- Email: [email protected]
Behavior of Enterococci in Egg Processing Operations
Behavior of Enterococci in Egg Processing Operations c. IMAI Laboratory of Q. P. Corporation, Sengawa-Cho, Chofu-Shi, Tokyo 182, Japan (Received for publication July 7, 1979)
INTRODUCTION Enterococci, as well as coliforms, have been suggested as indicators of fecal contamination of foods. Several papers on die presence and growth of enterococci in eggs and egg p r o d u c t s have been published. Bhargava et al. ( 1 9 7 5 ) studied the microflora of the shell surface of eggs and reported t h a t enterococci were less c o m m o n than coliforms and staphylococci. Nowicki (1976) reported t h a t enterococci were detected in frozen, pasteurized, b r o k e n - o u t whole eggs, and Konecny ( 1 9 7 1 ) stated t h a t the growth of enterococci in unpasteurized eggs was rapid at b o t h r o o m t e m p e r a t u r e and refrigerator t e m p e r a t u r e . Gravani et al. ( 1 9 7 2 ) studied the growth of Streptococcus faecalis var. liquefaciens in egg white and yolk at 25 and 3 2 C; Dabbah et al. ( 1 9 7 1 ) reported t h a t small n u m b e r s of S. faecalis remained after the pasteurization of whole egg at 6 0 C for 180 m i n ; Kraft et al. ( 1 9 6 7 ) studied the growth of enterococci in yolk at I O C . Ozawa (1951a,b) detected enterococci in naturally fermented dried egg white and studied the fermentation of egg white with S. faecalis. However, little literature concerning the presence of enterococci in dried or frozen egg p r o d u c t s and their behavior in egg processing operations seems t o be published. The purpose of the present study was to isolate enterococci from dried and frozen egg p r o d u c t s , to pursue their origin, and
to investigate their behavior in egg processing operations. MATERIALS AND METHODS Eggs and Egg Products. Dried and frozen egg p r o d u c t s (whole egg and egg yolk) of d o m e s t i c and foreign origin were furnished by an egg processing c o m p a n y in Japan. Unwashed and washed shell eggs were gathered from several parts of J a p a n and were used for e x p e r i m e n t s within 2 days after arriving at the l a b o r a t o r y . Isolation and Identification of Enterococci from Eggs and Egg Products. The azide esculin agar (AE agar) m e t h o d (Horie et al, 1 9 7 4 ) was used for the detection of enterococci from eggs and egg p r o d u c t s . Acid formation from mannitol, sorbitol, arabinose, glycerol, melezitose, and melibiose, liquefaction of gelatin, requirem e n t of folic acid, reduction of tetrazolium, and resistance to .04% tellurite were used for the identification of enterococci (Horie et al., 1 9 7 7 ) . The enterococci on the shell surface of eggs were measured by the swabbing m e t h o d , using moistened sterilized c o t t o n . In egg p r o d u c t s , total bacteria counts with standard plate agar and coliform counts with desoxycholate agar were measured for comparison. Decrease of Enterococci with Disinfectants. Cationic surfactant (Vantocil IB, Ueno Seiyaku Co., Ltd., Osaka), quaternary a m m o n i u m salts (Egg Clean, T a m u r a Seiyaku Co., Ltd., T o k y o ) ,
1767
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
ABSTRACT Since enterococci were detected in dried and frozen egg products (whole egg and egg yolk), the origin of the enterococci and their behavior in the different stages of egg processing were surveyed. The bacterial survey of unwashed and washed eggs, gathered from several parts of Japan, showed the presence of an average of 60 enterococci per egg on the shell surface of unwashed eggs. Smaller numbers of enterococci were detected on the shell surface of washed eggs. Most of the detected enterococci were Streptococcus faecalis and S. faecalis var. liquefaciens. The contents of two eggs were contaminated with enterococci when 120 washed eggs were examined. Enterococci were destroyed to some extent by the disinfectants used for washing the shell eggs, but they seemed to have greater resistance than Escherichia coli and Pseudomonas fluorescens used for comparison. In whole egg and egg yolk, enterococci grew rapidly at 25 C, slowly at 10 C, and very slowly at 2.5 C. The frozen storage of both whole egg and egg yolk at —20 C for 3 months only slightly decreased the number of enterococci. They were decreased only slightly by normal pasteurization but were destroyed in the desugaring process by the glucose oxidase method. Enterococci numbers were only slightly decreased by spray-drying whole egg and yolk. Considering these characteristics of enterococci, it appears to be difficult to produce enterococci-free egg products in a normal production line. (Key words: chlorine, chlorine dioxide, chiller water, poultry, shell-life, broilers) 1980 Poultry Science 59:1767-1772
1768
IMAI
Growth of Enterococci in Whole Egg and Yolk. One milliliter each of the suspensions of S. faecalis, S. faecalis var. liquefaciens, S.
T A B L E 1. Enterococci
faecium, and S. durans isolated from shell surfaces was added to 200 g each of whole egg and yolk aseptically prepared and mixed well. The whole eggs and yolks were divided into 4 portions and were stored at 25, 10, 2.5, and —20 C. The enterococci counts were periodically measured with yeast glucose agar. Decrease of Enterococci by Pasteurization. Whole eggs and yolks, inoculated with the above mentioned enterococci, E. coli, and P. fluorescens, were prepared so that the number of each bacterium might be approximately 10 6 /g. These egg preparations were then pasteurized in test tubes at 60, 62, 64, and 66 C for 3.5 min. The bacterial counts before and after pasteurization were measured using the same procedure as above. Behavior of Enterococci in Desugaring and Spray-Drying Processes. Four kilograms each of whole egg and yolk were aseptically prepared and inoculated with 1 ml each of the suspensions of S. faecalis (2.3X 10 /ml), S. faecalis var. liquefaciens (1.7X 10 /ml), S. faecium (3.4xl0 8 /ml), and S. durans (2.6x 10 8 /ml). One kilogram each of the whole egg and yolk was desugared by the glucose oxidase method (Ayres, 1958), and the enterococci counts in the desugaring process were measured with yeast glucose agar. The remaining whole egg and yolk, before desugaring, were dried in a Niro spray-dryer with a 12,000 rpm atomizer at 150 C (inlet air), and the enterococci counts of the dried products were measured.
in •whole egg and egg yolk
products Enterococci
Product
Origin
Dried Whole egg Whole egg Whole egg Whole egg Egg y o l k Egg y o l k Egg y o l k
Israel New Zealand Denmark China US Denmark China
<10 1X10 <10 <10 2.6X102 3X10 8X10
Japan
1.3X10 2 9X10 1.0X10 2 7.0X102 1.4X10 2
Frozena Whole egg Whole egg Yolk with 10% salt Yolk with 10% salt Yolk with 20% sugar
Australia US Japan Canada
Count/g
All the frozen products were pasteurized before freezing.
Identification
Tested strains S. faecalis S. faecalis var. Irregular t y p e
Tested strains S. faecalis S. faecalis var. S. faecium Irregular t y p e
liquefaciens
liquefaciens
26 50% 35% 15%
42 83% 7% 3% 7%
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
nonionic surfactant (Linda E-100, Yokohama Yushi Co., Ltd., Yokohama), benzalkonium chloride (Osuban, Takeda Seiyaku Co., Ltd., Osaka), and sodium hypochlorite (Riko, Riko Kyosan Co., Ltd., Tokyo) were used for this experiment at two levels of concentration. One milliliter each of suspensions of S. faecalis, S. faecalis var. liquefaciens, S. faecium, and S. durans isolated from egg shells, Escherichia coli (ATCC 10356), and Pseudomonas fluorescens (IFO 3081) was inoculated into 100 ml each of disinfectant solutions and mixed well. After standing for 60 sec, 1 ml each of these solutions was added to 9 ml of sterilized peptone buffer solution, and bacterial counts were measured with yeast glucose agar (polypeptone 1%, yeast extract .5%, glucose .5%, NaCl .5%, agar 1.5%, pH 7.0). For the disinfection of the shell surface of eggs, 20 liters of each disinfectant solution were added to a water bath with a shaking apparatus, and the temperature of the solution was controlled at 30 C. Forty unwashed eggs were placed in a metal cage, and the cage was submerged in the solution. After shaking for 5 min, the eggs were taken out and rinsed in running water. The surface of the eggs were swabbed with moistened sterilized cotton, and enterococci counts were measured with AE agar and total bacteria counts with standard plate agar.
BEHAVIOR OF ENTEROCOCCI
1769
TABLE 2. Enterococci on the shells of unwashed and washed eggs Enterococci Count/egg Egg producer
Minimum
Washed G H I
J K L
<20 2.8X102 4X10 <20 2X10 <20
3.1X10 2 1.0X10 4 6.3X103 1.0X10 2 1.4X10 3 1.8X10 2
4X10 9.3X102 6.0X102 2X10 1.7X10 2 <20
<20 <20 <20 <20 <20 <20
9X10 <20 1.4X10 2 <20 <20 <20
<20 <20 <20 <20 <20 <20
Identification
Tested strains S. faecalis S. faecalis var. S. faecium S. durans Irregular t y p e
Tested strains S. faecalis S. faecalis var.
liquefaciens
liquefaciens
167 41% 42% 4% 5% 8%
16 50% 50%
The average means logarithmic average.
RESULTS AND DISCUSSION Table 1 shows t h e presence of enterococci in whole egg and y o l k p r o d u c t s . In these products, no coliforms were detected (less than 10/g) and total bacteria counts ranged from 70/g to 2.5X 10 /g. F e w enterococci were detected in dried whole eggs, b u t they were detected t o some e x t e n t in t h e other products, while coliforms were absent and t h e bacterial counts were low. Dried whole eggs e x p o r t e d t o J a p a n are usually desugared before drying, but dried yolks are n o t . T h e egg processing c o m p a n y t h a t furnished t h e samples of egg p r o d u c t s t o t h e
laboratory imports only desugared p r o d u c t s as dried whole egg. It was supposed t h a t the absence of enterococci in dried whole eggs was caused by the disinfectant action of hydrogen p e r o x i d e as an oxygen source for the oxidation of glucose. Most of t h e isolated enterococci were S. faecalis and S. faecalis var. liquefaciens. Table 2 shows the distribution of enterococci on the shells of 360 unwashed eggs and 360 washed eggs. A m u c h larger c o u n t of enterococci was detected on the unwashed eggs t h a n on the washed eggs. Most of t h e detected e n t e r o c o c c i were S. faecalis and S. faecalis var.
TABLE 3. Effect of disinfectants on enterococci and gram negative bacteria Bacteria c o u n t / m l Concentrationa Disinfectant Control Cationic surfactant Quaternary a m m o n i u m salts Nonionic surfactant Benzalkonium chloride Sodium h y p o c h l o r i t e
s.
(%)
S. faecalis
faecalis var. liquefaciens
S. faecium
S. durans
E. coli
p. fluorescens
.2 .02
6.3X106 <10 1.4X10 4
8.8X105 1.0X10 2 4.0X102
7.7X105 <10 <10
3.1X105 <10 <10
2.0X106 <10 <10
6.9X 1 0 s <10 <10
.1 .01 .1 .01 .3 .03 .02 .002
<10 <10 <10 4.2X10" <10 <10 <10 <10
3.2X103 2.0X103 <10 6.7X103 <10 <10 <10 <10
2.1X102 2.2X103 <10 <10 <10 <10 <10 7.1X103
6.8X103 7.9X103 1X10 4.2X103 <10 <10 <10 <10
3X10 <10 <10 4X10 <10 <10 <10 <10
<10 3X10 <10 <10 <10 <10 <10 <10
The larger concentration of each disinfectant is recommended by its producer.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
Unwashed A B C D E F
Average 1
Maximum
1770
IMAI TABLE 4. Effect of disinfectants on total bacteria and enterococci on the surface of egg shells Enterococci/egg
Total bacteria/egg Disinfectant
Minimum
Maximum
Average 1
Minimum
Control
1.3X10 6
7.2X106
3.8X10 6
1X10
3X10
2.5X102
1.3X10 2
<10
.1% Quaternary a m m o n i u m salts
1.3X10"
2.2X105
6.9X104
<10
.1% Anionic surfactant
1.6X10 4
3.1X10"
2.5X10"
<10
<10
<10
.3% Benzalkonium chloride
1X10
3.4X10 2
8X10
<10
<10
<10
.02% Sodium hypochlorite
4X10
9.4X103
6.3X102
<10
<10
<10
.2% Cationic surfactant
Average
7.8X102
1.1X10 2
<10
<10
3X10
<10
The average means logarithmic average.
liquefaciens. Small percentages of S. faecium and S. durans were detected on the unwashed eggs. When the contents of 120 washed eggs were examined, enterococci were found in only two eggs. In these two eggs, the enterococci counts were 6 . 5 x l 0 3 / g and 20/g, and they were mainly S. faecalis var. Hqurfaciens (62%) and S. faecalis (3 3%). Table 3 shows the effect of disinfectants on enterococci, E. coli, and P. fluorescens. Benzalkonium chloride was the most effective of five kinds of disinfectants, and the next was sodium hypochlorite. Other disinfectants, which are used for cleaning shell eggs, were effective to some extent. In this experiment, it was found that enterococci had greater resistance to disinfectants than E. coli and P. fluorescens. This tendency was in agreement with the previous report on the resistance of group D streptococci and E. coli to chorination (Deibel, 1964). Table 4 shows the effect of disinfectants on total bacteria and enterococci on the shell surface of eggs. Quaternary ammonium salts and nonionic surfactant were not very effective in reducing total bacteria and enterococci counts. The other three kinds of disinfactant showed strong action on these bacteria. In this experiment, eggs were submerged in disinfectant solutions for 5 min. In egg processing plants, however, a shorter time is used for disinfecting eggs, so it was thought that the presence of enterococci on the shell surface of washed eggs was not uncommon. Figure 1 shows the increase of enterococci in
whole egg at 25 and 10 C. All the enterococci grew rapidly at 25 C but slowly at 10 C. The number of enterococci remained practically unchanged at 2.5 C after 7 days, and they decreased slightly at —20 C after 3 months. Figure 2 shows the increase of enterococci in egg yolk at 25 and 10 C. These enterococci grew more rapidly in egg yolk than in whole egg. At 2.5 C, S. faecalis showed a slight increase, but others did not show any increase or decrease for a week. At —20 C, all the enterococci showed only a slight decrease after 3 months. Figure 3 shows the decrease of enterococci in the desugaring process of whole egg and yolk by the glucose oxidase method. The enterococci disappeared in this process after 5 to 6 hr. It was supposed that this extinction
• S faecalis
12 24 Hours of storage
var liquefaciens
*—*
S.duran.
48 Days of storage
FIG. 1. Increase of enterococci in whole egg at 25 and IOC.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
1
Maximum
BEHAVIOR OF ENTEROCOCCI • S . faecalis * ' S faecium ' S. faecalis var. liquefaciens *---* S. durans
D
1771
• - • W h o [e egg
»v
— Egg yolk S N
\
\ •Sv
s
* \ ^ •
FIG 2. Increase of enterococci in egg yolk at 25 and 10 C.
0 was caused b y t h e disinfectant action of h y d r o gen peroxide added into egg c o m p o n e n t s as an oxygen source for t h e oxidation of glucose. This finding explains the absence of entercocci in the samples of commercially dried whole eggs in Table 1. Table 5 shows t h e effect of pasteurization on enterococci, E. coli, and P. fluorescens in whole egg and yolk. E. coli and P. fluorescens were destroyed a t 6 0 C for 3.5 min, b u t t h e pasteurization at 64 C for 3.5 min was n o t effective against enterococci. Enterococci were destroyed at 66 C for 3.5 min. However, at this t e m p e r a t u r e there is a slight d e n a t u r a t i o n of egg proteins and, consequently, pasteurization at
1
%
2 3 A 5 Hours of desugaring
6
FIG 3. Decrease of enterococci in desugaring process of whole egg and yolk by the glucose oxidase method.
6 6 C c a n n o t be r e c o m m e n d e d for commercial operation. T h e enterococci ( 2 . 5 x l 0 5 / g ) a d d e d i n t o whole egg and egg yolk were decreased to 1 . 8 x l 0 5 / g in dried whole egg and 1 . 0 x l O s / g in dried egg yolk after spray-drying at 1 5 0 C. Based on this result, it was assumed that it was difficult to destroy enterococci by spray-drying
TABLE 5. Effect of pasteurization on enterococci and gram negative bacteria in whole egg and egg yolk Bacteria count/g Species of bacteria Whole egg S. faecalis S. faecalis var. liquefaciens S. faecium S. durans E. coli P. fluorescens Egg y o l k S. faecalis S. faecalis var. liquefaciens S. faecium S. durans E. coli P. fluorescens
Pasteurizing temperature (C)
Before pasteurization
60
62
64
2.8X106
3.1X10 5
1.2X104
1.6X102
6
3.0X10 3.2X10 6 3.0X106 2.7X106 5.2X105
5
7.2X10 4.2X103 8.3X103 <10 <10
5
2.1X10 4.1X102 2.0X102 <10 <10
5.1X10 4X10 <10 <10 <10
3
2.3X106
2.3X10 5
2.1X10 4
1.9X10"
3.0X106 2.7X106 2.6X106 2.9X106 5.4X105
5.8X105 5.2X10 5 3.2X10 S <10 <10
5.7X105 1.7X10 5 3.6X10 4 <10 <10
2.5X104 <10 <10 <10 <10
66
<10 <10 <10 <10 <10 <10
4X10 <10 <10 <10 <10 <10
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
%
1772
IMAI
REFERENCES Ayres, J. C , 1958. Methods for depleting glucose from egg albumen before drying. Food Technol. 12:186-189. Bhargava, D. N., U. Mandokhot, R. S. Panghal, K. G. Naryan, and 1. P. Singh, 1975. Quantitative and qualitative microflora on the poultry egg shell surface, Indian J. Poultry Sci. 10:19-23. Dabbah, R., W. A. Moats, and V. M. Edwards, 1971. Survivor curves of selected Salmonella enteritidis serotypes in liquid whole egg homogenates at 60
C. Poultry Sci. 50:1772-1776. Deibel, R. H., 1964. The group D. streptococci. Bacteriol. Rev. 28:330-366. Gravani, R. B., J. P. Preziose, and D. V. Vadehra, 1972. The effect of the growth of Streptococcus faecalis var. liquefaciens on egg yolk. Poultry Sci. 51:1812-1813. Horie, S., H. Ando, and M. Abe, 1977. Distribution of enterococci species in commercial frozen foods.J. Food Hyg. Soc. Japan. 1 8 : 3 9 - 4 3 . Horie, S., S. Sato, T. Morita, H. Inoue, T. Izumi, and M. Yamagata, 1974. Azide esculin agar plate method for determination of enterococci in frozen foods. J. Food Hyg. Soc. Japan. 15: 105-109. Konecny, S., 1971. Study of microbial and chemical indices in non-pasteurized egg contents. Hidinarsky Priemysel. 13:175-186. Kraft, A. A., J. C. Ayres, R. H. Forsythe, and J. R. Schultz, 1967. Keeping quality of pasteurized liquid egg yolk. Poultry Sci. 46:1282. Nowicki, L., 1976. Extent of bacterial contamination of pasteurized broken-out whole eggs. Medycyna Weteryarjna. 32:275-277. Ozawa, Y., 1951a. Chemical and bacteriological studies on the egg white products. II. Streptococci of the fermenting egg white (Streptococci isolated from dried egg albumen, fresh egg white, egg shells, hen's feces and its intestinal tracts). Nat. Inst. Agr. Sci. (Japan). Series G, (2):19-25. Ozawa, Y., 1951b. Chemical and bacteriological studies on the egg white products. III. The resistance and the fermentability of various streptococci and other saprogenous bacteria in the fresh egg white. Nat. Inst. Agr. Sci. (Japan). Series G , ( 2 ) : 2 7 - 3 1 .
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
at this temperature. As dried whole egg and yolk are not heat treated under dry circumstances, the enterococci contained in raw materials will survive in final products unless the materials are desugared by the glucose oxidase method. If enterococci are utilized as indicators of fecal contamination in the future, very severe control will be needed in the processing operations of frozen and dried egg products. This is because enterococci exist widely on the shell surface of eggs, remain sometimes in egg contents, have stronger resistance to disinfectants and pasteurization than gram negative bacteria such as E. coli and P. fluorescens, grow rapidly in whole egg and yolk at room temperature, and are not destroyed by freezing or spraydrying.
INTRODUCTION Enterococci, as well as coliforms, have been suggested as indicators of fecal contamination of foods. Several papers on die presence and growth of enterococci in eggs and egg p r o d u c t s have been published. Bhargava et al. ( 1 9 7 5 ) studied the microflora of the shell surface of eggs and reported t h a t enterococci were less c o m m o n than coliforms and staphylococci. Nowicki (1976) reported t h a t enterococci were detected in frozen, pasteurized, b r o k e n - o u t whole eggs, and Konecny ( 1 9 7 1 ) stated t h a t the growth of enterococci in unpasteurized eggs was rapid at b o t h r o o m t e m p e r a t u r e and refrigerator t e m p e r a t u r e . Gravani et al. ( 1 9 7 2 ) studied the growth of Streptococcus faecalis var. liquefaciens in egg white and yolk at 25 and 3 2 C; Dabbah et al. ( 1 9 7 1 ) reported t h a t small n u m b e r s of S. faecalis remained after the pasteurization of whole egg at 6 0 C for 180 m i n ; Kraft et al. ( 1 9 6 7 ) studied the growth of enterococci in yolk at I O C . Ozawa (1951a,b) detected enterococci in naturally fermented dried egg white and studied the fermentation of egg white with S. faecalis. However, little literature concerning the presence of enterococci in dried or frozen egg p r o d u c t s and their behavior in egg processing operations seems t o be published. The purpose of the present study was to isolate enterococci from dried and frozen egg p r o d u c t s , to pursue their origin, and
to investigate their behavior in egg processing operations. MATERIALS AND METHODS Eggs and Egg Products. Dried and frozen egg p r o d u c t s (whole egg and egg yolk) of d o m e s t i c and foreign origin were furnished by an egg processing c o m p a n y in Japan. Unwashed and washed shell eggs were gathered from several parts of J a p a n and were used for e x p e r i m e n t s within 2 days after arriving at the l a b o r a t o r y . Isolation and Identification of Enterococci from Eggs and Egg Products. The azide esculin agar (AE agar) m e t h o d (Horie et al, 1 9 7 4 ) was used for the detection of enterococci from eggs and egg p r o d u c t s . Acid formation from mannitol, sorbitol, arabinose, glycerol, melezitose, and melibiose, liquefaction of gelatin, requirem e n t of folic acid, reduction of tetrazolium, and resistance to .04% tellurite were used for the identification of enterococci (Horie et al., 1 9 7 7 ) . The enterococci on the shell surface of eggs were measured by the swabbing m e t h o d , using moistened sterilized c o t t o n . In egg p r o d u c t s , total bacteria counts with standard plate agar and coliform counts with desoxycholate agar were measured for comparison. Decrease of Enterococci with Disinfectants. Cationic surfactant (Vantocil IB, Ueno Seiyaku Co., Ltd., Osaka), quaternary a m m o n i u m salts (Egg Clean, T a m u r a Seiyaku Co., Ltd., T o k y o ) ,
1767
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
ABSTRACT Since enterococci were detected in dried and frozen egg products (whole egg and egg yolk), the origin of the enterococci and their behavior in the different stages of egg processing were surveyed. The bacterial survey of unwashed and washed eggs, gathered from several parts of Japan, showed the presence of an average of 60 enterococci per egg on the shell surface of unwashed eggs. Smaller numbers of enterococci were detected on the shell surface of washed eggs. Most of the detected enterococci were Streptococcus faecalis and S. faecalis var. liquefaciens. The contents of two eggs were contaminated with enterococci when 120 washed eggs were examined. Enterococci were destroyed to some extent by the disinfectants used for washing the shell eggs, but they seemed to have greater resistance than Escherichia coli and Pseudomonas fluorescens used for comparison. In whole egg and egg yolk, enterococci grew rapidly at 25 C, slowly at 10 C, and very slowly at 2.5 C. The frozen storage of both whole egg and egg yolk at —20 C for 3 months only slightly decreased the number of enterococci. They were decreased only slightly by normal pasteurization but were destroyed in the desugaring process by the glucose oxidase method. Enterococci numbers were only slightly decreased by spray-drying whole egg and yolk. Considering these characteristics of enterococci, it appears to be difficult to produce enterococci-free egg products in a normal production line. (Key words: chlorine, chlorine dioxide, chiller water, poultry, shell-life, broilers) 1980 Poultry Science 59:1767-1772
1768
IMAI
Growth of Enterococci in Whole Egg and Yolk. One milliliter each of the suspensions of S. faecalis, S. faecalis var. liquefaciens, S.
T A B L E 1. Enterococci
faecium, and S. durans isolated from shell surfaces was added to 200 g each of whole egg and yolk aseptically prepared and mixed well. The whole eggs and yolks were divided into 4 portions and were stored at 25, 10, 2.5, and —20 C. The enterococci counts were periodically measured with yeast glucose agar. Decrease of Enterococci by Pasteurization. Whole eggs and yolks, inoculated with the above mentioned enterococci, E. coli, and P. fluorescens, were prepared so that the number of each bacterium might be approximately 10 6 /g. These egg preparations were then pasteurized in test tubes at 60, 62, 64, and 66 C for 3.5 min. The bacterial counts before and after pasteurization were measured using the same procedure as above. Behavior of Enterococci in Desugaring and Spray-Drying Processes. Four kilograms each of whole egg and yolk were aseptically prepared and inoculated with 1 ml each of the suspensions of S. faecalis (2.3X 10 /ml), S. faecalis var. liquefaciens (1.7X 10 /ml), S. faecium (3.4xl0 8 /ml), and S. durans (2.6x 10 8 /ml). One kilogram each of the whole egg and yolk was desugared by the glucose oxidase method (Ayres, 1958), and the enterococci counts in the desugaring process were measured with yeast glucose agar. The remaining whole egg and yolk, before desugaring, were dried in a Niro spray-dryer with a 12,000 rpm atomizer at 150 C (inlet air), and the enterococci counts of the dried products were measured.
in •whole egg and egg yolk
products Enterococci
Product
Origin
Dried Whole egg Whole egg Whole egg Whole egg Egg y o l k Egg y o l k Egg y o l k
Israel New Zealand Denmark China US Denmark China
<10 1X10 <10 <10 2.6X102 3X10 8X10
Japan
1.3X10 2 9X10 1.0X10 2 7.0X102 1.4X10 2
Frozena Whole egg Whole egg Yolk with 10% salt Yolk with 10% salt Yolk with 20% sugar
Australia US Japan Canada
Count/g
All the frozen products were pasteurized before freezing.
Identification
Tested strains S. faecalis S. faecalis var. Irregular t y p e
Tested strains S. faecalis S. faecalis var. S. faecium Irregular t y p e
liquefaciens
liquefaciens
26 50% 35% 15%
42 83% 7% 3% 7%
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
nonionic surfactant (Linda E-100, Yokohama Yushi Co., Ltd., Yokohama), benzalkonium chloride (Osuban, Takeda Seiyaku Co., Ltd., Osaka), and sodium hypochlorite (Riko, Riko Kyosan Co., Ltd., Tokyo) were used for this experiment at two levels of concentration. One milliliter each of suspensions of S. faecalis, S. faecalis var. liquefaciens, S. faecium, and S. durans isolated from egg shells, Escherichia coli (ATCC 10356), and Pseudomonas fluorescens (IFO 3081) was inoculated into 100 ml each of disinfectant solutions and mixed well. After standing for 60 sec, 1 ml each of these solutions was added to 9 ml of sterilized peptone buffer solution, and bacterial counts were measured with yeast glucose agar (polypeptone 1%, yeast extract .5%, glucose .5%, NaCl .5%, agar 1.5%, pH 7.0). For the disinfection of the shell surface of eggs, 20 liters of each disinfectant solution were added to a water bath with a shaking apparatus, and the temperature of the solution was controlled at 30 C. Forty unwashed eggs were placed in a metal cage, and the cage was submerged in the solution. After shaking for 5 min, the eggs were taken out and rinsed in running water. The surface of the eggs were swabbed with moistened sterilized cotton, and enterococci counts were measured with AE agar and total bacteria counts with standard plate agar.
BEHAVIOR OF ENTEROCOCCI
1769
TABLE 2. Enterococci on the shells of unwashed and washed eggs Enterococci Count/egg Egg producer
Minimum
Washed G H I
J K L
<20 2.8X102 4X10 <20 2X10 <20
3.1X10 2 1.0X10 4 6.3X103 1.0X10 2 1.4X10 3 1.8X10 2
4X10 9.3X102 6.0X102 2X10 1.7X10 2 <20
<20 <20 <20 <20 <20 <20
9X10 <20 1.4X10 2 <20 <20 <20
<20 <20 <20 <20 <20 <20
Identification
Tested strains S. faecalis S. faecalis var. S. faecium S. durans Irregular t y p e
Tested strains S. faecalis S. faecalis var.
liquefaciens
liquefaciens
167 41% 42% 4% 5% 8%
16 50% 50%
The average means logarithmic average.
RESULTS AND DISCUSSION Table 1 shows t h e presence of enterococci in whole egg and y o l k p r o d u c t s . In these products, no coliforms were detected (less than 10/g) and total bacteria counts ranged from 70/g to 2.5X 10 /g. F e w enterococci were detected in dried whole eggs, b u t they were detected t o some e x t e n t in t h e other products, while coliforms were absent and t h e bacterial counts were low. Dried whole eggs e x p o r t e d t o J a p a n are usually desugared before drying, but dried yolks are n o t . T h e egg processing c o m p a n y t h a t furnished t h e samples of egg p r o d u c t s t o t h e
laboratory imports only desugared p r o d u c t s as dried whole egg. It was supposed t h a t the absence of enterococci in dried whole eggs was caused by the disinfectant action of hydrogen p e r o x i d e as an oxygen source for the oxidation of glucose. Most of t h e isolated enterococci were S. faecalis and S. faecalis var. liquefaciens. Table 2 shows the distribution of enterococci on the shells of 360 unwashed eggs and 360 washed eggs. A m u c h larger c o u n t of enterococci was detected on the unwashed eggs t h a n on the washed eggs. Most of t h e detected e n t e r o c o c c i were S. faecalis and S. faecalis var.
TABLE 3. Effect of disinfectants on enterococci and gram negative bacteria Bacteria c o u n t / m l Concentrationa Disinfectant Control Cationic surfactant Quaternary a m m o n i u m salts Nonionic surfactant Benzalkonium chloride Sodium h y p o c h l o r i t e
s.
(%)
S. faecalis
faecalis var. liquefaciens
S. faecium
S. durans
E. coli
p. fluorescens
.2 .02
6.3X106 <10 1.4X10 4
8.8X105 1.0X10 2 4.0X102
7.7X105 <10 <10
3.1X105 <10 <10
2.0X106 <10 <10
6.9X 1 0 s <10 <10
.1 .01 .1 .01 .3 .03 .02 .002
<10 <10 <10 4.2X10" <10 <10 <10 <10
3.2X103 2.0X103 <10 6.7X103 <10 <10 <10 <10
2.1X102 2.2X103 <10 <10 <10 <10 <10 7.1X103
6.8X103 7.9X103 1X10 4.2X103 <10 <10 <10 <10
3X10 <10 <10 4X10 <10 <10 <10 <10
<10 3X10 <10 <10 <10 <10 <10 <10
The larger concentration of each disinfectant is recommended by its producer.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
Unwashed A B C D E F
Average 1
Maximum
1770
IMAI TABLE 4. Effect of disinfectants on total bacteria and enterococci on the surface of egg shells Enterococci/egg
Total bacteria/egg Disinfectant
Minimum
Maximum
Average 1
Minimum
Control
1.3X10 6
7.2X106
3.8X10 6
1X10
3X10
2.5X102
1.3X10 2
<10
.1% Quaternary a m m o n i u m salts
1.3X10"
2.2X105
6.9X104
<10
.1% Anionic surfactant
1.6X10 4
3.1X10"
2.5X10"
<10
<10
<10
.3% Benzalkonium chloride
1X10
3.4X10 2
8X10
<10
<10
<10
.02% Sodium hypochlorite
4X10
9.4X103
6.3X102
<10
<10
<10
.2% Cationic surfactant
Average
7.8X102
1.1X10 2
<10
<10
3X10
<10
The average means logarithmic average.
liquefaciens. Small percentages of S. faecium and S. durans were detected on the unwashed eggs. When the contents of 120 washed eggs were examined, enterococci were found in only two eggs. In these two eggs, the enterococci counts were 6 . 5 x l 0 3 / g and 20/g, and they were mainly S. faecalis var. Hqurfaciens (62%) and S. faecalis (3 3%). Table 3 shows the effect of disinfectants on enterococci, E. coli, and P. fluorescens. Benzalkonium chloride was the most effective of five kinds of disinfectants, and the next was sodium hypochlorite. Other disinfectants, which are used for cleaning shell eggs, were effective to some extent. In this experiment, it was found that enterococci had greater resistance to disinfectants than E. coli and P. fluorescens. This tendency was in agreement with the previous report on the resistance of group D streptococci and E. coli to chorination (Deibel, 1964). Table 4 shows the effect of disinfectants on total bacteria and enterococci on the shell surface of eggs. Quaternary ammonium salts and nonionic surfactant were not very effective in reducing total bacteria and enterococci counts. The other three kinds of disinfactant showed strong action on these bacteria. In this experiment, eggs were submerged in disinfectant solutions for 5 min. In egg processing plants, however, a shorter time is used for disinfecting eggs, so it was thought that the presence of enterococci on the shell surface of washed eggs was not uncommon. Figure 1 shows the increase of enterococci in
whole egg at 25 and 10 C. All the enterococci grew rapidly at 25 C but slowly at 10 C. The number of enterococci remained practically unchanged at 2.5 C after 7 days, and they decreased slightly at —20 C after 3 months. Figure 2 shows the increase of enterococci in egg yolk at 25 and 10 C. These enterococci grew more rapidly in egg yolk than in whole egg. At 2.5 C, S. faecalis showed a slight increase, but others did not show any increase or decrease for a week. At —20 C, all the enterococci showed only a slight decrease after 3 months. Figure 3 shows the decrease of enterococci in the desugaring process of whole egg and yolk by the glucose oxidase method. The enterococci disappeared in this process after 5 to 6 hr. It was supposed that this extinction
• S faecalis
12 24 Hours of storage
var liquefaciens
*—*
S.duran.
48 Days of storage
FIG. 1. Increase of enterococci in whole egg at 25 and IOC.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
1
Maximum
BEHAVIOR OF ENTEROCOCCI • S . faecalis * ' S faecium ' S. faecalis var. liquefaciens *---* S. durans
D
1771
• - • W h o [e egg
»v
— Egg yolk S N
\
\ •Sv
s
* \ ^ •
FIG 2. Increase of enterococci in egg yolk at 25 and 10 C.
0 was caused b y t h e disinfectant action of h y d r o gen peroxide added into egg c o m p o n e n t s as an oxygen source for t h e oxidation of glucose. This finding explains the absence of entercocci in the samples of commercially dried whole eggs in Table 1. Table 5 shows t h e effect of pasteurization on enterococci, E. coli, and P. fluorescens in whole egg and yolk. E. coli and P. fluorescens were destroyed a t 6 0 C for 3.5 min, b u t t h e pasteurization at 64 C for 3.5 min was n o t effective against enterococci. Enterococci were destroyed at 66 C for 3.5 min. However, at this t e m p e r a t u r e there is a slight d e n a t u r a t i o n of egg proteins and, consequently, pasteurization at
1
%
2 3 A 5 Hours of desugaring
6
FIG 3. Decrease of enterococci in desugaring process of whole egg and yolk by the glucose oxidase method.
6 6 C c a n n o t be r e c o m m e n d e d for commercial operation. T h e enterococci ( 2 . 5 x l 0 5 / g ) a d d e d i n t o whole egg and egg yolk were decreased to 1 . 8 x l 0 5 / g in dried whole egg and 1 . 0 x l O s / g in dried egg yolk after spray-drying at 1 5 0 C. Based on this result, it was assumed that it was difficult to destroy enterococci by spray-drying
TABLE 5. Effect of pasteurization on enterococci and gram negative bacteria in whole egg and egg yolk Bacteria count/g Species of bacteria Whole egg S. faecalis S. faecalis var. liquefaciens S. faecium S. durans E. coli P. fluorescens Egg y o l k S. faecalis S. faecalis var. liquefaciens S. faecium S. durans E. coli P. fluorescens
Pasteurizing temperature (C)
Before pasteurization
60
62
64
2.8X106
3.1X10 5
1.2X104
1.6X102
6
3.0X10 3.2X10 6 3.0X106 2.7X106 5.2X105
5
7.2X10 4.2X103 8.3X103 <10 <10
5
2.1X10 4.1X102 2.0X102 <10 <10
5.1X10 4X10 <10 <10 <10
3
2.3X106
2.3X10 5
2.1X10 4
1.9X10"
3.0X106 2.7X106 2.6X106 2.9X106 5.4X105
5.8X105 5.2X10 5 3.2X10 S <10 <10
5.7X105 1.7X10 5 3.6X10 4 <10 <10
2.5X104 <10 <10 <10 <10
66
<10 <10 <10 <10 <10 <10
4X10 <10 <10 <10 <10 <10
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
%
1772
IMAI
REFERENCES Ayres, J. C , 1958. Methods for depleting glucose from egg albumen before drying. Food Technol. 12:186-189. Bhargava, D. N., U. Mandokhot, R. S. Panghal, K. G. Naryan, and 1. P. Singh, 1975. Quantitative and qualitative microflora on the poultry egg shell surface, Indian J. Poultry Sci. 10:19-23. Dabbah, R., W. A. Moats, and V. M. Edwards, 1971. Survivor curves of selected Salmonella enteritidis serotypes in liquid whole egg homogenates at 60
C. Poultry Sci. 50:1772-1776. Deibel, R. H., 1964. The group D. streptococci. Bacteriol. Rev. 28:330-366. Gravani, R. B., J. P. Preziose, and D. V. Vadehra, 1972. The effect of the growth of Streptococcus faecalis var. liquefaciens on egg yolk. Poultry Sci. 51:1812-1813. Horie, S., H. Ando, and M. Abe, 1977. Distribution of enterococci species in commercial frozen foods.J. Food Hyg. Soc. Japan. 1 8 : 3 9 - 4 3 . Horie, S., S. Sato, T. Morita, H. Inoue, T. Izumi, and M. Yamagata, 1974. Azide esculin agar plate method for determination of enterococci in frozen foods. J. Food Hyg. Soc. Japan. 15: 105-109. Konecny, S., 1971. Study of microbial and chemical indices in non-pasteurized egg contents. Hidinarsky Priemysel. 13:175-186. Kraft, A. A., J. C. Ayres, R. H. Forsythe, and J. R. Schultz, 1967. Keeping quality of pasteurized liquid egg yolk. Poultry Sci. 46:1282. Nowicki, L., 1976. Extent of bacterial contamination of pasteurized broken-out whole eggs. Medycyna Weteryarjna. 32:275-277. Ozawa, Y., 1951a. Chemical and bacteriological studies on the egg white products. II. Streptococci of the fermenting egg white (Streptococci isolated from dried egg albumen, fresh egg white, egg shells, hen's feces and its intestinal tracts). Nat. Inst. Agr. Sci. (Japan). Series G, (2):19-25. Ozawa, Y., 1951b. Chemical and bacteriological studies on the egg white products. III. The resistance and the fermentability of various streptococci and other saprogenous bacteria in the fresh egg white. Nat. Inst. Agr. Sci. (Japan). Series G , ( 2 ) : 2 7 - 3 1 .
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
at this temperature. As dried whole egg and yolk are not heat treated under dry circumstances, the enterococci contained in raw materials will survive in final products unless the materials are desugared by the glucose oxidase method. If enterococci are utilized as indicators of fecal contamination in the future, very severe control will be needed in the processing operations of frozen and dried egg products. This is because enterococci exist widely on the shell surface of eggs, remain sometimes in egg contents, have stronger resistance to disinfectants and pasteurization than gram negative bacteria such as E. coli and P. fluorescens, grow rapidly in whole egg and yolk at room temperature, and are not destroyed by freezing or spraydrying.