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Esiwgré Jiaseph W’Zaéyka

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LIBRARY 1
Michigan State .
University

    
   
   

   

ABSTRACT

THE INFLUENCE OF ULTRASONIC TREATMENT
ON EGG QUALITY

by Edward Jeseph Wladyka

This study was undertaken to evaluate the influence
of ultrasonic treatment on egg cleanliness, albumen quality
and the location and incidence of bacteria. The eggs used
in four experiments were randomly selected as they were
collected from the nest, and artificial soil was applied
to such eggs in one experiment.

Artificially soiled eggs which had been subjected to
ultrasonic treatments of 25, 42 and 85 Kc per sec for 3
minutes in water and water plus a detergent were visually
compared with similarly soiled eggs washed in a water-
detergent solution in an immersion type washer and with
artificially soiled control eggs. Eggs subjected to ultra-
sonic treatment at all three frequencies were cleaner than
eggs washed in an immersion type washer (by visual compari-
son) and the inclusion of a detergent in the liquid medium
increased the effectiveness of soil removal.

Randomly selected eggs were subjected to ultrasonic
frequencies of 25, 42 and 85 Kc per sec for periods of 10
minutes and stored at 15.50 C (600 F) for periods up to 28

Edward Joseph Wladyka

days. Albumen quality of the eggs was determined by measur-
ing Haugh scores and pH values at O, 7, l4 and 28 days fol-
lowing treatment. Ultrasonic treatment did not affect the
albumen quality of eggs as measured by Haugh score and pH.
Eggs were subjected to 42 Kc per sec ultrasonic
treatments for periods of 3, 5, 10 and 15 minutes in a solu-
tion containing a water soluble dye. Visual evaluations of
results indicate that dye penetration occurred in some of
the eggs and appeared most often in the small end of the egg.
Increasing the length of ultrasonic treatment resulted in
increased concentration of dye where penetration occurred.
Eggs were treated for periods of five minutes in one
of three treatments (water; water + Pseudomonas; water +
Pseudomonas + fecal material) with and without the addition
of ultrasonic energy. Bacterial counts were obtained out-
side the egg shell, the shell membrane area and inside the
shell membrane. Bacterial counts on the outside shell sur-
face were greatly reduced by all treatments. Counts in the
shell membrane area and inside the shell membrane were in-

creased by treatments which included ultrasonic energy.

THE INFLUENCE OF ULTRASONIC TREATMENT
ON EGG~QUALITY

By

Edward Joseph Wladyka

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science

1962

ACKNOWLEDGMENTS

My most sincere gratitude and appreciation are
extended to Dr. L. E. Dawson for his professional counsel,
guidance and encouragement throughout the course of this
study and develOpment of this material.

Acknowledgments are due to Dr. B. S. Schweigert for
his cOOperation in.making facilities available for this
study; to Professor J. A. Davidson, Dr. C. L. Bedford and
Dr. R. C. Nicholas for their critical review of the manu-
script; and to Dr. W. L. Mallmann for his professional
advice.

An appreciation of gratitude is also extended to
Robert W. Walker for his assistance in bacterial analysis
and to Mrs. Maurice Ritchey for her aid in the preparation
of the manuscript. Special gratitude is due to my parents
Mr. and Mrs. A. Wladyka for their continued encouragement

and understanding throughout this study.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . .

Bacteriology of Shell Eggs . . . . .

Egg Washing . . . . . . . . . . . . .
Ultrasonic Cleaning . . . .

PRmEDURE O O O O O O O O O O O O O O O 0

Experiment 1
Experiment 2
Experiment 5
Experiment 4

Cleaning Effectiveness
Albumen Quality . . .
Dye Penetration . . .
Bacterial Analysis . .

RESULTS 0 O O O O O O O O O O O O O O O 0

Cleaning Effectiveness
Albumen Quality . . . .
Dye Penetration . . . .
Bacterial Analysis . .

DISCUSSION 0 O O O O O O O O O O C O O 0
SUMMARY AND CONCLUSIONS . . . . . . . . .
LITERATURE C ITED O O C O O O O O O O O C

APPENDH O O O O O O O O O O O O O O O O

111

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030103 01 H 4

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12
15
15
19

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29
45
48
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54

LIST OF FIGURES

Figure Page
1. Photograph of artificially soiled eg 3 before
and after treatments (Experiment I . . . . . 20

Photograph of inner surface of egg shells after
42 Kc per sec ultrasonic treatment periods of

3,5,10 and 15 minutes in dye solution
(Experiment 3). .

2.

. . 28

iv

Table
1.

10.

11.

LIST OF TABLES

Influence of 25 Kc per sec ultrasonic energy
on egg quality, Trial 1, Experiment 2 . . .

Influence of 42 Kc per sec ultrasonic energy
on egg quality, Trial 1, Experiment 2 . . .

Influence of 85 Kc per sec ultrasonic energy
on egg quality, Trial 1, Experiment 2 . . .

Influence of ultrasonic energy on egg quality

of eggs treated within 6 hours after gather-

ing, Trial 2, Experiment 2. . . . . . . . .

Influence of ultrasonic energy on initial egg
quality, Trial 5, Experiment 2. . . . . . .

Effects of 25 Kc per sec ultrasonic treatment
on bacterial numbers on the outside surface
of shell eggs l-day after treatment,
Experiment 40 O O O O O O O O O O O O O O 0

Effects of 25 Kc per sec ultrasonic treatment
on bacterial numbers on the outside surface
of shell eggs 7-days after treatment,
Experiment 4. . . . . . . . . . . . . . . .

Effects of 25 Kc per sec ultrasonic treatment
on bacterial numbers in the shell membrane
area 1-day after treatment, Experiment 4. .

Effects of 25 Kc per sec ultrasonic treatment
on bacterial numbers in the shell membrane
area 7-days after treatment, Experiment 4 .

Effects of 25 Kc per sec ultrasonic treatment
on bacterial numbers inside the shell mem-
brane l-day after treatment, Experhment 4 .

Effects of 25 Kc per sec ultrasonic treatment
on bacterial numbers inside the shell mem-
brane 7-days after treatment, Experiment 4.

Page

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24

25

27

27

30

51

52

32

34

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Table

12-

15.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Effects of 42 Kc per sec ultrasonic treatment
on bacterial numbers on the outside surface
of shell eggs l-day after treatment,
Experiment 40 O O O O O O O O O O O O O O 0

Effects of 42 Kc per sec ultrasonic treatment
on bacterial numbers on the outside surface
of shell eggs 7-days after treatment,
Experiment 40 o o o o e e e o o o o o o e 0

Effects of 42 Kc per sec ultrasonic treatment
on bacterial numbers in the shell membrane
area l-day after treatment, Experiment 4. .

Effects of 42 Kc per sec ultrasonic treatment
on bacterial numbers in the shell membrane
area 7~days after treatment, Experiment 4 .

Effects of 42 Kc per sec ultrasonic treamment
on bacterial numbers inside the shell mem-
brane area l-day after treatment,
Experiment4oeeoeeooeoeoooco

Effects of 42 Kc per sec ultrasonic treatment
on bacterial numbers inside the shell mem-
brane 7-days after treatment, Experiment 4.

Effects of 85 Kc per sec ultrasonic treatment
on bacterial numbers on the outside surface
of shell eggs l-day after treatment,
Experiment4ooeoeooeeooeoeeo

Effects of 85 Kc per sec ultrasonic treatment
on bacterial numbers on the outside surface
of shell eggs 7-days after treatment,
Experiment 40 c e o o o e e o o o e e e o 0

Effects of 85 Kc per sec ultrasonic treatment
on bacterial numbers in the shell membrane
area l-day after treatment, Experiment 4. .

Effects of 85 Kc per sec ultrasonic treatment
on bacterial numbers in the shell membrane
area 7-days after treatment, Experiment 4 .

Effects of 85 Kc per sec ultrasonic treatment
on bacterial numbers inside the shell meme
brane l-day after treatment, Experiment 4 .

Effects of 85 Kc per sec ultrasonic treatment
on bacterial numbers inside the shell mem-
brane 7-days after treatment, Experiment 4.

vi

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35

57

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59

4O

4O

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42

INTRODUCTION

A problem of major concern in the marketing of shell
eggs is the heavy financial loss suffered annually by pro-
ducers, processors, and consumers due to soiled eggs. This
may be primarily attributed to three factors: (1) the cost
of cleaning eggs; (2) a reduction in the producers' income
due to a lowering of product quality and grade; and (3) to
the spoilage of eggs in marketing channels as a result of
the handling of soiled eggs by producers and processors.

An economically effective means of removing the soil
from dirty eggs would not only present a visibly cleaner
product, but should also reduce the incidence of bacterial
spoilage of soiled eggs.

The washing of eggs has become an accepted practice.
In general, eggs cleaned by existing commercial procedures
have not been as satisfactory in.marketing channels as
naturally clean eggs. For this reason new or improved
techniques should be investigated.

In a relatively short period of time ultrasonic
cleaning equipment has become important in the field of
industrial cleaning. Applications in the field of cleaning
agricultural products, however, have received very little
attention. The limited use of ultrasonic energy in egg

washing indicates that naturally soiled eggs can be

effectively cleaned, but that the effects on egg quality have
not been adequately evaluated.

The purposes of this study, therefore, were to use
ultrasonic frequencies of 25, 42, and 85 Kc per sec, (1) to
evaluate the cleaning ability of ultrasonic energy; (2) to
determine the effects of ultrasonic energy on albumen quality
and stability; (3) to determine the influence of ultrasonic
treatment on bacterial incidence on the shell of the egg,
and (4) to determine the influence of ultrasonic treatment

on bacterial penetration of the egg shell.

LITERATURE REVIEW
Bacteriology of Shell Eggs

According to Stuart and McNally (1945), shell eggs
are generally sterile when laid. Bacterial contamination on
the shells of newly laid eggs can therefore be attributed to
contact with the nesting material, the feet and body of the
bird, and subsequent handling procedures. Microbial spoil-
age of eggs encountered in commercial distribution channels
is, therefore, mainly due to the invasion of the egg by
spoilage-producing organisms after the egg has been laid
(Haines and Moran, 1940). These authors also reported that
bacterial penetration was related to (a) the inherent poros-
ity of the egg shell, and (b) the treatment (washing) the
egg received after laying. The microbial population in the
environment also contributes to bacterial contamination.

Several investigators have suggested that the pores
provide a means of microbial entry into the egg (Haines and
Moran, 1940; Fromm and Margolf, 1955; and Stuart and McNally,
1945). Kraft gt_gl, (1958) reported that shell porosity is
a useful index for determining the susceptibility of egg to
bacterial contamination.

Mountney and Vanderzant (1958) reported that there

was little if any correlation between egg shell permeability

and bacterial contamination. However, Kraft et_513 (1958)
reported a significant relationship between egg shell per-
meability and bacterial contamination. Fromm and Munroe
(1960) reported that interior quality and rate of bacterial
contamination are related to the permeability of the egg
shell. Due to the evidence indicating a correlation between
shell permeability and bacterial penetration, it may be
assumed that a direct relationship between such contamina-
tion and egg shell permeability does exist.

Stuart and McNally (1945) reported on the ”bacteri-
cidal activity" of the shell membrane, and indicated that it
may be more important as an agent in preventing contamina-
tion of the interior of the egg than the mucoid layer or
"bloom” covering the shell. Walden gt_gl, (1956) reported
tracer experiments in which the egg shell membrane restrained
the passage of microorganisms for periods of time up to 20
hours.

The existence of lysozyme in the whites of eggs and
its bacterial inhibition is well established, and a decrease
in the number of contaminated eggs can be attributed to this
enzyme (Fleming, 1922).

Since the shells of most eggs are sterile when the
eggs are laid, it is necessary to keep the shells clean to
prevent contamination. According to Funk (1957), 99 percent
of all eggs are clean at the time they are laid. At the

time of gathering, however, the percentage of soiled eggs

in the state of Michigan has varied from a high of 55 per-
cent to a low of 51 percent (Dawson and Watts, 1952). These
factors have led to an increase in the practice of cleaning
eggs at the farm level.

Eggs vary considerably in degree of soiling when
gathered from nests. Dawson and Watts (1952) classified
dirty eggs into five groups according to the amount of dirt
present on the shell. Approximately 45 percent of the eggs
examined were clean or had a very slight spot of dirt and 8
percent were classified as dirty or very dirty. This varia-
bility in the degree of dirtiness in naturally soiled eggs
necessitates the use of artificially soiled eggs in studies
comparing egg washing techniques. The use of an artificial
soil for dirtying eggs was reported by Sabet (1955). The
adhering properties of the artificial soil were improved

by the inclusion of egg albumen.
Egg Washing

Many reports have been written about the advantages
and disadvantages of washing eggs. Some investigators have
reported that no harm results to the eggs due to washing if
the procedure is properly carried out (Miller 2£_313, Mc-
Nally, 1952). According to Pino (1950), eggs submerged for
one minute in a detergent solution at 1400 F, rinsed by a
spray of tap water at 140° F, and air dried by a fan main-
tained quality equal to untreated eggs kept under similar

conditions. Other investigators have reported the possibil-
ity of harmful effects to eggs due to washing (Lorenz and
Starr, 1951; Starr gt_al,, 1951).

Winter and Clements (1956) reported that "properly
washed" eggs kept as well as unwashed eggs. Furthermore,
the removal of bacteria from the surface of the washed eggs
resulted in a more "sanitary" product. The eggs were washed,
using an ”approved" detergent, within 24 hours of the time
of lay, and were then dried by placing the basketful of
eggs in front of an electric fan.

Forsythe gt_al, (1952) stated that prOper washing of
all eggs, clean or dirty, was very desirable as long as the
washing was carried out before the organisms penetrated to
the egg contents.

Recent trends have indicated a preference for clean-
ing soiled eggs prior to candling (by the first receiver) as
opposed to farm washing (Anon., 1957). This enables the
plant manager to have better control of the washing proce-
dures. If bacteria and dirt can be removed more effectively
by the processor, and the rate of bacterial develOpment
curtailed, it is possible that considerable savings can be

realized in the marketing of eggs.
Ultrasonic Cleaning

Ultrasonic sound waves have been used successfully

in the field of industrial cleaning for a number of years.

In this respect it has usually been associated with the
cleaning of impervious metals in the form of intricate parts
or assemblies of complex design which are too delicate to
clean by established methods.

Thomas (1961) reported the application of ultrasonic
cleaning to 7/32 inch and 1/4 inch diameter steel balls
which are used as valves in hydraulic valve lifters. The
removal of a protective oil film.and also minute residual
particles was effected in an immersion time of three minutes
at a frequency of 590 Kc per sec.

High frequency sound waves transmitted throughout a
cleaning fluid cause cavitation to take place in the liquid.
The cavitation, as reported by Jehnson (1929), consists of
the formation of thousands of microscopic bubbles within a
liquid cleaning medium. The formation and collapse of these
cavitations produce a powerful scrubbing action on soil
(Carlin, 1949). Since the liquid medium.is inelastic with
respect to sound, it ruptures or cavitates when sound waves
pass through it. Vacuum pockets are created which almost
immediately collapse. The implosion of thousands of these
cavitations results in a scrubbing action by which the soil
particles are removed from the product.

Crawford (1955) reported that water was an excellent
medium for ultrasonic cleaning. Murdock (1956) suggested
that frequencies just above human audibility (20 Kc/sec)

were the most effective for soil removal. Ultrasonic waves

usually refer to those waves at a frequency above 15,000
cycles per second.

Ultrasound can be divided into two frequency ranges,
a lower and a higher. The lower frequency range extends
from about 15 Kc to 50 Kc, the higher frequency range in-
cludes those frequencies above 50 Kc.

For a given power input, cavitation is more intense
at a lower frequency than at a higher frequency since parti-
cle displacement for a given power input is inversely pro-
portional to the frequency (Thomas, 1961). In addition, if
frequency is too high, the implosions do not attain a suita-
ble magnitude to develop an appreciable energy upon collapse.

The two important functioning parts of an ultrasonic
device are the generator and the transducer. The generator
produces high frequency current; the transducer changes the
electrical impulses into high frequency sound waves. Trans-
ducers at present are of two principal types, the piezo-
electric and the magnetostrictive (Anon., 1959).

For many years ultrasonic energy has been used as a
laboratory instrument in various fields of endeavor. Harvey
and Loomis (1929) destroyed "luminous bacteria" with sound
waves of 400 Kc.

According to Thornley (1955) long time treatments of
high temperature would be necessary for the destruction of
most species of bacteria. Each bacteria would require dif-

ferent treatment times, in some cases up to 90 minutes.

Mackeprang 33 El. (1957), in studying the bacterial
effects of ultrasonics, noted that exposure for short peri-
ods of time at low intensity caused agglutination of the
bacteria with.a corresponding fall in colony count. Longer
exposure at higher frequencies resulted in increased pulver-
ization of the bacteria. However, after twenty-six hours,
15 percent of the bacteria were still viable.

Dawson g§_§13 (1960) reported cleaning eggs with
the aid of ultrasonic energy. Eggs were subjected to treat-
ments of 400 Kc/sec, 40 Kc/sec and 10 Kc/sec. Results indi-
cated that eggs could be cleaned by the use of ultrasonic
energy, and that treatments of one or more minutes were
effective. Although the lower frequencies appeared to exhib-
it the greatest cleaning effect, the 10 Kc/sec sound waves
were audible and objectionable.

PROCEDURE

The eggs used in this study were randomly selected
as they were collected from the nests and treated within 24
hours after they were laid. All of the eggs were obtained
from the Michigan State University Poultry Farm.

Experiment 1 - Cleaning Effectiveness

Uniform soiling techniques were used in which the
artificial soil consisted of 200 gms of tap water, 100 gms
of chicken feces and 15 gms of powdered egg white. Ninety-
six (96) eggs were coated with this artificial soil. The
eggs were dipped in the liquid soil mixture by means of a
wire holder, placed in an incubator at 58.6° 0 (100° F)
until dry to the touch (2-5 min) and then held at 15.500
(600 F) for 2 days until washed. The cleaning effectiveness
of the washing procedure was evaluated by visually comparing
the washed eggs with artificially soiled control eggs.

Trial 1 - Ultrasonic Energy in Water

Tap water at a temperature of 49 :_l° C was placed in

three transducerized cleaning tanks connected to ultrasonic

generators at 25 Kc, 42 Kb and 85 Kc per second and degassedl

 

1The tap water used as the liquid medium contains a
certain.amount of dissolved gases (air). The rapid removal
of these gases from solution with the beginning of cavitation

is termed de-gassing.

10

11

by subjecting the water to ultrasonic radiations for periods
of five minutes. Six artificially soiled eggs were placed
in each of the 25 Kc and 42 Kc cleaning tanks. Four eggs
were placed in the smaller 85 Kc cleaning tank. These soiled
eggs were subjected to an ultrasonic treatment of three min-
utes, removed and dried at room temperature. Twelve of the
artificially soiled eggs were washed in this manner in each
of the ultrasonic cleaning units.
Trial 2 - Ultrasonic Energy in Water Plus Detergent

A commercial detergent (Sparkleen)2 was added to
degassed tap water at a temperature of 49 1.10 C and an
appr0priate amount of solution was placed in each trans-
ducerized tank (25 Kc; 42 Kc; 85 Kc). Six artificially
soiled eggs were placed in each of the 25 Kc and 42 Kc tanks.
Four eggs were placed in the 85 Kc tank. All eggs were sub-
jected to an ultrasonic treatment of three minutes and then
removed and dried. Twelve of the soiled eggs were washed in
this manner in each of the ultrasonic cleaning units. The
wash.water was changed at the end of each treatment.
Trial 5 - Immersion Type Washer

Twelve artificially soiled eggs were coded and dis-
tributed throughout an egg basket containing approximately

ten dozen eggs. The basket of eggs was washed for three

 

zsparkleen - (sodium hexametOphosphate) a detergent
manufactured by Calgon 00., a division of Hogan Chemicals &
Controls Inc., Pittsburgh, Pa.

12

minutes in an immersion type egg washer3 in which a commercial
detergent was used. The eggs were allowed to dry, and the
coded eggs were removed from the basket. A visual comparison
was made of the eggs which had been washed by the different

methods.
Experiment 2 - Albumen Quality

Trial 1

Tap water at 50-550 C was placed in each of the
transducerized cleaning tanks (25, 42, and 85 Kc per sec)
and degassed. Five groups of six eggs were placed in the
water at 49 1.10 C and soaked for ten minutes. The eggs
were air dried and placed in cartons. At the end of each
group treatment, the water in each tank was replaced, and
the procedure repeated.

Five additional groups of six eggs were similarly
placed in water (49 _+_ 1° c) in the transducerized tanks (25
Kc; 42 Kc; 85 Kc). In this instance the groups of eggs were
subjected to ultrasonic treatments of ten.minutes. The eggs
were agitated periodically during the treatments by means of
a glass rod. At the conclusion of the treatments, the eggs

were dried and placed in cartons. The tap water in each of

the transducerized tanks was replaced at the end of each

 

3Big Dutchman Ni-Egg-Ra (Model A) egg washer manufac-
tured by Big Dutchman Automatic Poultry Feeder Co., Zeeland,
Michigan.

15

group treatment.

Five groups of six eggs were placed in cartons to
serve as controls.. One group (6 eggs) from each of the
three treatments was then evaluated for quality. Three
eggs of each group were used in the detennination of Haugh
score4 by using the procedure reported by Kilpatrick gt_§1,
(1958). The pH of the albumens of each of the three remain-
ing eggs in each group was measured using a Beckman glass
electrode pH meter (Model H-2).5

The remaining eggs were held at 15.50 C, and albumen
quality evaluations (Haugh score; pH) were made atseven day
intervals for a twenty-eight day period.

Trial 2

The initial Haugh scores and pH values of the ultra-
sonically treated eggs in Trial 1 were determined after a
holding period of approximately 48 hours. Since pH changes
rapidly, eggs were obtained within one hour of the gathering
time to obtain a more precise initial determination of Haugh
score and pH. Four groups of 4 eggs were placed in water
(49 1 1° c) in the 42 Kc transducerized tank and subjected
to ultrasonic treatment for ten.minutes. The eggs were

agitated periodically throughout the treatment and air dried

 

4Haugh score is an evaluation of interior egg quality
and is determined by the measurement of egg weight and albumen
height.

5Manufacturedby Beckman Instrwments, Inc., South
Pasadena, California, U.S.A.

14

after treatment.

Sixteen eggs (used as controls) were held at room
temperature for a period of time equal to that which the
treated eggs were held. Twelve eggs were used for Haugh
score determinations and four were used for measuring pH.
Haugh score and pH determinations were made on the ultra-
sonically treated eggs.

Four groups of four eggs were subjected to ultra-
sonic treatments of 85 Kc/sec for ten.minutes and were eval-
uated for quality. Twelve eggs were used in the determination
of Haugh score, and pH of the albumen of each of the four
remaining eggs was measured.

Trial'5

Forty-two eggs were obtained within one hour of the
gathering time and 28 of the eggs were randomly sorted into
groups of 4 eggs. Seven groups were placed in water (49 :_
10 C) in the 85 Kc transducerized tank and subjected to
ultrasonic treatments for ten minutes. After each treatment
the treated eggs were evaluated for quality. Five groups
were used to obtain Haugh scores and two groups were used
for pH values.

The quality of fourteen additional eggs from the
same gathering was measured after the eggs were held at room
temperature for a period of time equal to that which the
treated eggs were held. Ten eggs were used to obtain Haugh

scores and four were used for pH values.

15

Experiment 5 - Dye Penetration

An alizarin dye6 was added to tap water at 49 :_10 C

in the transducerized tank of the 42 Kc ultrasonic unit.

The water in the tank was degassed by passing ultrasonic
waves through it for a period of five minutes. Four groups
of six eggs were placed in the dye solution in a stainless
steel perforated basket and subjected to ultrasonic treat-
ments of 5, 5, 10 and 15 minutes. The eggs were agitated
periodically during the treatments. After treatment the
eggs were broken open and the contents removed. A visual

evaluation was made of dye penetration of the egg shell.
Experiment 4 - Bacterial Analysis

In this eXperiment the effects of ultrasonic treat-
ment on bacterial incidence on the shell of eggs, and bacte-
rial penetration of the egg shells were evaluated.

The eggs used in this study were randomly selected
as they were collected from the nest. The tap water used
in all treatments had been previously degassed, and all eggs
were washed at a temperature of 49 :_10 0.

Eggs were treated in six different ways to assist in

evaluating the influence of ultrasonic energy on bacterial

 

6Alizarin Blue Black, manufactured by National
Aniline Division, Allied Chemical & Dye Corp., 40 Rector St.,
New York 6, New York.

16

incidence and penetration. Treatments consisted of washing
eggs in:

1) Water only - 4 eggs

2) Water + ultrasonic energy - 8 eggs

5) Water + Pseudomonas - 4 eggs

4) Water + Pseudomonas + ultrasonic energy - 8 eggs

5) Water + Pseudomonas + fecal material - 4 eggs

6) 'Water + Pseudomonas + fecal material + ultrasonic

energy - 8 eggs.

Eight eggs were used in treatments 2, 4, 6 and 8 to
provide additional data on the bacterial analysis of ultra-
sonically treated eggs. Eggs were subjected to all six
treatments using each of the three ultrasonic cleaning units
(25, 42, and 85 Kc/sec).

The source of bacteria for treatments 5-6 was from

eggs which had been previously inoculated with Pseudomonas

 

fluorescens and Which showed positive bacterial development

 

under black light. For treatments 5 and 4 the contents of
one of the inoculated eggs was added to the water in the
transducerized tanks and stirred.

For treatments 5 and 6 fecal material was added to
the liquid medium of water + Pseudomonas which was prepared
as in treatment 4 (25 gms of fecal material per liter of
water). Ultrasonic waves were then passed through the liquid

for three minutes to aid in the disintegration of the fecal

17

material. Chicken feces were used as a source of organic
matter and additional contamination.

The eggs for all of the treatments were washed for
five minutes and were agitated periodically (with a glass
rod) during the treatments. After washing the eggs were
removed from the liquid medium, air dried, and placed in
cartons. Eggs from treatments 5 and 6 were carefully removed
from the wash solution in such a manner as to leave a.minimum
amount of organic matter on the egg shell.

The eggs from each treatment were randomly divided
into two groups, and each group was held at 15.50 C until
further analysis. The first group was analyzed one day
after treatment and the second was analyzed 8 days after
treatment.

The bacterial analysis consisted of:

1) Outside shell surface. Each egg was placed in a

 

sterile beaker to which sterile water was added and swirled
in the water for approximately five seconds. By the aid of
a sterile l-ml pipette, l-ml of the wash water was transfer-
red to a plate of Tryptoneglucose-extract agar (TGE). The
plates were incubated at 200 C for 48 hours.

2) Inside shell membrane, A circle approximately one
inch in diameter was cut through the large (air cell) end of
the egg with the aid of a Carborundum disc attached to a
chuck of an electric motor. The shell section was removed

with sterile forceps. Egg contents were removed from the

18

shell, and lO-ml of sterile water was pipetted inside the
shell. The water was mixed with the albumen adhering to the
inner shell membrane, and l-ml of this rinse was pipetted
into a plate of TGE agar. The plates were incubated at 200 C
for 48 hours.

5) Shell membrane area. The shell membrane was scraped

 

free from the shell by means of a sterile scapula in the
lower half (small end) of the egg in order to minimize the
possibility of any contamination at the open end of the egg.
The shell membrane was torn away from the shell, and the
water remaining from the rinse test was swirled inside the
scraped egg shell. One (1)-ml of the wash water was pipetted
into a plate of TGE agar; the plates were incubated at 20° C
for 48 hours.

Bacteria on the plates were counted at the end of
the incubation period. Aseptic techniques were followed as

closely as possible throughout the test.

RESULTS

Cleaning Effectiveness

Figure l is a photograph of artificially soiled
eggs before and after each of the washing treatments in
Experiment 1. In Figure 1-A, the cleanliness of artificially
soiled control eggs (top row) can be compared with similarly
soiled eggs after ultrasonic treatment of 25 Kc per sec in
water (middle row) and in water plus a detergent (bottom row).

Ultrasonic frequencies of 42 and 85 Kc per sec were
similarly used in washing treatments and results are shown
in Figures l-B and l-C. The addition of a detergent to the
ultrasonic cleaning solution resulted in.more thoroughly
cleaned eggs (bottom row vs middle row in Figures 1-A, 1-8,
and l-C).

In Figure 1-D, artificially soiled control eggs can
be compared with similarly soiled eggs after they were washed
in an immersion type washer with a detergent. The ultrasonic
washing treatments at each of the frequencies (25, 42 and 85
Kc/sec) were more effective in cleaning the soiled eggs (on
a visual basis) than the washing treatment in the immersion
type washer.

The eggs washed with ultrasonic waves of 25 Kc per

sec in water plus a detergent (Figure l-A, bottom row) were

19

20

 

Fig. 1 Photographs of artificially soiled eggs before and
after treatment.

In Photographs A, B, and C, the top row of eggs
represents controls, the second row represents ultrasonic
treatment in water, and the third row represents water plus
a detergent. In Photograph D, the top row represents control

eggs and the second row represents eggs washed by a commer-
cial washer, Experiment 1.

A) 25 Kc per sec ultrasonic generator.
B) 42 Kc per sec ultrasonic generator.
C) 85 Kc per sec ultrasonic generator.
D) Commercial immersion type egg washer.

21

the most effectively cleaned.
Albumen Quality

Albumen quality values for the control and treated
eggs in Experiment 2 are presented in Tables 1-5. The
effects of ultrasonic treatments of 25 Kc per sec on albumen
quality are reported in Table l. The mean Haugh score of
the treated eggs was 54.0 at the end of the 28 day holding
period at 15.50 C. This was essentially the same as the
mean Haugh score of the control eggs (55.0). The eggs which
had been immersed in water without ultrasonic energy had a
mean Haugh score of 58.6 at the end of this holding period.
The variability between individual eggs and between these
groups of eggs prior to storage was such that treatment
influences were not shown. The greater recorded loss in
Haugh units for the control eggs (26.7 as compared to 15.4
and 17.0 for the treated lots) showed up because of the
abnormally high.mean Haugh score for the control eggs. This
was attributed to one egg and is a hen influence not treat-
ment influence.

The pH values for the treated and control eggs were
essentially the same at each evaluation time. The relatively
high initial pH values may be attributed to the time elapsed

between lay and initial pH determinations.

22

TABLE l.--Inf1uence of 25 Kc per sec ultrasonic energy on
egg quality, Trial 1,

-—_

(Experiment 2)

 

r

 

Storage time (days)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment L Deiiine
J 0 '— 7 J 14 21 28 Storage
Mean Haugh Score
Control 81.0 60.5 67.0 57.7 55.0 26.0
Immersed in
Water 74.0 71.5 65.7 57.7 58.6 15.4
Immersed in
Water +
Ultrasonic
Energy 71.0 66.5 58.5 60.0 54.0 17.0
pH Values
Control 8.9 ~9.1 9.1 9.1 9.1
Immersed in
Water 900 900 901 901 900
Immersed in
Water +
Ultrasonic
Energy 900 901 901 900 900

 

 

 

 

 

 

 

Data showing variability among individual eggs are

reported in Appendix Table l.

23

The influence of 42 Kc per sec ultrasonic energy on
egg quality for Experiment 2 is shown in Table 2. The change
in egg quality, as measured by Haugh units, during the 28 day
holding period, was similar for the control and treated eggs.
Decline in Haugh units during the 28 days was 15.7, 15.7 and
16.5 for the three groups respectively.

The pH of the egg albumen throughout the holding
periods was not affected by water immersion or ultrasonic
treatment at 25 Kc per sec.

The albumen qualities of eggs treated with 85 Kc per
sec ultrasonic waves in Trial 1 of Experiment 2 are shown in
Table 5. The control eggs declined 22.5 Haugh units in 28
days compared with 15.0 for each of the treated groups. This
greater decline, which showed up in the control eggs only,
was attributed to one egg with an abnormally low Haugh score.
This could have been due to the individual bird influence
and is not attributed to treatment affect.

The pH values for the control and treated eggs in
Experiment 2 (Tables 1, 2.and 5) show little variation
throughout the 28 day holding period.

The influence of ultrasonic energy on initial egg
quality for Trial 2, Experiment 2 is shown in Table 4. The
average Haugh score of the eggs subjected to ultrasonic
treatments of 42 Kc per sec was 78.7. This was essentially
the same as the mean.Haugh score of the controls, 79.0. The

eggs treated with ultrasonic waves of 85 Kc had a slightly

24

TABLE 2.--Inf1uence of 42 Kc per sec ultrasonic energy on
egg quality, Trial 1, (Experiment 2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Storage time (days) Decline
Treatment ‘ ’ in
O '7 I 14 J 21 I 28 1 Storage
Mean Haugh Score
Control 77.7 75.0 67.5 55.0 62.0 15.7
Immersed in
Water 85.0 70.0 72.7 61.7 67.5 15.7
Immersed in
Water +
Ultrasonic
Energy 80.5 62.5 56.0 62.7 64.0 16.5
pH Values
Control 8.9 9.0 9.1 9.1 9.1
Immersed in
Water 809 901 901 900 900
Immersed in
water +
Ultrasonic
Energy 8.9 9.1 9.1 9.1 9.0
Data showing variability among individual eggs are

reported in Appendix Table 2.

TABLE 5.--Inf1uence of 85 Kc per sec ultrasonic energy on
egg quality, Trial 1,

  

(Experiment 2)

-_—.—_—___-_‘ . --- _. .- _.

Decline

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment in
0 7 14 21 28 Storage
Mean Haugh Score
Control 75.0 64.5 60.5 58.5 52.7 22.5
Immersed in
Water 74.7 70.0 67.0 59.0 59.7 15.0
Lmnersed in
Water +
Ultrasonic
Energy 75.7 69.5 68.5 59.7 60.7 15.0
pH Values
Control 9.0 9.1 9.1' 9.1 9.1
Immersed in
Water 900 901 902 901 900
Immersed in
Water +
Ultrasonic
Energy 9.0 9.1 9.1 9.1 9.0
Data showing variability among individual eggs are

reported in.Appendix Table 5.

26

lower mean Haugh score of 74.9.
The pH values for the eggs treated with 42 and 85 Kc
ultrasonic waves and the controls are essentially the same.
Table 5 shows the influence of 85 Kc per sec ultra-
sonic energy on initial egg quality in Trial 5, Experiment 2.
Little variation is evident between the mean Haugh scores
and pH values for the control and treated eggs which were
evaluated within 2 hours after gathering. These eggs had
higher Haugh scores and lower pH values than the eggs eval-

uated in Trials 1 and 2 of Experiment 2 (Tables 1-4).
Dye Penetration

Figure 2 is a photograph of the inner surface of egg
shells which were treated with ultrasonic waves of 42 Kc per
sec in water containing a water soluble dye (Experiment 5).

Eggs treated for 5 minutes displayed some evidence
of dye penetration through the pores of the egg shell. As
the treatment time was increased to 5, 10 and 15 minutes, a
greater concentration of dye was observed on the inside of
the egg shell. The penetration did not occur in all of the
eggs treated, and was more concentrated in the small end of
the eggs.

Since dye was forced through the shells, it was

believed that bacteria might behave in a similar manner.

27

TABLE 4.--Influence of ultrasonic energy on quality of eggs
treated within 6 hours after gathering, Trial 2, Experiment 2

 

 

 

 

 

 

 

 

 

 

Number Average Number
Treatment of Eggs Haugh Score of Eggs pH
Controls 12 79.0 4 8.5
42 Kc/sec 11 78.7 4 8.5
85 Kc/sec 12 74.9 4 8.4
TABLE 5.--Influence of ultrasonic energy on initial egg

quality, Trial 5, Experiment 2

W m

Number Average Number
Treatment of Eggs Haugh Score of Eggs pH
Controls 10 80.2 4 8.5
85 Kc/sec 20 81.5 e 8.5

 

 

 

 

 

V . .
a. “‘- ;" , 3".O'
fro

4'0
,1

(”a
.8"

.5
F.
7..

”- e
. .-
it:

'

t}-

28

 

Fig. 2 Photograph of inner surface of egg shell after 42 Kc
per sec ultrasonic treatment periods of 5,
utes in dye solution, Experiment 5.

Upper
Upper
Lower
Lower

right
left
right
left

5 minute
5 minute
10 minute
15 minute

5, 10 and 15 min-

treatment
treatment
treatment
treatment.

29

Bacterial Analysis

The influence of ultrasonic energy on bacterial
incidence on shell eggs and incidence inside of the treated
eggs is reported in Tables 6-25 for Experiment 4.

Tables 6 and 7 show the effects of ultrasonic treat-
ments of 25 Kc per sec on the bacterial numbers on the out-
side of the egg shells.

The number of organisms per egg one day after treat-
ment (Table 6) indicates a very marked reduction in numbers
of bacteria on the shell surface of treated eggs when com-
pared with controls. The addition of ultrasonic energy to
water, water + Pseudomonas, and water + Pseudomonas + fecal
material did not consistently lower the numbers of organisms
on the treated eggs as compared with the treatments without
the ultrasonic energy.

Table 7 presents the number of organisms found per
egg seven days after treatment. The treated eggs, both
immersed and immersed plus ultrasonic energy, had much lower
bacterial counts than the controls. The addition of ultra-
sonic energy to the washing treatment did not consistently
reduce the number of organisms found.

In Tables 8 and 9 are presented the effects of ultra-
sonic treatments of 25 Kc per sec on bacterial numbers found
in the shell membrane area. The results, one and seven days

after treatment, indicate that bacterial numbers did not

30

TABLE 6.--Effects of 25 Kc per sec ultrasonic treatment on
bacterial numbers on the outside surface of shell eggs l-day

after treatment, Experiment 4

 

r-

 

Treatment Number of Organisms per Egg1
Water 200 100 100 2,900
Water + U.E.2 0 0 2,500 100
O 500 500 ---
Water + Pseud. 1,500 200 200 100
Water + Pseud. + U.E. 100 0 200 200
O 400 100 600
Water + Pseud. + Feces 600 500 1,000 400
Water + Pseud..+ Feces 400 1,400 800 4,400
+ U.E. 100 800 400 600
Control 66,000 10,000 8,000 49,500

 

 

 

 

 

lEach figure represents the number of bacteria from

one egg.

2Ultrasonic energy.

5Broken egg shell.

51

TABLE 7.--Effects of 25 Kc per sec ultrasonic treatment on
bacterial numbers on the outside surface of shell eggs 7-days
after treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg
Water --- 0 400 0
Water + U.E. 0 0 0 100
100 100 0
Water + Pseud. 100 100 200 0
Water + Pseud. + U.E. 0 100 0 0
700 0 0 100
Water + Pseud. + Feces 0 --- 0 100

Water + Pseud. + Feces

+-U.E. 500 0 0 400
200 100 0 600
Control 55,000 92,000 5,000 2,500

 

 

 

 

 

52

TABLE 8.--Effects of 25 Kc per sec ultrasonic treatment on

bacterial numbers in the shell membrane area l-day after

treatment, Experiment 4

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Number of Organisms per Egg
Water 0 0 0 0
Water + U.E. O 0 l l
0 0 0 0
Water + Pseud. 0 0 0 1
Water + Pseud. + U.E. 0 0 1 Tnc.
0 6 Tnc. 55
Water + Pseud. + Feces 0 0 0 1
Water + Pseud. + Feces 20 l 10 0
+ U.E. 85 7 Tnc. Tnc.
Control 0 O O 0
TABLE 9.--Effects of 25 Kc per sec ultrasonic treatment on
bacterial numbers in the shell membrane area 7-days after
treatment, Experiment 4
Treatment Number of Organisms per Egg
Water w-- 5 0 0
Water + U.E. 0 2 0 0
0 l 202 0
Water + Pseud. 0 0 0 5
Water + Pseud. + U.E. 4,950 6 Tnc. 0
Tnc. 0 Tnc. 5
Water + Pseud. + Feces --- --- 605 40
Water + Pseud. + Feces 15 0 565 Tnc
+ U.E. Tnc. 0 Tnc. 880
Control 0 0 0 0

 

 

 

 

 

55

increase in the control eggs. Eggs treated without ultra-
sonic energy showed some microbial penetration; however, the
addition of ultrasonic waves to the treatments resulted in

an increase in bacteria found. This is especially evident
when ultrasonic energy was added to water + Pseudomonas and
water + Pseudomonas + feces. In a number of the eggs treated
in this manner, the microorganisms were too numerous to count
with the dilution used.

The effects of ultrasonic treatments of 25 Kc per sec
on the bacterial numbers found inside the shell membrane of
the eggs are shown in Table 10 and 11. One day after treat-
ment (Table 10), there was some evidence of microbial growth,
but only in four of the eggs subjected to treatments which
included ultrasonic energy. Similar results were found
seven days after treatment (Table 11). However, the incidence
of microbial penetration through the shell membrane was de-
tected in 7 0f the ultrasonically treated eggs, and there was
an increase in the number of organisms found.

The effects of ultrasonic treatments of 42 Kc per sec
on bacterial numbers from shell eggs for Experiment 4 are
shown in Tables 12-17. In Tables 12 and 15 are presented the
number of organisms outside the shell one and seven days
after treatment. At both.eva1uation dates the number of
organisms on the treated eggs were lower than on the controls.
The microbial counts one day after treatment were lower on

the eggs subjected to ultrasonic energy than on eggs treated

54

TABLE 10.--Effects of 25 K0 per sec ultrasonic treatment on
bacterial numbers inside the shell membrane l-day after
treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg
Water 0 0 0 0
Water + U.E. 0 0 0 0
0 0 0 0
Water + Pseud. 0 0 0 0
Water + Pseud. + U.E. 0 0 0 164
0 0 25 0
Water + Pseud. + Feces 0 0 0 0
Water + Pseud. + Feces 0 0 0 0
+-U.E. 2 O 4 0
Control 0 0 0 0

 

 

 

 

 

TABLE ll.--Effects of 25 Kc per sec ultrasonic treatment on
bacterial numbers inside the shell membrane 7-days after
treatment, Experiment 4.

 

 

 

 

Treatment Number of Organisms per Egg
Water --- 0 0 0
Water + U.E. 0 5 l 0
0 0 O 0
Water + Pseud. 0 0 0 2
Water + Pseud. + U.E. 0 0 550 0
50 0 585 0
Water + Pseud. + Feces --- --- 660 0
Water + Pseud. + Feces 0 0 605 0
+ U.E. O 0 600 0
Control 0 0 O O

 

 

 

 

55

TABLE 12.--Effects of 42 Kc per sec ultrasonic treatment on
bacterial numbers on the outside surface of shell eggs l-day
after treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg
Water 0 200 100 200
Water + U.E. 200 0 100 100
0 100 100 0
Water + Pseud. 1,800 1,500 500 200
Water + Pseud. + U.E. 1,700 0 0 100
0 0 0 100
Water + Pseud. + Feces 27,500 1,500 1,000 400
Water + Pseud. + Feces 0 100 700 100
+ U.E. 1,200 900 100 200
Control 110,000 140,000 165,000 58,500

 

 

 

 

 

TABLE 15.--Effects of 42 Kc per sec ultrasonic treatment on
bacterial numbers on the outside surface of shell eggs 7-days
after treatment, Experiment 4

 

-_

 

-;

 

Treatment Number of Organisms per Egg
Water 100 0 1,000 200
Water + U.E. 0 500 0 400
0 0 100 100
Water + Pseud. 0 0 700 0
Water + Pseud. + U.E. O 0 400 900
0 0 900 200
Water + Pseud. + Feces 5,800 0 100 1,900
Water + Pseud. + Feces 0 400 200 10,200
+ U.E. 0 100 400 400
Control 44,000 85,000 95,000 41,200

 

 

 

 

 

56

without ultrasonic energy.

Tables 14 and 15 show the effects of 42 Kc per sec
ultrasonic energy on the bacterial count in the shell membrane
area. One day after treatment (Table 14), bacteria were
present in three eggs treated with ultrasonic energy in com-
bination with water + Pseudomonas and water + Pseudomonas +
feces. Seven days after treatment, the number of eggs con-
taining bacteria and the number of organisms per egg in-
creased in the ultrasonically treated eggs. No bacteria were
found in this area of the eggs which did not receive ultra-
sonic treatment.

The data presented in Table 16 indicate the absence
of organisms inside the shell membrane of the control and
treated eggs one day after treatment. Seven days after
treatment (Table 17) bacteria were found inside the shell
membrane of two of the eggs that were subjected to treat-
ments Which included ultrasonic energy at 42 Kc per sec. No
bacteria were found in the other treated eggs or controls.

The effects of 85 Kc per sec ultrasonic treatments
on the bacterial numbers of shell eggs for Experiment 4 are
presented in Tables 18-25. Tables 18 and 19 report the
number of organisms on the outside of the egg shells. These
data indicate considerable variation in numbers of bacteria
found in control eggs, among treatments and within treatments.

The number of organisms found in the shell membrane

area of each egg are shown in Tables 20 and 21. The control

57

TABLE 14.--Effects of 42 K0 per sec ultrasonic treatment on

bacterial numbers in the shell membrane area l-day after

treatment, Experiment 4

 

 

Treatment

Number of Organisms per Egg

 

Water

Water + U 0E0

Water + Pseud.

Water + Pseud. + U.E.

Water + Pseud. + Feces

Water + Pseud. + Feces
+ U.E.

Control

 

O OO O OO O OO O

 

O

O OO O OO O OO

 

(D
K) <0
O HO O OO O OO O

 

01
CO
0 OO 0 OCT! 0 OO O

 

TABLE 15.--Effects of 42 Kc per sec ultrasonic treatment on

bacterial numbers in the shell membrane area 7-days after

treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg

Water 0 0 0 0
Water + U.E. 0 --- 0 605
0 0 0 1

Water + Pseud. 0 0 O 0
Water + Pseud. + U.E. 0 0 96 Tnc.
--- 0 --- Tnc.

Water + Pseud. + Feces 0 0 0 0
Water + Pseud. + Feces 0 --- Tnc. 0
+ U.E. 600 0 0 0
Control 0 0 O 0

 

 

 

 

 

58

TABLE 16.--Effects of 42 Kc per sec ultrasonic treatment on

bacterial numbers inside the shell membrane l-day after

treatment, Experiment 4

 

 

Treatment

Number of Organisms per Egg

 

Water

Water + U 0E 0

Water + Pseud.

Water + Pseud. + U.E.

Water + Pseud. + Feces

Water + Pseud. + Feces
+'U.E.

Control

 

O OO O OO O OO O

 

O

O OO O OO O OO

 

O

O OO O OO O OO

 

O OO O OO O OO O

 

TABLE 17.--Effects of 42 Kc per sec ultrasonic treatment on

bacterial numbers inside the shell membrane 7-days after

treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg
Water 0 0 0 0
Water + U.E. 0 --- 0 0
0 O 0 0
Water + Pseud. 0 0 0 0
Water + Pseud. + U.E. 0 0 0 0
0 28 0 0
Water + Pseud. + Feces 0 0 0 0
Water + Pseud. + Feces 0 0 4 0
+ U.E. 0 O 0 0
Control 0 0 0 0

 

 

 

 

 

59

TABLE 18.--Effects of 85 Kc per sec ultrasonic treatment on
bacterial numbers on the outside surface of shell eggs 1-day

after treatment, Experiment 4

Treatment A Number of Organisms per Egg

 

Water

Water + U.E.

Water + Pseud.

Water + Pseud. + U.E.

Water + Pseud. + Feces

Water + Pseud. + Feces
+‘U.E.

Control

 

400

100
100

800

200
100

9,200

220,000
35,000

6,000

 

400

800
500

100

400
200

44,000

27,500
58,500

12,500

 

200

500
200

1,000

1,000

700
800

49,500

 

600

200
5,200

10,000

100
800

500
800

140,000

 

TABLE 19.--Effects of 85 Kc per sec ultrasonic treatment on

bacterial numbers on the outside surface of shell eggs 7-days
after treatment, Experiment 4

A

-:

Treatment

 

 

 

 

 

 

Number of Organisms per Egg
Water 0 0 400 200
Water + U.E. 0 200 100 0
100 100 400 700
Water + Pseud. 0 0 600 56,800
Water + Pseud. + U.E. 0 100 900 52,000
500 51,000 1,500 100
Water + Pseud. + Feces 55,000 5,600 1,800 500
Water + Pseud. + Feces 10,000 400 1,100 400
+ U.E. 8,400 15,700 200 500
Control 15,200 7,100 62,000 2,900

 

40

TABLE 20.--Effects of 85 K0 per sec ultrasonic treatment on

bacterial numbers in the shell membrane area 1-day after

treatment, Experiment 4

 

 

Treatment Number of Organisms per Egg
Water 0 O O 0
Water + U.E. 0 165 O 88
0 40 O 0
Water + Pseud. 0 0 O 0
Water + Pseud. + U.E. O 5 0 0
1,400 91 0 0
Water + Pseud. + Feces 0 0 0 0
Water + Pseud. + Feces 2 5 0 0
+ U.E. 825 52 l 4
Control 0 0 O 0

 

 

 

 

 

TABLE 21.--Effects of 85 Kc per sec ultrasonic treatment on
bacterial numbers in the shell membrane area 7-days after
treatment, Experiment 4

 

 

 

 

Treatment Number of Organisms per Egg
Water --- 0 0 0
Water + U.E. 0 --- 184 0
0 5 10 Tnc.
Water + Pseud. 0 0 116 0
Water + Pseud. + U.E. Tnc. O 10 0
Tnc. 5,500 Tnc. 0
Water + Pseud. + Feces O 0 --- 0
Water + Pseud.'t Feces O 180 O 2
0 Tnc. Tnc. 124
‘Control 0 0 0 O

 

 

 

 

41

eggs and the eggs treated without ultrasonic energy (with

one exception) were free from bacteria at both one and seven
days following treatments. Organisms were found in the shell
membrane area of 26 eggs which were treated with 85 Kc per

sec ultrasonic waves. Twelve eggs evaluated one day after
treatment contained bacteria and fourteen eggs contained
bacteria seven days after treatment. In addition, the numbers
of organisms in six of these eggs were too numerous to count
with the dilutions used.

Numbers of organisms inside the shell membrane of
eggs subjected to 85 K0 per sec ultrasonic waves are pre-
sented in Tables 22 and 25. One day after treatment none of
the control or treated eggs contained bacteria inside the
shell membrane. Only the group of eggs treated with ultra-
sonic energy contained bacteria inside the shell membrane.

Of the eggs treated with the ultrasonic waves, eight con-
tained bacteria in the albumen. In four of these eight eggs,
the numbers of organisms were too numerous to count with the
dilutions used.

The data from the bacterial determinations were
analyzed statistically by the method of difference from rank
sums (Kramer, 1960). The number of bacteria per egg inside
the shell membranes and in the shell membrane areas were
significantly higher (1 percent level) in the eggs treated
with ultrasonic energy than in all other eggs. No significant
differences were found in numbers of bacteria per egg on the

shell surfaces due to ultrasonic treatment.

42

TABLE 22.--Effects of 85 Kc per sec ultrasonic treatment on
bacterial numbers inside the shell membrane 1-day after
treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg
Water 0 0 0 0
Water + U.E. 0 0 0 0
0 0 0 0
Water + Pseud. 0 0 0 0
Water + Pseud. + U.E. 0 0 0 0
0 0 O 0
Water + Pseud. + Feces 0 0 0 0
Water + Pseud. + Feces 0 0 O 0
+ U.E. O O O 0
Control 0 0 O O

 

 

 

 

 

TABLE 25.-~Effects of 85 Kc per sec ultrasonic treatment on
bacterial numbers inside the shell membrane 7-days after
treatment, Experiment 4

 

 

 

Treatment Number of Organisms per Egg
Water 0 0 0 0
Water + U.E. O Tnc. 0 5
0 0 0 Tnc.
Water + Pseud. 0 0 0 0
Water + Paella. + U.E. O O 7 O
456 O Tnc. 0
Water + Pseud. + Feces O 0 --- 0
Water + Pseud. + Feces 0 0 0 0
O 0 Tnc. 244
Control 0 0 0 0

 

 

 

 

DISCUSSION

The artificially soiled eggs which were washed by
means of ultrasonic energy in the medium containing a deter-
gent were cleaner, regardless of the frequency of the ultra-
sonic treatment, than eggs washed by an immersion type washer
currently used for egg washing. The superior cleaning abil-
ity of the ultrasonic cleaning units was probably due to the
effects of cavitation 0n the shell of the egg.

A comparison of the cleaning effectiveness of 25 Kc,
42 Kc, and 85 Kc ultrasonic cleaning units indicated that
the 25 Kc unit was the most effective in cleaning artifi-
cially soiled eggs. Therefore, it would appear that the
frequencies just above human audibility (20 Kc) are the
most effective for cleaning purposes, as reported by murdock
(1956).

Artificially soiled eggs Which were subjected to
ultrasonic waves in water containing a detergent were more
effectively cleaned than eggs subjected to ultrasonic energy

in water alone. The detergent apparently aided in soil dis-

persion and in the prevention of soil redeposition as reported

by Murdock (1956).
Albumen quality was not adversely affected by ultra-
sonic treatment since the mean Haugh scores of eggs held up

to 28 days at 15.5° c (60° F) were similar for the treated

45

44

and control eggs. These results are similar to those re-
ported by Dawson gt El, (1960) in which ultrasonic treat-
ment did not affect the albumen quality of eggs held for
14 and 28 days at 15.5° 6.

Quality variations between eggs within treatments
were greater than variations between treatments. Quality
differences between eggs can be attributed to individual
hen differences and to the difference in holding time from
the time of lay to the time of quality evaluation of the
eggs.

The relatively high initial pH readings for the
eggs in Trial 1 (Experiment 1) can be attributed to the time
interval from lay to quality evaluation. The initial pH of
eggs was reduced in Trials 2 and 5 (Experiment 1) in which
the holding period from lay to treatment was reduced. Treat-
ment did not influence pH values of egg albumen in any of
the trials.

The albumen quality (Haugh score) of eggs which.had
been immersed in water and subsequently held for 28 days at
15.50 C was slightly higher than the albumen quality of eggs
treated with ultrasonic energy and the unwashed control eggs.
This could be attributed to a partial thermostabilization of
the eggs immersed in water. Funk (1944) reported that eggs
thermostabilized in water (15 minutes at 1500 F) retained
their albumen quality much better than untreated eggs. The

temperatures of the water in which the eggs used in this

45

present study were immersed was 49 :_1° C (approximately
1200 :_2° F). Therefore, it is possible that the immersion
of the eggs in water at this temperature did result in a
partial thermostabilization and would account for the higher
albumen quality of the immersed eggs as compared to the con-
trols and the ultrasonically treated eggs.

Bacteria and dirt are located on the shell surface
of the eggs prior to cleaning. To minimize bacterial decom-
position it is essential that the bacteria and dirt be re-
moved before they penetrate through the egg shell into the
inner contents of the egg. The pores of egg shells could
provide a means of entry into the egg for the bacteria and
dirt. Almquist and Holst (1951) demonstrated a means of
determining egg shell porosity by the immersion of eggs for
2 minutes in a solution of methylene blue in 95 percent
alcohol.

The results of this study indicate that a water
soluble dye can be forced through the pores of the egg shell
by subjecting eggs to ultrasonic treatments of 42 Kc per sec.
Dye penetration was observed in some of the eggs. Lengthen-
ing the period of ultrasonic treatment increased the concen-
tration of dye where penetration did occur. This difference
in dye penetration between eggs may be attributed to the
differences in porosity between eggs from different hens as
reported by Almquist and Holst (1951).

Most of the dye penetration was observed in the small

46

end of the egg. This phenomenon is probably a result of a
focusing effect caused by the shell of the egg. These
results are in accordance with the work of Dawson gt_gl, (1961)
who reported that a water soluble dye was forced through the
pores of eggs using 10 Kc and 40 Kc ultrasonic generators and
appeared most often in the small end of the eggs.

Since dye was forced through the shells, it was
believed that bacteria.might behave in a similar manner.

In this study the numbers of organisms on the outside of the
shells of the treated eggs were greatly reduced in comparison
with the numbers on the unwashed controls. This would be
expected from any means of physically removing dirt includ-
ing immersion in water or treatment with ultrasonic radia-
tion.

Considerable variation in numbers of organisms is
evident among eggs as well as among treatments; this may be
attributed to variables such as egg size and initial shell
cleanliness. Increased numbers of organisms found on eggs
treated with water + Pseudomonas + fecal material in combina-
tion with ultrasonic energy may be due to the separation of
clumps of bacteria as a result of the ultrasonic treatment.
This separation would change the distribution of the bacteria
in the liquid medium and thus increase the possibility of
bacterial deposition on the outside shell surface of treated

eggs.
Analysis of the bacteria found in the shell membrane

47

area indicates an absence of organisms in the control eggs.
However, there were some organisms in some of the eggs
treated by immersion in water. Of primary interest is the
increase in numbers of organisms found in eggs treated by
ultrasonic radiation. This increase, especially in the eggs
examined seven days after treatment, can be attributed to
the direct effect of ultrasonic energy on the cuticle of the
egg or on the bacteria themselves. Coagulation or removal
of the cuticle as a result of the ultrasonic treatment could
reduce the effectiveness of this protective material. Ultra-
sonic radiations may cause changes in or dislodging of the
pore filling plaque, and thus alter the penetrability of the
egg shell.

It is noteworthy that relatively few organisms were
detected in the albumen of the control or the treated eggs.
However, the albumen of those eggs which were subjected to
ultrasonic treatments of 85 Kc did show some evidence of the
presence of bacteria. This indicates the possibility of
increased bacterial penetration which may occur with fre-

quencies of 85 Kc per sec.

SUMMARY.AND CONCLUSIONS

Artificially soiled eggs were subjected to ultra-
sonic treatments of 25 Kc, 42 Kc and 85 Kc per sec for peri-
ods of three minutes in water and water plus a detergent.
Similarly soiled eggs were washed in a water-detergent solu-
tion in an immersion type washer. A visual comparison of
cleanliness between the washed eggs and artificially soiled
control eggs indicated that the eggs subjected to ultrasonic
treatment at all three frequencies were cleaner than the
eggs washed in the immersion type washer. Eggs treated at
a frequency of 25 Kc were the most effectively cleaned (by
visual comparisons) and the inclusion of a detergent in the
liquid medium.increased the effectiveness of soil removal
from the shell of the eggs.

Eggs which were randomly selected as they were col-
lected from the nests were subjected to ultrasonic treatment
at frequencies of 25 Kc, 42 Kc and 85 Kc per sec for a peri-
od of 10 minutes. These eggs, as well as controls, were
held at 15.50 C (600 F) for periods up to 28 days. Albumen
quality (Haugh score; pH) of the eggs was determined at 0,
7, 14 and 28 days following treatment. The results indicate
that ultrasonic treatment does not affect the albumen quality
of eggs measured by Haugh score and pH.

A water soluble dye was added to water in the 42 Kc

48

49

transducerized tank. Eggs were subjected to ultrasonic
treatments (42 Kc) for periods of 5, 5, 10, and 15 minutes
in this solution. After the treatment a visual evaluation
was made of the egg shells. The results indicate that dye
penetration occurred in some of the eggs and appeared.most
often in the small end of the egg. Increasing the period of
ultrasonic treatment resulted in an increased concentration
of dye where penetration occurred.

Experiments were conducted to determine the effects
of ultrasonic energy on bacterial penetration of the egg
shell and membrane. Eggs were randomly selected as they were
collected from the nest and tested for periods of five min-
utes in one of three treatments (water; water + Pseudomonas;
water + Pseudomonas + fecal material) with or without the
addition of ultrasonic energy. Ultrasonic frequencies of
25 Kc, 42 Kc and 85 Kc per sec were used with each of these
treatments. Bacterial analysis consisted of total counts
on the outside shell surface, the shell membrane area and
inside the shell membrane.

The bacterial counts on the outside shell surface
were greatly reduced by all of the treatments. Counts in-
side the eggs were increased by treatments which included
ultrasonic energy; this indicates a possible effect of the
ultrasonic treatment on the porosity of egg shell.

The results of this study indicate that eggs were

effectively cleaned by ultrasonic energy in the ranges

50

studied, that albumen quality was not adversely affected by
ultrasonic treatment, that the number of bacteria on the
shells of the eggs was reduced by ultrasonic treatment, but
the bacterial count inside the eggs was increased by ultra-
sonic treatment. Thus the use of ultrasonic energy for
washing eggs can not be recommended due to this increase
in numbers of organisms found in the eggs.

Further studies are necessary to fully determine
the relationship between ultrasonic energy and bacterial
incidence inside the shell membrane of ultrasonically treated
eggs and to develop means of destroying bacteria or pre-
venting the entrance of bacteria into the interior of ultra-

sonically treated eggs.

LITERATURE CITED

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Anonymous, 1957. Marketing Eggs. Agric. Marketing Service,
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Carlin, B., 1949. Ultrasonics. McGraw-Hill Book Co., Inc.,
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Crawford, A. E., 1955. Ultrasonic engineering with partic-
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Dawson, L. E., C. W. Hall and E. H. Farmer, 1961. The use
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Dawson, L. E., and J. P. Watts, 1952. Effect of nest litter
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., 1944. Thermo-stabilized eggs. U. S. Egg and
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51

52

Haines, R. B., and T. Moran, 1940. Porosity of, and bacte-
rial invasion through the shell of the hen's egg. J.
Hygiene 40:455-461.

Harvey, E. N., and A. L. Loomis, 1929. The destruction of
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Johnson, C. H., 1929. The lethal effects of ultrasonic
radiation. J. Physiol. 67:556-559.

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Kraft, A. A., E. H. McNally, and A. W. Brant, 1958. Shell
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Kramer, A., 1960. A rapid method for determining signifi-
cance of differences from rank sums. Food Technol.
14:576-581.

Lorenz, F. W., and P. B. Starr, 1951. Spoilage of washed
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Mackeprang, B., 1957. The dispersing and bacterial affect
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McNally, E. H., 1952. Effect of drying after washing on
the incidence of eggs infected by spoilage microorganisms.
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55

Sabet, T. Y., 1955. Studies on egg washing and preservation.
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Starr, P. B., F. W. Lorenz, and F. X. Ogasawara, 1951.
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APPENDIX

TABLE l.--Influence of 25

55

Kc per sec ultrasonic energy on

egg quality, Trial 1, (Experiment 2)

L L

V_‘_: -

Storage time (days)

 

 

O I 7 I 14 21 I 28

 

 

Treatment
Haugh Scores of Individual Eggs

Control 88 65 55 48 54
77 46 77 65 65
78 72 69 62 48
Immersed in Water 75 71 62 62 57
71 80 65 56 64
76 65 64 55 55
Immersed in Water + 67 .72 42 60 58
Ultrasonic Energy 64 62 59 59 55
82 65 74 61 51

 

 

 

 

 

 

56

TABLE 2.--Influence of 42 Kc per sec ultrasonic energy on
egg quality, Trial 1, (Experiment 2)

 

 

 

 

 

 

 

Store 6 time da s
Treatment 5 ( y ) -
0 7 ] 14 21 If 28
Haugh Scores of Individual Eggs
Control 77 78 76 54 57
7O 72 65 52 70
85 75 65 55 59
Immersed in‘Water 79 64 70 66 66
84 69 75 65 62
86 77 75 56 74
Immersed in Water + 77 60 55 60 55
Ultrasonic Energy 86 61 57 65 69
78 66 60 65 71

 

 

 

 

 

 

57

TABLE 5.--Influence of 85 Kc per sec ultrasonic energy on
egg quality, Trial 1, (Experiment 2)

 

* L 1 f
“I .— r—fi

Storage Time (days)

 

Treatment * ‘
0 I 7 I 14 I 21 II

 

Haugh Scores of Individual Eggs

 

Control 66 57 47 58
78 77 61 47

81 79 75 7O

Immersed in Water 72 66 75 69
66 69 71 66

86 75 57 52

Immersed in Water + 75 76 61 59
Ultrasonic Energy 77 65 74 61
74 67 70 59

 

 

 

 

 

46
64
48

62
66
60

59
65

 

 

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