Comparison of the Efficacies of Gaseous Ozone and Sodium Hypochlorite in Cleaning Stainless Steel Particles Fouled with Proteins

Biocontrol Science, 2003, Vol. 8, No. 2, 87-91

Note

KAZUHIRO TAKAHASHI', KUNIHIKO KOIKE2, AND SATOSHI FUKUZAKI1*

Industrial Technology Center of Okayama Prefecture,1 5301 Haga, Okayama 701-1296, and 2Shiga Technology Center, lwatani International Corp., 4-5-1 Katsube, Moriyama, Shiga 524-0041, Japan

Received 16 July 2002/Accepted 21 September 2002

The efficacies of gaseous ozone and sodium hypochlorite as an oxidizing agent for cleaning were compared in a laboratory model system using stainless steel particles fouled with proteins. Exposure of the protein-fouled stainless steel particles to 0.5% (v/v) gaseous ozone for 30 min enhanced the removal of the various proteins during subsequent alkali cleaning with NaOH solution to the same degree as the NaOH solution containing sodium hypochlorite at 0.2 to 0.4 g/l. The effect of ozone pretreatment on the removal of the proteins depended on the concentration of gaseous ozone. Pretreatment with a highly concentrated gaseous ozone of 20% for 30 min resulted in the almost complete removal of the proteins from stainless steel particles.

Key words : Cleaning/Gaseous ozone/Hypochlorite/Protein/Stainless steel.

In the food industry, the cleanliness and sterility of 1972). There are many advantages of using ozone as

food-processing equipment is one of the most impor- an oxidizing agent in the food industry. Ozone is a tant factors in manufacturing wholesome and safe more powerful oxidant than chlorine and can kill a products. The combined use of detergent and chlo- large number of microorganisms (Graham, 1997; Kim, rine such as sodium hypochlorite has been practiced et al. 1999; Moore, et al. 2000). In addition, ozone can

extensively in the dairy industry (Clegg, 1962). The be applied as a gas or in water, and it leaves no resid- main action of hypochlorite is due to the oxidation of ual toxicity after decomposition to oxygen. However, organic substances of soils and microorganisms. few published reports are available on the possible However, the repeated use of chlorinated alkaline de- use of gaseous ozone in the cleaning process. tergent results in the abrasion and corrosion of metal- The aim of this work is to compare the efficacy of

lic materials. In addition, chlorination brings health gaseous ozone pretreatment with that of sodium hazards to human beings and wildlife through the pro- hypochlorite in the cleaning of stainless steel particles duction of harmful compounds in the environment fouled with proteins that have different properties, (Richardson et al., 1998). Therefore, alternative e.g., thermal stability, solubility, and structure. cleaning agents to chlorine are being explored for Nonporous stainless steel particles (SUS 304: 2-10 food-processing equipment. One such compound is m) were obtained from The Nilaco Corp. (Tokyo);u ozone (O3) which has been utilized as a sanitizing their specific surface area, as determined by BET

agent in European water treatment plants (Gomella, method, was 0.43 m2/g. Crystalline BSA and ƒÀ -lacto-

globulin ( ƒÀ -Lg) were purchased from Nacalai *Corresponding author . Tel : +81-82-286-9600, Fax:+ 81-82- Tesque Inc. (Kyoto). Casein and gelatin were pur- 286-9630 chased from Kanto Chemical Co., Inc. (Tokyo) and 88 K. TAKAHASHI ET AL.

Wako Pure Chemical Ind. Ltd. (Osaka), respectively. The model protein-fouled stainless steel particles were prepared at 40•Ž by introducing a 50-ml aliquot of a protein solution (3g/l, 10-3 M KNO3) and 10 g of stainless steel particles into a 125-ml glass vial, which was then laid on its side in a water bath at 40

and reciprocally shaken (140 oscillations per min)•Ž for 2 h. The pH values of the protein solutions were adjusted to 5.6 for BSA and gelatin (at the point of zero charge) , and 6.8 for ƒÀ -Lg and casein (pH of milk). After being shaken, the protein-fouled stainless steel particles were collected and washed with 40 ml of 10-3M KNO3 solution by centrifugation (2300 •~g for 1 min). The washed protein-fouled particles were dried at 40•Ž for 16h and stored in a desiccator be- fore use for the cleaning experiments. The amounts of BSA, gelatin, ƒÀ -Lg, and casein adsorbed were 2.7,

1.8, 1.6, and 2.7 mg/m2, respectively. Gaseous ozone of 0.5 to 6.0% (v/v) was gener- ated from pure oxygen (99.999%, v/v) by a silent discharge ozonizer (M001; OHNIT Co. Ltd.,

Okayama) equipped with an ozone monitor (PG-620;

Ebara Jitsugyo Co. Ltd., Tokyo). The above gaseous FIG. 1 . Effects of ozone pretreatment (0.5% for 30 min) ozone was concentrated to 15 and 20% using the and sodium hypochlorite (0.2 g AC//) on the removal of adsorption-desorption technique on silica gel (Koike proteins from stainless steel particles during cleaning with NaOH solutions of different pHs at 40•Ž. (A) BSA, (B) et al., 2000). Prior to cleaning, a 5-g portion of -Lg , (C) casein, (D) gelatin. Batchwise cleaning experi-ƒÀ protein-fouled stainless steel particles was treated ment was conducted at 40•Ž for 2 h with shaking (140 oscil- with gaseous ozone of 0.5 to 20% at room tempera- lations per min). Symbols: A, NaOH alone; O, ozone ture for 30 min. pretreatment; ^, sodium hypochlorite. Batchwise cleaning was conducted by introducing a

1.16-g portion of protein-fouled stainless steel parti- stainless steel particles, and that of the protein in so- cles (ca. 0.5m2) and 5 ml of cleaning solution into a lution were measured with a total organic carbon ana-

25-ml glass vial, which was then placed horizontally in lyzer (TOC-5000; Shimadzu Co., Kyoto) equipped a water bath (40 to 80•Ž) and reciprocally shaken with a solid sample module (SSM-5000; Shimadzu

(140 oscillations per min) for 2 h. The pH values of Co., Kyoto). cleaning solutions were adjusted to pH 9.0 to 13.5 Figure 1 shows the effects of gaseous ozone

with NaOH (1 M solution). After each cleaning experi- pretreatment (0.5% for 30 min) and sodium ment, the particles bearing the remaining proteins hypochlorite (0.2 g AC/l) on the removal of the pro- were collected by centrifugation (2300 xg for 1 min). teins when used in conjunction with the NaOH solu- To determine the effects of oxidizing agents, protein- tions of different pHs at 40•Ž. In the case of cleaning fouled particles were cleaned at 40•Ž for 2 h with with NaOH solution alone, the removal efficiency was NaOH solutions of pH 9.0 to 13.5 with and without so- markedly higher at pHs above 11 and increased with

dium hypochlorite at a final concentration of 0.2 g increasing pH. This indicates that protein removal de-

available chlorine (AC) per liter, respectively, as de- pends on the hydroxide ion concentration (Koopal, scribed above. Continuous cleaning was conducted at 1985; Takehara et al., 2001). It was noted that the re- 40•Ž in a stainless steel column (4 mm ƒ³ •~ 50 mm) moval of the proteins occurred markedly at the alka- by feeding 10-3 M KNO3 solution (pH 5.6) for 120 line pH region above the intrinsic dissociation

min followed by feeding NaOH solution of pH 12 with constant (pKint) of a side-chain amino group of, for in- or without sodium hypochlorite (0.4g AC/l) for 180 stance, lysine, i.e., 10.8. The use of sodium min at a flow rate of 0.25 ml / min (Urano and hypochlorite and ozone significantly enhanced the re- Fukuzaki, 2001). The alkali eluate was fractionally moval of the proteins. The cleaning effects of ozone

collected, and its protein concentration was meas- pretreatment against all the proteins were comparable ured. to those of alkaline hypochlorite solution at 0.2 g AC

The amount of protein adsorbed or remaining on /l although small differences in the effects on various CLEANING EFFICACY OF GASEOUS OZONE 89

proteins were observed. The performance of hypochlorite and ozone in removing proteins is asso- ciated with the decomposition of proteins by their strong oxidative power (Merrill et al., 1962; Urano and

Fukuzaki, 2001). As a result of decomposition, large fractions of the proteins could be removed even when

using a low-pH NaOH solution. It is known that the effects of the combination of NaOH solution and temperature are also to solubilize adsorbed proteins through the chemical reaction be-

tween NaOH and proteins, i.e., hydrolysis of proteins

(Twomey, 1968). Figure 2 shows the effect of tem-

perature on the protein cleaning efficiency by the NaOH solutions of different pHs. All the proteins were

removed more efficiently by increasing the temperature from 20 to 80•Ž at pHs especially above 12. Although BSA and p -Lg, which contain sulfhydryl (

SH) groups and are respectively denatured at tem-

peratures above 60•Ž and 70•Ž at a neutral pH (De Wit and Swinkels, 1980; Steim, 1965), high temperatures under strongly alkaline conditions (pH>12) can enhance the removal of proteins irrespective of the Cleaning time (min) thermal stability of the proteins. As shown in Figs. 1 and 2, in the case of cleaning with a low-pH NaOH so- FIG.3. Time courses for the removal of proteins from stain- lution below 11, the effects of ozone and sodium less steel particles during continuous cleaning with the hypochlorite on the removal of the proteins were NaOH solution of pH 12 alone, NaOH solution (pH 12) combined with ozone pretreatment (0.5% for 30 min), or NaOH solution (pH 12) with hypochlorite of 0.4g AC/i. (A) BSA, (B) p -Lg, (C) casein, (D) gelatin. Continuous cleaning was conducted in a stainless steel column at 40•Ž by a continuous feeding of 10-3 M KNO3 solution (pH 5.6) followed by alkali solutions. Symbols: •¢, NaOH alone; • sodium hypochlorite; •Z, ozone pretreatment.

greater than those at temperatures at 60 and 80•Ž. Figure 3 compares the effects of sodium hypochlorite (0.4g ACM and ozone pretreatment

(0.5%, 30 min) on the removal of the proteins from stainless steel particles during continuous alkali cleaning (pH 12). The times of - 120 and 0 min denote the times when 10' M KNO3 (prerinse solution) and cleaning solution started to flow in the column, respectively. Higher initial rates and protein removal efficiencies were achieved by cleaning with alkaline

hypochlorite solution or by combined ozone / NaOH cleaning than by cleaning with NaOH solution alone. The cleaning efficiencies obtained for combined ozone/NaOH cleaning were approximately equal to those obtained for cleaning with alkaline hypochlorite

solution, except for the relatively larger residual FIG.2. Effects of temperature on the removal of proteins amounts of BSA and casein at 180 min. In the case of from stainless steel particles during cleaning with NaOH so- cleaning with alkaline hypochlorite solution, the resid- lutions at different pHs. (A) BSA, (B) ƒÀ -Lg, (C) casein, ual amounts of the four proteins were reduced to less (D) gelatin. For cleaning conditions, see the legend for Fig. 1. Symbols: •›, 20•Ž; •¢, 40•Ž; š˜, 60•Ž; •ž, 80 than 2% (w/w). On the other hand, the advantage of ozone pretreatment was the fact that 25 to 52%

•Ž. 90 K. TAKAHASHI ET AL.

fractions of adsorbed proteins on stainless steel parti- decrease in TOC might be attributed to the formation

cles could be removed during the prerinse process of volatile gaseous products, e.g., CO2, during ozone through partial decomposition of protein molecules. oxidation. In addition, the amount of BSA removed

Greater amounts were removed during the prerinse during prerinse process increased from ca. 30 to 70%

process after ozone pretreatment in the order: gelatin (w/w) by increasing ozone concentrations from 2 to > casein > ƒÀ-Lg > BSA. Gelatin and casein are 20%. This implies that adsorbed BSA was partially

characterized by high contents of proline and decomposed into low-molecular fragments by gase-

hydroxyproline residues which contribute to an ex- ous ozone (Urano and Fukuzaki, 2001), resulting in

panded disordered structure (Mohammed et al., the facilitation of the removal of BSA from stainless

2000). On the other hand, BSA and ƒÀ -Lg are in a steel surfaces. Pretreatment with ozone of 20% re-

compact folded conformation and the magnitudes of sulted in the almost complete removal of BSA from

intramolecular interactions on their molecules are stainless steel particles during alkali cleaning.

thought to be greater than those of gelatin and casein. The results presented here showed that the com-

This may be the reason for the small amounts of BSA plementary actions of alkalinity and oxidizing agents and ƒÀ -Lg removed during the prerinse process after can provide an adequate cleaning efficacy against

ozone pretreatment. protein soiling. The results also suggested that gase-

Figure 4 shows the effect of ozone concentration in ous ozone could be potentially used as an alternative

the pretreatment on the removal of BSA, which cleaning agent to sodium hypochlorite for removing

showed a larger residual amount in Fig. 3, from stain- proteins soiling stainless steel surfaces. less steel particles during continuous alkali cleaning

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