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Some Characteristics of Psychrophilic Bacteria Isolated from Green Rotten Eggs
Some Characteristics of Psychrophilic Bacteria Isolated from Green Rotten Eggs C. IMAI
Laboratory of Q.P. Corporation, Sengawa-Cho, Chofu-Shi, Tokyo 182, Japan (Received for publication June 11,1975)
ABSTRACT To investigate the origin of "fishy" odour detected from frozen whole egg products imported from some European companies, 21 strains of psychrophilic bacteria isolated from green rotten eggs were identified, and the behaviour of these bacteria in egg white and yolk were examined. Of 21 identified strains, 19 were presumably Pseudomonas, one was Flavobacterium, and another was Chromobacterium. In egg white these bacteria produced fluorescence, very little volatile basic nitrogen (TVB-N) and trimethylamine nitrogen (TMA-N), and no hydrogen sulfide or putrid odour. In egg yolk these bacteria produced "fishy," sulfuric, or ammoniac odour; hydrogen sulfide; TVB-N and TMA-N; and increased acidity of ether extract. It was assumed that these decomposed products, such as TMA and hydrogen sulfide, caused "fishy" odour in frozen whole egg. Inoculation of a strain to whole egg reproduced a frozen whole egg with "fishy" odour. POULTRY SCIENCE 55: 606-610, 1976
INTRODUCTION N frozen whole egg products imported from a few European factories, "fishy" odour was sometimes detected in spite of the small bacterial count, whereas in frozen egg white produced in the same factories such odour was never detected. The investigation of these factories showed that the shell eggs were not responsible for this odour, but long holding time in the chilling tank due to some accident such as pasteurizer trouble was suspected as the origin of this odour. Haines (1938) showed that typical coliform organisms were found to cause a "fishy" flavour in eggs without producing definite rotting, and Habson-Frohock et al. (1973) showed that trimethylamine carried from the diet caused "fishy" eggs. According to Shimizu (1972), Pseudomonas bacteria, detected frequently in eggs, grow at low temperature, decompose protein and fat, and can be easily destroyed by pasteurization. Nath and Baker (1973) showed that Pseudomonas fluorescens grew rapidly in fresh egg white diluted with water, growth of the bacteria was stimulated by the addition of carbohydrate and ovomucoid-rich egg exudute, and the egg white inoculated with the organism
I
suffered extensive proteolysis. However, little literature concerning the behaviour of these psychrophilic bacteria in egg yolk seems to be published. The purpose of the present study was to investigate the behaviour of psychrophilic bacteria isolated from green rotten eggs, in egg white and yolk, in order to determine the origin of "fishy" odour detected in frozen whole egg, and to roughly identify these bacteria. MATERIALS AND METHODS Ten samples of rotten egg detected in the breaking room when cool stored eggs were processed were brought to the laboratory. After the bacterial counts were measured by plate count agar at 18° C. for 48 hr., 1 to 3 strains of representative colonies picked up from each plate were used for the following examinations. Properties of Bacteria for Identification. Cowan and Steel's methods (1965) were used. Heat Resistance of Bacteria. method (1967) was used.
Frazier's
Behaviour of Bacteria in Egg White and 606
607
PSYCHR0PH1LIC BACTERIA
Yolk. To each 200 g. of egg white and yolk aseptically broken, separated, and taken into a homogenizer cup, 1 ml. of suspension of these bacteria was added and well mixed. Each egg white and yolk were removed into sterilized glass bottles with stoppers, and incubated at 18° C. Periodically the bacterial count and fluorescence (only for egg white) were measured. After incubating for 10 days, pH by glass electrode, production of hydrogen sulfide by lead paper (Cowan and Steel, 1965), TBN-N and TMA-N by microdiff usion method (Conway, 1950), and acidity of ether extract (A.O.A.C., 1970) were determined and odour was observed. Measurement of Fluorescence. Fluorescence of egg white was determined with a HITACHI fluorescence spectrophotometer 204 at an exciting wavelength of 418 m\x. and a measuring wavelength of 465 m\x., using a stock solution of 1% quinine in N sulfuric acid for standardization of the instrument. Reproduction of Frozen Egg with "Fishy" Odour. One ml. of suspension of strain 6-A, which produced strong "fishy" odour and grew at low temperature, was inoculated into 500 g. of aseptically broken and homogenized whole egg and incubated at 10° C.; the bacte-
rial count, TMA-N, and odour were measured. After 48 hr. the whole egg was pasteurized at 60° C. for 4.5 min., frozen at -20° C , and the bacterial count, TMA-N and odour determined. Twenty g. of defrosted whole egg were well mixed with 20 ml. of ether, 20 ml. of saturated KC1 solution, and 40 ml. of 5% trichloroacetic acid, and centrifuged. Then the ether and water layers were filtered. Fluorescence of ether and KC1 extracts were determined by the above method. RESULTS AND DISCUSSION Green rotten eggs detected in the egg processing factory had strong putrid odours such as sulfuric, ammoniac, or "fishy," and bacterial counts of 107 to 10 8 /g. (Table 1). The properties of the bacteria isolated from these rotten eggs are shown in Table 2. It was assumed from the definitions of Cowan and Steel (1965) that there were 19 strains of Pseudomonas,one Flavobacterium(3-A), and one Chromobacterium (3-B). Most of these strains could grow at the low temperature of 0° to 5° C , but the growth rate at 0° C. was considerably delayed in some strains. Sixteen strains grew rapidly at 5° C , so there is a possibility that these bacteria multiply in a chilling tank with difficulty. Heat resistance of these strains (Table 3)
TABLE 1.—Appearance, odour, and bacterial count of green rotten eggs* detected in egg processing factory Sample egg 1 2 3 4 5 6 7 8 9 10
Appearance of yolk
Odour
White White Pink Pink Brown Normal White Pink Normal White
Sulfuric Sulfuric Ammoniac "Fishy" Sulfuric "Fishy" "Fishy" Sulfuric "Fishy" Ammoniac
*Egg whites of all rotten eggs showed visible green.
Bacterial count/g. 8.7 5.0 8.0 2.7 6.3 2.3 9.6 7.8 4.3 2.6
x x x x x x x x x x
107 108 108 108 107 107 106 107 107 108
Symbol of isolates 1-A 2-A and 2-B 3-A and 3-B 4-A and 4-B 5-A and 5-B 6-A, 6-B, and 6-C 7-A and 7-B 8-A and 8-B 9-A and 9-B 10-A, 10-B, and 10-C
608
C. IMAI
TABLE 2.—Properties of isolated strains* Symbol of isolates 1-A 2-A 2-B 3-A 3-B 4-A 4-B 5-A 5-B 6-A 6-B 6-C 7-A 7-B 8-A 8-B 9-A 9-B 10-A 10-B 10-C
Motility + + +
— + + + + + + +
—
+ + + + + + + + +
Gelatin liquefaction + + + +
+ + + + + + + + +
-
+ + + + + + +
Catalase + + + + + + + + + + + + + + + + + + + + +
Oxidase + + + +
Pigment by
Glucose
— + + + + + + + + + + + + + + + +
0 + + + + + + + + + + + +
F -
-
-
+ + + + + + + +
-
t _)_•)*#
G***(+) G (+) Br (+) Br (-) — (+) Br (-) Br (-) — (±) - (±) G (+) Bl (+) G (+) G (+) G (+) Br (-) G (+) Br (-) Br (+) B (+) Br (±)
-
-
Growth temperature °C. 0-37 0-37 0-37 0-37 5-37 0-37 5-37 0-37 0-37 0-37 0-37 15-37 0-42 12-37 5-37 5-37 5-37 5-37 5-37 5-37 5-37
*A11 strains were Gram negative rod, and grew in aerobic condition. **(+): Fluorescence was produced. ***G: Green, Br: Brown, Bl: Blue. was generally weak. It was presumed that, if these bacteria grew in the factory processing, they should be largely destroyed at the stage of pasteurizing. 104 to 10 5 /g. of these
strains inoculated into egg white did not grow so rapidly in the first 4 days, but thereafter grew rapidly to reach 10s to 10 7 /g. after 7 days. This lag time in the first stage seemed
TABLE 3.—Heat resistance of isolated strains" Symbol of isolates 1-A 2-A 2-B 3-A 3-B 4-A 4-B 5-A 5-B 6-A 6-B 6-C 7-A 7-B 8-A 8-B 9-A 9-B 10-A 10-B 10-C
45° C. + + + + + + + + + + + + + + + + + + + + +
50° C. + + + + + + + + + + + + + +
-
+ + + + + +
Heating temperature 55° C. 60° C. + —
—
—
—
—
—
— — — — —
+ + +
+ + +
+ + + +
— + + + + +
65° C.
70° C.
+ + + +
+
— — + +
— — +
*In pH 7.0 buffer solution, heating time: 4.5 min., bacterial number in buffer solution: 105-106/ml.
609
PSYCHROPHILIC BACTERIA
> Bacterial count of strain 6-A ' B a c t e r i a l count of strain 9-A -oFluorescence of strain 6-*Fluorescence of strain 9-
30
d> 7
20 «
$ 6
o
u
10
o 5 _i
A
2 4 6 8 Days of i ncubation
10
FIG. 1. Bacterial counts and fluorescences of egg white inoculated with representative strains.
to be caused by lysozyme as shown in the report of Gardner and Nikoopour (1969). Eighteen strains identified with Pseudomonas and 1 strain of Flavobacterium gave fluorescence to egg white. In the speed of fluorescence production each strain showed a little difference. Generally, fluorescence was recognized when the bacterial counts reached 10 5 /g., and after that increased with great force. It was 4 to 10 days after inoculating that visible green colour in egg white was recognized. Variations of bacterial count and fluorescence of egg white inoculated with two representative strains (6-A and 9-A) are shown in Fig. 1. All bacteria inoculated into yolk grew rapidly without lag time, and the bacterial counts reached to 105 to 10 7 /g. after incubating for 2 days, and to 107 to 10Vg. after 4 days. The properties of egg white and yolk incubated for 10 days are shown
TABLE 4.—Properties of egg white and yolk inoculated with each isolate and incubated for W days* Egg yolk Symbol of isolates 1-A 2-A 2-B 3-A 3-B 4-A 4-B 5-A 5-B 6-A 6-B 6-C 7-A 7-B 8-A 8-B 9-A 9-B 10-A 10-B 10-C Control***
Egg white TVB-N mg.
TMA-N
mg.
pH
%
7c
pH
8.8 8.6 8.6 8.6 8.7 8.6 8.7 8.7 8.6 8.6 8.6 8.8 8.6 8.6 8.7 8.6 8.6 8.7 8.6 8.6 8.6 8.9
0 0 0 0 0 0 0 0 0 0 0 0 0 0 22 0 0 0 4 3 0 0
1.47 0.00 0.00 0.00 0.27 0.00 1.14 0.00 1.44 0.00 0.00 0.00 1.26 1.44 0.00 0.72 0.82 1.01 0.00 0.73 0.00 0.00
6.0 5.8 6.2 6.4 6.0 5.7 6.1 6.2 6.3 6.2 5.8 5.8 6.0 6.4 5.8 6.4 6.3 6.4 6.4 6.7 5.6 5.6
Hydrogen sulfide + + + + + + + + + + + + + +
-
+ + + + + +
-
%
%
Acidity of ether extract
88 38 44 25 29 37 36 44 40 68 22 28 30 38 34 46 71 96 93 45 21 8
2.34 2.07 1.44 1.17 1.36 15.18 1.53 2.52 4.13 6.83 1.08 2.14 2.34 2.34 1.53 2.07 3.43 3.23 6.90 3.68 4.32 0.00
3.18** 7.46 3.09 4.03 3.26 3.18 2.61 1.48 3.56 3.28 5.13 3.19 3.49 5.33 1.83 3.51 3.36 4.18 3.42 2.60 5.32 1.32
TVB-N mg.
TMA-N mg.
*Hydrogen sulfide and putrid odour were not detected from any samples of egg white. **ml. of 0.05 N sodium ethylate per 1 g. of extract. ***Not inoculated.
Odour "Fishy" "Fishy" "Fishy" "Fishy" Sulfuric "Fishy" Sulfuric Sulfuric "Fishy" "Fishy" Sulfuric "Fishy" "Fishy" Moldy Moldy "Fishy" "Fishy" "Fishy" Ammoniac "Fishy" "Fishy"
—
610
C. IMAI
in Table 4. In egg white there was no production of putrid odour and hydrogen sulfide, little decrease of p H , and little production of T M A - N . Most strains did not produce T V B - N . In yolk, strong putrid odours such as " f i s h y , " moldy, and a m m o n i a c developed; p H increased little; and the production of hydrogen sulfide, T V B - N , and T M A - N were recognized. Acidity of ether extract w a s considerably increased by lipase action of these bacteria. As the result of inoculation of whole egg with strain 6-A, formation of T M A - N and " f i s h y " odour in whole egg w a s recognized after 36 to 48 hr. at 10° C. (Fig. 2). After pasteurizing at 60° C. for 4.5 min. and freezing at - 2 0 ° C , the bacterial count was decreased to less than 1 0 / g . ; but the content of T M A - N hardly decreased (1.38 m g . % ) , and
Bacterial
count 2.0
TMA-N
1.5
en
E 1.0
0.5
12 24 36 Hrs. of i n c u b a t i o n
48
FIG. 2. Bacterial count and TMA-N in whole egg inoculated with strain 6-A.
the " f i s h y " o d o u r was well retained. T h e fluorescences of KCl and ether extracts were 3.1 (control 2.0) and 0.3 (control 0.3), respectively, and the differences between sample and control were not marked. Steffen et al. (1943) showed that variable amounts of free fatty acid in commercial egg p o w d e r s presumably indicated b r e a k d o w n prior to drying. A s a method of investigation for the origin of " f i s h y " o d o u r caused by psychrophilic bacteria, it is assumed that determinations of T M A - N in whole egg and acidity of ether extract should be significant. REFERENCES Association of Official Analytical Chemists, 1970. Official Methods of Analysis of Association of Official Analytical Chemists, 11th ed. Association of Official Analytical Chemists, Washington, D.C. Cowan, S. T., and K. J. Steel, 1965. Manual for the Identification of Medical Bacteria. The University Press, Cambridge, UK. Conway, E. J., 1950. Microdiffusion Analysis and Volumetric Error. Crosby Lockwood and Son Ltd., London. Frazier, W. C , 1967. Food Microbiology, 2nd ed. McGraw-Hill Book Company, New York. Gardner, F. A., and H. Nikoopour, 1969. Growth characteristics of Pseudomonas fluorescens on conalbumin and lysozyme substrates. Poultry Sci. 48: 43-48. Habson-Frohock, A., D. G. Land, N. M. Griffiths and R. F. Curtis, 1973. Egg-taint: Association with trimethylamine. Nature, 243: 304-305. Haines, R. B., 1938. Observation on the bacterial flora of the hen's egg with a description of a new species of Proteus and Pseudomonas causing rots in eggs. J. Hyg. 38: 338-355. Nath, K. R., and R. C. Baker, 1973. Factors affecting the growth of Pseudomonas fluorescens in liquid egg white. Appl. Microbiol. 25: 442-446. Shimizu, U., 1972. Shokuhin Eiseigaku Jiten. Ishiyaku Shuppan Co. Ltd., Tokyo. Steffen, A. H., E. W. Hopkins, R. W. Kline and G. H. Wetzel, 1943. A chemical method for scoring dried whole egg. U.S. Egg Poultry Mag. 49: 308310, 334-336.
AUGUST 4-6, 1976. NUTRITION AND DRUG INTERRELATIONS INTERNATIONAL SYMPOSIUM, NUTRITION FOUNDATION AND THE IOWA STATE UNIVERSITY NUTRITION SCIENCES COUNCIL, IOWA STATE UNIVERSITY, AMES, IOWA
Laboratory of Q.P. Corporation, Sengawa-Cho, Chofu-Shi, Tokyo 182, Japan (Received for publication June 11,1975)
ABSTRACT To investigate the origin of "fishy" odour detected from frozen whole egg products imported from some European companies, 21 strains of psychrophilic bacteria isolated from green rotten eggs were identified, and the behaviour of these bacteria in egg white and yolk were examined. Of 21 identified strains, 19 were presumably Pseudomonas, one was Flavobacterium, and another was Chromobacterium. In egg white these bacteria produced fluorescence, very little volatile basic nitrogen (TVB-N) and trimethylamine nitrogen (TMA-N), and no hydrogen sulfide or putrid odour. In egg yolk these bacteria produced "fishy," sulfuric, or ammoniac odour; hydrogen sulfide; TVB-N and TMA-N; and increased acidity of ether extract. It was assumed that these decomposed products, such as TMA and hydrogen sulfide, caused "fishy" odour in frozen whole egg. Inoculation of a strain to whole egg reproduced a frozen whole egg with "fishy" odour. POULTRY SCIENCE 55: 606-610, 1976
INTRODUCTION N frozen whole egg products imported from a few European factories, "fishy" odour was sometimes detected in spite of the small bacterial count, whereas in frozen egg white produced in the same factories such odour was never detected. The investigation of these factories showed that the shell eggs were not responsible for this odour, but long holding time in the chilling tank due to some accident such as pasteurizer trouble was suspected as the origin of this odour. Haines (1938) showed that typical coliform organisms were found to cause a "fishy" flavour in eggs without producing definite rotting, and Habson-Frohock et al. (1973) showed that trimethylamine carried from the diet caused "fishy" eggs. According to Shimizu (1972), Pseudomonas bacteria, detected frequently in eggs, grow at low temperature, decompose protein and fat, and can be easily destroyed by pasteurization. Nath and Baker (1973) showed that Pseudomonas fluorescens grew rapidly in fresh egg white diluted with water, growth of the bacteria was stimulated by the addition of carbohydrate and ovomucoid-rich egg exudute, and the egg white inoculated with the organism
I
suffered extensive proteolysis. However, little literature concerning the behaviour of these psychrophilic bacteria in egg yolk seems to be published. The purpose of the present study was to investigate the behaviour of psychrophilic bacteria isolated from green rotten eggs, in egg white and yolk, in order to determine the origin of "fishy" odour detected in frozen whole egg, and to roughly identify these bacteria. MATERIALS AND METHODS Ten samples of rotten egg detected in the breaking room when cool stored eggs were processed were brought to the laboratory. After the bacterial counts were measured by plate count agar at 18° C. for 48 hr., 1 to 3 strains of representative colonies picked up from each plate were used for the following examinations. Properties of Bacteria for Identification. Cowan and Steel's methods (1965) were used. Heat Resistance of Bacteria. method (1967) was used.
Frazier's
Behaviour of Bacteria in Egg White and 606
607
PSYCHR0PH1LIC BACTERIA
Yolk. To each 200 g. of egg white and yolk aseptically broken, separated, and taken into a homogenizer cup, 1 ml. of suspension of these bacteria was added and well mixed. Each egg white and yolk were removed into sterilized glass bottles with stoppers, and incubated at 18° C. Periodically the bacterial count and fluorescence (only for egg white) were measured. After incubating for 10 days, pH by glass electrode, production of hydrogen sulfide by lead paper (Cowan and Steel, 1965), TBN-N and TMA-N by microdiff usion method (Conway, 1950), and acidity of ether extract (A.O.A.C., 1970) were determined and odour was observed. Measurement of Fluorescence. Fluorescence of egg white was determined with a HITACHI fluorescence spectrophotometer 204 at an exciting wavelength of 418 m\x. and a measuring wavelength of 465 m\x., using a stock solution of 1% quinine in N sulfuric acid for standardization of the instrument. Reproduction of Frozen Egg with "Fishy" Odour. One ml. of suspension of strain 6-A, which produced strong "fishy" odour and grew at low temperature, was inoculated into 500 g. of aseptically broken and homogenized whole egg and incubated at 10° C.; the bacte-
rial count, TMA-N, and odour were measured. After 48 hr. the whole egg was pasteurized at 60° C. for 4.5 min., frozen at -20° C , and the bacterial count, TMA-N and odour determined. Twenty g. of defrosted whole egg were well mixed with 20 ml. of ether, 20 ml. of saturated KC1 solution, and 40 ml. of 5% trichloroacetic acid, and centrifuged. Then the ether and water layers were filtered. Fluorescence of ether and KC1 extracts were determined by the above method. RESULTS AND DISCUSSION Green rotten eggs detected in the egg processing factory had strong putrid odours such as sulfuric, ammoniac, or "fishy," and bacterial counts of 107 to 10 8 /g. (Table 1). The properties of the bacteria isolated from these rotten eggs are shown in Table 2. It was assumed from the definitions of Cowan and Steel (1965) that there were 19 strains of Pseudomonas,one Flavobacterium(3-A), and one Chromobacterium (3-B). Most of these strains could grow at the low temperature of 0° to 5° C , but the growth rate at 0° C. was considerably delayed in some strains. Sixteen strains grew rapidly at 5° C , so there is a possibility that these bacteria multiply in a chilling tank with difficulty. Heat resistance of these strains (Table 3)
TABLE 1.—Appearance, odour, and bacterial count of green rotten eggs* detected in egg processing factory Sample egg 1 2 3 4 5 6 7 8 9 10
Appearance of yolk
Odour
White White Pink Pink Brown Normal White Pink Normal White
Sulfuric Sulfuric Ammoniac "Fishy" Sulfuric "Fishy" "Fishy" Sulfuric "Fishy" Ammoniac
*Egg whites of all rotten eggs showed visible green.
Bacterial count/g. 8.7 5.0 8.0 2.7 6.3 2.3 9.6 7.8 4.3 2.6
x x x x x x x x x x
107 108 108 108 107 107 106 107 107 108
Symbol of isolates 1-A 2-A and 2-B 3-A and 3-B 4-A and 4-B 5-A and 5-B 6-A, 6-B, and 6-C 7-A and 7-B 8-A and 8-B 9-A and 9-B 10-A, 10-B, and 10-C
608
C. IMAI
TABLE 2.—Properties of isolated strains* Symbol of isolates 1-A 2-A 2-B 3-A 3-B 4-A 4-B 5-A 5-B 6-A 6-B 6-C 7-A 7-B 8-A 8-B 9-A 9-B 10-A 10-B 10-C
Motility + + +
— + + + + + + +
—
+ + + + + + + + +
Gelatin liquefaction + + + +
+ + + + + + + + +
-
+ + + + + + +
Catalase + + + + + + + + + + + + + + + + + + + + +
Oxidase + + + +
Pigment by
Glucose
— + + + + + + + + + + + + + + + +
0 + + + + + + + + + + + +
F -
-
-
+ + + + + + + +
-
t _)_•)*#
G***(+) G (+) Br (+) Br (-) — (+) Br (-) Br (-) — (±) - (±) G (+) Bl (+) G (+) G (+) G (+) Br (-) G (+) Br (-) Br (+) B (+) Br (±)
-
-
Growth temperature °C. 0-37 0-37 0-37 0-37 5-37 0-37 5-37 0-37 0-37 0-37 0-37 15-37 0-42 12-37 5-37 5-37 5-37 5-37 5-37 5-37 5-37
*A11 strains were Gram negative rod, and grew in aerobic condition. **(+): Fluorescence was produced. ***G: Green, Br: Brown, Bl: Blue. was generally weak. It was presumed that, if these bacteria grew in the factory processing, they should be largely destroyed at the stage of pasteurizing. 104 to 10 5 /g. of these
strains inoculated into egg white did not grow so rapidly in the first 4 days, but thereafter grew rapidly to reach 10s to 10 7 /g. after 7 days. This lag time in the first stage seemed
TABLE 3.—Heat resistance of isolated strains" Symbol of isolates 1-A 2-A 2-B 3-A 3-B 4-A 4-B 5-A 5-B 6-A 6-B 6-C 7-A 7-B 8-A 8-B 9-A 9-B 10-A 10-B 10-C
45° C. + + + + + + + + + + + + + + + + + + + + +
50° C. + + + + + + + + + + + + + +
-
+ + + + + +
Heating temperature 55° C. 60° C. + —
—
—
—
—
—
— — — — —
+ + +
+ + +
+ + + +
— + + + + +
65° C.
70° C.
+ + + +
+
— — + +
— — +
*In pH 7.0 buffer solution, heating time: 4.5 min., bacterial number in buffer solution: 105-106/ml.
609
PSYCHROPHILIC BACTERIA
> Bacterial count of strain 6-A ' B a c t e r i a l count of strain 9-A -oFluorescence of strain 6-*Fluorescence of strain 9-
30
d> 7
20 «
$ 6
o
u
10
o 5 _i
A
2 4 6 8 Days of i ncubation
10
FIG. 1. Bacterial counts and fluorescences of egg white inoculated with representative strains.
to be caused by lysozyme as shown in the report of Gardner and Nikoopour (1969). Eighteen strains identified with Pseudomonas and 1 strain of Flavobacterium gave fluorescence to egg white. In the speed of fluorescence production each strain showed a little difference. Generally, fluorescence was recognized when the bacterial counts reached 10 5 /g., and after that increased with great force. It was 4 to 10 days after inoculating that visible green colour in egg white was recognized. Variations of bacterial count and fluorescence of egg white inoculated with two representative strains (6-A and 9-A) are shown in Fig. 1. All bacteria inoculated into yolk grew rapidly without lag time, and the bacterial counts reached to 105 to 10 7 /g. after incubating for 2 days, and to 107 to 10Vg. after 4 days. The properties of egg white and yolk incubated for 10 days are shown
TABLE 4.—Properties of egg white and yolk inoculated with each isolate and incubated for W days* Egg yolk Symbol of isolates 1-A 2-A 2-B 3-A 3-B 4-A 4-B 5-A 5-B 6-A 6-B 6-C 7-A 7-B 8-A 8-B 9-A 9-B 10-A 10-B 10-C Control***
Egg white TVB-N mg.
TMA-N
mg.
pH
%
7c
pH
8.8 8.6 8.6 8.6 8.7 8.6 8.7 8.7 8.6 8.6 8.6 8.8 8.6 8.6 8.7 8.6 8.6 8.7 8.6 8.6 8.6 8.9
0 0 0 0 0 0 0 0 0 0 0 0 0 0 22 0 0 0 4 3 0 0
1.47 0.00 0.00 0.00 0.27 0.00 1.14 0.00 1.44 0.00 0.00 0.00 1.26 1.44 0.00 0.72 0.82 1.01 0.00 0.73 0.00 0.00
6.0 5.8 6.2 6.4 6.0 5.7 6.1 6.2 6.3 6.2 5.8 5.8 6.0 6.4 5.8 6.4 6.3 6.4 6.4 6.7 5.6 5.6
Hydrogen sulfide + + + + + + + + + + + + + +
-
+ + + + + +
-
%
%
Acidity of ether extract
88 38 44 25 29 37 36 44 40 68 22 28 30 38 34 46 71 96 93 45 21 8
2.34 2.07 1.44 1.17 1.36 15.18 1.53 2.52 4.13 6.83 1.08 2.14 2.34 2.34 1.53 2.07 3.43 3.23 6.90 3.68 4.32 0.00
3.18** 7.46 3.09 4.03 3.26 3.18 2.61 1.48 3.56 3.28 5.13 3.19 3.49 5.33 1.83 3.51 3.36 4.18 3.42 2.60 5.32 1.32
TVB-N mg.
TMA-N mg.
*Hydrogen sulfide and putrid odour were not detected from any samples of egg white. **ml. of 0.05 N sodium ethylate per 1 g. of extract. ***Not inoculated.
Odour "Fishy" "Fishy" "Fishy" "Fishy" Sulfuric "Fishy" Sulfuric Sulfuric "Fishy" "Fishy" Sulfuric "Fishy" "Fishy" Moldy Moldy "Fishy" "Fishy" "Fishy" Ammoniac "Fishy" "Fishy"
—
610
C. IMAI
in Table 4. In egg white there was no production of putrid odour and hydrogen sulfide, little decrease of p H , and little production of T M A - N . Most strains did not produce T V B - N . In yolk, strong putrid odours such as " f i s h y , " moldy, and a m m o n i a c developed; p H increased little; and the production of hydrogen sulfide, T V B - N , and T M A - N were recognized. Acidity of ether extract w a s considerably increased by lipase action of these bacteria. As the result of inoculation of whole egg with strain 6-A, formation of T M A - N and " f i s h y " odour in whole egg w a s recognized after 36 to 48 hr. at 10° C. (Fig. 2). After pasteurizing at 60° C. for 4.5 min. and freezing at - 2 0 ° C , the bacterial count was decreased to less than 1 0 / g . ; but the content of T M A - N hardly decreased (1.38 m g . % ) , and
Bacterial
count 2.0
TMA-N
1.5
en
E 1.0
0.5
12 24 36 Hrs. of i n c u b a t i o n
48
FIG. 2. Bacterial count and TMA-N in whole egg inoculated with strain 6-A.
the " f i s h y " o d o u r was well retained. T h e fluorescences of KCl and ether extracts were 3.1 (control 2.0) and 0.3 (control 0.3), respectively, and the differences between sample and control were not marked. Steffen et al. (1943) showed that variable amounts of free fatty acid in commercial egg p o w d e r s presumably indicated b r e a k d o w n prior to drying. A s a method of investigation for the origin of " f i s h y " o d o u r caused by psychrophilic bacteria, it is assumed that determinations of T M A - N in whole egg and acidity of ether extract should be significant. REFERENCES Association of Official Analytical Chemists, 1970. Official Methods of Analysis of Association of Official Analytical Chemists, 11th ed. Association of Official Analytical Chemists, Washington, D.C. Cowan, S. T., and K. J. Steel, 1965. Manual for the Identification of Medical Bacteria. The University Press, Cambridge, UK. Conway, E. J., 1950. Microdiffusion Analysis and Volumetric Error. Crosby Lockwood and Son Ltd., London. Frazier, W. C , 1967. Food Microbiology, 2nd ed. McGraw-Hill Book Company, New York. Gardner, F. A., and H. Nikoopour, 1969. Growth characteristics of Pseudomonas fluorescens on conalbumin and lysozyme substrates. Poultry Sci. 48: 43-48. Habson-Frohock, A., D. G. Land, N. M. Griffiths and R. F. Curtis, 1973. Egg-taint: Association with trimethylamine. Nature, 243: 304-305. Haines, R. B., 1938. Observation on the bacterial flora of the hen's egg with a description of a new species of Proteus and Pseudomonas causing rots in eggs. J. Hyg. 38: 338-355. Nath, K. R., and R. C. Baker, 1973. Factors affecting the growth of Pseudomonas fluorescens in liquid egg white. Appl. Microbiol. 25: 442-446. Shimizu, U., 1972. Shokuhin Eiseigaku Jiten. Ishiyaku Shuppan Co. Ltd., Tokyo. Steffen, A. H., E. W. Hopkins, R. W. Kline and G. H. Wetzel, 1943. A chemical method for scoring dried whole egg. U.S. Egg Poultry Mag. 49: 308310, 334-336.
AUGUST 4-6, 1976. NUTRITION AND DRUG INTERRELATIONS INTERNATIONAL SYMPOSIUM, NUTRITION FOUNDATION AND THE IOWA STATE UNIVERSITY NUTRITION SCIENCES COUNCIL, IOWA STATE UNIVERSITY, AMES, IOWA