vegetables (such as tomatoes) are the main fresh fruits and vegetables vulnerable to microbial contamination, and the foodborne diseases caused by them account for 22 - 30% and 6 - 8% of the foodborne diseases related to fruits and vegetables respectively. The main foodborne pathogenic bacteria easily infected in fresh fruits and vegetables include Salmonella typhimurium, Escherichia coli O157:H7, Listeria, Pseudomonas, etc. Salmonella belongs to Gram-negative rod-shaped intestinal pathogenic bacteria. Among all foodborne pathogenic bacteria, the number of food poisoning incidents caused by Salmonella ranks first every year. Millions of people are infected with Salmonella in countries around the world, and in the United States, the infection rate of fruits and vegetables caused by Salmonella accounts for 33.7%. Its harm is very serious. Therefore, prevention in terms of food safety is crucial for preventing Salmonella infection. At the same time, Escherichia coli O157:H7, as a Gram-negative bacterium, also has relatively widespread foodborne hazards, with an outbreak trend and strong pathogenicity. It has the characteristic of being resistant to low temperatures and can survive in natural water for several months. These factors reduce its time and space limitations during the infection process.

[0003] Various emerging non-thermal disinfection technologies applied to the sterilization of agricultural products have been widely studied. However, due to the high cost of equipment or high cost, it is difficult to be widely applied on a large scale. Chemical disinfectants have the advantages of low cost, good disinfection performance, and can be mixed with water in different proportions, and are thus widely used. Currently, chlorine-based disinfectants are the most commonly used fruit and vegetable cleaning agents. However, using sodium hypochlorite in fruit and vegetable washing water will produce low-concentration carcinogenic compounds, such as trihalomethanes and chlorite residues, etc., which cause certain harm to the human body. In order to prevent cross-contamination of pathogens or cleaning water sources, a relatively high free chlorine concentration is required for the disinfectant. Peracetic acid has received attention as an alternative to chlorine-based disinfectants. However, due to its strong oxidizing and corrosive properties, cleaning may cause chemical damage to nutrients such as vitamin C in fruits and vegetables, affecting the quality and nutritional value of fruits and vegetables. Using organic acids to compound with such common disinfectants can enhance the antibacterial and disinfection effects of sodium hypochlorite when used alone, and at the same time reasonably reduce the dosage of chlorine-based disinfectants to achieve a more ideal purpose of removing bacteria on the surface of fresh fruits and vegetables.

[0004] Organic acids such as citric acid are good substitutes in current fruit and vegetable cleaners. Before dissociation, it has lipophilicity. After passing through the cell membrane and entering the cell, the organic acid will dissociate, and the charged anions and protons cannot pass through the plasma membrane, resulting in a decrease in the intracellular pH value, thereby inducing cell damage and changes in the functions of enzymes, structural proteins, and DNA. Among them, citric acid has good broad-spectrum antibacterial ability and can inhibit a variety of foodborne pathogenic bacteria such as Escherichia coli O157:H7 and Staphylococcus aureus, and shows good synergistic effects in the compounding with different types of substances. Tartaric acid is a naturally occurring organic acid that widely exists in fruits such as grapes and citrus fruits. Its low toxicity makes it have good safety in the food industry, and its good antioxidant function makes its application in fruit and vegetable preservation more prominent. However, the cost of using organic acids alone is relatively high, and the acidic odor of organic acids themselves will affect the sensory properties of fruits and vegetables. In summary, this study aims to explore whether the compounding of organic acids and common disinfectants can play and synergistically improve their respective bactericidal advantages to achieve a higher cleaning and bactericidal effect on fruits and vegetables through compounding. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an efficient surface cleaning disinfectant for fresh fruits and vegetables, which can be used as a new compound disinfectant for surface cleaning and disinfection of fresh fruits and vegetables. This new compound disinfectant has high inhibitory effects on foodborne pathogenic bacteria Salmonella typhimurium and Escherichia coli O157:H7 that are easily infected by fresh fruit and vegetable products. Its specific compounding formula is: a mixed solution of low-concentration sodium hypochlorite and citric acid.

[0006] The concentration of citric acid in the surface cleaning disinfectant for fresh fruits and vegetables is 3.5 - 4.5 mM;

[0007] The available chlorine content in the surface cleaning disinfectant for fresh fruits and vegetables is 70.0 - 80.0 ppm.

[0008] Further preferably, the concentration of citric acid in the surface cleaning disinfectant for fresh fruits and vegetables is 3.8 - 4.2 mM; the available chlorine content in the surface cleaning disinfectant for fresh fruits and vegetables is 73 - 77 ppm. Most preferably, the concentration of citric acid in the surface cleaning disinfectant for fresh fruits and vegetables is 4.0 mM; the available chlorine content in the surface cleaning disinfectant for fresh fruits and vegetables is 75.0 ppm.

[0009] A new and convenient method applicable to the surface cleaning and disinfection of fresh fruits and vegetables can be applied in the pretreatment process of fresh fruits and vegetables to play the role of efficiently inhibiting foodborne pathogenic microorganisms and long-term preservation. The optimized formula obtained by screening is:

[0010] The compound disinfectant is a mixed solution of low-concentration sodium hypochlorite and citric acid (a mixed solution containing a citric acid solution with a molar concentration of 4.0 mM and sodium hypochlorite with an available chlorine content of 75.0 ppm).

[0011] In this invention, two typical organic acids, citric acid and tartaric acid, and a common food disinfectant, sodium hypochlorite solution, are used as raw materials. The compounding ratio is optimized, and the compound combination with the best antibacterial synergistic effect is screened. The research applies this method to the actual cleaning of fruits and vegetables, effectively reducing the residual amount of foodborne pathogenic bacteria on the surface of cherry tomatoes and lettuce. At the same time, the mechanism research provides a theoretical basis for the practical application of compound sterilization to give full play to the sterilization advantages of organic acids and common disinfectants more efficiently. The technical solution of this invention provides support for realizing a new, safer and greener method for preventing and controlling microorganisms on the surface of fruits and vegetables, and has important research significance, great economic value and market demand.

[0012] A preparation method of a disinfectant for cleaning the surface of fresh fruits and vegetables includes the following steps:

[0013] (1) Uniformly dissolve the solid chemical citric acid in water to prepare a citric acid solution as mother liquor 1;

[0014] (2) Dilute sodium hypochlorite with water as mother liquor 2;

[0015] (3) Mix mother liquor 1 and mother liquor 2 to obtain a disinfectant for cleaning the surface of fresh fruits and vegetables, which is used for cleaning and disinfecting the surface of fresh fruits and vegetables.

[0016] Further preferably, a new method for cleaning and disinfecting the surface of fresh fruits and vegetables includes the following steps:

[0017] (1) Uniformly dissolve the solid chemical citric acid in pure water to prepare a citric acid solution with a concentration of 8.0 mM as mother liquor 1.

[0018] (2) Dilute the chemical agent sodium hypochlorite solution with pure water to make its available chlorine content 150.0 ppm as mother liquor 2.

[0019] (3) Mix mother liquor 1 and mother liquor 2 in an equal volume ratio of 1:1 to obtain a new compound disinfectant that can be applied to the cleaning and disinfection of the surface of fresh fruits and vegetables.

[0020] In step (1), the citric acid solution with a molar concentration of 8.0 mM is to prepare a citric acid solution containing 1.537 g / L of citric acid by mass fraction.

[0021] In step (2), that is, to prepare a sodium hypochlorite solution with an available chlorine content of 150.0 mg / L.

[0022] In step (2), the mass concentration of sodium hypochlorite (mg / L) = the available chlorine content (mg / L) / the chlorine content (%) in the sodium hypochlorite solution. The specific chlorine content of the chemical agent sodium hypochlorite solution can be accurately measured by the iodometric method or the Amplite fluorescence method hypochlorous acid detection kit.

[0023] In step (3), mix in a volume ratio of 1:1 to ensure that the final compound disinfectant contains a mixed solution of 4.0 mM citric acid solution and sodium hypochlorite with an available chlorine content of 75.0 ppm. This compound disinfectant should be prepared and used immediately to ensure the best effect.

[0024] Specifically, the research on the preparation method, application effect and action mechanism of a new compound disinfectant used for surface cleaning and disinfection of fresh fruits and vegetables was screened and optimized, mainly including the following steps:

[0025] (1) Determination of minimum inhibitory concentration and bactericidal concentration: First, prepare citric acid solutions, tartaric acid solutions and sodium hypochlorite solutions with different concentration gradients. The minimum inhibitory concentration (MIC) of chemical disinfectants against two harmful pathogenic bacteria, S.typhimurium and E.coli O157:H7, was determined by the two-fold dilution method. After treating with the above MIC concentration solution, the minimum bactericidal concentration (MBC) was determined by culturing on PCA medium.

[0026] (2) Determination and screening of the synergistic effect of different compounding methods: With reference to the MIC values of the three chemical disinfectants in step (1), citric acid solutions and tartaric acid solutions with different concentration gradients were compounded with sodium hypochlorite solutions with different concentration gradients. The FICI method was used to determine whether there was a synergistic effect, additive effect, etc. when the two reagents were used in combination. The antibacterial effects of various compound combinations were compared, and four compound combinations with better antibacterial synergistic effects were screened and optimized.

[0027] (4) Determination of the time-kill curve of the four compound combinations against two foodborne pathogenic bacteria: To verify the bactericidal effect of the screened compounding method, a time-dependent bactericidal experiment was carried out by the colony counting method. Prepare a bacterial suspension, inoculate it into different sample culture solutions, incubate, and spread and count the colonies on the plate on time. Draw a curve of the relationship between the average colony (log CFU / mL) and time.

[0028] (5) Evaluation of the bactericidal effect of the compound combination in the cleaning of fresh fruits and vegetables: Using two types of fresh fruits and vegetables, cherry tomatoes and lettuce, which are easily contaminated by foodborne pathogenic bacteria, as experimental objects, the two main foodborne pathogenic bacteria were mixed in equal proportions. Inoculate the fruits to make the pathogens adhere. Prepare control groups, water groups and sample groups respectively, and let them dry naturally after cleaning, and finally count the remaining number of pathogenic bacteria (CFU / cm 2 ).

[0029] (6) Observation of bacterial morphology after treatment with the compound solution: Observe the morphological changes of bacteria cells after treatment using a scanning electron microscope. Select the optimal compound combination solution of citric acid and sodium hypochlorite to treat S.typhimurium (ATCC 14028) cells, and perform pre-treatment before electron microscopy observation. Store the samples in 100% ethanol, perform gold plating treatment after drying, and then observe using a scanning electron microscope.

[0030] (7) Analysis of functional changes in bacterial cell membranes: Analyze the changes in cell membrane integrity after the action of the compound cleaning disinfectant by measuring the leakage of intracellular nucleic acids and proteins, and using the PI fluorescence staining method; at the same time, evaluate the effect on the bacterial cell membrane potential; use CLSM laser confocal microscopy to observe the damage of the cytoplasmic membrane after fluorescence staining, and explore the bactericidal mechanism of the optimized compound cleaning disinfectant from the perspective of changes in cell membrane morphology and properties.

[0031] Preferably, in the exploration of antibacterial effects, the two main harmful pathogenic bacteria in the two fresh fruits and vegetables are selected as S.typhimurium (ATCC 14028) and E.coli O157:H7 (ATCC 35150).

[0032] Preferably, in step (1), in order to obtain an accurate MIC, after measuring the range where the MIC is located by the two-fold dilution method, a concentration gradient within a specific small range is further configured for measurement. The MBC identification standard is that the compound concentration with less than 5 colonies growing on the PCA medium is the MBC value.

[0033] Preferably, in step (2), when using the FICI method to measure the synergistic effect of multiple drugs when used in combination, using the checkerboard method and the single variable method, the different compound ratios of the required citric acid, tartaric acid and sodium hypochlorite solutions are FIC 有机酸 Take 1 / 3; 1 / 2; 2 / 3, FIC 消毒剂 Take between 1 / 4 and 2 / 3.

[0034] Preferably, in step (4), the concentration of the bacterial suspension is 10 6 CFU / mL, the control group is 0.85% saline by mass fraction, the culture temperature is 37 °C, and the incubation times are 0, 2, 4, 6, 12, 24 h.

[0035] Preferably, in step (5), the cherry tomatoes need to be wiped with alcohol to remove the surface wax, and the cherry tomatoes and lettuce are rinsed with pure water in turn and placed in a ultra-clean bench for 2 h of ultraviolet sterilization to eliminate the existence of natural flora on the fruit peel.

[0036] Preferably, in step (5), the control group, the water group and the sample group are naturally dried after cleaning, placed in a homogenization bag containing 10 mL of 0.85% saline by mass fraction for 2 min of homogenization treatment, and then the final number of pathogenic bacteria is calculated using the plate dilution coating counting method.

[0037] Preferably, the pretreatment in step (6) should be carried out strictly in accordance with the requirements, glutaraldehyde solution fixation, phosphate buffer (PBS) washing, 1% osmium acid fixation, PBS continued washing, gradient concentration ethanol solution dehydration. The scanning electron microscope was used for observation at 40,000× and 20,000× magnifications, and the marking units were 2μm and 1μm respectively.

[0038] Preferably, in step (7), the determination of various indicators of the functional changes of the bacterial cell membrane of the optimized composite disinfectant should be carried out strictly in accordance with the specific implementation methods in the specification.

[0039] The present invention has the following technical effects:

[0040] Against the backdrop of the high incidence of foodborne diseases worldwide, surface cleaning and disinfection of fresh fruits and vegetables has become a key technical research topic. The long-term use of common chlorine-based disinfectants on fresh fruits and vegetables poses potential hazards to the human body. Organic acids such as citric acid and tartaric acid that can be used to disinfect food systems meet the GRAS standard. The study selected two organic acids, citric acid and tartaric acid, to compound sodium hypochlorite solution, a commonly used disinfectant, and attempted to reduce the amount of sodium hypochlorite used in actual production, making surface cleaning of fresh fruits and vegetables greener and safer while reducing costs.

[0041] Citric acid, tartaric acid and sodium hypochlorite solution were compounded in proportion to screen and optimize the combination with the best antibacterial synergistic effect. The high antibacterial effect against two typical foodborne pathogens commonly found in fruits and vegetables, S. typhimurium and E. coli O157:H7, was evaluated in the cleaning of two types of fresh fruits and vegetables, cherry tomatoes and lettuce. The molecular mechanism of the antibacterial effect of the compound cleaning and disinfectant was further analyzed from the perspective of changes in cell membrane morphology and properties.

[0042] This technology is intended to be used in the cleaning and antibacterial treatment of fresh fruits and vegetables. This study found that the combination of citric acid and sodium hypochlorite disinfectants has a good synergistic antibacterial effect. At the same time, the combined cleaning disinfectant obtained by optimizing the ratio of the two has a significant bactericidal effect on foodborne pathogens on the surface of cherry tomatoes and lettuce. This technology solves the problem of pathogenic bacteria contamination of fruits and vegetables in a safer and greener way, and effectively ensures the quality and food safety of fruits and vegetables. At the same time, the exploration of the synergistic antibacterial mechanism also provides a theoretical basis for the technology. DETAILED DESCRIPTION

[0043] In order to more clearly express the purpose and technical scheme of the present invention, the present invention will be described in detail below in conjunction with specific implementation cases and drawings. Unless otherwise defined, the technical and scientific terms used in the present invention have the same meaning as those in the field to which the present invention belongs. In addition, the following specific implementation cases are only used to explain the present invention and do not limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0044] The reagents and materials used in the present invention are all commercially available. Description of the Drawings

[0045] Figure 1 Time - bactericidal curves of the optimal compounding combination against S.typhimurium (ATCC 14028) and E.coli O157:H7 (ATCC35150);

[0046] Figure 2 Scanning electron microscope images of S.typhimurium (ATCC 14028) under different disinfectant treatments;

[0047] Figure 3 Nucleic acid and protein leakage in bacterial cells after different treatments;

[0048] Figure 4 Changes in PI staining fluorescence level and cell membrane potential after different sample treatments;

[0049] Figure 5 Fluorescence observation under a CLSM laser confocal microscope after DAPI / FM4 - 64 staining.

[0051] Example 1

[0052] Determination of MIC and MBC

[0053] The minimum inhibitory concentration of sodium hypochlorite and organic acids against S.typhimurium (ATCC 14028) and E.coli O157:H7 (ATCC 35150) was determined by the two - fold dilution method. The MIC value range was initially obtained using the two - fold dilution method, and further experiments were carried out by configuring specific concentration gradients to obtain a more accurate MIC. The judgment criterion was that the lowest concentration at which the liquid in the 96 - well plate did not become turbid compared to the corresponding control group was the MIC value. 100 μL of the solution was taken from the above - mentioned MIC value determination solution and spread on a PCA medium, and cultured overnight at 37°C to measure the MBC value. The judgment criterion was that the compounding concentration with less than 5 colonies growing on the PCA medium was the MBC.

[0054] The measurement results are shown in Table 1:

[0055] Table 1 Determination of the minimum inhibitory concentration and minimum bactericidal concentration of organic acids and sodium hypochlorite used alone against two food - borne pathogenic bacteria

[0056]

[0057] Result analysis:

[0058] As can be seen from the table, for organic acids and common disinfectants, S.typhimurium (ATCC 14028) showed higher tolerance than E.coli O157:H7 (ATCC 35150). In addition, the MIC and MBC values of the two strains against sodium hypochlorite solution were equal, indicating the strong bactericidal properties of the sodium hypochlorite solution.

[0059] Example 2

[0060] Determination and screening of the synergistic effects of different compound combinations

[0061] To verify the compounding effect and further explore the synergistic effect of compounding. FICI is an index used to evaluate the synergistic effect of antibacterial drug combinations on bacteria. It is usually used to determine whether two or more drugs have synergistic, additive, antagonistic or indifferent effects when used in combination. According to the results of the previous experimental data, different compounding concentrations and ratios of citric acid, tartaric acid and sodium hypochlorite solution required for measuring FICI were set (FIC for organic acids was taken as 1 / 3; 1 / 2; 2 / 3, and FIC for disinfectants was taken between 1 / 4 and 2 / 3). Using the single variable method, two different concentration gradient compounding combinations were set to measure their inhibitory effects on S.typhimurium (ATCC14028) and E.coli O157:H7 (ATCC 35150), and finally their synergistic effects were determined by the FICI value.

[0062] The synergistic effects of different compounding combinations obtained from the study are shown in Table 2:

[0063] Table 2 Synergistic bactericidal effects of different compounding combinations on two foodborne pathogenic bacteria

[0064]

[0065] Note: CA is citric acid; TA is tartaric acid; NaClO is sodium hypochlorite solution;

[0066] FICI < 1.0: Synergism; FICI = 1: Commutative effect;

[0067] 1 < FICI ≤ 2: Indifference; FICI > 2: Antagonism.

[0068] Result analysis:

[0069] When organic acids at sub-MIC concentrations are used in combination with sodium hypochlorite solution, the usage concentration of sodium hypochlorite can be significantly reduced. In the determination of antibacterial synergy against E. coli O157:H7, 0.5 < FICI(CA+Na) < 1, indicating that there is a synergistic effect when citric acid and sodium hypochlorite are combined; in the determination of antibacterial synergy against S. typhimurium, 0.5 < FICI(CA+NaClO) < 1, indicating that there is a synergistic effect when citric acid and sodium hypochlorite are combined. At the same time, it can be seen from the table that the combined effect of citric acid and sodium hypochlorite solution is better than that of tartaric acid combination.

[0070] When the FIC value of citric acid is 0.5 and the FIC value of sodium hypochlorite solution is 0.25, the compounding method of cleaning and disinfection is the best compounding ratio.

[0071] Example 3

[0072] Determination of the time-kill curve of the best compounding combination against two foodborne pathogenic bacteria

[0073] First, prepare before the experiment, make NB150 culture solution, PCA plate, normal saline with a mass fraction of 0.85%, and bacterial suspension. Using the colony counting method, time-dependent bactericidal experiments were carried out in 6 treatment groups: blank control group (normal saline with a mass fraction of 0.85%); CA(MIC); NaClO(MIC); CA(1 / 2MIC); NaClO(1 / 4MIC); and CA(1 / 2MIC)+NaClO(1 / 4MIC).

[0074] Inoculate the logarithmic-phase S. typhimurium (ATCC 14028) and E. coli O157:H7 (ATCC 35150) into NB containing antibacterial agents, with normal saline with a mass fraction of 0.85% as the control group. Place it in a natural room temperature environment for 0, 2, 4, 6, 12, 24 h, and detect regularly. Dilute continuously in 0.85% NaCl solution, take 100 μL of the diluted bacterial suspension for plate coating. Incubate at 37 °C for 24 h and count the colonies. Draw the curve of the relationship between the average colony (logCFU / mL) and time as Figure 1 shown, the time-kill curve of citric acid and sodium hypochlorite alone or in combination against S. typhimurium (ATCC 14028) (A); the time-kill curve of citric acid and sodium hypochlorite alone or in combination against E. coli O157:H7 (ATCC 35150) (B). CK is the blank control group; NaClO represents sodium hypochlorite; CA represents citric acid; Combination1 and Combination2 are antibacterial compounding combinations (1 / 4MIC NaClO + 1 / 2MIC CA) against two foodborne pathogenic bacteria respectively.

[0075] Result analysis:

[0076] The compounding of citric acid and sodium hypochlorite solution can effectively inhibit the growth of foodborne pathogenic bacteria, and there are significant differences from the blank group and the sub-treatment group. The bactericidal effect of the compounding group over time is similar to that of using sodium hypochlorite solution (300 ppm) alone, while significantly reducing the dosage of sodium hypochlorite solution by 4.0 times. The compounding group is superior to or similar to the antibacterial effect of using citric acid (8.0 mM) alone within 0 - 24 hours, indicating that compounding can better achieve the bactericidal effect and effectively reduce the dosage of organic acid by 2.0 times, reducing the cleaning cost and improving the cleaning effect.

[0077] Through the verification study on the antibacterial synergistic effect, a mixed solution of 4.0 mM citric acid solution and sodium hypochlorite with an available chlorine content of 75.0 ppm was optimized as a new compound disinfectant formula.

[0078] Example 4

[0079] Bactericidal effect of the compound disinfectant on foodborne pathogenic bacteria on the surface of cherry tomatoes

[0080] Prepare PCA culture medium plates before the experiment; configure physiological saline with a mass concentration of 0.85% and NB nutrient solution; prepare solutions of E. coli O157:H7 (ATCC 35150) and S. typhimurium (ATCC 14028); prepare fresh cherry tomatoes and store them refrigerated at 4°C.

[0081] First, eliminate the natural flora existing on the surface of cherry tomatoes. Wipe the cherry tomatoes with alcohol to remove the surface wax, rinse them successively with pure water and place them in the ultra-clean bench for ultraviolet sterilization for 2 hours. Secondly, inoculate on the surface of cherry tomatoes. Mix the solutions of E. coli O157:H7 (ATCC 35150) and S. typhimurium (ATCC 14028) in a ratio of 1:1 (v / v). Mark a piece (1.0 cm × 1.0 cm) on the fruit surface with a marker pen, and evenly apply 50 μL of the bacterial suspension to 5 points on the marked section with a micropipette. The inoculated fruits are placed at room temperature for 2 h to allow the pathogens to attach. Then, conduct sample treatment. In the water group, immerse the cherry tomatoes in 200 mL of pure water; in the sample group, select CA (MIC); NaClO (MIC); CA (1 / 2 MIC); NaClO (1 / 4 MIC) and CA (1 / 2 MIC) + NaClO (1 / 4 MIC) solutions to soak the cherry tomatoes in 200 mL. After washing for 3 min, take them out, air-dry naturally, cut off the marked area, and put it into a homogenization bag containing 10 mL of physiological saline for homogenization treatment. Finally, use the plate dilution coating counting (CFU / g) to calculate the number of pathogenic bacteria in the sample group.

[0082] The results are shown in Table 3 as follows:

[0083] Table 3 Bactericidal effects of compound disinfectants on mixed foodborne pathogenic bacteria on the surface of cherry tomatoes

[0084]

[0085] Note: The inoculation amount on the surface of cherry tomatoes was maintained at 7.86 ± 0.48 log CFU / cm 2 ; CA is citric acid; NaClO is sodium hypochlorite solution Experimental analysis:

[0086] Compared with the blank control and the sub-treatment group, the bacteriostatic effect of the compound solution of citric acid and sodium hypochlorite solution can significantly and effectively kill the growth of foodborne pathogenic bacteria on the surface of tomatoes, and it is 6.06 log CFU / cm less than that of the control group 2 . When the inactivation effect > 5 log CFU, it is the recognized target pathogen bactericidal inactivation level (FDA, 2001). At the same time, the cleaning effect of the compound solution is better than that of using citric acid alone, indicating that the compound can achieve a good bacteriostatic effect and effectively reduce the dosage of organic acid by 2.0 times. The bacteriostatic effect is also similar to that of using sodium hypochlorite solution alone, and significantly reduces the dosage of chlorine-based disinfectant by 4.0 times, with good practical application value

[0087] Example 5

[0088] Bactericidal effects of compound disinfectants on foodborne pathogenic bacteria on the surface of Romaine lettuce

[0089] Among fresh fruit and vegetable products, leafy vegetables (such as Romaine lettuce) are the most severely contaminated by foodborne pathogenic bacteria. Therefore, the experiment further verified the application of compound disinfectants in the cleaning and disinfection of Romaine lettuce. First, prepare the bacterial solution by mixing the solutions of S.typhimurium (ATCC 14028) and E.coli O157:H7 (ATCC 35150) in a ratio of 1:1 (v / v) (about 8 log CFU / mL). Then, prepare the materials and perform pre-treatment. Fresh and flawless Romaine lettuce was purchased one day before the experiment and stored overnight in a 4°C refrigerator. On the day of the experiment, it was placed at 22°C to adapt to room temperature. The outer layer of the Romaine lettuce was discarded to avoid possible previous contamination, rinsed with sterile water, and then placed in a laminar flow hood for 2 h of ultraviolet sterilization. Cut into square pieces of equal area (2.0 cm × 2.0 cm) with sterile stainless steel scissors, pipette 50 μL of the bacterial suspension and evenly spread it on the marked sections. After inoculation, dry the leaves for about 3 h. Two leaves were used for each treatment in each replicate. Then, prepare the sample solution. Prepare 100 mL each of the blank control group and the sample group, rinse for 3 min and then air dry naturally, put them into a homogenization bag containing 10 mL of normal saline for 2 min of homogenization treatment. Use the plate dilution coating counting method (CFU / cm 2) Finally, calculate the number of pathogenic bacteria on the lettuce after treatment for each sample group.

[0090] The results are shown in Table 4:

[0091] Table 4 Bactericidal effects of the compound disinfectant on mixed foodborne pathogenic bacteria on the surface of lettuce

[0092]

[0093] Note: The inoculation amount on the surface of lettuce was maintained at 7.86 ± 0.48 log CFU / cm 2 ; CA is citric acid; NaClO is sodium hypochlorite solution Experimental analysis:

[0094] Compared with the blank and sub-MIC treatment groups, the compound solution can significantly and efficiently kill pathogenic bacteria on lettuce, effectively reducing the number of pathogenic bacteria by 5.18 log CFU / cm 2 . The cleaning effect of the compound is similar to that of using organic acid and sodium hypochlorite alone (2.20 ± 0.13 log CFU / cm 2 ) and slightly better than both, indicating that the compound can achieve a good antibacterial effect and effectively reduce the dosage of organic acid by 2.0 times, and significantly reduce the dosage of sodium hypochlorite by 4.0 times. At the same time, it was found that using clean water to wash the contaminated lettuce still had 6.43 ± 0.27 log CFU / cm 2 of pathogenic bacteria remaining, indicating that microorganisms adhere relatively firmly to lettuce, and once contaminated, clean water washing cannot effectively remove the attached foodborne pathogenic bacteria.

[0095] Example 6

[0096] SEM scanning electron microscope cell morphology observation

[0097] First, prepare the control group; CA (1 / 2 MIC); NaClO (1 / 4 MIC) and CA (1 / 2 MIC) + NaClO (1 / 4 MIC) solutions for sample treatment of S. typhimurium (ATCC 14028) cells. Then, place the samples. Take 1 mL of each sample and centrifuge at 8000 r for 5 min, add 1 mL of 2.5% glutaraldehyde solution, and place it in a refrigerator at 4 °C overnight. Wash with 0.1 M, pH 7.4 phosphate buffer solution (PBS), fix the samples with 1% osmium tetroxide, wash with PBS, then dehydrate the samples successively with gradient ethanol solutions, and finally store the samples in 100% ethanol, coat them after drying. Finally, observe the cell morphology, and observe with a scanning electron microscope at magnifications of 40,000× and 20,000× respectively. The results are as Figure 2Scanning electron microscope images of S.typhimurium (ATCC 14028) cells under different sample treatments are shown. Among them, (A-a) is the control group; (B-b) is the group treated with citric acid CA (1 / 2 MIC); (C-c) is the group treated with sodium hypochlorite NaClO (1 / 4 MIC); (D-d) is the compound disinfectant (1 / 4 MIC NaClO + 1 / 2 MIC CA). The magnification of A, B, C, D is 40,000×, and the scale bar is 1 μm; the magnification of a, b, c, d is 20,000×, and the scale bar is 2 μm.

[0098] Result analysis:

[0099] It can be observed that Figure 2 in the control group, the bacterial cell morphology is oval and the cells are round. The damage of organic acids to cells is mainly reflected in the perforation and damage of the cell membrane. Some bacterial cell membranes are ruptured, and there is a little sign of content leakage. Compared with the sodium hypochlorite treatment group, the cell morphology and structure change little. In the group treated with sodium hypochlorite alone, it can be observed that most cell centers are sunken inward, and the cell morphology changes greatly, while the cell membrane shows almost no perforation or rupture. The compound disinfectant causes the most serious damage to cell morphology and cell membrane. It is observed that the vast majority of cell morphologies are severely deformed, the cells are ruptured and the contents flow out, and there are multiple perforations in the cell membrane.

[0100] Example 7

[0101] Analysis of intracellular nucleic acid and protein leakage

[0102] The leakage of two types of biological macromolecules, proteins and nucleic acids, in S.typhimurium (ATCC 14028) cells treated with different sample solutions of CA (MIC); NaClO (MIC); CA (1 / 2 MIC); NaClO (1 / 4 MIC) and CA (1 / 2 MIC) + NaClO (1 / 4 MIC) was measured. The bacterial suspension treated with the sample solution was collected, centrifuged at 8000 rpm and 4 °C for 10 min, 5 mL of the supernatant was taken, and the absorbance values of the supernatant at 260 nm and 280 nm were measured by an ultraviolet spectrophotometer, which respectively represent the release of nucleic acids and proteins. Bovine serum albumin BSA was used as a standard product, and a series of BSA solutions with concentration gradients were prepared. Their absorbances were measured and a standard curve was plotted. The protein release amount of bacteria was calculated through the bovine serum albumin standard curve. The results are as Figure 3As shown, the intracellular nucleic acid (A) and protein leakage (B) of S.typhimurium (ATCC 14028) after different sample treatments. CK is the control group; CA is citric acid; NaClO is sodium hypochlorite solution; Combination is the compound combination (1 / 4MIC NaClO + 1 / 2MIC CA). The values are expressed as mean ± standard deviation; different letters represent significant differences between the data (P<0.05).

[0103] Result analysis:

[0104] As can be seen from the figure, as the concentration of sodium hypochlorite increases, the content of nucleic acid and protein flowing out of the cells increases. After treatment with NaClO (MIC), the protein leakage amount reaches as high as 0.823 mg / mL, indicating that sodium hypochlorite has a greater impact on the level of intracellular macromolecule leakage. After treatment with the compound disinfectant, the fluorescence level indicating nucleic acid leakage and the extracellular protein content are much higher than those of the 1 / 4MIC sodium hypochlorite treatment group and the 1 / 2MIC citric acid treatment group, and there are significant differences. The fluorescence value and extracellular protein content of the compound group are significantly higher than those of citric acid alone by about 2 times, indicating that citric acid alone has a certain impact on the cell membrane integrity. The addition of a small amount of sodium hypochlorite can further significantly improve the damage of organic acids to the cell membrane integrity and increase the leakage degree of biological macromolecules.

[0105] Example 8

[0106] PI fluorescence staining analysis

[0107] The integrity of the cell membrane of S.typhimurium (ATCC 14028) after treatment was determined using methicillin-resistant propidium iodide (PI). After centrifuging the bacterial cells for 10 min, they were resuspended in sterile saline. After treatment with different samples for three minutes, they were centrifuged and resuspended again. The OD 600 of each solution was 0.2. The bacterial suspension was mixed with PI at a final concentration of 10 μM and incubated in the dark at 37 °C for 30 min. The fluorescence intensity was monitored using a fluorescence spectrophotometer with an excitation wavelength of 535 nm and an emission wavelength of 617 nm. Each test was independently repeated three times. The experimental results are as Figure 4 shown, the changes in the PI staining fluorescence intensity (A) and cell membrane potential changes (B) of S.typhimurium (ATCC 14028) after different sample treatments. CK is the control group; CA is citric acid; NaClO is sodium hypochlorite solution; Combination is the compound combination (1 / 4MIC NaClO + 1 / 2MIC CA). The values are expressed as mean ± standard deviation; different letters represent significant differences between the data (P<0.05).

[0108] Result analysis:

[0109] As can be seen from the graph, the fluorescence value treated with sodium hypochlorite is higher than that treated with citric acid alone, indicating that it is easier for PI dye to leak through the cell wall and membrane after treatment with sodium hypochlorite, which is consistent with the analysis results of the release of intracellular nucleic acids and proteins. The fluorescence value of the compound group is about 3 times higher than that of the group treated with citric acid alone, further verifying that the addition of a small amount of sodium hypochlorite in the compound combination significantly enhances the degree of damage to the integrity of the bacterial cell membrane.

[0110] Example 9

[0111] Cell membrane potential analysis

[0112] Metabolically active bacteria have a normal membrane potential, which is crucial for the physiology of bacteria. Therefore, analyzing the changes in membrane potential after treatment with the compound solution can illustrate the mode of the antibacterial mechanism from another perspective. Rhodamine 123 fluorescent probe was used in the study to detect the changes in membrane potential.

[0113] First, dissolve rhodamine in phosphate buffered saline solution to prepare a stock solution of 1 mg / mL. Second, centrifuge the bacterial cells for 10 min and resuspend them in sterile saline. Treat with different samples for three minutes, wash the bacterial cell precipitate twice with 0.85% sterile saline, then add rhodamine 123 solution (1 mg / mL) with a final concentration of 2 μg / mL, incubate in the dark for 30 min, centrifuge the stained samples, wash them twice with 0.85% sterile saline, and then resuspend them in sterile saline solution. Finally, transfer the treated bacterial suspension to a black 96-well plate and perform fluorescence measurement using an M5 all-wave multifunctional microplate reader with excitation and emission wavelengths of 480 nm and 530 nm, respectively. The experimental results are as Figure 4 (B) shows.

[0114] Result analysis:

[0115] After treatment with different treatment groups, the cell membrane potential basically undergoes hyperpolarization. Hyperpolarization is generally caused by the stress response of bacteria to external stress. Under the action of changes in pH value or antibacterial substances, harmful anions accumulate in the cell, resulting in changes in the membrane permeability to ions (especially K+). To maintain the potential balance, the cell will perform automatic regulation, leading to hyperpolarization of the membrane potential.

[0116] Among them, the fluorescence value treated with NaClO (1 / 4 MIC) is slightly higher than that treated with CA (MIC), indicating that the treatment with sodium hypochlorite has a greater impact on the potential. The fluorescence value of the compound group is significantly higher than that of the group treated with citric acid alone, indicating that the compound of sodium hypochlorite and citric acid can significantly enhance the level of membrane potential hyperpolarization.

[0117] In addition, the high-concentration sodium hypochlorite solution caused depolarization of the membrane potential. This may be because most bacteria in the high-concentration sodium hypochlorite treatment group had ruptured and died, losing their normal potential regulation function, and the membrane potential had completely dissipated.

[0118] Example 10

[0119] Observation by CLSM laser confocal microscopy fluorescence staining

[0120] The damage effect of the selected compound disinfectant on S.typhimurium (ATCC14028) cells was evaluated using visual fluorescence imaging technology. After culturing, centrifuging, and resuspending the bacteria, the cells were incubated with various sample solutions. The treated strains were mixed with 20 μg / mL DAPI and incubated for 30 min, and then incubated with 20 μg / mL A at room temperature for 60 min. 5 μL of concentrated cells were placed on a slide coated with 1.5% agarose for microscopic observation. Images were acquired using a Zeiss 980 super-resolution laser confocal microscope (ZEISS LSM 980, Carl Zeiss Microscopy GmbH, Jena, Germany) with an oil immersion objective lens. The experimental results are as Figure 5 shown. After treatment with different sample solutions, after staining the cell membrane lipids and intracellular DNA of S.typhimurium (ATCC14028) with DAPI / FM4-64 respectively, fluorescence observation under a laser confocal microscope was carried out. CK was the control group; CA was citric acid; NaClO was sodium hypochlorite solution; Combination was the compound combination (1 / 4MIC NaClO + 1 / 2MIC CA), and the marking unit = 2 μm.

[0121] Result analysis:

[0122] In the blank control group, the red fluorescence emitted by the staining of the cell membrane by FM4-64 and the blue fluorescence of intracellular DNA stained by DAPI were obvious; in the CA (1 / 2MIC) treatment group, the two fluorescences weakened, and the red fluorescence of some cells appeared segmented, possibly indicating that the cell membrane of the damaged part had ruptured; in the NaClO (1 / 4MIC) treatment group, the red fluorescence weakened and the blue fluorescence basically disappeared, indicating that the treatment with sodium hypochlorite had a great impact on DAPI staining and the intracellular substances were damaged; in the CA (MIC) treatment group, the two fluorescences weakened, and compared with the CA (1 / 2MIC) treatment group, the number of red fluorescences decreased, indicating that the cell membrane was severely damaged and only a small part was intact; in the NaClO (MIC) treatment group, the two fluorescences basically disappeared and no complete bacterial cell structure could be seen. The fluorescence intensity of the compound disinfectant treatment group was the weakest, and the number of both fluorescences decreased significantly, indicating that the compound synergistically enhanced the effects of the two disinfection methods on membrane integrity and intracellular substances, resulting in damage to membrane components and structural lysis.

[0123] Examples 5 to 10 explored the molecular mechanism of the synergistic antibacterial effect of the compound of citric acid and low-concentration sodium hypochlorite from the perspectives of bacterial microscopic morphology analysis, cell membrane integrity, membrane potential and membrane functionality, providing theoretical support for the practical application of a new surface cleaning disinfectant for fresh fruits and vegetables.

[0124] The above are only the preferred embodiments of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A fresh fruit and vegetable surface cleaning and disinfecting agent, characterized in that: Made from a mixture of citric acid, sodium hypochlorite and water; The concentration of citric acid in the fresh fruit and vegetable surface cleaning and disinfecting agent is 3.5-4.5 mM; The effective chlorine content in the fresh fruit and vegetable surface cleaning and disinfecting agent is 70.0-80.0 ppm.

2. The fresh fruit and vegetable surface cleaning and disinfecting agent according to claim 1, characterized in that: The concentration of citric acid in the fresh fruit and vegetable surface cleaning and disinfecting agent is 3.8-4.2 mM; the effective chlorine content in the fresh fruit and vegetable surface cleaning and disinfecting agent is 73-77 ppm.

3. The fresh fruit and vegetable surface cleaning and disinfecting agent according to claim 2, characterized in that: The concentration of citric acid in the fresh fruit and vegetable surface cleaning and disinfecting agent is 4.0 mM; the effective chlorine content in the fresh fruit and vegetable surface cleaning and disinfecting agent is 75.0 ppm.

4. The method for preparing the fresh fruit and vegetable surface cleaning and disinfecting agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) uniformly dissolving solid chemical citric acid in water to prepare a citric acid solution as mother solution 1; (2) diluting sodium hypochlorite with water to obtain mother solution 2; (3) Mixing the mother liquor 1 with the mother liquor 2 to obtain a fresh fruit and vegetable surface cleaning and disinfecting agent.

5. Use of the fresh fruit and vegetable surface cleaning and disinfecting agent as claimed in any one of claims 1 to 3 in cleaning and disinfecting the surface of fresh fruits and vegetables.

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