KR101536936B1 - Sterilization Method for Fresh Vegetables of Slightly Acidic Electrolyzed Water Using Ultrasonication and Water - Google Patents

Abstract

The present invention relates to a method of sterilizing fresh vegetables of microacid electrolytic water in which fresh vegetables such as Chinese cabbage, lettuce, sesame, and spinach are treated with ultrasonic waves and wash water. (3 min) and water washing (150 rpm, 1 min) after concurrent treatment with acidic electrolytic water in order to measure the bactericidal activity by treating the acidic electrolytic water and to enhance the bactericidal effect of the acidic electrolytic water. Invented the condition.
The concurrent treatment method of the present invention is characterized in that the treatment is carried out at a temperature of 23 ± 2 ° C. in comparison with the treatment with acidic electrolytic water (SAEW), the treatment with ultrasound and acidic electrolytic water (SAEW + US) and the treatment with acidic electrolytic water (SAEW + WW) Yeast and mold in each sample, total bacterial count, Escherichia coli O157 : H7 , Listeria The monocytogenes were measured to have a reduction range of 1.76 log cfu / g to 2.80 log cfu / g.

Description

A method for sterilization of fresh vegetable of microacid electrolytic water in combination with ultrasonic wave and washing water treatment. {Sterilization Method for Fresh Vegetables of Slightly Acidic Electrolyzed Water Using Ultrasonication and Water}

The present invention relates to a method for sterilizing fresh vegetables of micro-acidic electrolytic water in combination with ultrasonic waves and water treatment. More specifically, in order to enhance the bactericidal activity of micro-acidic electrolyzed water by treating micro-acidic electrolytic water to fresh vegetables such as Chinese cabbage, lettuce, sesame and spinach, and to enhance the bactericidal effect of ultrasonic treatment, The inventors invented the optimal condition of water washing after 2 to 4 minutes of water treatment and 100 to 200 rpm of water treatment for 30 seconds to 90 seconds. The concurrent treatment method of the present invention is characterized in that the treatment is carried out at a temperature of 23 ± 2 ° C. in comparison with the treatment with acidic electrolytic water (SAEW), the treatment with ultrasound and acidic electrolytic water (SAEW + US) and the treatment with acidic electrolytic water (SAEW + WW) Yeast and mold in each sample, total bacterial count, Escherichia coli O157 : H7 , Listeria The monocytogenes were measured to have a reduction range of 1.76 log cfu / g to 2.80 log cfu / g.

SAEW (Slightly Acidic Electrolyzed Water) is one kind of electrolytic water having a pH of 5.0 ~ 6.5 containing high concentration of hypochlorous acid. It has been proved that the antimicrobial effect caused by the presence of hypochlorous acid is present. This electrolytic water is made by electrolysis of diluted hydrochloric acid (HCl) and / or NaCl solution in a membrane-free electrolyzer. The acidic electrolytic water has the advantage of being able to have antimicrobial activity by minimizing potential damage to human health and environment and by using less chlorine which shows reduced surface corrosion. Therefore, there is a growing interest in new applications for the sterilization action of micro-acid electrolytic water in the food industry as an environmentally friendly sanitizing method.

Ultrasound is a form of energy generated by the very high-frequency sound waves that are perceived in the human ear. Although the introduction of ultrasound in the food industry is more recent, the introduction of ultrasound as a method of another environmentally friendly antimicrobial treatment method has a long history. Ultrasonography was performed using Escherichia coli , Listeria monocytogenes and other pathogens have been shown to have an antimicrobial effect. Inactivation of this microorganism is mainly due to cavitation that disrupts cell structure and function. It has been reported that it is not effective to inactivate bacteria in food when used singly because it is carried out in salt solution and water. However, ultrasound combined with other sanitary procedures will probably have a more lethal effect. Therefore, when defective with a disinfectant for decontamination of fresh products, the function of the ultrasonic wave needs to be further investigated. As mentioned above, acidic electrolytic water has been extensively studied. However, the combination of micro-acid electrolytic water, ultrasound, and post-treatment of water washing has not been reported as best. In addition, studies from other studies of ultrasound combined with other bactericides due to the application of different bactericides, other samples and other laboratory conditions are difficult to compare. Important changes in physical parameters such as ultrasonic properties, size and shape of the ultrasonic bath, depth of the liquid, volume, temperature and processing time make the comparison even more difficult. According to the prior art related to the present invention, Korean Patent Laid-Open Publication No. 2009-0016956 (micro acidic electrolytic water production method and apparatus) can reduce the hydrochloric acid in the electrolytic solution by using electrolytically prepared hydrochloric acid, which is contained in the microacid electrolytic water, As the electric conductivity of the acidic electrolytic water is higher than that of water, the amount of hydrochloric acid can be reduced, and the pH of the electrolytic water is increased. However, these prior arts have different technical constructions from those of the present invention.

Despite the strengthening of food safety regulations and advances in food processing methods, vegetables have been associated with many foodborne illnesses caused by a variety of pathogenic microorganisms. The fact that lettuce, spinach, cucumber, and other vegetables are frequently reported to cause food poisoning proves that today's commercial hygiene practices are not sufficient to ensure product safety. Thus, complete inactivation or removal of pathogens from fresh products is still a challenge for the food industry, and it is essential to develop today's technologies as well as develop new sanitizing approaches. Radiation therapy, two other types of electrolytic water, lukewarm water, chlorine dioxide, ultrasound, organic acids, and hydrogen peroxide are some examples of today's sanitation technologies that have been used and studied for hygienic treatment of fresh products. The combination of these methods has also been studied in the search for higher efficiency. Although all of these methods can be used to sanitize fresh products, their efficacy is different and there is a difficulty in ensuring that no product can completely remove microorganisms.

(A) evaluating the efficacy of acidic electrolytic water in the sterilization of four most common fresh vegetables (Chinese cabbage, lettuce, true sesame leaf, spinach) in Korea under the same laboratory conditions; (B) developing a hurdle approach that is simple, environmentally friendly, and easy to perform using micro-acidic electrolyzed water and ultrasonic, wash water to maximize decontamination effectiveness based on the fact that each hurdle application may increase antimicrobial activity; (C) study the effects of product types on sanitizing effects; (D) As one of the two most important pathogens in fresh vegetables, Escherichia coli , Listeria Study the effects of sanitizing treatment on monocytogenes .

The newly developed hurdle sterilization method of the present invention is environmentally friendly, inexpensive and easily applicable. The efficacy of acidic electrolytic water for decontamination in fresh foods was confirmed in four different vegetables. This method provided a synergistic effect of microacid electrolytic water and ultrasonic waves by including a simple water wash in the last step of hurdle treatment.

Figure 1 compares the microbial deactivation effects of Chinese cabbage at different treatments.
Figure 2 compares microbial inactivation at different treatments in the supernatant.
Figure 3 compares the microbial inactivation of the sesame leaf with different treatments.
Figure 4 shows microbial reduction in different treatments of spinach.
Figure 5 shows the effect of the manufacturing form on the sterilization effect in the treatment of 5 groups.

In order to measure the bactericidal activity of fresh vegetables by treatment with acidic electrolytic water and to enhance the bactericidal effect of acidic electrolytic water, optimum conditions of ultrasonic treatment and water washing after acidic electrolytic water treatment were developed.

The following Table 1 shows the degree of microbial reduction of Chinese cabbage, lettuce, true sesame leaf, and spinach after the acidic electrolytic water treatment.

Decrease in number of bacteria when treated with natural groups and pathogens when immersed for 3 minutes at room temperature (23 ± 2 ° C) sample Yeast mold Total number of bacteria Escherichia coli L. monocytogenes Chinese cabbage 1.4 ± 0.27 1.53 ± 0.2 1.22 ± 0.11 1.19 ± 0.08 Moderate 1.41 ± 0.1 1.33 + - 0.12 1.23 + 0.06 1.20 + 0.24 Cherry leaves 1.24 0.32 1.26 + - 0.47 1.15 ± 0.31 1.31 + 0.28 spinach 1.19 ± 0.19 1.08 + - 0.24 1.12 ± 0.17 1.48 0.33

* Decrease (log cfu / g) is reported in the sense of triple multiple ± standard deviation. All reductions represent a significant difference from the control (untreated) (p? 0.05).

≪ Experimental Example 1 > Preparation of sample and inoculum

Chinese cabbage, lettuce, sesame leaf, and spinach were purchased from local supermarkets in Chuncheon, Korea. Chinese cabbage and lettuce were removed from 3 ~ 4 leaves, damaged part and inner part. Chamomile leaves and spinach were used as a sample to select non - injured parts. Then, 10 g of the appropriate sample was selected and placed in the polyethylene sterilization pack and stored at 4 캜. The E. coli O157: H7 (B0259, B0273, B0265) used in the present invention was obtained from the Food Science Department of the University of Georgia. L. monocytogenes (ATCC 19115, ATCC 19111, Scott A) was obtained from Gyeonggi Provincial Health and Environment Research Institute. These fungi are not only widely distributed in the environment but also pathogenic, and have been selected because of their occurrence and detection in the human body. Each stored pathogen was transferred to TSB medium and cultured at 35 ° C for 18 hours. 10 ml of the cultured pathogen was washed with 10 ml of 0.1% peptone water after centrifugation (3000 × g, 10 minutes, 4 ° C.) to obtain a final cell concentration of 8 log cfu / ml. Thereafter, E. coli and L. monocytogenes Each strain was mixed with each strain to obtain a suspension containing the strains. Each suspension was plated on TSA medium by decidual dilution method and the number of bacteria was measured by incubating at 37 ° C for 24 hours.

≪ Experimental Example 2 >

10 g samples were placed in a sterile tray and maintained at a distance of 30 cm in a UV apparatus (Entkeimungssssrank, 220 V, Ernst Schuttjun Laboratorotebau, 3400, Göttingen, Germany) to remove other germs contaminated with the sample. , TUV 15W) on both sides for 15 minutes each. After treatment, the number of bacteria present was reduced to a minimum detection limit of less than 10 cfu / g. To inoculate the treated specimens, the specimens were placed on sterilized aluminum foil in a biosafety hood, and 0.1 ml of each pathogen suspension was inoculated into at least 20 drops on the abaxial-side of the leaf surface. In order to attach the bacteria to the leaf surface, it was dried in a laminar flow hood at 23 ± 2 ℃ for 30 minutes. Finally, the initial bacterial inoculum concentration was set at a level of about 6 log cfu / g.

≪ Experimental Example 3 > Preparation and sanitization of microacid electrolytic water

The acidic electrolytic water used in the present invention was electrolyzed by 6% HCL diluted in a membrane-free electrolytic apparatus (BC-360, Cosmic Round 149 Korea Co. Ltd., Seongnam, Korea) set at 2.5 A and 22.8 V It was made. The electrolyzed high concentration of hypochlorous acid is 1/4 per minute to produce the final acidic electrolytic water with an oxidation-reduction potential (ORP) of 500 to 600 mV, an effective chlorine content (ACC) of 21 to 22 mg / At the flow rate of the water. The acidic electrolytic water was sampled 20 minutes after the start of the apparatus when a stable current was reached. The pH, redox potential, and effective chlorine content of the acidic electrolytic water solution were measured with a dual-scale pH meter (Accumet 155 model 15, Fisher Scientific Co., Fair Lawn, N.J.) associated with pH and redox potential. The effective chlorine content (ACC) was determined by colorimetry using a digital chlorine test kit (RC-3F, Kasahara Chemical 157 Instruments Corp., Saitama, Japan). The detection range for the measurement is 0 to 300 mg / l. After the acidic electrolytic water production, it was stored in the polypropylene container until it was used for the experiment.

Fresh samples and inoculated samples (Chinese cabbage, lettuce, sesame leaf, spinach) were divided into five treatments and untreated controls. For experimental treatment, samples were transferred to a sterile container and immersed in the treatment group solution. All treatments except sonication were performed at 200 ml of the appropriate solution for 3 minutes at a temperature of 23 ± 2 ° C. The immersion time of the washing water was 1 minute. The wire mesh was used so that all samples could be completely immersed during processing.

Samples of groups 1 and 2 were treated with distilled water and microacid electrolytic water, respectively. Group 3 consisted of samples immersed in SAEW after washing with distilled water for 1 minute at 100 rpm (MMS-3010nMulti 170 Shaker, EYELA, Tokyo, Japan) (SAEW-WW). In group 4, the samples were immersed in microacid electrolytic water and ultrasonicated together (SAEW + US). Finally, Group 5 was treated with SAEW and ultrasound simultaneously with water (SAEW + US-WW) as in Group 3. After each microacid electrolytic water treatment, the sample was immersed in 200 ml of neutralizing solution for 1 minute to stop the microacid electrolytic water activity.

Ultrasonic processing was performed using a bench-top ultrasonic cleaner (JAC-4020, KODO Technical Research Co., Ltd., Hwaseong Co., Ltd.) set at an acoustic energy density (AED) of 400 W / l and a fixed frequency of 40 kHz at a temperature of 23 + Gyeonggi-do, South Korea). The square tank equipment (721x451x297mm) was filled with 6L of acidic electrolytic water before the experiment. After each experiment, the acidic electrolytic water was removed and the tank was sterilized by UV irradiation for 10 minutes, washed with 70% ethanol and dried. The aseptic environment was maintained during all experimental procedures to ensure accurate evaluation of fresh products.

<Experimental Example 4> Microbiological analysis and sanitization analysis

All samples were sterilized and immediately transferred to a stomacher bag (Nasco Whirl-Pak, Janesville, WI) containing 90 mL of BPW (Difco, Sparks, MD) and run on a sewerd stomocher (400 Circulator, Seward, London, UK) Homogenized for 3 minutes. After homogenization, 1 ml of sample was serially diluted in sterile 9 ml of peptone water to measure surviving microorganisms, and 0.1 ml of diluted solution was plated on each of the selective media. Yeast and mold were plated on potato dextrose agar and incubated for 5 days at 25 ° C. Total bacterial counts were measured on TSA medium and cultured for 24 hours at 35 ° C. E. coli and L. monocytogenes were cultured in Oxford Agar Base (OAB; Difco) medium supplemented with Sorbitol MacConkey (SMAC; Difco) medium and antimicrobial supplement (BactoTM 195 Oxford Antimicrobic Supplement, Difco) for 24 hours at 37 ° C . The number of surviving bacteria was expressed as log cfu / g. All analyzes were carried out in triplicate equally for each experiment.

The mean of the number of bacteria (log cfu / g) measured from each treatment was analyzed using IBM SPSS Statistics Version 19 (SPSS Inc., An IBM Company). Tukey's multiple range test was used, and a significant difference test is valid at p≤0.05.

<Example 1> Changes in the number of viable cells in a fresh product by SAEW treatment

Table 1 shows the degree of microbial depletion of Chinese cabbage, lettuce, sesame leaf, and spinach after acidic electrolytic water treatment. There was a significant difference in the degree of reduction between 1.08 and 1.53 log cfu / g when compared to the untreated group (P? 0.05). The results showed that yeast, mold (YM) and total bacterial count (TBC) were 1.40 and 1.53 log cfu / g in Chinese cabbage, respectively. The microbial reduction of cabbage in YM and TBC after treatment with acidic electrolytic water of pH 6.1 and ACC 20 mg / l is 1.30 and 1.50 log cfu / g, respectively, similar to those of Koide et al. (2009). The results are also similar to those reported by Hao et al. (2011). They were immersed in fresh water for 5 minutes in micro-electrolytic water and showed a decrease in total aerobic bacteria of 1.56 and 1.64 log cfu / g in YM and TBC, respectively. Although we observed a decrease in the number of E. coli 1.33 and 1.35 log cfu / g microbes in TBC and lettuce, respectively, but very different from 2.50 and 2.80 log cfu / g reported by Issa-Zacharia et al. (2011) Respectively. The 3 min immersion time for microacid electrolytic water was selected based on the previously reported studies. Zhang et al. (2011) also showed a destructive feature that indicates the possibility of accelerating pectin degradation, which results in ACC being more effective in decontaminating effect compared to treatment time, and ultrasound treatment results in smoothness of vegetable texture Keep the minimum processing because you have it. Regardless of the type of product, the minimum number of microbes decreased by 1 log cfu / g in the same experimental conditions was obtained from the acidic electrolytic water treatment, which indicates that the acidic electrolytic water has the potential to improve product stability Able to know.

Example 2: Sterilization effect of different treatments in Chinese cabbage

Figure 1 shows the relative microbial deactivation effect of different treatments in Chinese cabbage. YM, TBC, and initial bacterial counts of inoculated pathogens were 5.50, 6.40, and 6.00 log cfu / g, respectively. In the treatment group 3, the addition of water washing after immersing the acidic electrolytic water increased the microbial reduction more than the acidic electrolytic water alone treatment. Treatment group 4 treated with microacid electrolytic water and ultrasonic treatment showed strong antimicrobial effect when compared with microacid electrolytic water alone treatment. Interestingly, however, only microacid electrolytic water treatment showed negligible decrease in YM at 0.22 log cfu / g compared with the group. A similar degree of reduction was also observed in some of the experimental groups on other samples. Results showed that the simultaneous treatment of bactericides with ultrasonic treatment showed various effects compared with the previous reports, except that the results of the decrease of the number of microorganisms in the combination of microacid electrolytic water and ultrasonic treatment were obtained. Brilhante SJos and Dantas Vanetti (2012) reported synergistic effects of simultaneous treatment of commercial bactericides and ultrasonication in the inactivation of pathogens diluted in lettuce. Huang et al. (2006) suggested that ultrasonication increases the chlorine dioxide reduction of microorganisms. Furthermore, Zhou et al. (2009) is a cleaning agent for ultrasonic treatment comprises only fungicide to the 0.7 ~ 1.10 log all experimental groups with the cfu / g (clorine, acidified sodium cholrite, peroxyacetic acid, acdic electrolyzed water) E. coli in spinach for And a significant increase in the number of patients. However, Ajlouni et al. (2006) reported that the reduction of microorganisms was due to the effect of bactericides, not ultrasound treatment. The number of higher microbial reductions after the combination of ultrasound and micro-electrolytic water is due to the activation mechanism of ultrasound, the formation of high pressure and temperature conditions, the cell wall damage of microorganisms and the high utilization of micro-electrolytic water resulting in maximum cell damage As a result. The micromechanical impact produced in regions of high pressure and temperature will also physically eliminate the number of cells and make the weaker surface adhesion of some other organisms (Ellery and Schleyer 1984). As a result, these cells would probably be more available as hypochlorous acid. Ultrasonic processing may also have the potential to extend the shelf life of the product (Cao et al. 2010). On the other hand, even though ultrasound can usually kill or eliminate some cells, the number of cells will probably decrease but will not completely separate from the surface of the product. As a part, more than 1 cfu from a single source may reattach to the surface. This may be the reason why we observe a slight reduction in microbial depletion in some of the experimental groups compared to the experimental group with microacid electrolytic water treatment and the experimental group with microacid electrolytic water and ultrasound treatment. This reduction will not be concluded from experimental errors observed in two different products, and the experiments are repeated independently. It is also restricted to YM and TBC populations, not diluted hospitals. This is due to the fact that ultrasonic waves are difficult to remove completely, and at the same time, natural bacteria attach more strongly to the surface of the product. As a result, the hurdle-treated group (Group 5) showed the greatest reduction of microbial contamination. YM, TBC, E. coli and L. monocytogenes were observed at 2.50, 2.54, 2.60 and 2.80 log cfu / g, respectively. This reduction was noticeably different from the other two groups combined with the treatment groups (p? 0.05). The addition group (group 3) or the ultrasonically treated group (group 4) to increase the decontamination effect of microacid electrolytic water was not very satisfactory, but the groups combining the two steps showed a lot of increase effect. According to the results, the wash water can not normally be used to properly remove the amount of microorganisms remaining after the acidic electrolytic water treatment. On the other hand, ultrasonic treatment usually has a synergistic effect with acidic electrolytic water, but also has negative effects in two cases. However, the combination of micro-acid electrolytic water and ultrasound treatment in a two-step hurdle that has shown good results is that the wash water may make it easier to remove the amount of surviving microbes physically loosened.

&Lt; Example 3-1 > Sterilization effect of different treatments in lettuce.

Figure 2 shows the inactivation effect of relative microorganisms in the other treatments in lettuce. The initial numbers of YM and TBC were 4.5 and 5.90 log cfu / g, respectively. Treatment group 3 showed a slight increase in all microbial population reductions compared to group 2. Group 4 showed a marked increase in the reduction of E. coli and L. monocytogenes . However, the decrease of YM in lettuce was slightly decreased compared to group 2, which was treated only with acidic electrolytic water such as Chinese cabbage. Compared with two different combined experimental groups, the more prominent group 5 showed the maximum reduction of 2.25, 2.40, 2.50 and 2.60 log cfu / g in YM, TBC, E. coli and L. monocytogenes , respectively.

&Lt; Example 3-2 > Sterilization effect of different treatments on sesame leaf

Figure 3 shows the effect of different treatments on the inactivation of the relative microorganisms in the sesame leaf. The initial numbers of YM and TBC were 4.60 and 5.70 log cfu / g, respectively. Group 5 was more effective in reducing microbes than group 3, Group 3 and Group 4 only (P0.05), and YM, TBC, E. coli , L. monocytogenes 1.76, 1.88, 2.33 and 2.40 log cfu / g, respectively.

&Lt; Example 3-3 > Sterilization effect of different treatments in spinach

Figure 4 shows the degree of reduction of microorganisms in different treatments in spinach. The initial counts of YM and TBC were 5.20 and 6.80 log cfu / g, respectively. Groups 3, 4, and 5 showed the same pattern as the result of sesame leaf. The reduction of the optimal hurdle treatment by group 5 was 1.97, 2.08, 2.41 and 2.49 log cfu / g for YM, TBC, E. coli and L. monocytogenes, respectively.

Example 3-4 Effect of Product Type on Germicidal Effect of Optimal Treatment

Figure 5 shows the effect of the product type on the germicidal effect of group 5. The decrease of microorganisms was lower in all treatments than that of cabbage and lettuce. Statements were confirmed by YM and TBC (P ≤ 0.05). This difference can be predicted by the fact that the surface of true sesame leaves and spinach is more imbalanced than Chinese cabbage and lettuce. This reflects not only the reflection or refraction of the wavelength of the ultrasonic waves but also the final combination of the low efficacy of the wash water and the hurdle treatment effect.

&Lt; Example 3-5 > Effect on the diluted bacteria in the sterilization effect of the highest treatment

The results showed that E. coli showed strong resistance against L. monocytogenes as well as optimal hurdle treatment. E. coli showing reduced levels of 0.50 ~ 2.40 log cfu / g in the treatment of mushrooms with electrolytic water was Salmonella typhimurium, Bacillus cereus , and L. monocytogenes (Ding et al., 2011).

The sterilization method of the present invention has a synergistic effect of microacid electrolytic water and ultrasonic wave by including a simple water washing in the test for confirming the efficacy of microacid electrolytic water for decontamination in fresh vegetables. Therefore, it is environmentally friendly, inexpensive, and easily applicable, so that it is industrially applicable.

Claims (5)

Characterized in that the microbicidal effect is enhanced by the ultrasonic treatment and the water washing in the microacid electrolytic water in parallel,
Wherein the microacid electrolytic water exhibits an oxidation-reduction potential (ORP) of 500-600 mV of hypochlorous acid at a high concentration, an effective chlorine content (ACC) of 21-22 mg / l, and a pH of 5.2-5.5. Disinfection method of fresh vegetable of mixed acidic electrolytic water
The method of claim 1, wherein the ultrasonic treatment is performed for 2 to 4 minutes and the water washing is performed for 100 to 200 rpm for 30 seconds to 90 seconds to enhance the sterilizing effect. How to sterilize fresh vegetables
The method of claim 1, wherein the fresh vegetable is Chinese cabbage, lettuce, sesame or spinach. Method of sterilizing fresh vegetable of micro-acid electrolytic water in combination with ultrasonic wave and washing water treatment
delete [2] The method according to claim 1, wherein the concurrent treatment method comprises the steps of treating micro-acidic electrolytic water (SAEW) at a temperature of 23 ± 2 ° C, concurrent treatment of SAW + US with ultrasound and micro- And a method of sterilizing fresh vegetable of micro-acidic electrolytic water in combination with ultrasonic wave and washing water treatment

Images