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 Studies an 966d   5’         

‘ Heat inhibiting Activity 0f Various
Gompaunds and wmmcrcia/ Pradutts

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TEXAS AGRICULTURAL EXPERIMENT STATION

R. D. LEWIS. DIRECTOR. COLLEGE STATION. TEXAS

DIGEST

Several compounds which offered some promise as inhibitors of heating and the growth of molds in
mixed feeds and feed ingredients were tested for their fungistatic activity in corn meal containing various
amounts of moisture. Calcium propionate and sorbic acid at a level of 0.3 percerlt prevented heating
completely in all feed ingredients tested. Smaller amounts of the inhibitors only delayed heating; Propion-
ic acid and propionic anhydride. each at a level of 0.1 percent. prevented heating. Sodium propionate was
not effective as a fungistatic agent at a level of 0.6 percent. Propionamide and propionanalide also were
not effective at a level of 0.1 percent which was the maximum level tested. l

Short-chain fatty acids——butyric. valeric and caproic acids—inhibited or delayed heating when they
were added to com meal at a level of 0.1 to 0.2 percent. The heat-inhibiting activity of the fatty acid
decreased as the number of carbons in the fatty acids increased. Citric and succinic acid did not inhib-
it heating. Acetyl acetone and benzyl mucochlorate inhibited heating when added to corn meal at a
level of 0.1 percent. Compounds which inhibited heating were more effective when the moisture content of
the ingredient was only slightly above the critical level. They were less effective at higher moisture con-
tents.

Four products which the manufacturer claimed inhibited heating were tested for their fungistatic activ-
ity. None of these materials was active at a level several times that recommended by the manufacturer as
being effective.

ACKNOWLEDGMENTS

This manuscript was adapted in part from a dissertation submitted to the Graduate School of the Ag-
ricultural and Mechanical College of Texas by Iohn V. Halick in partial fulfillment of the requirement for
the Degree of Doctor of Philosophy.

Acknowledgment is made to A. I. Kaplan. president of Southwestern Sugar and Molasses Company.
New York. New York. for a grant-in-aid which supported this study in part; to L. T. Spence. Merck and
Company. Rahway. New Iersey. for acetyl acetone and benzl mucochlorate; to F. M. Iornlin. E. I. du Pont de
Nemoures G Company. Wilmington. Delaware. for propionic acid and calcium propionate.

CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v . . . . . v . . . . 3

Experimental, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v . . . . . . 3

Propionates as Inhibitors of Heating . . . v . . . . . . . . . . . v . . v . . . . . . . . . . . . . . . . . . . . . . . . . , . , . v . . . . . 4  . . v v . . 3

Heat-inhibiting Action of Various Compounds. . . . . . . . . . . v v . , v . v . . . . . . . . . . v v v . . . . . . . . . . . . . v . . . . v v 4

Propionate and Sorbic Acid as Inhibitors of Heating in Different Ingredients . 4 . . . . . v . v v . . v . . . . . . . 4

Heat-inhibiting Action of Propionate in the Presence of Sodium Acetate and Yeast Extract . , . . . . . . .. 5

Heat-inhibiting Activity of Commercial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v . . . . . . . . v . . v . . . . . v 5

Use of Inhibitors in Feeds . . . . . . . . . . , . . . . . . v . . . v v v v . . . . . . v v . . v . . . . . . v . . . . . . . . . . . . _ . . . . . . v . . _ . . . . . 5

References . . . . v . v . v . . v . . . . . . . . . . . . . . . . . v . v . v . . . . . . . . v . . . . . . . . . . . . . . . . v . v v v . . . . . . . . . . . . . . . . v . . . v . 6

  
   
   
 
   
   
   
   
   
   
  
  
   
  
  
 
 
   
  
  
   
   
  
   
   
 
   
   
  
   
 
   

5' HE DISCOVERY OF A LOW-COST compound that
i would inhibit the growth of molds and the
" companying heating, which would not decrease
he value of grain or feed for animal consump-
'on, would have great economic importance in
“e grain, feed, animal and human food indus-
les.

Milner, Christensen and Geddes (1947) tested
pproximately 100 compounds for their mold in-
'bitory action on wheat stored with a moisture
ntent of 1'6 to 25 percent. On the basis of
1 toxicity to seed with a moisture content of
0 percent and a fair to high toxicity to molds,
'ght compounds were tested extensively as mold
hibitors. The order of their decreasing ac-
'vity were: 8-hydroxyquinoline sulfate, thio-
rea, p-aminobenzoic acid, sulfonilamide, benzene
lfonamide, 2-aminothiazole, chloramine B and
lcium propionate.

Wolford (1945) used sodium propionate to
uce the growth of micro-organisms in fruits
d vegetables, and Lenhart and Cosens (1949)
.1. d propionic acid to inhibit the growth of molds
sugar solutions.

i‘ Dubos (1950) reported that short-chain or-
nic acids, such as acetic, propionic, butyric,
proic and capric, inhibited the growth of mam-
-lian tubercle bacilli to a marked degree. More
T ‘ently sorbic acid has been used extensively to
' ibit the growth of molds in various foods,
illips and Mundt (1950), Jones and Harper
V952), Smith and Rollin (1954a) (1954b) ; Mel-
' k, Luckmann and Gooding (1954) ; Deuel, A1-
l-Salter, Wei] and Smyth (1954); Boyd and
rr (1955); Beneke and Fabian (1955), Mel-
k, Valteich and Hackett (1956).

a It has now become common practice for com-
rcial bakeries and other food processors to
'- inhibitors to prevent the growth of molds
various food products. Sorbic acid has been
a. recently in human foods to a greater extent
n propionate because it is a more effective
'bitor and it does not impair the flavor of
's as much as propionate.

Halick and‘ Richardson (1953) used calcium
pionate to prevent heating and the growth
F olds in corn meal that contained 17.4 percent

pectively, professor, Department of Biochemistry and
trition; and chemist, USDA, Substation No. 4, Beau-
t, Texas, formerly graduate student, Department of
- hemistry and Nutrition. -

Studies on 9m! Spill/aye . . .

ifleat Inhibiting Activity 0/ Various éampounds and fiommcrcia/ Products
‘ ‘L. R. RICHARDSON and IOHN V. HALICK*

moisture. These studies were carried out at a
temperature of 90° F and a relative humidity
of 770 percent. Corn meal with 0.3 percent cal-
cium propionate did not heat in 42 days. With-
out propionate and stored under the same con-
ditions, it started to heat in 2 days and reached
a maximum temperature of 126° F in 3 days.

The studies using various inhibitors to pre-
vent heating in feed ingredients were continued
and the results are reported in this bulletin.

EXPERIMENTAL

Propionates as Inhibitor oi Heating

Since earlier tests have shown that calcium
propionate prevented heating in corn meal, a test
was carried out to determine whether other pro-
pionates also were effective inhibitors. The pro-
pionates tested were propionic acid, propionic
anhydride, calciumpropionate, sodium propionate,
propionamide and propionanalide. The com-
pounds were mixed with the corn meal and heat-
ing studies were carried out in the apparatus
previously described by Halick and Richardson
(1953). The effect of adding various amounts
of these propionates on the growth of molds and
the accompanying heating in corn meal is sum-
marized in Table 1. Propionic acid and propionic
anhydride at a level of 0.1 percent inhibited the

TABLE 1. EFFECT OF VARIOUS PROPIONATES ON HEAT-
ING AND THE GROWTH OF MOLDS IN YELLOW CORN

MEAL
. Mold
 Inhibitor P??? $3221’. 
orn meal. ‘Y a e days '
c ° per gm
15.2 Propionic acid None 7 525.0
15.1 " " 0.1 1.8
14.9 Propionic anhydride None 7 360.0
14.8 " " 0.1
15.2 Calcium propionate None 7 525.0
15.1 " " 0.1 7 525.0
15.0 " " 0.3 3.5
14.8 Sodium propionate 0.1 7 565.0
14.8 " " 0.3 7 245.0
13.4 " " 0.6 14 184.0
15.6 Propionamide 0.1 5 800.0
14.4 " 0.3 6 665.0
15.9 l Propioncmalide 0.1 6 650.0
15.8 " 0.3 ll 205.0

growth 0f molds and heating in corn meal con-
taining approximately 15 percent moisture. The
same meal without an inhibitor started to heat
in 7 days and reached a maximum temperature
0f 120° F in 10 days. Calcium propionate in-
hibited the growth 0f molds and heating at a
level of 0.3 percent, but was not effective at a
level of 0.1 percent. Sodium propionate did not
inhibit the growth of molds and heating at a
level of 0.6 percent. Both propionamide and pro-
pionanalide were not effective as mold inhibitors
at a top level of 0.3 percent under the conditions
used in these experiments. Why calcium pro-
pionate was more effective than sodium propio-
nate is not understood. It is well known that
propionates are more effective inhibitors of the
growth of molds in an acid environment and it
is possible that calcium propionate may provide
a more acid environment than the sodium salt.

Heat-inhibiting Action oi
Various Compounds

Short-chain fatty acids of the same homolo-
gous series as propionic acid have been reported
to inhibit the growth of various microorganisms.
In view of these reports, several fatty acids and

TABLE 2. EFFECT OF VARIOUS COMPOUNDS ON HEAT-
ING AND THE GROWTH OF MOLDS IN YELLOW CORN

MEAL
Moisture Percent started 
content oi Inhibitor added to heat, 10,000
corn meal. ‘Y, d‘!!! per gm
15.2 Butyric acid None II 525.0
15,2 " " 0.10 60.0
15.2 " " 0.30 0.3
18.3 " " None 2 1550.0
18.9 " " 0.10 I2 650.0
18.0 " " 0-30
14.0 Valeric acid None 8
14.0 " " . 0-10
13.4 Caproic acid None 12
13.4 " " 0.10 22
13.4 ” " 0-20
16.3 Sorbic acid None 3
16.3 " ” 0.10 17
16.3 " " 0.20 33
16.3 " " 0.30
15.2 Acetyl acetone None 9 100.0
15.2 " " 0.02 l0 360.0
15.2 " " 0.05 24 325.0
15.2 " " 0.10 V 5.0
15.2 Benzyl mucochlorate None 10 550.0
15.2 " " 0.02 12 235.0
15.2 " " 0.05 19 155.0
15.2 " " 0.10 1.5
15.7 Citric acid None 6
15.6 " " 0.25 7
15.5 " " 0.50 7
15.3 " " 0.75 7
15.5 " " 1.00 7
15.4 Succinic acid None 7
15.6 " " 0.25 7
15.5 ” " 0.50 7
15.6 " " 0.75 7
15.7 " " 1.00 7

other compounds which offered some promise
were tested as inhibitors of heating in corn meal.
The results are summarized in Table 2.

Butyric acid at a level of 0.1 percent prevented 
heating in corn meal containing 15 percent mois- 
ture. When the moisture content of the meal .
was increased to 18 percent, 0.1 percent of butyric _
acid delayed the onset of heating, but 0.3 percent i;
was required to prevent it completely._- Valerie 
acid at a level of 0.1 percent also prevented heat- ;
ing in corn meal that contained 14 percent mois- ;
ture. This acid was not tested with meals that :
contained a larger amount of moisture. Caproic .
acid did not prevent heating at a level of 0.1 1.
percent in corn meal containing 13.4 percent .
moisture. At a level of 0.2 percent, however, it "
prevented heating. Caproic acid was less effec- Q
tive than butyric or valeric acid. The heat in- g
hibiting activity of these acids appeared to de- _
crease as the number of carbons in the chain,

increased.

. Sorbic acid at a level of 0.3 percent prevented ’
heating for at least 42 days when the corn meal
contained 16.3 percent moisture. At a level of I
0.1 and 0.2 percent, sorbic acid delayed heating.
from 3 to 17 and 33 days, respectively. Acetyl
acetone and benzyl mucochlorate, each at a level 
of 0.1 percent, prevented heating and the growth ’
of molds completely in corn meal containing 15.2 "
The latter two compounds
were not effective inhibitors of heating at a level a
of 0.02 or 0.05 percent. Citric and succinic‘
acids did not prevent heating and the growth of
molds when added to corn meal at a level of 1.0 _

percent moisture.

percent.

Propionate and Sorbic Acid as Inhibitors
oi Heating in Diiierent Ingredients

Tests were carried out to determine whether;
calcium propionate and sorbic acid prevented heat-
ing in a variety of feed ingredients. Both com- i
pounds were added separately to the ingredient}
at a level of 0.3 percent. The flasks containing T
the ingredients with and without the inhibitor
were then stored in the heating apparatus under f
the same conditions used in other studies. The g
ingredient used and days required for the var-i
ious treatments to start heating are given in

Table 3.

The data show that calcium propionate and
sorbic acid prevented heating in every ingredi-
ent used when its moisture content was only .1
slightly above the critical level. When the mois- 
ture content of an ingredient was 1 to 3 percentf
above the critical level, the inhibitors were much
For example, meat and bone ’
scraps have a critical moisture level of 10.0 per-y
cent. When the moisture content was increased
to 11.1 percent, it started to heat in 17 days.
without the inhibitors, and did not heat in 42:
days with either inhibitor. When the moisture
was increased to 12.5 percent, the sample con-

less effective.

  
 
   
  
  
 
   
   
   
     
   
   
    
  
   
   
  
  
 
  
   
   
  
    
  
    
   
 

TABLE 3. INHIBITION OF HEATING IN FEED INGREDIENTS
A ONTAINING VARIOUS AMOUNTS OF MOISTURE WITH
. ' PROPIONATE AND SORBATES

I Days to start heating
. _ With inhibitors
Moisture

lnsredieni Qgnfent, 7° 1 _Wi§h9u1 Calcium Sorbic
- mhlbllm‘ propionate. acid.
0.3% 0.3%

Wheat shorts 15.3 20

aked barley 14.9 -1l

‘I ound oats 14.9 12

a 1.1. meal 13.6 12

orghum gluten meal 11.9 19

i ' eed meal 12.4 19

I eat 6. bone scraps o 11.1 17

f. eat 6. bone scraps 12.5 9 15 I5

‘I ybean meal 15.0 30 30

‘Cottonseed meal 12.4 21

Cottonseed meal 14.7 8 6 l0

itaining the inhibitors started to heat in about
the same time as the sample containing 11.1 per-
: ent moisture and no inhibitor.

' 1 The results with soybean and cottonseed meals
ontaining large amounts of water were similar
~ 1 those with meat and bone scraps. The results
how that calcium propionate and sorbic acid
inhibit the growth of molds and heating i11 prac-
ically all feed ingredients. However, the ac-
ivity of both compounds depended on the amount
f inhibitor present and on the moisture content
if the ingredient.-

i eat-inhibiting Action oi Propionate in the
i’ esence oi Sodium Acetate and Yeast
tract

It was observed in studies which were carried
ut in a nutrient medium with an isolated mold,
spergillus niger, that the addition of sodium
‘cetate or a yeast extract to the medium de-
‘troyed the mold inhibiting action of the propio-
ate. As a result of this observation, sodium
lcetate and a yeast extract were each added to
’ rn meal containing 0.3 percent calcium propio-
ate. The meal contained 15.8 percent moisture
nd heated readily without the propionate. When
he propionate was present, the mixture did not

CABLE 4. HEAT INHIBITING ACTION IN CORN MEAL BY

EXTRACT
i Moisture C l . st rt c“
_ content oi l! mum Supplement a e °
M meal, 7° prozpionate. °/o heat. days
' 15.8 None None 4
15.8 0.3 None ‘
15.8 0.3 Yeast extract 1%
15.8 0.3 Sodium acetate 1%

i‘ - heating observed

VOPIONATE IN THE PRESENCE OF ACETATE AND YEAST '

heat even though sodium acetate or yeast ex-
tract was present. These data show that the
sodium acetate or yeast extract did not destroy
the inhibiting action when the propionate was
added to an ingredient. These results are sum-
marized in Table 4.

Heat-inhibiting Activity oi
Commercial Products

Several commercial products which were
claimed by the manufacturer to inhibit heating
and the growth of molds were tested. These
data are summarized in Table 5. None of the
products tested was effective even at levels that
would make their use prohibitive.

Use oi Inhibitors in Feeds

The question may be asked whether the use
of inhibitors in mixed feeds would be practical.
Calcium propionate and sorbic acid are allowed
by the Food and Drug Administration to be used
as fungistatic agents in human foods and pre-
sumably are permissible for animal feeds. There-
fore, it appears that these two compounds could
be used to inhibit heating in animal feed, pro-
vided the cost would not be too great. Whether
the cost is too great can be determined only
by the feed manufacturer and the manufacturer
of the inhibitor. In private conversations with
one of the authors (LRR), manufacturers have
expressed doubt that it would be feasible to use
propionates as inhibitors of heating in mixed
feeds under present conditions. It appears that
sorbic acid would be prohibitive at the present
time for the same reason.

TABLE 5. ACTIVITY OF COMMERCIAL PRODUCTS WHICH
THE MANUFACTURER CLAIMED WOULD INHIBIT HEATING

Motistlzrei Amount oi Started to
60521152611? 7° product added heat. days
15.47 None 3
15.26 3/4 lb per ton‘ 4
15.51 5 lb per ton 4
15.44 1 lb per ton‘ 4
15.43 5 1b per ton 3
15.64 None 3
16.00 1.0% 2
16.25 1.5% 3
16.19 2.0% 3
16.64 2.5% 3
16.18 None 4
15.93 300 ppm 4
16.14 350 ppm 4
16.11 400 ppm 4
16.02 500 ppm 4
16.91 None 4
16.73 0.00$[, 4
16.6 0.01 o 4
16.72 0.05% 4
16.55 0.10% 4

‘Amount recommended as being eiiective.

The cost of using acetyl acetone at the rate
of 0.1 percent would be approximately $3.50 per
ton and the cost of using benzyl mucochlorate
would be about the same or more at the present
time. Therefore, these two compounds and fatty
acids would be too expensive.

It appears that the use of materials with
moisture contents below the critical level is the
only practical procedure to prevent heating in
mixed feeds and feed ingredients. The critical
moisture contents of approximately 30 feed in-
gredients are given in Texas Station Bulletin 860.

REFERENCES

Beneke, E. S. and F. W. Fabian 1955. Sorbic acid as
a fungistatic agent at different pH levels for molds iso-
lated from strawberries and tomatoes. Food Tech. 9:486.

Boyd, J. W., and H. L. A. Tarr 1955. Inhibition of mold
and yeast development in fish products. Food Tech. 9:411.

Deuel, H. J. J12, R. Alfin-Salter, C. S. Weil and H. F.
Smyth Jr. 1954. Sorbic acid as a fungistatic agent for
foods. I. Harmlessness of sorbic acid as a dietary com-
ponent. Food Research 19:1.

Dubos, R. J., 1950. The effect of organic acids on mam-
malian tubercle bacilli. J. Exptl. Med., 92:319.

Halick, J. V., and L. R. Richardson 1953. Influence of
goisture on heating in feeds. Tex. Agri. Expt. Sta.
ul. 768.

  
  
   
  
  
 
 
 
 
  
  
 
   
 
 
 
 
  
  
  

Jones,  H., and G’. S. Harper 1952. A preliminary study
of factors effecting the quality of pickles on the Canadian
market. Food Tech. 6:304. *

Lenhart, J. and K. W. Cosens 1949. Molds and bacteria
curbed in immersion freezing. Food Ind. 21:442. I

Melnick, 1)., H. W. Valteich, and A. Hackett 1956. Sorbic
acid as a fungistatic agent for foods. “XI. Effectiveness
of ascorbic acid in protecting cakes-i; Food Research
21:133. ' i’ »

Melnick, D., F. H. Luckmann, and C. M. Gooding 1954. Sor-
bic acid as. a fungistatic agent for foods. VI: Metabolic
degradation of sorbic acid in cheese by molds and mech-
anism of mold inhibition. Food Research 19:44. .

Milner, M., o. M. Christensen, and W. F. Geddes 1947*
Grain storage studies. VII. Influence of certain mol
inhibitors on respiration of moist wheat. Cereal Chem
24:507.

Phillips, G. F., and J. O. Mundt 1950. Sorbic acid a.
an inhibitor of scum yeasts in cucumber fermentations.
Food Tech. 4:291. a

Smith, D. P., and N. J . Rollin 1954a. Sorbic acid as a fungi
static agent for foods. VIII. Need and efficacy in pro
tecting packaged cheese. Food Tech. 8:133. ‘

Smith, n. P., and N. J. Rollin 1954b. Sorbic acid as a fungi
static agent for foods. VII. Effectiveness of sorbic aci
in protecting cheese. Food Research 19:59. '»

Wolford, E. R., 1945. The effect of sodium propiona q
on microorganisms. J. Bact. 50:235. .

[Blank Page in Original Bulletin]

     

k um srmou
Q us: stosrmous

I n m» luminous:
4 ooorwmo srmnus

Location of field research units oi the Texas
Agricultural Experiment Station and cooperating
agencies

IN THE MAIN STATION, with headquarters at College Station, are 16 subj»
matter departments, 2 service departments, 3 regulatory services and .
administrative staff. Located out in the major agricultural areas of Texas =
21 substations and 9 field laboratories. In addition, there are 14 cooperafr
stations owned by other agencies. '
Forest Service, Came and Fish Commission of Texas, Texas Prison Syst
U. S. Department of Agriculture, University of Texas, Texas Technologi,
College, Texas College of Arts and Industries and the King Ranch. So‘
experiments are conducted on farms and ranches and in rural homes. a

ORGANIZATION

THE TEXAS STATION is conducting about 400 active research projects, grou
in 25 programs, which include all phases of agriculture in Texas. Amo

these are:

Conservation and improvement of soil
Conservation and use of water
Grasses and legumes

Grain crops

Cotton and other fiber crops
Vegetable crops

Citrus and other subtropical fruits
Fruits and nuts

Oil seed crops

Ornamental plants

Brush and weeds

OPERATION

Insects

Two additional programs are maintenance and upkeep, and central service’

Research results are carried to Texas farmers,
FGILChITLGIZ ‘and homema/cers by county agents
and specialists of the Texas Agricultural Ex-

tension Service

jOl/[Cly 2 KQJQCLPCA n95 jOWLOPFOl/Ui’) I"OgI"€55

State-wide Research

i‘ i 4y

‘k

The Texas Agricultural Experiment Station
is the public agricultural research agency
of the State of Texas. and is one of ten

parts of the Texas AcSM College System

    
   
   
  
   
 

Cooperating agencies include the Te

Beef cattle

Dairy cattle

Sheep and goats

Swine

Chickens and turkeys
Animal diseases and parasites
Fish and game

Farm and ranch engineering
Farm and ranch business
Marketing agricultural products j
Rural home economics '5
Rural agricultural economics
Plant diseases

AGRICULTURAL RESEARCH seeks the WHATS. the
WHYS, the WHENS. the, WHERES and the HOWS oi
hundreds oi problems which confront operators of farms
and ranches. and the many industries depending on
or serving agriculture. Workers of the Main Station
and the field units of the Texas Agricultural Experiment
Station seek diligently to find a solutions to these
problems. -