Peracetic acid type sterilizing composition solution and method for sterilizing containers

Abstract

A peracetic acid type sterilizing composition solution containing peracetic acid, acetic acid, hydrogen peroxide and catalase enzyme, wherein said sterilizing solution has a pH in a range of 2.6 to 5.0, said catalase enzyme is contained in an amount of 0.1 to 10 μg/ml, the concentration of said peracetic acid is 500 to 10,000 ppm, and the concentration of said hydrogen peroxide is less than 500 ppm. The concentration of the hydrogen peroxide can be efficiently decreased due to the catalase enzyme that is added in small amounts thereby suppressing the bubbling caused by the addition of the catalase enzyme.

Claims

1. A sterilizing composition solution containing peracetic acid, acetic acid, hydrogen peroxide and catalase enzyme, wherein said sterilizing solution has a pH in a range of 3.0 to 5.0, said catalase enzyme is contained in an amount of 0.1 to 10 μg/ml, a concentration of said peracetic acid is 500 to 10,000 ppm, and a concentration of said hydrogen peroxide is less than 500 ppm.

2. The sterilizing composition solution according to claim 1, wherein a concentration of chloride ions is not more than 10 μg/ml.

3. The sterilizing composition solution according to claim 2, wherein said chloride ions are stemming from sodium chloride.

4. The sterilizing composition solution according to claim 1, wherein said catalase enzyme is stemming from fungi.

5. The sterilizing composition solution according to claim 1, wherein during the preparation said sterilizing composition. solution, said catalase enzyme is capable of decomposing not less than 35% of said hydrogen peroxide in less than 10 minutes.

6. A method for preparing the sterilizing composition solution of claim 1 by adjusting the pH of the sterilizing solution that contains peracetic acid, acetic acid and hydrogen peroxide and, thereafter, adding the whole amount of the catalase enzyme thereto in a divided manner.

7. A method for sterilizing containers by adjusting the temperature of the sterilizing composition solution of claim 1 to be 40 to 75° C. and applying said sterilizing composition solution to surfaces of the containers.

Description

EXAMPLES

(1) (Preparation of the Peracetic Acid Type Sterilizing Composition Solution)

(2) The peracetic acid type sterilizing composition solution was prepared by using, as the peracetic acid type sterilizer, the Oxyper 100 (manufactured by Nihon Peroxide Co.: 10.2% by weight of peracetic acid, 20.6% by weight of acetic acid, 17.2% by weight of hydrogen peroxide), diluting it with water so that the concentrations of the peracetic acid were as shown in Tables 1 to 4, and adding a 5N aqueous solution of sodium hydroxide so that the pH was 2.0 to 5.5. Thereafter, the Leonet F Plus (manufactured by Nagase Chem-Tech Co., stemming from the Aspergillus niger) was added as the catalase enzyme in such an amount that the final concentration of the catalase enzyme was 0.1 to 10 μg/ml relative to the whole amount of the peracetic acid type sterilizing composition solution.

(3) (Method for Measuring the Components of the Peracetic Acid Type Sterilizing Composition Solution)

(4) 1. Amount of the Peracetic Acid.

(5) After the reaction with the catalase enzyme, the amount of the peracetic acid was measured by the potassium permanganate—iodine method. That is, the sample was titrated with the potassium permanganate under an acidic condition of sulfuric acid to measure the concentration of the hydrogen peroxide and was, thereafter, titrated with the sodium thiosulfate while adding the potassium iodide and a starch indicator thereto to measure the concentration of the peracetic acid.

(6) As for the catalase enzyme reaction end time, measurement was taken for every 5 minutes after 5 minutes have passed from when the catalase enzyme was added to the peracetic acid type sterilizing composition solution, and the moment of when there was seen no reduction in the concentration of the hydrogen peroxide was regarded to be the end of the reaction, and the concentration at this moment was measured. 2. Amount of the Hydrogen Peroxide.

(7) The amount of the hydrogen peroxide includes two concentrations, the one being a value of after the peracetic acid type sterilizer was diluted with water but before the catalase enzyme was added (value calculated from the concentration of the starting peracetic acid type sterilizer by taking the dilution into account) and the other being a value measured after the above-mentioned catalase enzyme reaction has been finished. The method of measurement complied with the method for measuring the amount of the peracetic acid described in 1. above. 3. Amount of the Catalase Enzyme.

(8) The amounts of the catalase enzyme were measured according to the Experimental Protocol using the Nano Orange (registered trademark) Protein Quantitation Kit (manufactured by Invitrogen Co.) to be as shown in Tables 1 to 4.

(9) (Method of Evaluation)

(10) 1. Power for Decomposing the Hydrogen Peroxide.

(11) The peracetic acid type sterilizing composition solution was measured for its concentration of the hydrogen peroxide (I.sub.0) just after the preparation thereof and for its concentration of the hydrogen peroxide (I) after 10 minutes have passed from the start of preparation, and from which a reduction ratio [(I.sub.0−I)/I.sub.0]×100 (%/10 min.) was calculated. The power for decomposing the hydrogen peroxide per 10 minutes was evaluated to be ◯ if it was not less than 35% and X if it was less than 35%. 2. Peracetc Acid Decomposition Percentage.

(12) The peracetic acid type sterilizing composition solution was measured for its concentration of the peracetic acid (I.sub.0) just after the preparation thereof and for its concentration of the peracetic acid (I) after one hour has passed at 25° C. from the start of preparation, and from which a reduction ratio [(I.sub.0−I)/I.sub.0]×100 (%/hour) was calculated. The peracetic acid decomposition percentage per hour was evaluated to be ◯ if it was not more than 1%, evaluated to be Δ if was 1 to 3%, and was evaluated to be X if it exceeded 3%. 3. Surface State of the Sterilizing Composition Solution in the Tank.

(13) The surface of the peracetic acid type sterilizing composition solution was confirmed with the naked eye 5 minutes after the start of preparation of the peracetic acid type sterilizing composition solution. The surface was evaluated to be ◯ if there was no bubbling or formation of aggregates and X if they were formed. 4. Effect for Sterilizing the Containers.

(14) A 500 ml polyethylene terephthalate bottle (hereinafter referred to as bottle) was used as the container, and a bacteria solution was prepared by using Bacillus cereus ATCC 9139 as sample bacteria. The bacteria solution was uniformly blown from a sprayer onto the inner surface of the bottle such that the density thereof was 10.sup.6 cfu/bottle. Thereafter, the bottle was dried to obtain a bottle for evaluation. Next, the peracetic acid type sterilizing composition solution just after the catalase enzyme reaction has been finished was adjusted for its temperature to be 65° C., and was sprayed into the bottle for evaluation so as to be brought in contact therewith for 8 seconds. Next, the inner surface of the bottle was washed with the aseptic water. By using the standard agar culture medium, the aseptic water used for washing was measured for the number of living bacteria based on the membrane filter method. From the initial number of bacteria and the number of living bacteria, the sterilization effect (D) was found in compliance with the following formula. The testing was conducted a number of times n=3, and the sterilizing effect was evaluated with the average number of living bacteria as the number of living bacteria.
D=LOG(N.sub.0/N) wherein N.sub.0 is the initial number of bacteria and N is the number of living bacteria.

(15) If the D-value was not less than 6D, the sterilizing effect was high and was evaluated to be ◯, if the D-value was not less than 5D but was less than 6D, the sterilizing effect was exhibited and was evaluated to be Δ and if the D-value was less than 5D, the sterilizing effect was low and was evaluated to be X.

Examples 1 to 3, Comparative Examples 1 and 2

(16) The power for decomposing the hydrogen peroxide and the peracetic acid decomposition percentage were examined depending on the pH values. The peracetic acid type sterilizer was diluted such that the compositions were nearly the same except the pH value, i.e., such that the amount of the peracetic acid was about 3,000 to about 3,300 ppm and the initial amount of the hydrogen peroxide was about 4,500 to about 5,000 ppm. Thereafter, a 5N aqueous solution of sodium hydroxide was added thereto such that the pH values were as shown in Table 1, and the catalase enzyme was added thereto in an amount of 0.4 μg/ml to evaluate. The results were as shown in Table 1.

(17) TABLE-US-00001 TABLE 1 Sterilizing composition solution Results of evaluation Amount of Peracetic hydrogen peroxide Hydrogen acid Amount of Before After Amount Method peroxide decomposition peracetic adding adding of of decomposing percentage at acid catalase catalase catalase adding power 25° C. (ppm) (ppm) (ppm) pH (μg/ml) catalase (%/10 min.) (%/hour) Ex. 1 3060 4682 136 ◯ 2.6 0.4 one time 40 ◯ 0 ◯ Ex. 2 3154 4889 0 ◯ 4.0 0.4 one time 88 ◯ 0 ◯ Ex. 3 3358 5373 0 ◯ 5.0 0.4 one time 99 ◯ 1 Δ Comp. 3094 4827 561 X 2.1 0.4 one time 28 X 0 ◯ Ex. 1 Comp. 3300 5214 0 ◯ 5.5 0.4 one time 99 ◯ 10 X Ex. 2

Examples 4 to 7, Comparative Examples 3 to 6

(18) To compare the surface state of the sterilizing composition solution in the preparation tank and the power for decomposing the hydrogen peroxide depending on the amount of the catalase enzyme and on the blending method, the peracetic acid type sterilizer was diluted such the amount of the peracetic acid was about 3,000 to about 3, 300 ppm and the initial amount of the hydrogen peroxide was about 4,500 to about 5,000 ppm, and the pH was adjusted to be 2.0 to 4.0. Thereafter, the catalase enzyme was added thereto in amounts of 0.12 to 20 μg/ml as shown in Table 2 to evaluate.

(19) In Examples 4 to 6 and in Comparative Examples 3 to 6, the catalase enzyme was added at one time. In Example 7, however, the whole amount of the catalase enzyme was added in two times being divided into half just after the pH was adjusted and another half after 5 minutes have passed, and the concentrations of the peracetic acid and the hydrogen peroxide were measured just before the catalase enzyme was added in the second time (value marked with * in Table 2). In other respects, the evaluation was made in the same manner as in Example 4. The results were as shown in Table 2.

(20) TABLE-US-00002 TABLE 2 Results of evaluation Sterilizing composition solution Surface state Amount of hydrogen of the peroxide sterilizing Hydrogen Amount of Before After Amount Method composition peroxide peracetic adding adding of of solution in decomposing acid catalase catalase catalase adding the tank power (ppm) (ppm) (ppm) pH (μg/ml) catalase *1 *2 (%/10 min.) Ex. 4 3116 5141 374  ◯ 4.0 0.12 one time ◯ ◯ 65 ◯ Ex. 5 3230 4684 0 ◯ 4.0 1.2 one time ◯ ◯ 88 ◯ Ex. 6 3353 4962 0 ◯ 4.0 10 one time ◯ ◯ 100 ◯ Comp. 3326 5122 1224   X 4.0 0.04 one time ◯ ◯ 34 X Ex. 3 Comp. 3298 4947 0 ◯ 4.0 15 one time X ◯ 100 ◯ Ex. 4 Comp. 3281 5053 0 ◯ 4.0 20 one time X X 100 ◯ Ex. 5 Comp. 3000 4590 0 ◯ 2.0 20 one time X X 100 ◯ Ex. 6 Ex. 7 3362 5043  0* ◯ 4.0 0.48 *3 ◯ ◯ 100 ◯ *1: Bubbling *2: Aggregates *3: divided into 2 times

Examples 8 to 11, Comparative Examples 7 and 8

(21) How the effect for sterilizing the containers would be affected was examined depending on the amount of the peracetic acid and the amount of the hydrogen peroxide. As shown in Table 3, the peracetic acid type sterilizer was diluted such the amount of the peracetic acid was about 300 to about 15,000 ppm and the initial amount of the hydrogen peroxide was about 400 to about 24,000 ppm, and the pH was adjusted to be 4.0. Thereafter, the catalase enzyme was added thereto in amounts of 0.4 to 10 μg/ml as shown in Table 3 to evaluate.

(22) As described above, since the amount of the catalase enzyme has been varied, the surface states of the sterilizing composition solutions in the preparation tanks were also evaluated. The results were as shown in Table 3.

(23) TABLE-US-00003 TABLE 3 Results of evaluation Sterilizing composition solution Surface state Amount of hydrogen of the peroxide sterilizing Amount of Before After Amount Method composition peracetic adding adding of of Effect for solution in acid catalase catalase catalase adding sterilizing the tank (ppm) (ppm) (ppm) pH (μg/ml) catalase containers *1 *2 Ex. 8 527 812 0 ◯ 4.0 0.4 one time Δ ◯ ◯ Ex. 9 1105 1790 0 ◯ 4.0 0.4 one time ◯ ◯ ◯ Ex. 10 5270 8327 0 ◯ 4.0 1 one time ◯ ◯ ◯ Ex. 11 9880 14919 0 ◯ 4.0 2.5 one time ◯ ◯ ◯ Comp. 289 448 0 ◯ 4.0 0.4 one time X ◯ ◯ Ex. 7 Comp. 15810 24347 986 X 4.0 10 one time ◯ X X Ex. 8 *1: Bubbling *2: Aggregates

Examples 12 and 13, Comparative Example 9

(24) How the effect for sterilizing the containers would be affected was confirmed depending on the concentration of the hydrogen peroxide in the peracetic acid type sterilizing composition solution in which the amount of the peracetic acid was in a low-concentration region. As shown in Table 4, the peracetic acid type sterilizer was diluted such the amount of the peracetic acid was about 1,000 ppm and the initial amount of the hydrogen peroxide was about 1,600 ppm, and the pH was adjusted to be 4.0. Thereafter, the catalase enzyme was added thereto in amounts of 0.4 μg/ml to decrease the amount of the hydrogen peroxide to 0 ppm. The sterilizing composition solution was then heated at 85° C. for 5 minutes to deactivate the catalase enzyme and to which the hydrogen peroxide as added to be about 200 to about 700 ppm as shown in Table 4 to evaluate. The results were as shown in Table 4.

(25) TABLE-US-00004 TABLE 4 Sterilizing composition solution Amount of hydrogen peroxide Results of Amount of Before After evaluation peracetic adding adding Amount of Method of Effect for acid catalase catalase catalase adding sterilizing (ppm) (ppm) (ppm) pH (μg/ml) catalase containers Ex. 12 1064 1628 235 ◯ 4.0 0.4 one time ◯ Ex. 13 1064 1628 480 ◯ 4.0 0.4 one time Δ Comp. 1064 1628 682 X 4.0 0.4 one time X Ex. 9

Examples 14 to 17, Comparative Examples 10 and 11

(26) The effect upon the sterilizing pipelines was examined depending on the difference in the concentration of the chloride ions in the peracetic acid type sterilizing composition solution.

(27) The peracetic acid type sterilizer was diluted such that the compositions were nearly the same except the concentration of the chloride ions in the peracetic acid type sterilizing composition solution, i.e., such that the amount of the peracetic acid was 3,000 ppm and the initial amount of the hydrogen peroxide was 4,800 ppm. Thereafter, the pH was adjusted to be 4.0. To the above peracetic acid type sterilizing composition solution was added a catalase enzyme-containing solution that has been desalted by using a UF membrane (manufactured by Asahi Kasei Co.: Microza UF Module, ACV-3010) so that the concentration of the catalase enzyme was 0.4 μm/ml to decrease the amount of the hydrogen peroxide to 0 ppm. Next, the sodium chloride was so added that the concentration of the chloride ions in the peracetic acid type sterilizing composition solution was 0 to 20 μm/ml to thereby obtain an initial peracetic acid type sterilizing composition solution for evaluation.

(28) The concentrations of the chloride ions were measured as described below. Table 5 shows the concentrations of the chloride ions in the peracetic acid type sterilizing composition solutions.

(29) (Measuring the Concentrations of the Chloride Ions)

(30) Concentrations of the chloride ions were measured by using the Ion Chromatograph, DX-320, manufactured by DIONEX Co. under the following conditions. Column: AS11-HC (4×250 mm) Guard Column: AG11-HC (4×50 mm) Temperature: 35° C. Eluent: KOH aqueous solution (gradient) 0-8 min. (1 mmol/L) 8-10 min. (1-7 mmol/L) 10-19 min. (7-14 mmol/L) 19-23 min. (14-23 mmol/L) 23-28 min. (23-45 mmol/L) 28-35 min. (45 mmol/L) Flow rate: 1.1 mL/min. Amount of injection: 25 μL Detector: Electrical conductivity detector (using suppressor) Suppressor: External mode, 170 mA

(31) An SUS stainless steel pipe (4 cm in diameter and 10 cm in length) used as a sterilizer pipeline was dipped in the peracetic acid type sterilizing composition solution, was stored at 40° C. for 6 months, and was evaluated. The amount of the peracetic acid varies with the passage of time. Therefore, the peracetic acid type sterilizing composition solution was replaced by the new one at a moment when the amount of the peracetic acid decreased down to 2800 ppm or less to maintain the amount of the peracetic acid to be 2800 to 3000 ppm.

(32) The inner and outer surfaces of the stainless steel pipe were observed with the naked eye, and were evaluated to be ◯ if there was no corrosion such as rust or pitting, evaluated to be Δ if they were discolored though there was no corrosion such as rust or pitting, and evaluated to be X if they were corroded. The results were as shown in Table 5.

(33) TABLE-US-00005 TABLE 5 Concentration of chloride ions (μg/ml) Corrosion Example 14 0 ◯ 15 1 ◯ 16 5 ◯ 17 10 Δ Comp. Ex. 10 12 X 11 20 X

INDUSTRIAL APPLICABILITY

(34) The peracetic acid type sterilizer composition solution of the invention is capable of efficiently decomposing the hydrogen peroxide in the sterilizing composition solution despite the content of the catalase enzyme is as very small as 0.1 to 10 μg/ml. Therefore, the peracetic acid type sterilizer composition solution of the invention is excellent in economy, can suppress the formation of bubbling or aggregates caused by the addition of the catalase enzyme, eliminates the need of frequency cleaning the tanks and the sterilizing injectors enabling the sterilization to be efficiency carried out and, therefore, can also be effectively used for the method for sterilizing the products in large quantity.

(35) Moreover, the concentration of the hydrogen peroxide has been decreased down to be less than 500 ppm and no defoaming agent has been used, either. Therefore, the hydrogen peroxide does not remain despite the washing is conducted for only a short period of time after the sterilization, suppressing the hygienic effect on the contents.

(36) By using the catalase enzymatic preparation having a decreased concentration of the chloride ions, further, it is made possible to suppress the pipes from corroding providing usefulness specifically for the method for sterilizing the products in large quantity.