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Antimicrobial Activity of Compounds Containing Active Chlorine and Iodine in the Presence of Egg Solids
Antimicrobial Activity of Compounds Containing Active Chlorine and Iodine in the Presence of Egg Solids w. A. MOATS Meat Science Research Laboratory, USDA, SEA, AR, Beltsville, Maryland 20705 (Received for publication January 15, 1981)
1981 Poultry Science 60:1834-1839 INTRODUCTION
Some commercial egg washers use chlorinated egg cleaning formulations. Also, plants operating under Federal Grading Service (USDA, 1974) are required to use a chlorinated rinse following washing. The effectiveness of these chlorinated compounds under commercial egg washing practices has not been established. Commercial egg washers are usually of the recirculating type and usual practice is to change washwater after approximately 4 hr of operation. During this period considerable buildup of organic matter occurs in the wash water (Hamm et al, 1974; Harris and Moats, 1975) from dirt washed from the eggs and from the contents of eggs broken during washing. The present experiments were run to determine whether or not active chlorine compounds incorporated into egg washwater would retain
1 Mention of specific trade names is for purpose of identification only and does not imply endorsement by die US Department of Agriculture over similar products not mentioned.
appreciable antimicrobial activity when washwaters were contaminated with egg solids. MATERIALS AND METHODS
To obtain sterile liquid egg, an egg was dipped in absolute alcohol and the surface ignited to burn off the excess alcohol to sterilize the surface. The shell was carefully chipped from one end with a sterile scalpel and the contents drained into a sterile beaker. The egg was mixed with a sterile stirring rod and pipetted aseptically into the test media. Bacterial Cultures. Streptococcus faecalis and Salmonella typhimurium strains were selected from stock cultures maintained in our laboratory. Cultures used were grown 24 hr in tryptic-soy broth (TSB; Difco) at 37 C. Washing and Sanitizing Chemicals. Egg cleaning formulations used were: Super-chlorital (Pennwalt Corp.), a chlorinated cleaner; Zep D (Zep Chemical Co.), an alkaline cleaner; CDB Clearon (FMC Corp.), active ingredient sodium dichloro-s-triazine trione dihydrate (sodium dichloroisocyanurate), a stable dry powder; and Du-San (Dubois Chemical Co.), an iodophor sanitizer . The sodium hypochlorite used was
1834
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ABSTRACT The antimicrobial activity of two chlorinated sanitizers, sodium hypochlorite and sodium dichloroisocyanurate, a chlorinated cleaner, and an iodophor were determined in the presence and absence of egg solids. Sodium hypochlorite and sodium dichloroisocyanurate were tested against Salmonella typhimurium using concentrations of about 270 ppm active chlorine near pH 7. The test organism was killed rapidly by the sanitizers in water. When 1.0% egg solids was added, antimicrobial activity rapidly decreased and was slight after 1 hr, although a chemical test showed 17 to 30 ppm residual active chlorine. The commercial chlorinated cleaner gave a pH of 11.2, and Streptococcus faecalis was used as the test organism since salmonellae do not survive at this pH. This cleaner at 240 ppm active chlorine was effective in water but was ineffective against the test organism after incubation 30 min with 1% egg solids despite the presence of 52 ppm residual active chlorine by the chemical test. Tests in a commercial plant demonstrated that active chlorine chemicals added to wash water were completely inactivated in a few minutes. An iodophor was tested at a molar concentration equivalent to that used with chlorine compounds (949 ppm I 2 ) and also at 25 ppm I 2 , both at acid pH's since iodophors were ineffective at neutral or alkaline pH's. At 25 ppm I 2 , the iodophor was ineffective when egg solids were added. At 949 ppm I2 and pH 3.52 the iodophor rapidly killed the salmonellae even after incubation for 1 hr with 1% egg solids and 710 ppm I2 remained. Maintaining bactericidal levels of residual chlorine in egg washwater appears impractical. Use of a iodophor might be effective but would be costly and would require major changes in washing procedures. (Key words: egg washing, chlorinated sanitizers, iodophors, antimicrobial activity, egg solids)
EGG SOLIDS EFFECT ON SANITIZERS
1835
TABLE 1. Effect of sodium hypochlorite on Salmonella typhimurium in the absence and presence of egg solids at neutral pH at 40 C
Medium NaOCl in water NaOCl and 1.0% egg solids Immediately after mixing2 30 min after mixing 60 min after mixing 1.0% egg solids in water
Active chlorine (ppm)
Log10 bacterial counts after 0 min 1
30 min
60 min
0*
265 144 35 30 0
Mean
1.45 7.20 7.20 7.18
0
0
5.20 7.00 7.18
2.51 6.48 7.26
.48a 4.97b 6.89c 7.21c
1 Samples withdrawn and plated immediately after addition of 2 ml of S. typhimurium soy broth to 200 ml test medium. 2
Egg added just prior to addition of bacterial culture and sampling.
3
household bleach (5.25% sodium hypochlorite according to the label). The active chlorine content of the sanitizing compounds and residual active chlorine were determined iodometrically by adding an excess of sodium thiosulfate and titrating the residual thiosulfate with standard iodine as described in Standard Methods for the Examination of Water and Waste Water (1976). Iodine was determined by titration with standard sodium thiosulfate. After determination of active chlorine or iodine content of the sanitizing
culture in Tryptic-
chemicals, appropriate amounts were used in preparation of the test media, which were restandardized. For the experiments, 200 ml of test media were placed in screw top jars of 450 ml capacity. Distilled water and the alkaline cleaner (4 g/liter of test medium) were sterilized by autoclaving the jars. Solutions of various sanitizers were assumed to be self-sterilizing. The test media in the jars were equilibrated to 40 C in a shaking water bath prior to addition of test bacteria, and all tests were conducted at
TABLE 2. Effect of sodium dichloro-s-triazinetrione on Salmonella typhimurium in the absence and presence of egg solids at neutral pH at 40 C
Medium Sodium dichloro-s-triazine trione in water 1.0% egg solids Immediately after mixing2 30 min after mixing 60 min after mixing 1.0% egg solids in water
Active chlorine (ppm)
Log10 bacterial count after 0 min 1
30 min
60 min
4.93 7.18 7.20 7.08
0
0
1.78 6.08 7.08
1.18 4.30 7.18
Mean
273
96 81 17 0
1.64ab 3.38bc 5.86cd 7.lid
' ' ' Numbers followed by the same superscript letter are not significantly different according to Duncan's multiple range test (Duncan, 1955). 1 Samples withdrawn and plated immediately after addition of 2 ml of S. typhimurium soy broth to 200 ml test medium. 2
Egg added just prior to addition of bacterial culture and sampling.
culture in Tryptic-
Downloaded from http://ps.oxfordjournals.org/ at New York University on May 18, 2015
' ' Numbers followed by the same superscript letter are not significantly different according to Duncan's multiple range test (Duncan, 1955).
1836
MOATS
40 C. For jars containing egg, 5 ml of the egg melange was added aseptically to the sanitizer solutions and the water and detergent controls giving an egg solids concentration of about 1.0%. Two ml of the test bacterial culture grown in i ars was aaaeu as specified to the test media and controls, mixed, and an initial sample withdrawn and plated in duplicate immediately on Tryptic-soy agar (TSA, Difco) containing . 1 % sodium thiosulfate. This re-quired from 2 to 3 min from the time the sample was taken to the time the plates were poured, which finally inactivated any residual chlorine present. Plates were counted after incubation for 48 h at 37 C. A sample for testing for residual chlorine was also withdrawn and immediately pipetted into an excess standard of sodium thiosulfate solution. Other samples were withdrawn at specified times after holding the test media in the water bath at 40 C. A statistical analysis of the data was prepared using a general linear models procedure.
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RESULTS AND DISCUSSION
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Several similar experiments were run to determine to what extent chlorine based sanitizers retained antimicrobial activity in the presence of egg solids. The results were generally similar in each experiment and typical results with each of the three sanitizers tested are shown. Results with sodium hypochlorite and sodium dichloro-s-triazine trione, two compounds commonly used as a source of active chlorine in sanitizing rinses in commercial egg washers are summarized in Tables 1 and 2. Salmonella typhimurium was the test organism. The experimental procedures were the same for each compound. The temperature used (40 C) is in the range (40 to 50 C) used in commercial eggwashers. The egg solids concentration of 1.0% is in the range found in egg washers after the washers have been in operation for several hours (Hamm et al, 1974; Harris and Moats, 1975). Although the pH's of the test media were not recorded, both the compounds and egg solids give pH's in the general range 7 to 8 or roughly neutral. The chlorine concentrations (about 270 ppm) were higher than is usually used in commercial egg washing where 50 ppm chlorine is required in the sanitizing rinse (USDA, 1974).
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EGG SOLIDS EFFECT ON SANITIZERS TABLE 4. Comparison of bactericidal effectiveness of chlorinated
Sodium hypochlorite Sodium dichloroisocyanurate Chlorinated alkaline cleaner
Mean logic survivors
15 15 15
3.91 b 3.60° 5.01* .05 according to Duncan's
(log 1.54) were found immediately after inoculation. When bacteria were inoculated immediately after addition of egg, some survived 6 0 min. Residual chlorine was also reduced immediately after addition of egg. When bacteria were inoculated 30 min after addition of egg, the chlorinated cleaner at pH 11 was totally ineffective in killing the test organism even t h o u g h considerable residual chlorine was detectable chemically at up to 60 m i n . N u m bers of bacteria were stable in t h e alkaline control with egg. However, t h e r e was a three log reduction in t h e test organism in t h e egg in water at neutral pH, presumably because of the lysozyme activity of egg (Board, 1 9 7 3 ) . The results indicate t h a t u n d e r the alkaline conditions used t o wash eggs, chlorinated comp o u n d s show little antimicrobial activity once egg solids are introduced into t h e w a s h w a t e r . When all observations for each chlorinated c o m p o u n d were combined (Table 4 ) , t h e alkaline cleaner was f o u n d to be significantly less bactericidal than t h e o t h e r t w o , assuming t h a t t h e response of t h e t w o test organisms t o chlorine was similar. It has been reported t h a t active chlorine is converted t o chloramines in t h e presence of amino groups such as are present in proteins. Chloramines are less bactericidal t h a n o t h e r sources of active chlorine b u t give a chemical test for residual chlorine (White, 1 9 7 2 ) . It seems probable t h a t this is t h e explanation for t h e loss of antimicrobial activity observed in t h e presence of egg. Active chlorine as h y p o chlorite has been f o u n d t o b e less bactericidal at alkaline than at neutral or acid pH's (White, 1972). This would a c c o u n t for t h e further loss of activity observed at p H 1 1 . The persistence of residual chlorine u n d e r actual commercial conditions was d e t e r m i n e d in a plant using chlorinated cleaner. This plant
Downloaded from http://ps.oxfordjournals.org/ at New York University on May 18, 2015
Since commercial egg washers c o m m o n l y use alkaline egg cleaning c o m p o u n d s and operate in t h e pH range 10 to 1 1 , t h e experiments were repeated using alkaline egg cleaners t o simulate commercial conditions (Table 3). Since previous studies (Kinner and Moats, 1981) had shown t h a t salmonellae were killed rapidly at 4 0 C at pH 10 or above, an organism, Streptococcus faecalis, which was k n o w n to survive well under these conditions was used. A commercial chlorinated and a c o m m e r cial alkaline egg cleaner were used, b o t h of which gave pH's above 11 when freshly mixed with water. Again, t h e initial inoculum as shown by t h e controls w i t h o u t chlorine was a b o u t 1 0 7 / m l . The chlorinated cleaner proved effective in water, although a few survivors
compounds
Number of observations
a 'b Numbers followed by the same superscript letter are not different at P Multiple Range Test (Duncan, 1955).
bacteria were found immediately after inoculations of t h e sanitizer solutions. When bacteria were inoculated immediately after addition of egg t o t h e sanitizer solutions, some survivors were found in samples withdrawn immediately after inoculation, log 1.45 in sodium h y p o chlorite and log 4.93 in t h e sodium dichloro-striazinetrione. None were found after holding 30 min, however. Addition of egg solids resulted in an immediate reduction in available chlorine t o less than half the initial values. When inoculation of bacteria was delayed 30 m i n after addition of egg, t h e rate of kill was markedly reduced with some survivors present 6 0 min after inoculation. T h e rate of kill was even slower when inoculation was delayed until 60 min after addition of egg. Considerable residual chlorine was detected chemically u p to 6 0 min after addition of t h e egg even t h o u g h its antimicrobial activity was slight at this t i m e . Bacterial n u m b e r s in t h e controls containing egg solids only remained stable. The results with t h e t w o sanitizers were quite similar with t h e same initial level of active chlorine.
1837
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3
2
1
a
28 4
6 >1 >1 0
6.17 6.17 6.17 6.30
869 754 711 0
3.52 3.52 3.52 6.56
5.2
949 2
act. (ppm)
2.42
PH
7.26 7.36 7.25 7.20
0 0 0 7.23
0 min1
None detectable after 90 min standing, pH fell to 4:7.
Egg added just prior to addition of bacterial culture and sampling.
Equivalent to 265 ppm Cl 2 .
Samples withdrawn and plated immediately after addition of 2 ml of S. typbimurium
culture in Tryptic-soy br
' Numbers followed by the same superscript letter are not significantly different according to Duncan's multip
Experiment 2 Iodophor in water Iodophor in 1.0% egg solids Immediately after mixing 30 min after mixing 60 min after mixing 1.0% egg in water
Experiment 1 Iodophor in water Iodophor in 1.0% egg solids Immediately after mixing 3 30 min after mixing 60 min after mixing 1.0% egg solids in water
Medium
TABLE 5. Effect of an iodophor sanitizer on Salmonella typbimurium in presence of egg solids at 40 C
m http://ps.oxfordjournals.org/ at New York University on May 18, 2015
EGG SOLIDS EFFECT ON SANITIZERS
The results with the iodophor are interesting since they show that iodine was more stable than chlorine in the presence of egg solids when used on an equimolar basis. The iodophor solutions were strongly acid, however. It would appear technically feasible to maintain bactericidal levels of an iodophor in egg washwater if an acid cleaner were used. However, several questions need to be answered before use of an acid cleaner can be advocated. How rapidly would it be neutralized by calcium carbonate in egg shells? Would egg solids precipitated under acid conditions interfere with washer operation? What would be the cost of using an iodophor? Would cleaning efficiency be improved
over that of alkaline cleaners now in use? The strongly alkaline washwater used in present commercial operation has been demonstrated to be effective in killing many types of bacteria including salmonellae (Kinner and Moats, 1981). This, coupled with proper attention to equipment cleanliness, has been found effective in maintaining low bacterial levels in washwater and on washed eggs without the need for costly sanitizers (Moats, i981). ACKNOWLEDGEMENT
The author thanks J. I. Schultz, Jr., and T. M. Brennan for technical assistance and the Pennwalt Corp., Zep Chemical Co., FMC Corp., and Dubois Chemical Co. for providing samples of chemicals used in this study and Bernard Weinland for preparing the statistical analyses of the data. REFERENCES Atherton, H. V., and J. A. Newlander, 1977. Chemistry and testing of dairy products. 4th ed. Avi Publ. Co., Westport, CT. Board, R. G. 1973. The microbiology of eggs. In Egg science and technology. W. J. Stadleman and O. J. Cotterill, ed. Avi Publ. Co., Westport, CT. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics 11:1—42. Hamm, D., G. K. Searcy, and A. J. Mercuri. 1974. A study of the waste washwater from egg washing machines. Poultry Sci. 53:191-197. Harris, C. E., and W. A. Moats, 1975. Recovery of egg solids from waste-waters of egg-grading and -breaking plants. Poultry Sci. 54:1518-1523. Kinner, J. A., and W. A. Moats, 1981. Effect of temperature, pH, and detergent on survival of bacteria associated with shell eggs. Poultry Sci. 60: 761-767. Moats, W. A., 1981. Factors affecting bacterial loads on shells of washed eggs. Poultry Sci. 60. (In press). Standard Methods for the Examination of Water and Waste Water, 1976. 14th ed. M. C. Rand, A. E. Greenberg, M. J. Taras, and M. A. Franson, ed., 1976. Amer. Publ. Health Ass., Washington, DC. United States Department of Agriculture, Agricultural Marketing Service, 1974. Regulations governing the grading of shell eggs and United States standards, grades and weight classes for shell eggs. (7CFR part 56). White, G. C , 1972. Handbook of chlorination. Von Nostrand Reinhold Co., New York, NY.
Downloaded from http://ps.oxfordjournals.org/ at New York University on May 18, 2015
added fresh chlorinated detergent together with supplemental chlorine in the form of bleaching powder at the midpoint of a 4 hr run. Considerable egg solids were evident in the washwater at this time. After the machine had run for 2 min following recharging, a sample of washwater was withdrawn and pipetted into an excess of sodium thiosulfate solution. No residual chlorine was found. Apparently, inactivation of chlorine is more rapid and complete under commercial conditions than in our laboratory experiments. It does not, therefore, appear realistic to attempt to maintain bactericidal levels of chlorine in egg washwaters as washers are now operated. An experiment was also run using an iodophor sanitizer (Table 5). Iodophors are considered to be effective only at acid pH, 5.0 or below (Atherton and Newlander, 1977). When used at the recommended concentration of 25 ppm I 2 , the iodophor was ineffective when egg solids were present. To be equivalent to the chlorine solutions used in the other experiments on an equimolar basis, a much higher concentration, 949 ppm I 2 , was also tested. This gave a strongly acid (pH 3.52) solution, even when egg solids were present. However, the reduction in available iodine in the presence of egg was relatively slow and bacteriological effectiveness was maintained for 60 min. From the rate of reduction of iodine, it probably would have remained effective for several hours longer.
1839
1981 Poultry Science 60:1834-1839 INTRODUCTION
Some commercial egg washers use chlorinated egg cleaning formulations. Also, plants operating under Federal Grading Service (USDA, 1974) are required to use a chlorinated rinse following washing. The effectiveness of these chlorinated compounds under commercial egg washing practices has not been established. Commercial egg washers are usually of the recirculating type and usual practice is to change washwater after approximately 4 hr of operation. During this period considerable buildup of organic matter occurs in the wash water (Hamm et al, 1974; Harris and Moats, 1975) from dirt washed from the eggs and from the contents of eggs broken during washing. The present experiments were run to determine whether or not active chlorine compounds incorporated into egg washwater would retain
1 Mention of specific trade names is for purpose of identification only and does not imply endorsement by die US Department of Agriculture over similar products not mentioned.
appreciable antimicrobial activity when washwaters were contaminated with egg solids. MATERIALS AND METHODS
To obtain sterile liquid egg, an egg was dipped in absolute alcohol and the surface ignited to burn off the excess alcohol to sterilize the surface. The shell was carefully chipped from one end with a sterile scalpel and the contents drained into a sterile beaker. The egg was mixed with a sterile stirring rod and pipetted aseptically into the test media. Bacterial Cultures. Streptococcus faecalis and Salmonella typhimurium strains were selected from stock cultures maintained in our laboratory. Cultures used were grown 24 hr in tryptic-soy broth (TSB; Difco) at 37 C. Washing and Sanitizing Chemicals. Egg cleaning formulations used were: Super-chlorital (Pennwalt Corp.), a chlorinated cleaner; Zep D (Zep Chemical Co.), an alkaline cleaner; CDB Clearon (FMC Corp.), active ingredient sodium dichloro-s-triazine trione dihydrate (sodium dichloroisocyanurate), a stable dry powder; and Du-San (Dubois Chemical Co.), an iodophor sanitizer . The sodium hypochlorite used was
1834
Downloaded from http://ps.oxfordjournals.org/ at New York University on May 18, 2015
ABSTRACT The antimicrobial activity of two chlorinated sanitizers, sodium hypochlorite and sodium dichloroisocyanurate, a chlorinated cleaner, and an iodophor were determined in the presence and absence of egg solids. Sodium hypochlorite and sodium dichloroisocyanurate were tested against Salmonella typhimurium using concentrations of about 270 ppm active chlorine near pH 7. The test organism was killed rapidly by the sanitizers in water. When 1.0% egg solids was added, antimicrobial activity rapidly decreased and was slight after 1 hr, although a chemical test showed 17 to 30 ppm residual active chlorine. The commercial chlorinated cleaner gave a pH of 11.2, and Streptococcus faecalis was used as the test organism since salmonellae do not survive at this pH. This cleaner at 240 ppm active chlorine was effective in water but was ineffective against the test organism after incubation 30 min with 1% egg solids despite the presence of 52 ppm residual active chlorine by the chemical test. Tests in a commercial plant demonstrated that active chlorine chemicals added to wash water were completely inactivated in a few minutes. An iodophor was tested at a molar concentration equivalent to that used with chlorine compounds (949 ppm I 2 ) and also at 25 ppm I 2 , both at acid pH's since iodophors were ineffective at neutral or alkaline pH's. At 25 ppm I 2 , the iodophor was ineffective when egg solids were added. At 949 ppm I2 and pH 3.52 the iodophor rapidly killed the salmonellae even after incubation for 1 hr with 1% egg solids and 710 ppm I2 remained. Maintaining bactericidal levels of residual chlorine in egg washwater appears impractical. Use of a iodophor might be effective but would be costly and would require major changes in washing procedures. (Key words: egg washing, chlorinated sanitizers, iodophors, antimicrobial activity, egg solids)
EGG SOLIDS EFFECT ON SANITIZERS
1835
TABLE 1. Effect of sodium hypochlorite on Salmonella typhimurium in the absence and presence of egg solids at neutral pH at 40 C
Medium NaOCl in water NaOCl and 1.0% egg solids Immediately after mixing2 30 min after mixing 60 min after mixing 1.0% egg solids in water
Active chlorine (ppm)
Log10 bacterial counts after 0 min 1
30 min
60 min
0*
265 144 35 30 0
Mean
1.45 7.20 7.20 7.18
0
0
5.20 7.00 7.18
2.51 6.48 7.26
.48a 4.97b 6.89c 7.21c
1 Samples withdrawn and plated immediately after addition of 2 ml of S. typhimurium soy broth to 200 ml test medium. 2
Egg added just prior to addition of bacterial culture and sampling.
3
household bleach (5.25% sodium hypochlorite according to the label). The active chlorine content of the sanitizing compounds and residual active chlorine were determined iodometrically by adding an excess of sodium thiosulfate and titrating the residual thiosulfate with standard iodine as described in Standard Methods for the Examination of Water and Waste Water (1976). Iodine was determined by titration with standard sodium thiosulfate. After determination of active chlorine or iodine content of the sanitizing
culture in Tryptic-
chemicals, appropriate amounts were used in preparation of the test media, which were restandardized. For the experiments, 200 ml of test media were placed in screw top jars of 450 ml capacity. Distilled water and the alkaline cleaner (4 g/liter of test medium) were sterilized by autoclaving the jars. Solutions of various sanitizers were assumed to be self-sterilizing. The test media in the jars were equilibrated to 40 C in a shaking water bath prior to addition of test bacteria, and all tests were conducted at
TABLE 2. Effect of sodium dichloro-s-triazinetrione on Salmonella typhimurium in the absence and presence of egg solids at neutral pH at 40 C
Medium Sodium dichloro-s-triazine trione in water 1.0% egg solids Immediately after mixing2 30 min after mixing 60 min after mixing 1.0% egg solids in water
Active chlorine (ppm)
Log10 bacterial count after 0 min 1
30 min
60 min
4.93 7.18 7.20 7.08
0
0
1.78 6.08 7.08
1.18 4.30 7.18
Mean
273
96 81 17 0
1.64ab 3.38bc 5.86cd 7.lid
' ' ' Numbers followed by the same superscript letter are not significantly different according to Duncan's multiple range test (Duncan, 1955). 1 Samples withdrawn and plated immediately after addition of 2 ml of S. typhimurium soy broth to 200 ml test medium. 2
Egg added just prior to addition of bacterial culture and sampling.
culture in Tryptic-
Downloaded from http://ps.oxfordjournals.org/ at New York University on May 18, 2015
' ' Numbers followed by the same superscript letter are not significantly different according to Duncan's multiple range test (Duncan, 1955).
1836
MOATS
40 C. For jars containing egg, 5 ml of the egg melange was added aseptically to the sanitizer solutions and the water and detergent controls giving an egg solids concentration of about 1.0%. Two ml of the test bacterial culture grown in i ars was aaaeu as specified to the test media and controls, mixed, and an initial sample withdrawn and plated in duplicate immediately on Tryptic-soy agar (TSA, Difco) containing . 1 % sodium thiosulfate. This re-quired from 2 to 3 min from the time the sample was taken to the time the plates were poured, which finally inactivated any residual chlorine present. Plates were counted after incubation for 48 h at 37 C. A sample for testing for residual chlorine was also withdrawn and immediately pipetted into an excess standard of sodium thiosulfate solution. Other samples were withdrawn at specified times after holding the test media in the water bath at 40 C. A statistical analysis of the data was prepared using a general linear models procedure.
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RESULTS AND DISCUSSION
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Several similar experiments were run to determine to what extent chlorine based sanitizers retained antimicrobial activity in the presence of egg solids. The results were generally similar in each experiment and typical results with each of the three sanitizers tested are shown. Results with sodium hypochlorite and sodium dichloro-s-triazine trione, two compounds commonly used as a source of active chlorine in sanitizing rinses in commercial egg washers are summarized in Tables 1 and 2. Salmonella typhimurium was the test organism. The experimental procedures were the same for each compound. The temperature used (40 C) is in the range (40 to 50 C) used in commercial eggwashers. The egg solids concentration of 1.0% is in the range found in egg washers after the washers have been in operation for several hours (Hamm et al, 1974; Harris and Moats, 1975). Although the pH's of the test media were not recorded, both the compounds and egg solids give pH's in the general range 7 to 8 or roughly neutral. The chlorine concentrations (about 270 ppm) were higher than is usually used in commercial egg washing where 50 ppm chlorine is required in the sanitizing rinse (USDA, 1974).
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EGG SOLIDS EFFECT ON SANITIZERS TABLE 4. Comparison of bactericidal effectiveness of chlorinated
Sodium hypochlorite Sodium dichloroisocyanurate Chlorinated alkaline cleaner
Mean logic survivors
15 15 15
3.91 b 3.60° 5.01* .05 according to Duncan's
(log 1.54) were found immediately after inoculation. When bacteria were inoculated immediately after addition of egg, some survived 6 0 min. Residual chlorine was also reduced immediately after addition of egg. When bacteria were inoculated 30 min after addition of egg, the chlorinated cleaner at pH 11 was totally ineffective in killing the test organism even t h o u g h considerable residual chlorine was detectable chemically at up to 60 m i n . N u m bers of bacteria were stable in t h e alkaline control with egg. However, t h e r e was a three log reduction in t h e test organism in t h e egg in water at neutral pH, presumably because of the lysozyme activity of egg (Board, 1 9 7 3 ) . The results indicate t h a t u n d e r the alkaline conditions used t o wash eggs, chlorinated comp o u n d s show little antimicrobial activity once egg solids are introduced into t h e w a s h w a t e r . When all observations for each chlorinated c o m p o u n d were combined (Table 4 ) , t h e alkaline cleaner was f o u n d to be significantly less bactericidal than t h e o t h e r t w o , assuming t h a t t h e response of t h e t w o test organisms t o chlorine was similar. It has been reported t h a t active chlorine is converted t o chloramines in t h e presence of amino groups such as are present in proteins. Chloramines are less bactericidal t h a n o t h e r sources of active chlorine b u t give a chemical test for residual chlorine (White, 1 9 7 2 ) . It seems probable t h a t this is t h e explanation for t h e loss of antimicrobial activity observed in t h e presence of egg. Active chlorine as h y p o chlorite has been f o u n d t o b e less bactericidal at alkaline than at neutral or acid pH's (White, 1972). This would a c c o u n t for t h e further loss of activity observed at p H 1 1 . The persistence of residual chlorine u n d e r actual commercial conditions was d e t e r m i n e d in a plant using chlorinated cleaner. This plant
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Since commercial egg washers c o m m o n l y use alkaline egg cleaning c o m p o u n d s and operate in t h e pH range 10 to 1 1 , t h e experiments were repeated using alkaline egg cleaners t o simulate commercial conditions (Table 3). Since previous studies (Kinner and Moats, 1981) had shown t h a t salmonellae were killed rapidly at 4 0 C at pH 10 or above, an organism, Streptococcus faecalis, which was k n o w n to survive well under these conditions was used. A commercial chlorinated and a c o m m e r cial alkaline egg cleaner were used, b o t h of which gave pH's above 11 when freshly mixed with water. Again, t h e initial inoculum as shown by t h e controls w i t h o u t chlorine was a b o u t 1 0 7 / m l . The chlorinated cleaner proved effective in water, although a few survivors
compounds
Number of observations
a 'b Numbers followed by the same superscript letter are not different at P Multiple Range Test (Duncan, 1955).
bacteria were found immediately after inoculations of t h e sanitizer solutions. When bacteria were inoculated immediately after addition of egg t o t h e sanitizer solutions, some survivors were found in samples withdrawn immediately after inoculation, log 1.45 in sodium h y p o chlorite and log 4.93 in t h e sodium dichloro-striazinetrione. None were found after holding 30 min, however. Addition of egg solids resulted in an immediate reduction in available chlorine t o less than half the initial values. When inoculation of bacteria was delayed 30 m i n after addition of egg, t h e rate of kill was markedly reduced with some survivors present 6 0 min after inoculation. T h e rate of kill was even slower when inoculation was delayed until 60 min after addition of egg. Considerable residual chlorine was detected chemically u p to 6 0 min after addition of t h e egg even t h o u g h its antimicrobial activity was slight at this t i m e . Bacterial n u m b e r s in t h e controls containing egg solids only remained stable. The results with t h e t w o sanitizers were quite similar with t h e same initial level of active chlorine.
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4
3
2
1
a
28 4
6 >1 >1 0
6.17 6.17 6.17 6.30
869 754 711 0
3.52 3.52 3.52 6.56
5.2
949 2
act. (ppm)
2.42
PH
7.26 7.36 7.25 7.20
0 0 0 7.23
0 min1
None detectable after 90 min standing, pH fell to 4:7.
Egg added just prior to addition of bacterial culture and sampling.
Equivalent to 265 ppm Cl 2 .
Samples withdrawn and plated immediately after addition of 2 ml of S. typbimurium
culture in Tryptic-soy br
' Numbers followed by the same superscript letter are not significantly different according to Duncan's multip
Experiment 2 Iodophor in water Iodophor in 1.0% egg solids Immediately after mixing 30 min after mixing 60 min after mixing 1.0% egg in water
Experiment 1 Iodophor in water Iodophor in 1.0% egg solids Immediately after mixing 3 30 min after mixing 60 min after mixing 1.0% egg solids in water
Medium
TABLE 5. Effect of an iodophor sanitizer on Salmonella typbimurium in presence of egg solids at 40 C
m http://ps.oxfordjournals.org/ at New York University on May 18, 2015
EGG SOLIDS EFFECT ON SANITIZERS
The results with the iodophor are interesting since they show that iodine was more stable than chlorine in the presence of egg solids when used on an equimolar basis. The iodophor solutions were strongly acid, however. It would appear technically feasible to maintain bactericidal levels of an iodophor in egg washwater if an acid cleaner were used. However, several questions need to be answered before use of an acid cleaner can be advocated. How rapidly would it be neutralized by calcium carbonate in egg shells? Would egg solids precipitated under acid conditions interfere with washer operation? What would be the cost of using an iodophor? Would cleaning efficiency be improved
over that of alkaline cleaners now in use? The strongly alkaline washwater used in present commercial operation has been demonstrated to be effective in killing many types of bacteria including salmonellae (Kinner and Moats, 1981). This, coupled with proper attention to equipment cleanliness, has been found effective in maintaining low bacterial levels in washwater and on washed eggs without the need for costly sanitizers (Moats, i981). ACKNOWLEDGEMENT
The author thanks J. I. Schultz, Jr., and T. M. Brennan for technical assistance and the Pennwalt Corp., Zep Chemical Co., FMC Corp., and Dubois Chemical Co. for providing samples of chemicals used in this study and Bernard Weinland for preparing the statistical analyses of the data. REFERENCES Atherton, H. V., and J. A. Newlander, 1977. Chemistry and testing of dairy products. 4th ed. Avi Publ. Co., Westport, CT. Board, R. G. 1973. The microbiology of eggs. In Egg science and technology. W. J. Stadleman and O. J. Cotterill, ed. Avi Publ. Co., Westport, CT. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics 11:1—42. Hamm, D., G. K. Searcy, and A. J. Mercuri. 1974. A study of the waste washwater from egg washing machines. Poultry Sci. 53:191-197. Harris, C. E., and W. A. Moats, 1975. Recovery of egg solids from waste-waters of egg-grading and -breaking plants. Poultry Sci. 54:1518-1523. Kinner, J. A., and W. A. Moats, 1981. Effect of temperature, pH, and detergent on survival of bacteria associated with shell eggs. Poultry Sci. 60: 761-767. Moats, W. A., 1981. Factors affecting bacterial loads on shells of washed eggs. Poultry Sci. 60. (In press). Standard Methods for the Examination of Water and Waste Water, 1976. 14th ed. M. C. Rand, A. E. Greenberg, M. J. Taras, and M. A. Franson, ed., 1976. Amer. Publ. Health Ass., Washington, DC. United States Department of Agriculture, Agricultural Marketing Service, 1974. Regulations governing the grading of shell eggs and United States standards, grades and weight classes for shell eggs. (7CFR part 56). White, G. C , 1972. Handbook of chlorination. Von Nostrand Reinhold Co., New York, NY.
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added fresh chlorinated detergent together with supplemental chlorine in the form of bleaching powder at the midpoint of a 4 hr run. Considerable egg solids were evident in the washwater at this time. After the machine had run for 2 min following recharging, a sample of washwater was withdrawn and pipetted into an excess of sodium thiosulfate solution. No residual chlorine was found. Apparently, inactivation of chlorine is more rapid and complete under commercial conditions than in our laboratory experiments. It does not, therefore, appear realistic to attempt to maintain bactericidal levels of chlorine in egg washwaters as washers are now operated. An experiment was also run using an iodophor sanitizer (Table 5). Iodophors are considered to be effective only at acid pH, 5.0 or below (Atherton and Newlander, 1977). When used at the recommended concentration of 25 ppm I 2 , the iodophor was ineffective when egg solids were present. To be equivalent to the chlorine solutions used in the other experiments on an equimolar basis, a much higher concentration, 949 ppm I 2 , was also tested. This gave a strongly acid (pH 3.52) solution, even when egg solids were present. However, the reduction in available iodine in the presence of egg was relatively slow and bacteriological effectiveness was maintained for 60 min. From the rate of reduction of iodine, it probably would have remained effective for several hours longer.
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