Effect of Pelleting of Poultry Feed on the Activity of Molds and Mold Inhibitors 1,2

ZHANET TABIB, FRANK T. JONES, and PAT B. HAMILTON

Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27650

(Received for publication February 18, 1983)

ABSTRACT Paired samples of mash and pellets made from the mash were taken at feed mills and
compared for their mold count and fungal activity as measured by metabolic CO 2 production.
Four paired samples contained Mold-X, a commercial mold inhibitor, of which the major active
ingredient is propionic acid, and four paired samples did not contain a mold inhibitor. PeIIeting
reduced the mold counts by a factor of about 100 to 10,000, depending upon the sample. In-
cubation of the samples at 29 C after adjusting the water content to 20, 25, or 30% caused an
approximate doubling of the time required for onset of metabolic CO 2 production by the pelleted
feed as compared to the mash. Incorporation of the mold inhibitor into mash appeared to give a
similar lag in onset of CO 2 production. Pellets made from mash containing the mold inhibitor had
an additional doubling in the delay of onset of CO 2 production. In a model system composed of
corn meal to which graded doses of propionic acid were added, the concentration of inhibitor that
provided 50010 inhibition was found to increase with time of incubation. The lag in onset of CO 2
production in this system was found to be 1 or 2 days depending on whether the corn meal was
heated for 5 min at 60 or 70 to 85 C, respectively. Heat and propionic acid also interacted to
reduce the mold count of corn meal in the model system. It would appear that the effectiveness of
propionic acid as a mold inhibitor can -be greatly increased by the pelleting process and that a
decrease in the fungal burden of feed is an attribute of pelleting.
(Key words: mold count, feed, pelleting, CO 2 production, Mold-X, propionic acid, heating)
1984 Poultry Science 63 :70-75

INTRODUCTION poultry salmonellosis (MacKenzie and Bains,

The pelleting of poultry feed has been
widely practiced for decades because it in-
creases uniformity of feed consumed by the
bird and because it improves the digestibility of
the feed (Calet, 1965; McEllhiney, 1980). The
results have been enhanced growth rate and
feed conversion efficiency in segments of the
poultry industry that have adopted the prac-
tice. Indeed, the usefulness of this practice has
rest.:lted in even the starter rations for chickens
being pelleted and then crumbled to a man-
ageable particle size with a consequent de-
creased cost of production of birds (Runnels et
al., 1976). A commonly overlooked aspect of
the pelleting process is that it influences the
microbiological burden of the poultry feed. The
high temperature during the pelleting process
has been important in breaking the cycle of
1 Paper No. 8476 of the J oumal Series of the
North Carolina Agricultural Research Service, Raleigh,
NC.
2 The use of trade names in this article does not
imply endorsement by the North Carolina Agricul-
tural Research Service nor criticism of similar ones
not mentioned.
1976; Patterson, 1969). An approximate
1000-fold reduction in the numbers of En-
terobacteriaceae during pelleting of feed has
been reported (Mossel, 1971; Stott et al., 1975;
Tabib et al., 1981). The reduction of the mold
count of feed during the pelleting process has
also been reported (Tabib et al., 1981). How-
ever, the importance, if any, qf this reduction
in mold count has not been investigated.
The relationship of pelleting to the microbial
status of poultry feed may be doubly im-
portant, because mold inhibitors such as sorbic
and benzoic acids have been reported to in-
teract with heat to produce a greater killing of
microorganisms in broth cultures (Beuchat,
1981a,b; Robinson and Hills, 1959; Shibasaki
and Tsuchido, 1973). Although there is low
correlation between number of microorganisms
and their acti~ity in feed and ingredients (Tabib
et al., 1982), the necessity for control of
mycotoxins in the poultry industry suggested
that the effect of pelleting on the activity of
molds and mold inhibitors should be investi-
gated.
The present communication reports on the
microbial status of pelleted and unpelleted feed
as influenced by propionic acid and a com-

70
 PELLETING AND MOLD
mercial inhibitor, Mold-X, which contains
propionic acid.
MATERIALS AND METHODS
Samples. Paired samples of mash and the
pellets being made from the mash were col-
lected within 5 min of each other at different
feed mills making broiler chicken feed. The
samples were placed in plastic bags and brought
to the laboratory where they were comminuted
in a high speed blender for 3 min prior to
assaying for mold count and fungal activity.
Four of the paired samples contained Mold-X, a
commercial inhibitor, while samples from four
other mills did not contain any added mold
inhibitor.
Mold Counts. One gram of the comminuted
feed samples was added to 10 ml of .85% NaCI
containing .05% Triton X-100 (Fisher Scientific
Co., Raleigh, NC) and shaken for 20 min on a
wrist action shaker. Further dilutions were
made in .005% Triton X-100 in .85% saline
after permitting larger particles to settle for 1
min. The high concentration of the nonionic
detergent (Triton X-100) in the initial suspen-
sion was required to obtain maximal counts.
The dilution series utilized a lower concen-
tration for the same reason. The molds were
counted in pour plates of Sabouraud's Dextrose
Agar (Difco). The plates were incubated 48 hr
at 29 C before counting on a Quebec colony
counter (New Brunswick Scientific Co., New
Brunswick, NJ).
Assessment of Fungal Activity. The moisture
content of the feed and corn meal was de-
termined gravimetrically by drying overnight at
110 C. The moisture content was then adjusted
to the desired level by the addition of deionized
water. There were four replicates per treatment
and each replicate consisted of 5.5 cm 3 of feed
or corn meal in culture tubes (16 x 150 mm)
which were capped with rubber septa designed
for use with serum bottles (Dixon and Hamil-
ton, 1981). The septa prevented the exchange
of headspace gas of the tube with the ambient
atomosphere so that CO 2 produced as a result
of microbial metabolism could be assessed in a
quantitative fashion. The replicates were
incubated at 29 C for 3 days in the case of feed
samples without Mold-X or for 6 days in the
case of samples that did contain the mold
inhibitor. The C02 content of the headspace
gas was determined daily by gas chromatog-
raphy (Model GC-6AM, Shimadzu Scientif-
71
ic Co., Baltimore, MD) using a thermal con-
ductivity detector, a stainless steel column (2 m
X 5.3 mm i.d.) with a packing of Poropak-Q,
and helium as the carrier gas (40 m1!min).
Injection port and detector temperatures were
120 C and the column temperature was 100 C.
Sampling of headspace gas and injection into
the column was accomplished with a dis-
posable tuberculin syringe and needle. The
amount of CO 2 in the sample of headspace gas
was quantitated by the peak height method.
Inhibitors. Propionic acid (Fisher Scientific
Co., Raleigh, NC) was diluted to 5% in de-
ionized water and the amount calculated to
yield the desired final concentration was added
to corn meal before final adjustment of the
moisture content. Mold-X, a commercial mold
inhibitor (AgResearch Mfg. Co., Inc., Wheeling,
IL 60050) composed of propionic, acetic,
sorbic, and benzoic acids, was purchased by the
integrated broiler operations for their own use
in feed.
Interaction of Inhibitor and Heat. After
determining gravimetrically the moisture con-
tent of corn meal from the university feed mill,
the moisture content was adjusted to 25% by
the addition of a calculated amount of de-
ionized water containing propionic acid to yield
a final concentration of 0, .125, .25, .5,1, and
2 mg/g of corn meal. Replicate tubes (8 per
treatment) were placed in a water bath at 60,
70, or 85 C for 5 min prior to cooling in a
running tap water bath. After cooling, 4 tubes
per treatment were used in determining their
mold count. The other 4 tubes per treatment
were incubated at 29 C for 6 days and the
concentration of propionic acid that inhibited
the CO 2 production by the corn meal by
one-half (IC so ) was determined daily by graphi-
cal estimation.
RESULTS
In four paired samples of mash and pellets
free of a mold inhibitor and whose moisture
content in subsamples had been adjusted to 20,
25, and 30% water (Fig. 1), the onset of micro-
bial activity as measured by release of metabol-
ic CO 2 from the feed was dependent on both
the moisture content and the time of incuba-
tion. In general, there appeared to be a delay of
at least one day in the onset of CO 2 production
by pellets as compared to the time of onset in
the mash from which the pellets were made.
This delay was generally independent of mois-
72 TABIB ET AL.

ture content. Once CO 2 production by the Reduction in mold count by the pelleting
pellets started, the rate of production roughly process (Table 1) was more dramatic than the
paralleled that obtained with the mash. The decrease in activity as measured by CO 2 pro-
results obtained from samples contammg duction. The presence of Mold-X in the feed
Mold-X are shown in Fig. 2. The onset of CO 2 appeared to have little effect on the reduction
production in mash containing Mold-X was of mold count associated with the pelleting
delayed by approximately one day past the process since mold counts were reduced to
time observed with mash that was free of about 100 cfu/g of pelleted feed, regardless of
Mold-X (Fig. 1). Pelleting of the mash that the presence or absence of inhibitor. The
contained Mold-X caused another lag in the influence of heating to various temperatures on
onset of CO 2 production (Fig. 2). This lag, the IC so of propionic acid in corn meal is
depending upon the sample and moisture shown in Figure 3. This model system, which
content, ranged from 1 to 2 days. In agreement was intended to approximate the heating that
with the results obtained with paired samples occurs during the pelleting process, showed a
free of Mold-X (Fig. 1), once CO 2 production delay of 1 day in the onset of microbial activity
commenced in pelleted material containing
Mold-X, the rate was approximately identical
to that of the untreated material (Fig. 2).

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TIME (days)

0
0 3 0 2
TIME (days)
FIG. 1. Comparison of metabolic CO 2 production
by four paired samples (A, B, C, and D) of untreated
mash (0-0) and pellets (e-e) adjusted to have a mois-
ture content of 20, 25, and 30% of water. Data
points are means of four replicates incubated at 29 C
and the CO 2 production was measured daily for
3 days. Vertical bars on points are SE; absence of
bars on points indicates the SE was smaller than
the space occupied by the data point.
3 0 3 FIG. 2. Comparison of metabolic CO 2 production
by four paired samples (E, F, G, and H) of mash
(0-0) and pellets (e-e) containing Mold-X (1 mg/g).
Moisture content of the paired samples was adjusted
to 20, 25, and 30% moisture content prior to in-
cubation at 29 C. The metabolic CO 2 produced was
measured daily for 6 days. Each data point is the
mean of four replicates and vertical bars on points
are SE. Absence of bars on a point indicates the
SE was smaller than the space occupied by the data
point. Note that the time scale is twice that of Figure
1.
 PELLETING AND MOLD 73

TABLE 1. Reduction of mold counts in chicken in material heated to 60 C for 5 min, a lag
feed by the pelleting process' of 2 days in material heated to 70 C, and a
delay of about 2.5 days in material heated to
Paired Inhibi- 85 C. The IC so in both heated and unheated
samples tor Mash Pellets
corn meal was dependent upon the time of
incubation, and the IC so increased with in-
A 5X 10' 7 X 10 2
B 2 X 10 6 1 X 10 2 creasing time of incubation. Once the microbial
C 8 X 10' .5 X 10 2 activity commenced in the heated material, the
D 4X 10 5 1 X 10 2 rate of increase in the IC so approximately
E + 1X 106 1 X 10 2 paralleled that in the unheated material.
F + 4 X 10 6 .2 X 10 2
2X 105 1 X 10 2
The influence of propionic acid (2 mg/g of
G +
H + 2X 105 1 X 10 2 corn meal) and of heating on the mold count of
corn meal is shown in Table 2. In general, the
'Paired samples of mash and the pellets made higher the moisture content of the corn meal,
from it were collected from feed mills. Tabular values the greater was the effect of the heat exposure
are mold counts per gram feed and are the means in reducing the mold count. The higher the
of duplicate determinations. temperature, the greater was the decrease in
mold count. There was a strong interaction
between temperature and propionic acid in
reducing the mold count. This interaction on
mold count between heat and mold inhibitor
was more readily apparent in the model system
than it was in feed samples taken from the
field.

DISCUSSION

The pelleting process reduced the mold

0
Q)
count in chicken feed by factors from 100 to
E 10,000 times depending upon the sample
co
~
0
(Table 1). Because a mold count does not
u
2.0 distinguish spores, sporangia, and hyphal
'"
""0 fragments, a mold count is a count of prop-
u agules or colony forming units that may have
0
u a very low correlation with total fungal biomass
co
0
a.
0
5.
TABLE 2. Interaction of heat and propionic acid
'"
E on the mold count of corn meal'
2.0
0
85°C Moisture
'" 1.0 Tempera- Propionic
U
ture acid 15 20 25
.50

(C) (%)
60 8X 105 7 X 10' 2 X 10'
+ 6X 10' 6 X 10 2 2 X 10'
70 2X 10' 1 X 10' 1 X 10 2
+ 1 X 10' <10 <10
85 2 X 10' 2 X 10 2 <10
FIG. 3. Influence of heating on the IC,o of pro- + <10 <10 <10
pionic acid in corn meal. The moisture content of the
meal was adjusted to 25% and after heating 5 min 'Corn meal with a total mold count of 8 X 10s/g
at the indicated temperature the heated (e-e) and was adjusted to the indicated moisture content and
unheated (0-0) replicates were incubated at 29 C. heated in a water bath at the indicated temperature
CO 2 production was measured daily and the IC so for 5 min prior to cooling and plating. Propionic
calculated graphically from the means of four repli- acid concentration was 0 or 2 mg/g of feed. Tabular
cates per treatment. values are means of four replicates.
74 TABIB ET AL:
(Tabib et al., 1982). More important than this
dramatic reduction in mold count was the
decrease in fungal activity of the pelleted
feed (Fig. 1). The pelleting process caused a
delay of about 1 day in the onset of fungal
metabolic activity under the conditions tested.
A comparison of the results obtained with
the mash samples in Figure 1 with those of the
mash samples in Figure 2 may not be valid
because they are not paired samples; never-
theless, it is interesting that the onset of CO 2
production was delayed approximately 1 day
by the addition of inhibitor. This suggested that
the inhibitor was effective in inhibiting fungal
activity. The combination of mold inhibitor
plus pelleting resulted in an even greater delay
in the onset of microbial activity (Fig. 2). These
results, which were confirmed in a model in
vitro system (Fig. 3 and Table 2), suggest that
1) reduction of the fungal burden may be
added to the list of positive attributes of the
pelleting process, 2) there is a synergistIc
interaction between organic acid mold in-
hibitors and heat in feed, and 3) a large part of
the apparent effectiveness of organic acids as
mold inhibitors resides in the fact that they
interact with heat during the pelleting process.
Interaction between heat and organic acid
inhibitors has previously been reported in
broth cultures offungi (Beuchat, 1981a,b).
Another interesting aspect of the present
results was that the primary effect of mold
inhibitors and pelleting was a time lag before
the onset of activity. Once microbial activity
began in treated samples, the activity approxi-
mately paralleled that observed in untreated
samples. One explanation for this apparent
disappearance of the inhibitory activity would
be disappearance of the inhibitor itself as a
result of microbial metabolism. Such metab-
olism of antifungal agents and consequent relief
of inhibition has been reported for gentian
violet (Hall and Hamilton, 1981), sorbic acid
(Doyle and Marth, 1975), ammonia (Lancaster
and Bothast, 1976), and propionic acid (Burrell
et al., 1973).
A practical yet fundamental question is
whether the reduction in fungal metabolic
activity observed in the present study results in
less production of mycotoxins and a conse-
quent lessened deleterious effect in poultry.
Certainly, there can be no formation of my-
cotoxins in the absence of respiration with the
possible exception of a theoretical nonoxida-
tive and noncarboxylative transformation of
preformed precursors which would occur only
in minute quantities. Hence, the delayed onset
of respiratory CO 2 production associated with
the use of mold inhibitors clearly suggests
freedom from mycotoxin production during
the lag period. Once respiration commences,
the effect of the mold inh"ibitor lessens and the
freedom from mycoto~in production would be
a matter of degree. On a practical basis, freedom
from mycotoxin production could be assured
by 1) increased concentration of inhibitor (Fig.
3), 2) decreased moisture content (Fig. 1
and 2), 3) decreased time between production
and consumption (Fig. 1 and 2), 4) increased
heat exposure during pelleting (Fig. 3), and 5)
as seems likely, a combination of these prac-
tices.
In the highly competitive poultry industry,
managers examine all feed ingredients to try to
eliminate every unnecessary expense. All too
frequently, economizing has resulted in the
elimination of mold inhibitors from the feed
because evidence of a beneficial effect was
presumably lacking. The present results would
appear to offer proof that organic acid mold
inhibitors have a detectable beneficial effect
under commonly used or attainable conditions
when included in feed. A final proof would be
to document under field conditions the findings
elucidated here. Despite these promising results,
it should not be forgotten that a low mold
count does not make feed immune to fungal
attack. Pelleting as it is normally practiced
results III net addition of water to feed and
makes it more easily spoiled (Tabib et al.,
1981).
ACKNOWLEDGMENTS
We thank Scott Johnson and Nancy Bailey
for technical assistance. This work was sup-
ported in part by the AgResearch Mfg. Co.,
Inc., Wheeling, IL 60090, whose help is greatly
appreciated.
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 PELLETING AND MOLD
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