J

"
ll

1 3

III

5

WI

”1|er

WW“:

'

H

[H
;
II

J
I

am;
1034:.
CD\IU'|

 

i“ t
1

F‘,

uu-a :onzn‘ \l ' t! .- “‘22.
‘ » ”P v (at? £0" C.J ‘.!;:f‘?“ 0

&;:\‘.5:\L

I: J 0‘ -"\~“ f"¢ 81 W (“t I. L H r' {‘3- 'F‘ j
v- ' -‘ 0 Uh- 0*n-v-ox -.v c. r. ‘
x. :‘xh an... arm} L. a; t... I \..’\.u'

)yr paw?» L's-v” 2.1"": 3. 3992193: 99:99-33
’W Vodoas‘; W...—o" ch‘u-UI- vil1laa; ‘ . Lu

lslp p 3:47: f‘jtfifi‘“
.t ;‘\" I MWJII‘H‘."

3:5 for h! a D9339 cf 5%. S.

'4“ ‘ "2 “"1 f“ “'35":
L‘fiJHfiLGALJ :13th- s: hmav‘é.

, + -~. :~_.- - .
t".."<‘.,"*¢f I. 32"- ' 9‘}
fib‘hC’é-‘L (‘10 L3 . ‘lauLz-l'

-?
‘ ‘raj

(127:
GP.”

 

SIS

 

.- ___ ——_

This is to certify that the

thesis entitled
The inhibitory action of various organic acids on

some food-poisoning organisms when tested in soft

cus tard .
presented by

Joseph Alfred Stevens

has been accepted towards fulfillment
of the requirements for

Master's degree in Bacteriology

 

Majoi professor

Date MW

0-169

 

 

‘—

THE INHIBITCHY ACTIJN OF VARIOUS OR&&IIC
ACIDS Oh SOLE FOCD-POISOEIEG ORGANISMS

Iii-iii.” TASTED LI SOFT CUSTARD

By

Joseph A. Stevens

(O-

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTEd OE SCIENCE

Department of Bacteriology and Public Health
1953

\\
\
1.4"

F

"‘x, 1.3 3

AC KN OWLEDG EMEN T

The writer wishes to express his sincere
appreciation to Dr. H. L. Mallmann for his advice
and guidance throughout this work. He wishes also

to thank Dr. Anita Leavitt for her cooperation with

this work.

‘fj—eu .

V' kid] I

INTHOUVCTION . . . . . .
HISTORY . . . . . . . . .
EXPERIMENTAL . . . . . .
RESULTS AhD DISCUSSION ,
SUIVTMARY . . . . . . . . .

BIBLIOGRAPHY . . . . . .

LIST OF TABLES

Table Page
1. Growth of organisms in peptone broth containing

various concentrations of organic acids . . . 1h

2. Control tests on basic substrate . . . . . . . l7

3. Growth of Micrococcus pyogenes, variety aureus
in custard containing graded amounts of organ-
ic acids 0 O O O O O O O 0 O O O O O O O O O 18

 

h. Growth of Salmonella typhimurium in custard
containing graded amounts of organic acids . 2O

 

5. Growth of Salmonella schottmuelleri in custard
containing graded amounts of organic acids . 22

 

6. Growth of food poisoning organisms in custard
containing graded amounts of DHA-S . . . . . 2h

7. Palatability of acid-treated soft custard . . . 25

INTRODUCTION

Aside from the intoxication known as botulism, the
majority of cases of food poisoning that come to the attention
of the public is caused by the enterotoxic substance produced
by micrococci, or is infection by certain members of the
salmonella group. Of these cases, the foods which we have
most frequently observed containing the causative agent are
pies and pastry, eSpecially those containing custard (16).

The basic custard formula is used to produce such desserts
as puddings and pies, and is used as a filling for cream
puffs and 5c1airs.

The causative organisms can enter the custard from a
number of sources: the milk (16, 23, 31), eggs (9), or
sugar used to make the custard, the water supply (16) used
to wash the utensils, the persons involved in its prepar-
ation (2A), or the air.

Those strains of Micrococcus pyogenes, var. aureus and
glbgg which produce the enterotoxin that causes food poisoning
symptoms may be found in abundance in nature. In man they
are carried in the throat (2h), and are frequently found in
the postnasal discharge following common colds, and can be
isolated from pimples, carbuncles, boils, and other infections.

The percentage of the total number of strains of micrococci

which are capable of producing enterotoxin is not known because

of testing difficulties. However, the number of strains of
micrococci which produce enterotoxin is small as compared
to the total number (12).

The symptoms of micrococcal food poisoning usually appear
about three hours after the individual has partaken of the
food containing the enterotoxin. The first symptom to appear
is salivation, followed by nausea, vomiting, retching, abdom-
inal cramping and diarrhea. All of these symptoms need not
appear in order to confirm the presence of enterotoxin. Mi-
crococcal food poisoning in otherwise normal individuals is
usually not fatal, although a few fatal cases have occurred in
which inadequate treatment was given.

Many different types of fbods have been involved in micro-
coccal—type food poisoning. Kelly and Dack (23) mention out-
breaks owing to contamination of cheese, cream-filled cake,
chicken gravy, cream-filled‘eclairs (7) and tarts, custard-
filled’doughnuts, chocolate eclairs, milk (8), and tongue
sandwiches. Ice cream (2h), pineapple pudding (1h), and bread
pudding (28), also have been incriminated.

In upstate New Ybrk from 1935 to 1939, l? outbreaks (that
gave rise to 12h6 caseS) (7) of micrococcal food poisoning were
reported that were due to contaminated cream-filled bakery goods.

The Species of Salmonella most often responsible for pro-

 

ducing food poisoning infections in man are §._paratyphi, §,

schottmuelleri, S. typhimurium, §. choleraesuis, and §.

enteritidis.

 

Although contaminated meat is most often incriminated

(ll), many Salmonella infection outbreaks have been due to

 

foods other than meat (2, 28, 33, 3h). Milk and milk
products may become contaminated from a diseased cow or
subsequently upon handling. The pasteurization process is
effective in destroying salmonella organisms in such media.

Rodents (36), houseflies (26), and human carriers are
the vectors by which salmonella organisms may gain entrance
to foods such as custards.

The symptoms of Salmonella gastrointestinal infections
are characterized by nausea, vomiting, abdominal pain and
diarrhea. The symptoms usually appear 12 to 2k hours after
the food is ingested.

At the present writing, the evidence points to the
view that Salmonella food poisoning is actually an infection

and that toxins or toxic substances play no role (10).

”—7...-L

 

 

HISTORY

This writer's interest in the problem of diminishing
the number of possibilities of obtaining food poisoning or
food infections by ingesting contaminated soft custard pas-
tries was stimulated by a suggestion from Dr. W. L. Mallmann.
This suggestion was based on a bakery's claim in an advertise-
ment in a Chicago newspaper that it had perfected a sugar
cream filling for pies which inhibited the growth of all food
poisoning or food infection type bacteria. The filling had

the following formula:

10 lbs. water

5 lbs. granulated sugar

3 oz. pure vanilla

1 oz. salt

1 lb. ’ cereal starch

h lbs. h oz. whipped, fresh-frozen egg whites

It was decided to prepare this pie filling, sterilize it, and
inoculate it with organisms of the type most frequently found
(15, 2l) in food poisoning or food infection cases. Therefore,
one micrococcus and two salmonella species were used.

On hand in our laboratories was a known enterotoxin pro-
ducing strain (Brail) of Micrococcus pyogenes, var. aureus
which was used as the food poisoning type test organism. Cul-
tures of Salmonella typhimurium and Salmonella schottmuelleri

were used as the food infecting type test organisms.

None of the constituents of the cream filling formula
was ever known to be of an inhibitory nature to microorgan-
isms. Furthermore, the presence of the egg whites would
supply the nitrogen requirements which Surgalla and Hits (35)
found to be necessary for enterotoxin production by micrococci.

After 2h hours incubation at 37 C, 5 ml of this cream
filling, inoculated with E. pyogenes, var. aureus, str. Brail,
was swallowed rather hesitantly by this writer. Within 100
minutes, the bakery's advertisement was diSproved. A head-
ache, followed by a violent case of vomiting, and a severe
case of diarrhea of six hours duration, firmly convinced
this writer that the cream filling did support bacterial growth.

Since this type of pie filling did not inhibit micro-
biological growth, it was decided to investigate certain
substances which might do so.

It followed, logically, to look into the use of various
organic acids whose bacteriostatio prOperties (or those of
their salts) were known; and whose previous use in foods was
allowed by the Federal Food and Drug Act. It was also decided
to use the sodium salt of dehydroacetic acid, which has only
recently been develOped (6). This acid and its salts have been
used as mycostatic agents in bread and dairy products (3, 38).

Dehydroacetic acid” (3-acetyl-6-methy1-l,2-pyran 2,4 (3)-

dione) and its sodium salt are white crystalline powders.

 

*Chemicals kindly supplied by the Dow Chemical Company,
Midland, Michigan.

__—_ E,

They have been designated as DHA and DHA-S, reSpectively, by
the Dow Chemical Company. DHA, molecular weight 168.1, is

only slightly soluble in water (0.1 g per 100 g water at 25 C),
very soluble in organic solvents, and moderately soluble in
oils and waxes. DHA-S, molecular weight 208.1, is very soluble
in water, and only slightly soluble in organic solvents.

These compounds were found to be stable When heated to 120-

130 C for one hour (38, hO), and are odorless and tasteless

in all concentrations suitable for use.

Extensive toxicological and pharmacological work has
been done on DHA and DHA—S by Spencer (32), Seevers (29),
Shideman (30) and Woods (39), 33 E1. The majority of the
work was done on animals, little on humans. The results of
their work indicated that DHA and DHA-S would only be toxic
when used in concentrations much higher than those necessary
to produce stasis. Because of the solubility factor, only
DHA-S was tested.

These chemicals were to be placed into soft custard in
graded amounts, and their effectiveness noted.

It was decided to employ an adaption of the plate count
method to observe the effect of the chemicals tested, as there
is little or no agreement (17, 20) in the literature on the
effectiveness of the kitten test as a means of determining the
presence of enterotoxin in food. Further disagreement in the

literature on the production of antibodies (19, 2S) and the

use of a serological test (1, h, 5) induced this writer to

seek the more simplified plate count method to determine the
presence or inhibition of microorganisms. Additional factors
which prompted the use of the plate count method were the
difficulty in feeding a definite amount of custard to kittens,
and the amount of work in purifying the enterotoxin (13, 22,

37) and subsequent antibody production.

EXPERIMENTAL

The organic acid stock solutions were made by dissolving
one equivalent weight of the acid in one liter of distilled

water to produce a l N solution.

 

 

 

Acid Molecular weight Equivalent weight
Acetic 60.5 60.5
Citric 192.0 6h.0
Lactic 90.08 90.08
Maleic 116.03 58.015
Malic l3u.05 67.025
Propionic 7 .08 78.08
Succinic 11 .05 59.025
Tartaric 150.09 75.0%

The DEA-S stock solution was a five percent solution, prepared
by dissolving 0.60 gram.DHA-S in 12.0 ml of distilled water.
These solutions were autoclaved at 15 pounds pressure for 20
minutes.

The broth medium employed in the primary work was a one
percent solution of dehydrated Bacto-peptone (commercially
produced by the Difco Laboratories).

The soft custard employed in the second phase of the work

was made up in multiples of the following formula:

Milk 2hh grams
Eggs #8 grams
Sugar 25 grams

The soft custard medium was always freshly prepared.

Both the peptone medium and the soft custard were dis-
pensed into a series of ten test tubes in portions of exactly
5 ml. In the cases where one of the organic acids was to be
tested, the next step was to add exactly 5 m1 of the 1 N
acid solution into the first test tube in the series. The
acid and the medium were then thoroughly mixed. (This pro-
cedure was standardized by aSpirating the mixture into a
pipette and then discharging it back into the test tube. This
cycle was repeated five times to assure complete mixing.) The
second of ten serial dilutions was then made by withdrawing
5 ml of the mixture from the first test tube and mixing it
in the prescribed manner with the 5 m1 of medium contained
in the second test tube. Eight further serial dilutions were
prepared in like manner.

In the case of DHA-S, the test tubes were prepared as

follows:

Final concentration

 

 

Medium 5% stock solution of DHA-S of DHA-S in medium
u.l ml 0-9 m1 0'9 %
11-3 ml 0.7 ml 0-7 7°
LL05 ml 0.5 ml 0.5 70
4.7 ml 0.3 m1 0-3 3”")

Where the peptone medium was involved, sterilization
was accomplished by autoclaving the test tubes containing the
peptone medium and the organic acids or DHA—S at 15 pounds
pressure for 15 minutes.
Where the soft custard medium was involved, sterilization

was accomplished by the process known as inspissation, which

10

is usually employed in autoclaving media that contain eggs.
This is done in the following manner: the autoclave is pre-
heated, then loaded with the test tubes containing the soft
custard medium and the organic acids or DEA-S. The door is
closed, and all the outlet valves are closed tightly. The
stem inlet valve is opened just a crack. The steam is allowed
to enter the chamber slowly so that the pressure does not ex-
ceed 5 pounds in 15 minutes. At this rate, at the end of a
h5-minute period, the pressure within the autoclave should be
15 pounds. This pressure is then maintained for a 20-minute
period, at the end of which time the steam valve is closed,
and the pressure within the autoclave is allowed to return to
normal. When this procedure is followed carefully, the me-
dium will appear as a firm custard, with no bubbles or frothi-
ness. Because of the possibility of air pockets occurring in
the chamber, sterility tests were made, employing test tubes
which contained only 5 m1 of the soft custard medium.

After the media were steriliZed, they were allowed to
cool to room temperature and inoculated. The inoculum used
was the third of three successive Zh-hour cultures of either
Micrococcus pyogenes, var. aureus, Salmonella typhimurium, or
Salmonella schottmuelleri, grown in 10 ml of peptone broth.

In the cases Where the peptone medium was involved, the
size of the inoculum was standardized by using one loopful of

the organisms suspended in the peptone broth.

11

In the cases where the saft custard medium was involved,
a straight wire was used as the inoculating needle. For
standardization purposes a single stab into the soft custard
medium was made after dipping the inoculating needle into
the 2h-hour old organisms suspended in peptone broth.

The inoculated test tubes were then incubated at 37 C

for 2h hours.

Three groups of controls were set up, when using the

soft custard medium, as follows:

Group I - the medium was inoculated, then immediately
plated, i.e., an endeavor was made to deter-
mine the average number of organisms
inoculated into each test tube.

Group II - the medium was inoculated, then incubated
for 24 hours at 37 C, and plated. This
would demonstrate whether the organism
had grown in the medium.

Group III - controls were checks on the sterility of the
medium. Number 1 consisted of 5 ml of soft
custard plated immediately after inspissation.
Number 2 consisted of 5 ml of soft custard
that was incubated after it had been inspis-
sated, at 37 C for 2h hours, then plated.
Should either #1 or #2 show any bacterial
growth, it would serve as an indication that
the inspissation process had not served its

purpose of sterilization.

12

At the end of the incubation period, the growth in the
tubes was observed. In the cases of the test tubes containing
peptone broth, this was done by holding the test tube up to
a light source and classifying the growth according to the
turbidity present, the range being - (negative indicating no
apparent growth) to +h (very turbid).> Prior to reading the
test tubes, they were all rotated in a manner that would re-
suspend any cells that had settled to the bottom of the test
tubes, or any that grew in the manner of a pellicle on the
surface of the peptone broth.

In the cases of the test tubes containing soft custard,
the plate count method was employed. This was accomplished
in the following manner: 5 ml of sterilized peptone broth
was added aseptically to each test tube. Then the soft
custard medium was broken up into small pieces, using a
sterile 5 m1 pipette. The soft custard medium and the
peptone broth were more or less homogenized by aSpirating
the mixture into the 5 ml pipette, and then discharging it
back into the test tube. This cycle was repeated five times
to assure uniformity of the suSpension.

One ml of this suspension was then placed into a water
dilution blank bottle containing 99 ml of sterile distilled
water, and shaken 25 times. This produced a 1:100 dilution.
Ten ml of this 1:100 dilution was added to a second water
dilution blank bottle containing 90 ml of sterile distilled

13

water (to make a 1:1000 dilution of the original material)
and shaken 25 times. This step was repeated once more, in
order to establish a 1:10,000 dilution of the original mater-
ial. One ml of each dilution was transferred aseptically

into one of three sterile petri dishes; cooled liquid nutrient
agar was then addedh the plate swirled, and the agar allowed
to solidify. The plates were then incubated at 37 C for h8
hours. At the end of the incubation period, the number of

colonies per plate was read macroscopically.

TABLE 1

GROWTH OF ORGANISMS IN PEPTONE BROTH
CONTAINING VARIOUS CONCENTRATIONS OF ORGANIC ACIDS
(Read at 24 hours)

 

 

Cone. of acid in

M. pyogenes, ‘S. t hi- .§° schott-
g/mlbggtgeptone 'var. aureus murIum mueIIerI PH

 

Acetic acid

 

 

 

1 Normal

(.605 g/lO ml acid) 2.50
.03025 - - - 2.92
001512 ‘ ‘ ' 3030
000756 - ' ‘ 3058
000378 ' “ “ 3086
000189 ' - - #017
.00094 - - - hogs
0000”? ‘ - ’ “0 0
000021... ++ + " 5018
.00012 ++++ $4+ + 5.68
.00006 ++++ ++++ :4 6.20
.0000 ++++ ++++ ++++ 7.0

Citric acid

1 Normal

(.640 g/lO m1 acid) 1.89
.03200 - - - 2.15
.01600 - - - 2. 9
000800 "' " " 20
.00400 - - - 3.2
000200 - “ - 3078
.00100 - - - 4.30
.00050 - + - 4.73
.00025 ++ +++ - 5.20
.00013 +++ ++++ + 5.70
.00006 ++++ ++++ ++ 6.15

Lactic acid

1 Normal

(.9008 g/lO ml acid) 1.83
0014501.}. " "' - 2025
000252 " " " 2059
001126 " " " 2089
000563 ‘ ' ' 3025
000282 "' - " 3058
0001u1 ' ‘ ' 3099
000070 - "‘ - (4,011.8
.00035 +++ ++ - 5.00
.00018 ++++ ++++ + 5.60
.00009 ++++ ++++ 6.15

TABLE 1 (Cont.)

 

 

Cone. of acid in
H. pyggenes, ‘S. t ohi- g. schott-
utter—

 

 

 

 

 

g/mlbggtgeptone var. aureus m mueIIerI
Maleic acid
1 Normal
(.58015 g/lO m1 acid) 0.82
.02901 - - - 1.10
001h50 - - - 1.40
000725 ' ‘ ‘ 1075
000363 ‘ ‘ - 2031
.00181 - - - 3.18
000091 ' “ ' neon
.00045 - ++ + 4.80
.00023 ++ ++++ ++ 5.60
.00011 ++ ++++ ++++ 6.31
.00006 ++++ ++++ ++++ 6.75
Malic acid
1 Normal
(.67025 g/lO ml acid) 1.65
003351 - ' ‘ 201“
.01676 - - - 2.55
000838 - I- I- 2.83
000419 - - ' 3032
.00210 - - - 3.80
.00105 - - - 4.29
.00052 - ++ ++ 4.89
.00026 ++++ ++++ +++ 5.95
.00013 ++++ ++++ ++++ 6.55
.00007 ++++ ++++ ++++ 6.83
PrOpionic acid
1 Normal
(.7408 g/lO ml acid) 2.49
00370“ - ' ‘ 300M
001852 - - _ BOAO
.00926 - - - 3.70
0004,63 " " " 3097
000232 - - - #022
.00116 - - .. 17.50
000058 - - - 4.80
000029 + + - 5.15
.00015 +++ ++++ - 5.60
.00007 ++++ ++++ + 6.10

TABLE 1 (Cont.)

 

 

Cone. of acid in

 

 

 

M. ogenes, S. tthi- S. schott-
g/mlbggtgeptone '?ar. aureus 'fmurium "mueIIeri pH
Succinic acid

1 Normal

(.59025 g/lO ml acid) 1.99
.02951 - - - 2.55
001h76 ' ' ‘ 3005
000738 "' "' " 3020
.00369 - - - 3.50
000185 ' ' ' 3090
000092 ' ' ' “020
.00006 - + ++ n.65
.00023 ++++ +++ ++++ 5.25
.00012 ++++ ++++ ++++ 6.20
.00006 ++++ ++++ ++++ 6.60

Tartaric acid

1 Normal

(.750h 3/10 m1 acid) 1.66
003752 - “ “ 1095
001876 II " - 2026
.00938 - - - 2.60
000u69 ‘ “ ' 2096
000235 ‘ ' ‘ 3038
000118 ' ' ‘ 3090
.00059 - - - u.uo
000029 " ++ II 14.92
.00015 ++ ++++ + 5.52
.0000? ++++ ++++ ++ 6.03

 

 

17

TABLE 2

CONTROL TEsTs or BASIC sUBsTRATE
(Read at LB hours)

 

 

 

 

 

 

 

 

 

Dilution
Organism
1:100 1:1,000 1:10,000
Group I
M. oaenes, var. aureus TNTC ( 65) 62 7
s. typhimurium '"“ ” ' TNTC ( 13) AS u
Ԥ. schottmuelleri 2&2 28 h
Group II
M. pyogenes var. aureus TNTC TNTC TNTC
E. cyphimurium. ““"" TNTC TNTC TNTC
é. schottmuelleri TNTC TNTC TNTC
Group III
No. 1 (immediately plated
out) 0 0 0
No. 2 (incubated 2i hrs.,
37 C) 0 o o

TNTC - Too numerous to count

“Group I - Inoculated, then immediately plated out (to
determine the number of organisms inoculated
into each tube)

Group II - Inoculated, incubated for 2h hours at 37 C,
then plated out (to determine if the organism
grew in custard)

Group III - Sterility controls (to determine that no air
pockets were present in the autoclave)

18

TABLE 3
GROWTH OF KICROCOCCUS PYOGEHES, VARIETY AUREUS IN CUSTARD
CONTAINING GRADED AMOUNTS OF OHGAHIC ACIDS
(Read at L8 hours)

 

 

 

 

 

 

 

 

 

Cone. of acid in Dilutibns
g/ml of custard 1:100 1:1,000 1:1o,000 pH
Acetic acid
.03025 0 0 0 3.91
.01512 0 0 0 8.51
.00756 15 2 o 5.15
.00378 187 12 1 5.80
.00189 TNTC (315 37 h 6.18
.00094 TNTC TNTC TNTC 6.80
Citric acid
.03200 0 0 o 2.69
.01600 0 o 0 .52
.00800 8 1 0 .30
.00800 TNTC TNTC TNTC (3300 5.15
.00200 TNTC TNTC TNTC 5.80
Lactic acid
.08508 0 0 0 2.75
.02252 2 o o .33
.01126 1 o 0 .08
.00563 8 1 0 5.00
.00281 TNTC TNTC TNTC 5,70
Maleic acid
.02900 0 0 0 1.23
.01850 0 0 0 1.87
.00725 28 h 0 3.83
.00362 TNTC TNTC TNTC 8.76

19

TABLE 3 (Cont.)

 

 

Cone. of acid in Dilutions
g/ml of custard 1:100 1:1,000 1:10,000 pH

 

Malic acid

 

 

 

 

.03351 0 o 0 2.%0
.01675 0 0 o 2. 8
.0083? 8 1 o .62
.00818 TNTC TNT TNTC i,88
Propionic acid
.03708 O O O 3.79
.01852 0 0 o 8.31
.00926 3 5 o 8.80
.00863 11 1“ 2 5.32
.00231 TNTC (398) 38 3 5.85
.00115 TNTC TNTC TNTC 6.20
Succinic acid
.02951 0 0 0 3.15
.01875 19 2 0 .59
.0073? 28 2 o .09
.00368 TNTC TNTC TNTC .55
Tartaric acid
.03752 0 2.30
.01876 0 3.01
.00869 1 5.00
.00238 TNTC 5.68

 

TNTC - Too numerous to count

GR OWTH 01“

T T

<7 7». ..
L401LJJI.

TABLE 8

'GWBLLA TYPHIKURIUE IN CUSTAHD

 

CONTAINING GHADED AMOUNTS OF ORGAKIC ACIDS
(Read at 88 hours)

20

 

 

Conc. of acid in

Dilutions

 

 

 

 

 

 

g/ml of custard 1:100 1:1,000 1:10,000 pH
Acetic acid
.03025 0 0 0 3.91
.01512 2 0 0 8.51
.00756 0 0 0 5.15
.00378 56 7 2 5.80
.00189 TNTC (387) 36 2 6.15
.00008 TNTC TNTC TNTC 6.80
Citric acid
.03200 0 0 0 2.69
.01600 1 1 0 3.52
.00800 8 0 0 8.30
.00800 TNTC TNTC TNTC (1780) 5.15
Lactic acid
.08508 0 0 0 2.75
.02252 0 0 0 P°33
.01126 0 o o 1.08
.00563 0 0 0 5.00
.00281 TNTC TLTC TNTC (1900) 5.70
Maleic acid
.02900 0 0 o 1.23
.01850 0 0 0 1.87
.00725 38 1 0 3.83
.00362 TNTC TNTC TNTC 8.76

TABLE 8 (Cont.)

 

Cone. of acid in Dilutions

 

 

 

 

 

 

 

g/ml of custard 1:100 1:1,000 l:l0,000
Malic acid
.03351 0 0 2.80
.01675 0 0 2.88
.00837 8 0 .62
.00818 TNTC TNTC i-hB
Prqgionic acid
.03708 0 0 .79
.01852 1 0 8.31
.00926 6 0 8.80
.00863 32 0 5.32
.00231 TNTC (688) 56 5.85
.00115 TNTC TNTC 6.20
Succinic acid
.02951 0 0 3.15
.01875 0 0 13.59
.00737 0 0 .09
.00368 TNTC TNTC 8.55
Tartaric acid
.03752 0 0 2.30
.01876 0 0 3.01
.00938 1 1 3.92
.00869 TNTC (989) 108 5.00
.00238 TNTC TNTC 5.68
TNTC- T00 numerous to count

GROWTH OF
CONTAINING GRADED AMOUNTS OF
(Read at 88 hours)

TABLE 5

SALHONELLA SCHOTTMUELLERI IN

 

CUSTARD
OHGAHIC ACIDS

 

 

 

 

 

 

 

 

Cone. of acid in Dilutions

g/ml of custard 1:100 1:1,000 1:10,000 pH

Acetic acid
.03025 0 0 0 3.91
.01512 0 0 0 8.51
.00756 0 0 0 5.15
.00378 16 2 1 5.80
.00189 TNTC 168 18 6.18

Citric acid

-/ .03200 0 0 0 2.69
.01600 0 0 0 i.52
.00800 12 2 1 .30
.00800 TNTC TNTC TNTC (782) 5.15

Lactic acid
.08508 0 0 0 2.75
.02252 0 0 0 3.33
.01126 0 0 0 (.08
.00563 3 0 0 5.00
.00281 TNTC TNTC TNTC 5.70

Maleic acid
.02900 0 0 0 1.23
.01850 0 0 0 1.87
.00725 0 0 0 .83
.00362 TNTC TNTC TNTC .76

23

TABLE 5 (Cont.)

 

 

 

 

 

 

 

 

Cone. of acid in Dilutions
g/ml of custard 1:100 1:1,000 1:10,000 pH
Malic acid
.03351 0 2.80
.01675 0 2. 8
.00837 18 .62
.00818 TNTC .88
Propionic acid
.03708 0 0 0 fi.79
.01852 0 0 0 .31
.00926 0 0 0 8.80
.00231 282 33 8 g.85
.00115 TN 0 TNTC NT .20
Succinic acid
.02951 0 0 0 3.15
.0187; o o o 3.59
.0073? 0 0 0 8.09
.00368 TNTC TNTC TNTC 8,55
Tartaric acid
.03752 0 0 0 2.30
.01876 0 0 0 3.01
.00938 0 0 0 .3.92
.00869 52 7 1 5.00
.00238 TNTC TNTC 60 5,68

TNTC- Too numerous to count

TABLE 6

GROWTH OF FOOD POISONING ORGANISMS IN
CUSTARD CONTAINING GHADED ALOUNTS OF DNA-S
(Read at 88 hours)

 

Cone. of DHA-S
in custard
(percent) 1:100 1:1,000 l:l0,000 pH

Dilutions

 

 

Micrococcus‘pyogenegl
var. aureus, Str. Brail

 

 

 

0 3 TNTC TNTC TNTC 7.60
0.5 TNTC TNTC TNTC 7.55
0.7 TNTC TNTC TNTC 7.57
0.9 TNTC TNTC TNTC 7. 2
1.1 TmTc 286 38 7.8
Salmonella typhimurium
0.3 TNTC TNTC TNTC 7.60
0.5 TNTC TNTC TNTC 7.55
0.7 TNTC TNTC TNTC 7.57
0.9 TNTC 250 33 7.52
1.1 TNTC 180 21 7.86
Salmonella schottmuelleri

0.3 TNTC TNTC NTC 7.60
0.5 TNTC TNTC ThTC 7.55
0.7 TNTC TNTC 222 7.57
0.9 TNTC 163 13 7.52
1.1 278 31 2 7.86

 

TABLE 7

PALATABILITY OF ACID-TREATED SOFT CUSTARD

 

 

m

25

 

 

 

Acid Palatability pH
Acetic
.03025 - 3.91
001512 " “-051
.00756 - 5.15
000378 "' 5080
.00189 i 6.1“.
.00098 + 6.80
Citric
.03200 ~ 2.69
.01600 - 3.52
.00800 - 8.30
.00800 + 5.15
Lactic
.08508 - 2.75
.02252 - 3.33
.01126 - .08
.00563 i 5.00
.00281 + 5.70
Maleic
.02900 - 1.23
00114.50 " 1087
000725 - 3083
.00362 - 8.76
.00181 + 5.36

TABLE 7 (Cont.)

26

 

 

 

 

 

Acid Palatability pH
Malic
003351 “ 20 0
.01675 - 2.88
.0083? - 3062
.00818 - 8.88
.00209 + 5.18
Propionic
00370“ ‘ 3079
.01852 - “-031
.00926 - 8.80
.00863 - 5.32
.00231 - 5.85
.00115 - 6.20
000057 + 6.80
Succinic
.02951 — 3.15
.01875 ' 3059
000737 ' “009
000368 i, 8.55
00018“ + 5013
Tartaric
.03752 - 2.30
.01876 "' 3001
000938 ’ 3092
.00869 "' 5.00
.00238 + 5.68
- Unpalatable

able

+ Presence of acid still detectable and slightly disagree-

+ Palatable, even though the flavor of the acid was at

times apparent

RESULTS AND DISCUSSION

The results of the primary work (Table l) where the
actions of various organic acids in peptone broth were tested
on food poisoning and food infecting bacteria at first appear
to be vague. However, closer over-all examination shows that
‘g. schottmuelleri is on the whole much more susceptible to
inhibition by the organic acids tested than.fl, pyogenes, var.

aureus and §. typhimurium. This means that a much smaller

 

concentration of the acid is required to keep down the growth.
On the other hand, the other salmonella tested, g. gzgg_-
murium, proved to be the most resistant to the inhibitory
actions of the organic acids. At times this resistance was
rather great, as in the cases of citric and prOpionic acids,
where it grew in concentrations four times as great as those
which almost completely inhibited the growth of g. schott-
muelleri.

The general effect appears to indicate that acetic,
prOpionic and tartaric acids give the best results, closely
followed by citric and lactic acids.

It is difficult to come to any sound conclusions on the
basis of the presence of carboxyl groups (since three of these
acids are monocarboxylic, one is di-carboxylic and the remaining
is tri-carboxylic) except to point out that the monocarboxylic

acids gave a much better result than the di-carboxylic acids

tested. It would be grossly unrealistic to make any assump-
tions on the part of tri-carboxylic acids from the results

of this work as citric acid was the only one tested. A simi-
lar attempt to draw any definite conclusions at this point,

on the basis of pH, is rather difficult since the range that
induced stasis was tremendous. For example, in the maleic
acid results, a pH of 8.08 is apparently necessary to produce
inhibition of all three organisms tested, whereas in the

cases of acetic and propionic acids, an acidity of only pH
8.80 was required to achieve the same effect. In addition,
the results on the basis of pH varied even more greatly when
the organisms were considered individually, e.g., M. o enes,
var. aureus, str. Brail was inhibited at a pH of 8.88 using
lactic acid, whereas the same effect was achieved at a pH of
8.92 using tartaric acid. Similarly, a pH of 8.08 using
maleic acid inhibited.§. typhimurium, while on certain occasions,
acetic acid at a pH of 5.18 produced the same results. The

pH range of inhibition regarding §. schottmuelleri is even

 

more pronounced: inhibition occurred at a pH of 8.08 using
maleic acid, and occasionally acetic acid inhibited the or-
ganism at a pH of 5.68. The controls for this portion of the
work were tubes of peptone broth inoculated with the respective
organisms. The growth that was achieved within 28 hours was
considered to be ++++, and all subsequent readings of the acid
dilutions tubes were based upon this control, (i.e., no mech-

anical reading of turbidity was employed). The dearth of

29

DHA-S necessitated its non-inclusion in this primary phase.
However, the results of the work done by Brunner and Mall-
mann (3) provided information comparable to that obtained
in this primary phase on the organic acids.

Table 2 shows the types and results of the controls used
in the second phase. The Group I controls indicate that the
average numbers of organisms inoculated into the test tubes
of soft custard were as follows:

E, pyogenes, var. aureus, str. Brail 630,000

‘g. typhimurium 862,000

 

S. schottmuelleri 618,000

 

The Group II controls indicate that the organisms grew in
the soft custard, i.e., there were no natural inhibitory
agents present in the custard. The Group III controls
prove that the inspissation process not only "set" the
custard, medium, but also simultaneously sterilized it.

In Table 3, the effects of the organic acids tested on
g, pyogenes, var. aureus, str. Brail, in soft custards, are
recorded. Although it has been previously stated that ten
serial dilutions were prepared, only those concentrations
showing inhibition, and the first (and sometimes second) con-
centrations Showing lack of inhibition, are listed. ‘When mixed
with the soft custard medium, a much greater concentration of

the organic acids was necessary to produce stasis of the

organism than when the acids were mixed with the peptone broth.

30

The pH values indicate that the constituents of the soft
custard medium either buffered or neutralized some of the
effects of the acids on the organism. Acetic and propionic
acids produced the best results, i.e., lower concentrations
were necessary to produce inhibition. Next in value are lac-
tic and tartaric acids. The greatest concentrations of acids
needed to inhibit the growth of E. pvopenes, var. aureus, str.
Brail, were demonstrated in the cases of the remaining acids:
citric, maleic, malic and succinic. One startling difference
is that citric acid when mixed with the soft custard medium
gave nowhere near the (proportional) results one was led to
expect after examining Table 1. It is difficult to evaluate
the exact role pH plays in the inhibition of the organism.

This becomes apparent in comparing the results observed when
using maleic and acetic acid. Inhibition occurred in the
former at the concentration where the pH of the soft custard
medium was 3.83, whereas in the latter the pH was 6.18. When
comparing the results of the acids on the basis of the number
of carboxyl groupings present, it is noted that the monocar-
boxylic acids yielded the best results.

In Table 8, the effects of the organic acids tested on
§.Atyphimurium, in soft custard, are recorded.

In Table S, the effects of the organic acids tested on
é. schottmuelleri in soft custard, are recorded.

Against both §. typhimurium and'é. schottmuelleri, as

 

well as previously mentioned in the case of‘fl. pyogenes, var.

\

31

aureus, str. Brail, acetic and prepionic acids gave the best
results, closely followed by lactic and tartaric acids.

In Table 6, the effect DHA-S had on all three organisms,
tested in soft custard, is recorded. Ԥ. schottmuelleri and
§. typhimurium proved to be the more susceptible to static
action of DEA-S, as a concentration of only 0.9 percent was
required. A concentration of 1.1 percent was necessary to
achieve the same effect against E, pyogenes, var. aureus, str.
Brail. The pH of these concentrations was above neutrality,
and this would indicate that the action of DHA-S is not based
upon the hydrogen ion concentration but rather upon the chem-
ical structure of the compound. This has previously been
mentioned by Coleman and Wolf (6). It has been observed by
Geiger and Conn (18) and.later by Roblin (27) that such anti-
biotics as clavicin and penicillic acid were alpha, beta
unsaturated ketones. The structural grouping found in these
antibiotics, i.e., the -CH=9-8=O grouping, is also observed
in DHA-S. Geiger and Conn (18) observed that it is this
grouping that in all probability is reSponsible for the anti-
bacterial activity of clavicin and penicillic acid. Before
concluding, it is necessary to add that in the concentrations
of acetic, lactic, prepionic and tartaric acids and DHA-S
that proved to be effective against bacterial reproduction,
the appearance of the custard was quite normal. The matter
of taste, however, was quite a different story. The DEA-S

apparently did not alter the flavor or taste of the soft custard,

32

but the organic acids certainly did, especially acetic and
prOpionic acids. The concentrations that induced bacterio-

stasis were all unpalatable. The least objectionable in

taste was citric acid. These data are contained in Table 7.

1.

2.

3.

8.

5.

6.

7.

SUMMARY

Eight organic acids and DHA~S were tested in peptone
broth and a soft custard medium for their effectiveness
against food poisoning and food infecting organisms.
Acetic and prepionic acids, closely followed by lactic
and tartaric acids proved to be the best inhibitors in
soft custard.

A 0.9 percent concentration of DHA-S was required to

inhibit the Salmonella tested, while a 1.1 percent

 

concentration.was required to inhibit the enterotoxin-
producing strain tested.

No correlation of results could be made on the basis of
pH.

Of the organic acids tested, the consistently best
results were achieved with the monocarboxylic acids.
The concentrations of organic acids that induced
bacteriostasis rendered the soft custard unpalatable.
Soft custard containing a bacteriostatic concentration

of DHA-S was completely palatable.

.-.__ -_ -l

10.

ll.

BIBLIOGMAPHY

Blair, J. E. 1939. The pathogenic Staphylococci. Bact.
Rev. 3:97e186.

Brown, E. G., G. a. Combs and E. Wright. 1980. Food-
borne infection with Salmonella aertrycke. J.A.M.A.

118:682-688.

Brunner, J. R. and W. L. Mallmann. 1950. Effectiveness
of dehydroacetic acid as a mold inhibitor on butter and
cheese surfaces. Mich. 832} Exp. Sta, Quar. Bull. 33:
127-1LL2. ‘

 

 

 

Burnet, F. M. 1929. The exotoxins of Staphyloccus .
pyggenes aureus. ‘i. Path. Bact. 32:717-738.

 

Burnet, F. M. 1931. The interactions of Staphyloccus
toxin anatoxin and antitoxin. g. Path. Bact. 38:

871-892.

Coleman, G. H. and P. A. Wolf. Making proteinaceous and
fatti foods resistant to microorganisms. U. S. Patent
7

# 2, 8,228.

Coughlin, F. E. and B. Johnson, Jr. 1981. Gastroenter—

itis outbreaks from cream-filled pastry. Amer. 3. Pub.
Health 31:285-250.

 

Crabtree, J. A. and W. Litterer. 1938. Outbreak of milk
poisoning due to a toxin-producing Staphylococcus found
in the udders of two cows. Amer. g. Egg. Health 28:
1116-1122.

 

Crowe, M. 1986. A localized outbreak of Salmonella food
poisoning apparently transmitted by a hefiTs egg. ‘1.

gig ° (414-: 314-2-314-5-

Dack, G. M., W. E. Cary and P. H. Harmon. 1928. Unsuccess-
ful attempt to produce Salmonella intoxication in man.
J. Prevent. Med. 2:879é883.

 

Dack, G. M. 1987. Problems and errors in assigning
causes of bacterial food poisoning. Amer. g. Pub. Health

37:360-368.

 

12.

13.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Dack, G. M. 1989. Food poisoning. Rev. ed. Chicago:
The Univ. of Chicago Press. 188 pp.

 

Davison, E. and G. M. Dack. 1939. Some chemical and
physical studies of Staphylococcus enterotoxin. 9g.
Infect. Diseases 68: 302- ~306.

 

Doyle, H. S. 1986. An outbreak of food poisoning in
Prince Albert, Saskatchewan. Canad. J. Pub. Health
37: 65-68.

Draper, F. 1985. Gastro- enteriti s and dysentery in
children. The bacterioloay of cases caused by Sal-
monella and dysentery organisms. LTEed. J. Austra 11 la

32'E17-825.

Fuchs, A. W. 1981. Disease outbreaks from water, milk
anduother foods in 1939. Pub. Health Re .56: 2277-
228 .

 

Fulton, F. 1983. Staphylococcal enterotoxin - with
special reference to the kitten test. Brit. g. Expt.
Path. 28: 65- -72.

Geiger, w. B. and J. E. Conn. 1985. The mechanism of
the antibiotic action of clavicin and penicillic acid.
J. Amer. Chem. Soc. 67: 112- 116.

Glenny, A. T. and M. F. Stevens. 1935.’ Staphylococcus
toxins and antitoxins. g. Path. Bact. 80:201-210.

Hammon, W. MCD. 1981. Staphylococcus enterotoxin: An
improved cat test, chemical and immunological studies.
Amer. g. Pub. Health 31:1191e1198.

Haynes, W. C. and G. J. Hucker. 1985. Studies on
Coccaceae XVIII. The enterotoxin producing micrococci.
New York (Geneva) Agr. Exp. Sta. Tech. Bull. 275 :1—82.

 

Holt, L. B. 1936. The purification of Staphylococcus
toxoid. Brit. g. Expt. Path. 17:318-323.

 

Kelly, F. C. and G. h. Dack. 1936. Experimental Sta h lo-
coccus food poisoning. Amer. J. Puo. Health 26: 1077-
1082.

'McCastline, W. B., R. Thompson and M. L. Isaacs. 1937.

A food poisoning outbreak due to Staphylococcus. g.
Baoto 33:50-51.

 

3o

25. Minett, F. C. 1938. EXperiments on Staphylococcus food
poisoning. J. qu. 38:623-637.

 

26. Ostrolenk, M. and H. Welch. 19h2. The house fly as a
vector of food poisoning organisms in food producing
establishments. Amer. J. Pub. Health 32:h87-h9&.

 

27. Roblin, R. 0., Jr. 19h6. Metabolite antagonists. Chem.
Rev. 38:255-377 0

28' Rosenau, M' J' and H' Weiss. 1921. Food infections.
i-é-an- 77:19118-1950.

29. Seevers, M. H., F. E. Shideman, L. A. Woods, J. R. Weeks
and W. T. Kruse. 1950. Dehydroacetic acid. II.
General pharmocology and mechanism of action. ‘g. Phar-
macol. Expt. Therap. 99:69-83.

 

30. Shideman, F. B., L. A. Woods and M. H. Seevers. 1950.
Dehydroacetic acid. IV. Detoxication and effects on
renal function. 'g. Pharmacol. Expt. herap. 99:98-111.

 

31. Slocum, G. G. and B. A. Linden. 1939. Food poisoning
due to Staphylococcus. Amer. g. Pub. Health 29:1326-1330.

 

 

32. Spencer, H. Co, V. K. Rowe and D. D. McCollister. 1950.
Dehydroacetic acid (DHA) I. Acute and chronic toxicity.
J. Pharmacol. Bxpt. Therap. 99:57-68.

 

33. Spray, R. s. 1926. An outbreak of food poisoning probably
due to rat virus. J.A.M.A. 86:109-111.

3h. Stewart, H. C. and N. Litterer. 1927. An outbreak of
gastro-enteritis. J.A.m.A. 89:158h-1587.

3S. Surgalla, M. J. and K. E. Hite. 19h6. Production of
staphylococcal enterotoxin in chemically defined media.
Proc. Soc. Expt. Biol. Med. 61:2hh-2h5.

36. Welch, H., M. Ostrolenk and M. T. Bartram. 19hl. Role
of rats in the Spread of food poisoning bacteria of the
Salmonella group. Amer. g. Pub. Health 31:332-340.

 

37. Wittler, R. G. and L. Pillemer. l9h8. The immunochem-
istry of toxins and toxoids. V. The solubility of
Staphylococcal toxin in methanol-water mixtures under
controlled conditions of pH, ionic strength and tempera-
ture. ‘J. Biol. Chem. 17h:23-29.

 

38. Wolf, P. A. 1950. Dehydroacetic acid a new microbio-
logical inhibitor. Food Tech. #:29h-297.

39. Woods, L. A., F. E. Shideman, M.-H. Seevers, J. B. Weeks
and W. T. Kruse. 1950. Dehydroacetic acid (DHA).
III. Estimation, absorption, and distribution. 9g.
Pharmacol. Expt. Therap. 99:84-;7.

 

 

MO. Summary 2; Toxicological Studies 2_ DEA. The Dow Chemical
Company, Midland, Michigan.