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This is to certify that the

thesis entitled

Relationship Between Some Insecticides and
Lactic Culture Organisms in Milk

presented bg

Sun C. Kim

has been accepted towards fulfillment
of the requirements for

Ph.D. Food Science

 

degree in

béLCC’L/Wpci“ (\ Lflfb’wl’fijb/

Major pic/lessor

Date June 14, I969

 

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ABSTRACT
RELATIONSHIP BETWEEN SOME INSECTICIDES
AND LACTIC CULTURE ORGANISMS IN MILK
By

Sun C. Kim

Sterile whole milks containing from <O.l to 100 ppm
of dieldrin and heptachlor were inoculated with Strepto—

coccus lactis A254, S. lactis A62, Streptococcus cremoris

 

E—8, Streptococcus diacetilactis 18—16 and Lactobacillus

 

case; and incubated for H8 hr at appropriate temperatures.
In addition, milks containing similar amounts of
methoxychlor and malathion were inoculated with S. lactis
A25“ and E. Eéiil and incubated for M8 hr at 32 C. The
insecticides had no effect on the growth or production of
lactic acid by the streptococci. Heptachlor, methoxychlor
and malathion had neither adverse nor stimulative effect
on the growth and lactic fermentation ability of E. case;
in milk at 32 C. Dieldrin did not influence the lactic
fermentation ability of g. EEEEE but resulted in slight
reduction in viable cell count in the milks containing
10 and 100 ppm.

In order to study the susceptibility of aldrin, DDT
and lindane, to the biological action of lactic starter
cultures, sterile whole milks containing 1 ppm of aldrin,

DDT or lindane were inoculated with S. lactis A25u,

Sun C. Kim

S. lactis A62, S. cremoris E—8, S. diacetilactis 18-16,

 

 

S. diacetilactis DRCI and S. casei. After two weeks of

 

incubation at apprOpriate temperatures, the insecticides
were extracted from the milks, cleaned, concentrated, and
analyzed by gas chromatography using an electron capture
detector. None of the organisms modified or degraded
aldrin, DDT or lindane. This proves that commonly used
lactic starter cultures neither help detoxify DDT or
lindane, nor enhance the toxicity of aldrin in milk.

Since the growth of S. BEES; was slightly inhibited
by 10 and 100 ppm of dieldrin in milk, possible uptake of
dieldrin by S. Eggs; was studied using luC—dieldrin. When
S. EEEEE was grown in Trypticase Soy Broth (TSB) containing

0.01—0.02 ppm of luC—dieldrin for 18 hr at 32 C the amount

of lac-dieldrin retained with the harvested and washed

cells was less than 0.2% of the total lLlC—dieldrin in the
TSB. Since less than 0.2% of such a low concentration as
0.01—0.02 ppm of dieldrin was retained with the harvested
cells, the count of the radioactivity obtained from the
cells could be attributed to the residual radioactivity
due to incomplete washing rather than to true uptake of

lLlC—dieldrin into the cells. The population was about

108 cells/ml.

RELATIONSHIP BETWEEN SOME INSECTICIDES

AND LACTIC CULTURE ORGANISMS IN MILK
By

Sun Ct Kim

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Food Science

1969

ACKNOWLEDGMENTS

The author wishes to express her sincere apprecia—
tion to her major professor, Dr. L. G. Harmon, for his
guidance and understanding throughout this study; to
Dr. P. Markakis for the use of his laboratory facilities;
to H. L. Sadoff, H. A. Lillevik, J. R. Brunner and C. M.
Stine as members of the guidance committee.

Appreciation is extended to Dr. B. S. Schweigert,
Chairman, Department of Food Science, for his interest
in this program and to Michigan State University for the

facilities which were provided.

ii

TABLE OF CONTENTS

ACKNOWLEDGMENTS
LIST OF TABLES.

LIST OF FIGURES

INTRODUCTION
LITERATURE REVIEW.

Pesticide Residues in the Human Diet

Toxicity of Insecticides

Metabolism of Chlorinated Hydrocarbon
Insecticides .

Storage of Chlorinated Hydrocarbon Insecti—
cides in the Human Body. . . .

Effect of Insecticide Residues on Micro-
organisms

Transformation of Chlorinated Hydrocarbon
Insecticides by Microorganisms

Effect of Processing on Insecticides in Milk.

Comments and Objectives.
MATERIALS AND METHODS

Effect of Insecticide Residues on Growth and
Fermentation Ability of Lactic Culture
Organisms

Effect of Lactic Culture Organisms on
Aldrin, DDT, and Lindane in Milk.

Preparation of samples
Extraction of insecticides from milk

Separation of insecticide and milk- fat.

Partitioning with acetonitrile, water
and petroleum ether
Cleanup of extracted insecticide.
Analysis of insecticide.
The Uptake of Dieldrin by Lactobacillus
casei. . . . . . . . .

 

iii

Page

ii

vii

CDNKJ'IUUUU

10

12
Ill
l6

l9

19

21
21
22
22

23
214
25

26

RESULTS

Effect of Insecticide Residues on Growth and
Fermentation Ability of Lactic Culture
Organisms . . .

Effect of Lactic Culture Organisms on
Aldrin, DDT nd Lindane in Milk . .

The Uptake of l c— dieldrin by Lactobacillus
casei. . . . . . . . . . . .

 

DISCUSSION

SUMMARY AND CONCLUSIONS.
LITERATURE CITED
APPENDIX.

Chemical Nomenclature of the Insecticides.
Tables 2 through 15

iv

Page
29

29
140
149
51
65
69
77

78
80

Table

LIST OF TABLES

Page
Calculation of uptake of l[JO—dieldrin by
Lactobacillus casei when grown in Trypticase
Soy Broth (TSB) containing 1 C—dieldrin for
18 hr at 32 C . . . . . . . . 50

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when

the milk was inoculated with Streptococcus

lactis A25U and incubated at 32 C . . 80

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when

the milk was inoculated with Lactobacillus

casei and incubated at 32 C . . . . . . 81

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated,

when the milk was inoculated with Strepto-

coccus lactis A25A and incubated at 32 C . . 82

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated,

when the milk was inoculated with Lacto—

bacillus casei and incubated at 32 C . . . 83

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of methoxychlor as indicated,

when the milk was inoculated with Strepto~

coccus lactis A25“ and incubated at 32 C . . 8A

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of methoxychlor as indicated,

when the milk was inoculated with Lacto—

bacillus casei and incubated at 32 C . . . 85

 

 

Table

10.

11.

12.

13.

1M.

15.

Page

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of malathion as indicated,

when the milk was inoculated with StreEto-

coccus lactis A25u and incubated at 32 C . . 86

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of malathion as indicated,

when the milk was inoculated with Lacto-

bacillus casei and incubated at 32 C . . . 87

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when

the milk was inoculated with Streptococcus

lactis A62 and incubated at 32 C. . . . . 88

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when

the milk was inoculated with Streptococcus
diacetilactis 18-16 and incubated at 32 C. . 89

 

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated, Whaq
the milk was inoculated with Streptococcus

lactis A62 and incubated at 32 C. . . . . 90

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated,

when the milk was inoculated with Strepto-

coccus diacetilactis 18—16 and incubated at

32 C . . . . . . . . . . . . . . 91

 

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when

the milk was inoculated with Streptococcus
cremoris E-8 and incubated at 25 C . . . . 92

Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated,

when the milk was inoculated with Strepto—

coccus cremoris E-8 and incubated at 25 C. . 93

 

vi

LIST OF FIGURES

Figure Page

1. Effect of different concentrations of
dieldrin on viable cell count, pH and % of
lactic acid in whole milk when the milk was
inoculated with Streptococcus lactis A254 or
Lactobacillus casei and incubated at 32 C
for 24 hr . . . . . . . . . . . . 31

 

 

2. Effect of different concentrations of hepta-
chlor on viable cell count, pH and % of lactic
acid in whole milk when the milk was inocu-
lated with Streptococcus lactis A254 or
Lactobacillus casei and incubated at 32 C for
24 hr. . . . . . . . . . . . . . 32

 

 

3. Effect of different concentrations of
methoxychlor on viable cell count, pH and %
lactic acid in whole milk when the milk was
inoculated with Streptococcus lactis A254 or
Lactobacillus casei and incubated at 32 C
for 24Ihr . . . . . . . . . . . . 33

 

 

4. Effect of different concentrations of
malathion on viable cell count, pH and % lac—
tic acid in whole milk when the milk was inocu—
lated with Streptococcus lactis A254 or Lacto-
bacillus casei and incubated at 32 C for
24 hr. . . . . . . . . . . . . . 35

 

 

5. Effect of different concentrations of
dieldrin on viable cell count, pH and %
lactic acid in whole milk when the milk was
inoculated with Streptococcus lactis A62 or
Streptococcus diacetilactis 18-16 and incu-
bated at 32 C for 24 hr. . . . . . . . 36

 

6. Effect of different concentrations of hepta—
chlor on viable cell count, pH and % lactic
acid in whole milk when the milk was inocu-
lated with Streptococcus lactis A62 or Strep-
tococcus diacetilactis 18-16 and incubated
at 32 C for 24 hr. . . . . . . . . . 37

 

vii

Figure

10.

ll.

12.

13.

14.

Effect of different concentrations of
dieldrin on viable cell count, pH and %
lactic acid in whole milk when the milk was
inoculated with Streptococcus cremoris E-8
and incubated at 25 C for 24 hr.

 

Effect of different concentrations of
heptachlor on viable cell count, pH and %
lactic acid in whole milk when the milk was
inoculated with Streptococcus cremoris E-8
and incubated at 25 C for 24 hr. .

 

Chromatogram of noninoculated milk contain-
ing 1 ppm of aldrin.

Chromatogram of milk containing 1 ppm of
aldrin, inoculated with S. lactis A62 and
incubated at 32 C for two weeks. .

Chromatogram of noninoculated milk contain-
ing 1 ppm of DDT.

Chromatogram of milk containing 1 ppm of
DDT, inoculated with S. lactis A62 and
incubated for two weeks at 32 C.

Chromatogram of a noninoculated milk contain—
ing 1 ppm of lindane

Chromatogram of milk containing 1 ppm of

lindane, inoculated with S. lactis A62 and
incubated for two weeks at 32 C. .

viii

Page

38

39

41

43

45

46

47

48

INTRODUCTION

Pesticides are substances intended for preventing,
destroying, repelling mitigating or regulating unwanted
organisms or plants. Pesticides include insecticides,
fungicides, herbicides, rodenticides, mematocides and
plant regulators.

The President's Science Advisory Committee (1963)
states that according to chemical composition, pesticides
can be classified into (a) chlorinated hydrocarbons,

(b) organic phosphorus compounds, (c) other organic com—
pounds and (d) inorganic substances.

During the past twenty-five years pesticides have
had an important role in improving the quality and quantity
of man's food and fiber. The increased production and
widespread use of pesticides have unintentionally caused
new problems. Pesticides are designed to kill or inter—
fere with the metabolism of unwanted target organisms.
However, because of biochemical similarities among many
species and inadequate selective specificity of the toxi-
cant, pesticides may be destructive, not only to pests,
but also to other plant and animal bodies. Consequently,
extensive research has been performed to learn the effect

of pesticides on man and his total living environment.

The acute toxicity of pesticides to human beings, animals
and wildlife is well documented in the literature.
However, still more studies are necessary to deter—
mine the effects of pesticides on various other benefi—
cial organisms which are utilized by man, and to deter-

mine the fate of pesticides in biological systems.

LITERATURE REVIEW

Pesticide Residues in the Human Diet

 

Pesticides can enter the body by ingestion, absorp-
tion through the intact skin, and by inhalation as indi—
cated by the President's Science Advisory Committee
(1963). Among these three routes of entrance, ingestion
of food is the most important source of pesticide resi-
dues for the general public according to Richardson and
Foter (1966), and Duggan and Dawson (1967). Crops may be
contaminated with pesticide residues by direct applica-
tion, by uptake from the treated soil, or by drift or
runoff from applications to adjacent fields. Residues
in milk and meat may result from direct application of
insecticide to the animal, from the animal's ingestion
of residue in feed and water, inhalation of toxic vapors,
and absorption from applications to the animal's housing
as stated by Lisk (1966), and Richardson and Foter (1966).

Dietary intake of pesticide residues in human food
has been discussed by several investigators including
Campbell §£_él° (1965), DuBois (1965), Cummings (1966),
Richardson and Foter (1966), Duggan and Dawson (1967),
Duggan and Weatherwax (1967), Duggan (1968) and Lindgren
eS_§S. (1968). The most frequently occurring pesticides

3

in the human diet are chlorinated hydrocarbon insecti-
cides. Duggan (1968) pointed out that nearly 75 per cent
of the total daily intake of chlorinated organic insecti-
cides is from dairy and other animal products. Organic
phosphate insecticides are not found as frequently in
food as chlorinated hydrocarbon insecticides. Duggan

and Weatherwax (1967) indicated that malathion is the
most common organic phosphate insecticide in the diet and
accounts for more than 80 per cent of the daily intake

of organic phosphate pesticides. Although the tolerance
for pesticides in milk is zero, the Food and Drug Admin—
istration (FDA) has established a series of actionable
levels for the more commonly used insecticides such as
aldrin, dichloro-diphenyl—trichloroethane (DDT), dieldrin,
heptachlor, lindane and methoxychlor as pointed out by
Campbell eS_§S. (1965). Presence of various insecticides
in milk has been discussed by Marth and Ellickson (1959a,
b, c), Henderson (1965) and Heineman (1966).

The amount of residues in food products varies.
According to the Food and Agriculture Organization (FAO)
Working Party of the United Nations and the World Health
Organization (WHO) Expert Committee on Pesticide Resi—
dues (1967) the "tolerance level” is the permitted con—
centration of a pesticide residue in or on a food. The
FDA is responsible for establishing safe tolerances of

pesticide residues on food products and for enforcing

 

such tolerances by preventing interstate and foreign
transport of foods not meeting these tolerances. As
an administrative principle, tolerances are set by the
FDA at one hundredth of the lowest level which causes
adverse effects in the most sensitive test animals
whenever data on human toxicity are not available. After
reviewing the data on which tolerances have been based,
experimental evidence has proved inadequate in certain
instances. In such cases the FDA has reassessed toler-
ance levels according to additional information available.
In deliberations on the toxicity of pesticide resi—
dues in the human diet, the FAO Working Party on Pesti-
cide Residues (1967) pointed out the importance of
sufficient information on pesticide losses, degradation
or transformation during storage or processing of food
before consumption. Certain changes in pesticides may
result in either detoxification or increased toxicity.
The FAO Working Party reported that in many instances
little, if any, information was available on the post—

harvest fate of pesticides on many crops.

Toxicity of Insecticides
Organic phosphorus compounds inhibit cholinesterase
activity and thereby interfere with the transmission of
impulses from nerve to ganglion and nerve to muscle.
Most organic phosphorus insecticides have relatively high

acute toxicities and have caused many fatal and nonfatal

poisonings in man. Many of them are degraded rapidly
and thus seldom persist in the environment.

Acute toxicity of chlorinated hydrocarbon insecti-
cides is associated with the central nervous sytem but
toxic mechanisms are unknown. The toxic effects result
in generalized convulsion, excitability, trembling, or
paralysis, occasionally followed by death.

The LD50 values for individual insecticides are
given in the Agriculture Handbook No. 313, ”Suggested
Guide for The Use of Insecticides to Control Insects
Affecting Crops, Livestock, Households, Stored Products,
and Forest Products" by Agricultural Research Service
and Forest Service, U.S.D.A (1966), and in the "Guide to
the Analysis of Pesticide Residues” published by the U.S.
Public Health Service (1965). There are wide differences
among species as well as sex of the same species in
susceptibility to insecticides.

The President's Science Advisory Committee (1963)
reported the chronic effect of insecticides based on the
FDA feeding experiments. Mice fed as little as 0.5 ppm
of dieldrin in the diet developed histological liver
damage, and an increase to 10 ppm caused a fourfold in-
crease in the frequency of liver tumors. One hundred
per cent mortality of 14 nursing puppies occurred when
several pregnant dogs were fed dieldrin at 0.6 mg/kg of

body weight during pregnancy. No dieldrin was included

in the ration during lactation. Reproduction studies

in rats showed that 2.5 ppm dieldrin and 50 ppm DDT in
the diet reduced the number of young that survived the
nursing period. With rats, 5 ppm of DDT in the diet
produced histOIOgical changes in the liver, but there

was no evidence of tumor induction. Similar results were
obtained by many other researchers and these reports have
been reviewed in "Evaluation of Some Pesticide Residue in
Food" by the FAO Working Party on Pesticide Residues and
the WHO Expert Committee on Pesticide Residues (1967).

Kupfer (1967) stated that the administration of
organo—chlorine insecticides to various species of
animals stimulated both the hepatic microsomal oxida-
tions of drugs and the microsomal hydroxylation of
steroids.

Boyd and Chen (1968) found that rats fed a low pro—
tein diet from the time of weaning were twice as suscep—
tible to the toxic effect of lindane.

Metabolism of Chlorinated
Hydrocarbon Insecticides

The literature on the metabolism of various chlori—
nated hydrocarbon insecticides in biological systems has
been reviewed in "Scientific Aspects of Pest Control" by
the National Academy of Sciences and National Research
Council (1966), and in ”Evaluation of Some Pesticide

Residues in Food” by the FAO Working Party and the

 

 

 

WHO Expert Committee on Pesticide Residues (1967). The
known metabolic products of DDT are dichloro—diphenyl—
dichloroethane (DDD), dichloro—diphenyl—dichloro-ethene
(DDE), and Dichloro—diphenyl—acetic acid (DDA). The
liver and the intestinal flora are believed to be
associated with the metabolism of DDT in animals. Con-
version of DDT to DDD, DDE, and DDA is a detoxication
process. Lindane is converted to pentachlorocyclohexane.
Aldrin and heptachlor are epoxidized to dieldrin and
heptachlor epoxide, respectively. Rat liver microsomes
epoxidize aldrin and heptachlor SS £1239. Epoxidation
of aldrin or heptachlor is a toxicative process since the
epoxides are more toxic than their precursors according
to Westlake and San Antonio (1960), Krzeminski (1962)
and Brooks (1967).
Storage of Chlorinated Hydrocarbon
Insecticides in the Human Body

Due to the hydrophobic nature of chlorinated hydro-
carbon insecticides, storage of these chemicals is mostly
in adipose tissue. There are many reports on the storage

of DDT and DDE in human fat including Hayes et al. (1956),

 

Dale eS_SS. (1963), Hunter eS_§S. (1963), Hoffman eS_§S.
(1964), Egan eS_SS. (1965) and Robinson eS_§S. (1965).
The amount of DDT reported varied from 2 to 30 ppm. The
insecticide that has been studied most in regard to

storage in the human body is DDT.

Durham (1965) studied the storage and excretion of
DDT and its metabolites in volunteers who took known daily
doses of DDT for 21 of the 48 months they were under
observation. Men who received 3.5 mg/man/day stored in—
creasing amounts of DDT and DDE and excreted increasing
amounts of DDA in their urine for several months. They
then reached a steady state of storage and excretion with
no further change until the dosage was discontinued, after
which the excretion diminished. The same phenomenon was
observed in men who received 35 mg/man/day except they
stored and excreted a great deal more DDT—derived material
than the men given the lower level. After dosage was
stopped, excretion decreased very slowly. It was also
reported that no clinical effect associated with dosage
was detected by the men themselves or by physical exami—
nation and laboratory testing.

However, the FAO Working Party and the WHO Expert
Committee on Pesticide Residues (1967) stated that the
possibility for DDT stored in the body to have deleterious
consequences later in life could not be ruled out. In
addition, there is no proof yet to refute that histolog—
ical changes observed in the liver cells of test animals
might also take place in man.'

Several reports, including those of Dale gS_§S.
(1963), Hunter eSSSS. (1963), Hoffman §S_SS. (I964),

Egan et a1. (1965) and Robinson et a1. (1965) are

10

available on the storage of dieldrin in human fat in the
general population. The average concentration ranged
between 0.03 and 0.3 ppm.

Hoffman eS_SS. (1964) reported an average of 0.57
ppm of lindane in the analyses of 282 samples of human
fat obtained from the general population of the United
States. In the United Kingdom an average of 0.015 ppm
of lindane was found in 20 human fat samples according
to Robinson eS_§S. (1965).

Even human milks contain some insecticides such as
DDT, dieldrin and lindane as reported by West (1964),
Egan §£_§l- (1965), Quinby eS_§S. (1965), and Curley
and Kimbrough (1969).

Hayes (1966, 1968) stated that DDT, lindane,
dieldrin and heptachlor epoxide were found not only in
the adipose tissue of humans, but also in blood and in
organs. Presence of these insecticides in blood was
also reported by Dale eS_§S. (1966, 1967).

Effect of Insecticide Residues
on Microorganisms

 

 

The influence of insecticide residues on micro-
organisms has been studied by Lewis and Hammer (1946),
Wilson and Choudhri (1946), Carlyle and Thorpe (1947),
Smith and Wenzel (1947), Wilson (1948), Angelotti eS_§S,
(1954) and Bollen eS_§S. (1954). With few exceptions,

the studies have been limited to organisms important in

ll

soil microbiology. The reports, in general, indicated
that bacteria are not affected by the various insecti-
cides. Further studies on the effect of chlorinated
hydrocarbon pesticides on soil microorganisms were re—
viewed by Bollen (1961), Martin (1963) and Marth (1965),
who also indicated that normal applications of the common
insecticides have little effect on soil microbes.

Some fungi and protozoa were affected by some
insecticides according to Kirkwood and Phillips (1946),
Smith and Wenzel (1947), Wilson (1948), Bollen eS_§S,
(1954), Johnston (1957), Ukeles (1962) and Williams
Et—Q- (I963).

Painter and Kilgore (1963) reported that none of
13 insecticides added to grape musts in wine fermenta—
tion had any measurable effect on the fermentation
normally accomplished by the inoculated yeast Saccharomyces

cerevisiae var. ellipsoideus.

 

Shaw and Robinson (1960) and Bartha §£_él° (1967)
found that nitrification was not influenced adversely
by chlorinated hydrocarbon insecticides.

Collins and Langlois (1968) observed inhibition of
Pseudomonas fluorescens by 50 and 100 ppm of DDT in
Trypticase Soy Broth (TSB), but no inhibition of S.

fluorescens by corresponding amounts of DDT in skim milk.

 

Dieldrin and heptachlor did not inhibit S. fluorescens

in TSB or in skim milk. Heptachlor had a bactericidal

l2

effect on Stappylococcus aureus in TSB but no adverse

 

effect on S. aureus in skim milk. DDT reduced the growth
of S. aureus in TSB but not in skim milk. Dieldrin
showed no effect on S. aureus in TSB or in skim milk.
Transformation of Chlorinated Hydrocarbon
Insecticides by Microorganisms

Kallman and Andrews (1963), reported the formation
of DDD from DDT in the presence of S. cerevisiae from
commercial yeast cakes in buffered sucrose medium. When
DDE was also incubated with S. cerevisiae in order to
validate a hypothesis that formation of DDD is preceded
by the formation of DDE from DDT, no DDD was formed.
Therefore, the hypothesis was invalidated and it was
concluded that DDD is produced directly from DDT by
reductive dechlorination.

Barker eS_§S. (1965) observed the conversion of
DDT to DDD by Proteus vulgaris, a bacterium isolated from
the intestinal flora of a mouse. It was also suggested
that DDE did not appear to be an intermediate in the pro—
duction of DDD from DDT.

Stenersen (1965) isolated Aerobacter aerogenes,

 

Alcaligenes faecalis, Escherichia coli, Bacillus brevis

 

and Serratia marcescens from the excreta of flies and

 

studied transformation of DDT by these organisms in
meat extract bouillon. When growing anaerobically or

under oxygen deficiency, the facultative anaerobes

13

S. marcescens, S. coli and one unidentified strain,

 

converted to DDT to DDD and DDE. In aerated cultures
neither the facultative anaerobes nor the obligate aerobes
converted any DDT to DDD or other products.

From the study on dechlorination of DDT to DDD by

Aerobacter aerogenes, Wedemeyer (1966) and Plimmer et a1.

 

(1968) indicated that reduced cytochrome oxidase was
responsible for DDT dechlorination and that the presence
of oxygen hindered dechlorination.

Mendel and Walton (1966) demonstrated that the
normal flora of the gastrointestinal tract, rather than
the liver as others have suggested, was the major source
of the DDD that is found in animals fed DDT. When rats
were given DDT by stomach tube, DDD occurred in the feces
and livers of rats, but not in the feces and livers of
rats injected intraperitoneally with DDT. Coliform
bacteria isolated from feces of control animals could
affect reductive dechlorination of DDT to DDD.

Johnson eS_SS. (1967) found many pathogenic and
saprophytic bacteria associated with plants which could
convert DDT to DDD. Chacko eS_SS. (1966) reported that
several streptomyces species degraded DDT. Anaerobic
dechlorination of DDT to DDD by soil microorganisms was
studied by Guenzi and Beard (1967).

Gamma-pentachlorocyclohexane was identified as a

decomposition product of lindane in soil. Yule et a1.

14

(1967) stated that this product was 1000 times less toxic
to insects than lindane and that soil microorganisms and
alkaline soils are involved in the degradation process.

Bartha eS_§S. (1967) studied the stability of
aldrin, dieldrin, endrin, telodrin, DDT, DDD, and
methoxychlor in soil and found no indication of micro—
bial degradation during 30 days. However, Matsumura and
Boush (1967) isolated Pseudomonas and Bacillus species,
after examining more than 500 soil isolates from dif—
ferent locations which could degrade dieldrin. Partial
hydrolysis of dieldrin by S. aerogenes in TSB was ob—
served by Wedemeyer (1968).

Effect of Processing on Insecticides
in Milk

Any process or manufacturing operation which de—
toxifies or reduces insecticide residues in milk would be
of significant value. Some researchers investigated the
possibility of removing insecticides form milk by ordinary
manufacturing process.

In 1950 Mann found that pasteurization had little
effect on the amount of DDT in milk. Langlois eS_SS.
(1964, 1965) noted that DDT and lindane in milk were
essentially stable during pasteurization, homogenization,
condensation and storage for four months. The only
significant loss of DDT and lindane was caused by spray

drying in the manufacture of spray dried whole milk

15

powder. During the manufacture of Swiss—type cheese
detectable amounts of endrin, dieldrin, heptachlor, and
lindane were removed in whey, resulting in less insecti—
cide in the cheese product. Condensing of milk caused
significant loss of heptachlor epoxide and dieldrin but
no loss of heptachlor and endrin. Loss of heptachlor,
heptachlor epoxide, dieldrin and endrin was observed
during spray drying and drum drying of milk.

According to the study by Stemp and Liska (1966)
40—50 per cent of the telodrin residues were destroyed
in evaporated milk and 10—20 per cent of the residues
were destroyed during processing of milk for dry whole
milk. Methoxychlor was stable throughout the processing
treatments.

Kroger (1968) observed that freeze—drying and milk
deodorization were not effective on the reduction or
elimination of heptachlor epoxide, dieldrin and DDT
from butteroil.

Ledford eS_gS. (1968) also indicated that commercial
steam distillation—vacuum processing removed a noticeable
amount of lindane, but little heptachlor, dieldrin and

DDT.

16

Comments and Objectives

 

With the persistent appearance of insecticides in
milk it seemed desirable to understand the effect and
fate of insecticides in milk in conjunction with the pro—
cessing of dairy products. Marth and Ellickson (1959c)
pointed out that little research had been done concerning
the effects of insecticide residues on the processes
employed in the manufacture of various dairy products and
Vice versa. .

In the manufacture of fermented milk products such
as sour cream, yoghourt, cultured buttermilk and cheese,
inoculation of milk with lactic culture organisms and
subsequent fermentation are essential processes. The
main functions of lactic cultures are: to produce lactic
acid and some flavor compounds. If milk happens to con—
tain any antimetabolite or antimicrobial agent the lactic
cultures may not grow or function properly. For example,
antibiotics or sanitizing compounds are known to occur
in milk sometimes and cause inhibition or retardation of
growth and fermentation ability of lactic cultures as
pointed out by Foster eS_SS. (1957).

Being aware of (a) the presence of insecticides in
milk, (b) inadequate selective toxicity of the insectides,
and (c) the important role of lactic cultures in the manu—

facture of cultured dairy products, some people working

17

with lactic culture organisms have expressed concern about
the effect of insecticides on these cultures.

As reviewed in the preceding section several re—
ports are available regarding the effect of processing
and storage of dairy products on insecticides from the
physical point of View. From the microbiological stand-
point, there is very little information concerning the
effect of the fermentation process on insecticide resi—
dues in the manufacture of cultured dairy products.

In general, microorganisms are well known for their
ability to decompose or modify various organic compounds.
Furthermore, several organisms were reported to modify
some insecticides. Consequently, it appeared conceivable
that lactic culture organisms might also have the ability
to modify some insecticides. Since transformation of
insecticides does not necessarily reduce the toxicity,
it would be meaningful to understand how some insecti-
cides are affected by lactic culture organisms during
fermentation in milk.

Knowledge of any chemical alteration in insecticide
residues in food is essential to evaluate the chronic
effect of the residues, and to assess realistic tolerance
levels of the residues in foodstuffs.

The objectives of this research were to:

a) determine the effect of some insecticides on

the growth and fermentation ability of com—

mercially important lactic culture organisms.

18

(b) determine the ability of the lactic culture
organisms to transform aldrin, DDT and
lindane in milk, and

(c) determine the uptake of dieldrin by SggSg-

bacillus casei.

 

MATERIALS AND METHODS

Effect of Insecticide Residues on Growth
and Fermentation Ability of Lactic
Culture Organisms

Dieldrin,l heptachlor,2 and methoxychlor3 were
chosen as the chlorinated hydrocarbon insecticides for
this study. Also included was one organophosphate
insecticide, malathion.)-I

Stock solutions containing 10% of these insecti-
cides were prepared by dissolving dieldrin, heptachlor,
or methoxychlor in acetone, and by dissolving malathion
in 95% ethyl alcohol. Subsequent lO—fold dilutions of
the stock solutions were prepared using the solvents men-
tioned above so that 1.0 ml added to 100 ml of sterile
whole milk would give milk containing 100, 10, l, or 0.1
parts per million (ppm) of the insecticide. Preliminary

experiments indicated that these solvents had no effect

on the growth of the test organisms when used at

 

lDieldrin, 99+% pure, Shell Chemical Co., New York,
New York.

2Heptachlor, 99+% pure, Applied Science Labs, Inc.,
State College, Pennsylvania.

3Methoxychlor, 100% pure, Shell Chemical Co., New
York, New York.

uMalathion, 96% pure, American Cyanamid Co.,
Princeton, New Jersey.

19

20

concentrations up to 1% in the growth medium. The origi-
nal milk usually contained less than 0.01 ppm, and always
less than 0.05 ppm of insecticide.

The milk samples containing various amounts of
insecticides were inoculated with 0.1% of an active litmus

culture of Streptococcus lactis A62, S. lactis A254,

 

Streptococcus cremoris E—8, Streptococcus diacetilactis

 

18—16, or Lactobacillus casei and were incubated at 32 C

 

for 48 hr, except for samples inoculated with S. cremoris
which were incubated at 25 0. Two control samples of milk
with no added insecticides were included with each trial,
one inoculated and one non-inoculated. The non—inoculated
controls were included to check for contamination of
samples throughout the experiment.

At 0, 24, and 48 hr of incubation, aliquots were
withdrawn aseptically to determine the total plate count,
pH and titratable acidity. Duplicate plates for a proper
dilution were prepared using Plate Count Agar (Difco).l
A Beckman pH Meter was used for pH measurement. For the
determination of titratable acidity, a 9 ml aliquot from
each sample was transferred to a "Mann Acidity Cup” using
a 9 m1 milk pipette, and diluted with 18 m1 COe—free
deionized water. After addition of 1 ml of phenolphthalein

solution (1% in ethyl alcohol), the diluted milk sample was

 

lPlate Count Agar, Difco Laboratories, Detroit,
Michigan.

21

titrated with 0.1N NaOH to first persistent pink. The
acidity was calculated as per cent lactic acid.
Effect of Lactic Culture Organisms on
Aldrin, DDT and Lindane in Milk

Preparation of samples.—-To each of seven Erlen—
meyer flasks, containing 25 m1 sterile milk, was added
25 ug of aldrinl dissolved in acetone. Six individual
samples were inoculated with 0.25 ml litmus milk culture
of one of the following organisms: S. lactis A254, S.
lactis A62, S. cremoris E-8, S. diacetilactis 18—16,
S. diacetilactis DRCI or S. eegei. The above cultures
had been activated in litmus milk by four consecutive
daily transfers prior to use. The 18 hr old cultures
of the last transfer were used for inoculation. One
remaining sample was not inoculated and was used as a
control. The milk inoculated with S. cremoris E—8 was
incubated at 25 C for two weeks. The remaining six milk
samples were incubated at 32 C for two weeks. Samples
containing DDT 2 and lindane3 were prepared in the same
manner.

After two weeks of incubation the insecticides were
extracted from milk samples and cleaned. The techniques

for the extraction and cleaning were adopted from the

 

lAldrin, 99+% pure, Applied Science Labs, Inc.,
State College, Pennsylvania.

2’3DDT, and lindane, 99+% pure, Applied Science
Labs, Inc., State College, Pennsylvania.

22

methods recommended by Shell Chemical Company (1964) and
by the FDA Pesticide Manual (1965).

Procedure for extraction, separation, partitioning
and cleanup were performed separately but identically for
each sample. Reference will be made to only one sample
for the procedures mentioned above.

Extraction of insecticides from milk.—-To the

 

Erlenmeyer flask containing a milk sample, 30 m1 of 95%
ethyl alcohol and 15 ml of 5% potassium oxalate solution
were added with thorough mixing after each addition. The
milk fat containing the insecticide was then extracted
from the milk with 100 m1 anhydrous ethyl ether by shak-
ing the mixture for 20 min on a Burrell Wrist—Action
Shakerl with an automatic timer. The ethyl ether layer
was decanted into a 600 ml beaker and the milk was re-
extracted twice with 100 m1 of hexane-ethyl ether (1:1,
v/v). The extractions were combined and the solvent was
evaporated from the extracted sample on a steam bath
leaving fatty materials and the insecticide in the beaker.

Separation of insecticide and milk-fat.--The fatty

 

materials along with the insecticide, were transferred to
a 125 ml separatory funnel by washing the beaker five

times, each time with 5 ml of hexane. Thirty milliliters

 

lBurrell Wrist—Action Shaker with a timer, Burrell
Corp., Pittsburgh, Pennsylvania.

23

of acetonitrile1 were added to the separatory funnel, and
the mixture was shaken vigorously for one minute. After
the layers were separated, the lower layer (acetonitrile)
was transferred into a one liter separatory funnel con-
taining 700 ml of 2% NaCl solution and 100 ml of petroleum
ether.2 Partitioning with 30 ml of acetonitrile was re—
peated three more times, combining the acetonitrile ex—
tracts each time in the one liter separatory funnel. At
this point fatty materials remained in the hexane layer
and the insecticide was extracted into acetonitrile.
Partitioning with acetonitrile, water and petroleum
eSSeS.——By swirling the one liter separatory funnel gently
in an inverted position, the insecticide was recovered in
petroleum ether and the acetonitrile was removed in the
aqueous layer (2% NaCl solution). The aqueous layer was
transferred into another one liter separatory funnel and
extracted with 100 m1 of petroleum ether for 15 sec. The
aqueous layer was discarded. The petroleum ether layer
was added to the first one liter separatory funnel. The
combined petroleum ether extracts were washed twice with
100 ml of 2% NaCl solution and the washings (salt solu—

tion of lower layer) were discarded. The petroleum ether

 

lAcetonitrile, Grade for pesticide analysis, Fisher
Scientific Company, Detroit, Michigan. Saturated with
redistilled hexane before use.

2Petroleum ether, Nanograde for pesticide analysis,
Mallinckrodt Chemical Works, St. Louis, Missouri.

 

24

extract was transferred to a 600 ml beaker. The separa—
tory funnel was rinsed three times with 10 ml of petroleum
ether and washings were combined in the beaker. The
petroleum ether was evaporated on a steam bath, leaving
the insecticide in the beaker.

Cleanup of extracted insecticide.——In addition to

 

the removal of fatty materials by partitioning the milk—
fat containing the insecticide between acetonitrile and
hexane, further cleanup of the extracted insecticide was
required to remove other impurities prior to testing the
sample by gas chromatography. Adsorption column chroma—
tography was used for cleanup. The adsorbent was a
Florisill—Celite 5452 mixture. Florisil was activated
by heating at 130 C for 5 hr or longer before use. The
Florisil—Celite 545 mixture consisted of 100 parts of
activated Florisil, 12 parts of distilled water and 20
parts of Celite 545.

3

The chromatographic column of 20 mm x 400 mm was

packed, in order, with l in. of granular anhydrous sodium

 

lFlorisil, 60—100 mesh, Floridin Company, Talla—
hassee, Florida. A synthetic selective adsorbent,
magnesia—silica gel.

2Celite 545, Johns—Manville Corporation, New York,
New York. Celite 545 is a processed diatomite product.

3Chromatographic column, with Lab—Crest teflon
stopcock, sintered glass disc, detachable joint with
teflon seal. Made by the Lab-Crest Scientific Division
of Fisher and Porter. Distributed by Brinkmann Instru—
ments, Inc., Des Plaines, Illinois.

 

 

 

25

sulfate (Na2SOu), 10 g of Florisil—Celite 545 mixture,
and 1 in of granular anhydrous NaZSOu. The packed column
was washed with 50 ml of hexane. When the hexane reached
the top layer of Na2SOM, the insecticide extract was
transferred to the column using 5 ml of hexane and a
pasteur pipette. A 500 m1 Kuderna-Danish concentratorl
was placed under the column to collect the effluent and
the insecticide extract was allowed to enter the column.
The beaker was rinsed three times with 5 ml, and one time
with 30 ml of hexane. Each rinse was added to the column
and allowed to enter the column. The insecticide extract
was eluted from the column with an additional 350 m1 of
hexane at a rate of 4—5 ml per min. The hexane effluent
in the Kuderna—Danish concentrator was evaporated to a
final volume of 4-5 ml. The concentrate was transferred
to a 25 ml volumetric flask and the final volume was
adjusted to 25 ml with hexane which had been used to rinse
the Kuderna—Danish concentrator.

The cleaned insecticide was saved for later analysis
by gas liquid chromatography.

Analysis of insecticide.-—Final analysis was made

 

by gas liquid chromatography (GLC) using a F & M Hewlett—

Packard Model 5750B Research Gas Chromatograph2 equipped

 

lKuderna—Danish concentrator, Kontes Glass Company,
Vineland, New Jersey.

2Gas Chromatograph, Hewlett—Packard Aanlytical
Instruments, Palo Alto, California.

 

   

26

with an electron capture (EC) detector. The column for
GLC was a 4' x 1/4” coiled glass column which was pre—
packed with 3.8% UC—W98 (a silicone rubber) on 80/100
mesh Diatoport S, and pretested by Hewlett—Packard. The
carrier gas was a 90% argon—10% methane mixture 1 with
a flow rate of 60—70 ml/min. Temperatures at the injec—
tion port, column, and EC detector were 255 C, 195-200 C
and 193-196 C, respectively. The pulse interval on
the EC detector was 50 u see and the chart speed of the
recorder was 0.25”/min or 0.5"/min. The injection volume
was 2—5 ul, depending on the kind of insecticide under
investigation, and the volume of the samples injected
was always the same as that of the controls. The effect
of lactic starter culture organisms on insecticides was
checked by comparing the chromatograms of insecticides
from fermented milks with the Chromatogram of insecti—
cides from a non—fermented milk control.
The Uptake of Dieldrin by
Lactobacillus casei

S. eeSeS was activated by three daily transfers in

Trypticase Soy Broth (TSB)2 at 32 C. At the end of 18 hr

incubation after the last transfer, 1 m1 of culture was

 

l90 argon—10% methane, The Matheson Co., East
Rutherford, New Jersey.

2Trypticase Soy Broth, BBL, Division of BioQuest,
Division of Becton, Dickinson and Company, Cockeysville,
Maryland.

27

added to 100 ml of TSB containing luC—dieldrinl with a

specific activity of 72.4 milli curie/milli As a
control, the same amount of S. eeeeS culture was added
to 100 m1 of TSB containing no dieldrin. The samples
were incubated at 32 C for 18 hr.

In order to measure the activity of luC—dieldrin
in TSB, 0.5 m1 of TSB containing lLiC—dieldrin, 14.5 ml
of scintillator solution, and Cab—O—Sil2 were added to
a glass liquid scintillation vial. The amount of
Cab-O—Sil was just enough to obtain a stable homogeneous
gel. Then the contents were shaken vigorously and
counted for radioactivity using a Packard—Tri—Carb
Liquid Scintillation Spectrometer Model 3310.3 The
composition of the scintillation solution was 6 g/l of
2,5-diphenyloxazole (PPO), and 0.2 g/l of 1,4—bis—2—
(4—methyl—5—phenyloxazolel(POPOP) in toluene. The
number of viable cells was determined on Plate Count
Agar.

Twenty—five milliliters of the 18 hr culture in
TSB containing lliC—dieldrin were washed with 25 m1 of

hexane in a stainless steel centrifuge bottle by shaking

 

l lLlC-dieldrin, Amersham/Searle Corporation, Des
Plaines, Illinois.

2Cab—O—Sil, New England Nuclear Corp., Pilot
Chemicals Division, Boston, Massachusetts.

3Packard Tri—Carb Liquid Scintillation Spectrometer
Model 3310, Packard Instrument Company, Inc., Downers
Grove, Illinois.

 

 

28

the mixture for 30 sec. Then the cells were harvested by
centrifuging at 10,400 x g for 20 min using a Sorvall
automatic refrigerated centrifuge.l The harvested cells
were washed twice with 50 ml of physiological saline
(0.89% NaCl), transferred to a liquid scintillation vial
using 0.5 ml TSB, and prepared for radioactivity counting
as described previously. The culture in the control TSB
was treated in the same manner. Results are reported on

a count per minute (cpm) per milliliter basis.

 

lSorvall Superspeed, Model RC-2, Ivan Sorvall,
Irlc., Norwalk, Connecticut.

RESULTS

Effect of Insecticide Residues on Growth
and Fermentation Ability of
Lactic Culture Organisms

 

 

 

After individual cultures were inoculated into the
milks containing 0, 0.1, 1, 10, and 100 ppm of the
insecticide under study, the milks were incubated for
48 hr at appropriate temperatures as indicated in the
Materials and Method section.

The tables 2 through 15 in the Appendix contain
all the data on viable cell count, pH of the milks and
per cent lactic acid in the milks at 0, 24 and 48 hr,
for all of the combinations of cultures and insecticides
tested.

Using the data obtained after the cultures were
exposed to the insecticides in milk for 24 hr, the log of
the viable cell count/m1, pH,and per cent lactic acid of
the inoculated milks were plotted against the concentra—
tion of each insecticide tested. Because of the over—
lapping of the graphs, no more than two different lactic
cultures could be represented in one figure, even when
four different cultures were tested against the same kind

of insecticide.

29

30

The graphs in Figure 1 describe the effect of
different concentrations of dieldrin on Viable cell count,
pH and per cent lactic acid in whole milk when the milk
was inoculated with S. lactis A254 or S. eeeeS and incu—
bated at 32 C for 24 hr.

The data in Figure 1—A show that 0.1 to 100 ppm of
dieldrin in milk had no effect on the viable cell count
of S. lactis A254. However, a slight reduction in the
viable cell count of S. eeeei was observed in the milks
containing 10 and 100 ppm of dieldrin. Figures l—B and
1—0 indicate that the presence of dieldrin in amounts up
up 100 ppm in milk did not interfere with the reduction
of pH or the production of lactic acid in milk by S.
lactis A254 or S. eeeeS.

As noted in Figure 2, 100 ppm of heptachlor in
milk had neither an adverse nor a stimulative effect on
viable cell counts or fermenting ability of S. lactis
A254 or S. eeSeS when inoculated into milk and incubated
at 32 C for 24 hr.

In the milks containing 0.1, l, 10, and 100 ppm of
methoxychlor, the viable cell count, pH and per cent
lactic acid were about the same as in the control milk
containing no methoxychlor, when the milks were inocu—
lated with S. lactis A254 or S. eeeeS and incubated at

32 C for 24 hr. The results are depicted in Figure 3.

31

 

 

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When S. lactis A254 or S. eeeeS was grown for 24 hr
at 32 C in milks containing 0.1 to 100 ppm of malathion,
the malathion had no influence on the viable cell count
or fermentation ability of the organisms. The results
are illustrated in Figure 4.

Three additional strains of Streptococcus were ex—
posed to dieldrin and to heptachlor to determine the
uniformity of the resistant characteristic within the
Streptococcus genus. As represented in Figures 5 and 6,
both dieldrin and heptachlor at concentrations of 100 ppm
in milk had no effect on the viable cell count or fermen—
tation ability when the milks were inoculated with S.
lactis A62 or S. diacetilactis 18—16 and incubated at
32 C for 24 hr.

Figures 7 and 8 show that 0.1 to 100 ppm of dieldrin
or heptachlor in milk did not hinder the growth and lactic
acid producing ability of S. cremoris E—8 when the
organism was grown in the milk for 24 hr at 25 C.

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rarior to inoculation with the lactic cultures. For ex—
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35

 

 

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37

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38

 

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39

 

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3. o. . ..o o oo. o. . ..o o oo. o. . ..o o
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40

at 0 hr of incubation. The lower pH in the milk contain—
ing 100 ppm of heptachlor was not due to the presence of
the heptachlor since the preliminary study showed that
addition of 100 ppm of an insecticide to milk did not
change the pH of the milk. After S. eeSeS was allowed to
grow and ferment milk for 24 hr, the pH of the inoculated
milk containing 100 ppm of heptachlor was 6.10 and the pH
of the inoculated control was 6.14. However, the differ—
ence in pH in this case could not be attributed to the
effect of the 100 ppm of heptachlor in the milk since
there was initially the same degree of difference in pH
between the two milks.

Although viable cell count, pH and per cent lactic
acid were also determined at 48 hr of incubation, the
data after 48 hr of incubation did not provide any more
or different information that the data at 24 hr of incu—
bation in respect to the effect of the insecticides on
the growth and lactic acid fermentation ability of the
cultures. Therefore, only the data at 24 hr of incuba—
tion are included in the graphs in Figures 1 through 8
in order to show the effect of different concentrations
of insecticides on the growth and lactic acid producing
ability of the cultures.

Effect of Lactic Culture Organisms on
Aldrin, DDT and Lindane in Milk
Figure 9 shows the Chromatogram of aldrin ex—

fsracted from the noninoculated control milk. The milk

 

 

Recorder Response, % Full Scale

90

80

7O

6O

50

4O

30

2O

10

 

41

 

 

 

 

Sample size 5ul
-' Solvent Chart speed 0.5 in/min
Aldrin
ji-
1—
| I I I I
I I l l I
2 4 6 8 10
Retention Time (min.)
Figure 9. Chromatogram of noninoculated milk containing

1 ppm of aldrin.

42

contained 1 ppm of aldrin. Figure 10 shows the chromato—
gram of aldrin extracted from a milk sample which con—
tained 1 ppm of aldrin and was inoculated with S. lactis
A62. All the milks including the control were incubated
at 32 C for two weeks.

When aldrin converts to dieldrin (a metabolic pro—
duct of aldrin)or any other analog, the size of the peak
representing aldrin becomes smaller and a new peak rep—
resenting dieldrin or the analog appears on the chromato—
gram. Comparison of the chromatogram in Figure 10 with
the chromatogram of the control in Figure 9 shows no
difference between the two chromatograms. This indicates
that aldrin was not degraded or converted to an analog
when present in a milk substrate inoculated with S. lactis
A62 and incubated at 32 C for two weeks.

The chromatograms of aldrin extracted from other
milk samples containing aldrin and inoculated with S.
lactis A254, S. diacetilactis 18—16, S. diacetilactis
DRCI, S. cremoris E-8 or S. eeeeS were all the same as
those of the milk inoculated with S. lactis A62. This
indicates that none of the above lactic culture organ—
isms modified or degraded aldrin in milk. Since these
chromatograms are all identical, only the chromatogram
of the milk inoculated with S. lactis A62 is presented

as a representative of the entire group.

 

 

9O

8O

7O

60

50

4O

3O

Recorder Response, % Full Scale

20

10

“F

 

 

43

Sample size 5ul

Solvent Chart speed 0.5 in/min

Aldrin

 

 

 

l I l l l

 

I l I r l

2 4 6 8 10

Retention Time (min.)

Figure 10. Chromatogram of milk containing 1 ppm of aldrin,
inoculated with S. lactis A62 and incubated at 32 C for two weeks.

44

The effect of lactic culture organisms on DDT and
lindane were also studied. The lactic culture organisms
included S. lactis A62, S. lactis A254, S. diacetilactis
18-16, S. diacetilactis DRCI, S. cremoris E—8 and S.
eeeeS. Figure 11 shows the chromatogram of DDT from the
noninoculated control milk. Figure 12 shows the chromato-
gram of DDT extracted from the milk sample inoculated
with S. lactis A62. All the samples were incubated for
two weeks at 32 C except the milk sample which was inocu-
lated with S. cremoris and incubated at 25 C. In Figure
12 the height of the DDT peak is 37% full scale, which is
the same as the height of the DDT peak in the chromato-
gram of the control in Figure 11. Thus it was apparent
that DDT was stable even after two weeks of incubation
with S. lactis A62. DDT was also stable in the presence
of other lactic culture organisms, S. lactis A254, S.

diacetilactis 18—16, S. diacetilactis DRCI, S. cremoris

 

 

E-8 and S. eeeeS.

The chromatogram of lindane extracted from a non—
inoculated control milk is shown in Figure 13. The
chromatogram of lindane extracted from a milk sample
inoculated with S. lactis A62 and incubated for two
weeks is illustrated in Figure 14.

Chromatograms performed on the extracts from the
milk samples inoculated with S. lactis A254, S.

diacetilactis 18—16, S. diacetilactis DRCI, S. cremoris E—8

 

 

“5

 

 

 

 

 

 

 

 

90 “ q
80 -_ Sample Size 3ul
Chart speed 0.25 in/min
70 “P Solvent
(1)
2?;
o 60 ‘*‘
U)
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& 50 4—
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(1)
m
p
(D
o
‘5 30 -.
8 DDT
m
20 it
10 -r
0 g "L 1l V’T ##1“
M 8 12 16

Retention Time (min.)

Figure ll. Chromatogram of noninoculated milk containing
1 ppm of DDT.

90

8O

70
(D
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m
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& 50
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Figure 12.

1+6

 

 

 

 

 

Sample size 3ul
-L Chart speed 0.25 in/min
'_ Solvent
-- DDT
I .. ———+
I r V l I
u 8 12 16

Retention Time (min.)

Chromatogram of milk containing 1 ppm of DDT,

inoculated with S. lactis A62 and incubated for two weeks at 32 C.

 

 

 

 

 

“7

 

 

 

 

 

 

 

 

 

8o -- '1
Sample size 2ul

O .1-

7 Chart speed 0.25 in/min
o
H
m
c?) 60 --
r-l
H
5
m
R
a 50" Lindane
m
U)
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o.
8 40-~
m K
$4
(1)
o
‘5
o 30"“?
o
m \

20--

101f

o E i

f
4 8 12

Retention Time (min.)

Figure 13. Chromatogram of a noninoculated milk con—
taining 1 ppm of lindane.

 

 

48

 

 

 

 

 

 

 

80 q- '1
Sample size 2ul
70-- Chart speed 0.25 in/min
I
o
g 60 " Lindane
O
m
H
r—{
5
m 50-"
K"
a)“
8
o MO-m
Q
U)
o
m
8
"a 30 4-
o
O
m
m
20 +-
10 -—u..
0 § § %

H 8 12

Retention Time (min.)

Figure lu. Chromatogram of milk containing 1 ppm of lin—
dane, inoculated with S. lactis A62 and incubated for two weeks
at 32 C.

 

 

L r. . ,. .

a . . . . e . I. c . 1
cs. ..... w... a". ... . .. . r
...r .. r . .. .

 

“9

or S. EEEEE were the same as those obtained from the milk
inoculated with S. lactis A62. A comparison of the
chromatograms in Figures 13 and 1“ indicates that lindane
was also inert to the biological action of the lactic
culture organisms.

When degradation or modification of DDT or lindane
occurs the peak size representing DDT or lindane de—
creases and a new peak appears in the chromatogram to
represent the newly formed analog. Such phenomena did
not occur in the milk samples containing DDT or lindane
when the milk samples were inoculated with the above

lactic culture organisms and incubated for two weeks.

The Uptake of luC-dieldrin by

Lactobacillus casei

 

 

When a study was performed on the effect of dieldrin,
heptachlor, methoxychlor and malathion on the growth of
S. gaggl, it was found that only dieldrin inhibited the
growth of S. gaggl as illustrated in Figure l-A. There-
fore, possible uptake of dieldrin into the cells of S.
gaggl was studied using luC-dieldrin. Table 1 contains
data and calculations indicating the amount of uptake of

luC—dieldrin in TSB by S. casei. Cells were grown for

18 hr at 32 C in TSB containing luC—dieldrin. After the
cells were harvested and washed, the cells of S. casei
retained less than 0.2% of the total lU'C-dieldrin in the

TSB. For example, when there was about 68,000 cpm of

 

 

v

  

 

 

 

 

 

50
lL‘C—dieldrin in the TSB the harvested cells retained about

1M

140 cpm of C—dieldrin, which is 0.16% of 68,000 cpm.

When the TSB growth medium contained 1N2,000 cpm of 1MC—
dieldrin, the cells harvested from the TSB retained
280 cpm, which is 0.18% of the 142,000 cpm. The amount of

luC—dieldrin remaining with the harvested cells was even

less than 0.2% of the total lLLC—dieldrin in the growth
medium. These results negate the idea that dieldrin may

be absorbed into the cells of S. casei. The radio—

activity counted from the harvested cells is probably

 

due to the residual radioactivity resulting from incom—

plete washing.

TABLE 1.——Calcu1ation of uptake of lLLC—dieldrin by

Lactobacillus casei when grown in in
Trypticase Soy Broth (TSB) containing C—
dieldrin for 18 hr at 32 C.

 

 

Trial 1 Trial 11

 

(a) *cpm of 1 m1 TSB culture contain-

ing lac-dieldrin. 2,118 5,670
(b) cpm of 25 ml TSB culture contain—

ing lac-dieldrin = (a) x 25 67,966 1M1,750
(c) cpm of cells harvested from 25

m1 of TSB containing l“C—dieldrin 136 279
(d) cpm of cells harvested from 25 ml

of TSB containing no 1 C—dieldrin 30 30
(e) per cent of lLlC—dieldrin retained

by S. casei

= ——$917%7£92—— x 100 0.16 0.18

(f) viable cell count per m1 2M x 107 22 x 107

 

*count per minute

 

 

 

DISCUSSION

Lactic starter cultures are essential in the manu—
facture of cheese and fermented milk products. When
lactic cultures fail to grow or produce lactic acid, the
failure is usually attributed to the presence of anti-
biotics, bacteriophages, free fatty acids, sanitizing
chemicals in milk, or to some unknown factor. Failure of
lactic starter cultures can be avoided by excluding milk
containing inhibitory quantities of the above substances.
Therefore, personnel in any culture company or dairy
plant working with lactic starter cultures should know
all the possible causes which can interfere with the
activity of lactic starter cultures.

Some people even suspected that certain insecti—
cides in milk, unintentionally present in excess of the
actionable level set by the Food and Drug Administration
(FDA), might inhibit the growth and fermentation ability
of lactic starter cultures. However, there has been no
experimental proof to refute or support such an opinion.

The purpose of the first part of the experiment was
to study the effect of some insecticides on the growth and
fermentation ability of several lactic starter cultures.

Since lactic cultures produce lactic acid through the

51

 

52

fermentation of lactose in milk, the pH and per cent
lactic acid developed in the inoculated milk are used as
indices of the culture's fermentation ability. The growth
was measured by viable cell count.

Data in Figures 1 through 4 show that none of the
dieldrin, heptachlor, methoxychlor, and malathion in
milk, ranging from 0.1 to 100 ppm had any effect on the
viable cell count, pH and per cent lactic acid when the
milk was inoculated with S. lactis A254 and incubated at
32 C.

When three additional Streptococci, S. lactis A62,

S. diacetilactis 18-16 and S. cremoris E—8 were also ex-

 

posed to 0.1 to 100 ppm of dieldrin and heptachlor, their
growth and lactic acid fermentation ability were not
affected.

Commercially important streptococci utilized as lactic

starter cultures include S. lactis, S. diacetilactis,

 

S. cremoris and S. thermophilus. In this study two

 

strains of S. lactis, one strain of S. diacetilactis and

 

one strain of S. cremoris were tested against two to four
different insecticides. The chlorinated hydrocarbon
insecticides can be classified into the following four
groups: (1) aldrin, dieldrin and endrin; (2) DDT, DDD,
DDE and methoxychlor; (3) heptachlor and heptachlor
epoxide; and (U) lindane and BHC. The insecticides in

each group are very closely related in their chemical

 

53

composition and structure. Three different groups are
represented by dieldrin, heptachlor and methoxychlor,
which were the insecticides selected for testing in this
experiment.

Since all the lactic streptococci were not tested
against all the insecticides which can possibly occur in
milk, no absolute conclusion can be made concerning the
effect of insecticide residues on lactic cultures. How—
ever, the results of this work prove that concentrations
of insecticides far in excess of the amount normally
occurring in milk will not interfere with normal growth
and lactic fermentation ability of streptococci used in
the manufacture of cultured dairy products.

In the current edition of Bergey's Manual of
Determinative Bacteriology by Breed §S_§S. (1957) S.
diacetilactis is not listed as a separate species and
some people designate it as S. lactis var. diacetilactis.
However, research in recent years has established S.
diacetilactis as a separate species. According to Foster
EE_§l- (1957) and Hammer and Babel (1956), S. diaceti—
lactis is similar in most characteristics to S. lactis
and differs from S. lactis in its ability to ferment
citrate. Also S. diacetilactis uses more oxygen and pro—
duces much more CO2 than S. lactis, and possesses a
vigorous diacetyl reductase enzyme which is absent in

S. lactis.

 

 

 

 

54

The results in Figures 2, 3, and A prove that up to
100 ppm of heptachlor, methoxychlor and malathion had
neither an adverse nor a stimulative effect on the growth
and lactic fermentation ability of S. 22521 in milk at
32 C. Dieldrin did not influence the lactic fermenta—
tion ability of S. EEEEE but resulted in slight reduc—
tion in Viable cell count (about one half log cycle) in
the milks containing 10 and 100 ppm, as shown in Figure
1—2. Since the reduction of the viable cell count did
not occur in the milks containing 0.1 and 1 ppm of
dieldrin, and the concentrations between 1 and 10 ppm
were not tested, it is not known exactly what is the
lowest limiting concentration of dieldrin to cause re—
duction in viable cell count of S. ga§§i_in milk. How-
ever, the reduction in growth of S. EEEEE at both 10 and
100 ppm was very slight and does not seem significant
from a practical standpoint. S. gggg; is seldom used in
lactic starter cultures but does have an important role
in the ripening of Cheddar and some other varieties of
cheese.

In general, lactic starter cultures are mixed
strains of S. lactis and/or S. cremoris plus one strain
of Leuconostoc to ferment citric acid. Some Lactobacilli
are used as lactic starter cultures only for special
occasions. Lactobacillus acidophilus is used to make

acidophilus milk. Lactobacillus bulgaricus is used for

 

 

.7.
41‘

 

 

55

the manufacture of Bulgarian milk, and is also used in
combination with S. thermophilus for the manufacture of
yoghourt and Swiss cheese. Only the most commonly used
lactic starter cultures were included in this study and
it is not known how insecticides would affect the growth
and lactic fermentation of S. acidophilus and S.
bulgaricus, but presumably their response would be simi—
lar to that of S. gaSgS.

In contrast to the results of this study, Bradley
and Li (1968) reported that the presence of dieldrin in
milk reduced acid production by lactic cultures in the
manufacture of Cheddar cheese. According to their data,
the reduction of titratable acidity due to the presence
of dieldrin was 0.01—0.02 per cent lactic acid at the
stage of draining. However, prior to inoculation, the
titratable acidity of the milk containing dieldrin was
lower than that of the control milk by 0.01—0.05 per cent
lactic acid. In this case, the initially lower acidity
in milk containing dieldrin seems to account for the
lower acidity of the same milk after inoculation and
incubation, compared with the acidity of the control milk.

This study was restricted to the effect of some
insecticides on the growth and lactic fermentation ability
of culture organisms commonly used in dairy products.

Thus it cannot be predicted, from the results of this

 

 

56

work, how lactic cultures would react to the presence of
insecticides in substrates other than milk.
Collins and Langlois (1968) found that heptachlor

and DDT showed no effect on Staphylococcus aureus in skim

 

milk, although heptachlor was bactericidal and DDT was
inhibitory to S. aureus in Trypticase Soy Broth (TSB)

at 100 ppm. Pseudomonas fluorescens was inhibited by 100

 

ppm of DDT in TSB but not in skim milk. Dieldrin had no
effect on the growth of S. aureus and S. fluorescens
either in TSB or in skim milk. Speculation based on the
observation by Collins and Langlois (1968) suggests that
lactic starter cultures would not be affected by insecti—
cides in milk, although they might be adversely affected
by some insecticides in certain broth media. Furthermore,
100 ppm of heptachlor or DDT in milk is an unrealistic
concentration.

There are no other reports with which the results
of this study can be compared. The soil bacteria were
not affected by various chlorinated hydrocarbon insecti-
cides as discussed in the Literature Review. The observa—
tion by Collins and Langlois (1968) and the results of
this study indicate that insecticides certainly do not
have a strong or a wide spectrum of antibacterial
activity.

Because of the widespread use, various insecticides

have persistently appeared in nearly all the milk, and it

 

U.

' ~<
‘L J

 

57

is extremely difficult to obtain milk from any species
which is completely free of all insecticides. The
actionable amount of DDT, DDE, DDD, lindane, BHC and
methoxychlor in milk is 0.05 ppm in milk or 1.25 ppm in
milk fat; and for aldrin, dieldrin, heptachlor, hepta—
chlor epoxide and endrin the actionable amount is 0.01
ppm in milk or 0.25 ppm in milk fat. When no test
insecticide was added to milk used in this investigation
the milk was designated as the control, containing less

than 0.05 ppm of the test insecticide under study and

 

confirmed by chromatographic analysis.

It is unlikely that any insecticide will occur in
milk in concentrations much above the actionable level.
One hundred parts per million of dieldrin or heptachlor
in milk is 10,000 times higher than the actionable level
recognized by the FDA and 100 ppm of methoxychlor in
milk is 5,000 times higher than the actionable level.
Therefore, the effect of concentrations above 100 ppm of
insecticide was not routinely tested in this study.

Malathion was included in this study since it is
the most widely used organophosphate insecticide. There
is no actionable level for organophosphate insecticides
in milk. This is because organophosphate insecticides
seldom survive the biochemical reactions of the mammalian

system to appear in milk. Consequently the organophosphate

 

 

 

58

insecticides are of little concern in milk, except when
absorbed into milk from the environment.

When the susceptibility of bacteria to a chemical
agent is tested, if the initial population is very high,
the bacteria may overcome the effect of the chemical
agent. In this experiment, 0.1% of culture was inoculated
into milk samples although frequently 0.5% to 1% of lactic
starter culture is inoculated into milk for the produc—
tion of fermented milk products.

In this experiment, the samples inoculated with
S. cremoris were incubated at 25 C and the samples inocu—
lated with S. lactis, S. diacetilactis or S. 9222; were
incubated at 32 C. The incubation temperature of 32 C
and 25 C were chosen for convenience within the tempera—
ture range which allows active growth of the cultures.

In commercial practice milks inoculated with lactic cul—
tures are incubated at temperatures ranging from 21 to
U5 0, depending on the organism used and product de-
sired. The optimum growth temperature for S. lactis,

S. diacetilactis and S. EEEEE is about 30 C but tempera—

tures of 20 to 25 C are more favorable to Leuconostoc

 

dextranicum and Leuconostoc citrovorum which are used
along with the lactic starter cultures.

In one group of experiments methoxychlor was dis-
solved in butteroil, which was then mixed with non-fat

dry milk solids and water, reconstituted into milk and

 

 

.2-
..Su-

 

 

59

used as a growth medium. When this milk was inoculated
with S. lactis A254 and S. gaggi there was no effect by
methoxychlor on the growth and lactic acid producing
ability of the cultures. When the same experiments were
performed in cereal milk (half and half), similar results
were obtained even at concentrations up to 1000 ppm of
methoxychlor in cereal milk.

Chlorinated hydrocarbon insecticides are chemically
and physically most stable and persistent among the
pesticides. Presently, only slight modification of a
few chlorinated hydrocarbon insecticides is known to
occur after they are sprayed in commercial application.
In spite of the continuous effort, there is little pro-
gress in finding a way to remove or destroy the unwanted
residual insecticides which pollute human food and
environment.

As discussed in the Literature Review, aldrin, DDT,
heptachlor, and lindane are the few chlorinated hydro—
carbon insecticides which are known to be susceptible to
the metabolism of some living organisms. Metabolism of
aldrin to dieldrin, and heptachlor to heptachlor epoxide
is a toxicative process. Modification of DDT and lindane
to their analogs is a detoxication process.

The second part of this experiment was to determine
if lactic starter cultures would be able to modify

aldrin, DDT or lindane in milk and alter the toxicity of

 

 

60

the insecticides. When five lactic streptococci and
S. EEEEE were allowed to grow in the milks containing
1 ppm of aldrin, DDT or lindane for two weeks, none of
the organisms modified or degraded aldrin, DDT or lindane
This proves that commonly used lactic starter cultures
neither help detoxify DDT or lindane in milk, nor en—
hance the toxicity of aldrin.

Stenerson (1965) observed the conversion of DDT to

DDD by Escherichia coli in meat extract bouillon. Mendel

 

and Walton (1966) and Langlois (1967) demonstrated the
ability of S. 99;; to dechlorinate DDT to DDD in TSB
but Langlois (1967) found that when S. Eflli was inocu-
lated into skim milk containing DDT there was little
conversion of DDT to DDD. According to the observation
by Langlois (1967) milk seems to protect an insecticide
from the action of bacteria, even when an organism has
the potential to react on the insecticide. Studies on
the modification of insecticides by microorganisms have
been mainly devoted to DDT in synthetic broth media
rather than in food substrate. The lactic starter cul-
tures are Gram—positive, whereas most of the bacteria
which have been reported to alter DDT are Gram-negative
organisms such as S. coli, Aerobacter aerogenes, Proteus

vulgaris, Serratia marcescens, and some species of

 

 

Pseudomonas, Erwinia, Achromobacter and Xanthomonas.

 

 

 

 

Wedemeyer (1966) and Johnson (1969) suggested that the

 

 

 

 

61

cytochrome system in microorganisms is directly responsible
for the reductive dechlorination of DDT to DDD. Lactic
streptococci and lactobacilli do not have cytochrome sys—
tems.

The results of this research, in combination with
observations of other investigators, lead to the conclu—
sion that modification or degradation of insecticides in
milk by lactic starter cultures is not likely to occur.
Subsequently, the effect of the fermentation process on
insecticides in milk should be of little concern in assess-
ing a reasonable ”tolerance level” for insecticides in
milk and in the evaluation of acceptable daily intake.

With the analytical techniques used in this study,
0.01 ug/ml of the insecticides and related analogs could
be detected.

Since the growth of S. ggggl was slightly inhibited
by 10 and 100 ppm of dieldrin in milk, uptake of dieldrin
by S. BEBE; was studied using 1uC—dieldrin. Studying the
uptake of lLlC—dieldrin by S. EEEEE in milk rather than in
TSB might have given better correlation between the re—
sults of this part of the work and the results of the
study on the effect of dieldrin on S. ggggl. However, no
practical method was available to harvest S. SEES; cells
from coagulated milk samples. When S. Eéiél was grown in

TSB containing 0.01 to 0.02 ppm of luC-dieldrin for 18

hr the amount of lLlC—dieldrin retained with the harvested

 

 

 

i'1~

 

62

and washed cells was less than 0.2% of the total luc—

dieldrin in the TSB. In handling radioactive material,
several washings do not accomplish complete removal of
residual radioactivity. Since less than 0.2% of such a
low concentration as 0.01—0.02 ppm of dieldrin was re—
tained with the harvested cells, the count of the radio—
activity obtained from the cells could be attributed to
the residual radioactivity due to incomplete washing
rather than to true uptake of luC—dieldrin into the cells.
The population of the cells was about 108 cells/ml.

It is not known why chlorinated hydrocarbon insecti—
cides have little effect on lactic starter cultures and
why they were stable against the activity of lactic cul—
tures. From the known information, however, some specu—
lation can be made. Although the mechanism of the toxicity
by chlorinated hydrocarbon insecticides is not elucidated
yet, these chemicals are known to be nerve poisons. Bac—
teria are not known to possess a nerve system and there
is no experimental evidence to prove that the insecti—
cides have antibacterial activity.

Kostenbauder (1968) explained that (a) the degree of
antimicrobial activity of a chemical agent can often be
correlated with the rate at which the chemical agent gains
access to the site at which it acts and (b) the distribu—
tion of antimicrobial agents between aqueous and non—

aqueous phases is an important factor.

63

The hydrOphobic nature of chlorinated hydrocarbon
insecticides and their negligible solubility in water
limit their availability in the aqueous phase of milk.
Wettability of bacteria indicates that bacterial cell
walls are more hydrophilic than hydrophobic. Actually,
when the TSB cultures of S. lactis A62 and S. 22221 were
skaken with hexane and the layers allowed to separate, no
organisms were observed in the hexane layer when subjected
to microscopic examination. Salton (1964) mentioned in
his discussion of ”solubility properties" of the bacterial

cell wall, that walls of many Gram-positive bacteria are

 

insoluble in many organic solvents including alcohols,
ethers and acetone. Salton (196“) also cited that the

isolated walls of Streptococcus faecalis and Corynebac-

 

terium diphtheriae were insoluble in 90% (w/w) phenol,

 

while the isolated cell wall of S. 32;; was soluble.

All the lactic starter cultures are Gram—positive
and there seems to be little chance for such cultures to
stay in the lipid phase in which the insecticides are
present.

With high concentrations of insecticides in milk
there could be some contact between lactic cultures and
insecticides. However, several points should be con-
sidered: (a) the cell wall of Gram—positive bacteria
contains about 2% lipid compared with about 20% lipid in

the cell wall of Gram—negative bacteria (Luria, 1960),

6A

(b) the nature of the cell wall of Gram—positive bacteria
as discussed above and (c) the hydrophobic nature of
chlorinated hydrocarbon insecticides. In view of these
facts an assumption could be drawn that the insecticides

would have little access into the cells of lactic starter

cultures.

 

 

 

SUMMARY AND CONCLUSIONS

The purpose of the first part of this research was
to study the effect of some insecticides on the growth
and fermentation ability of the lactic starter cultures.
Streptococcus lactis A25“, S. lactis A62, Streptococcus

cremoris E—8, Streptococcus diacetilactis 18—16 and

 

Lactobacillus casei were grown for “8 hr in sterile

 

 

whole milks containing 0, 0.1, 1, 10 and 100 ppm of
dieldrin, and heptachlor. S. lactis A25H and S. gggg;
were also grown for A8 hr in sterile whole milk contain-
ing 0, 0.1, l, 10 and 100 ppm of methoxychlor and mala—
thion. Observation of viable cell count, pH and per cent
lactic acid in the milks at 2A and A8 hr incubation indi—
cated that dieldrin, heptachlor, methoxychlor and mala-
thion had no effect on the growth and lactic fermentation
ability of the streptococci tested.

Since all the lactic streptococci were not tested
against all the insecticides which can possibly occur in
milk, no absolute conclusion can be made concerning the
effect of insecticide residues on lactic cultures. How—
ever, the results of this work prove that concentrations
of insecticides far in excess of the amount normally

occurring in milk will not interfere with normal growth

65

66

and lactic fermentation ability of streptococci commonly
used in the manufacture of cultured dairy products.

Heptachlor, methoxychlor and malathion had no in—
fluence on the growth and lactic acid producing ability
of S. EEEEE- Dieldrin did not affect the lactic ferment—
ing ability of S. SEES; but caused slight reduction in
viable cell count of S. EEEEE in the milks containing 10
and 100 ppm. However, the reduction in growth of S.
ggggl was very slight and does not seem significant from
a practical standpoint. S. SEES; is seldom used in lactic
starter cultures but does have an important role in the
ripening of Cheddar and other varieties of cheese.

The second part of this research was to determine
if lactic starter cultures would be able to modify
aldrin, DDT or lindane in milk and alter the toxicity of
the insecticides. When S. lactis A62, S. lactis A254,

S. cremoris E—8, S. diacetilactis 18—16, S. diacetilactis
DRCI, and S. SEES; were allowed to grow in the milks
containing 1 ppm of aldrin, DDT or lindane for two weeks,
none of the organisms modified or degraded aldrin, DDT

or lindane. This proves that commonly used lactic
starter cultures neither help detoxify DDT or lindane in
milk, nor enhance the toxicity of aldrin. Degradation

of DDT or lindane is a detoxication process and modifica—
tion of aldrin and heptachlor to their epoxides is a

toxicative process. Among chlorinated hydrocarbon

 

 

67

insecticides, aldrin, DDT, lindane and heptachlor are the
few which are known to be susceptible to the metabolism
of some living organisms. Thus the results of this re-
search lead to the conclusion that modification or de-
gradation of insecticides in milk by lactic starter cul-
tures are not likely to occur. Consequently the effect
of the fermentation process on insecticides in milk should
be of little concern in assessing a reasonable tolerance
level for insecticides in milk and in the evaluation of
acceptable daily intake.

In order to study the possible uptake of dieldrin
by S. Eéiél the organism was grown for 18 hr in Trypticase
Soy Broth containing 0.01—0.02 ppm of luC—dieldrin. The
harvested and washed cells retained less than 0.2% of
the total luC—dieldrin in the TSB. Such a low level of
radioactivity counted from the harvested cells could be
attributed to the residual radioactivity due to incomplete

1A

washing rather than to true uptake of C-dieldrin into

the cells.

 

   

LITERATURE C ITED

 

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.i‘i: I":
I."
I‘.
A
. i '
U: .

 

 

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APPENDIX

 

 

CHEMICAL NOMENCLATURE OF THE INSECTICIDES

Aldrin

BHC

DDA
DDA (TDE)
DDE

DDT

Dieldrin

Endrin

Heptachlor

Heptachlor
epoxide

Lindane

l, 2, 3, 4, lO—hexachloro—l,4, 4a, 5, 8,
8a—hexahydro-l, 4—gSSg—ggg—5, 8—
dimethanonaphthalene.

l, 2, 3, 4, 5, 6—hexachlorocyclohexane
consisting of several isomers and con—
taining a specified percentage of gamma
isomer.

2,2—bis(pfchlorophenyl) acetic acid.

1, l-dichloro—2, 2—bis(p—chlorophenyl)ethane
l, l—dichloro—2, 2—bis(R—chlorophenyl)ethane
l, l, lO—trichloro—2,2-bis(p—chlorophenyl)
ethane

l, 2, 3, 4, 10, l——hexachloro—6, 7—epoxy—l,
4, 4a, 5, 6, 7, 8, 8a—octahydro-l, 4-endo—
exo—5, 8—dimethanonaphthalene.

l, 2, 3, 4, 10, lO—hexachloro—6, 7—epoxy—1,
4, 4a, 5, 6, 7, 8, 8a—octahydro—l, 4—endo—
endo-5, 8—dimethanonaphthalene.

l, 4, 5, 6, 7, 8, 8—heptachloro—3a, 4, 7,
7a, tetrahydro—4, 7—methanoindene.
2,3—eopxyul, 4, 5, 6, 7, 8, 8—heptachloro—
hexahydro—4, 7—endomethanoindene.

ggmmg isomer of l, 2, 3, 4, 5, 6—hexachloro—
cyclohexane.

78

 

 

79

Malathion S—[l, 2—bix(ethoxycarbony1)ethyl] S,
S—dimethyl phosphorodithioate.
Methoxychlor l, l, l—trichloro-2, 2-bis(pfmethoxyphenyl)

ethane.

 

 

80

TABLE 2.——Viable cell count per m1, pH, and per cent
lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when
the milk was inoculated with Streptococcus
lactis A254 and incubated at 32 C.

 

ppm of dieldrin added to milk

 

Incubation

time (hr) 0 0.1 l 10 100

 

viable cell count per ml of milk

 

 

 

 

 

 

0 71 x 10Ll 53 x 10“ 51 x 10“ 64 x 10” 71 x 10“
24 63 x 107 50 x 107 58 x 107 56 x 107 48 x 107
48 57 x 107 54 x 107 58 x 107 71 x 107 59 x 107

pH of milk

0 6.42 6.42 6.42 6.42 6.44
24 5.22 5.22 5.22 5.22 5.22
48 5.02 5.03 5.04 5.04 5.02

% lactic acid in milk

0 0.14 0.14 0.14 0.14 0.14
24 0.56 0.57 0.57 0.57 0.58
48 0.69 0.70 0.69 0.68 0.70

 

 

 
 

81

TABLE 3.--Viable cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when
the milk was inoculated with Lactobacillus
casei and incubated at 32 C.

 

 

ppm of dieldrin added to milk

 

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 18 x 10“ 18 x 10“ 82 x 103 13 x 10“ 14 x 10”
24 12 x 107 13 x 107 10 x 107 31 x 106 37 x 106
48 27 x 107 24 x 107 20 x 107 20 x 107 13 x 107
pH of milk
0 6.48 6.49 6.49 6.49 6.48
24 6.12 6.13 6.12 6.14 6.13
48 5.51 5.50 5.50 5.53 5.53
% lactic acid in milk
0 0.14 0.15 0.15 0.15 0.15
24 0.26 0.26 0.26 0.27 0.27

48 0.44 0.45 0.44 0.43 0.43

 

 

82

TABLE 4.——Viable cell count per m1, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated,

when the milk was inoculated with Streptococcus
lactis A254 and incubated at 32 C.

 

Incubation

ppm of heptachlor added to milk

 

 

 

 

 

 

 

 

time (hr) 0 0.1 1 10 100
viable cell count per m1 of milk
0 73 x 10Ll 69 x 10L1 62 x 10L4 65 x 10“ 70 x 104
24 42 x 107 34 x 107 41 x 107 39 x 107 40 x 107
48 48 x 107 47 x 107 63 x 107 61 x 107 65 x 107
pH of milk
0 6.45 6.45 6.45 6.54 6.45
24 5.21 5.21 5.22 5.22 5.22
48 5.05 5.05 5.06 5.07 5.06
% lactic acid in milk

0 0.15 0.15 0.15 0.15 0.15
24 0.61 0.62 0.61 0.61 0.62
48 0.67 0.67 0.67 0.66 0.68

 

 

83

TABLE 5.—-Viable cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of heptachlor as indicated,
when the milk was inoculated with Lactobacillus
casei and incubated at 32 C.

 

ppm of heptachlor added to milk

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 39 x 10” 38 x 104 39 x 104 31 x 10“ 38 x 10”
24 11 x 107 11 x 107 92 x 106 96 x 106 11 x 107
48 35 x 107 34 x 107 38 x 107 37 x 107 40 x 107
pH of milk
0 6.46 6.47 6.45 6.46 6.42
24 6.14 6.14 6.13 6.13 6.10
48 5.42 5.42 5.42 5.42 5.40
% lactic acid in milk
0 0.14 0.14 0.14 0.15 0.16
24 0.21 0.22 0.20 0.20 0.21

48 0.46 0.46 0.46 0.45 0.46

 

 

 

84

TABLE 6.——Viable cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of methoxychlor as indicated,
when the milk was inoculated with Lactobacillus
lactis A254 and incubated at 32 C.

 

ppm of methoxychlor added to milk

 

Incubation

 

 

 

 

 

 

 

time (hr) 0 0.1 l 10 100
Viable cell count per ml of milk
0 28 X 10” 37 X 104 29 X 10“ 39 X 10“ 44 X 10“
24 40 x 107 40 x 107 43 x 107 39 x 107 35 x 107
48 62 x 107 55 x 107 49 x 107 63 x 107 65 x 107
pH of milk
0 6.48 6.48 6.48 6.48 6.48
24 5.02 5.02 5.02 5.02 5.02
48 4.78 4.78 4.78 4.78 4.78
% lactic acid in milk

0 0.14 0.14 0.14 0.14 0.14
24 0.58 0.58 0.59 0.58 0.57
48 0.64 0.65 0.65 0.64 0.64

 

 

 

85

TABLE 7.——Viab1e cell count per m1, pH, and per cent
lactic acid of whole milk containing various
concentrations of methoxychlor as indicated,
when the milk was inoculated with Lactobacillus
casei and incubated at 32 C.

 

 

ppm of methoxychlor added to milk-

 

Incubation 0

time (hr) 0.1 1 10 100

 

viable cell count per m1 of milk

 

 

 

 

0 24 x 10Ll 25 x 10” 23 x 10” 27 x 101‘l 27 x 10L1
24 16 x 107 18 x 107 12 x 107 16 x 107 16 x 107
48 68 x 107 80 x 107 68 x 107 74 x 107 78 x 107

pH of milk

0 6.45 6.45 6.45 6.45 6.45
24 5.92 5.92 5.92 5.92 5.92
48 5.06 5.02 5.01 5.01 5.05

 

% lactic acid in milk

 

0 0.13 0.13 0.13 0.13 0.13
24 0.21 0.22 0.22 0.22 0.21

48 0.49 0.50 0.51 0.51 0.49

 

86

TABLE 8.——Viab1e cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of malathion as indicated,
when the milk was inoculated with Streptococcus
lactis A254 and incubated at 32 C.

 

ppm of malathion added to milk

 

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 24 x lO11L 24 x 10” 24 x 10“ 24 x 104 24 x 10“
24 16 x 107 17 x 107 16 x 107 17 x 107 15 x 107
48 12 x 107 12 x 107 12 x 107 14 x 107 14 x 107
pH of milk
0 6.4 6.4 6.4 6 4 6.4
24 5.0 4.98 4.98 4.98 4.98
48 4.75 4.75 4.78 4.73 4.75
% lactic acid in milk
0 0.16 0.16 0.16 0.16 0.16
24 0.58 0.58 0.59 0.58 0.58
48 0.66 0.66 0.65 0.66 0.66

 

 

87

TABLE 9.——Viable cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of malathion as indicated,
when the milk was inoculated with Lactobacillus
casei and incubated at 32 C.

 

ppm of malathion added to milk

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 24 x 10” 24 x 10“ 24 x 10“ 24 x 10“ 24 x 10“
24 22 x 107 24 x 107 21 x 107 21 x 107 19 x 107
48 26 x 107 34 x 107 38 x 107 28 x 107 24 x 107
pH of milk
0 65 65 6.5 65 65
24 61 61 6.1 61 61
48 53 53 5.3 53 53
% lactic acid in milk
0 0.14 0.14 0.14 0.14 0.14
24 0.19 0.20 0.20 0.18 0.20

48 0.40 0.42 0.40 0.41 0.41

 

 

88

TABLE 10.——Viable cell count per m1, pH, and per cent
lactic acid of whole milk containing various
concentrations of dieldrin as indicated,
when the milk was inoculated with Streptococcus
lactis A62 and incubated at 32 C.

 

ppm of dieldrin added to milk

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 86 x 1011l 98 x 10“ 96 x 104 87 x 10Ll 88 x 10Ll
24 57 x 107 50 x 107 55 x 107 54 x 107 54 x 107
48 55 x 107 61 x 107 61 x 107 64 x 107 47 x 107
pH of milk
0 6.43 6.43 6.43 6.43 6.43
24 5.27 5.25 5.25 5.27 5.26
48 5.01 5.01 5.00 5.00 5.00
% lactic acid in milk
0 0.14 0.14 0.14 0.14 0.15
24 0.55 0.55 0.56 0.56 0.55

48 0.63 0.64 0.65 0.64 0.64

 

89

TABLE ll.-—Viable cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when
the milk was inoculated with Streptococcus
diacetilactis 18—16 and incubated at 32 C.

 

ppm of dieldrin added to milk

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 84 x 104 82 x 104 79 x 104 85 x 104 72 x 10“
24 51 x 107 55 x 107 48 x 107 57 x 107 57 x 107
48 36 x 107 45 x 107 37 x 107 33 x 107 46 x 107
pH of milk
0 6.43 6.44 6.43 6.43 6.44
24 5.44 5.43 5.43 5.44 5.44
48 5.28 5.28 5.28 5.28 5.28
% lactic acid in milk
0 0.16 0.16 0.16 0.16 0.16
24 0.50 0.50 0.52 0.51 0.52

48 0.56 0.57 0.56 0.56 0.56

 

 

 

. ..——— m—vm- ~

 

90

TABLE l2.——Viab1e cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated, when
the milk was inoculated with Streptococcus

lactis A62 and incubated at 32 C.

 

ppm of heptachlor added to milk

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 10 x 105 10 x 105 10 x 105 11 x 105 11 x 105
24 61 x 107 72 x 107 70 x 107 71 x 107 60 x 107 --
48 65 x 107 63 x 107 61 x 107 59 x 107 64 x 107
pH of milk
0 6.42 6.42 6.42 6.42 6.42
24 4.80 4.80 4.80 4.80 4.80
48 4.64 4.60 4.63 4.65 4.65
% of lactic acid in milk
0 0.14 0.14 0.15 0.15 0.15
24 0.56 0.56 0.55 0.55 0.56
48 0.58 0.59 0.59 0.58 0.59

 

 

 

91

TABLE l3.——Viable cell count per ml, pH, and per cent
lactic acid of whole milk containing various
concentrations of heptachlor as indicated,
when the milk was inoculated with Streptococcus
diacetilactis 18-16 and incubated at 32 C.

 

 

 

 

 

 

 

 

 

 

 

ppm of heptachlor added to milk 1
Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 95 x 10Ll 89 x 10” 93 x 10” 10 x 105 84 x 10Ll
24 65 x 107 77 x 107 60 x 107 72 x 107 70 x 107
48 69 x 107 57 x 107 56 x 107 77 x 107 78 x 107
pH of milk
0 6.43 6.43 6.42 6.43 6.43
24 5.47 5.47 5.47 5.45 5.46
48 5.32 5.32 5.32 5.31 5.31
% lactic acid in milk
0 0.14 0.14 0.14 0.13 0.14
24 0.49 0.49 0.49 0.50 0.49

48 0.55 0.56 0.54 0.54 0.55

 

 

 

 

 

92

TABLE l4.——Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of dieldrin as indicated, when
the milk was inoculated with Streptococcus
cremoris E—8 and incubated at 25 C.

 

Incubation

ppm of dieldrin added to milk

 

 

 

 

 

 

 

time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 51 x 10“ 50 x 101l 48 x 10Ll 57 x 107 56 x 10“
24 96 x 107 88 x 107 96 x 107 95 x 107 93 x 107
48 15 x 107 12 x 107 14 x 107 16 x 107 13 x 107
pH of milk
0 6.44 6.43 6.43 6.42 6.42
24 4.99 4.97 4.98 4.99 4.99
48 4.99 4.98 4.99 4.99 4.99
% lactic acid in milk
0 0.15 0.14 0.15 0.14 0.14
24 0.66 0.66 0.66 0.66 0.64
48 0.66 0.67 0.67 0.66 0.66

 

 

93

TABLE 15.——Viable cell count per ml, pH, and per cent

lactic acid of whole milk containing various
concentrations of heptachlor as indicated,
when the milk was inoculated with Strepto—
coccus cremoris E—8 and incubated at 25 C.

 

ppm of heptachlor added to milk

 

 

 

 

 

 

 

Incubation
time (hr) 0 0.1 1 10 100
viable cell count per ml of milk
0 90 X 10Ll 92 X 10}4 85 X 10“ 92 X 10“ 86 X 10Ll
24 81 x 107 91 x 107 82 x 107 78 x 107 87 x 107
48 61 x 107 48 x 107 44 x 107 68 x 107 62 x 107
pH of milk
0 6.42 6.43 6.43 6.41 6.41
24 4.83 4.82 4.83 4.84 4.83
48 4.45 4.45 4.45 4.46 4.46
% lactic acid in milk

0 0.13 0.14 0.14 0.13 0.13
24 0.68 0.69 0.68 0.69 0.68
48 0.72 0.72 0.72 0.72 0.71

 

 

 

 

 

 

 

“1
Nl
All
ml
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