THE EFFECT OF SORBIC ACID ON YEASTS ASSOCIATED
WITH CUCUMBER FERMENTATIONS

By
William Elliott Ferguson

A THESIS

submitted to the School of Graduate Studies of Michigan
State Univeraity of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Microbiology and Public Health

1955

THEbi-

 

ACKNOWLEDGMENTS

The author is deeply grateful to Dr. R. N. Costilow
for his guidance throughout the course of these studies.
Thanks are also due Dr. L. J. Wickerham of the Northern
Utilization Research Branch, U.S.D.A., Peoria, Illinois,
filo graciously supplied.many of the yeast cultures used
and the Carbide and Carbon Chemical Company, New York,

for their generosity in providing the sorbic acid.

William.Elliott Ferguson

THE EFFECT OF SORBIC ACID ON YEASTS
ASSOCIATED WITH CUCUMBER FERMENTATIONS

ABSTRACT

The influence of sorbic acid on 2h cultures of yeasts representing
11 different species which are often isolated frcm cucumber fermentations
was determined. The effectiveness of the acid as a yeast inhibitor de-
pended greatly on.the hydrogen.ion concentration of the substrate. .At
pH 5 .0 all yeasts tested were completely inhibited by 0.1 percent. sorbic
acid; but, as the pH was increased toward neutrality, a number of the
species were found to grow. The addition of salt to the substrate in
addition to sorbic acid aided the inhibition of some species. Survival
studies of the yeasts in the presence of 0.05 and 0.1 percent sorbic
acid at pH h.5 showed that the death rate of most species was more rapid
with the higher concentration. The results indicated.that sorbic acid
may be somewhat fungicidal as well as fungistatic at low pH levels. Sor-
bic acid.was found.to be quite stable in a laboratory medium. Spectro-
photometric analysis of a medium inoculated with a yeast and of uninocu-
1ated.medium.after 2dweeks incubation showed no significant change in

concentration of sorbic acid.

WZZW

VRalph N. Costilow
Associate Professor

TABLE OF CONTENTS

Page
INTRODUCTIONOOOOO00000000000.000.00.000.000000000000 1
REVIEW OF LITERATURE

Microbiology of cucumber fermentations......... 2
Effect of sorbic acid on microorganisms........ 2
EXPERIMENTAL

Cultures used and method of handling........... 7

Source of sorbic acid and method of addition... 7

Methods of estimating growth................... 8
RESULTS

The effect of sorbic acid and sodium sorbate
on growth of pure cultures of yeasts at pH 6.0. 9

Relationship of size of inoculum to effective-
ness of sorbic acid at pH 6.0 ................. 12

The relationship of pH to inhibitory action of
sorbic 8°1dooeoecoo0.900009000000000...oeeoeoea 14

The inhibition of yeasts by sorbic acid in the
presence of sodium chloride.................... 18

The effect of different concentrations of
sorbic acid on inhibition of yeasts............ 23

Survival of yeasts in the presence of sorbic

ac1d00000..00....0.0.0....COOOCCOOOOOOOOOOOOOOO 24

Fate of sorbic acid in media inoculated with

ye as ts O O O O O O O O O C O O O O O O I C O O O O C O O O O O O O O O O O O O C O O O O 28
DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O O 34
SWEMARY AND C ONC LUSIONS 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O C 36

BIBLIOGRAPHY-0.0.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 38

INTRODUCTION

Yeasts are normally present in cucumber curing brines.
The types present can be divided into 2 classes, sub-sur-
face and film-forming yeasts. The former are gas produc-
ers and often cause a type of spoilage referred to as
"bloaters" (hollow cucumbers). The film-forming yeasts,
while not considered responsible for "bloater" spoilage, are
undesirable in that they utilize the lactic acid produced
during fermentation; thereby, partially destroying the pre-
serving quality of the brine.

Unlike most other organisms associated with spoilage
in brines, yeasts are both salt and acid tolerant. There-
fore, some other means of control is required to prevent
their develOpment. A survey of the effect of sorbic acid,
an alpha-beta unsaturated fatty acid, on pure cultures of
the predominant species of these yeasts was made in this

study.

REVIEW OF LITERATURE

Microbiology of cucumber fermentations. The identifi-
cation of predominant types of microorganisms associated
with the fermentation of cucumbers has been studied by
different workers in recent years. Etchells _e_t_ a}... (5, 6)
identified the film-forming and sub-surface yeasts normally
found in such fermentation brines. Costilow and Fabian (1)
in their studies of commercial cucumber fermentations found
only 2 groups of active microorganisms -- acid forming
bacteria and yeasts. The acid forming bacteria were found
to multiply rapidly and reach maximum numbers in S or 6
days. Yeasts decreased in numbers for 2 or 3 days after
brining but then increased gradually with peak numbers found
in 10 to 20 days. Rosen and Fabian (15) found a similar
pattern of microorganism deve10pment in laboratory studies
on cucumber fermentations. In addition to these 2 groups
of microorganisms, Etchells _e_t_ 9:1; (7) in their studies of
commercial cucumber fermentations in North Carolina noted a
. hydrogen fermentation due to Aerobacter. This occured very

frequently in high salometer brines.

Effect of sorbic acid on microorganisms, Gooding (8)

in 1915 found that sorbic acid could be incorporated into

noted that this compound in concentrations effective
against such organisms was tasteless, odorless and non-
toxic. Smyth and Carpenter (18) confirmed the non-toxic-
ity factor and more recently Deuel gt gl.(2, 5) proved its
harmlessness as a dietary component. They found from.rat
feeding studies, that intermediary metabolism of sorbic
acid is identical with such normally occurring fatty acids
as caproic and butyric and that under normal conditions of
alimentation sorbic acid it completely oxidized to 002 and
H20. In these same studies they found sorbic acid consid-
erably less toxic than sodium benzoate, a preservative used
in many food products.

Phillips and Mundt (14) apparently were the first to
suggest the use of sorbic acid to control the development
of scum yeasts on pickle brines. They found that this com-
puund in a concentration of 0.1 per cent would prevent mold
and scum.yeast development under conditions extremely favor-
<able for their growth without harmful effect to the ferm-
entation process. Jones and Harper (9) confirmed these
findings. These papers dealt primarily with the film-form-
ing yeasts in general. Apparently no work was done to eval-
uate this compound's effectiveness against pure cultures of
yeasts including the sub-surface types considered more im-

portant as Spoilage yeasts in the pickle industry.

14.
Further indication that sorbic acid might be useful in

cucumber fermentations was presented by Emard and Vaughn

(h) who stated that this compound can be used advantageously
in.the isolation of lactic acid bacteria, particularly those
belonging to the genus Lactobacillus. Their findings showed
this compound to be selective for the catalase negative
bacteria such as Lactobacillus, Leuconostoc, and Clostridium
genera. All catalase positive organisms tested including
yeasts, molds, actinomycetes and bacteria were inhibited by a
concentration of 0.12 per cent sorbic acid when the pH of the
medium.was between 5.0 and 5.5 in the absence of phosphate
salts. The degree of effectiveness of sorbic acid depended
upon its concentration, the type of basal medium.and the pH
of the medium. ‘York and Vaughn (19) found that sorbic acid
could be used as an enrichment for media to isolate the more
fastidious species of Clostridium if the hydrogen.ion con,
centration of the medium was increased to pH 6.0.

Recently, extensive studies have been published on the
efficiency of sorbic acid in controlling mold growth on
cheese. Smith and Rollin (16, 17) found that 0.05 per cent
sorbic acid mixed into processed cheese inhibited mold
growth and that the application of this compound to cheese
wrappers offered a practical method for packaging cheese.

They estimated that its use would save 13 million pounds of

cheese annually in the United States. No mold growth and
no organoleptic difficulties were encountered when % to i -
pound packages of cheese were wrapped in a thermoplastic
coated cellophane wrapper treated with 2.5 to 5.0 grams of
sorbic acid per 1,000 square inches of paper. This concenp
.tration will furnish not more than 0.1 per cent sorbic acid
to the cheese.

Although oxidizable by air, as are other polyunsaturated
C6 fatty acids, Melnick gt 31. (11, 12, 13) found that no
oxidative deterioration of sorbic acid occurred in packages
so wrapped during relatively long storage periods at h50F.
However, when cheese was wrapped with only one surface
protected by the sorbic acid treated wrapper, molds grew on
the other surfaces and the concentration of sorbic recover-
able decreased. From this and other studies they concluded
that in cheese with a high ratio of mold to sorbic acid
concentration, the sorbic acid was utilized by the mold as a
source of carbon. If the ratio was reversed, the sorbic
acid effectively inhibited the dehydrogenase enzyme system
of the molds; thereby, exhibiting fungistatic activity.

Melnick and Luckmann (10) presented a precise
spectrOphotometric method for the estimation of sorbic acid
in cheese and on cheese wrappers. The sorbic acid was dis-

tilled at atomospheric pressure and the sorbic acid in the

absorption curve.

EXPERIMENTAL

Cultures used and method of handlinglr The pure cult-
ures of yeasts used in this study were isolated by Etchells
and Bell (5), Etchells 22 31. (6) and Costilow and Fabian
(1) during their work on the identification of yeasts from
cucumber brines. Cultures isolated by Etchells and Bell
(5) and Etchells 22 El! (6) were obtained from Dr. L. J.
Wickerham of the Northern Utilization Research.Branch,
United States Department of Agriculture, Peoria, Illinois.

All cultures were carried on V-8 agar slants and fresh
transfers were made at about 2-week intervals. Two or
three strains of each species were studied.

For each experiment, young actively-growing cultures
used for inoculation purposes were obtained by repeated
transfers in dextrose broth. The dextrose broth consisted
of 0.5 per cent peptone, 1.0 per cent dextrose and 0.1 per
cent yeast extract. All yeasts used in these studies grew
well in this medium.at pH levels between h.5 and 7.0.

Source of sorbic acid and method of addition. The
sorbic acid was obtained from the Carbide and Carbon
Chemicals Company, New York 17, New'York.

Sorbic acid was added to the media prior to pH adjust-
ments and sterilization. The concentrations were calculated

on a moisture free basis and added as per cent by weight per

8
on a moisture free basis and added as per cent by weight per

unit volume of media.

Methods 3: estimating growth. In the preliminary work,
yeast growth was estimated by visual observation of turbid-
ity and scum formation as compared to uninoculated controls.
Later the growth of the sub-surface yeasts was determined
by measuring per cent light transmission in a photelometer.

In the study of yeast survital both plate counts on
dextrose agar (Difco) plus 0.1 per cent yeast extract and
acidified with 3 m1. of 5 per cent tartaric acid per 100 m1.

and most probable numbers in dextrose broth were run.

RESULTS

The Effect of Sorbic Acid and Sodium Sorbate on Growth of
Pure Cultures of Yeasts at_pH 6.0.

Phillips and Mundt (1h) stated that sorbic acid and
its sodium.salt appeared equally effective in laboratory
tests in controlling pure cultures of film-forming yeasts
on artificial media but showed no data to substantiate
this statement. Due to the limited solubility of sorbic
acid in water, the use of the soluble sodium salt, if
equally effective, would obviously offer certain advantages.
Deuel gtflgl. (2) compared by rat feeding experiments the LDSO
for sorbic acid and sodium.sorbate and found the latter
considerably more toxic. However, sodium sorbate has not
been available commercially and these workers have not yet
given their method of preparation of the compound.

To compare the relative effectiveness of sorbic acid
and sodium sorbate in controlling growth.of yeasts from
cucumber fermentations, a sample of sodium sorbate obtained
from.Mr. J. Sheneman, Department of Bacteriology, Michigan
State University was used. This was p*epared by Mr.

Sheneman in the following manner:

Sodium hydroxide, sorbic acid and distrilled water were

mixed in the ratio of 5:1h:50. This mixture was then.thor-

10
oughly dried in a vacuum oven to remove all free water and
leave sodium sorbate. Theoretically, this should yield
16.75 grams sodium sorbate when calculated according to the
following reaction:

NaOH + CsH7COOH ~9 CSH7COONa. + H20

Since 1h grams of sorbic acid was used to make this
16.75 grams of sorbate, then 1.0 gram of sorbic acid is
equivalent to approximately 1.2 grams of sodium.sorbate.

To compare the relative effectiveness of the 2 comp
pounds 0.1 per cent sorbic acid and 0.12 per cent sodium
sorbate were added to separate lots of dextrose broth. A
third lot of dextrose broth was used as a control medium.
All 3 lots were dispensed in 10 ml. amounts into test tubes,
sterilized, inoculated with 1 drop of each yeast culture
studied and incubated at 30°C. Growth was assessed by visual
examination only.

As shown in Table l, neither sorbic acid nor sodium
sorbate was very effective at pH 6.0 in inhibiting yeast
growth. However, visible growth occurred consistently
earlier in those tubes containing sodium.sorbate for all
cultures except strain'Y-35 of,3hgdgtgzgla sp. This
culture showed no growth in the presence of either compound.
Sorbic acid under these conditions apparently caused
complete inhibition of growth of both cultures of

Rhodotorula and of Debggyomyces membranaefaciens var.

11

Tahlel

Effect of Sorbic Acid an! Sodiu Sorbete on Ieeete at pH 6.0

TIILTHIIT

 

 

 

 

 

 

 

Control 0.1330rb1e Acid 0.12180diul Sex-bate
Daye Days
011m 1 2 3 1 2 J k 6 1h 1 2 )_ I; 6 LL
WWW (Y 11.6) t 5.
F. vereatms - + - - - - + 2+ - 9 3+ 3+ In 50
‘ E. ”NEE (1.176) O 50 C O O O O 29 ' . 3‘. 3‘. h. '1.
(1-72) So - - . e e . - 2. 2. 3. 3. 5.
(1.58) 5. - Q n u C O O O 2* 2. 3. 3.
(1.61;) So - - o - e e c e e 2e 3. 3.
3300mm
m sp. (I-35) 2+ In 5+ - - - - - - - - - - -
m up. (1403) 3+ 5+ - - - - - - - + + 2+ 3+
‘I'CRUIASPGIA
1'. rose! (F392) 5+ + 3+ 3+ 3+ In 3+ In In In In 5+
T. rose! (1407; 5+ + 3+ 3+ 3+ In In 3+ In 5+ 5+ 5° 5*
g. "Troo- (1.237 5» 2+ 3+ 3. 3» 1.. In In In I» I.» 5. 5»
(1-109) 5+ 2+ In In In In In In 5+ 5+ 5+ 5+ 5+
(1-277) 5+ In In In In In In In In In In In In
(Y-IIJJ) 5+ 3+ In In In In In In In 5' 5+ 5+
ZMACGIARMCB 0.3.
. ome C. (I-lOOO) 3+ 5. - - + . . 2. - In In I» In In
2', 51003315551111; (IS-86h) 2+ 5+ - - e e e e n e be he he 5.
Z. ep. 15-559.? - 5+ - + + 2+ 3+ 3+ - 2+ 3+ In 5+ 5+
% ( 11.8 ) 5+
, mm. 7 2. - 0 2+ In In In - + In 5+ 5+ 5+
g. E (ma-3m) In so . 2+ 2. 3. 3. 3. . 1.. 1.. 5. 5. 5.
Inmmcrs
D. membranaefaciem var. 5*
' m—R 3 - - - - - - - 2+
D. aziSranagmfa ciene w. . + . 5. 5‘. 5.
3 meridians (NH-72 + 3+ 5+ - - - + + + - - 2+ 3+ 5+ 5+
BNDOIYCOPSIS< )
E. 051'! ”-1 So - - e e e 0 2+ In 5+ 5+ St
E- 053 (”'15) 5+ - - - e e e 20 In 5* 5+ 5* 55:

b ung-h...- of plus signs indicates the extent of turbidity observed; 5+ - mm turbidity; - - no growth observed.

12
Hollandicus culture NFY-32. Partial inhibition of

Brettano ces, Hansenula, zygosaccharomyces and Endomypgpe

 

gig’ species was evident but little or no inhibition occurred
in those tubes inoculated with Torulaspora, Torulopsis and
Candida species. However, later studies have proved that
sorbic acid.is much.more effective against growth of all
yeasts in a more acid medium. Also, it is quite conceivable
that sodium sorbate might be more effective if the pH of the
medium.were lowered. This has not been confirmed at the

present time.

Relationship of Size of Inoculum.to Effectiveness
of Sorbic Acid at pH 6.0

Melniok‘gt‘gl. (13) showed that high.mold populations
would grow in the presence of sorbic acid while lower popup
lations would not. Therefore, it was possible that lower
yeast inocula might result in complete inhibition at pH 6.0.
To test this possibility, serial dilutions were made of
representative strains of the yeasts and the above experhment
repeated.

Results of this study (Table 2) showed that the ability
to survive and develop in the presence of 0.1 per cent
sorbic acid varied considerably with different species of
yeasts. However, it was apparent that with all species

studied if growth occurred in the 10"2 dilution Of the inoc-

stle2

Relatiomhip of Sise of Inoculu to the Inhibita'y Action of 0.1 Percsm Sorbic Acid at pH 6.0

Dilution of Culture Used for Inoculation

13

 

 

Cells/n1.

inc I- Days 30'
mm x :3 1 2 2 I. a A l 2 ”I.” a n, 2 We g 1 DI.” 8
am menu 32 .0 5+ - + 3+ 5+ 5+ - - + I» I» - - 3+ 3+ - - 3+
m.” ”flmm ha 3‘. 5. C O 9 3+ 30 C O C 2* a. o I O Q Is 0 0
"7m
Motel-uh III. 9 2. 5. - .. - - - - - - - - - .. - - .. - -
W 11.0 5+ Ia+Is+h+Is+ 3+h+b+h+h+ h+h+h+h+- InIn
Torn]. is new 180 5+ In In In In In In In 3+ In In In - In In
W Menu 25 2+ 5. - . . 3. 3. - - . . . - - . . - - .
Candida kneel 62 5+ 1+ 3+ 3+ In + 3+ 3+ In In + 2+ 3+ In - e 2+
‘11“: F3117

Wad“.
ver. cus - 2 + 5+ - - - - - - - - - - - - - - - - -
5+ + + + 3+ 3+ - - - 2+ 2. - - 2. 2. - - .

“$.0F1s obi-1 180

I Controls were inoculated with the highest dilution of inoculu (10-5) in each instance.
If later of plus signs indicates the extent of turbidity observed; 5+ equals unsu- turbidity; - equals no with observed.

1h
ulum, then it also occurred in the 10'"5 dilution. The

time required for growth to appear in the 10-5 dilution
varied according to the reproduction rate of the particular
yeast. Species of Torulaspora and Torulopsis which re-
produce very quickly showed comparable growth in all
dilutions tested within.h days. Brettanomyces, Hansenula,
Eygosaccharggyces and Endomycopsis species reproduce more
slowly and required a longer time to show visible growth in.
the higher dilutions of inoculum. The species of Rhodotorula
and Debagyonyces studied were inhibited regardless of the size
of inoculum.used. These results indicate that some factor
other than size of inoculum.was responsible for the inability

of 0.1 per cent sorbic acid to control growth of these yeasts.

The Relationshipgofgpfl to Inhibitory
Action of Sorbic Acid

The inefficiency of sorbic acid in inhibiting growth of
yeasts at pH 6.0 even when the inoculum.was greatly diluted
suggested that sorbic acid, like benzoic acid, might be use-
less as an inhibitory agent in low acid.media. This was,
also, indicated in the results of Emard and Vaughn (h). To
test the pH effect, the yeasts were inoculated into the basal
medium.with and without sorbic acid at three pH levels -—
6.0, 5.5, and 5.0.

15

In Table 3 the growth of one culture of each species
at 3 pH levels is compared after incubation for 7 days.
Growth in all control tubes was heavy. At pH 6.0, as pre-
viously found, growth was evident in all cultures except those
of Rhodotorulg in the presence of 0.1 per cent sorbic acid .
As the pH of the medium.decreased, greater inhibition occurred.
At pH 5.5 only 3 of the 10 species (Torulaspgra roseiI
Torulopsis holmii and Candida krusei) grew to the extent that
turbidity could be detected by visual examination. All
cultures were apparently inhibited completely at pH 5.0.

Realizing the inaccuracy of evaluating growth of sub-
surface yeasts by visual examination, turbidity measurements
were made of tubes inoculated with these yeasts using a
Cenoo-Sheard-Sandford photelometer with a filter having a
maximum.central transmission of 610 mu. The pH5.0 and 6.0
series of tubes were thus measured and compared for per cent
light transmission. The photelometer was standardized for
each series of tubes against a tube of similar medium.which
had not been inoculated. Since growth in the control
medium.at all pH levels was heavy, readings were taken on
the more acid series only. As seen in Table h, the trans-
mission of light through this medium inoculated with the
different yeasts varied from 5 to 13.5 per cent. Photel-
ometer readings on media containing 0.1 per cent sorbic

acid and inoculated with these yeasts confirmed the find-

16

Table 3
Effect of pH on the Inhibition of Yeasts by 0.1 Percent Sorbic Acid
pH 6e° PH 505 PH 500

 

Oultm'e Control 0.}! SA: Contra]. Oi: SJ: Combo]. 0g 8.1.
W versatilis 5+, 2+ 5+ - 5. -
Hansen]: subge_1£culose 5* In 5* - 5* -
Rbodota-ule sp. 5+ - 5+ - 5. -

T roeei 5* 5* In 5* -
Torul 1201-11 5* In 5* 3* 5* '-
W 5* 3+ 5. - 5. -
cums. krusei 5+ In 5+ 2+ 5+ -

‘11!!! FWD
9. 2* 54' O 5* Q

 

Maggie cheeri 5* 2+ 5+ - 5+ -

I Number of plus signs indicates the extent of turbidity observed; 5+ indicates
uni-n turbidity; - indicates no growth observed.
All readings taken after 7 days incubation.

17

Table II
Effect of [ii on Inhibition of Yeasts by 0.1 Percent Sorbic Acid

1 mint Transmission‘

Control 0.1% Sorbic Acid
th PH 5.5 PH e e

 

 

 

 

Brettenmes versatilis (I-lh6) 12.5 98.5 7h.0
Ramona]; subglliculosa (1-72) 6.0 99.0 8h.0
Rhodoturula sp- (1-35) 13.5 98.5 96.0
Torulaspors £933; (Ir-392) 5.0 98.0 h8.0
Torulopsis carolirnsm (RY-199) 7.5 99.0 92.5
Torulmis & (1-109) . 5.5 98.5 62.0
Monaccharms halasenbranis (153-861;) 5.0 98.0 73.5
e

Readings made after 7 days incubation

18
ings. At pH 6.0 all cultures with the possible exception
of ghodotorula grew under these conditions but to a lesser
degree than in the absence of the inhibitory compound. At
pH 5.0 no significant change in light transmission was ob-
served.-‘The loss of 1.0 to 1.5 per cent transmission was
attributed to the added inoculum and to variations in the
photelometer cells. These results show very definitely that
the hydrogen ion concentration of the substrate is critical

in the inhibition of yeast growth by sorbic acid.

The Inhibition of Yeasts by_80rbic Acid in the
Presence of Sodium Chloride

Since these yeasts are primarily a problem in cucumber
fermentations, it was thought advisable to test the effect-
iveness of sorbic acid in controlling their growth in a sub-
strate containing similar salt concentrations.

Salt was added to culture media in concentrations of
5 and 10 per cent and the media then adjusted to pH 5.0.
Growth determinations were again made by photelometer
measurements for sub-surface yeasts and by visual examin-
ation for the film-forming species. Readings were taken at
the time of inoculation and at 2,6,9,12,16,24,50 and 60 hours.

The results of this study may be noted in Tables 5 and
6 and growth patterns of representative fast growing and

slow growing yeasts are shown in Figure 1. The addition

19

m5
IrreototOJmantioAcmonIeestOnltmeinelbdiuconteinuuShmentSelt ($5.0).

 

c ‘ gm man- 0 3.1
o n l' O l g ‘
£122 026°9‘r'1216m60 0269O‘r15162560
mm
'0 nm% (“7:11.63 1” 9905 9800 9600 98.0 ”00 98.5 3800 1m 9905 . 9905 ' 9905 9905
g. verse 176 m 9905 9805 ”05 9805 ”05 ”.5 3600 m ”05 - 99.5 - 9905 9905
Islam
. culoee (1-72; 98.5 99.0 98.5 98.0 93.0 also 35.5 6.0 99.5 99.5 - 99.5 - 99.0 99.5
m “'9 9905 990° ”00 9705 950° 9100 5605 80° 9905 9900 " 990° ' 9900 9905
g. c (14.1.) 98.5 98.5 98.5 98.5 97.0 9h.5 8L.5 13.0 99.5 99.5 - 99.0 - 99.0 99.5
mam-cam
m». (1-35) 100 99.0 98.0 97.5 97.0 97.5 88.5 36.0 100 99.0 - 99.0 - 99.0 99.5
m... (I-lOJ) 100 99.0 98.5 98.0 96.0 88.5 7M 35.5 10:. 99.5 - 99.5 - 99.0 99.5
mm
'!. mu! (1-392 99.5 98.5 96.0 93.5 815.0 58.0 12.0 5.5 99.5 99.0 - 99.0 - 99.0 98.0
1'. We (14:07 98.5 98.0 97.0 92.5 82.0 56.0 11.0 5.5 99.0 98.0 - 98.0 - 98.0 98.0
1'. To?! (me? 99.5 98.0 98.0 96.5 89.5 72.5 18.5 5.5 99.5 99.0 - 99.0 - 98.5 98.0
moms
. 1. (mm 99.0 98.5 98.0 96.0 92.5 87.0 51..5 10.0 99.0 98.0 - 99.0 «- 98.5 98.0
1.5133 (1-277 99.0 98.0 98.0 97.0 97.0 97.5 9h.o 9.0 99.0 98.5 - 98.5 - 98.0 98.5
LEE; (14.13 99.0 99.0 97.5 97.5 95.0 92.0 83.0 1.1.0 99.0 99.0 - 96.5 - 98.5 99.0
,0 OII‘OIJJM (HY-199) 9905 9600 9705 9“ 8500 5705 05 80° 9905 9905 - 990$ - 990° 990°
:0 mm (3.“) ”0° 98.5 9600 9305 8300 5°00 1600 705 9905 99.5 ‘ 990° " 990° 990°
momenta ea.
0 manil(f-1m0 1m 9900 980° 990° ”.0 ”05 ”.0 7.5 m 99.0 - 99.5 . ”as ”as
zmus-osug 100 98.5 98.5 99.0 98.5 98.5 98.5 7.5 100 99.5 - 99.5 99.5 99.5

I
.3
O
.3
“I

Ma ”0 (13.“9) 1m 9905 990° ”05 9805 990° 9805 65.0 1m 9905 ' 9905

Table 6

20

Effect of 0.1 Percent Sorbic Acid on but cum in a sodiu- Containing 10 Percent Salt (1:8 5.0)

Cult!!!

BEETIIICHICIS
"B:';;F;=Ellis (Iilhé)
1L.versa£III; (13176)

30 E 111L041 1.72
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HHODOTGRULL

m . .35)
m 3. g-ioa)

 

 

 

 

 

 

0 407
.0 £3.51 $.28?

rosutorsxs
It £71533? 'I-277
T’ o ,( )

1': 2mm... (xx-199)
_, c .nians (HI-200)

ZIUOSACCHAROHICES le‘e
"1E71EDEEEEEFIEIH (151000;
e mtg-IE (MI.
I: '9- 05-5557

 

 

 

 

99.5

99.5 100

98.0
99.0

99.5
99.5

98.0
98.0
98.5

97.0
97.5

98.0
98.0

99.0
99.0
99.5

98.0
99.0

99.0
99.5

96.0
98.0
98.0

9h.0
97.0
97.0
97.0
97.0

99.0
99.5

Control
Iiiiif“

98.5
99.0

98.0
98.5
98.0

98.0
97.0

9h.0
97.5

9h.c
97.0

97.0 96

89.0

98.5
98.5

99.0
99.0

98.0

Li t Transmission

16 30 60

99

97.5

5 97.0

97.0

98.0
95.0

69.0
78.0
91.0

93.5
96.0
95.0
60.0
h5.o

97.5
97.0
98.0

98.5

.0
98.5 98 5 hi.

81.5
82.5
97.0

86.0
85.0

39.5
0

99.5

98.0
98.0
98.0

99.5
100

99.0
99.0

98.0
98.0

97.5
98.0

98.0
98.0
98.0

98.0
97.0
98.0
97.5
97.0

9900
93-5
9900

98.5
99.0

98.0
98.0

97.5
98.0

98.0
98.0
9805

98.0
97.0
97.5

97.0
99.0

98.5
98.5

99.C
990C

98.C
98.(

97.5
98.(

98.!
98.(
96.(
98.(

93.(

5 97.'

97.2

99.‘
99.‘
99.‘

Per cent Light Transmission

21

 
  

   
  

 

 

 

o F-
Controls
20 .—
40 i— Controls
BRETTANOMYCES VERSATILIS
. . 5% “I. (slow growing)
I, 0 ----- 0 10% salt
63 r— ”
80 r—
TORULASPORA ROSEI
(lost growing)
0.1%
100 Sorbic acid Oolfi Sorbic acid
0 10 20 30 40 50 60 o 10 20 30 40 so 60

Time (hours)

Fig. I. Ellect of 0.] Per cent Sorbic Acid on Representative Yeast Cultures in Media Containing Salt

22
of salt did not reduce the inhibitory capacity of sorbic
acid. Complete inhibition of all yeasts occurred in the
presence of both salt concentrations in the sorbic acid
medium. Also it was observed that the presence of 5 per
cent salt in the dextrose broth without sorbic acid offered
no retarding effect on the growth of these yeasts. With
the exception of Brettanomyces and Zygosaccharomyces
species growth of all yeasts was observed between 9 and 2h
hours of incubation. Under similar conditions, but with the
salt increased to 10 per cent, the growth pattern of most
yeasts was similar but somewhat slower. Candida krusei and

TorulOpsis holmii, however, failed to grow in the higher salt

 

concentration. Etchells and Bell (5) stated that the former
yeast is susceptible to high salt concentrations with 10 per
cent possibly the maximum concentration permitting growth.

or greater interest perhaps was the fact that all 3 cultures
of Torulopsis holmii failed to grow in the higher salt broth.
According to Etchells.ggflgl. (6) and Costilow and Fabian (1)
this yeast predominates in the early fermentation of cucump
bers. These results indicate that this may be due to its
inability to tolerate the higher salt concentrations of the
later stages. At any rate these findings indicated that

salt did not interfere with the inhibitory action of the
sorbic acid and, in some instances, may have aided the sorbic

acid by exerting an inhibitory action of its own.

23
The Effect of Different Concentrations of Sorbic Acid

on Inhibition of Yeasts

Phillips and Mundt (1h) stated that 0.1 per cent sorbic
acid in pickle brines effectively controlled filmrforming
yeasts whereas 0.05 per cent failed to do so. Assuming the
0.1 per cent concentration equally effective against sub-
surface spoilage yeasts, its use would serve to eliminate
some of the hazards of cucumber curing. However, the cost
of adding this amount to large vats of cucumbers would dis-
courage its use by some pickle manufacturers.

The fact that 0.1 per cent sorbic acid in the basal
medium.adjusted to pH 5.0 so effectively inhibited all these
yeasts suggested that in the more acid pickle brines with a
pH range of 3.9 to u.3 within 3 days after brining and cone
taining 7 to 8 per cent salt a lesser concentration might
serve as efficiently. This possibility was investigated by
comparing the inhibitory effect of 0.05 per cent sorbic acid
in.a medium containing 8 per cent salt and adjusted to pH h.5.
Again growth of subsurface yeasts was determined by photel-
ometer measurements and of filmeforming'yeasts by visual
examination.

This experiment showed that both 0.05 and 0.1 per cent
sorbic acid completely inhibited all yeasts tested under
these conditions. The results were quite similar to those
in Table 6. Representative results for two of the yeasts

24
these conditions. The results were quite similar to those
in Table 6. Representative results for two of the yeasts
are given in Table 7 which shows complete inhibition by
both 0.05 and 0.1 per cent sorbic acid.

After 50 days incubation, the flasks containing sorbic
acid medium were re-inoculated with the yeasts and incubated
for an additional 50 days. No growth was evident in any of

the flasks.

Survival 2; Yeasts in Presence 2; Sorbic Acid

This phase of study was carried out to determine the
type of effect which sorbic acid has on yeast cells-fung-
istatic or fungicidal. A 1 ml. inoculum of a 24 hour cult-
ure of each yeast was inoculated into 5 flasks of dextrose
broth plus 8 per cent NaCl (pH 4.5). One flask contained
0.05 and one 0.1 per cent sorbic acid and the other served
as a control. Viable cell counts were made by the stand-
.ard plate count and most probable numbers techniques.

Tables 8 and 9 show similar results obtained by plate
counting and most probable numbers techniques. In the con-
trol tubes the numbers of yeasts increased in a manner typi-
cal of a normal growth curve. Flasks and tubes containing
sorbic acid showed a reduction in viable yeast cells for
both methods of counting. As seen in the plate counting

method 0.1 per cent sorbic acid reduced the yeast count more

25

Table 7

Effect of Different Concentrations of Sorbic Acid on (north of Toasts

Per cent Ligbtrmuion

g. 52; g. versatilis
11.! some gas as some as: an
Initial 99.0 99.0 99.0 100 99.5 99.5
I: hours 98.5 99.0 99.0 100 100 99.0
8 hours 98.5 99.0 97.5 99.5 99.0 98.5
12 hour. 93.5 98.0 98.0 99.0 98.0 98.0
16 hours 87.0 99.0 98.0 93.5 98.5 99.0
21. hours 51.5 97.5 97.5 86.5 97.5 99.0
35 hours 13.0 98.0 98.0 $5 98.5 99.0
Isa hours 8.5 98.0 97.5 16.0 98.5 99.0
60 hours 6.0 98.0 98.0 8.0 98.0 98.0 '

120 hour. 505 9705 9800 l-leo 9805 ”05

26

Table 8

Survival of Ieasts in lbdia Containing Different Concentrations of Sorbic
Acid as Detendned by Standard Plate Counting Procedures

 

 

 

 

 

 

 

 

 

 

 

‘1' i n 0
Culture treat-ant Initial 6 hrs 12 hrs 1 5 ‘15?
(x 135) (x 13) (x a) (2 fi) (1: ) (x ) (x 1 )
Brettan Control .3 .3 .I. 1.8 1.h a ho
versafi 0.05% S.A. .3 0 0 0 0 0 O
W 0.11 3.1. .2 0 0 0 0 0 0
Hamenula Control 15 20 81. 620 52 33 6
cum‘ 0005‘ 80‘. 8 1.2 .3 01 O O O
- 0.15 3.1. 10 .1 0 0 0 0 0
Rhodotoa'ula sp. Control 11.1. 1.6 83 880 5.1; 15 22
- 0.05% S.A. 8.I; 1.I1 1 .1 0 0 O
0.15 5.1. 7.5 .7 0 0 0 0 0
fin)» Control 8?? 33 100 102 16 a? 16
1‘0” 0.05% 50‘. 8.6 3.8 .5 1.7 0 O 0
m 0.15 5.1. 8.3 h.0 .5 .1 0 0 0
mo . Control 23 7f‘ ‘150 9,300 257? .01. .0003
mo 0.05% 50‘s 2]. 18 19 17.6 .01 O O
1W 0.15 3.1. 19.6 21 16 8 0 0 0
Eula is Control 3.6 h.7 111.5 611 20 7.2 .19
16%" 0.055 s... 2.2 0 0 0 0 0 0
FEST 0.15 5.1. 1.2 0 0 0 0 0 0
z harm. Control .3 1.3 11.5 78 8.3 3.7 1.8
assabrmis 0.05% S.A. .2 .2 .2 .l 0 0 0
0.01% 5.1. .3 .3 .1 0 0 0 0
Esta. come as 31 32 50 1.5 8.8 h
0.05% 3.1. 50 18 11.8 1 0 0 0
muff-3m. 0,1; 54. 28 17 1 .1 0 0 0
2% w Control .9 .3 3 8 .3: 1.6 7
In ranae aciens 0.051 S.A. l .2 0 0 0 0 0
var. Rmmcus 0.1} S.A. .5 0 0 0 0 0 0
m-
End 0 is Control 120 11.3 570 5.300 77 53 51.
‘W 0.05% 5.1. 80 17 3.3 .2 0 0 0
”-1 0.15 SJ. 9h 3.? .1 0 0 0 0

Table?

27

Survival of Yeast Cells in Medium Containing 0.1 Per cent Sorbic Acid
Using H.P.ll. Techniqm

 

 

 

 

 

 

 

 

 

 

 

 

'1' i n 0
Culture Treatment Initial l d ds 8 d
(x 133) (x 1§> (x 1 (x 1 ) (x {3%)
BIN*«Wmtifllflwfl Control 25 1.30 25 .92 .25
versatilin 0.1% 8.1. 25 0 0 0 0
Hansel-111111 Control 1.3 11,700 11.7 92 1:3
sufigmculou 0.1% SA. 92 0 0 0 0
mzodotomh sp. Control 9.2 9,300 .92 .025 .00113
- 0.1% 5.1. 7.1; 0 0 0 0
Torulss Control 92 9, 200 25 25 11;.7
'56.on 0.1% 5.1. 143 25 0 0 0
Torulo is Control 920 11,300 25 1:3 .1117
WEI-1n- 0.1% 3.1. 1.30 o 0 0 o
RY-Ig
Torulo s Control 92 92,000 1.30 250 25
o 0.15 3.1. 92 25 o o o
E— aaccharolwces Control 2.5 250 25 9.2 2.5
%mbrafis 0.1: 3.1. ‘ 2.5 0 0 o 0
Candida krusei Control 1.3 25,000 92 ha 92
wry-38r— 0.1¢ 5.1. 9.2 2.5 0 0 0
Raw Control .9 2.5 .025 .0h3 .0113
m ranae acieg 0.11 8.1. .14 0 0 0 0
var. Hollandicus
-?

Ext! 0 is Control 113 25,000 114? 113 711
’fiE-o 0.1% 5.1. 1.3 0 0 0 0

 

28
duction in viable yeast cells as compared with normal growth
curves is illustrated in Figure 2 for cultures of Torulopsis

caroliniana and Brettangglpes versatilis. The former yeast

 

was apparently the most resistant and the latter the most
susceptible to sorbic acid.

a To determine whether the lack of growth was due to the
absence of viable cells or to fungistatic action of sorbic
acid carried over into the growth medium, further studies
were made. Tubes used in the M.P.N. technique in the 8th
day planting which contained 1, 0.1, 0.01 and 0.001 ml.
inoculum respectively of the dextrose broth cultures of $232;-
aspora £2E2$ and Candida krusei were emptied into Petri plates
and the plates poured heavily with dextrose agar. Plates
were incubated at 30°C for h to 6 days after which time it
was found that all plates were completely void of growth.

This indicated that the yeast cells were destroyed rather
than.merely inhibited by residual sorbic acid.

Fate of Sorbic Acid in.Media Inoculated with Yeasts

Melnick'g§.al. (13) noted that sorbic acid was oxidized
by air and its loss due to metabolic degradation by molds in
cheese was complete when the ratio of molds to sorbic acid
was initially high. A parallel case with yeasts in cucumber
brines would not normally exist since the initial load or

yeast cells would be small and the sorbic acid concentrat-

Log of Numbers per ml.

 

 

29

    
     

BRETTANOMYCES VERSATILIS

o——o

Control
0.05% Sorbic Acid
0.1% Sorbic Acld

l l I 1 J I

TORULOPSIS CAROLINIANA

DAYS
Fig. 2 Survival of Yeasts In the Presence cl Sorbic Acid

30
ion would be uniform throughout the brine. However, to
determine whether or not there was any loss of sorbic acid
due to oxidation during incubation or by a normal inoculum
of yeasts sorbic acid determinations were made of the
following media;

1. Freshly prepared basal medium (dextrose broth) without
sorbic acid; pH n.60.

2. Freshly prepared medium.containing 0.1 per cent sorbic
acid; pH n.51. .

3. Sterile medium.incubated 3 weeks at 30°C which contained
initially 0.1 per cent sorbic acid; th.63.

h. Medium similar to 3 except inoculated with 3 drops of
an active culture of Torulopsis holmii per 100 ml.
medium prior to incubation.

Measurements were made on a 1:250 dilution of each.of
the above media by the spectrophotometric method of Melnick
and Luckmann (10). This method involves the plotting of
ultraviolet absorbency readings of the different media for a
series of wavelengths with.the maximum.absorbancy readings
converted to concentration of sorbic acid.

Results of these studies on the samples listed above
are given in Table 10 and the absorbency readings in Figure
3. Results from.medium.l give irrevelant sorbic acid
readings since no sorbic acid as such had been added to this

sample. Therefore, this reading was subtracted from.absorb-

31

Table 10

Effect of Time and Organisms on Retention of Sorbic
Acid in Culture Media

 

 

 

 

591° ta: .._ 12:21am“ ........ 32:22:21:
1 Fresh 1.60 0.0% non. .0025
2 Fresh 14.51 0.12: none .105
3 3 weeks 13.63 0.1% none .106
11 3 weeks 11.66 0.15 ToruloEis M .116

t Concentrations of sorbic acid were calculated from urine:- absorbnnce
st 258 - 260 91.

52

 

 

 

 

   

1.? - '—
l-O — _'
.8 -— .—
6 _ r—
-‘ " Treatment 1: _ Treatment 2:
Fresh F""‘
No sorbic acid 0.1% sorbic acid
2 Oil. 1:250 Dil. 1:250
.3 W 1 l I I #L
c
.5 230 240 250 210 270 230
1.2 — —
1.0
.8
.6
.4 __ _. Treatment 4:
Treatment 3: lncubated
lncubated 0.1% sorbic acid
0.1% sorbic acid Inoculated
-2 pl? 4.63 pH 4.66
DII. 1:250 Dil. 1:250
I l I A l 1 l l l l

 

 

 

 

Wavelength (mu)

Fig. 3 Ultraviolet Absorption Curves of Media

55
readings since no sorbic acid as such had been added to
this sample. Therefore, this reading was subtracted from
absorbency readings for the other media to correct for this
error.

According to the method of calculating percentage sorbic
acid from these ultraviolet light absorbency readings as
used by Melnick and Luckmann (10) it was apparent that no
loss of sorbic acid occurred during incubation in any of the
media. In fact concentrations of sorbic acid appeared
slightly higher than those added. This apparent increase
may have been due to inaccurate weighing of ingredients and
to evaporation of water in the process of media sterilizat-
ion. Further evaporation may have occurred in flasks (3)

and (4) during the 5 week incubation period at 50 C.

54
DISCUSSION

Results of these studies are in complete agreement
with reports of Emard and Vaughn (4) who state that the pH
of the substrate is of major importance in the inhibition
of yeasts by sorbic acid. However, in cucumber fermentations
this factor requires no consideration since the hydrogen
ion concentration of a cucumber brine reaches pH 4.0 or
possibly lower within 2 or 5 days after brining. Yeast
activity during these first few days is negligible in any
case.

In contrast to reports by Phillips and Mundt (14)
that 0.05 per cent sorbic acid was ineffective in the con-
trol of surface yeasts in fermentation brines, this concen-
tration of sorbic acid in a medium.containing 7.5 to 8 per
cent salt was found to be as effective in inhibiting yeast
growth as the 0.1 per cent concentration of sorbic acid.

That sorbic acid is fungistatic to yeasts, as Melnick
2£_gl.(13) found it was to molds, is unquestionable in
media of pH 5.0 or lower. Survival studies of these yeasts
in media containing 0.1 and 0.05 per cent sorbic acid show-
ed a complete absence of viable cells in both media by the
7th day. The reduction, although faster in the 0.1 per
concentration, was rather slow to claim much in the way of

fungicidal qualities although Smith and Rollin (17) have

35

reported that 0.05 to 0.066 per cent sorbic acid in cheese
actually kills molds.

SpectrOphotometric studies showed that there was no
loss of sorbic acid in media either from oxidation or yeast
inoculation under the conditions of these experiments.

Again this is in contrast to reports of Melnick 92 51. (12)
who found sorbic acid oxidizable by air to form peroxides
and aldehydes. These same workers (13) also found a degrad-
ation of sorbic acid when the mold population was high.
Apparently yeast cultures do not use sorbic acid as a source
of carbon which, according to York and Vaughn (19), some
organisms are capable of doing.

Based on the excellent inhibition of yeasts obtained
by the low concentrations of sorbic acid used in these
studies and the apparent stability of sorbic acid under these
conditions it would appear that its use in cucumber ferment-

ations is practical and desirable.

36
SUMMARY AND CONCLUSIONS

The effectiveness of sorbic acid in the inhibition of
yeasts normally associated with cucumber fermentations was
primarily dependent on the hydrogen ion concentration of the
substrate. At pH 6.0 sorbic acid and its sodium salt were
ineffective in the control of yeasts although reproduction
was slowed considerably. The size of inoculum was of minor
significance since growth occurred in most instances whether
this inoculum contained a few hundred or several million
yeast cells. At pH 5.5 most yeasts were inhibited by a 0.1
per cent concentration of sorbic acid and at pH 5.0 or lower
all yeasts were completely inhibited. At these lower pH
levels a lower concentration (0.05 per cent) of sorbic acid
proved equally effective.

The survival of these pure cultures of yeasts in media
containing 0.05 per cent aid 0.1 per cent sorbic acid re-
spectively at 3000 showed that the death rate of most species
tested was slower in the lower concentration but within 7 days
viability was zero in both instances. Further platings from
these media were void of growth indicating that the yeasts
were non-viable rather than inhibited by residual sorbic acid.

Common salt in concentrations of 5, 8 31d 10 per cent did
not lessen the efficiency of the sorbic acid. In fact it was
found that the 10 per cent concentration of salt exerted an

inhibitory action of its own on pure cultures of Candida krusei

37
fact, it was found that the 10 per cent concentration of
salt exerted an inhibitory action of its own on pure cultures
of Candida krusei and TorulOpsis holmii. Further studies are
indicated on the effect of various concentrations of salt and
sorbic acid in combination.

The effectiveness of sorbic acid in inhibiting yeast .
growth apparently is not transitory. Spectr0photometrio
studies showed that no loss of sorbic acid occurred in cottonp
stOppered flasks of sterile or inoculated media over a 3-week
period at room temperature. Furthermore, flasks of media of
pH 5.0 containing sorbic acid and inoculated with pure
cultures of the individual yeasts showed no growth of any
yeast in the presence of either 0.05 per cent or 0.1 per cent
sorbic acid after incubation for 30 days at 30°C. No growth
was evident even after reinoculation and a further 30 days

incubation period of these flasks.

l.

3.

5.

BIBLIOGRAPHY

Costilow, R. N., and Fabian, F. W. Microbiological study
of cucumber fermentations. Appl. dicrobiol., l; 514 -
519. 1955.

Deuel, H. J., Jr., Alfin-Slater, R., Well, 0. S., and
Smyth, H. F., Jr. Sorbic acid as a fungistatic agent for
foods. I. Harmlessness of sorbic acid as a dietary com-

ponent. Food Research, 12: l. 1954.

Deuel, H. J., Jr., Calbert, C. F., Anisfeld, L., McKeehan,
H ., and Blunden, H. S. Sorbic acid as a fungistatic
agent for foods. II. Metabolism of alpha-beta unsat-
urated fatty acids with emphasis on sorbic acid. Food
Research, 12: 13. 1954.

Emerd, L. 0., and Vaughn, R. H. Selectivity of sorbic
acid media for the catalase negative lactic acid bacteria

and clostridie. J. Bact., fig: 487, 1952.

Etchells, J. L., and Bell, T. A. Film yeasts on

commercial cucumber brines. Food Technol., 4; 77, 1950.

Etchells, J. L., Costilow, R. N., and Bell, T. A.
Identification of yeasts from commercial cucumber fermen-
tations in northern brining areas. Farlowia, g: 249,

1952.

7.

10.

11.

12.

15.

59

Etchells, Je Le, Fabian, Fe We, Ed Jones, Is De T116
Aerobacter fermentation of cucumbers during salting.

Mich. Agr. Expt. Sta. Tech. Bull. 200, 1945.

Gooding, C. M. Process for inhibiting growth of molds.
U. A. Patent Number 2,379,294 (1945) assigned to Best

Fbods Inc.

Jones, A. H., 81d Harper, G. S. A preliminary study of
factors affecting the quality of pickles on the Canadian
market. Food Technol., g; 504, 1952.

Melnick, D., ald Luckmenn, F. H. Sorbic acid as a
fungistatic agent for foods. III. Spectrophotometric
determination of sorbic acid in cheese and in cheese

wrappers. Food Research 19: 28. 1954.

Melnick, D., and Luckmann, F. H. Sorbic acid as a
fungistatic agent for foods. IV. Migration of sorbic
acid from wrapper into cheese. Food Research, 19: 28.

1954.

Melnick, D., Luckmann, F. H., and Gooding, C. M. Sorbic
acid as a fungistatic agent for foods. V. Resistance
of sorbic acid in cheese to oxidative deterioration.

Fbod Research, lg: 55. 1954.

Melnick, D., Luckmann, F. H., and Gooding, C. M. Sorbic
acid as a fungistatic agent for foods. VI. Metabolic

14.

15.

16.

17.

18.

19.

4O

degradation of sorbic acid in cheese by molds and the
mechanism of mold inhibition. Fbod Research, 19; 44.
1954.

Phillips, G. F., and Mundt, J. O. Sorbic acid as an
inhibitor of scum yeast in cucumber fermentations. Food

Technol.,‘g: 291. 1950.

Rosen, 8., 81d Fabian, F. W. The importance of biotin,
niacin and pantothenic acid in cucumber fermentations.

Food Technol., 1; 244, 1955.

Smith, D. P., and Rollin, N. J. Sorbic acid as a
fungistatic agent for foods. VII. Effectiveness of
sorbic acid in protecting cheese. Fbod Research, lg:

1. 1954.

Smith, D. P., 81d Rollin, N. J. Sorbic acid as a
fungistatic agent for foods. VIII. Need and efficacy
in protecting packaged cheese. Fbod Technol., 9: 133,

1954.

Smyth, A. F., and Carpenter, C. P. Further experience
with.the range finding test in the industrial toxicology
laboratory. J. Ind. Hyg. Toxicol., g9: 65. 1948.

Y ork, K. Y., and Vaughn, R. H . Use of sorbic acid
enrichment media for species of Clostridium. J. Bact.,

58: 759, 1954.

R5311 USE UiiLl

MAR 30 1981 “s

 

 

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