MICROBIAL COUNTS AND ORGANIC
ACID QUANTITATIIJN AS QUALITY
INDIBES RF EGG PRODUCTS

THESIS FOR THE DEGREE 0F PH. D.
MICHIGAN STATE UNIVERSITY
LAWRENCE RRGER YORK
19'”

   
   

“.151
LIST-EAR; Y 5
Michigan State
UBSVCXST t)“

mm

TIMES:g

This is to certify that the

thesis entitled

MICROBIAL COUNTS AND ORGANIC
ACID QUANTITATION AS QUALITY
INDICES OF EGG PRODUCTS

presented by
Lawrence Roger York
has been accepted towards fulfillment

of the requirements for

Ph. D. degreein Food Science

 

Date May 19, 1971

0-7639

ABSTRACT
MICROBIAL COUNTS AND ORGANIC
ACID QUANTITATION As QUALITY
INDICES 0F EGG PRODUCTS
By

Lawrence Roger York

Factors relating to quality indices of liquid, frozen
and dried whole egg products were evaluated. These included
the relationship between the microscopic counts and organic
acid content of commercial egg and egg inoculated with
certain species of bacteria. Also, the effect of spray
drying on the organic acid content of whole egg and the
shelf—life of commercial liquid whole egg were determined.

Gas-liquid chromatographic procedures were first
developed which were more rapid than those previously used.
A 10 min centrifugation was used to remove the precipitated
protein from the egg and an enlarged liquid-liquid extractor
was develOped to eliminate condensing the sample prior to
recovering the acids. By this procedure, formic, acetic,
propionic, butyric, lactic and succinic acids were determined
from a single sample.

A column of 10 per cent FFAP on Chromosorb W, A/W,
80/100 mesh, was demonstrated to rapidly separate the
organic acids. Formic acid was detected as butyl formate,
with butyl valerate as the internal standard. Acetic,
propionic and butyric acids were detected as the acids per

se, with butyl caprate as the internal standard. Lactic and

Lawrence Roger York

succinic acids were detected as butyl lactate and dibutyl
succinate, with butyl caprate as the internal standard.
Commercial liquid, frozen and dried eggs were sampled
by the Michigan Department of Agriculture as a part of their
food inspection program. The direct microscopic counts of
the whole egg products ranged from less than 20,000 to
A,600,000 organisms per g. Lactic acid (2 to 5 mg per 100 g
of egg) was the only acid detected. No correlation existed
between the microbial counts and the lactic acid content
of the egg.
Samples of aseptically prepared liquid whole egg were

inoculated with a single species of bacteria (Pseudomonas

 

fluorescens, Achromobacter xerosis, Escherichia coli,

 

 

 

Salmonella choleraesuis, Streptococcus faecalis or Staphy-

 

lococcus aureus) and incubated for periods up to 22 hr.

 

At frequent intervals, the samples were analyzed for their
microbial count and organic acid content.

The control samples of liquid whole egg contained both
acetic acid (0.3-2.5 mg per 100 g egg) and lactic acid
(l.A—7.6 mg per 100 g egg), as did all of the inoculated
samples. Succinic acid (0.2—0.6 mg per 100 g egg) was

present in egg in which Streptococcus faecalis, Salmonella

 

 

choleraesuis and Escherichia coli had grown. Formic acid

 

 

was found only in egg which had been decomposed by

Escherichia coli and even then, in small amounts.

 

At a total plate count of 110,000,000 per ml, the

Escherichia coli inoculated egg contained 1.0 mg of formic

 

Lawrence Roger York

acid, l6.A mg of acetic acid, 13.1 mg of lactic acid and
2A.8 mg of succinic acid per 100 g of egg. When the total
plate count increased to 5A0,000,000 per ml, the organic
acid content decreased to 0.5 mg of formic acid, 13.6 mg of
acetic acid, 6.6 mg of lactic acid and 6.6 mg of succinic
acid per 100 g of egg.

The effect of spray drying on the organic acid content
of liquid whole egg was determined. Five and 50 mg of
formic, acetic, propionic, butyric, lactic and succinic
acids were added per 100 g of liquid whole egg and the
product spray dried.

Control samples of the dried egg contained 2 to A.l mg
of lactic acid (calculated per 100 g of liquid egg). When
5 mg (per 100 g liquid egg) of acid were added, 36-58 per
cent of the volatile acids were lost but only about 35 per
cent of lactic and succinic acids were lost during drying.
When 50 mg (per 100 g liquid egg) of acid were added, less
than five per cent of the lactic and succinic acids were
lost but more than 60 per cent of the formic, acetic,
propionic and butyric acids were lost.

High quality, pasteurized liquid whole egg was found
to remain wholesome for five and 13 days when stored at
9 and 2 C, respectively. The pasteurized egg contained no

Escherichia coli, salmonella, streptococci or staphylococci

 

organisms.
These data indicate that acetic, lactic and sometimes

succinic acid may be found in small amounts in wholesome

Lawrence Roger York

liquid whole egg. Also, organic acid accumulation may vary

as microbes both produce and utilize the organic acids.

MICROBIAL COUNTS AND ORGANIC
ACID QUANTITATION AS QUALITY
INDICES OF EGG PRODUCTS

By

Lawrence Roger York

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Food Science
and Human Nutrition

1971

ACKNOWLEDGMENTS

Appreciation is expressed to Dr. L. E. Dawson for
his guidance during my study at Michigan State University.
Appreciation is also expressed to Dr. R. V. Lechowich,

Dr. C. L. Bedford, Dr. J. F. Price and Dr. D. E. Ullrey,
members of my committee.

I am grateful to the Department of Food Science and
Human Nutrition for providing academic facilities; to the
Department of Health, Education and Welfare, United States
Public Health Service for providing financial support
through Grant No. FD-00220; and to the Michigan Department
of Agriculture for their cooperation in data collection.

Appreciation is extended to my wife, Mary, for her
assistance in the preparation of this thesis and for her
encouragement and support throughout my academic studies.

Thanks are expressed to my parents and family, who
provided motivation and support to make this achievement

possible.

ii

TABLE OF CONTENTS

LIST OF TABLES.
LIST OF FIGURES
INTRODUCTION. . . . . . . . . . . . . . . . . . .
REVIEW OF LITERATURE.
Quality Evaluation of Egg Products
Microbiology of Eggs
Microorganisms as Sources of Organic Acids .
Effect of Processing on Organic Acid Formation
Methods of_Determining Organic Acids
Volatile Fatty Acids.
Lactic and Succinic Acids
EXPERIMENTAL METHODS.
Equipment and Reagents
Gas-Liquid Chromatograph and Columns.
Liquid—Liquid Extractor
Spray Drier . . . . . . . . . . . . .
Reagents.
Organic Acid Recovery.
Protein Denaturation. . . . . . . . . . . .
Liquid and Frozen Egg.
Dried Egg.

iii

11
15
16
l6
19
22
22
22
23
23
23
2A
2A
2A
2A

Page

Centrifugation. . . . . . . . . . . . . . . . . 2A
Liquid-Liquid Extraction. . . . . . . . . . . . 25

Preparation of Volatile Acids for GLC
Injection. . . . . . . . . . . . . . . . . . 25

Preparation of Butyl Esters for GLC
Injection. . . . . . . . . . . . . . . . . 27

Calibration of GLC R-Values. . . . . . . . . . . . 29
Volatile Acids. . . . . . . . . . . . . . . . . 29
Butyl Esters. . . . . . . . . . . . . . . . . . 30

Calculations for Organic Acid Quantitation . . . . 31

Determination of Liquid- -Liquid Extractor
Efficiency. . . . . . . . . . . . . . . 3A

Determination of Butyl Esterification
Efficiency. . . . . . . . . . . . . . . . . . . 35

Eggs Evaluated . . . . . . . . . . . . . . . . . . 35
Commercial Egg Products . . . . . . . . . . . . 35
Inoculated Liquid Whole Egg . . . . . . . . . . 36
Spray Dried Whole Egg . . . . . . . . . . . . . 37
Pasteurized Liquid Whole Egg. . . . . . . . . . 39

Microbiological Procedures . . . . . . . . . . . . A0
General . . . . . . . . . . . . . . . . . . . . A0

Total Plate Count . . . . . . . . . . . . . . . A0

Coliforms . . . . . . . . . . . . . . . . . . . A0
Presumptive Test . . . . . . . . . . . . . . A0
Confirmed Test . . . . . . . . . . . . . . . A0

E. 9913 . . . . . . . . . . . . . . . . . . . . Al

Fecal Streptococci. . . . . . . . . . . . . . . Al
Presumptive Test . . . . . . . . . . . . . . Al

iv

Confirmed Test
Staphylococci
Salmonella.
RESULTS AND DISCUSSION.

Organic Acid Recovery and Esterification
Extraction of Organic Acids
Esterification Efficiency

GLC Columns and Calibration. . . . . . . . . .
Columns
Internal Standards.
R—Values.

Analysis of Egg Products
Commercial Egg Products
Inoculated Liquid Whole Egg
Spray Dried Whole Egg
Shelf-Life of Pasteurized Liquid Whole Egg.

BIBLIOGRAPHY.

Table

10.

ll.

12.

LIST OF TABLES

The percentage of formic and acetic acids
recovered using 2A hr liquid-liquid
extraction.

The percentage of propionic and butyric
acids recovered using 2A hr liquid-
liquid extraction

The percentage of lactic and succinic
acids recovered using 2A hr liquid-
liquid extraction

Butyl esterification efficiency of formic,
lactic and succinic acids . .

R—values of acetic, propionic and butyric
acids

R-values of butyl formate and butyl acetate.
R—values of butyl propionate and butyl
butyrate. . . . . . . . . . . . . .

R-values of butyl lactate and dibutyl
succinate . . . . . . . . .

Microbial counts and organic acid content of
commercial liquid and frozen whole egg.

Microbial counts and organic acid content
of commercial dried whole egg

Total plate count and organic acid content
of control samples of liquid whole egg.

Total plate count and organic acid content
of liquid whole egg inoculated with
Pseudomonas fluorescens . . .

 

vi

A7

A8

A9

57
58

59

60

62

65

67

68

Table

13.

1A.

15.

l6.

l7.

18.

19.

20.

Total plate count and organic acid content
of liquid whole egg inoculated with
Achromobacter xerosis . . . .

 

Total plate count and organic acid content
of liquid whole egg inoculated with
Staphylococcus aureus . . . .

 

Total plate count and organic acid content
of liquid whole egg inoculated with
Streptococcus faecalis. . . . .

 

Total plate count and organic acid content
of liquid whole egg inoculated with
Salmonella choleraesuis . . . . . .

 

Total plate count and organic acid content
of liquid whole egg inoculated with
Escherichia coli. . . . . . . .

 

Organic acid content of control samples
of spray dried whole egg.

Organic acid content of spray dried whole
egg which had five mg of each acid
added per 100 g of liquid egg

Organic acid content of spray dried whole

egg which had 50 mg of each acid added
per 100 g of liquid egg . . .

vii

70

72

73

75

77

78

80

 

 

..I 9
II o.-. TIT“
V

1“!
Il‘l

Figure

LIST OF FIGURES

Chromatogram of butyl formate and butyl
valerate. . . . . . . . . . . .

Chromatogram of acetic, propionic, and
butyric acids and butyl caprate

Chromatogram of butyl lactate, dibutyl
succinate, and butyl caprate. .

Total plate count of pasteurized commercial
liquid whole egg held at 2 C and 9 0.

viii

53

5A

55

82

INTRODUCTION

More than seven billion lbs of liquid, frozen and dried
egg products were produced in the United States in 1970.
The quality indices of these food products include functional
characteristics, odor, microbial counts and the accumulation
of formic, acetic, lactic and succinic acids. While some
lactic acid occurs in newly-laid eggs, an accumulation of
lactic acid and the presence of formic, acetic and succinic
acids have been demonstrated to occur in decomposed liquid
egg as a result of microbial growth.

This study was conducted to assess the validity of

some of the indices of quality of egg products, and relate
these to product wholesomeness and shelf-life. Specific
objectives were:

1. To develop improved procedures for the gas-liquid-
chromatographic determination of organic acids in
egg products.

2. To tabulate the microbial counts and the organic
acid contents of official samples of commercial egg
products; to determine if a correlation exists
among these factors; and to establish the amount of

acids commonly found in wholesome egg products.

To study the role that specific bacteria have in

producing organic acids during their growth in

liquid whole
To study the
acid content
To determine

whole egg.

egg.
effect of spray-drying on the organic
of egg.

the shelf—life of pasteurized liquid

REVIEW OF LITERATURE

Quality Evaluation of Egg Products

To standardize chemical methods for the examination of
frozen and liquid egg products, Lourie (1922) recommended
that the procedures reported by the U.S.D.A. (1920) be
tentatively adopted as official methods. These procedures
included the determination of the acidity of fat. Initially,
the committee on recommendations of referees did not approve
this recommendation, but suggested that collaborative work
on the composition of eggs be done first. However, in 1923,
the method of determining acidity of fat was adopted as
official and it was immediately recommended that the desig-
nation be changed from acidity of fat to acidity of ether
extract (Callaway, 1936; White, 1936).

As a result of research done at various U.S.D.A. labora-
tories on measuring the decomposition of eggs, it was estab-
lished that newly-laid eggs of healthy fowl are essentially
free from bacteria and that spoilage of eggs in the shell is
due to the development of the embryo or to the invasion of
bacteria, which multiply and result in decomposition of the
constituents of the egg (Callaway, 1936). Decomposition
changes also occur in broken out egg unless prevented by

freezing or drying.

Mitchell (19A0) reported that volatile acid number may
be of value in detecting decomposition in yolks since good
yolks contain little or no volatile acids, while yolks in
advanced stages of decomposition contain appreciable quanti—
ties of volatile acids. He cautioned that decomposition may
involve many types of metabolically diverse microorganisms.
Thus, numerous end products can be expected and it is doubt-
ful that any one chemical method can be developed to detect
decomposition from all causes.

Lepper et a1. (19AA) found that the acidity of ether
extract was not adequate to verify egg spoilage and proposed
that viable count, direct microscopic count and the occurence
of formic, acetic and lactic acids be used as quality
indices. None of the frozen egg products they examined had
a direct microscopic count greater than 5,000,000 per g,
and dried egg products did not exceed 100,000,000 per g,
when they were prepared from wholesome shell eggs. In all
cases where these counts were exceeded, decomposed eggs had
been added to the product or the product had been stored
under conditions that allowed decomposition to occur. They
concluded that the presence of decomposed egg in liquid egg
was demonstrated by a direct microscopic count exceeding
5,000,000 per g, with detectable amounts of either formic
or acetic acid, or lactic acid in excess of seven mg per
100 g of egg. The presence of decomposed eggs in dried egg
was verified by a direct microscopic count greater than

100,000,000 per g with detectable amounts of formic acid,

and acetic acid over 65 mg and lactic acid over 50 mg per
100 g of egg (on a dry basis). The odor of liquid egg and
the taste of dried egg were used as initial indices of
decomposition, and were correlated with the microbiological
and chemical indices. Acetic acid was permitted since it
apparently formed in appreciable quantities during drying.
In addition, traces of formic acid may have formed during
drying. It was noted, however, that certain types of
decomposed egg might be present without being detected by
bacteriological and chemical methods. The odor test proved
reliable for establishing a decomposed condition in liquid
and frozen eggs in the absence of other criteria.

Lepper and Hillig (19A8) reported that succinic acid
was not found in shell eggs of acceptable quality or in
frozen or dried egg prepared from acceptable quality shell
eggs. Succinic acid formed during the process of egg
decomposition, either in the shell or after separation from
the shell. Succinic acid was not found in odorless eggs
which had high microbial counts, indicating that the type
of decomposition will determine its occurrence.

Lepper et a1. (1956) and Hillig et a1. (1960) studied
the reliability of determining decomposition by odor, and
noted that eggs with a direct microscopic count of 200,000
to 16,800,000 bacteria per g contained no formic, acetic
or succinic acids but contained five to 15 mg lactic acid
per 100 g egg. Samples with direct microscopic counts of

100,000,000 to A00,000,000 organisms per g contained

significant amounts of each acid studied. In both studies
it was concluded that the criteria of decomposition, either
Chemical or bacteriological, are not present in sound, whole-
some eggs, promptly frozen after breaking and mixing. The
work of Lepper et a1. (19AA) was also confirmed, since a
direct microscopic count of more than 5,000,000 per g of egg
with determinable amounts of either formic or acetic acids,
or lactic acid in excess of seven mg per 100 g of egg demon-
strated decomposed egg. Succinic acid was found in spoiled,
"off-odor" eggs but not in fresh eggs, thus, it was
considered an additional chemical index of decomposition.
They also concluded that odor is a reliable index of whole-
someness when used by experienced examiners.

Steinhauer et a1. (1967) stored liquid whole egg up to
eight days at 16 C and during that time the number of
bacteria increased from 800 to 330,000,000 per g of liquid
egg. Lactic acid increased from 0.A8 mg to 18.5 mg per 100
g of liquid egg and succinic acid was detected in each
sample of egg evaluated. Only traces of formic and acetic
acids were found, even in samples with very high bacterial
numbers. They concluded that although some relationship
exists between numbers of bacteria and quantities of acids
present, the detection of organic acids does not appear to
be a valid index of egg quality without supporting data.

While developing a gas-liquid chromatographic method
for organic acid detection, Steinhauer (1968) permitted

liquid whole egg to decompose from natural microbial

 

-—l
'l' 5

 

 

 

contamination. The total plate count of the egg was
1,700,000,000 microorganisms per g. The organic acid
content was found to be 21A.9 mg of lactic acid, 35.6 mg

of acetic acid, 29.8 mg of succinic acid, 17.8 mg of formic
acid and A.6 mg of propionic acid per 100 g of liquid whole
egg. No butyric acid was detected.

Landes and Dawson (1971) used silicic acid column
chromatography to analyze liquid whole egg and found that
a number of different acids were present. Those present
in the greatest quantities were formic, acetic and lactic
acids.

A court case involving the shipment of frozen whole egg
from Tennessee to Illinois established a precedent for the
validity of organoleptic tests, microbial count and the
presence of certain organic acids in frozen whole egg as
indices of wholesomeness of egg suspected of being decomposed
or of containing decomposed egg (Anon., 1959). The following
conclusions of law applicable to this study were made:

1. "Organoleptic tests by the use of the sense of smell
are determinative of the presence of decomposed
substances in frozen eggs when the odor of decom-
position is present. Decomposition can exist,
however, even when no odor is obtained but this
decomposition will be detected by bacteriological
and chemical analyses."

2. "The presence of bacteria in frozen whole egg in an

amount in excess of 5,000,000 per gram of egg by

 

‘ lv\‘l

 

....5.\I

 

8

direct microscopic count is determinative of the
presence of decomposed substance in eggs."

3. "The presence of acetic, formic or succinic acid in
any measurable quantity in frozen whole egg is
determinative of the presence of decomposed
substances in the eggs."

A. "The presence of lactic acid in excess of seven
mg per 100 g of egg in combination with a direct
microscopic bacterial count of 5,000,000 or more
is determinative of the presence of decomposed
substances in frozen eggs."

Microbiology of Eggs

Most newly laid eggs are bacteriologically sterile
(Haines, 1939; Gibbons et al., 19AA; and Winter et al.,
19A6). More recently, Brooks and Taylor (1955) reported
that about 90 per cent of newly laid eggs are free of
microorganisms, and the true value may be even higher.
Forsythe et a1. (1953) reported that egg shells and shell
membranes harbored 10,000 to 1,000,000 (with a mean of
63,000) organisms per egg, yet egg contents averaged fewer
than two organisms per egg.

Stuart and McNally (19A3) reported that the shells of
only a few eggs were contaminated when passing through the
cloaca and that the main contamination apparently occurred
after laying. The extent of external contamination is a
function of the cleanliness of the nesting area and the

manner in which the eggs are handled after laying (Harry,

1963b). Eggs are also extremely susceptible to contamination
by high microbial count wash water (Penniston and Hedrick,
19A7; Brown et al., 1966). Board et a1. (196A) reported
that the number of bacteria on shell eggs varies greatly,
even on clean eggs. Kraft et a1. (1967) stressed that the
main source of contamination in broken out eggs is the
breaking process.

Since the potential sources of contamination are
numerous, they provide a great variety of microorganisms
that may be found on eggs. Evidence indicates that some

pathogens such as Salmonella species pass from the alimentary

 

canal via the blood to the ovaries (Rettger, 1913; May, 192A;
and Gordon and Tucker, 1965) but there appears to be no such
migration by microorganisms capable of decomposing eggs.
Rot producing organisms are of external origin and Brooks
and Taylor (1955) and York et a1. (1970) observed that less
than one per cent of naturally clean eggs decomposed during
prolonged storage. Gillespie et al. (1950) found that most
of the bacteria present on egg shells were Gram-positive
cocci and rods that were not involved in rotting. Relatively
few Gram-negative bacteria, some of which cause rots, were
present unless large numbers were added by improper washing
methods.

Gram—positive bacteria were numerically predominant on
clean or lightly soiled eggs, while Gram-negative bacteria
were predominant on badly soiled eggs (Zagaevsky and

Lutikova, 19AA; and Board et al., 196A). These contaminants

10

were probably derived from dust, soil and feces. Forsythe
et a1. (1953) found few molds on shell eggs.

The distribution of microflora in washings from shell
eggs was studied by Haines (1939). He noted a distribution
on nonspore forming rods, 38 per cent; sporing rods, 30 per
cent; cocci, 25 per cent; yeast, four per cent; and molds,
three per cent. More recently, Zagaevsky and Lutikova (19AA)
and Gunderson and Gunderson (19A5) reported similar micro-
flora on shell eggs.

The most common contaminants of the contents of
unbroken, newly—laid eggs were found to be micrococci which
grow poorly, if at all, at the body temperature of the hen
(Hadley and Caldwell, 1916; Miller and Crawford, 1953).
They have also been recovered from ova removed from hens by
dissection (Harry, 1963a).

Even though newly-laid, sound-shelled eggs are rela-
tively free of microorganisms, nearly all commercially
prepared liquid, frozen and dried egg products contain
several hundred to several million bacteria per g (Redfield,
1920; Johns, l9AA; McFarlane et al., 19A7; and Winter and
Wrinkle, 19A9). The dirt on shell eggs and the practices
used in egg-breaking plants account for most of the sources
of bacteria found in liquid, frozen and dried egg products
(Haines, 1939; Rosser, l9A2; Schneiter et al., 19A3;
Zagaevsky and Lutikova, l9AA; McFarlane et al., l9A5; Johns

and Berard, l9A5; Gunderson and Gunderson, l9A5; Solowey

11

et al., l9A6; Penniston and Hedrick, 19A7; Winter and
Wrinkle, l9A9; and Kraft et al., 1967).

The dust on shell eggs, which consists of fecal
material, soil, nest and feather particles, may provide a
heterogeneous and variable microflora in egg products.
Microorganisms are also present in the air, on equipment
and on workers in the breaking plants. Haines (1939) and
Zagaevsky and Lutikova (19AA) reported a number of species
found on egg shells. These included species of Achromo-

bacter, Pseudomonas, Streptococcus, Staphylococcus,

 

 

Alcaligens, Flavobacterium, Proteus, Bacillus, Sarcina,

 

 

 

Serratia, Micrococcus, coliform bacteria and molds. Florian

 

 

and Trussell (1957) identified species of Pseudomonas,

 

Alcaligens, Proteus, Flavobacterium, Paracolobactrum,

 

 

 

Achromobacter, Aerobacter, and Escherichia as spoilage

 

 

 

organisms of shell eggs.
Microorganisms as Sources of Organic Acids

As previously discussed, early work on the development
of chemical methods to evaluate egg quality included a
measurement of volatile acids. At the same time, studies
on the metabolism of microorganisms were conducted which
noted the ability of certain microorganisms to produce the
volatile organic acids found in decomposed egg.

Lepper et a1. (19AA) reported that formic and acetic
acids were formed and lactic acid was substantially increased
when liquid egg decomposed. This development of acids was

accompanied by a great increase in the microbial population.

12

Thus, the association between bacterial count and organic
acid content as indices of egg quality was apparent.

Later studies on the metabolism of microorganisms
demonstrated that numerous microorganisms are capable of
producing short-chain organic acids. Many of these same
types of microorganisms are found in egg products.

Many microorganisms ferment glucose mainly to lactic
acid with an accompanying formation of trace amounts of
formic and acetic acids (Prescott and Dunn, l9A0). These
include certain bacilli and molds, a large number of lacto-

bacilli and all members of the genera of Streptococcus,

 

Pediococcus and Microbacterium.

 

 

Homofermentative bacteria, which include Streptococcus,

 

Pediococcus and certain Lactobacillus, produce lactic acid

 

 

as virtually the only end product of glucose fermentation.
On the other hand, heterofermentative bacteria such as

Leuconostoc and certain Lactobacillus, ferment glucose to

 

 

lactic acid, carbon dioxide, ethyl alcohol and sometimes
acetic acid (Stanier et al., 1963). Thimann (1955) reported

that homofermentative Thermobacterium, Streptobacterium

 

 

and Streptococcus produce lactic acid from sugar in an 80 to

 

90 per cent yield with only traces of by—products formed.

The heterofermentative Betabacterium and Leuconostoc convert

 

 

about one—half of the sugar to lactic acid with the remainder
converted to carbon dioxide, hydrogen, ethyl alcohol, formic

acid and acetic acid.

13

The by-products of glucose fermentation by species of

the genus Streptococcus have been extensively studied

 

(Hammer, 1920; Foster, 1921; Langwill, 192A; Long and
Hammer, 1936; Friedmann, 1939; Gunsalus and Umbreit, 19A5;

and White et al., 1955). The Streptococcus liquefaciens

 

strain studied by Gunsalus and Niven (19A2) was known to
produce lactic acid in excess of 90 per cent of the glucose
fermented. At a pH of 6.5 or higher, the production of
lactic acid decreased to A0 per cent and the combined
production of formic acid, acetic acid and ethyl alcohol
increased to account for 25 to A0 per cent of the sugar
fermented. Platt and Foster (1958) reviewed reports that
products other than lactic acid may be produced by homo-
fermentative streptococci. These substances include small
quantities of formic, acetic, propionic and butyric acids,
ethyl alcohol, carbon dioxide, 2,3-butanediol, acetoin

and biacetyl.

According to Gallagher and Stone (1939), Knaysi and
Gunsalus (19AA) and Puziss and Rittenberg (1957), many
members of the genus Bacillus produce formic, acetic, lactic
and succinic acids in variable amounts from the fermentation
of glucose. Traces of butyric acid were sometimes detected.
Wood (1961) reported that certain clostridia and bacilli
produce butyric acid as a characteristic product of carbo-
hydrate fermentation.

Several species of Lactobacillus and Leuconostoc were

 

 

studied by Nelson and Werkman (1935), Gibbs et a1. (1950)

1A

and DeMoss et al. (1951) and found to produce lactic and
acetic acids as principle end products.

Stokes (19A9) observed that Escherichia coli fermented

 

glucose to formic, acetic, lactic and succinic acids and
ethyl alcohol. Blackwood et a1. (1956) studied several

strains of Escherichia coli and reported that formic, acetic,

 

lactic and succinic acids and ethyl alcohol were the major
products of glucose fermentation. The amount of each acid
produced was different at pH 6.2 than at pH 7.8. Butyric
acid was produced at either the high or low pH but not at
both. Similarly, the pH has been observed to influence the

2,3—butanediol fermentation of Aerobacter (Mickelson and

 

Werkman, 1938). Above pH 6.3, acetic and formic acids
accumulated and below pH 6.3, only formic acid accumulated.
A mixed-acid glucose fermentation is characteristic of

the genera Escherichia, Erwinia, Salmonella, Shigella and

 

Proteus. Formic, acetic, propionic and butyric acids are
produced, along with ethyl alcohol, carbon dioxide and
hydrogen gas. Only small amounts of formic acid accumulate
since most of the formic acid is converted to carbon dioxide
and hydrogen gas. However, Shigella and a few Salmonella
lack the enzyme formic hydrogenlyase and, therefore, are
unable to cleave formic acid to carbon dioxide and hydrogen.
Consequently, formic acid accumulates and no gas is formed
(Stokes, 1956; Stanier et al., 1963).

Members of the genera Aerobacter, Klebsiella and

Serratia ferment glucose by a characteristic butylene glycol

l5

fermentation. The products formed are the same as those
from a mixed acid fermentation, in addition to 2,3-butylene
glycol. However, the organic acids may occur in only small
quantities or not at all (Peterson and Breed, 1928; Wood,
1961; and Stanier et al., 1963).

Certain facultative anaerobic bacteria of the genus

Achromobacter have been observed to possess a mixed acid

 

type of fermentation similar to that of Escherichia coli

 

(Doudoroff, 19A2).

Stanier (l9A7) studied strains of fluorescent pseudo-
monads and reported that some of them oxidize alcohol to
acetic acid. He also noted that pseudomonads cannot tolerate
media which are more acidic than pH 5.0, and that they
vigorously attack proteins and peptones. Crawford (195A)

studied members of the genus Pseudomonas and found that

 

formic, acetic, lactic and succinic acids were the products
of glucose fermentation.
Effect of Processing on Organic Acid Formation

Studies concerning the quality of egg products indicated
that when shell eggs were broken-out and made into liquid
and frozen egg, there was no change in the organic acid
content as long as the eggs were handled properly and not
allowed to decompose (Callaway, 1936; Lepper et al., l9AA;
Lepper and Hillig, 19A8).

Reagan (1970) observed the concentration of short-
chain fatty acids in liquid whole egg before and after the

egg had been pasteurized. He reported that pasteurization

 

I I‘ll
.3. .Q)...

 

‘

16

of liquid egg caused no change in the amount of acid present.
Furthermore, he added formic, acetic, propionic, butyric,
lactic and succinic acids to liquid egg prior to pasteur-
ization and found that the concentrations of the added acids
were not altered by the pasteurization process. He used a
continuous pasteurization process which held the liquid
egg at 60 C for 3.5 min.

Callaway (1936) reported that the acidity of the ether
extract of dried egg was directly related to the acidity
of the ether extract of the corresponding liquid egg.

Lepper et a1. (19AA) spray-dried batches of liquid
egg which had been judged, by odor, to be high grade, low
grade and inedible. During the drying process there was no
change in the amount of lactic acid present, however, traces
of formic acid and appreciable amounts of acetic acid were
formed. Over-heating was found to cause no further increase
of these acids. Clayborn and Patterson (19A8) and Lepper
and Hillig (19A8) observed that dried egg prepared from
fresh liquid egg did not contain succinic acid. However,
succinic acid was found in dried egg when the dried egg was
prepared from decomposed liquid egg.

Methods of Determining Organic Acids

Volatile Fatty Acids

 

 

Early procedures for the determination of volatile
fatty acids were generally of two types; steam distillation,
and partition of the acids between water and a suitable

solvent.

1?

Dyer (1917) proposed the first distillation procedure
in which steam was used to isolate the fatty acids. He
reported that when a solution of a volatile acid is steam-
distilled at a constant rate, a definite percentage of the
acid is recovered in a given volume of distillate. Clark
and Hillig (1921) noted that the size and design of the
distillation apparatus affected the quantity of acid
recovered per volume of distillate and they proposed a
distillation model that was easily constructed from available
laboratory equipment and was operated under specified
conditions.

Hillig and Knudsen (19A2) studied the distillation
rates of one, two, three and four-acid systems using formic,
acetic, propionic and butyric acids. When the percentage of
a single acid in the distillate was plotted against the
volume of distillate, a straight line was obtained. However,
a solution of two or more acids appeared as a curved line
when plotted. This indicated that, in a mixture of acids,
the percentage of acid recovered in any single volume of
distillate was not the same for each acid.

In its present form, the steam distillation procedure
is an official A.O.A.C. procedure for the quantitation of
volatile fatty acids.

Werkman (1930) partitioned the volatile acids between
water and isopropyl ether and reported satisfactory results
when the quantity of each acid was high. A procedure for

detecting micro-amounts of organic acids, including volatile

l8

fatty acids, was developed by Ramsey and Patterson (19A5)
and Ramsey (1963). The acids were recovered by steam
distillation, separated by silicic acid partition column
chromatography and quantitated by titration with sodium
hydroxide.

Landes and Dawson (1971) adapted the procedure used
by Ramsey (1963) to determine the presence of organic acids
in liquid whole egg.

Paper chromatography was used by Stark et a1. (1951)
and Kennedy and Barker (1951) to separate and identify
volatile fatty acids but quantitation was poor.

The development of gas-liquid chromatography (GLC)
offered speed, improved sensitivity and refined separation
of volatile fatty acids in analytical research. Using
GLC procedures, James and Martin (1952) reported a method
for the separation and estimation of straight and branched
chain fatty acids from formic to dodecanoic. Hawke (1957)
chromatographed volatile acids recovered from oxidized
butterfat, and Hankinson et a1. (1958) developed a GLC
procedure for determining short-chain fatty acids in milk.

Shelley et a1. (1963) reported a quantitative GLC
procedure for detecting formic, acetic, propionic and
butyric acids recovered from food products by steam distil—
lation. They demonstrated the usefulness of this procedure
for analyzing fatty acids in eggs and fish. A similar

procedure was developed by Steinhauer and Dawson (1969b)

19

for detecting acetic, propionic and butyric acids in liquid
whole egg.

It became known that rapid GLC analysis of long—
chain fatty acids was possible by preparing esters, usually
methyl esters, of the fatty acids. Using this information,
Horrocks et a1. (1961) and Gehrke and Lamkin (1961) demon—
strated the preparation of methyl esters of short-chain
fatty acids for GLC analysis.

The GLC procedure used by Shelley et a1. (1963) to
detect volatile acids required the use of an all—glass
injection and column system to prevent decomposition of
formic acid which occurred in metallic systems. Steinhauer
and Dawson (1969b) reported that formic acid could be
detected as its butyl ester without loss when using an
all-metal injection and column system.

Lactic and Succinic Acids

 

One procedure for determining lactic acid in biological
materials was to oxidize the lactic acid to acetaldehyde
and determine the quantity of the latter (Friedmann and
Graeser, 1933). Barker and Summerson (19Al) used sulfuric
acid to oxidize the lactic acid and quantitated the ace—
taldehyde by colorimetric technique. The color was developed
by reacting p-hydroxydiphenyl with acetaldehyde in the
presence of cupric ions.

Hillig (1937a, b, 0) reported a colorimetric procedure
for the direct determination of lactic acid in which the

acid is recovered by liquid-liquid extraction and reacted

20

with ferric chloride to produce a quantitative color. This
procedure was used by Lepper et a1. (19AA) to detect lactic
acid in eggs, and in its present form is an official
A.O.A.C. procedure.

A method to determine both lactic and succinic acids
in food was developed by Clayborn and Patterson (19A8).

They separated the acids by silicic acid column chroma-
tography and quantitated the acids by titration. They

also prepared zinc lactate and barium succinate crystals

for crystallographic identification. This type of procedure
is currently an official A.O.A.C. procedure for determining
succinic acid content.

Lactic and succinic acids were among the acids isolated
from blood and identified by silicic acid chromatography
(Ramsey, 1963). Landes and Dawson (1971) applied a variation
of this procedure to identify acids, including lactic and
succinic, from liquid whole egg.

Paper chromatography has been used to separate and
identify lactic and succinic acids (Magasanik and Umbarger,
1950). However, as with volatile acids, the procedure is
not satisfactory for accurate quantitation.

Gehrke and Goerlitz (1963) reported a gas chromato—
graphic procedure for the determination of lactic and
succinic acids, isolated from biological materials, as
their methyl esters.

A method to detect lactic and succinic acids as butyl

esters, from liquid whole egg, was developed by Steinhauer

D: ‘I 1?

VI.’

21

and Dawson (1969a). Quantitation by this procedure was
comparable to that by A.O.A.C. (1965) procedures. Reagan
et a1. (1971) modified the method reported by Steinhauer
and Dawson (1969a) to make it more rapid.

Salwin and Bond (1969) reported a GLC procedure for
detecting lactic and succinic acids as propyl esters. This
esterification technique is advantageous in that it is very

rapid and does not require heating.

EXPERIMENTAL METHODS

Equipment and Reagents

Gas-Liquid Chromatograph and Columns

 

A dual column, flame ionization, model 810 F and M
Scientific Co. gas-liquid chromatograph (GLC) equipped with
a model 929AN Honeywell recorder was used in this study.

The columns were six feet long, one-fourth inch diameter
stainless steel packed with 10 per cent FFAP (Free Fatty
Acid Phase) on Chromosorb W, A/W, 80/100 mesh. The carrier
gas was helium at a flow rate of A0 ml per min. The air
flow rate was 370 ml per min and the hydrogen flow rate was
62 ml per min. Detector temperatures were 270 C and injec-
tion port temperatures were 230 C. The chart speed was
15 in per hr and the size of the injected sample was 1 ul.

An isothermal column temperature was maintained during
the analysis of each injected sample. The column temperature
was 170 C for the detection of acetic, propionic and butyric
acids; 200 C for the detection of butyl lactate and dibutyl
succinate; and 100 C for the detection of butyl formate,
butyl acetate, butyl propionate and butyl butyrate. For
analyses at 170 C and 200 C, butyl caprate was the internal
standard. At 100 C, butyl valerate was the internal

standard.

22

I} I‘ll-TITIIII
I I. IPA ‘Illllllll .1". l

23

Liquid—Liquid Extractor

 

The liquid-liquid extraction apparatus was similar in
design to that used in A.O.A.C. (1965) procedure 15.012 and
illustrated in A.O.A.C. (1965) Fig. 15:1. However, the
sample holding portion of the extractor used in this study
was enlarged to hold 500 ml of liquid. Also, a 100 ml
round-bottom flask was attached to the side-arm of the
extractor rather than a 250 ml round—bottom flask.

Spray Drier

 

A Swensen Pilot Plant Model spray drier was used to
dehydrate the egg samples for this study. The drier was of
the vertical-upright, concurrent flow type with a cyclone
powder collector and had the capacity to evaporate 12 to
16 lbs of water per hr. An outlet temperature of 85 C was
maintained as closely as possible.

Reagents

Reagent grade chemicals and distilled water were used
throughout this study. The chemicals were checked for
purity using the gas—liquid chromatograph and, when neces-
sary, were redistilled.

To determine the exact normality and mg concentration
of each acid solution, 20 ml aliquots were pipetted into a
flask and titrated with an accurately standardized 0.1 N
solution of sodium hydroxide to a phenolphthalein end point.

Since the butylated acids could not be titrated, they
were weighed to the nearest 0.1 mg on a Mettler balance to

determine the mg concentration of each solution.

2A

Organic Acid Recovery

Protein Denaturation

 

Liquid and Frozen Egg. Frozen egg samples were thawed

 

in a bath of cold, running tap water for about one and one-
half hr.

A 200 g aliquot of liquid egg was weighed into a tared,
1,000 m1 wide-mouth erlenmeyer flask. Three hundred grams
of water were added and the contents mixed by swirling. To
the egg-water mixture, 75 g of l N sulfuric acid and 125 g
of 20 per cent (w/w) phosphotungstic acid were added. The
flask was stoppered and shaken vigorously for one min.

Egleg Egg. Fifty grams of dried egg were weighed into
a tared, 1,000 m1 wide-mouthed erlenmeyer flask. The dried
egg was reconstituted by adding 150 g of water and stirred
until the rehydrated egg was well mixed. An additional
300 g of water were added, the flask stoppered with a rubber
stopper and the solution mixed by shaking. Fifty grams of
l N sulfuric acid and 75 g of 20 per cent (w/w) phospho-
tungstic acid were stirred into the egg-water mixture.
Approximately 75 m1 of water were added to make the mixture
weigh 700 g. The flask was stoppered and shaken vigorously
for one min.

Centrifugation

 

Following protein denaturation, a 225 g portion of the
liquid, frozen or dried egg sample was poured into each of

three 250 m1 polyethylene centrifuge bottles. These were

25

placed into a Sorvall RC-2B centrifuge and centrifuged for
10 min at 10,000 rpm (16,300 X gravity).

Liquid—Liquid Extraction

 

The filtrates in the three bottles were combined
following centrifugation and A50 g were transferred to the
sample holding portion of a liquid-liquid extractor. An
inner tube was placed into the extractor and a sufficient
amount of diethyl ether was added to raise the total volume
of liquid to the extractor side-arm level.

Eighty milliliters of diethyl ether and a glass boiling
bead were placed into a 100 ml round-bottom flask and the
flask connected to the extractor side-arm. The flask was
heated by a preheated heating mantle. Preheating the mantle
was necessary to prevent superheating the ether. The heating
mantle was attached to a powerstat set at 97 (on a scale
of 100). This setting provided sufficient heat to produce
a steady flow of ether through the extractor. An extraction
time of 2A hr was used and timing commenced as the first
drops of ether flowed through the sample. At the end of the
extraction period, the heat was turned off and the ether
allowed to cool before the round—bottom flask was dis-
connected.

Preparation 93 Volatile Acids for GLC Injection

 

 

Several drops of 5 N ammonium hydroxide (in acetone)
were added to the extraction flask following removal from
the extraction assembly to make the acid-ether mixture basic.

The boiling flask was placed over a 65—70 C steam bath and

26

the sample concentrated to a volume of two-three m1. A few
drops of dichloroacetic acid solution (12.5 g per 100 ml
acetone) were added to the condensed sample to make it acidic
and liberate the ammonium salts. The sample was quantita-
tively transferred to a 10 ml volumetric flask using a micro-
funnel. The round—bottom flask and microfunnel were rinsed

a minimum of three times, with diethyl ether, into the
volumetric flask. Additional ether was added to the flask

to make the solution total 8—8.5 ml. The flask was stoppered
and its contents were mixed.

A one ul aliquot was injected into the GLC to determine
the peak heights of acetic, propionic and butyric acids.
Based upon the peak heights of the acids and the sensitivity
of the GLC apparatus, the previously determined R-value
(detector response ratio of the compound and the internal
standard) data were used to calculate the approximate concen-
tration of each acid in the 8-8.5 ml sample. With this
information, the necessary amount of internal standard was
determined to provide a peak height commensurate with the
peak heights of the acids. The proper amount of internal
standard was added and the contents of the flask were
brought to 10 ml with diethyl ether. The solution was mixed
and one ul aliquots were injected into the GLC to quantitate
for acetic, propionic and butyric acids.

The internal standard was butyl caprate. A stock
solution of butyl caprate was made by weighing, to the

nearest 0.1 mg, approximately 0.75 g of the ester into a

27

100 m1 volumetric flask and making to volume with diethyl
ether. Various dilutions of the stock solution were prepared
so that exactly one ml of one of the dilutions would provide
a peak height commensurate with the peak height of the acids
when added to the sample mixture.

Preparation 9: Butyl Esters for GLC Injection

 

 

The sample remaining after volatile acid analysis was
quantitatively transferred to a 50 m1 round-bottom flask and
the ether was evaporated over a 65-70 C steam bath. Approx—
imately 1.3 m1 of 1.25 N HCl in n-butanol and 500 mg of
anhydrous sodium sulfate were added to the sample. The
flask was connected to a water cooled condenser and posi-
tioned in a heating mantle. The mixture was refluxed for
two hr with the heating mantle connected to a powerstat set
at A5 (on a scale of 1A0). The top of the condenser was
equipped with a drying tube filled with dessicant to aid in
keeping the esterification system free of water.

The heating mantle was disconnected and allowed to cool
at the end of the esterification period. The sides and
joint of the condenser were rinsed with one m1 of diethyl
ether into the round-bottom flask. The contents of the
flask were quantitatively transferred to a 10 ml volumetric
flask and additional ether was added, when necessary, to
make the volume of solution total 8-8.5 ml.

One microliter aliquots were injected into the GLC to
determine the peak heights of butyl formate, butyl lactate

and dibutyl succinate. Based upon the peak heights of the

28

butyl esters and the sensitivity of the GLC apparatus, the
previously determined R-value data were used to calculate
the approximate concentration of each butyl ester in the
8-8.5 ml sample. With this information, the necessary
amounts of internal standards were determined to provide
peak heights commensurate with the peak heights of the butyl
esters. The proper amounts of the internal standards were
added and the contents of the flask were brought to 10 ml
with diethyl ether. The solution was mixed and one ul
aliquots were injected into the GLC to quantitate for butyl
formate, butyl lactate and dibutyl succinate.

The internal standard for the determinations of butyl
lactate and dibutyl succinate was butyl caprate. Since
butyl caprate had been previously added as the internal
standard for volatile acids, this was considered in the
total amount added.

The internal standard for the determination of butyl
formate was butyl valerate. A stock solution of butyl
valerate was made by weighing, to the nearest 0.1 mg,
approximately 1.5 g of the ester into a 100 ml volumetric
flask and making to volume with diethyl ether. Various
dilutions of the stock solution were prepared so that exactly
one m1 of one of the dilutions would provide a peak height
commensurate with the peak height of butyl formate when

added to the 10 ml volumetric flask.

 

C.

‘IIIT

l/NL

29

Calibration of GLC R-Values

Volatile Acids

 

Individual stock solutions of acetic, propionic and
butyric acids were prepared by weighing, to the nearest
0.1 mg, approximately 1.5 g of each acid into a 100 ml
volumetric flask and making to volume with diethyl ether.

A stock solution of butyl caprate, the internal standard,
was prepared in a similar manner using approximately
0.75 g of the ester.

Twenty milliliters of each stock solution were pipetted
into a 100 ml volumetric flask and mixed. Dilutions of
this mixture were made by consecutively pipetting together
20 ml of diethyl ether and 20 m1 of acid—ether mixture to
provide a range of acid concentrations.

Triplicate one ul injections of each concentration were
made into the GLC and the proper GLC sensitivity for each
concentration was noted. The peak heights of acetic,
propionic and butyric acids and of butyl caprate were
measured in mm. The R—value was calculated for each acid
using the formula reported by Shelley et a1. (1963):

(hA)(CIS)

(hIS)(CA)

RA =

 

RA is the detector response ratio of the acid and the

internal standard; hA and hIS are the peak heights of the

acid and of butyl caprate, respectively; and cA and cIS are

the concentrations (mg per 10 m1 of ether solution) of the

acid and of butyl caprate, respectively.

:1.

 

U

30

Butyl Esters

 

Individual stock solutions of butyl formate, butyl
lactate, dibutyl succinate and butyl valerate were prepared
by weighing, to the nearest 0.1 mg, approximately 3.0 g of
the butyl ester into a 100 m1 volumetric flask and bringing
to volume with diethyl ether. A stock solution of butyl
caprate was prepared in a similar manner using approximately
1.5 g of the ester.

Twenty milliliters of each stock solution were pipetted
into a 200 ml volumetric flask and mixed. Dilutions of this
mixture were made by consecutively pipetting together 20 ml
of diethyl ether and 20 ml of ester-ether mixture to provide
a range of butyl ester concentrations.

Triplicate one ul injections were made into the GLC
at each temperature-concentration combination and the prOper
GLC sensitivity for each concentration was noted. The peak
heights for each butyl ester were measured in mm. The
detector response value was then calculated for each ester

using the formula reported by Shelley et a1. (1963):

(h )( )
R BE CIs

 

BE (hIS)(CBE)

R is the detector response ratio of the butyl ester and

BE

the internal standard; hBE and h are the peak heights of

IS
the butyl ester and the internal standard, respectively;

and CBE and CI are the concentrations (mg per 10 m1 of

S
ether solution) of the acid and the internal standard,

respectively.

31

Calculations for Organic Acid Quantitation
Rearrangement of the detector response value equation
gives the following:
For the volatile acids:
(hA)(CIS)

(h13><RA>

 

CA =
For the butyl esters:
(hBE)(CIS)

(“13)(Rss)

The peak heights for the acids and the butyl esters

CBE =

 

were measured from the chromatograms of the egg samples.

The R-value for each acid and butyl ester had been previously
determined and the amount of internal standard added was
known. Thus, the only unknowns in the two equations were

the concentrations of the acids and of the butyl esters.
These concentrations were calculated and their units were

in mg of acid or butyl ester per 10 m1 of ether. To convert
the units to mg acid per 100 g egg, the concentration was

multiplied by a conversion factor.

The calculations used to determine the conversion

factors for liquid and frozen egg were as follows:

32

 

 

 

Column

A_ci_d A a 9 .12 _E_ E.

Formic 700 100 100 0.A507 100 .3611A
E50 85.5 97.0 200

Acetic 700 100 100 .8AA36
550 92 l 200

Propionic 700 100 100 .79305
550 98-5 200

Butyric 700 100 100 .77980
550 99 7 200

Lactic 700 100 100 0.6162 100 .59561
H50 90 1 89.0 200

Succinic 700 100 100 0.513A 100 .A60A8
H50 96.8 9.0 200

Where: Column A is the factor which corrects for the fact

Column B is the factor which accounts for the

were used.

liquid extraction.

that A50 g of the original 700 g of the egg-

water—phosphotungstic-sulfuric acid mixture

percentage of the acid recovered by liquid-

Column 0 is the factor which accounts for the butyl

Column D is the factor which converts the butyl

esterification efficiency.

ester to the acid.

33

Column E is the factor which converts mg acid per
200 g of egg to mg acid per 100 g of egg, where
a 200 g egg sample was used.

Column F is the conversion factor (product of
columns A-E) for each acid in liquid and frozen
egg.

The calculations used to determine the conversion

factors for dried egg were as follows:

 

Column

 

 

 

Acid E g E

Formic .3611A 52.8 0.39559
A8.2

Acetic .8AA36 52.8 0.92A9l
A8.2

Propionic .79305 52.8 0.86871
A8.2

Butyric .77980 52.8 0.85A19
ARTE

Lactic .59561 52.8 0.652A3
A8.2

Succinic .A60A8 52.8 0.50AA1
A8.2

Where: Column F is the conversion factor (product from

previous page) for each acid in liquid and
frozen egg.
Column G is the factor which converts 50 g of spray

dried egg (3.6 per cent moisture; A8.2 g of

3A

solids) to 52.8 g of solids (the amount of solids
in 200 g of liquid egs).
Column H is the conversion factor (product of
columns F and G) for each acid in dried egg.
Determination of Liquid—Liquid Extractor Efficiency

Individual solutions of formic, acetic, propionic,
butyric, lactic and succinic acids were prepared in 1 N
concentrations. A 0.05 N solution of alcoholic potassium
hydroxide was prepared by adding 2.8 g of potassium hydroxide
to one liter of ethyl alcohol. These solutions were used
to determine the percentage of each acid that was recovered
by the liquid-liquid extraction procedure.

Ten ml of a l N acid solution, AAO ml of water and five
drops of 50 per cent (v/v) sulfuric acid were placed into
the sample holding portion of an extractor. This solution
represented a A50 g egg filtrate sample and was extracted
by the procedure previously described in the section on
liquid-liquid extraction. At the same time, a solution
of A50 g of water and five drops of 50 per cent sulfuric
acid was extracted, as a control.

When the extraction was completed, each round-bottom
flask was cooled and disconnected from the extractor side-
arm. Its contents, containing the extracted organic acid,
were titrated with 0.05 N alcoholic potassium hydroxide to
a phenolphthalein end point. Ten m1 of the corresponding
acid stock solution were pipetted into each of three 125 ml

erlenmeyer flasks and titrated in the same manner. Using

35

the titration values, the percentages of the organic acids
recovered by the extraction procedure were calculated by

the equation:
ml alcoholic KOH to _ ml alcoholic KOH
titrate extracted sample to titrate control

ml alcoholic KOH to
titrate 10 ml stock solution

Determination of Butyl Esterification Efficiency

 

(100)

Formic, lactic and succinic acids were prepared in
separate 0.1 N solutions and five ml of each solution were
pipetted into a 100 ml round-bottom flask. The water was
evaporated from the mixture using a steam bath and a stream
of air. After drying, the acids were butylated using the
procedure previously described in the section on preparation
of butyl esters for GLC injection. The concentrations of
the butyl esters were determined using the procedure previ-
ously described in the section on calculations for organic
acid quantitation. The per cent esterification for each
acid was calculated by the equation:

(amount of butyl ester recovered)

 

(100)

theoretical amount of butyl ester
recovered if butylation was 100 per cent

Eggs Evaluated

Commercial Egg Products

 

Commercial liquid, frozen and dried eggs were sampled
according to A.O.A.C. (1965) procedures by the Michigan

Department of Agriculture, as a part of their food inspection

36

program. The containers of egg were obtained from egg
processors and users of egg products throughout the state
of Michigan.

A total of A1 whole egg samples were evaluated during
the period from July 1, 1969 to June 30, 1970.

Inoculated Liquid Whole Egg

 

Forty-four dozen eggs from a commercial poultry farm,
having one breed and age of hens, were used. The eggs were
all produced on the same day, washed at the farm and cooled
to 10 C. The next day the eggs were taken to the laboratory
where they were divided into seven lots of seventy—five eggs
each, submerged in 70 per cent alcohol for 10 min, placed
on sterile egg flats and allowed to drain dry.

The eggs were broken, one lot at a time, into a
1.5 gallon stainless steel Waring Blender and blended for
10 sec at low speed. The lots that were to be inoculated
with a specific microorganism were inoculated while still in
the blender, then blended for an additional 10 sec.

The seven lots were:
American Type Culture

 

 

 

 

 

Egg Inoculum Collection No.
1 none (control)
2 Pseudomonas fluorescens 13525
3 Achromobacter xerosis 1A780
A Salmonella choleraesuis 13312
5 Escherichia coli 11775
6 Streptococcus faecalis 19A33

 

7 Staphylococcus aureus (golden) 12600

 

37

The blended eggs were transferred to a sterile container
with a spout through which 250 g portions were dispensed
into sterile 18 oz Whirl-Pak bags. The bags were sealed
and the egg samples were incubated at room temperature for
approximately 0, 6, 9, 12, 15 and 18 hr.

At the end of each incubation period, three bags were
opened and microbial samples were taken. The bags were
sealed again and placed into a -18 C freezer where they were
held until the egg was used for the evaluation of its organic
acid content.

To prepare the inocula, the Pseudomonas, Achromobacter,

 

 

Escherichia and Salmonella organisms were grown on separate

 

 

Tryptone Glucose Yeast Extract Agar slants. The Strepto-

coccus and Staphylococcus organisms were grown on separate

 

Brain Heart Infusion Agar slants. Agar slants were incubated
for 2A hr, after which the microorganisms were washed with

10 ml of five per cent tryptose into 90 ml of five per cent
tryptose. Total plate counts were made of the tryptose
solutions to determine the amount needed to provide an
inoculum of approximately 5,000 organisms per g of egg.

While the total plate counts were being determined, the
tryptose solutions were stored in a A C refrigerator.

Spray Dried Whole Egg

 

Ten dozen fresh eggs from the Michigan State University
Poultry Farm were broken into a 1.5 gallon stainless steel
Waring Blender jar and blended for 10 sec at low speed. The

liquid egg was strained through a double cheesecloth and

38

divided into three lots of 2,000 g each. The first lot
served as a control with nothing added. To the second and
third lots, sufficient volumes of organic acids were used
to add 5 mg or 50 mg of each acid per 100 g of egg.

Each lot of egg was blended for 10 sec at low speed
after the acids were added. The liquid egg was then dried
in a Swensen Pilot Plant Model spray drier. The drier was
thoroughly cleaned after drying each lot of egg to prevent
possible contamination of the next lot.

Each lot of dried egg was collected from the spray
drier in a one gallon jar and held at -18 C until used for
organic acid analysis.

Individual solutions of formic, acetic, propionic,
butyric and lactic acids were made by weighing, to the
nearest 0.1 mg, approximately 15 g of each acid into a
50 ml volumetric flask and filling the flask to volume with
water. Twenty milliliters of each acid and 20 ml of water
were pipetted together and mixed, then a lO-fold dilution
of this mixture was made. Approximately 15 g of succinic
acid were weighed, to the nearest 0.1 mg, and added to a
500 ml volumetric flask and made to volume with water. The
10-fold dilution mixture and the succinic acid solution were
pipetted into the egg in amounts that provided the desired

concentrations of acids in the liquid egg.

39

The exact mg of organic acids added per 100 g of liquid
egg were as follows:

Lot Formate Acetate Propionate Butyrate Lactate Succinate

 

 

1 0.0 0.0 0.0 0.0 0.0 0.0
2 5.0 5.0 5.0 5.0 A.9 5.0
3 50.0 50.0 50.0 50.0 A9.0 50.0

Pasteurized Liquid Whole Egg

 

A 30—1b container of pasteurized liquid whole egg was
obtained from a commercial egg processor on the day of
preparation. The container was put into an insulated box
that contained dry ice to cool the egg during transport.
The liquid egg was divided at the laboratory between two
sterile 30-1b containers. One of these was stored at 2 C
and the other was stored at 9 0. Samples were taken at
daily intervals to determine the total plate count.

Six weeks later, a second container of pasteurized
liquid whole egg was obtained from the same commercial
processor and handled in the same manner.

The liquid was prepared from clean fresh shell eggs.
The shell eggs were broken out by "hand" under ultraviolet
light.

The liquid egg was pasteurized at 61 C for 3.5 min in a
continuous egg pasteurizer. Immediately following pasteur-

ization, the egg was cooled to approximately 6 C.

A0

Microbiological Procedures

General

The microbiological procedures selected for use were
adaptations of those described by U.S.D.A. (1969), Institute
of American Poultry Industries (1962) and American Public
Health Association (1966).

Standard sampling and plating techniques were used.
All egg samples were plated at each sampling period to
determine the total plate count.

The media and their use for each microbial count
procedure are discussed below.

Total Plate Count

 

Plate Count Agar (PCA) was incubated at 35 C for A8 hr.

Coliforms

 

Presumptive Test. One ml from each decimal dilution of

 

egg was pipetted into five Lauryl Sulfate Tryptose (LST)
Broth fermentation tubes. The tubes were incubated at 35 C
for 2A hr and observed for the presence of gas. The last
dilution in which all five tubes were positive and the next

two higher dilutions were noted. The number of positive

' tubes in each of the selected dilutions was recorded and a

Most Probable Number (MPN) table was used to determine the
MPN of coliforms per ml of egg.

Confirmed Test. Material from each of the fermentation

 

tubes of the three highest positive dilutions was streaked

onto the surface of Levine's Eosin-Methylene-Blue (EMB)

Al

Agar. The agar was incubated at 35 C for l8-2A hr and
observed for typical coliform colonies.
A-C_03_L_i

From each gassing tube used in determining the coliform
count, a loopful of material was inoculated into an EC Broth
tube. The EC Broth tubes were incubated in a A5.5 0 water
bath for 2A hr. Every tube producing gas was considered
positive and, consequently, every corresponding LST tube was
considered positive for E. egll. Using the number of
positive LST tubes in each dilution, the MPN of E. 99;;
was determined.

Fecal Streptococci

 

Presumptive Test. One ml from each decimal dilution of

 

egg was pipetted into five tubes of Azide Dextrose Broth
(ADB). The tubes were incubated at 35 C and observed for
growth at 2A and A8 hr. Each tube showing growth was
considered to be presumptive positive.

Confirmed Test. One loopful of broth was transferred

 

from each of the presumptive positive tubes into a tube of
Ethyl Violet Azide (EVA) Broth. The EVA Broth was incubated
at 35 C and observed for turbidity at 2A and A8 hr. The
number of confirmed positive tubes in each dilution and,
consequently, the number of corresponding positive ADB tubes
in each dilution was used to determine the MPN of fecal

streptococci.

A2

Staphylococci

 

One ml from each decimal dilution of egg was pipetted
into five Trypticase Soy Broth (TSB) (with 10 per cent
sodium chloride) tubes. The tubes were incubated at 35 C
for A8 hr and observed for growth.

A loopful of material from each growth—positive TSB
tube was streaked onto a previously prepared Vogel-Johnson
(V-J) Agar plate. The V—J Agar plates were incubated at
35 C for A8 hr.

Two colonies which reduced tellurite were picked from
the V—J Agar and streaked on Plate Count Agar slants. The
slants were incubated at 35 C for 18-2A hr and examined
microscopically for coccal forms. The number of TSB tubes
which contained coccal forms was noted and the MPN of
Staphylococci was determined.

Salmonella

 

One ml from each decimal dilution of egg was pipetted
into five Lactose Broth tubes. The broth was incubated at
35 C for 2A hr and a one—half m1 portion from each tube was
transferred to ten m1 of TT Broth (Difco). The TT Broth was
incubated at 35 C for l8—2A hr. A loopful of TT Broth was
streaked onto Brilliant Green Sulfa (BGS) Agar. The BGS
Agar was incubated at 35 C for 22—2A hr and observed for
typical Salmonella colonies. The three highest dilutions of
Lactose Broth which contained Salmonella were noted and used

to determine the MPN of Salmonella.

RESULTS AND DISCUSSION

Organic Acid Recovery and Esterification

Extraction 9E Organic Acids

 

 

The organic acids in egg products must be carefully
recovered and concentrated for accurate analyses. The
procedure includes protein precipitation, extraction,
concentration and analyses.

During the precipitation of the protein and the prepara-
tion of a 200 g egg sample for liquid-liquid extraction by
A.O.A.C. (1965) procedures, the sample is made to a total
weight of 1,000 g. The sample is then filtered and 500 g of
filtrate are collected for analysis. The filtration takes
90 min and it is not always possible to obtain more than
A75 g of filtrate.

A rapid (10 min) centrifugation procedure was developed
to replace the lengthy (90 min) filtration step. This
required an enlarged sample-holding portion on the liquid-
liquid extractor, since the amount of liquid recovered from
a 1,000 g sample by centrifugation was about 775 g. Results
obtained using an enlarged extractor indicated that it was
difficult to extract satisfactory percentages of the acids
from a sample this large. Therefore, a procedure was

developed to recover the acids from a smaller sample.

A3

AA

The protein in 200 g egg samples was most satisfactorily
denatured by using the amounts of sulfuric acid (75 g) and
phosphotungstic acid (125 g) solutions suggested by the
A.O.A.C. (1965) procedures. However, 300 g of water could
be used instead of 600 g. For optimum recovery of the acids,
it was necessary to add the full 300 g of water to the egg
before denaturation of the protein. During centrifugation,
about A80 g of liquid were obtained of which A50 g were
placed into the extractor.

Each enlarged extractor, developed for this study, had
a capacity of approximately 525 ml. However, only A50 m1
of sample could be placed into the extractor, since the
solution expanded as the ether flowed through it. It was
necessary to allow ample space for this expansion to prevent
water from spilling over into the side—arm of the extractor.

A 100 ml round-bottom flask was more satisfactory for
receiving the organic acids than a larger flask, since it
could be handled more easily and the organic acids could be
transferred to a 10 m1 volumetric flask with less chance of
sample loss. A 100 ml round-bottom flask was also used for
the butylation of the acids. Thus, when desired, the acids
could be extracted, the ether evaporated and the acids
butylated without transferring the acids from one container
to another.

An extraction time of 2A hr resulted in the recovery of
a satisfactory percentage of each organic acid. The per-

centages of formic, acetic, propionic, butyric, lactic and

Ila “.CII.||||||ITTII I.

 

‘ 2‘... tn‘lfii. a .Q I ”ill

 

45

succinic acids recovered by liquid-liquid extraction are
presented in Tables 1, 2 and 3. More than 95 per cent of
the butyric, propionic and succinic acids were recovered
while 90 to 95 per cent of the acetic and lactic acids were
recovered. Less than 90 per cent of the formic acid was
recovered (85.A per cent). All of the recoveries were
considered satisfactory and consistent enough to provide
reliable quantitation. Steinhauer and Dawson (1969a, b)
reported comparable recoveries, except that only 77 per cent
of the formic acid was recovered by steam distillation.

Since good recovery of the volatile acids was possible
with liquid-liquid extraction, all of the organic acids
studied could be determined from the same sample.

This procedure for the extraction of organic acids from
egg proved satisfactory for this study. Centrifugation
required a much shorter time than filtration (10 min vs
90 min), and it resulted in a larger percentage of the acids
being recovered since A50 g of a 700 g sample were used
instead of 500 g of a 1,000 g sample (A.O.A.C., 1965).
Liquid—liquid extraction of the A50 g sample eliminated the
need for a condensation step which required heating the
sample for several hr.

Esterification Efficiency

 

The efficiencies of the butyl esterifications of formic,
lactic and succinic acids are presented in Table A. These

efficiencies are comparable to those reported by Steinhauer

A6

Table l. The percentage of formic and acetic acids
recovered using 2A hr liquid—liquid extraction.

 

 

 

 

Replicate Formi; acid Aceti; acid
1 85.A2 93.66
2 86.31 93.17
86.80 93.70
83.68 90.30
5 8A.73 90.90
6 87.35 ' 92.00
7 85.75 91.85
8 8A.90 92.15
9 83.A0 91.85
10 85.90 91.85
Average 85.A2 92.1A

Standard deviation
of the mean 1.A7 l.A0

 

T IQLI|IIIIII

 

W‘hifl

.91 Marv"! .

‘I inn-I.

 

A7

Table 2. The percentage of propionic and butyric acids
recovered using 2A hr liquid—liquid extraction.

 

 

 

 

Replicate Propion;c acid Butyri; acid
1 97.65 98.00
2 97.33 96.51
99.29 99.22
99-05 99-61
5 99-76 95-74
6 100.2A 99-15
7 99.68 99.AA
8 101.20 98.87
9 100.25 99.72
10 102.A0 98.A5
Average 99.69 98.A7

Standard deviation
of the mean 2.0A 1.67

 

A8

Table 3. The percentage of lactic and succinic acids
recovered using 2A hr liquid—liquid extraction.

 

 

 

Replicate Lacti; acid Succin;c acid
1 89.AA 98.79
2 90.56 97.AA
3 92.20 96.76
A 91.67 93.60
5 90.56 97.AA
6 88.07 96.15
7 89.77 96.86
8 90.91 97.AA
9 89.20 96.76
10 88.66 96.76
Average 90.10 96.80

Standard deviation
of the mean 1.55 1.58

 

A9

Table A. Butyl esterification efficiency of formic,
lactic and succinic acids.

 

 

Percentage of acid esterified

 

 

 

 

 

Replicate Formate Lactate Succinate
1 97.1 91.6 91.5
2 96.5 90.3 90.8
99.3 89.1 89.8
A 9A.A 87.8 86.7
5 98.5 87.8 89.8
6 97.7 91.1 90.0
7 96.1 86.6 86.3
8 95.8 87.A 87.1
9 96.A 88.3 87.6
10 98.2 90.1 90.5
Average 97.0 89.0 89.0

Standard deviation
of the mean 1.9 2.6 3.2

 

50

(1968) and Reagan (1970). The variation was not excessive
and was similar for each acid.

Steinhauer (1968) found that butyl esters of formic,
acetic, propionic and butyric acids could be satisfactorily
prepared for GLC analysis. He also reported that attempts
to prepare methyl esters of these acids were not successful
because the esterification efficiencies were not consistent.

Salwin and Bond (1969) and Staruszkiewicz (1969)
reported the use of propyl esters for determining lactic and
succinic acids in eggs. The basic esterification procedure
was to react the acids with boron-trifluoride-propano1 for
10 min on a steam bath. Their procedure was used by this
author in a collaborative study on the detection of
B—hydroxybutyric acid in eggs and found to be a very good
procedure. Also, experimentation with this procedure
indicated that it might be adapted to the determination of
volatile acids in eggs.

GLC Columns and Calibration
Columns

Initially, columns of 20 per cent ethylene glycol
adipate (EGA) and 20 per cent diethylene glycol succinate
(DEGS) were compared for their ability to produce good
separation and short retention times of the fatty acids.
Both columns provided good separation, however, the retention
time for each acid was shorter with the DEGS columns than
with the BOA columns. All of the preliminary studies were,

therefore, conducted using DEGS columns.

51

While the EGA and DEGS columns functioned well in
separating the acids and the butyl esters, they were not
completely satisfactory. The retention time of dibutyl
succinate was as long as A0 min. Also, the Chromatogram
peaks produced by the acids were not as sharp as desired,
especially the dibutyl succinate peak. Steinhauer (1968)
used 20 per cent EGA columns and reported that succinic
acid was eluted A0 min after being injected. Reagan (1970)
used 20 per cent DEGS columns and programmed the temperature
to increase from 130 to 170 C to elute succinic acid in
about 25 min and to increase the sharpness of the acid peak.

Ten per cent FFAP columns were found to produce sepa-
ration of the fatty acids with short retention times. Also,
the peaks produced by the acids were quite sharp. Since
sharp peaks are more accurate than low, broad peaks when
using peak height as the unit of measurement, FFAP columns
were used in this study. However, these columns were not
completely free of problems. Butyl formate and butyl acetate
had a tendency to elute so rapidly that they partially over-
laped the diethyl ether peak. Also, acetic, propionic and
butyric acid peaks tended to overlap each other, especially
when the GLC was set at a high sensitivity. After a period
of experimentation, the proper GLC operating parameters were
established which minimized the overlaping tendencies, yet
retained the favorable characteristics of the columns. The
operating parameters have been presented in the section on

equipment and reagents.

52

Chromatograms of the acids and the butyl esters are
presented in Figures 1, 2 and 3. These chromatograms
illustrate the rapid elution times of all of the compounds
when using FFAP columns. The shortest elution time was
two and one—fifth min for butyl lactate, while the longest
time was 11.5 min for butyl caprate, an internal standard.

Acetic, propionic and butyric acid peaks were not as
well separated as desired, however, the amount of overlap
at the base of the peaks was small and did not affect the
quantitation of the acids. All of the butyl ester peaks
were well separated on the chromatograms.

With some gas chromatographs it requires very fine
adjustment of the instrument to program the column temper—
ature and maintain a steady base-line. This problem was
eliminated by using the FFAP columns since the analyses
could be conducted at isothermal temperatures.

Internal Standards

 

The GLC procedure described by Shelley et a1. (1963)
used an internal standard and R—value to assist in deter-
mining the amount of acid present in liquid egg. The R-value
is a ratio of the response of the GLC detector to a given
concentration of internal standard and acid. When there is
a slight variation in the 1 ul size aliquot injected into
the GLC, this is corrected for by the R—value formula
because the variation is the same for the internal standard

and the acid.

Recorder Response

100

50

53

 

 

 

 

 

 

 

 

l
l. Diethyl Ether
2. Butyl Formate
2 3. Butyl Valerate
3
l k \J
O A 8 12

Time (minutes)

Figure 1. Chromatogram of butyl
formate and butyl valerate.

Recorder Response

100

50

5A

Diethyl Ether
Acetic Acid
Propionic Acid
Butyric Acid
Butyl Caprate

U'l-t-‘LMTUI-J

 

 

 

 

 

 

 

 

 

 

 

 

o A 8 12
Time (minutes)
Figure 2. Chromatogram of acetic,

propionic, and butyric acids and butyl
caprate.

Recorder Response

100

50

55

 

 

 

 

 

 

 

 

 

l

I l. Diethyl Ether
2. Butyl Lactate
3. Butyl Caprate

2 A. Dibutyl Succinate
3
A
0 A 8 12

Time (minutes)

Figure 3. Chromatogram of butyl
lactate, dibutyl succinate, and butyl
caprate.

56

In this study, butyl valerate was found to be a good
internal standard for the detection of butyl formate
(Figure 1). At a column temperature of 100 C it was eluted
eight min after butyl formate was eluted. At column tem—
peratures of 170 C and 200 C it was eluted with diethyl
ether, thus its peak did not interfere with the other acid
peaks.

As illustrated in Figure 2, butyl caprate was a good
internal standard when determining the amount of volatile
acids present. It was eluted about five min after butyric
acid was eluted. Butyl caprate also served as an internal
standard for the determination of butyl lactate and dibutyl
succinate (Figure 3), and it was eluted between these two
esters. However, at a column temperature of 100 C it was
not eluted from the column, so it did not interfere with the
determination of butyl formate.

R-Values

The R-values for the acids and the butyl esters are
presented in Tables 5, 6, 7 and 8. The first three columns
in each table indicate the concentrations of acid or butyl
ester used to calibrate the GLC at the different GLC
sensitivities (range and attenuation).

The R-values were quite high for most of the sub—
stances, however, they were relatively low for acetic acid
and dibutyl succinate. High R-values provide more sensitive
quantitation than low R-values, when measuring peak heights

produced by the acids.

57

Table 5. R-values of acetic, propionic and butyric acids.

 

 

 

Mg acid per ml Chromatograph Standard
of calibration deviation
sample Range Attenuation R—value of the mean

 

Acetic acid

0.A15 10 2 0.18 0.000
0.831 10 A 0.25 0.000
1.661 10 8 0.35 0.001
3.32A 10 16 0.AA 0.000
3.32A 10 32 0.38 0.003

Propionic acid

0.A72 10 2 0.A9 0.000
0.9AA 10 A 0.56 0.000
1.889 10 8 0.61 0.000
3-779 10 16 0.75 0.001
3-779 10 32 0.67 0.001

Butyric acid

0.A72 10 2 0.5A 0.000
0.9A6 10 ' A 0.60 0.001
1.893 10 8 0.62 0.001
3.786 10 16 0.71 0.000
3.786 10 32 0.71 0.002

 

1Average of three replicates.

. .\IO

‘11 pl

 

III}. II In It‘.

.IT’... 4"

58

Table 6. R-values of butyl formate and butyl acetate.

 

 

 

Mg acid per m1 Chromatoggaph Standard
of calibration l deviation
sample Range Attenuation R-value of the mean

 

Butyl formate

0.089 10 2 2.89 0.017
0.179 10 A 2.62 0.013
0.358 10 8 2.63 0.007
0.717 10 16 2.51 0.00A
1.A35 10 32 2.51 0.027

Butyl acetate

0.089 10 2 2.75 0.003
0.179 10 A 2.59 0.013
0.359 10 8 2.58 0.016
0.718 10 16 2.A3 0.002
1.A36 10 32 2.5A 0.011

 

1Average of three replicates.

59

Table 7. R-values of butyl propionate and butyl butyrate.

 

 

 

Mg acid per ml Chromatogrgph Standard
of calibration deviation
sample Range Attenuation R-value of the mean

 

Butyl propionate

0.091 10 2 2.29 0.000
0.182 10 A 2.10 0.011
0.365 10 8 2.09 0.000
0.730 10 16 2.05 0.000
1.A61 10 32 2.00 0.000

Butyl butyrate

0.089 10 2 1.62 0.001
0.178 10 A 1.52 0.003
0.357 10 8 1.52 0.002
0.715 10 16 l.A7 0.002
1.A31 10 32 l.A7 0.000

 

1Average of three replicates.

60

Table 8. R-values of butyl lactate and dibutyl succinate.

 

 

 

Mg acid per ml Chromatograph Standard
of calibration l deviation
sample Range Attenuation R—value of the mean

 

Butyl lactate

0.156 10 2 1.2A 0.057
0.312 10 A 1.19 0.003
0.625 10 8 1.17 0.00A
1.251 10 16 1.2A 0.001
2.502 10 32 1.32 0.000

Dibutyl succinate

0.157 10 2 0.3A 0.001
0.31A 10 A 0.35 0.000
0.628 10 8 0.36 0.000
1.255 10 16 0.38 0.000
2.511 10 32 0.39 0.000

 

1Average of three replicates.

61

The standard deviation of the mean was very small at
each GLC sensitivity. This indicates that when replicate
injections of a sample were made, the detector response to
the acids and butyl esters was consistent.

Analysis of Egg Products

Commercial Egg Products

 

The microbial counts and the organic acid content of
commercial liquid and frozen whole eggs are presented in
Table 9. Most of the samples were frozen, but several of
them were liquid. Steinhauer (1968) demonstrated that no
organic acids were lost during the freezing and storage of
frozen egg, therefore, the analyses of liquid and frozen
egg may be compared without concern for the effect of
freezing on the organic acid content.

The direct microscopic counts of the Al samples ranged
from less than 20,000 to A,600,000 per g. The only acid
detected was lactic acid, and its concentration was generally
the same in samples with low direct microscopic counts as in
samples with high direct microscopic counts. Thus, there
was no correlation between the direct microscopic count and
the organic acid content of the egg.

Twelve of the samples contained relatively low numbers
of certain bacteria for which standard analyses are specif-
ically conducted. Of these bacteria, E. 39;; was the most
commonly present. However, the presence of these bacteria

did not appear to effect the organic acid content of the

egg.

Table 9.

whole egg

62

Microbial counts and organic acid content of commercial liquid and frozen

Hicroorganismsgper g of egg?/ (X 1000)

 

 

 

Mu/
100 g ”em“
3/ Micro- Total lytic Salmon-
i/ egg' scopic plate Coli— Fecal Strep.& ella &

Sample Lactate count count forms E.coli Strep. Staph. Shig.

1. L 3.5 4,600 < 3

2. L 1.8 4,200 98

3. F 4.4 3,500 3,000 1,000 1,000

4. L 3.9 2,100 2,000 1 l

5. L 4.2 520 320 10 10

6. F 4.5 500 6.5

7. L 2.6 320 6

8. F 4.6 160 15 .1 .1

9. F 4.0 120 18 .1 .1

10. F 1.6 100 < 3

11. L 4.4 80 < 3

12. F 3.4 60 39 23

13. F 2.8 60 18 .1 .1

14. P 6.5 40 37 .01 .01

15. L 3.6 40 21 .01 .01

16. L 4.4 20 9.8 3.8

17. F 7.8 20 3.3 Present
18. F 3.6 < 20 34
19. F 2.7 < 20 33
20. L 5.4 < 20 30 6.6
21. F 3.9 < 20 28
22. F 3.1 < 20 21
23. L 5.4 < 20 21
24. F 2.9 < 20 18
25. F 3.1 < 20 16
26. F 3.5 < 20 8.8
27. F 5.0 < 20 8.8
28. F 2.2 < 20 6.6
20. F 1.6 < 20 6.1
30. r 3.6 < 20 6.0
31. r 2.7 5 20 4.9
32. r 4.8 < 20 < 3
33. r 4.2 ’t 20 < 3
34. r 4.2 : 20 < 3
35. F 3.4 20 < 3
36. r 3.4 : 20 < 3
37. F 3.3 , 20 < 3
38. r 2.6 2 20 < 3
39. F 2.0 20 < 3
40. r 1.7 < 20 < 3
41. r 0.6 < 20 < 3
1, L = Liquid, F = Frozen; The odor of all samples was normal. ‘7—
2. No yeasts or molds were detected in any sample.
3. No formic, acetic, succinic, propionic or butyric acid was detected.

 

/
.r
5!'< .- ’.

63

The data in Table 9 indicate that the range of the
lactic acid content was generally from 2 to 5 mg per 100 g
of liquid egg. However, five samples contained less than
2 mg and four samples contained more than 5 mg of lactic
acid per 100 g of egg. The variation in the lactic acid
content was similar to that reported by Steinhauer et a1.
(1967).

Even though the direct microscopic and total plate
counts were low, sample 17 contained 7.8 mg of lactic acid
per 100 g of egg. This indicates that relatively high
amounts of lactic acid may sometimes be present even though
there may be no indication of microbial decomposition.

Sample one had a direct microscopic count of A,600,000
per g yet its total plate count was less than 3,000 per g.
This demonstrates the effectiveness of pasteurization for
reducing the number of viable microorganisms in liquid egg.
It also illustrates that the direct microscopic count should
be included when evaluating egg products of an unknown
history, to obtain a more complete microbial profile of the
egg. Reagan (1970) reported that a continuous pasteurization
process for liquid whole egg does not alter the organic acid
content of the egg, although it does reduce the total plate
count.

Michigan law requires that all liquid, frozen and dried
egg products for human consumption be free from Salmonella
organisms and other pathogenic bacteria. However, it does

not state that egg products must be pasteurized.

 

6A

Egg products which pass through interstate commerce are
required, by Federal law, to be pasteurized. Some of the
egg products discussed in Table 9 were obtained from
consumers and possibly were from interstate commerce.

On July 1, 1971, a new Federal egg law will become
effective. This law requires that all egg products be
pasteurized.

The odor of all of the samples was considered to be
normal, with no odor of decomposition present.

While these data are for whole egg products having
direct microscopic counts less than 5,000,000 per g, others
have reported an accumulation of organic acids when the
direct microscopic counts were considerably higher than
5,000,000 per g. Lepper et a1. (1956) and Hillig et a1.
(1960) examined packs of edible and inedible frozen egg and
found that when the direct microscopic counts were
118,000,000 to AA0,000,000 per g with a pronounced off—odor,
the egg contained the following acids in mg per 100 g of
egg: formic, 36 to 77; acetic, 63 to 108; lactic, A7 to 97;
and succinic, 19 to 37. Other samples with the odor of
decomposition and direct microscopic counts as high as
115,000,000 per g contained the following maximum amounts of
acids in mg per 100 g of egg: formic, A.9; acetic, 9.1;
lactic, 61.0; and succinic, 0. They also reported one
sample with a direct microscopic count of 63,000,000 per g
in which the only acid present was lactic acid, in the

amount of 10 mg per 100 g of egg.

65

Two samples of commercial dried whole egg were evalu-
ated for microbial counts and organic acid content (Table
10). The direct microscopic count of the first sample was
250,000,000 per g and was accompanied by large amounts of
formic, acetic and succinic acids. The second dried egg
sample had a direct microscopic count of 2,000,000 per g and
only lactic acid was detected (6.A mg per 100 g of dried
ess).

Lepper et a1. (19AA) reported that with dried eggs, a
direct microscopic count of over 100,000,000 per g with
detectable amounts of formic acid, and acetic acid over
65 mg and lactic acid over 50 mg per 100 g of dried egg
demonstrated the presence of decomposed egg. Thus, the
first dried egg sample in this study had multiple indices
of decomposition.

Table 10. Microbial counts and organic acid content of
commercial dried whole egg.

 

 

 

 

Direct
microscopic Total plate
count count Mg acid per 100 g dried egg
(org/g) (org/g) Formic Acetic Lactic Succinic
250,000,000 37,000 51.9 A3.0 116.8 28.8
2,000,000 3,000 0 0 6.A 0

 

Inoculated Liquid Whole Egg

 

The role that specific bacteria have in producing

organic acids in liquid whole egg was determined. The

66

bacteria inoculated into the samples were representative of
the bacteria that may be found in egg products. Uninoc-
ulated samples were used as control samples to establish
the normal level of organic acids present in the liquid egg.

The total plate count and the organic acid content of
the control samples of liquid whole egg are presented in
Table 11. After processing, fewer than 300 bacteria per m1
of egg were present. This number increased very little
until the 18 hr sampling time, at which time the total
plate count (TPC) was 2,700 per m1. Even this was a very
low TPC, especially when compared with the 18 hr TPC of the
inoculated samples.

The low total plate counts of the control samples
indicated that egg samples were prepared for inoculation
with only a small amount of contamination occurring. The
low numbers of contaminants present were believed to have
no influence on the results of the studies using inoculated
samples.

The only organic acids found in the control egg samples
were acetic and lactic acids. Lepper et al. (19AA) did not
detect acetic acid in fresh eggs. However, Steinhauer et a1.
(1967) and Reagan (1970) reported that small amounts of
acetic acid may be found in fresh liquid whole egg.

The total plate count and the organic acid content of
liquid whole egg samples inoculated with Pseudomonas

fluorescens, Achromobacter xerosis and Staphylococcus aureus

 

are presented in Tables 12, 13 and 1A respectively.

67

Table 11. Total plate count and organic acid content of
control samples of liquid whole egg.

 

 

Total plate

 

 

Hr at count Mg acid per 100 g egg
22 C (org/ml) Formic Acetic Lactic Succinic
0 < 300 0 0.3 5.5 O
6 < 300 0 2.0 1.A 0
9 < 300 0 0.A 5.6 0
12 350 0 0.8 7.6 0
15 300 0 0.7 5.3 0
18 2,700 0 2.5 1.6 0

 

68

Table 12. Total plate count and organic acid content of
liquid whole egg inoculated with Pseudomonas fluorescens.

 

 

 

Total plate

 

 

Hr at count Mg acid per 100 g egg

22 C (org/m1) Formic Acetic Lactic Succinic
0 < 3,000 0 0.1 3.1 0

6 3,200 0 0.9 3.8 0

9 28,800 0 0.9 A.2 0

13 670,000 0 0 A.1 0

15 17,000,000 0 o A.1 0

18 18,000,000 0 1.0 3.3 0

 

69

Table 13. Total plate count and organic acid content of
liquid whole egg inoculated with Achromobacter xerosis.

 

 

 

Total plate

 

 

Hr at count Mg acid per 100 g egg
22 C (org/ml) Formic Acetic Lactic Succinic
0 < 300 0 0.1 7.2 0
6 300 0 0.3 7.0 0
9 500 ‘ 0 0.2 6. 0
13 2,000 0 0.2 6.1 0
15 19,000 0 - 3.7 0
22 2,700,000 0 — A.5 0

 

70

Table 1A. Total plate count and organic acid content of
liquid whole egg inoculated with Staphylococcus aureus.

 

 

 

Total plate

A

 

 

 

Hr at count Mg acid per 100 g egg
22 C (org/ml) Formic Acetic Lactic Succinic
9 9,500 0 0 2.6 0
7 9,000 0 0.A 3.1 0
10 250,000 0 0.A 3.5 0
13 970,000 0 0.9 2.8 0
16 6,200,000 0 1.0 A.3 0

 

71

The total plate counts of the Achromobacter inoculated

 

samples were relatively low and increased to only 2,700,000
per ml during the incubation period. However, the Pseudo-

monas and Staphylococcus total plate counts were higher,

 

indicating that a greater degree of decomposition occurred
in these samples.

Acetic and lactic acids were the only organic acids
detected in the egg and their concentrations were comparable

to those of the control samples. Thus, Pseudomonas

 

fluorescens, Achromobacter xerosis and Staphylococcus

 

 

 

aureus organisms had no effect on the organic acid content
of the egg.

The total plate counts of Streptococcus faecalis

 

(Table 15) increased from 15,000 to A2,000,000 per ml.
While no formic acid was detected, acetic and lactic acids
were present in concentrations comparable to those of the
control samples. Very small amounts of succinic acid were
present in the samples that had total plate counts of
7,800,000 and A2,000,000 per ml with visual signs of
decomposition.

The total plate count and organic acid content of

liquid whole egg samples inoculated with Salmonella

 

choleraesuis are presented in Table 16. No formic acid was

 

detected and acetic and lactic acids were present in
amounts comparable to those of the control samples. Suc-
cinic acid was detected in small amounts in all of the

incubated samples, indicating that small amounts of succinic

72

Table 15. Total plate count and organic acid content of
liquid whole egg inoculated with Streptococcus faecalis.

 

 

 

Total plate

 

 

Hr at count Mg acid per 100 g egg
22 C (org/ml) Formic Acetic Lactic Succinic
0 15,000 0 0.1 6.A 0
6 120,000 0 0 6.5 0
9 900,000 0 0 6.7 0
12 7,800,000 0 0.A A.A 0.2

15 A2,000,000 0 l.A 3.5 0.A

 

Table 16.

73

Total plate count and organic acid content of
liquid whole egg inoculated with Salmonella choleraesuis.

 

 

 

Total plate

 

 

Hr at count Mg acid per 100 g egg
22 C (org/ml) Formic Acetic Lactic Succinic
0 12,000 0 0.2 u.8 0
7 60,000 0 0.3 6.5 0.2
9 l.200,000 0 0.5 6.3 0.5
12 3,500,000 0 0.5 7.5 0.1
15 u,900,000 0 0.5 u.u 0.2
18 7,000,000 0 1.5 5.2 0.6

7U

acid may be present as a result of the growth of Salmonella

 

choleraesuis in liquid egg. The TPC reached 1,200,000 per

 

ml after 9 hr of incubation and then increased to 7,000,000
per ml after 18 hr of incubation.

The liquid whole egg inoculated with Escherichia coli

 

had total plate counts from 7,700 to 540,000,000 per ml
(Table 17). The organic acid content was comparable to that
of control samples until the total plate count reached
9,000,000 per ml. At this time, a small amount of succinic
acid was detected. When the egg had decomposed to a TPC

of 110,000,000 per ml, all four of the acids were found in
the egg. The amount of formic acid was small. However,
acetic, lactic and succinic acids were present in apprecia-

ble quantities. Escherichia and similar coliform—type

 

microorganisms produce all four of the acids during fermen—
tation. However, formic acid is converted to carbon dioxide
and hydrogen has, in a one—to—one ratio. Therefore, its
accumulation is not as great as the accumulation of the
other acids (Stanier et al., 1963).

When the TPC of the E. 991i inoculated egg increased
to 540,000,000 from 110,000,000 per ml, there was a substan-
tial decrease in the quantity of each organic acid. This
demonstrated that microorganisms may utilize the organic
acids as well as produce them. This fact may account for
some of the variation that occurs in the organic acid

content of egg products.

75

Table 17. Total plate count and organic acid content of
liquid whole egg inoculated with Escherichia coli.

 

 

 

Total plate

 

 

Hr at count Mg acid per 100 g egg
22 C (org/m1) Formic Acetic Lactic Succinic
0 7,700 0 0.8 A.1 0
6 58,000 0 1.0 3.2 0
9 1,000,000 0 0.5 0.2 0
12 9,000,000 0 1.2 5 2 0 3
15 110,000,000 1.0 16.u 13.1 24.8
18 5M0,000,000 0.5 13.6 6 6 6.6

 

76

Spray Dried Whole Egg

 

Quality evaluation of egg products includes the
determination of the microbial count and organic acid
content of spray dried egg. This study was conducted to
determine the effect of spray drying on the organic acid
content of egg.

The organic acid content of the control samples of
spray dried whole egg are presented in Table 18. The only
acid detected was lactic acid. The amount of lactic acid
present in the dried egg was calculated as mg per 100 g of
liquid egg. Thus, when compared with the amount of lactic
acid commonly found in liquid egg, it appeared that little,
if any, lactic acid was lost during the drying process.

Lepper et a1. (194A) reported that acetic acid may be
formed during the drying of liquid whole egg, however, it
was not recovered from the dried control samples in this
study.

A specific amount of each organic acid was added to
liquid whole egg and the product dried. Analysis of the
treated egg provided information on the fate of these acids
during the drying process.

The organic acid content of spray dried whole egg
which had five mg of each acid added per 100 g of liquid
egg is presented in Table 19. Only 2.1 mg of formic acid
per 100 g egg (A2 per cent of that added) were recovered
from the dried egg. The maximum amount of acid recovered

was 3.“ mg per 100 g egg (68 per cent of that added) of

A.

77

Table 18. Organic acid content of control samples of
spray dried whole egg.

 

 

Mg acid per 100 g of egg (liquid egg basis)

 

 

Replicate Formic Acetic Propionic Butyric Lactic Succinic
1 0 0 0 0 2 0 0
2 0 0 0 0 3 O 0
3 0 0 O 0 A l 0
A O 0 0 0 3 8 0
5 0 0 O 0 3 A 0
Average 0 0 0 0 3.3 0
Standard
deviation 0 0 0 0 0.51 0

of the mean

 

78

Table 19. Organic acid content of spray dried whole egg
which had five mg of each acid added per 100 g of liquid egg.

 

 

Mg acid per 100 g of egg (liquid egg basis)

 

 

Replicate Formic Acetic Propionic Butyric Lactic Succinic
l 2 A 3 0 3.2 3 2 3 2 2 5
2 1.7 3 7 2.3 2 6 A.8 A 3
3 2 0 2 9 2.9 3 O 5 2 A 0
A 2.6 3 8 3.0 2 7 7.2 3 l
5 1.8 3 5 2 5 3.0 5 6 3 2
Average 2.1 3.A 2.8 2.9 5.2 3.A
Standard
deviation 0.12 0.1A 0.11 0.0A 1.66 0.A2

of the mean

 

79

acetic and succinic acids. Although 5.2 mg of lactic acid
per 100 g egg were recovered, it must be considered that
3.3 mg of lactic acid per 100 g egg were present in the
fresh liquid egg in addition to the A.9 mg per 100 g egg
that were added. Using average figures, the amount of
lactic acid in the liquid egg just prior to drying was 8.2
mg per 100 g egg. Of this, 5.2 mg per 100 g egg (63 per
cent) were recovered from the dried egg.

The organic acid content of spray dried whole egg which
had 50 mg of each acid added per 100 g of liquid egg is
presented in Table 20. The recovery of the volatile acids
(formic, acetic, prOpionic and butyric) averaged 3A.6 to
38.6 per cent. Succinic acid was recovered in the greatest
amount, 90.A per cent of that added. Lactic acid had a
reasonably high recovery of 87.5 per cent of the acid which
was in the egg Just prior to drying. The amount of lactic
acid in the egg just prior to drying was A9.0 mg of added
acid per 100 g egg plus 3.3 mg per 100 g egg of naturally
occurring acid.

These data indicate that a larger percentage of the
volatile acids, but a smaller percentage of lactic and
succinic acids were lost during spray drying when 50 mg
of acid were added per 100 g egg than when 5 mg of acid were
added per 100 g egg.

Shelf-Life g: Pasteurized Liquid Whole Egg

 

 

The shelf—life of pasteurized commercial liquid whole

egg was determined at storage temperatures of 2 C and 9 C.

Table 20.

80

Organic acid content of spray dried whole egg

which had 50 mg of each acid added per 100 g of liquid egg.

 

 

Mg acid per 100 g of egg (liquid egg basis)

 

 

Replicate Formic Acetic Propionic Butyric Lactic Succinic
1 16.A 16.A 17.1 18.3 A8.6 A6.3
2 18.7 17.5 18.0 20.0 A7.3 A7.6
3 16.1 19.A 17.9 18.8 A6.A A8.A
A 18.3 19.1 18.6 18.5 AA.3 A3.3
5 17.0 19.2 19.8 20.7 A2.A AO.9
Average 17.3 18.3 18.9 19.3 A5.8 A5.2
Standard
deviation 1.06 1.18 1.19 0.87 A.85 7.66

of the mean

 

81

These conditions represented one desirable temperature and
one higher than normally recommended.

As illustrated in Figure A, total plate counts of the
freshly prepared liquid egg were quite low (< 300 per ml).
At 2 C the TPC remained relatively constant for three days
of storage. After A-6 days of storage the bacteria began
to multiply and entered a logarithmic growth phase (days
6-13). After 13 days of storage, the total plate count was
nearly 1,000,000 and the egg decomposed rapidly.

At 9 C the growth phases of the bacteria were accel-
erated considerably compared to the growth phases at 2 C.
After five days of storage, the total plate count of the
egg was 600,000 and increased rapidly so that the egg was
noticeably decomposed after six days of storage.

The pasteurized liquid whole egg contained no E. 3911
salmonella, streptococci and staphylococci organisms.

These data indicate that high quality, pasteurized
liquid whole egg will remain wholesome for five and 13 days

when stored at 9 and 2 C, respectively.

Microorganisms (per ml of liquid egg)

1x106

1x105

1x10“

1x103

1x102

82

9 C
— — — 2 c

 

 

 

0 1. 2 3 A 5 6 7 8 9 10 11 12
Days Storage

Figure A. Total plate count of pasteurized
commercial liquid whole egg held at 2 C and 9 C.

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