JP2003206209A - Method for sterilizing toxin-producing bacterium and method for inactivating formed toxin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a treatment method with which sterilization of a toxin- producing bacterium and inactivation of a formed toxin produced by the toxin- producing bacterium are efficiently carried out by slight energy consumption without using a specific device. <P>SOLUTION: Electrolytically generated acidic water having strong acidity formed by diaphragmatic electrolysis using a diluted aqueous solution of an inorganic salt as the water to be electrolyzed is adopted as a treating solution for sterilizing a toxin-producing bacterium or a treating solution for inactivating a formed toxin produced by the toxin-producing bacterium so that the toxin- producing bacterium is sterilized and the formed toxin is inactivated by action of high-level effective chlorine and dissolved oxygen which the electrolytically generated acidic water has. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for sterilizing a toxin-producing fungus (which is referred to as a toxin-producing fungus in the present invention), and a toxin produced by the toxin-producing fungus (which is produced in the present invention). (Referred to as toxin).

[0002]

2. Description of the Related Art Ensuring the safety of foods and feeds is one of the extremely important factors for humans and livestock. Foods and feeds that are contaminated with toxic fungi (toxin-producing fungi) and their products (produced toxins) are harmful to the health of humans and livestock. Sterilizing the producing fungi and inactivating the produced toxin produced by the toxin-producing fungi are important means for ensuring the safety of foods, feeds and the like. There are many microorganisms that cause food poisoning by contaminating foods and feeds, and representative examples thereof include fungi, staphylococci, bacilli and the like.

Regarding the safety management of foods, in order to prevent the occurrence of large-scale food poisoning, importance is attached to the control of pathogenic fungi that cause food poisoning, suppression of growth, sterilization, etc. We have established evaluation criteria called control points and are requesting the development of technology that meets these evaluation criteria. Based on this request, the food industry has proposed techniques such as food ozone treatment and electron beam radiation treatment. In addition, for the identified fungus fungus and its produced toxin aflatoxin, treatment with ammonia gas at high temperature has been proposed.

[0004]

All of these safety measures already proposed have the main purpose of sterilizing toxin-producing fungi, and inactivate the toxin produced by the toxin-producing fungus. Is insufficient, or
Means that have a sufficient effect on the inactivation of the produced toxin may impair the quality of the food and are not always considered to be practical. Moreover,
In any of these means, the amount of energy required for processing is large and a special processing device is required, so there is a cost problem. Therefore, in recent years, as a measure for preventing contamination of foods, feeds, etc., further development of effective and energy-saving technology has been demanded.

The present inventor intends to cope with such a problem by focusing on the bactericidal action of electrolyzed acidic water, and its main purpose is to sterilize the toxin-producing fungus and to produce the toxin-producing fungus. It is an object of the present invention to provide a treatment method capable of efficiently inactivating a toxin without consuming a special device and consuming a small amount of energy.

[0006]

The present invention relates to a method for sterilizing a toxin-producing fungus and a method for inactivating a toxin produced by a toxin-producing fungus, and the sterilizing method for a toxin-producing fungus according to the present invention. In this case, as the sterilization treatment liquid, a strongly acidic electrolyzed acidic water produced by a diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water is adopted. Further, in the method for inactivating the produced toxin according to the present invention, the inactivation treatment liquid is a strongly acidic electrolyzed acid produced by diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water. It is characterized by adopting water.

The sterilization treatment liquid and the inactivation treatment liquid used in the sterilization method and the inactivation method according to the present invention include:
It is preferable that the acidic water is a strongly acidic electrolyzed acidic water that is generated by diaphragm electrolysis using sodium chloride or a dilute aqueous solution containing sodium chloride as a main component as electrolyzed water, and further, the pH is 2.0 to 3. In the range of 5, effective chlorine concentration is 2
More preferably, it is a strongly acidic electrolyzed acidic water in the range of 0 ppm to 50 ppm and the dissolved oxygen concentration in the range of 10 mg / L to 20 mg / L.

The method for sterilizing toxin-producing fungi and the method for inactivating the produced toxin according to the present invention are particularly useful for sterilizing fungi, staphylococci, bacilli, etc., and produced toxins produced by these fungi. , Ie, aflatoxin, staphylococcal enterotoxin A (S. aureus enterotoxin A ... S
EA), Bacillus cereus toxin, etc. can be suitably used for the inactivation treatment.

[0009]

In the method for sterilizing toxin-producing fungi according to the present invention, as a sterilizing solution, a strong acidic electrolyzed acid produced by diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water. Water is adopted, and the toxin-producing fungi can be rapidly and effectively sterilized by the action of the high level of available chlorine and dissolved oxygen contained in the electrolyzed acidic water. In addition, the effective chlorine and dissolved oxygen having the bactericidal ability contained in the electrolyzed acidic water do not remain in the object to be treated such as food, feed, etc. during the sterilizing treatment or immediately thereafter, and the treated object is not treated. Sufficient safety is ensured after processing the object.

Further, in the method of inactivating the produced toxin according to the present invention, the strongly acidic electrolysis produced by the diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water as the inactivation treatment liquid. Acidic water is adopted, and the produced toxin produced by the toxin-producing fungus is rapidly and effectively inactivated by the action of the high level of available chlorine and dissolved oxygen contained in the electrolytically produced acidic water. Is something that can be done. Further, the available chlorine and dissolved oxygen having an inactivating ability contained in the electrolyzed acidic water may be rapidly disappeared during or after the inactivation treatment and may remain in a food, feed or other object to be treated. In addition, the safety of the processed material after processing is sufficiently ensured.

In the sterilizing method and the inactivating method according to the present invention, the sterilizing solution and the inactivating solution are:
It is preferable to employ strong acidic electrolyzed acidic water generated by diaphragm electrolysis using sodium chloride or a dilute aqueous solution containing sodium chloride as a main component as electrolyzed water. Furthermore, pH is 2.0 to 3 It is preferable to employ the strongly acidic electrolyzed acidic water having an effective chlorine concentration of 20 ppm to 50 ppm and a dissolved oxygen concentration of 10 mg / L to 20 mg / L in the range of 0.5. If the electrolyzed acidic water is adopted, toxin-producing fungi can be sterilized quickly and effectively, and the toxin produced can be rapidly and effectively inactivated. Since it is a dilute aqueous solution containing sodium or sodium chloride as a main component, the safety of the processed material after the processing can be more reliably ensured.

The method for sterilizing a toxin-producing fungus and the method for inactivating a produced toxin according to the present invention are particularly applicable to fungi that cause acute liver injury and liver cancer, and toxins produced by fungi. It is a very effective method against staphylococci that produce large-scale food poisoning, and a toxin produced by staphylococci, and particularly against Staphylococcus aureus enterotoxin A. Is.

The method for sterilizing toxin-producing fungi and the method for inactivating produced toxins according to the present invention are also effective for sterilizing bacilli and inactivating Bacillus cereus toxins produced by bacilli. Is.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for sterilizing a toxin-producing fungus and a method for inactivating a toxin produced by a toxin-producing fungus. In the sterilization treatment of the toxin-producing fungi and the inactivation treatment of the produced toxin, the strong acid generated by the diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water is used as the sterilization treatment liquid and the inactivation treatment liquid. It uses electrolyzed acidic water. A preferred embodiment of the sterilization treatment solution and the inactivation treatment solution is sodium chloride or an inorganic salt containing sodium chloride as a main component, particularly, a diaphragm membrane electrolysis using a dilute aqueous solution of sodium chloride as electrolyzed water to form an anode of an electrolytic cell The strongly acidic electrolyzed acidic water generated in the electrolysis chamber on the side, the pH is in the range of 2.0 to 3.5, the effective chlorine concentration is 20 ppm to 50 ppm, and the dissolved oxygen concentration is 10 mg / It is in the range of L to 20 mg / L.

The fungus to be sterilized by the present invention is a fungus having the ability to produce a toxin, that is, a toxin-producing fungus, and the fungal type is not limited at all, and various types of toxins are produced. Fungi are subject to sterilization. The toxin produced by the inactivation treatment of the present invention is a toxin produced by a toxin-producing fungus to be sterilized. Examples of fungi targeted for sterilization treatment of the present invention include Aspergillus flavus, Aspergillus paratyticus and Staphylococcus (staphylococcus aureus), Vibrio, viruses, Salmonella, fungi that cause food poisoning such as Bacillus. be able to.

Among these toxin-producing bacteria, fungi such as Aspergillus flavus and Aspergillus paratyticus, which are one of the representative examples, produce the most potent aflatoxin of fungal toxins (mycotoxins), and aflatoxins are Causes acute liver damage and liver cancer. In addition, depending on Staphylococcus (staphylococcus aureus), which is another typical example, eight types of Staphylococcus enterotoxin (staphylococcal enterotoxin) A,
B, C ₁ , C ₂ , C ₃ , D, E and F are generated. Among Staphylococcus enterotoxins, Staphylococcus enterotoxin A (staphylococcal enterotoxin A ...
SEA) is the most powerful, and even a small amount causes food poisoning within a few hours.

According to the present invention, these toxin-producing fungi are sterilized by using a strongly acidic electrolyzed acidic water produced by a diaphragm electrolysis using a dilute aqueous solution of an inorganic salt such as NaCl as electrolyzed water, In addition, the electrolyzed acidic water is used as an inert treatment solution to inactivate the produced toxins produced by these toxin-producing fungi. The electrolyzed acidic water is produced by electrolytically operating a known electrolyzed water producing apparatus having a diaphragm electrolyzer, and is produced in an electrolysis chamber on the anode side that constitutes the electrolyzer. In the electrolysis operation, at the same time, electrolytically generated alkaline water is generated in the electrolytic chamber on the cathode side.

In the electrolysis operation of the electrolyzed water producing apparatus, electrolyzed water having different characteristics is produced under various operating conditions. A dilute aqueous solution of 0.1 to 0.5% by weight of NaCl is used as electrolyzed water. By applying a DC voltage to each electrode arranged in each partitioned electrolysis chamber, in the electrolysis chamber on the anode side, the pH is in the range of 2.0 to 3.5 and the effective chlorine concentration is 20 ppm to Within the range of 50 ppm and 10 mg /
Strongly acidic electrolyzed acidic water in the range of L to 20 mg / L is generated. The applied voltage to each electrode is different between continuous electrolysis in which electrolyzed water continuously flows and electrolyzes, and batch electrolysis in which electrolyzed water is stagnated in each electrolysis chamber and electrolyzes, but is several V to several tens. By setting appropriately in the range of V,
Electrolytically generated acidic water suitable for the sterilization treatment liquid and the inactivation treatment liquid can be obtained.

Table 1 shows the physicochemical parameters of electrolyzed water produced using the specified electrolyzed water producing apparatus, and Table 2 shows the physics of electrolyzed acidic water during electrolysis. The effect on chemical parameters is shown.
The electrolyzed water shown in Tables 1 and 2 has a two-chamber large-capacity electrolyzer having platinum chloride as an electrode and a diaphragm electrolyzer divided by an ion exchange membrane (Superox manufactured by Aoi Electric Co., Ltd.).
seed Labo JED-020 (Kanan, Shizuoka City) was used, and was produced by a batch-type electrolysis method,
The electrolyzed water shown in Table 1 is a 17.1 mM NaCl diluted aqueous solution prepared with ultrapure water and NaCl as electrolyzed water,
It was generated by electrolysis at room temperature (25 ° C.) at an electrolysis voltage of DC 9 to 12 V for 10 minutes. Further, the electrolytically generated acidic water shown in Table 2 is 0.1% prepared by ultrapure water and NaCl.
It was produced by electrolyzing a dilute aqueous solution of NaCl at wt% as electrolyzed water at room temperature (25 ° C.) at an electrolysis voltage of DC 9 to 11 V for up to 30 minutes.

[0020]

[Table 1]

[0021]

[Table 2] A large difference is observed in the physicochemical parameters of the electrolyzed acidic water, the electrolyzed alkaline water, and the ultrapure water used to prepare the electrolyzed water. The physicochemical parameters of the electrolyzed water shown in Table 1 are those produced by performing electrolysis of a 17 mM NaCl aqueous solution for 10 minutes with a diaphragm, but the electrolyzed acidic water is strongly acidic and has an effective chlorine concentration and Both dissolved oxygen concentration is high and ORP (oxidation-reduction potential) is also high. On the other hand, electrolytically generated alkaline water is strongly alkaline, has a low concentration of dissolved oxygen, has almost no available chlorine, and
RP is extremely low. Further, it is confirmed that these electrolyzed waters have physicochemical parameters that are clearly different from those of ultrapure water, and that the electrolyzed acidic water has a high oxidation rate and the electrolyzed alkaline water has a high reduction rate. The effective chlorine concentration is measured by "effective chlorine meter" manufactured by Central Chemical Co., Ltd. (Tokyo) based on the coulometric titration method.

In the strongly acidic electrolyzed acidic water, the available chlorine acting as an oxidizing component varies depending on the length of electrolysis as shown in Table 2, and the available chlorine gradually increases with time. In the present invention, electrolysis-generated acidic water having physicochemical parameters shown in Table 1 or physicochemical parameters similar thereto is adopted as a sterilization treatment liquid and an inactivation treatment liquid.

Since the strongly acidic electrolyzed acidic water contains a large amount of available chlorine and dissolved oxygen having a bactericidal ability to sterilize the toxin-producing fungi, the electrolyzed acidic water is used for sterilizing the toxin-producing fungi. By adopting it as the sterilization treatment liquid of (1), toxin-producing fungi can be sterilized quickly and effectively. In addition, effective chlorine and dissolved oxygen contained in a large amount of the electrolyzed acidic water have an inactivating ability to inactivate the toxin produced by the toxin-producing fungi, so that the electrolyzed acidic water is inactivated. By adopting it as the activation treatment liquid, the produced toxin can be rapidly and effectively inactivated.

In the electrolyzed acidic water, the available chlorine and dissolved oxygen contained in the acidic water disappear during the sterilization treatment and the inactivation treatment and immediately after the treatment, and remain in the food, feed, etc. to be treated. However, sodium chloride or sodium chloride containing sodium chloride as the main component is used as the electrolyzed water for producing the electrolytically generated acidic water. When using a dilute aqueous solution such as, foods that have been sterilized or inactivated,
There is no residual substance that may be harmful to health in the material to be treated such as feed, and there is an advantage that the safety of the material to be treated after treatment is more sufficiently ensured.

[0025]

Example 1 In this example, Aspergillus paratyticus, a type of fungus that causes food poisoning and causes acute liver damage and liver cancer, and aflatoxin, a type of fungal toxin produced by Aspergillus paratyticus. Were employed to carry out experiments to sterilize and inactivate them. In this experiment, electrolysis-generated acidic water and electrolysis-generated alkaline water were used as the sterilization treatment liquid and the inactivation treatment liquid, and the treatment effect was evaluated by high-performance thin-layer chromatographic analysis (HPTLC) of aflatoxin, Evaluation was performed by high performance liquid chromatography analysis (HPLC), histidine auxotrophic Salmonella mutant strain TA-98, and mutagenicity test against the same TA-100 strain. In conclusion, strongly acidic electrolyzed acidic water is extremely effective for sterilizing Aspergillus paratyticus and inactivating aflatoxin, and not so effective for electrolyzed alkaline water. It is speculated that this is due to the available chlorine and dissolved oxygen contained in the highly acidic electrolyzed acidic water at a high level.

For example, 10 in 0.01 mL³Spore concentration
50 times the amount of Aspergillus paratyticus (0.5
(mL) under strongly acidic electrolyzed acidic water, its growth
It was completely suppressed. Histidine-requiring Salmonella mutation
Afra against strains TA-98 and TA-100
Toxin B₁(AFB₁) Mutagenicity of AFB ₁
Remarkably weak when treated with strongly acidic electrolyzed acidic water
It was confirmed that it will become. Also, AFB₁Strong acid
When treated with degenerated acidic water, A in HPLC analysis
FB₁No peak corresponding to₁Is strongly acidic
Confirm that it was decomposed by treatment with electrolytically generated acidic water.
I confirmed. On the other hand, electrolytically generated alkaline water contains
The growth-suppressing effect of Pergillus paratyticus is also confirmed by AFB₁
No decrease in mutagenicity was observed.

From the results of this experiment, strongly acidic electrolyzed acidic water has a strong bactericidal action against fungi and a strong inactivating action against fungal toxins (mycotoxins). Was confirmed to be extremely effective in treating food and feed. The detailed conditions and results of this experiment are shown below. (1) Sterilization experiment of Aspergillus parasites: Using Aspergillus paratyticus IFO-30179 (corresponding to ATCC 15517) purchased from Osaka Fermentation Research Institute (Doshomachi, Osaka City), the antibacterial action of electrolyzed water against fungal toxin-producing fungi An experiment was conducted to confirm. Spores of lyophilized fungal, ¹⁰⁵ (100,000) in distilled water was sterilized spores /
After suspending to a concentration of mL, it was inoculated into a liquid medium (AFAP medium) specified for Aspergillus flavin and Aspergillus paratyticus. 2 fungi
After proliferating for 10 hours, a partial sample was taken to estimate the number of cells, and the culture was diluted to 10 ⁵ (100,000) cfu /
A strain cell suspension having a concentration of mL was prepared.

For sterilization experiments, 10 ³ (1000 pieces) c
A large number of 10 μL of a strain cell suspension containing fu was prepared, and 0.01 mL of 0.02 m of electrolyzed water produced by electrolysis using a dilute aqueous solution of NaCl as electrolyzed water was prepared.
L, 0.1 mL, 0.2 mL, and 0.5 mL were mixed separately, and cultured at room temperature for 12 hours (overnight). Then, sterilized distilled water was added to each suspension to a volume of 1.01 mL, and then 0.5 mL of each diluted sample solution was added to AF.
It was inoculated on PA agar medium and cultured at 25 ° C. for 5 days. The bactericidal effect was evaluated based on the generation state of bacterial colonies on the agar plate. In the sterilization experiment, all the experiment steps were performed in an isolated safety box in order to prevent biological damage. (2) Aflatoxin inactivation experiment: An inactivation experiment of aflatoxin, which is a secondary metabolite of Aspergillus paratyticus used in the sterilization experiment, was performed. Aflatoxins B ₁ , B ₂ , G ₁ , and G ₂ analyzed by high-performance thin layer chromatography (HPTLC) were mixed in an amount of 10 ng each for a total of 40
ng and high performance liquid chromatography (HPL
_{1 of} only aflatoxin B ₁ (AFB ₁ ) analyzed in C)
Electrolyzed water was added to 0 ng and these solutions were soaked in chloroform in order to investigate mutagenicity. Chloroform was carefully evaporated from the aflatoxin while flushing with nitrogen gas, and different amounts of electrolyzed water were added for 10 minutes,
Degradation of aflatoxin was confirmed. The AFB ₁ inactivation experiment was repeated at different treatment times at temperatures of 0 ° C, 20 ° C, 30 ° C, and 45 ° C, and two parameters of the treatment temperature and the treatment time affect the decomposition of aflatoxin by electrolyzed water. Confirmed the impact.

Since aflatoxin is highly toxic and induces liver cancer, it was handled in a safety cabinet and worn with rubber gloves and a protective mask so that it would not be taken into the body. After the experiment, aflatoxin was decomposed with excess electrolyzed acidic water, placed in a closed test tube, disinfected by autoclaving at 120 ° C. for 15 minutes, and then discarded.

Although the reactive species of electrogenerated water have not been fully specified, the major reactive species in electrogenerated acidic water is hypochlorous acid (HOCl). However, the molar mixing ratio of the aflatoxin solution to the electrolyzed acidic water depends on the effective chlorine concentration and / or the hypochlorous acid concentration n.
Since it was calculated based on the mol value, there is a range from 1: 1 to 1: 200. On the other hand, since the reactive species of electrolytically generated alkaline water are not known except for NaOH and dissolved hydrogen,
Zero times the amount of electrolyzed alkaline water was mixed with AFB ₁ . In each experiment of electrolyzed water, after culturing for 10 minutes, absolute ethanol was added to a final volume of 1 ml.

As a comparative example, in order to confirm the effect of NaOCl, which is currently used for sterilizing food materials, on the disappearance of CAFB ₁ spots by HPTL, NaFB was added to AFB ₁ .
An aqueous solution was mixed at a ratio of 8: 1 (molar concentration ratio of available chlorine to AFB ₁ ) at room temperature for 10 minutes under both alkaline and acidic conditions. Since NaOCl itself is strongly alkaline (pH 11.5), 0.01N HCl aqueous solution is carefully added to NaOCl to lower the pH to 3.0 to prepare a weakly acidic treatment solution, and AFN is added to this treatment solution.
B ₁ was mixed.

In the present experiment, the degradation state of AFB ₁ was first evaluated based on the change in the chromatographic pattern by HPTLC and HPLC, and then the histidine-requiring Salmonella mutant strain TA-98 and T-98.
Evaluation was based on the mutagenicity of strain A-100. (3) Effect of radical scavenger: Assuming that reactive chemical species such as OH groups and / or Cl groups generated from HOCl are involved in the sterilization treatment, before the treatment of AFB ₁ , OH group scavenger We confirmed the effect of pretreatment of electrolyzed acidic water with mannitol and thiourea, which are well known. Mannitol is a 1:10 ratio (A
The molar ratio of FB ₁ to available chlorine) and thiourea at a ratio of 0.1: 1 (the ratio of thiourea to available chlorine) were added to the electrolyzed acidic water. In this case, available chlorine corresponds to the molar concentration of HOCl. The treatment of AFB ₁ with each electrolyzed acidic water containing mannitol and thiourea was performed at room temperature for 10 minutes, the reaction product was extracted with CHCl ₃ (chloroform), and separated by HPLC to analyze the change in AFB _1. confirmed. (4) Chromatographic analysis of treated aflatoxin: (4a) High performance thin layer chromatography (HPTLC): After treating aflatoxin with electrolyzed water of different molar concentration ratio, ethyl alcohol was added up to 1.0 mL and 0.5 mL of ethyl acetate was added and shaken well, and the aflatoxin and the reaction product were extracted into the organic solvent phase. Ethyl acetate was carefully evaporated under a stream of N ₂ gas before adding 0.01 mL of fresh ethyl acetate. (Aflatoxin / electrolyzed water) 20 μL of ethyl acetate extracted from the reaction mixture solution and 20 μL of either the AFB ₁ solution or the aflatoxin mixed solution were treated with or without treatment with electrolyzed water to form an HPTLC plate (10 ×).
10 cm silica gel 60 coated plate)
Then, it was developed by 7 cm at a ratio of CHCl ₃ : acetone = 9: 1 (v / v). Aflatoxin is UV-A (3
65 nm) is detected as pale spots. (4b) High Performance Liquid Chromatography (HPLC): HP
LC analysis was carried out with and without derivatization. at first,
The AFB ₁ and the AFB ₁ was reacted in the electrolytic acid water, C
Reversed-phase octadecyl column (Zorbax ODS ... 4.6 × 25) eluted with a mixture of H ₃ CN: H ₂ O = 35: 65 (v / v) at a flow rate of 1.0 mL / min at room temperature.
0 mm, manufactured by DuPont Co) was used for analysis by HPLC. Aflatoxin is available from Hewlet Packard HP1046A (Hewlett Packard).
HP 1046A) 365 with programmable fluorescence detector
Fluorescence was detected by excitation at nm and emission at 412 nm. Before analysis by HPLC, the AFB _1, and the AFB ₁ was reacted in the electrolytic acid water into a trifluoroacetyl derivative.

As a control, 10 μL of ethanol
10 ng of AFB ₁ was put into the flask, 20 μL of trifluoroacetyl anhydride (TFA-anhydrin) was put into a bottle, shaken well in a sealed state, trifluoroacetylated, and then cultured in a dark place at room temperature for 15 minutes. Then, 180 μL of a mixed solution of acetone: water = 1: 9 (v / v) was added,
The solution was shaken well to mix and then subjected to HPLC. Similarly, the reacted AFB ₁ solution is evaporated under vacuum,
Trifluoroacetylation was performed by the procedure described above.

Next, the reversed phase column ODS-80 ^™ (4.6
× 150 mm: TSO-Gel manufactured by TOSO Co., Ltd., and analyzed without being chemically derivatized. Using the stepwise elution method,
From 0 minute to 40 minutes, 30% acetonitrile was allowed to flow in water at a flow rate of 1.0 mL / min, and then 40 minutes to 7 minutes.
Acetonitrile was linearly increased from 30% to 100% until 0 minutes. The eluate was measured by the fluorescence method described above. In the radical scavenger experiment, the elution peak was measured at 365 nm. (5) Histidine-requiring Salmonella mutant TA-98
And mutagenicity against TA-100: A
The mutagenicity of FB ₁ was evaluated using the histidine-requiring Salmonella mutants TA-98 and TA-100, and evaluated based on the conventional Ames test method. Cultures of a histidine-requiring Salmonella mutant TA-98 containing a histidine frameshift mutant and a histidine-requiring Salmonella mutant TA-100 containing a histidine-missense mutant are the Osaka Fermentation Research Institute (Osaka It was purchased from Doshu Town, Kita Ward).

200 ng of AFB ₁ (0.64 nmo
l), 100 μL of electrolytically generated acidic water containing 0.76 nmol equivalent of HOCl, or electrolytically generated alkaline water 10
After mixing 0 μL, a 20 μL aliquot was added to 500 μL of S9 mixture (pH 7.5) or phosphate buffer, followed by histidine auxotrophic Salmonella mutant TA-98 or the same containing 10 ⁷ cfu / mL. TA-100
100 μL was added. As a control, 100 μL of dimethyl sulfoxide was added instead of the electrolyzed water.

The mixture was shaken well and incubated at 37 ° C. for 20 minutes, then 2 mL of soft agar was added, and then Voge was added.
Inoculate l-Bonner E agar and incubate at 37 ° C for 4
It was left for 8 hours. Mutagenicity was evaluated by the number of revertant colonies generated in the presence of the S9 mixture. Other experimental conditions are AF by Swenson et al.
B _{1-2,3-dichloride} were the same as mutagenic analysis (two chloride). (6) Identification of radical species by ESR: Electrolyzed acidic water was analyzed by ESR spectroscopic examination to confirm whether hydroxyl groups (OH) were involved in bactericidal action. Electrolysis generated acidic water 200 μL, spin-restraining agent DMPO (5,5-dimethyl-1-pyrroline-oxide) 890 mM solution 10 μ
Mix L to make a spin adduct, JES-RE3X / E
SR data system ESPRIT330 (JEOL)
And the analysis by the ESR spectroscopic examination method was performed under the above experimental conditions. (7) Results and discussions: (7a) Sterilization effect of Aspergillus paratyticus by electrolyzed water: In Table 3, electrolysis-generated acidic water and electrolyzed alkaline water are used as treatment liquids and Aspergillus paratyticus is sterilized. The result of is shown. In the sterilization treatment, 10 μL of cell suspension (10 ³ cfu) was added to each treatment solution at various mixing ratios, and then the cells were inoculated on an AFAP agar plate and cultured at 25 ° C. for 5 days. The control shown in Table 3 means the case where the treatment liquid is not added. Since the growth of fungi cannot be accurately measured with cfu, the growth results are shown in a semi-quantitative state.

[0037]

[Table 3] That is, +++ indicates a state in which the fungus is completely growing on the plate, ± indicates a state in which a few colonies are formed, and − indicates a state in which no fungal growth is observed. ing. When strongly acidic electrolyzed acidic water is used as the treatment liquid, it can be sterilized almost completely with 50 times the amount of the cell suspension (10 ³ cfu). On the other hand, when a strongly alkaline electrolyzed alkaline water is used as the treatment liquid, Aspergillus paraticus cannot be sterilized.

FIG. 1 is a photograph showing the surface of a plurality of Petri dishes which had been cultured at 30 ° C. for 5 days under the above-mentioned conditions. In these photographs, the lower left photograph (Control) is a control treatment. The state of fungus culture on the surface of the Petri dish afterwards, the lower right photograph (× 10vol) is 10 of electrolytically generated acidic water.
Fungal culture on the surface of Petri dish after treatment with double the amount, the upper left photo (× 20vol) is the state of fungal culture on the surface of Petri dish after treatment with 20 times the amount of electrolyzed acidic water, upper center (× 50vol) is the state of fungal culture on the surface of a Petri dish after treatment with 50 times the amount of electrolyzed acidic water, and the upper right photo (× 100vol) is after treatment with 100 times the amount of electrolyzed acidic water. 3 shows the state of fungal culture on the surface of the Petri dish of FIG.

Among these photographs, the lower left photograph (Control) showing the surface of the petri dish after the control treatment, the lower right photograph showing the surface of the petri dish after the treatment with 10 times the amount of the electrolytically generated acidic water (x) 10 vol), a photograph in the upper left (× 20 vo) showing the surface of a Petri dish after treatment with 20 times the amount of electrolyzed acidic water.
In l), white fluffy mycelium is observed on the surface of the Petri dish. In contrast, a photograph in the upper center (× 50 vo) showing the surface of the Petri dish after treatment with 50 times the amount of electrolyzed acidic water.
l), in the upper right photograph (× 100 vol) showing the surface of the Petri dish after treatment with 100 times the amount of electrolytically generated acidic water, no mycelium is present.

The above results were obtained in a model experiment, and as a result of actually sterilizing the fungi attached to the surfaces of black pepper and turmeric with electrolytically generated acidic water, Aspergillus paratyticus was completely removed. The sterilization required a larger amount of electrolyzed acidic water than the results obtained in the model experiment. In this case, it was confirmed that the same treatment with the electrolytically generated alkaline water after the treatment with the electrolytically generated acidic water can sterilize and remove Aspergillus paratyticus extremely effectively. In the sterilization treatment of Aspergillus paratyticus, it is presumed that the combined use of electrolytically generated acidic water and electrolytically generated alkaline water having different characteristics is an effective means. (7b) HPTLC evaluation of aflatoxin degradation: after treating aflatoxin with electrolyzed water of different molar concentration ratios,
HPTLC analysis of the solution was performed. The result is HPTL
The photographs of the analysis state of C are shown in (a) to (d) of FIG.

FIG. 2A shows aflatoxins B ₁ , B ₂ , G ₁ and G _{2 with and} without treatment with electrolytically generated acidic water.
Shown are individual HPTLCs (10 ng each) and untreated HPTLCs of all mixtures of these (aflatoxin B ₁ , B ₂ , G ₁ , G ₂ Mix). The numbers indicate the molar mixing ratio of the aflatoxin mixture to the electrolytically generated acidic water. That is, (1) is 1: 1, (2) is 1: 4, (3) is 1: 6, (4) is 1: 8, (5) is 1:10, (6) is 1:20, (7) is 1:40,
(8) indicates a molar concentration mixing ratio of 1:60.

FIG. 2 (b) is a photograph showing the analysis state of HPTLC of AFB ₁ after the treatment with electrolytically generated water. 10 ng of AFB ₁ was added (1: 1) to (1:10)
Mix electrolyzed acidic water containing a molar equivalent amount of available chlorine, and since electrolyzed alkaline water does not contain active chlorine, mix ₁ to 10 times the amount of electrolyzed alkaline water with the AFB ₁ solution. did. Number 1 is control AF
B ₁ (10 ng), numbers 2 to 5 are 1 time, 3 times, 5 times,
AFB ₁ treated with electrolyzed acidic water containing 10 times the molar equivalent of available chlorine, the numbers 6 to 9 are 1 times, 3 times,
AFB treated with 5 times and 10 times the amount of electrolytically generated alkaline water
Indicates ₁ . The arrow indicates the spot of AFB ₁ .

FIG. 2C shows NaOC having a pH of 11.5.
2 is a photograph showing the HPTLC analysis state of AFB ₁ after treatment with an aqueous solution of 1 l. Add 10 ng of AFB ₁ to (1:
PH 1 containing 1) to (1: 8) molar equivalent of available chlorine
Mix with 1.5 NaOCl aqueous solution. The symbol C indicates a control AFB ₁ , and the numbers 1 to 8 are A treated with an aqueous NaOCl solution containing 1 to 8 molar equivalents of available chlorine.
This shows FB ₁ . The arrow indicates the spot of AFB ₁ .

FIG. 2D is a photograph showing the analysis state of HPTLC of AFB ₁ after the treatment with the aqueous NaOCl solution of pH 3. 10 ng of AFB ₁ (1: 1) to
(1: 8) Na at pH 3 containing molar equivalent amount of available chlorine
It was mixed with an aqueous solution of OCl. Reference C is control AFB
₁ and the numbers 1 to 8 represent AFB ₁ treated with an aqueous NaOCl solution containing 1 to 8 molar equivalents of available chlorine. The arrow indicates the spot of AFB ₁ .

As is clear from these figures, the fluorescent spots of AFB ₁ on the HPTLC plate and the fluorescent spots of the aflatoxin B ₁ , B ₂ , G ₁ , G ₂ mixture were 7 times the molar concentration. It disappeared by treatment with electrolyzed acidic water containing available chlorine (see Figure 2a). The treatment with the strongly acidic electrolyzed acidic water can completely eliminate the fluorescent spots of AFB ₁ , but the treatment of the strongly alkaline electrolyzed alkaline water cannot eliminate the fluorescent spots of AFB ₁ . It is confirmed that there is no effect on AFB ₁ (see b in FIG. 2). Be treated with AFB ₁ at 8 times the NaOCl aqueous solution pH11.5 molarity, fluorescent spots AFB ₁ was disappeared (see c in FIG. 2). Na adjusted to pH 3.0 with NaOCl aqueous solution
When AFB ₁ was treated with an aqueous solution of OCl, the fluorescent spots of AFB ₁ disappeared (see d in FIG. 2).

From the above results, the chemical species involved in the destruction of aflatoxin are the chemical species generated from HClO and HOCl which are presumed to act with reactive oxygen, not ClO ⁻ ions. It is estimated that (7c) HPLC evaluation of aflatoxin degradation: Aflatoxin was treated with electrolyzed acidic water at different molar concentration ratios and then HPLC analysis of the solution was performed. FIG. 3 shows 38.4n
3A is a chromatogram of AFB _1. FIG. 3A shows a chart (a) as a control, and FIG. 3B shows a chart (b) when treated with electrolytically generated acidic water having a molar concentration ratio of 3 times. c) is a chart of AFB ₁ peaks with time when treated with electrolytically generated acidic water having a molar concentration ratio of 7 times. Referring to AFB ₁ peak chart shown in FIG. 3, AFB ₁ peak eluting after 5.1 min decreases as the molar concentration ratio of the electrolytic acid water (based on the available chlorine) increases, 7 times the molar concentration It is confirmed that the AFB ₁ peak disappears completely when it is treated with the electrolytically generated acidic water having available chlorine. After 3.1 minutes and 8.3
The peak eluting after minutes has not yet been identified, but the peak after 8.3 minutes is presumed to be a product related to AFB ₁ formed after treatment with electrolyzed acidic water.

FIG. 4 is a graph showing the relationship between the amount of decrease in AFB _{1 and the} amount of electrolyzed acidic water. The amount of electrolyzed acidic water mixed with AFB ₁ is increased based on the HOCl molar concentration. And AFB ₁
It is confirmed that the weight loss is reduced. (7d) Effects of reaction time, temperature, and available chlorine concentration: Experiments of the effect of the processing time of electrolytically generated acidic water on AFB ₁ and its reactant were conducted and evaluated by HPLC. AFB ₁
(72.4 μM) at a pH of 2.0 and a Cl ⁻ concentration of 20 m
M in electrolyzed acidic water (effective chlorine 72.4 μM)
It was treated at 5 ° C. for 5 minutes and 60 minutes. The result (chromatogram) is shown in FIG. This result is based on the analysis by HPLC, but under the condition that the reaction product is not derivatized, and is different from the chromatogram shown in FIG. Referring to the chart a in FIG. 5, the AFB
₁ elutes as a single peak after 12.5 minutes, but this peak shows a large peak after 8 minutes and a small peak after 35 minutes in the treatment after 5 minutes and 60 minutes with the electrolytically generated acidic water at 0 ° C. It is recognized that the peak is replaced.

Graphs (a) and (b) shown in FIG.
It shows the relationship between the temperature and the treatment time with respect to the decomposition of B ₁ and the appearance of the reaction product eluted after 8 minutes. Of these graphs, graph (a) shows TR = 12.5 min.
It is a peak (AFB ₁ ) that appears later, and is a graph (b).
Is a peak (reaction product) appearing after TR = 8 min. In each graph, the vertical axis represents the peak area ratio of fluorescence intensity, and the horizontal axis represents the treatment time. Referring to each graph, it can be seen that as the amount of AFB ₁ (peak TR = 12.5 min) decreases, the amount of reactant (peak TR = 8 min) increases. Further, although the disappearance of AFB ₁ is somewhat delayed by the treatment at a low temperature, it is confirmed that almost 40% of AFB ₁ disappears even after the treatment at 0 ° C. for 5 minutes. The peak that elutes after 8 minutes is presumed to be hypochlorous acid contained in the reaction product of AFB ₁ or the electrolytically generated acidic water. This reaction is AF
Since B ₁ was largely eliminated by the treatment at 0 ° C., it is presumed that it was caused by free radicals.

FIG. 7 is a chromatogram showing the effect of the available chlorine concentration on the decomposition of AFB ₁ , which is the result obtained in the following experiment. That is, AFB ₁ (0.8 nmo
1) is a chlorine concentration of 2-200 times the available chlorine (1.6
˜160 nmol) electrolyzed acidic water is mixed while keeping the Cl ⁻ ion concentration at 20 mM, and the mixture is mixed at 30 ° C. for 1 hour.
It is a result of performing 0 minute treatment and then performing HPLC analysis. The chromatograms of the control, the molar concentration ratio of 2 times (× 2), and the molar concentration ratio of 10 times (× 10) are shown in 1/8 times the original size.

[0050] Treatment 5 minutes HOCl equimolar concentration of AFB ₁ electrolytic acid water, AFB ₁ is reduced 40%. Increasing the available chlorine electrolytic acid water, in the two volumes of effective chlorine, when treated for 10 minutes AFB ₁ at 30 ° C. is, AFB ₁ has disappeared completely. When AFB ₁ was treated with electrolytically generated acidic water containing 10 times the amount of available chlorine, the peak after 4 minutes, the peak after 8 minutes, and the peak after 32 minutes decreased. On the other hand, the peak eluting after 13 minutes increases as the available chlorine increases. AF
When B ₁ was treated with electrolytically produced acidic water containing 200 times the amount of available chlorine, all peaks on the chromatograph were significantly reduced. (7e) Effect of inhibition of AFB ₁ decomposition by radical scavenger: When AFB ₁ (0.128 mM) is treated with electrolyzed acidic water, either mannitol or thiourea is added to the electrolyzed acidic water with available chlorine (0 .64
It was added while increasing the molar concentration ratio to 0 mM). AFB ₁ (0.128 mM) in each of these electrolyzed acidic waters
Was treated at 30 ° C. for 10 minutes to evaluate the effect of inhibition of AFB ₁ decomposition by radical scavenger. The obtained results are shown in the graphs of FIGS. 8 and 9.

FIG. 8 shows that mannitol has an effective chlorine of 2-1.
It is a graph which shows the effect when it adds to 0 times the molar concentration, and FIG. 9 is a graph which shows the effect when thiourea is added to the molar concentration of 0.1 to 1.0 times the available chlorine. , The vertical axis represents H of AFB ₁ calculated as% of the sum of all peak ranges of control AFB ₁ observed at 365 nm
The intensity of the PLC peak (remaining AFB ₁ ) is shown, and the horizontal axis shows the molar concentration ratio of the radical scavenger to available chlorine.

When the molar concentration ratio of mannitol to available chlorine contained in the electrolyzed acidic water increases, the ratio of destroyed AFB ₁ decreases, and the same applies when the radical scavenger is thiourea. However, radicals
If the scavenger is thiourea, the amount of thiourea to the available chlorine is 1/10 in the amount of electrolytically generated acidic water.
Only 5% of AFB ₁ is decomposed, and when thiourea is used in the same proportion as available chlorine, AFB ₁ is not decomposed at all. The results of this experiment clearly show that the effect of mannitol and thiourea, especially thiourea, as a radical scavenger is due to the free radical mediated reaction of AFB ₁ quenching. Regarding mannitol, it is speculated that mannitol could not completely protect AFB ₁ from electrolyzed acidic water because the concentration of mannitol used in this experiment was not sufficient.
Therefore, it is presumed that a concentration close to the saturated state is required for mannitol to exert its effect as an OH group scavenger. (7f) Effect of electrolyzed acid water on aflatoxin mutagenicity: Aflatoxin mutagenicity experiments
Maron and Ames 19
83) according to His-histidine auxotrophic Salmonella mutants TA-98 and TA-100. Three
nmol AFB ₁ was treated with different amounts of electrogenerated acid water containing 40 ppm available chlorine for 10 minutes at room temperature. Then, the S-9 mixture was added to these solutions and precultured for 20 minutes, and then a histidine-requiring Salmonella mutant was added and cultured at 37 ° C. for 48 hours. The results are shown in Table 4. The data shown in Table 4 is [(sample cfu-blank cfu) / (blank cf)
u)] × 100, and is expressed as relative sudden induction percentage. NG means negative growth (cfu below control). The amount of electrolyzed acidic water indicates the nmol value of HOCl.

[0053]

[Table 4] As can be seen with reference to Table 4, 3 nmol of AFB
₁ is a histidine-requiring Salmonella mutant TA-98
Although it shows high mutagenicity to both of the same and TA-100, when AFB ₁ was treated with electrolyzed acidic water for 10 minutes, the mutagenicity of AFB ₁ was HOCl concentration of electrolyzed acidic water. It is observed that it decreases with an increase in The mutagenicity of AFB ₁ is significantly reduced when treated with an amount of available chlorine in electrolyzed acidic water 58 times equivalent to 173 nmol HOCl. The mutagenicity of AFB ₁ can be eliminated by further increasing the amount of available chlorine in the electrolyzed acidic water. (7g) ESR analysis of chemical species in electrolyzed acidic water: In order to identify radical species, spin restrainer DMPO (5,5'-
Dimethyl-1-pyrroline-N-oxide),
ESR analysis of electrolytically generated acidic water was performed. First, a weak spectrum was obtained indicating the formation of OH groups, and then 1 mM FeSO ₄ and 10 mL were added to enhance this spectrum.
DMPO was added to the electrolytically generated acidic water. When Fe ²⁺ ions are present, as shown in FIG.
ES showing typical reaction of MOP (Fenton reaction)
From the R spectrum, it can be seen that the electrolytically generated acidic water also contains H ₂ O ₂ . The above results indicate that OH groups and H ₂ O ₂ are present in the electrolytically generated acidic water. In addition, at acidic pH, HOC is generated in electrolytically generated acidic water.
The presence of l has also been confirmed by spectrophotometry. (8) Summary of consideration: Strongly acidic electrolyzed acidic water sterilizes Aspergillus paratyticus and aflatoxin A.
It was confirmed to eliminate the mutagenicity of FB ₁ . Further, it was confirmed by HPLC analysis that the aflatoxin AFB ₁ was decomposed by treating with electrolytically generated acidic water. However, the chemical formula of the reaction product generated by the treatment of electrolytically generated acidic water is specified by the LC-MS method and the NMR method.

[0054]

Example 2 In this example, staphylococcal enterotoxin A (SE produced by a typical staphylococcus causing food poisoning
Experiments were attempted to inactivate A). It is common practice to kill pathogenic bacteria that cause food poisoning.However, even if the pathogenic bacteria are killed, the toxin produced by the pathogenic bacteria may not completely lose its activity. I tried.

The toxin targeted for the inactivation treatment in this experiment is the strongest staphylococcal enterotoxin A (SEA) among the staphylococcal enterotoxins that cause enterotoxin type diseases, and SEA is
It was purchased from Sigma (St. Louis, Missouri, USA). SEA is only 0.4
Even at a concentration of ng / mL to 0.8 ng / mL, 3 hours to 5
It causes food poisoning in humans within a period of time, is extremely stable thermally, and its toxicity remains even after treatment at 100 ° C for 30 minutes, and has a strong resistance to strong acid and strong alkali. Have great toxicity.

The electrolytically generated water used as the inactivation treatment liquid in this experiment was a two-chamber large-capacity electrolysis apparatus (Aoi Electric Co., Ltd.) having a diaphragm electrolysis chamber partitioned by an ion exchange membrane using platinum chloride as an electrode. Company-made Superoxseed Labo
JED-020 (Kanan, Shizuoka City) was used to produce the product by a batch-type electrolysis method. As electrolyzed water,
It was produced by adopting a dilute aqueous solution of 0.1% concentration prepared by dissolving NaCl in deionized water and electrolyzing at room temperature (25 ° C.) for 12 minutes at an electrolytic voltage of DC 9 to 11V. According to the electrolysis, in the electrolysis chamber on the anode side of the electrolytic cell, the effective chlorine concentration is about 36.3 ppm, the oxidation-reduction potential (ORP) is +1180 mV, and the strongly acidic electrolyzed acidic water having a pH of 2.5 to 2.8 is generated. In the electrolysis chamber on the cathode side of the electrolyzer, the ORP was -880 mV and the pH was 11.
A strong alkaline electrolyzed alkaline water of 6 to 12.0 is obtained.

In this experiment, different amounts of strongly acidic electrolyzed acidic water (Example) and strongly alkaline electrolyzed alkaline water (in an example) were added to a solution containing a fixed amount of staphylococcal enterotoxin A (hereinafter referred to as SEA). Comparative Example) was added and stirred for 30 minutes to inactivate SEA, and the effect of the inactivation treatment on SEA was evaluated by RPLA method, immunoassay method, natural polyacrylamide gel electrophoresis (PAGE), It was evaluated by the amino acid analysis method.

In conclusion from the results obtained, when SEA was treated with a large amount (10 times or more) of strongly acidic electrolyzed acidic water, the immunoreactivity between SEA and the specific anti-SEA antibody disappeared, and SEA It was found that the protein splits and the content of amino acids, especially methionine (Met), tyrosine (Tyr), isoleucine (Ile), asparagine (Asn) and aspartic acid (Asp), decreases. These phenomena mean that SEA was inactivated. It has been found that SEA excreted in culture broth by Staphylococcus can also be inactivated by treatment with a large amount of strongly acidic electrolyzed acidic water. The detailed conditions and results of this experiment are shown below. (1) Inactivation experiment of staphylococcal enterotoxin A (SEA):
2.8 μg / m 2 in a model experiment with a medium without organic matter
L of a phosphate buffered saline solution (PBS: pH 7.4) was used as a base solution, and SEA purchased from Sigma (St. Louis, Missouri, USA) was suspended therein. A sample solution containing 70 ng of SEA in 25 μL of PBS was added to 0 μL, 75 μL, 125 mL, 250
It was mixed with mL of electrolyzed acidic water or electrolyzed alkaline water and incubated at room temperature for 30 minutes. Then 1% mannitol was added to 400 μL, 325 μL, 275 μL,
The reaction was stopped by the addition of 150 μL and a 25 μL aliquot was analyzed according to the RPLA method according to the manufacturer's instructions. Semi-quantitative analysis of SEA content was performed using standard SE
The solution A was compared with the result when the solution A was cultured at 37 ° C. for 24 hours.

A sample solution of a suspension containing 70 ng of SEA was added to 3 times the molar ratio of HOCl to SEA of about 230, 380 and 760, respectively.
Mix with 10 times the amount of electrolyzed acidic water and mix at room temperature for 3
It was left for 0 minutes. Similarly, a sample solution of a suspension containing 100 ng of SEA was mixed with electrolysis-generated alkaline water in the same volume ratio and left at room temperature for 30 minutes. The analysis of the sample solution showed that RPLA method and E for SEA
LISA method, natural PAGE method for proteins, and HPLC method for amino acid mixture. (2) Inactivation experiment of liquid culture broth containing SEA: SE released extracellularly into organic medium using broth containing SEA produced by Staphylococcus aureus
The inactivation experiment of A by the electrolytically generated acidic water was conducted. Brain-heart infusion SEA suspended in liquid medium (100 ng /
(mL) was regarded as the extracellularly released SEA in the culture broth (25 μL), which was calculated to be 5 times, 10 times, 25 times, 5 times.
0 times, 75 times, 100 times, 125 times the amount of electrolytically generated acidic water (effective chlorine 36.3 ppm, pH 2.31, ORP1
158 mV) and left for 5 minutes, then RPL
It was analyzed by Method A. 70ng SE as control
The supernatant containing A (25 μL) was mixed with 10 volumes of PBS rather than electrolyzed acidic water. The coagulation after reaction of SEA from the cell supernatant with anti-SEA bound to latex beads was assessed after 24 hours of incubation at 37 ° C. and compared with that of the control. (3) SEA condensation enzyme-linked immunosorbent assay (ELI
SA): Since the RPLA method is only a semi-quantitative measurement method, S
The denaturation of EA was also confirmed by the ELISA method. Electrolyzed acidic water 150 μL and SEA 150 μL were added to a multi-container microtiter plate (Falcon Microtest III Flexible Assay Plate, Becton Dickin
SEA was coagulated by culturing at 37 ° C. for 2 hours in addition to Son and Co (New Jersey, USA). Then, the electrolyzed acidic water and uncoagulated SEA are discarded, and the coagulated SEA is mixed with 2% bovine serum albumin (BSA) / PB.
Blocking (adhesion) was performed with S at room temperature for 30 minutes. B
Discard the SA / PBS solution and add 0.05% to the PBS in the container.
150 μL of Tween-20 was added and washed 3 times.

Next, 150 μL of anti-SEA antibody (Sig
ma Chemical, St. Louis, Mo., USA) at a dilution of 20,000 times the primary antibody and added.
The plate was incubated at 37 ° C for 1 hour. Then, the solution was discarded, and Tween-20 was added to PBS to wash the container. Then, in each container, a 1,000-fold diluted peroxidase affinity-labeled purified antibody against rabbit IgG (K
irkegaard & Perry Laborator
made by ies, Gettersburg, Maryland, USA) 150
μL was added, and the mixture was incubated at 37 ° C. for 1 hour. Further, this solution was discarded, and Tween-20 was added to PBS to wash the container. Subsequently, 0.03% of ethyl benzthiazoline 2,2'-azinodi-3-sulphonate and 0.03% were added to each container.
150 μL of a mixture of 1% hydrogen peroxide was added, and the mixture was incubated at 37 ° C. for 1 hour. The absorption of each container is
Leader (MT made by Corona Electric Co.
P-120, Tokyo) at 415 nm. (4) Natural PA of SEA and its reaction product after inactivation
GE analysis: Degradation of SEA treated with electrolyzed acidic water was confirmed. SEA before and after treatment with electrolyzed acidic water
25m of solution with 7.5% polyacrylamide gel
M Tris-190 mM glycine electrophoresis buffer (pH
It was confirmed by polyacrylamide gel electrophoresis (PAGE) using 8.3). The protein band is Com
Visualization with massie Brilliant Blue staining. (5) SEA amino acid composition analysis with or without treatment of electrolyzed acidic water: The amino acid composition of SEA after the treatment of electrolyzed acidic water was compared with that of untreated SEA. SEA
Was hydrolyzed with 6N HCl containing 1% phenol at 110 ° C. for 24 hours. Free amino acids released from SEA by hydrolysis were derivatized with phenyl isothiocyanate (PTC) before reverse phase PIC.
O-TAG column (3.9 x 150 mm, Waters
Ultraviolet absorption was observed at 254 nm using Associates) and analyzed by HPLC method. PTC
Amino acid derivatives were eluted from solution A and solution B with a programmed gradient. Solution A is 0.2% (v /
v) acetonitrile containing triethylamine in a ratio of 50: 3 (v / v), pH with 0.14M sodium acetate
Prepared at 6.8, solution B is 60% acetonitrile. (6) Results and discussion: (6a) Inactivation effect of Staphylococcal toxin A (SEA) by electrolyzed water (by RPLA method): 70 ng SEA
The graph of FIG. 11 shows the results of analysis by the RPLA method in the case where the above was treated with different amounts of electrolytically generated acidic water and electrolytically generated alkaline water for 30 minutes (model test without organic matter). In the graph, the vertical axis represents the amount of remaining SEA (ng /
mL), and the horizontal axis indicates the mixing ratio of electrolyzed water to SEA.

It was confirmed that the amount of electrolyzed acidic water used for inactivating SEA has a great influence on the experimental results. In a model test without organic substances, when SEA was treated with an equal amount of electrolytically generated acidic water, almost no effect of inactivating SEA was observed, but when treated with 3 times the amount of electrolytically generated acidic water. The amount of SEA was reduced by about 25%. The effect of inactivating SEA increases as the amount of electrolyzed acidic water increases, but when SEA is treated with 10 times the amount of electrolyzed acidic water, the coagulation of SEA with the anti-SEA antibody latex is observed. I couldn't do it. On the other hand, the treatment of SEA with the electrolytically generated alkaline water does not show the effect of inactivating SEA.

The graph of FIG. 12 shows the R when SEA was treated with different amounts of electrolyzed acidic water under nutrient broth conditions.
The analysis result by PLA method is shown. In the graph,
The vertical axis represents the amount of remaining SEA (ng / mL), and the horizontal axis represents the mixing ratio of electrolyzed acidic water to SEA.

For the treatment of SEA suspended in culture medium, 160 ng / mL SEA was added to the supernatant of cells grown by the brain-heart infusion liquid medium, where condensation was clearly visible, but SEA Was treated with 100 times the amount of electrolyzed acidic water, no condensation was observed. This result suggests that a large amount of electrolyzed acidic water can be used to denature the immune reaction site of the SEA protein even in the presence of organic matter. (6b) Inactivation effect of Staphylococcal toxin A (SEA) by electrolyzed water (by ELISA method): Since the RPLA method is a semi-quantitative measurement method and the sensitivity is not always sufficient, The result of the treatment of SEA with electrolyzed water was confirmed by the ELISA method (enzyme-linked immunosorbent assay method). The graph of FIG. 13 shows the results of treating SEA with electrolyzed acidic water. In the graph, the vertical axis is 4
Absorbance at 15 nm is shown, the horizontal axis is the amount of SEA (ng)
Is shown. In addition, in each graph, the plot 1 marked with □ is the control, and the plot 2 marked with ◇ is (1:
When treated with the electrolysis-generated acidic water of 5 v / v ratio, the graph 3 indicated by the plot ◯ is the case of treatment with the electrolysis-generated acidic water of (1:10 v / v) ratio.

In a control experiment (graph 1) in which SEA was not treated with electrolyzed acidic water, the SEA detection limit, determined by absorption at 415 nm, was found to be 3 ng. However, when SEA was treated with 5 times the amount of electrolytically generated acidic water (graph 2) and when SEA was treated with 10 times the amount of electrolytically generated acidic water (graph 3), S
SEA could not be detected by the ELISA method even if the original amount of EA was increased to 100 nm.

The above results indicate that treatment of SEA with electrolyzed acidic water denatures the antigenicity-determining site of SEA, or remodels the three-dimensional protein structure of SEA, resulting in an anti-SEA protein. It indicates that the immunoreactivity to the SEA antibody is lost. (6c) Analysis by natural PAGE method: FIG. 14 shows the patterns of electromigration of natural polyacrylamide gel when SEA was treated with electrolyzed water and without treatment. 14
Is a photograph showing a pattern of natural PAGE, in which the arrow 1 indicates the position of SEA, the arrow 2 indicates bovine serum albumin (BSA), and the lane 1 indicates standard SEA, Lanes 2-4 are 3
Lanes 5-7 are treated with electrolytically generated acidic water in an amount of 5 times, 10 times, and lanes 5 to 7 are treated with an electrolytically generated alkaline water in an amount of 3, 5, 10 times.

The natural SEA protein is approximately 30 kDa.
However, SEA treated with electrolyzed acidic water
Only a hazy band under and around the 30 kDa region is visible, indicating that electrolyzed acidic water caused degradation and division of protein molecules (lanes 2-4). The electric permanence pattern of SEA treated with electrolyzed alkaline water is
No change was seen (lanes 5-7). The above results indicate that the electrolyzed acidic water finely decomposes the SEA protein. (6d) Analysis by amino acid analysis method: Table 5 shows a comparison of amino acid composition between the case where SEA was treated with electrolytically generated acidic water and the case where it was not treated. In the table, (Asx) indicates aspartic acid + asparagine, and (Glx) indicates glutamic acid + glutamine. Further, the numerical values in the reference value column in the table are the numerical values showing the residual amino acid in the untreated complete SEA in mol value relative to SEA1Mol (Huang et al. 198
7), and the values in the column without treatment are values obtained from amino acid analysis by HPLC.

[0067]

[Table 5] When SEA is treated with 10 times the amount of electrolyzed acidic water, SE
It is recognized that the amino acid contained in A is clearly reduced. However, it should be noted that in the amino acid analysis in this experiment, data on tryptophan and cysteine are not accurate due to hydrolysis. The amino acid composition of SEA without treatment is in good agreement with the reference value, but the content of most amino acids is reduced in SEA when treated with electrolyzed acidic water. In particular, the contents of methionine, isoleucine, tyrosine, asparagine and aspartic acid are markedly reduced. Methionine and isoleucine are extremely susceptible to oxidation, especially methionine is sensitive to HOCl. However, the content of histidine, which is known to be easily oxidized, does not decrease as much as methionine. (7) Summary of Discussion: SEA can be inactivated by treating with electrolyzed acidic water. In particular, SEA secreted into the extracellular medium from Staphylococcus aureus cells grown in brain-heart infusion liquid medium can be inactivated by treatment with electrolyzed acidic water. Electrolytically generated acidic water modifies SEA by an oxidative chemical reaction with OH groups and reactive chlorine. The effect of the oxidation chemical reaction is generally suppressed in the presence of an organic substance such as an amino acid containing sulfur, and this action of electrolytically generated acidic water is remarkable.

The treatment with electrolyzed acidic water that eliminates or weakens the toxicity of pathogenic bacteria can be applied to hygiene control of foods and feeds. Even if it is difficult to completely kill the pathogenic bacterium, it is possible to reduce the pathogenicity by reducing the produced toxin produced by the pathogenic bacterium and prevent the occurrence of food poisoning.

The hypochlorite ion (effective chlorine 500
It has been reported that sterilization treatment with (ppm) is effective for inactivating prion, which is the cause of Creutzfeld-Jakob syndrome, and it is assumed that electrolyzed acidic water is effective for prevention of bovine spongiform encephalitis. To be done. In addition, it has been reported that sodium hypochlorite reduces hepatitis A virus by 99.9%, and it is speculated that electrolytically generated acidic water is effective for sterilization against hepatitis A virus.

[Brief description of drawings]

FIG. 1 is a copy of a photograph of the surface of a Petri dish showing the culture state of Aspergillus paraticticus.

FIG. 2 is a photocopy of a HPTLC analysis pattern of aflatoxin.

FIG. 3 is a chart of an aflatoxin chromatogram showing the relationship of the concentration ratio of electrolyzed acidic water.

FIG. 4 is a graph showing the relationship between the amount of electrolyzed acidic water mixed and the amount of aflatoxin reduction.

FIG. 5 is a chart of an aflatoxin chromatogram showing the relationship between the concentration of electrolyzed acidic water and the treatment time for the destruction of aflatoxin.

FIG. 6 is a graph showing the relationship between the treatment temperature and the temperature at which a reaction product appears after the decomposition of aflatoxin and the elution after 8 minutes.

FIG. 7 is a chart of an aflatoxin chromatogram showing the relationship between the concentration of electrolyzed acidic water and the treatment time for the decomposition of aflatoxin.

FIG. 8 is a graph showing the effect of radical scavenger (mannitol) on the decomposition of aflatoxin by electrolyzed acidic water.

FIG. 9 is a graph showing the effect of radical scavengers (thiourea) on the decomposition of aflatoxin by electrolyzed acidic water.

FIG. 10 DMPO spin-restrained ESR of electrolyzed acidic water
It is a chart figure which shows a signal.

FIG. 11 is a graph (RPLA method) showing the relationship (no organic matter) between the mixing amount of electrolyzed water and staphylococcal enterotoxin A.

FIG. 12 is a graph (RPLA method) showing the relationship between the mixing amount of electrolyzed water and staphylococcal enterotoxin A (under nutrient broth conditions).
Is.

FIG. 13 is a graph (ELISA method) showing the relationship between the concentration of staphylococcal enterotoxin A and the amount of decrease.

FIG. 14 is a photocopy of a natural PAGE analysis pattern of Staphylococcus enterotoxin A.

Claims (6)

[Claims] 1. A method for sterilizing toxin-producing fungi, wherein strong acid electrolyzed acidic water produced by diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water is adopted as the sterilization treatment liquid. A method for sterilizing toxin-producing fungi, which comprises: 2. The sterilization method for toxin-producing fungi according to claim 1, wherein the sterilization treatment liquid is produced by diaphragm electrolysis using sodium chloride or a dilute aqueous solution containing sodium chloride as a main component as electrolyzed water. Is a strongly acidic electrolyzed acidic water having a pH of 2.0 to 3.5, an effective chlorine concentration of 20 ppm to 50 ppm, and a dissolved oxygen concentration of 10 mg / L to 20 mg / L. A method for sterilizing toxin-producing fungi characterized by being in the range. 3. The method of sterilizing a toxin-producing fungus according to claim 1, wherein the toxin-producing fungus to be sterilized is a fungus, a staphylococcus or a bacillus. Sterilization method. 4. A method for inactivating a produced toxin produced by a toxin-producing fungus, wherein a strong acid produced by a diaphragm electrolysis using a dilute aqueous solution of an inorganic salt as electrolyzed water as an inactivation treatment liquid. A method for inactivating a toxin produced by a toxin-producing fungus, characterized in that the electrolyzed acidic water is used. 5. The method for inactivating a produced toxin according to claim 4, wherein the inactivation treatment liquid is a diaphragm electrolysis using sodium chloride or a dilute aqueous solution containing sodium chloride as a main component as electrolyzed water. The strongly acidic electrolyzed acidic water produced by the method has a pH of 2.0 to 3.5, an effective chlorine concentration of 20 ppm to 50 ppm, and a dissolved oxygen concentration of 10 mg / L to 20 mg. A method for inactivating a produced toxin produced by a toxin-producing fungus characterized by being in the range of / L. 6. The method of inactivating a produced toxin according to claim 4, wherein the produced toxin to be inactivated is a produced toxin produced by fungi, staphylococci or bacilli. A method for inactivating a produced toxin produced by a characteristic toxin-producing fungus.