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Physicochemical Changes Produced in Egg Yolk by Streptococcus faecalis var. liquefaciens
Physico chemical Changes Produced in Egg Yolk by Streptococcus faecalis var. liquefaciens ROBERT B. GRAVANI, DHARAM V. VADEHRA,1 and ROBERT C. BAKER Institute of Food Science, Cornell University, Ithaca, New York 14853 (Received for publication August 19, 1982)
1984 Poultry Science 63:654-660
INTRODUCTION
Freshly laid eggs are generally devoid of bacteria (Brooks and Taylor, 1955; Frazier and Westhoff, 1978). However, on exposure to environmental onslaughts, eggs became contaminated by a variety of microorganisms that pose spoilage and public health problems. Extensive work has been conducted on various aspects of microbial spoilage of eggs including a) the mechanism of penetration of microorganisms into the egg interior (Lifshitz and Baker, 1964; Lifshitz et al, 1964; Board, 1965a; Brown et al, 1965; Vadehra et al, 1970); b) antimicrobial properties of egg white proteins (Board, 1966, 1968, 1973; Yadav and Vadehra, 1977), c) the types of bacteria present in eggs (Board, 1965b, 1968, 1973 ; Frazier and Westhoff, 1978; Shafi etal, 1970); and d) control of microbial spoilage of shell and liquid eggs (Board, 1968, 1973; Frazier and Westhoff, 1978). However, not much work has been done on either the growth pattern or the changes produced by microorganisms in egg yolk. Gravani et al (1972), Gravani (1975), and Vadehra and Burley (1978) reported that Streptococcus faecalis var. liquefaciens and other organisms, when grown in egg yolk,
1 Department of Microbiology, Panjab University, Chandigarh, India.
caused separation of the homogeneous yolk into an upper layer that was clear and oily and a cloudy, viscous lower layer. This study was undertaken to determine the growth characteristics of S. faecalis var. liquefaciens in egg yolk and the compositional differences between the two layers produced from such growth. MATERIALS AND METHODS Egg Yolk Eggs less than 24 hr old from a single strain of White Leghorn hens were used throughout this study. Egg yolk was aseptically separated from albumen and transferred to sterile widemouth jars. The sterility of the egg yolk was determined using the standard plate count procedure (American Public Health Association, 1976) and by incubating uninoculated yolk at 25 C. Inoculation and Incubation S. faecalis var. liquefaciens was procured from the culture collection of the Microbiology Department at Cornell University. The organism was grown at 37 C for 18 to 24 hrin trypticase soy broth. Prior to inoculation, the broth culture was diluted with sterile water and it constituted the inoculum. The quantity of the diluted broth was varied to give an initial count of 5 X 10 3 to 1 X 10 4 cfu/ml of the
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ABSTRACT The growth of Streptococcus faecalis var. liquefaciens in sterile liquid egg yolk for 48 hr at 25 C resulted in the separation of the homogeneous yolk into two components, which appeared as layers. The upper layer is clear, oily, and has a higher amount of lipids and cholesterol as compared to the viscous lower layer, which contains greater quantities of protein, phosphorus, calcium, and iron. This separation is related to the production of an extracellular factor, probably a protease, which because of limited proteolysis causes destabilization of the egg yolk system. This destabilization results in, or is due to, aggregation of the granules, which because of size, weight, and compositional differences, settled to constitute the lower layer. Evidently, these granules are responsible for opacity, as the yolk devoid of this settled material is clear and makes up the upper layer. (Key words: egg yolk, separation factor, Streptococcus faecalis, physicochemical changes)
CHANGES IN YOLK BY STREPTOCOCCUS FAECALIS yolk. The inoculated egg yolk was incubated for 5 or 10 days at 25 C in sterile wide-mouth jars. Viable Count and pH
Preparation of Cell-Free Supernatant Two-liter flasks containing 1 liter of trypticase soy broth with .2 M phosphate buffer (pH 7.1) were inoculated with 10 ml of an overnight culture of Streptococcus faecalis var. liquefaciens and incubated for 18 hr at 37 C on a reciprocating shaker (80 rpm). The cell-free supernatant (CFS) was prepared by centrifugation of the cultures at 16,300 X g for 20 min at 4C. Enzyme Assays Proteolytic activity in the CFS was determined by modifying the method of Somkuti and Babel (1969). One-tenth milliliter of the test solution was added to 3 ml of a 1% skim milk powder solution in .05 M phosphate buffer (pH 7.45). The reaction mixture was thoroughly mixed and incubated at 37 C for 20 min. After adding an equal volume of 24% trichloroacetic acid (TCA), the tubes were mixed and allowed to stand at room temperature (25 ± 2 C) for 20 min. The precipitate was removed by centrifugation at 2,000 rpm for 5 min. The solution was then filtered through Whatman No. 41 filter paper and the absorbance determined at 280 nm. An absorbance of .01, under these experimental conditions, was defined as one "proteolytic unit." Lipolytic activity was determined by using the method described by Worthington (1972). Separation of Upper and Lower Layers For separation at the end of the incubation, the upper clear, oily layer was decanted and then aspirated while the lower, viscous layer was left in the jar. The separated layers were used for chemical analyses. Chemical Analysis Total protein. The micro-Kjeldahl procedure (Association of Official Analytical Chemists, 1970) and the modified Folin-Ciocalteu procedure (Lowry et al, 1951) were used.
Nonprotein nitrogen. The test sample (1.0 g) was added to 5.0 ml of 10% TCA, mixed, and centrifuged at 2,000 rpm for 20 min in a clinical centrifuge at room temperature. The centrifugate was filtered through Whatman No. 41 filter paper. The filtrate was neutralized with equal amounts of .625 M NaOH and the nonprotein nitrogen (NPN) was determined by the modified Folin-Ciocalteu procedure (Lowry et al., 1951) and quantitated using tyrosine as the standard. Lipids. The yolk samples were freeze dried in a Stokes freeze drier (Model No. 902241, Pennsalt Chemical Co., Philadelphia, PA) and extracted with a chloroform-methanol (2:1) solvent system using a Goldfisch fat extraction apparatus (Labconco Corp. of Kansas City, MO). Cholesterol. The procedure of LiebermannBurchard (Stadtman, 1957) was used. The test sample was diluted with water to give .1 to .5 mg of cholesterol, deproteinized with ethanolacetone (1:1), and analyzed for cholesterol. Ash and Mineral Analysis. The yolk samples were dry ashed at 600 C in a muffle furnace (Barber Coleman Model No. 293, Rockford, IL). The ash was dissolved in .1 N HCl and mineral content was analyzed by a photoelectric spectrophotometer (Applied Research Co., Model 2900, Dearborn, MI). Chromatographic
Analysis
Carboxymethyl cellulose (BioRad Labs, Richmond, CA) was used for the chromatography according to the procedure of Seideman etal. (1969). Ultracentrifugal
Studies
Whole yolk, upper and lower layers, was weighed into cellulose nitrate tubes and centrifuged at 81,500 X g in a swinging bucket rotor at 7 C for 10 hr (Model L2, Spinco Division, Beckman Instrument Co., Palo Alto, CA). RESULTS AND DISCUSSION Streptococcus faecalis var. liquefaciens grew extremely well in egg yolk and within 12 hr of incubation at 25 C the count reached 2.6 x 10 7 cfu/ml from an initial count of 8.9 X 10 3 cfu/ml (Table 1). The count after 36 hr was 4.2 X 10 9 cfu/ml. There was no decline in the viable count during the remaining incubation period (360 hr), indicating that the egg yolk is a good growth and maintenance medium for
Downloaded from http://ps.oxfordjournals.org/ at Rutgers University Libraries/Technical Services on June 1, 2015
The viable count of each yolk sample was determined using standard methods (American Public Health Association, 1976). The pH was measured at selected intervals by aseptically drawing a sample from the incubating egg yolk.
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TABLE 1. Viable count, pH, and visual changes produced by S. faecalis var. liquefaciens in egg yolk when incubated at 25 C for 360 hr Incubation time (hr)
Count
pH
Visual observations
6.10 5.60 5.60 5.60 5.60 5.60 5.58 5.60 5.60 5.60 5.60
Resembles stored control Resembles stored control Resembles stored control Resembles stored control Yolk beginning to separate into two layers Yolk separated into upper translucent and lower opaque layers Layers well-defined Layers well-defined Layers well-defined Layers well-defined Upper layer became opaque
(cfu/ml) 8.9 X 10 3 2.6 X 10 7 3.4 X 10' 4.2X10' 4.0 X 10 9 4.0 X 10 9 3.9 X 10 9 3.6X10' 3.2 X 10' 2.7X10' 2.6 X 10'
this organism. No drastic changes in pH were observed and this was due to the lack of ferm e n t a b l e c a r b o h y d r a t e s in egg yolk, because t h e organism otherwise ferments c a r b o h y d r a t e s t o p r o d u c e acid. T h e separation of egg yolk i n t o t w o layers was a b r u p t to start and was first noticed after 4 8 hr of g r o w t h . O n c e t h e separation was initiated, it was c o m p l e t e within 2 4 hr. T h e visual changes in the yolk samples inoculated with S. faecalis var. liquefaciens were c o m p a r e d with t h e u n i n o c u l a t e d fresh and incubated controls (Fig. 1). T h e p h o t o g r a p h s clearly show the separation of yolk into t w o layers. T h e e x t e n t of separation was m o n i t o r e d b y determining t h e a m o u n t of t h e u p p e r layer as a percentage of t h e total yolk. These determinations showed t h a t at t h e start of separation (48 hr), the u p p e r layer was a p p r o x i m a t e l y 10% of t h e total y o l k ; however, this figure increased t o 4 5 % in 72 hr and 59% in 5 days. Incubation b e y o n d this p r o d u c e d very limited settling, as t h e percentage of u p p e r layer after 10 days increased b y 4%. After t h e initiation, the separation is merely a gravitational p h e n o m e n o n , as the t w o layers could be separated by centrifugation much more quickly. Chemical analyses of t h e two layers s h o w e d t h a t t h e u p p e r layer c o n t a i n e d 1.5 times m o r e lipids and cholesterol while t h e lower layer had a b o u t twice the a m o u n t of protein and ash (Table 2). These differences in c o m p o s i t i o n , particularly that of cholesterol, show s o m e promise in producing a low cholesterol yolk fraction, provided there is no loss in functionality of this fraction. T h e c o m p o s i t i o n of
t h e layers isolated after 5 days of incubation were n o t significantly different from layers o b tained from samples incubated for 10 days. Mineral analyses of ash showed t h a t the K, Mg, Na, Zn, Mn, Cu, B, and Al c o n t e n t of t h e t w o layers was similar, b u t t h e lower layer had 1.5, 3.2, and 5.5 t i m e s m o r e P, Ca, and Fe as c o m p a r e d to the upper level. Burley and C o o k ( 1 9 6 1 ) have s h o w n t h a t most of t h e calcium, p h o s p h o r u s and iron in t h e egg yolk is associated with granules. T h e compositional differences of t h e layers relate to the n a t u r e of t h e egg yolk c o n s t i t u e n t s making up these layers. It w o u l d , therefore, appear t h a t granules constitute a major c o m p o n e n t of the lower layer. Assay of the C F S for e n z y m e activity revealed t h e presence of proteolytic b u t n o lipolytic activity.
ft
B
C
—
D
FIG. 1. Photographic representation of visual changes in egg yolk: uninoculated yolk incubated for 0 hr (A) and 5 days (B). Inoculated with S. faecalis var. liquefaciens and incubated for 5 (C) and 10 days (D).
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0 12 24 36 48 72 96 120 168 240 360
CHANGES IN YOLK BY STREPTOCOCCUS
657
FAECALIS
TABLE 2. The chemical composition of yolk and the upper and lower layers produced by the growth of S. faecalis var. liquefaciens after 5 and 10 days of incubation at 25 C Chemical analysis Incubation time
Sample
Lipid
Moisture
17.49 17.30 12.60 22.52 13.45 25.12
36.73 36.20 42.31 27.42 42.99 25.96
50.36 50.95 49.66 49.18 49.57 49.32
(days) Fresh uninoculated yolk (control) Stored uninoculated yolk (control) Upper layer Lower layer Upper layer Lower layer
0 5, 10 5 5 10 10
The NPN value (mg of tyrosine/g of yolk) of the upper layer was 1.59 at 5 days and 2.04 after 10 days of incubation at 25 C, while the corresponding values for the lower layer were 1.86 and 2.36, respectively. The control value of .80 remained constant throughout the study. The increase in NPN is obviously due to the proteolytic enzymes produced by the growth of S. faecalis var. liquefaciens and can be explained on the basis of the availability of a limited amount of free proteins (livetins) in the
2.0 r
(/o)
Ash
Cholesterol
1.35 1.40 1.40 2.53 1.64 2.99
9.65 9.45 12.85 9.77 16.39 9.62
—-
yolk. It appears that these soluble proteins are most vulnerable to proteolytic attack by the enzymes produced by S. faecalis var. liquefaciens. The remaining conjugated lipoproteins in yolk are probably not easily attacked by the proteases produced by this organism. The cromatographic pattern of the untreated egg yolk showed five major components (Fig. 2) and was similar to that reported by Seideman et al. (1969). The upper layer (Fig. 3) showed only two major components. It appears
A... <*-, B- and X- livetins B... Low density lipoproteins and some phosvitin C... Lipovitellin and some phosvitin D... Phospholipids and possibly some lipovitellin components E... Unidentified
.2
.4
.6
.8
1.0
e f f l u e n t v o l u m e (1) FIG. 2. Elution pattern of fresh uninoculated egg yolk, fractionated on a carboxymethyl cellulose column.
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Protein
GRAVANI ET AL.
658
2.0
r
A...<*-, B- and X- livetins B... Low density lipoproteins and some phosvitin C... Lipovitellin and some phosvitin D... Phospholipids and possibly some lipovitellin components E... Unidentified 5 day 10 day
E 1.5 CO CM
1.0 U C <0 JD
o
V) .Q CO
.6
1.0
1.2
effluent volume (1) FIG. 3. Effect of incubation time at 25 C on the carboxymethyl cellulose chromatographic pattern of upper layer separated from egg yolk inoculated with S. faecalis var. liquefaciens.
2.0
A...o<-, B- and X- livetins B... Low density lipoproteins and some phosvitin C... Lipovitellin and some phosvitin D... Phospholipids and possibly some lipovitellin components E - G Unidentified
E c o
5 day 10 day
CO CM CO
o c cc n o en
.o cc
T
.2
.4
.6 .8 1.0 effluent volume (1) FIG. 4. Effect of incubation time at 25 C on the carboxymethyl cellulose chromatographic pattern of lower layer separated from egg yolk inoculated with S. faecalis var. liquefaciens.
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c
o
CHANGES IN YOLK BY STREPTOCOCCUS
FAECALIS
659
REFERENCES
FIG. 5. Visual appearance of the ultracentrifugal pattern (81,500 X g for 10 hr) of a) fresh uninoculated egg yolk; b) uninoculated egg yolk incubated for 5 days; c) upper layer of separated yolk incubated for 5 days; and d) lower layer of separated yolk incubated for 5 days.
t h a t livetins and lipoproteins, which c o n s t i t u t e t h e u p p e r layer, have aggregated such t h a t these are eluting together as a single major c o m p o n e n t and the m i n o r peak is lipovitellin. T h e c h r o m a t o g r a p h i c analysis of t h e lower layer (Fig. 4) showed very small a m o u n t s of livetins and lipoproteins, while t h e major c o m p o n e n t s were the proteins of t h e granules (phosvitin and lipovitellin). T h e ultracentrifugal analysis (Fig. 5) showed t h a t t h e u p p e r layer had n o sedimenting material at 8 1 , 5 0 0 X g, confirming o u r h y p o t h e s i s t h a t the u p p e r layer consists of low density lipoproteins (Gravani et al, 1 9 8 2 ) . T h e lower layer on centrifugation showed a clear, oily s u p e r n a t a n t , which is p r o b a b l y the low density fraction a n d a g u m m y s e d i m e n t t h a t is derived from the granules.
American Public Health Assoc, Inc., 1976. Pages 1 — 701 in Compendium of Methods for die Microbiological Examination of Foods. M. L. Speck, ed. Am. Publ. Health Assoc., Inc., Washington, DC. Association of Official Analytical Chemists, 1970. Official Methods of Analysis. 11th ed. Assoc. Offic. Anal. Chem., Washington, DC. Board, R. G., 1965a. Bacterial growth on the penetration of the shell membranes of the hen's eggs. J. Appl. Bacteriol. 28:197-205. Board, R. G., 1965b. The properties and classification of the predominant bacteria in rotten eggs. J. Appl. Bacteriol. 28:437-453. Board, R. B., 1966. Review article: The course of microbial infection of the hen's egg. J. Appl. Bacteriol. 2 9 : 3 1 9 - 3 4 1 . Board, R. G., 1968. Microbiology of the egg: a review. Pages 133—162 in Egg Quality: A Study of the Hen's Egg. T. C. Carter, ed. Oliver and Boyd Ltd., Edinburgh, Scotland. Board, R. G., 1973. The microbiology of eggs. Pages 46—60 in Egg Science and Technology. W. J. Stadelman and O. J. Cotterill, ed. The Avi Publ. Co., Inc., Westport, CT. Brooks, J., and D. J. Taylor, 1955. Eggs and egg products. Spec. Rep. Food Invest. Board D.S.I.R. No. 6 0 : 1 - 1 0 4 . Brown, W. E., R. C. Baker, and H. B. Naylor, 1965. The role of the inner shell membrane in bacterial penetration of chicken eggs. Poultry Sci. 44: 1323-1327. Burley, R. W., and W. H. Cook, 1961. Isolation and composition of avian egg yolk granules and their constituents a- and B-lipovitellins. Can. J. Biochem. Physiol. 39:1295-1307. Frazier, W. C , and D. C. Westhoff, 1978. Contamination, preservation and spoilage of eggs. Pages 256—269 in Food Microbiology. 3rd ed. McGraw Hill, Inc., New York, NY. Gravani, R. B., J. P. Prezoise, and D. V. Vadehra, 1972. The effect of the growth of Streptococcus faecalis var. liquefaciens on egg yolk. Poultry Sci. 51:1812-1813. Gravani, R. B., 1975. The physico-chemical changes in egg yolk caused by the egg yolk separation factor of Streptococcus faecalis var. liquefaciens. Ph.D.
Downloaded from http://ps.oxfordjournals.org/ at Rutgers University Libraries/Technical Services on June 1, 2015
In conclusion, t h e egg yolk separation factor of S. faecalis var. liquefaciens p r o d u c e s several physical and chemical changes which c u l m i n a t e in t h e separation of t h e yolk into t w o layers. T h e factor, based on NPN analysis and the assay of cell-free s u p e r n a t a n t , appears to be a proteolytic e n z y m e . This e n z y m e evidently causes proteolysis, destabilization, and aggregation of t h e granules that c o m e o u t of suspension and settle as t h e lower layer. T h e remaining yolk, devoid of suspended material, c o n s t i t u t e t h e clear, oily upper layer. This hypothesis is s u p p o r t e d by c h r o m a t o g r a p h i c , ultracentrifugal, and mineral analyses.
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GRAVANI ET AL. Microbial flora of commercially pasteurized egg products. Poultry Sci. 4 9 : 5 7 8 - 5 8 5 . Somkuti, G. A., and F. J. Babel, 1969. Hydrolytic breakdown of casein by a proteinase of Streptococcus faecalis var. liquefaciens. J. Dairy Sci. 52: 1186-1191. Stadtman, T. C , 1957. Preparation and assay of cholesterol and egrosterol. Pages 392—394 in Methods in Enzymology. I. S. P. Colowick and N. O. Kaplan, ed. Academic Press, Inc., New York, NY. Vadehra, D. V., R. C. Baker, and H. B. Naylor, 1970. Role of cuticle in spoilage of chicken eggs. J. Food Sci. 35:5—6. Vadehra, D. V., and R. W. Burley, 1978. The "settling phenomenon" in egg yolk. What do bacteria do to eggs? CSIRO Food Res. Q. 38:40-45. Worthington Enzyme Corporation, 1972. Pages 1—216 in Worthington Enzyme Manual. Freehold, NJ. Yadav, N. K., and D. V. Vadehra, 1977. Mechanism of egg white resistance to bacterial growth. J. Food Sci. 4 2 : 9 7 - 9 9 .
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thesis, Cornell Univ., Ithaca, NY. Gravani, R. B., D. V. Vadehra, L. F. Hood, and R. C. Baker, 1982. Changes in the ultrastructure of egg yolk by the growth of Streptococcus faecalis var. liquefaciens. J. Food Sci. 47:1435—1437. Lifshitz, A., and R. C. Baker, 1964. Some physical properties of the egg shell membranes in relation to their resistance to bacterial penetration. Poultry Sci. 4 3 : 5 2 7 - 5 2 8 . Lifshitz, A., R. C. Baker, and H. B. Naylor, 1964. The relative importance of chicken egg exterior structures in resisting bacterial penetration. J. Food Sci. 29:94-99. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265— 275. Seideman, W. E., O. J. Cotterill, and C. W. Gehrke, 1969. Ion-exchange chromatography of egg yolk. I. Separation methods. Poultry Sci. 48:884— 894. Shafi, R., O. J. Cotterill, and M. L. Nichols, 1970.
1984 Poultry Science 63:654-660
INTRODUCTION
Freshly laid eggs are generally devoid of bacteria (Brooks and Taylor, 1955; Frazier and Westhoff, 1978). However, on exposure to environmental onslaughts, eggs became contaminated by a variety of microorganisms that pose spoilage and public health problems. Extensive work has been conducted on various aspects of microbial spoilage of eggs including a) the mechanism of penetration of microorganisms into the egg interior (Lifshitz and Baker, 1964; Lifshitz et al, 1964; Board, 1965a; Brown et al, 1965; Vadehra et al, 1970); b) antimicrobial properties of egg white proteins (Board, 1966, 1968, 1973; Yadav and Vadehra, 1977), c) the types of bacteria present in eggs (Board, 1965b, 1968, 1973 ; Frazier and Westhoff, 1978; Shafi etal, 1970); and d) control of microbial spoilage of shell and liquid eggs (Board, 1968, 1973; Frazier and Westhoff, 1978). However, not much work has been done on either the growth pattern or the changes produced by microorganisms in egg yolk. Gravani et al (1972), Gravani (1975), and Vadehra and Burley (1978) reported that Streptococcus faecalis var. liquefaciens and other organisms, when grown in egg yolk,
1 Department of Microbiology, Panjab University, Chandigarh, India.
caused separation of the homogeneous yolk into an upper layer that was clear and oily and a cloudy, viscous lower layer. This study was undertaken to determine the growth characteristics of S. faecalis var. liquefaciens in egg yolk and the compositional differences between the two layers produced from such growth. MATERIALS AND METHODS Egg Yolk Eggs less than 24 hr old from a single strain of White Leghorn hens were used throughout this study. Egg yolk was aseptically separated from albumen and transferred to sterile widemouth jars. The sterility of the egg yolk was determined using the standard plate count procedure (American Public Health Association, 1976) and by incubating uninoculated yolk at 25 C. Inoculation and Incubation S. faecalis var. liquefaciens was procured from the culture collection of the Microbiology Department at Cornell University. The organism was grown at 37 C for 18 to 24 hrin trypticase soy broth. Prior to inoculation, the broth culture was diluted with sterile water and it constituted the inoculum. The quantity of the diluted broth was varied to give an initial count of 5 X 10 3 to 1 X 10 4 cfu/ml of the
654
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ABSTRACT The growth of Streptococcus faecalis var. liquefaciens in sterile liquid egg yolk for 48 hr at 25 C resulted in the separation of the homogeneous yolk into two components, which appeared as layers. The upper layer is clear, oily, and has a higher amount of lipids and cholesterol as compared to the viscous lower layer, which contains greater quantities of protein, phosphorus, calcium, and iron. This separation is related to the production of an extracellular factor, probably a protease, which because of limited proteolysis causes destabilization of the egg yolk system. This destabilization results in, or is due to, aggregation of the granules, which because of size, weight, and compositional differences, settled to constitute the lower layer. Evidently, these granules are responsible for opacity, as the yolk devoid of this settled material is clear and makes up the upper layer. (Key words: egg yolk, separation factor, Streptococcus faecalis, physicochemical changes)
CHANGES IN YOLK BY STREPTOCOCCUS FAECALIS yolk. The inoculated egg yolk was incubated for 5 or 10 days at 25 C in sterile wide-mouth jars. Viable Count and pH
Preparation of Cell-Free Supernatant Two-liter flasks containing 1 liter of trypticase soy broth with .2 M phosphate buffer (pH 7.1) were inoculated with 10 ml of an overnight culture of Streptococcus faecalis var. liquefaciens and incubated for 18 hr at 37 C on a reciprocating shaker (80 rpm). The cell-free supernatant (CFS) was prepared by centrifugation of the cultures at 16,300 X g for 20 min at 4C. Enzyme Assays Proteolytic activity in the CFS was determined by modifying the method of Somkuti and Babel (1969). One-tenth milliliter of the test solution was added to 3 ml of a 1% skim milk powder solution in .05 M phosphate buffer (pH 7.45). The reaction mixture was thoroughly mixed and incubated at 37 C for 20 min. After adding an equal volume of 24% trichloroacetic acid (TCA), the tubes were mixed and allowed to stand at room temperature (25 ± 2 C) for 20 min. The precipitate was removed by centrifugation at 2,000 rpm for 5 min. The solution was then filtered through Whatman No. 41 filter paper and the absorbance determined at 280 nm. An absorbance of .01, under these experimental conditions, was defined as one "proteolytic unit." Lipolytic activity was determined by using the method described by Worthington (1972). Separation of Upper and Lower Layers For separation at the end of the incubation, the upper clear, oily layer was decanted and then aspirated while the lower, viscous layer was left in the jar. The separated layers were used for chemical analyses. Chemical Analysis Total protein. The micro-Kjeldahl procedure (Association of Official Analytical Chemists, 1970) and the modified Folin-Ciocalteu procedure (Lowry et al, 1951) were used.
Nonprotein nitrogen. The test sample (1.0 g) was added to 5.0 ml of 10% TCA, mixed, and centrifuged at 2,000 rpm for 20 min in a clinical centrifuge at room temperature. The centrifugate was filtered through Whatman No. 41 filter paper. The filtrate was neutralized with equal amounts of .625 M NaOH and the nonprotein nitrogen (NPN) was determined by the modified Folin-Ciocalteu procedure (Lowry et al., 1951) and quantitated using tyrosine as the standard. Lipids. The yolk samples were freeze dried in a Stokes freeze drier (Model No. 902241, Pennsalt Chemical Co., Philadelphia, PA) and extracted with a chloroform-methanol (2:1) solvent system using a Goldfisch fat extraction apparatus (Labconco Corp. of Kansas City, MO). Cholesterol. The procedure of LiebermannBurchard (Stadtman, 1957) was used. The test sample was diluted with water to give .1 to .5 mg of cholesterol, deproteinized with ethanolacetone (1:1), and analyzed for cholesterol. Ash and Mineral Analysis. The yolk samples were dry ashed at 600 C in a muffle furnace (Barber Coleman Model No. 293, Rockford, IL). The ash was dissolved in .1 N HCl and mineral content was analyzed by a photoelectric spectrophotometer (Applied Research Co., Model 2900, Dearborn, MI). Chromatographic
Analysis
Carboxymethyl cellulose (BioRad Labs, Richmond, CA) was used for the chromatography according to the procedure of Seideman etal. (1969). Ultracentrifugal
Studies
Whole yolk, upper and lower layers, was weighed into cellulose nitrate tubes and centrifuged at 81,500 X g in a swinging bucket rotor at 7 C for 10 hr (Model L2, Spinco Division, Beckman Instrument Co., Palo Alto, CA). RESULTS AND DISCUSSION Streptococcus faecalis var. liquefaciens grew extremely well in egg yolk and within 12 hr of incubation at 25 C the count reached 2.6 x 10 7 cfu/ml from an initial count of 8.9 X 10 3 cfu/ml (Table 1). The count after 36 hr was 4.2 X 10 9 cfu/ml. There was no decline in the viable count during the remaining incubation period (360 hr), indicating that the egg yolk is a good growth and maintenance medium for
Downloaded from http://ps.oxfordjournals.org/ at Rutgers University Libraries/Technical Services on June 1, 2015
The viable count of each yolk sample was determined using standard methods (American Public Health Association, 1976). The pH was measured at selected intervals by aseptically drawing a sample from the incubating egg yolk.
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TABLE 1. Viable count, pH, and visual changes produced by S. faecalis var. liquefaciens in egg yolk when incubated at 25 C for 360 hr Incubation time (hr)
Count
pH
Visual observations
6.10 5.60 5.60 5.60 5.60 5.60 5.58 5.60 5.60 5.60 5.60
Resembles stored control Resembles stored control Resembles stored control Resembles stored control Yolk beginning to separate into two layers Yolk separated into upper translucent and lower opaque layers Layers well-defined Layers well-defined Layers well-defined Layers well-defined Upper layer became opaque
(cfu/ml) 8.9 X 10 3 2.6 X 10 7 3.4 X 10' 4.2X10' 4.0 X 10 9 4.0 X 10 9 3.9 X 10 9 3.6X10' 3.2 X 10' 2.7X10' 2.6 X 10'
this organism. No drastic changes in pH were observed and this was due to the lack of ferm e n t a b l e c a r b o h y d r a t e s in egg yolk, because t h e organism otherwise ferments c a r b o h y d r a t e s t o p r o d u c e acid. T h e separation of egg yolk i n t o t w o layers was a b r u p t to start and was first noticed after 4 8 hr of g r o w t h . O n c e t h e separation was initiated, it was c o m p l e t e within 2 4 hr. T h e visual changes in the yolk samples inoculated with S. faecalis var. liquefaciens were c o m p a r e d with t h e u n i n o c u l a t e d fresh and incubated controls (Fig. 1). T h e p h o t o g r a p h s clearly show the separation of yolk into t w o layers. T h e e x t e n t of separation was m o n i t o r e d b y determining t h e a m o u n t of t h e u p p e r layer as a percentage of t h e total yolk. These determinations showed t h a t at t h e start of separation (48 hr), the u p p e r layer was a p p r o x i m a t e l y 10% of t h e total y o l k ; however, this figure increased t o 4 5 % in 72 hr and 59% in 5 days. Incubation b e y o n d this p r o d u c e d very limited settling, as t h e percentage of u p p e r layer after 10 days increased b y 4%. After t h e initiation, the separation is merely a gravitational p h e n o m e n o n , as the t w o layers could be separated by centrifugation much more quickly. Chemical analyses of t h e two layers s h o w e d t h a t t h e u p p e r layer c o n t a i n e d 1.5 times m o r e lipids and cholesterol while t h e lower layer had a b o u t twice the a m o u n t of protein and ash (Table 2). These differences in c o m p o s i t i o n , particularly that of cholesterol, show s o m e promise in producing a low cholesterol yolk fraction, provided there is no loss in functionality of this fraction. T h e c o m p o s i t i o n of
t h e layers isolated after 5 days of incubation were n o t significantly different from layers o b tained from samples incubated for 10 days. Mineral analyses of ash showed t h a t the K, Mg, Na, Zn, Mn, Cu, B, and Al c o n t e n t of t h e t w o layers was similar, b u t t h e lower layer had 1.5, 3.2, and 5.5 t i m e s m o r e P, Ca, and Fe as c o m p a r e d to the upper level. Burley and C o o k ( 1 9 6 1 ) have s h o w n t h a t most of t h e calcium, p h o s p h o r u s and iron in t h e egg yolk is associated with granules. T h e compositional differences of t h e layers relate to the n a t u r e of t h e egg yolk c o n s t i t u e n t s making up these layers. It w o u l d , therefore, appear t h a t granules constitute a major c o m p o n e n t of the lower layer. Assay of the C F S for e n z y m e activity revealed t h e presence of proteolytic b u t n o lipolytic activity.
ft
B
C
—
D
FIG. 1. Photographic representation of visual changes in egg yolk: uninoculated yolk incubated for 0 hr (A) and 5 days (B). Inoculated with S. faecalis var. liquefaciens and incubated for 5 (C) and 10 days (D).
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0 12 24 36 48 72 96 120 168 240 360
CHANGES IN YOLK BY STREPTOCOCCUS
657
FAECALIS
TABLE 2. The chemical composition of yolk and the upper and lower layers produced by the growth of S. faecalis var. liquefaciens after 5 and 10 days of incubation at 25 C Chemical analysis Incubation time
Sample
Lipid
Moisture
17.49 17.30 12.60 22.52 13.45 25.12
36.73 36.20 42.31 27.42 42.99 25.96
50.36 50.95 49.66 49.18 49.57 49.32
(days) Fresh uninoculated yolk (control) Stored uninoculated yolk (control) Upper layer Lower layer Upper layer Lower layer
0 5, 10 5 5 10 10
The NPN value (mg of tyrosine/g of yolk) of the upper layer was 1.59 at 5 days and 2.04 after 10 days of incubation at 25 C, while the corresponding values for the lower layer were 1.86 and 2.36, respectively. The control value of .80 remained constant throughout the study. The increase in NPN is obviously due to the proteolytic enzymes produced by the growth of S. faecalis var. liquefaciens and can be explained on the basis of the availability of a limited amount of free proteins (livetins) in the
2.0 r
(/o)
Ash
Cholesterol
1.35 1.40 1.40 2.53 1.64 2.99
9.65 9.45 12.85 9.77 16.39 9.62
—-
yolk. It appears that these soluble proteins are most vulnerable to proteolytic attack by the enzymes produced by S. faecalis var. liquefaciens. The remaining conjugated lipoproteins in yolk are probably not easily attacked by the proteases produced by this organism. The cromatographic pattern of the untreated egg yolk showed five major components (Fig. 2) and was similar to that reported by Seideman et al. (1969). The upper layer (Fig. 3) showed only two major components. It appears
A... <*-, B- and X- livetins B... Low density lipoproteins and some phosvitin C... Lipovitellin and some phosvitin D... Phospholipids and possibly some lipovitellin components E... Unidentified
.2
.4
.6
.8
1.0
e f f l u e n t v o l u m e (1) FIG. 2. Elution pattern of fresh uninoculated egg yolk, fractionated on a carboxymethyl cellulose column.
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Protein
GRAVANI ET AL.
658
2.0
r
A...<*-, B- and X- livetins B... Low density lipoproteins and some phosvitin C... Lipovitellin and some phosvitin D... Phospholipids and possibly some lipovitellin components E... Unidentified 5 day 10 day
E 1.5 CO CM
1.0 U C <0 JD
o
V) .Q CO
.6
1.0
1.2
effluent volume (1) FIG. 3. Effect of incubation time at 25 C on the carboxymethyl cellulose chromatographic pattern of upper layer separated from egg yolk inoculated with S. faecalis var. liquefaciens.
2.0
A...o<-, B- and X- livetins B... Low density lipoproteins and some phosvitin C... Lipovitellin and some phosvitin D... Phospholipids and possibly some lipovitellin components E - G Unidentified
E c o
5 day 10 day
CO CM CO
o c cc n o en
.o cc
T
.2
.4
.6 .8 1.0 effluent volume (1) FIG. 4. Effect of incubation time at 25 C on the carboxymethyl cellulose chromatographic pattern of lower layer separated from egg yolk inoculated with S. faecalis var. liquefaciens.
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c
o
CHANGES IN YOLK BY STREPTOCOCCUS
FAECALIS
659
REFERENCES
FIG. 5. Visual appearance of the ultracentrifugal pattern (81,500 X g for 10 hr) of a) fresh uninoculated egg yolk; b) uninoculated egg yolk incubated for 5 days; c) upper layer of separated yolk incubated for 5 days; and d) lower layer of separated yolk incubated for 5 days.
t h a t livetins and lipoproteins, which c o n s t i t u t e t h e u p p e r layer, have aggregated such t h a t these are eluting together as a single major c o m p o n e n t and the m i n o r peak is lipovitellin. T h e c h r o m a t o g r a p h i c analysis of t h e lower layer (Fig. 4) showed very small a m o u n t s of livetins and lipoproteins, while t h e major c o m p o n e n t s were the proteins of t h e granules (phosvitin and lipovitellin). T h e ultracentrifugal analysis (Fig. 5) showed t h a t t h e u p p e r layer had n o sedimenting material at 8 1 , 5 0 0 X g, confirming o u r h y p o t h e s i s t h a t the u p p e r layer consists of low density lipoproteins (Gravani et al, 1 9 8 2 ) . T h e lower layer on centrifugation showed a clear, oily s u p e r n a t a n t , which is p r o b a b l y the low density fraction a n d a g u m m y s e d i m e n t t h a t is derived from the granules.
American Public Health Assoc, Inc., 1976. Pages 1 — 701 in Compendium of Methods for die Microbiological Examination of Foods. M. L. Speck, ed. Am. Publ. Health Assoc., Inc., Washington, DC. Association of Official Analytical Chemists, 1970. Official Methods of Analysis. 11th ed. Assoc. Offic. Anal. Chem., Washington, DC. Board, R. G., 1965a. Bacterial growth on the penetration of the shell membranes of the hen's eggs. J. Appl. Bacteriol. 28:197-205. Board, R. G., 1965b. The properties and classification of the predominant bacteria in rotten eggs. J. Appl. Bacteriol. 28:437-453. Board, R. B., 1966. Review article: The course of microbial infection of the hen's egg. J. Appl. Bacteriol. 2 9 : 3 1 9 - 3 4 1 . Board, R. G., 1968. Microbiology of the egg: a review. Pages 133—162 in Egg Quality: A Study of the Hen's Egg. T. C. Carter, ed. Oliver and Boyd Ltd., Edinburgh, Scotland. Board, R. G., 1973. The microbiology of eggs. Pages 46—60 in Egg Science and Technology. W. J. Stadelman and O. J. Cotterill, ed. The Avi Publ. Co., Inc., Westport, CT. Brooks, J., and D. J. Taylor, 1955. Eggs and egg products. Spec. Rep. Food Invest. Board D.S.I.R. No. 6 0 : 1 - 1 0 4 . Brown, W. E., R. C. Baker, and H. B. Naylor, 1965. The role of the inner shell membrane in bacterial penetration of chicken eggs. Poultry Sci. 44: 1323-1327. Burley, R. W., and W. H. Cook, 1961. Isolation and composition of avian egg yolk granules and their constituents a- and B-lipovitellins. Can. J. Biochem. Physiol. 39:1295-1307. Frazier, W. C , and D. C. Westhoff, 1978. Contamination, preservation and spoilage of eggs. Pages 256—269 in Food Microbiology. 3rd ed. McGraw Hill, Inc., New York, NY. Gravani, R. B., J. P. Prezoise, and D. V. Vadehra, 1972. The effect of the growth of Streptococcus faecalis var. liquefaciens on egg yolk. Poultry Sci. 51:1812-1813. Gravani, R. B., 1975. The physico-chemical changes in egg yolk caused by the egg yolk separation factor of Streptococcus faecalis var. liquefaciens. Ph.D.
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In conclusion, t h e egg yolk separation factor of S. faecalis var. liquefaciens p r o d u c e s several physical and chemical changes which c u l m i n a t e in t h e separation of t h e yolk into t w o layers. T h e factor, based on NPN analysis and the assay of cell-free s u p e r n a t a n t , appears to be a proteolytic e n z y m e . This e n z y m e evidently causes proteolysis, destabilization, and aggregation of t h e granules that c o m e o u t of suspension and settle as t h e lower layer. T h e remaining yolk, devoid of suspended material, c o n s t i t u t e t h e clear, oily upper layer. This hypothesis is s u p p o r t e d by c h r o m a t o g r a p h i c , ultracentrifugal, and mineral analyses.
660
GRAVANI ET AL. Microbial flora of commercially pasteurized egg products. Poultry Sci. 4 9 : 5 7 8 - 5 8 5 . Somkuti, G. A., and F. J. Babel, 1969. Hydrolytic breakdown of casein by a proteinase of Streptococcus faecalis var. liquefaciens. J. Dairy Sci. 52: 1186-1191. Stadtman, T. C , 1957. Preparation and assay of cholesterol and egrosterol. Pages 392—394 in Methods in Enzymology. I. S. P. Colowick and N. O. Kaplan, ed. Academic Press, Inc., New York, NY. Vadehra, D. V., R. C. Baker, and H. B. Naylor, 1970. Role of cuticle in spoilage of chicken eggs. J. Food Sci. 35:5—6. Vadehra, D. V., and R. W. Burley, 1978. The "settling phenomenon" in egg yolk. What do bacteria do to eggs? CSIRO Food Res. Q. 38:40-45. Worthington Enzyme Corporation, 1972. Pages 1—216 in Worthington Enzyme Manual. Freehold, NJ. Yadav, N. K., and D. V. Vadehra, 1977. Mechanism of egg white resistance to bacterial growth. J. Food Sci. 4 2 : 9 7 - 9 9 .
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thesis, Cornell Univ., Ithaca, NY. Gravani, R. B., D. V. Vadehra, L. F. Hood, and R. C. Baker, 1982. Changes in the ultrastructure of egg yolk by the growth of Streptococcus faecalis var. liquefaciens. J. Food Sci. 47:1435—1437. Lifshitz, A., and R. C. Baker, 1964. Some physical properties of the egg shell membranes in relation to their resistance to bacterial penetration. Poultry Sci. 4 3 : 5 2 7 - 5 2 8 . Lifshitz, A., R. C. Baker, and H. B. Naylor, 1964. The relative importance of chicken egg exterior structures in resisting bacterial penetration. J. Food Sci. 29:94-99. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265— 275. Seideman, W. E., O. J. Cotterill, and C. W. Gehrke, 1969. Ion-exchange chromatography of egg yolk. I. Separation methods. Poultry Sci. 48:884— 894. Shafi, R., O. J. Cotterill, and M. L. Nichols, 1970.