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thesis entitled

Some Factors Influencing Production

of Aflatoxins by Aspergillus flavus

 

in Fermented Sausages
presented by

Valente Aivarez Barrera

has been accepted towards fulfillment
of the requirements for

M.S. Food Science

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SOME FACTORS INFLUENCING PRODUCTION OF
AFLATOXINS BY Aspergillus flavus IN FERMENTED SAUSAGES

By

Valente Alvarez-Barrera

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1981

ABSTRACT

SOME FACTORS INFLUENCING PRODUCTION OF AFLATOXINS
BY Aspergillus flavus IN FERMENTED SAUSAGES

By

Valente Alvarez-Barrera

Three types of sausages (smoked inoculated, unsmoked
inoculated and smoked uninoculated control) were produced.
They were stored at either 10 or 30°C at relative humidities
of either 79% or 89% for either 3 or 6 weeks.

After 3 weeks storage at 10°C, no aflatoxins were detec-
ted in any of the samples, but 0.26 pg/kg of aflatoxin 81
were detected in the unsmoked inoculated sausages held at
30°C and high relative humidity (89%). After 6 weeks
storage, mold growth was present on all sausages. In the
samples held at lOOC, however, aflatoxin B1 was detected only
on the unsmoked inoculated samples held at high relative
humidity. On the other hand, after incubation at 30°C afla-
toxins were found at higher levels (2.60-6.60 pg/kg) in all
samples stored at either low or high relative humidities.

Although application of smoke retarded mold growth and
aflatoxin formation, longer periods of time, high temperature
and high relative humidity were demonstrated to be important

determinants of aflatoxin production.

ACKNOWLEDGEMENTS

The author is grateful for the guidance, encouragement
and invaluable advise provided to him by Dr. A.M. Pearson
during the course of his academic program and research.

The author also wishes to express his gratitude and
sincere appreciation to the late Dr. R.M. Furtado for his
suggestions, invaluable assistance and encouragement during
the early phases of the research. Gratitude is also expressed
to Dr. J.F. Price and Dr. P. Markakis of the Department of
Food Science and Human Nutrition and to Dr. R.M. Luecke of
the Department of Biochemistry for serving on the Guidance
Committee and for reviewing the thesis. Special thanks are
expressed to Dr. J.F. Price for his cooperation and assistance
in the processing of the sausages. The author also wishes to
acknowledge the assistance of Mr. Sterling Thompson, Depart-
ment of Food Science for his help with the microbiological
work and Mr. Enrique Gonzalez who assisted with this project.

Special appreciation and gratitude goes to Gail Robinson
for her help, encouragement and effort in typing the original

copy of the thesis.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . v
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vi
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE. 3
Occurrence of Aflatoxins in Feeds and/or Foods 3
Chemistry of Aflatoxins. . 3
Presence of Aflatoxins in Meat Products. . . 6
Aflatoxin Carry Over into the Tissues of Animals
Fed on Contaminated Feeds. . . . 13
Methods for Aflatoxin Extraction and Identifica-
tion . . . . . . . . . . l5
General Extraction of Aflatoxins . . l5
Extraction and Identification of Aflatoxins. in
Meat Products. . . . . . . . . . . . . . . . l7
EXPERIMENTAL......................20

Testing the Aflatoxin- Producing Ability of Two
Different Strains of A. flavus . . . . . . . . 20

Inoculum Production and Harvesting . . . . . . . . 20
Culture. . . . . . . . . . . . . . . . . . 20
Aflatoxin Extraction . . . . 21
Identification, Quantification and Confirmatory.

Test for Aflatoxins. . . . . . . . . . . . . . . 22
Preparation of the Sausages. . . . . . . . . . . . 22
Inoculation of the Sausages. . . . . . . . . 23
Extraction of Aflatoxins from Sausages . . . . . . 28
Purification of the Aflatoxin Extract. . . . . . . 30

Liquid- Liquid Partition. . . . . . . . . . 3O
Silica Gel Column Chromatography . . . . . . . 30
Thin Layer Chromatography. . . . . . . . 32
Densitometric Analysis of Aflatoxins . . . . . 32
General Conformatory Test for Aflatoxin. . . . 34
Preparation of Aflatoxin Reference Standards . 35
Fat and Moisture Analysis. . . . . . . . . . . . . 35
Moisture Content . . . . . . . . . . . . . . . 35
Fat Content. , , . . . . . . . . . . . . . . . 35

Safety Procedures. . . . . . . . . . . . . . . 36

iii

RESULTS AND DISCUSSION.

Testing of Two Strains of A. flavus for Production

of Aflatoxins.

Visual Estimation of Mold Growth
Incubation for 3 Weeks
Incubation for 6 Weeks . .

Factors Influencing Aflatoxin Production . .
Aflatoxin Production During 3 Weeks Storage.
Aflatoxin Production During 6 Weeks Storage.

Other Factors Related to Aflatoxin Production.
Effects of pH. . . . . . . . . . . .
Effects of Smoking . .

Effect of Curing Ingredients

CONCLUSIONS
REFERENCES.

iv

Page

Table

LIST OF TABLES

Experimental conditions for holding sausages.

Salt solutions used to obtain desired relative
humidities for each temperature at which sausages
were aged . . . . . . .

Production of aflatoxin B] by A. flavus NRRL- 6649
on summer sausages, after 3 weeks of incubation
at 10°C and 30°C. Each value is the average of
three samples expressed in pg/kg.

Moisture content in summer sausages incubated for
3 weeks at l0o C and 30°C. Each value is the
average of three samples expressed as percent
moisture. . . . . . . . . . . . . . .

Production of aflatoxin B by A. flavus NRRL- 6549
on summer sausages after weeks of incubation at
l0°C and at 300 C. Each value is the average of
three samples expressed in ug/kg. . . .

Moisture content in summer sausages incubated for
6 weeks at l0o C and at 30°C. Each value is the
average of three samples expressed as percent

The pH of summer sausages incubated for 3 weeks
at 10°C and 30°C. Each value is the average of
three samples . . . . .

The pH of summ r sausages incubated for 6 weeks
at 10°C and 30 C. Each value is the average of
3 samples . . . . . . .

Page
25

27

41

44

47

49

51

52

LIST OF FIGURES

Figure Page
1 Structure of aflatoxins B], 82, G1, 62. M1 and M2 - 5

2 Design of controlled humidity 5 gallon plastic
pail for aging the sausages. . . . . 26

3 Spotting and scoring pattern for 2-dimensional
TLC plates . . . . . . . . . . . . . . . . . . . 33

vi

INTRODUCTION

Aflatoxins are a group of highly toxic secondary

metabolites produced by some strains of Aspergillus flavus

 

and by most, if not aTL strains of Aspergillus parasiticus.

 

Chemically, the aflatoxins are difuranocumarin derivatives,
are slightly soluble in water, are soluble in solvents of
intermediate polarity (including alcohols, chloroform, and
acetone) and are insoluble in hexane. Through studies with
experimental animals, strong evidence exists for involvement
of aflatoxins in induction of cancer. Aflatoxin B1 is the
most important in terms of occurrence and toxicity.

In addition to mold contaminated agricultural commodi-
ties, aflatoxins have been found in milk and milk products,
and in fresh, cured and smoked meat products. Most methods
that have been used for aflatoxin extraction from meat
products are based on methods used for extraction and
determination of aflatoxins in crop products. The methods
utilize extraction with suitable solvents, followed by puri-
fication, and a final separation and quantification of the
aflatoxins, which is generally carried out on thin layer
chromatography plates by visual comparison with aflatoxin

standards.

Two general methods have been reported for extraction
and identification of aflatoxins in cured meat products
(Bullerman gt _l., l969; Strzelecki, 1973). These methods
have been used by other workers for studying the presence of
aflatoxins on ham, country cured ham, salami, bacon and
fresh beef. However, they are not suitable for some meat
products since interfering materials cause problems in
spectrophotometric quantification of aflatoxins. Neverthe-
less, little is known about the growth and production of
aflatoxins by A. flavus on fermented sausages.

Thus, the present study was designed to investigate
the levels of aflatoxins produced by an aflatoxin producing
strain of A. flavus grown on fermented sausages under dif-
ferent conditions of manufacture and storage. The variables
investigated were the effects of temperature, relative
humidity, smoking versus no smoking and the length of

storage.

REVIEW OF LITERATURE

Occurrence of Aflatoxins in Feeds and/or Foods

Aflatoxins are produced by some strains of Aspergillus

 

flavus and by most, if not all, strains of Agpergillus para—

 

1., 1969; Bullerman, 1979). Studies on

siticus (Diener gt
aflatoxin production in mold contaminated food and agricul-
tural commodities began with the outbreak of Turkey X
disease in England in 1960, which has been reviewed by
Ciegler and Lillehoj (1968), Stoloff (1977), Wyllie and
Morehouse (1978), and Bullerman (1979).

Aflatoxins have been found in a number of foods/feeds,
such as peanuts and peanut products, cottonseed, corn,
Brazil nuts, pistachio nuts, almonds, pecans, and walnuts
(Stoloff, 1976). Other workers have studied the formation
of aflatoxins in inoculated cheeses and in cured and smoked
meat products (Lie and Marth, 1967; Van Walbeek gt 1.,

1969; Oldham gt a1., 1971; Shih and Marth, 1972).

Chemistty of Aflatoxins

 

Four compounds were originally isolated and were
designated as aflatoxins B], 82, G1 and G2. The specific
designation was due to their blue (B) and green (G) fluores-

cence under ultra violet light, respectively (Hartley gt gl.,

1963). Chemically, the aflatoxins are difuranocumarin
derivatives and have been characterized by a number of

1., 1963; Stoloff, 1977). In addition

researchers (Asao gt
to these four aflatoxins, two others are of significance,
M1, M2 which are hydroxylated derivatives of B1 and 82.
They are excreted in the urine, feces and milk as metabolic
products of B] and 82 following their consumption by mammals
(Frazier and Westhoff, 1978). Wyllie and Morehouse (1978)
reported 12 related compounds, whereas Beuchat (1978)

stated that there are 18 known aflatoxins. The chemical
structures for the six aflatoxins of significance are shown
in Fig. 1.

Beuchat (1978) pointed out that these compounds are
slightly soluble in water, soluble in solvents of inter-
mediate polarity, including alcohols, chloroform, and
acetone, and are insoluble in hexane. Stoloff (1977) repor-
ted that aflatoxins are remarkably stable in normal food and
feed systems, but are highly reactive at either end of the
pH scale (pH <3 and pH >10) or on exposure to ultraviolet
light in the presence of 02.

The site of unsaturation in the furan moiety and the
lactone structure are the reactive portions of the afla-
toxin molecule (Marth and Dyle, 1979). Several investigators
have demonstrated the sensitivity of aflatoxins to oxidizing
agents, particularly to alkaline oxidants, such as sodium

hypochlorite (Fischbach g: 31., 1965; Trager gt gl., 1967;

Bl B2
0 O O
C) ‘ C) C) I
O O OCH3 O OCH;
0
GI 2

 

Figure 1. Structure of aflatoxins B1, B2, G1, G2, M1 and M2.

Nartarajan gt 1., 1975). Studies on the mechanism of

detoxification with ammonia by Beckwith gt _1. (1975) showed
that the phenolic acid exposed on opening of the lactone
ring is particularly reactive. They also found that alcohol
in acid media reacted with the vinyl ether of the difuran
moiety, and resulted in hemiacetals that are very sensitive
to oxidative change. This is typified by the formation of

the water adduct, aflatoxin 32a (Beckwith gt gl., 1975).

Presence of Aflatoxins in Meat Products

Ayres gt _1. (1967) tested ninety samples of cured and
aged meats for characteristic fungal flora, representing
all major types of cured and aged meats produced in the
U.S. and Europe. They isolated 670 fungal strains (456
molds and 214 yeasts). The principal fungal flora of cured

and aged meat were species of Penicillium, Aspergillus,

 

 

Scopulariopsis, Pebaryomyces, Candida, and Torulopsis. They

 

 

 

observed that these genera had in common a tolerance for low
water activity. They showed that molds may be present in
large numbers on the surface of certain cured and aged meats,
especially on country cured hams and fermented sausages.

They isolated strains of A. giggt, A. tgggt, A. wentii, A.

flavus, A. frequentans, A. variable, and A. puberulum.

 

 

Bullerman and Ayres (1968) studied the potential
hazards to human health constituted by the presence of these
organisms on meat. He screened selected isolates from meat

for the production of aflatoxins. Of the mold strains

tested, only the A. flavus isolates produced chloroform
extractable fluorescent compounds, which coincided with
aflatoxin standards on thin layer chromatographic plates.

He demonstrated that A. flavus does indeed produce afla-
toxins. These results suggest that given the proper cir-
cumstances, a potential health hazard could exist from molds
growing on meat substrates.

Bullerman gt _1. (1969) attempted to determine the
quantities of aflatoxins produced on fresh and cured meats
during storage at various temperatures. Important factors
investigated included the effects of type of meat, strain of
mold, storage temperature, length of storage and number of
mold spores present. Aflatoxins were produced on three
different stored meat products, i.e., fresh beef, in which
bacterial spoilage was delayed with antibiotics, ham and
bacon. These products were stored at three different tem-
peratures, 15, 20 and 30°C. When stored at 10°C, the
products were spoiled by bacteria and yeast before detectable
levels of aflatoxins were produced. At 20°C, high levels of
aflatoxins (up to 630 ug/g of meat) were formed during

storage. Below 30°C, both A. flavus and A. garasiticus

 

produced more of aflatoxin G1 than 8,. Above 30°C, however,
A. flavus produced equal amounts of B1 and G], whereas A.

pgrasiticus continued to produce more G1 than B].

 

Bullerman gt 1. (1969) also determined the levels of

aflatoxins produced on salami by a known toxinogenic strain

of A. flavus under simulated conditions of manufacture. He
also studied aflatoxin production by known toxinogenic

strains of A. flavus and A. pgrasiticus on country cured

 

hams at different stages of aging. During aging of Italian-
type salami, A. flavus produced more aflatoxins than was
the case for smoked Hungarian-type salami held under the
same conditions. Temperatures below 15°C and humidities of
less than 75% were required to prevent aflatoxin development
during the aging of salami. A temperature of about 30°C
was required for maximum production of aflatoxins. The
presence of curing ingredients, especially pepper and sodium
nitrite, tended to reduce the amounts of aflatoxins in these
products.

Strzelecki gt g1. (1969) identified four strains of

A. f avus from a heavily mold contaminated country cured

 

ham. They concluded that the predominance of aspergilli

on country cured hams was due to its low moisture content
(approximately 30%). They grew molds on yeast extract
containing 20% sucrose (YES), and extracted the aflatoxins
with chloroform. A concentrate of the extract was resolved
on thin-layer chromatograms of silica gel, and the aflatoxin
concentration was determined visually by comparing with
aflatoxin standards. They found that out of 10 different
mold isolates, four of the five A. flavus isolates produced

aflatoxins.

To determine the incidence of mycotoxic molds in
domestic and imported cured and smoked meat, Bullerman (1977)
analyzed 171 samples. Analyses were carried out for visible
mold growth, isolation and classification of visible molds,
ability of mold isolates to produce known mycotoxins and
development of visible mold growth during simulated storage
of each sample. A total of 393 isolates were obtained from
domestic meat and 645 from imported meat. 0f the total

isolates, he found 68.1% were Penicillium, 2.4% Aspergillus,

 

 

4.9% Cladosporium, 5.8% Alternaria, 0.9% Fusarium and 17.9%

 

 

were from various other genera. He concluded that among
potentially toxigenic species of molds found on meat were

A. gyclopium, A. viridicatum, A. flavus and A. glaucus.

 

 

Five mycotoxins were identified on the meat, with aflatoxins
being one of them.

Oldham gt gt. (1971) investigated the growth of A.
flavus on certain perishible foods and the production of
aflatoxins at normal refrigerator temperatures. They inocu-
lated samples of cheddar cheese and luncheon meat with one
strain of A. flavus. The products were held for 12 days at
4.4, 7.2, and 25°C. All samples, except those inoculated
and incubated at 25°C, were negative for aflatoxin produc-
tion. Therefore, they reported that the mold isolated did
not produce detectable aflatoxins when kept at normal

refrigeration temperatures for up to 12 days.

lO

Mintzlaff gt, 1. (1972) analyzed Penicillium culture

 

isolated from mold-ripened sausages of European origin.

They found that the cultures produced nine different myco-
toxins, among which were aflatoxins B1 and G]. Some 88
isolates out of a total of 422 were found capable of toxin
synthesis. Fourty four of these cultures produced peni-
cillic acid, 17 ochratoxin A, ll tremortin, 10 citrinin,

and 6 patulin. However, toxins were not detected on
sausages containing mold producing mycotoxins during storage
up to 70 days. Although results indicated that consumption
of sausage containing mold is not generally a health hazard,
they recommended that manufacturers of these products could
utilize pure cultures of molds known to be toxicologically
safe for speeding up the aging process in meat.

Strzelecki (1973) followed the growth of aflatoxins in
raw and country cured hams, as well as in salami. He
reported that A. flavus will grow and produce aflatoxins
in country cured hams, with trace amounts being present
after 84 and 126 days at storage of 5 and 30.1°C, respec-
tively. In salami, aflatoxins were found at levels of
104.85 and 213.4 ug/lOO 9 after 13 and 78 days of storage,
respectively. Recovery of added aflatoxins from meat
products resulted in 16% recovery from raw ham after 6 weeks
storage, 7% recovery from country cured ham after 126 days,

and 19% in salami after 78 days.

11

In similar work, Murthay et a1., (1975) studied the
recovery of aflatoxin B1 injected into frozen beef at
different intervals of storage. 0n storing beef at ~18°C
for 20, 25, 32, 152 and 183 days, there was a diminution
in the recovery of injected aflatoxin B]. The author
discussed the possibility that a portion of the aflatoxin
was not recovered due to its combining with the meat con-
stituents as a consequence of physicochemical changes
occurring in the muscle during storage.

Strzelecki (1973) investigated the effect of the meat
curing ingredients on aflatoxin production. He reported that
aflatoxins were totally inhibited by potassium nitrite,
although sodium nitrite caused a slight stimulation. The
curing mixtures ggt gg, which included 5.71% NaCl, 2.16%
sucrose, 0.10% KNO3 and 0.05% NaNOz, resulted in somewhat
more aflatoxin production. Meier and Marth (1977) studied
the production of aflatoxins by mold grown in the presence
of curing salts. They reported that the effects of indi-
vidual curing salts or mixtures of curing salts on growth
and toxin elaboration was substrate dependent. A combina-
tion of NaNOZ and NaCl in yeast extract-sucrose (YES) broth
depressed growth and/or aflatoxin production. However,
biosynthesis of aflatoxin B1 was enhanced by presence of
1 and 4% NaCl in YES broth. Similar results were obtained
when working with sucrose-ammonium salts (SAS) broth. In

work on sausages, they found that 100 and 200 ppm of NaNO2

12

and NaCl, respectively, supported more mold growth and
aflatoxin production than was the case for control sausages
containing 3% NaCl and 100 ppm of NaNOZ. Addition of 2 and
3% NaCl without nitrite resulted in less aflatoxin production
than that of the control sausage. Overall results suggested
that high levels of 2 to 3% NaCl are inhibitory to aflatoxin
production.

van Walbeek gt__l, (1969) surveyed the influence of
refrigeration on aflatoxin production by unidentified
strains of A. flavus. They stated that aflatoxins were
produced in significant concentrations under conditions
simulating household refrigeration (7.5 to 10°C). The rate
of aflatoxin production at 10°C was markedly influenced by
preincubation culture at room temperature for 24 hours.

The authors found that toxin production was higher in solid
cultures than in liquid cultures.

Furtado _t _l. (1981) studied the effects of cooking
and/or processing upon levels of aflatoxins B1 and B2 in
meat from pigs fed a contaminated diet for 42 days. Fresh,
fresh-cooked, cured-smoked (hams only) and cured-smoked-
cooked products were analyzed. Some destruction of afla-
toxins B1 and 82 occurred during cooking and/or processing.
It was concluded that the maximum amount of inactivation did
not exceed 41% of the total and was generally in the range

of 15-30%. Thus, results suggested that aflatoxins are

quite stable during cooking and/or processing.

13

Aflatoxin Carrytpver into the Tissues of Animals Fed on
Contaminated Feeds

Since studies have shown that ingested aflatoxins may
be deposited in tissues as the original aflatoxin, or as one
of its metabolites, the problem of transmission of aflatoxins
through farm animals to human food has been studied by
several investigators (Newberne gt 1., 1955; Burnside

_t _l., 1957; Forgacs gt _l., 1958; Asplin and Carnaghan,
1961; Loosmore and Markson, 1961). Allcroft and Carnagham
(1963) were the first to investigate the presence of afla-
toxin residues from animals fed toxic material. Keyl and
Booth (1971) determined the adverse effects of graded
levels of aflatoxins in the rations of swine, beef cattle,
dairy cattle and poultry. They found no adverse effects of
aflatoxins at levels of 230, 300, and 450 ppm of aflatoxin.
Vanzytveld gt g1. (1970) studied the presence of afla-
toxins in the livers and skeletal muscle of chickens, which
had ingested a daily dose of aflatoxins over a six week
period. Aflatoxins were detected as a result of aflatoxin
ingestion. Mabee and Chipley (1973) investigated aflatoxin
carry over in broiler chickens by administering low levels

of 14

C-labeled aflatoxins. The radioactivity was detected
in the liver, heart, gizzard, breast and leg meat. Other
similar studies have been reported by Platonow (1965) and

Kratzer gt a1. (1969).

l4

Allcroft and Carnagham (1962) reported that extracts
of milk from cows fed aflatoxin contaminated rations and
aflatoxins administered directly induced identical lesions.
A relationship between aflatoxin intake and the concentration
of aflatoxin M in milk has been reported by several investi-
gators (Allcroft and Roberts, 1968; Keyl gt gl., 1968; Keyl
and Booth, 1971; Polan gt _l., 1974).

The effect of aflatoxins on pigs has been studied by
feeding them aflatoxin-contaminated diets (Krogh gt gl.,
1973). It has been reported that the response of swine to
aflatoxins depends on whether the aflatoxin-contaminated
protein is fed separately or is incorporated directly into
the total ration (Murthy gt gl., 1975). An appreciable
amount of aflatoxin B1 was found by Jacobson gt gt. (1978)
in the tissues of pigs fed aflatoxin B1. Furtado gt _1.
(1979) assessed the possibility of aflatoxin carry-over into
meat products from animals fed contaminated rations. They
reported no evidence of gross pathological lesions, however,
they detected residues of both aflatoxins B1 and B2.

These experiments demonstrated that ingested aflatoxins
are deposited as the original aflatoxin, or as one of its
metabolites. By using sensitive chemical methods, aflatoxin
residues were found in the organs and muscle of swine and
chickens, as well as in the organs and milk from cattle.

Since a recent study reported that aflatoxin B1 was detected

in liver samples from patients with primary hepatocellular

15

carcinoma (Siraj gt gl., 1980), the transmission of afla—
toxins through farm animals to human foods is an important

problem.

Methods for Aflatoxin Extraction and Identification

General Extraction of Aflatoxins. Sargent gt _1. (1961)
were the first to extract aflatoxins using mold-contaminated
peanut meal. They used exhaustive Soxhlet extraction of
the sample with methanol, followed by further extraction
with chloroform and defatting of the final extract with
petroleum ether. Simultaneous defatting and extraction was
used by Nesheim (1964), who blended the sample with aqueous
methanol and hexane. Acetone was utilized as the solvent
to extract aflatoxins from cottonseed, peanuts and other
similar products by several other workers (Pons and Gold-
blatt, 1965; Pons gt gl., 1966; Stoloff 23.11-1 1966).

Since the earlier methods yielded extracts having large
amounts of interfering materials, Coomes and Sanders (1963)
used liquid-liquid partition chromatography with methanol,
water and petroleum ether as solvents. Pons and Goldblatt
(1965) and Pons _t _l. (1966) used lead acetate to precipi-
tate interfering substances from the extracts.

A more efficient purification system in which the afla-
toxins were not destroyed or altered was utilized by DeIongh
gt 1. (1962). The method used silica gel column chromato-

-—-——

graphy to purify a crude extract that was sequentially

16

e1 uted with chloroform and methanol. Nesheim (1964) used
p23 rctition column chromatography of an aqueous methanol
ex tract of peanut meal on diatomaceous earth (celite) to
se parate aflatoxins». Then the aflatoxins were extracted
w‘i 12h a chloroformzhexane mixture (1:1, v/v). Pons gt a_l_.
(1 966) used a similar method, however, they eluted inter-
fe r‘ing substances with diethyl ether and the aflatoxins
w‘T th chloroform; methanol (97:3, v/v). Stoloff gt gt.
(1966) utilized partition chromatography on a cellulose
co'l umn, in which aflatoxins and the interfering substances
were eluted with hexane and chloroform (1:1, v/v).

The first analytical methods for separation of afla-
to>< ins used either paper chromatography or filter paper
(Sa rgent gt gl., 1961; Coomes and Sanders, 1963). Intro-
duc “tion of silica gel (Kiesegel G) for the separation of
sev eral fluorescent spots of a purified aflatoxin extract
from contaminated groundnut meal, was first accomplished by
081 Ongh gt 1. (1962). Later, TCL plates were adopted by

oth er workers with variations in the solvent developing

SYS tems (Broadbent gt a1., 1963; Hartley gt g_l_., 1963).
Although fluorescence of aflatoxins under UV light was widely
utl 1 ized for detection and estimation (Pons and Goldblatt,
‘95 9), until recently measurement had been by visual estima-
thn . Nabney and Nesbitt (1965) improved the accuracy and

pre cision of aflatoxin measurement by use of absorption

SpectrOphotometry at 366 nm. Ayres and Sinnhuber (1966)

17

re: paorted a more sensitive fluorodensitometric measurement
of: aflatoxin B1 directly on silica gel coated plates using
a 'r“ecording densitometer equipped for fluorescence emission
me asurements. This basic system has been widely adopted.
Extraction and Identification of Aflatoxins in Meat

Pr’¢:>»ducts. Methods for extraction, purification, determina-
ifi (:> n and measurement of aflatoxins in meat were briefly
men tioned by Murthay gt __1_. (1975). These methods are
moci i fications of those used in extraction and determination
of éi.flatoxins from cereals and other food products (Buller-
man __t _l., 1969; Strzelecki, 1973; Brown gt 1., 1973).

Bul ‘I erman _t__l, (1969) made the first attempt to extract
and analyze aflatoxins from meat. They extracted by blending
a 1 (I) 0 9 meat sample with chloroform. The water content of
the meat (ca 50-60%) and the chloroform, which contained
the aflatoxins, separated into two layers. They then
dec~an.nted the chloroform layer and filtered it before making
VIS»LJ a1 estimation of the aflatoxins. They then used this
"mil?! od for analyzing ham, bacon, salami and fresh beef.

A similar technique was utilized by Strzelecki (1973)
who ranalyzed salami, raw ham and country cured ham for
afléi‘toxins. He used 300 m1 of methanol in distilled water
(23" ) for extraction and a mixture of petroleum ether-
heXane-benzene (2:1:2, v/v) for defatting the samples. In

0rdE2r~ to eliminate emulsion formation, 10 g of soidum

C“)C>ride were added after filtering. Brown gt g1. (1973)

18

us ed a similar method but added 300 m1 5% NaCl solution.
Th ey did not need to defat the sample since they worked with
ra w tissues.

Furtado (1980) reported a suitable method for extrac-
ti on and analysis of aflatoxins from the tissues of pigs
an (1 meat products. The method was a modification of the
procedure of Trucksess and Stoloff (1979), which requires
ace- tone and NaCl for aflatoxin extraction. After filtering,
(NI—I4 )2504 and Pb(0AC)2 solutions were added to insure better
ext r‘action of the aflatoxins.

Neither Bullerman (1969) nor Brown (1973) used liquid-
liq t... id partitioning for removal of interfering lipids,
car‘ [3 ohydrates and pigments from the primary extracts. How-
eve r~ , Strzelecki (1973) defatted the samples by using a
mix 1:. ure of petroleum ether, hexane and benzene. The use of
liq L.- id-liquid partition systems was adopted and modified
by s everal workers (Coomes and Sanders, 1963; DeIongh
gt L1” 1964). Trucksess and Stoloff (1979) reported good
resu 1ts when using petroleum ether to remove the fat before
extr‘ action, cleanup, and quantitative determination of afla-
tOXi ns B1 and M1 from beef liver.

The use of chromatographic systems for partially
PWi ‘Fying crude or primary extracts offer an efficient

System, provided that aflatoxins are not destroyed or

alte red during the treatment. Therefore, different column

Systems were reviewed by Pons and Goldblatt (1969). A

19

a 1 uminum oxide and anhydrous sodium sulfate column has been
ut ‘ilized by Strzelecki (1973). Silica gel and celite columns
ha we been reported to provide good cleanup by other research-
er‘ s (Bullerman gt an 1969; Brown gt gin 1973; Trucksess and
St oloff, 1979).

Cleanup of aflatoxin extracts using TCL plates developed
in one or two dimensions, and quantification of the afla-
toxins by either visual estimation or densitometric analysis
have been widely utilized in studies ofmold contaminated
fee ds and other agricultural commodities (Nesheim, 1964;
Coo mes and Sanders, 1963; Pons and Goldblatt, 1965; Pons
gt _a__l_., 1966; Whitaker and Dickens, 1979). Modifications
of these procedures have been adopted to purify and quantify

afI atoxins in meat and meat products (Bullerman gt gl_.,

196 9; Brown gt 1., 1973; Strzelecki, 1973; Trucksess and

StO 1 off, 1979; Furtado, 1980).

EXPERIMENTAL

e sting the Aflatoxin-Producing Ability of Two Different

If

.4

r‘ains of A. flavus

A

if

(1’

Two strains of A. flavus obtained from the culture
co ‘1 'lection at the Northern Regional Research Laboratory of
the U.S. Department of Agriculture, Peoria, 111., were
exa mined for aflatoxin-producing ability. (1) NRRL-6549
A. flavus obtained from A. cieflar, Kulmback, Germany and
iso 1ated February, 1972 from raw ham; and (2) NRRL-6550
A. ‘flavus obtained from 3.6. Maxfield, Cullman, Alabama and

 

iso ‘Iated from the brain of a 14 day old broiler.

Ino culum Production and Harvesting
Inocula from the two molds were produced by growing
the molds at 28°C for 8 days on a thin layer of potato-
dex trose-agar in Roux bottles. The conidia were harvested
and suspended in 50 ml of sterile water, and gently agitated.
After harvesting, the spore suspensions were transferred to
a S terile bottle, enumerated with a hemocytometer and

dll uted to contain 1x106 conidia/ml.

Q!) ture
Demineralized water was used to prepare the basal

medium, which contained 2% yeast extract (Difco) and 20%

20

21

sucrose (YES). Flasks (500 ml) containing 50 ml of medium
peg r” flask were stoppered with foam plugs and autoclaved for
15 minutes at 20 psi. Media were inoculated with spores

fr' cm 8 day old cultures of the two strains. of A. flavus and
irj ¢::ubated 8 days at 28°C as stationary cultures. Experiments
we re replicated five times each and the results are reported

as averages.

AF 1 a toxin Extraction

 

Aflatoxins were extracted from cultures (mycelium

meci um) according to the techniques described by Buchanan

 

j;
gt ianfl. (1976). Chloroform (25 ml) was added to the culture,
whi ch was then agitated on a shaker (Model 6130, Eberbach
Cor‘pu Ann Arbor, Mich.) for 10 minutes. The liquid was
tMa r": tmansferred to 125 m1 separatory funnels and agitated
vigorously. The chloroform layer was drawn off into a 500
m1 if‘ound bottom flask. The extraction procedure was
reF’Eaeated twice with the three extracts from each replication
bei ng combined in the round bottom flask. Extracts were
evaporated almost to dryness using a Rotovapor R (Buchi,
Swi‘tzzerIand). The extracts were redissolved in chloroform
t0 i3 volume of 0.5 ml. They were then stored at -l6°C while

h01d i ng for analysis.

22

Identification, Quantification and Confirmatory Test for
gilatoxins

Aflatoxin assays were run by thin-layer chromatography
us ing precoated 20x20 cm silica gel plates (Sil-G-HR-25,

Br' ‘i'nkman Instruments Inc.). The plates were developed with

ch 1oroform-acetone (85:15, v/v) in the first direction and

et: her-methanol-water (95:4:1, v/v) for the second dimension.

The aflatoxins were quantitated by densitometric analysis

with a double beam scanning recording integrating Schoeffel

SD 3000-3 spectrodensitometer.

After quantification, general confirmatory tests for

afl atoxins were carried out according to the method of
Przybylski (1975). This procedure is described later herein.
Pregaration of the Sausages

Three types of summer sausages were prepared. They

were all prepared using the same formulation, but differed
in the following ways:
ted -

3

(a) lot l—control-smoked, uninocula-
(b) 1ot 2-smoked and inoculated; and (c) lot 3-not
51110 Red but inoculated.

The formula was made using 9.5 kg
of

“lean beef, 2.04 kg of lean pork, 2.04 kg of pork fat,
340 - 5 g of sodium chloride, 1.2 g of sodium nitrite, 2.4 9

0f Sodium nitrate, 136.2 g of sucrose, 33.9 g of coarse

black pepper, 12.6 g of coriander, 4.2 g of garlic powder,

4-2 g of allspice, 17.1 g of whole mustard seed, 17 g of

StaL\r‘ter culture (Lactacel Plus). The meat and fat were

23

ground separately in a Hobart meat grinder with a 4.8 mm

p1 ate. The spices, cure and starter culture were mixed

w‘i th the meat and fat in a 80 lb capacity paddle mixer

(M odel Buffalo, John E. Smith's Sons Co., Buffalo, N.Y).
The mixture was stuffed into 4.5 cm diameter natural

ca sings using an automatic stuffer (Model E-Z Pak water

pressure sausage stuffer, E-Zuber Engineering and Sales Co.,

Minneapolis, Minnesota). After stuffing, the sausages were

tie d into 8-8.5 cm lengths to give small sausages weighing

abo ut 100 g each. Following the smoking and fermentation

of the sausages for 13 hours, they were showered with hot

wat: er, tempered at room temperature, showered again with hot

wat: er, dried at room temperature and then chilled.

Ino culation of the Sausages
The sausages were inoculated with conidia of A. tlgygg

NRR L-6549, which was previously shown to produce aflatoxins.
Ino cula were produced according to the method described by
Bul Terman gt __1_. (1969). The mold was grown at 28°C for 10
day s on thin-layers of potato-dextrose-agar in Roux bottles.
The conidia were harvested and suspended in 2000 ml of

ste rile 0.05% Tween 80 solution. The inoculum level used
was 10° spores per sausage. The sausages were inoculated

by dipping individually for 30 seconds in a spore suspension

C0“ taining 106 spores per ml. The calculated number of

6

$130 res per sausage (10 spores/ml) was based on the assump-

thn that each dipped sausage had adsorbed an average of

24

1 lnl of inoculum.

The three lots of sausages were aged for 3 and 6 weeks
11'1: different conditions of smoking, temperature and humidity
a 2;. shown in Table 1. This gave the following treatments:
( 1 ) 10°C and 75% relative humidity, (2) 10°C and 87% rela-
t‘i’ ve humidity, (3) 30°C and 79% relative humidity, and
(4 ) 30°C and 89% relative humidity. About nine inoculated
SE! lasages were hung in fermentation pails similar to those
dee.s; cribed by Costilow and Uebersax (1978). The experiment
emp loyed twelve S—gallon plastic pails equipped with lids
mo cdl ified by cutting 2.5 cm off-centered holes to accommo-
da 1:: e #6 rubber stoppers. A small wire hook was attached
to the center of each stopper so that a wire rack could be
su £5: pended in the pail to support the free hanging sausages.
Cl <:>Ise to the hole in the center of the lid, another 2.5 cm
hop'l. e was made and was stoppered with a foam plug. The
whole system is shown in Figure 2.

A saturated salt solution (900 ml), selected to give
tWIEE' desired humidities at the temperatures used, was placed
in the bottom of each pail (Rockland, 1960). The saturated
53‘] t solutions and the relative humidities they provided
BEE? shown in Table 2. Each salt was added to the solutions
to ‘insure and maintain saturation, however, some sausages
10551: moisture as they aged, so salt was added when needed.

Thfe foam plugs provided sufficient gas exchange to get an

ae‘Y‘obic atmosphere in the pails. As in previous studies

25

Table 1. Experimental conditions for holding sausages.

 

Temperature (°C) 10 30
Lot No. Humidity Humidity

% %

Lot 1 a b 7 79

Control ’ 87 ‘ 89

Lot 2 c ’ 75 79

w/o smoke 87 89

Lot 3 a c 75 79

with smoke ’ 87 89

 

aSausages prepared under regular conditions of processing.
bSausages were not inoculated with A. flavus.

cSausages inoculated with A. flavus NRRL-6549 at about 106
spores/sausage.

26

1————n3/4"—-————1

#2 RUBBER STOPPER 6" RUBBER STOPPER

FOAM PLUG \\\ 1" HOLE
\ n

1 1 l

       

WIRE SAUSAGE
K

14“ RAC

SAUSAGES

 

PLASTIC SCREEN

 

RELATIVE
HUMIDITY
MEDIUM

 

 

 

 

Figure 2. Design of controlled humidity 5 gallon plastic
pail for aging the sausages.

27

Table 2. Salt solutions used to obtain desired relative
humidities for each temperature at which sausages

 

 

were aged.
Temperature Saturated Salt . .
(°C) Solution Used Hum1d1ty
%
10 NaCl 75
KCl 87
30 NH4SO4 79

BaCl 89

 

28

(Bullerman gt _l., 1969), it was assumed that this would not
permit exchange of air to alter the humidity of the environ-
ment within each pail.

After holding the sausages for either 3 or 6 weeks,
they were removed from the pails. They were placed in plas-
tic bags, frozen and stored at 416°C until they were removed

for analysis for aflatoxins.

Extraction of Aflatoxins from Sausages

 

Preliminary tests for extraction of aflatoxins from
sausages were carried out using the methods of Bullerman
gt gt. (1969) and Strzelecki (1973), which have been reported
to be suitable for the analysis of aflatoxins in meat, such
as ham, bacon, salami and some fresh meat. However, inter—
fering materials from the added smoke, the spices, and
perhaps from the high fat content of the sausages ggt gg
made the TLC manipulations very difficult. The plates
showed an unresolved streak without spot differentiation.
Thus, changes were made to eliminate the interfering
materials from the extracts but they were time consuming
and more complicated.

After considerable modification, extraction and quanti-
fication of the aflatoxins in the sausages were then carried
out using a modification of the procedure described by
Furtado gt 1. (1979, 1981), who reported a highly sensi-

tive method for determination of aflatoxins in tissues.

29

The modified procedure began with 100 g of sausage,
which was sliced and blended for two minutes in a Waring
blender at moderate speed with 42 ml of saturated NaCl
solution (40 g NaCl/lOO m1 H20) and 3 g of citric acid.
Then 300 ml of acetone were added to the'homogenate while
washing the sides of the blender jar. An additional blen-
ding was then performed at moderate and high speeds. After
blending, the material was filtered through fast filtering
prefolded paper (Whatman 114 V) and the filtrate was col-
lected in a 500 m1 Erlenmeyer flask. After complete
draining of the acetone from the residue on the filter
paper, the flask was stoppered with a cork stopper and
frozen at -16°C for 5 minutes. The precipitated fat was
removed by filtering, using the same type of paper.

After filtration was completed, the meat residue was
discarded. Then 150 m1 of water, 7.5 g of (NH4)ZSO4 and

20 m1 of Pb(OAC)2 solution (200 g Pb (OAC)2'3H O in 500 m1

2
H20 containing 3 ml of acetic acid and made to a volume of
one liter with H20) were added to the filtrate. The solu-
tion was stirred for 5 minute with a magnetic stirring
device. Then 10 g of diatomaceous earth were added to the
solution and stirring was continued for an additional %
minute. The solution was allowed to stand for about 5
minutes before filtering through fast filtering folded

paper. The final filtrate was collected in a 500 ml Erlen-

meyer flask.

3O

Purification of the Aflatoxin Extract

Liquid-Liquid Partition. The filtrate was transferred
to a 500 ml separatory funnel. Then 100 ml of petroleum
ether (BS-60°C b.p.) were added. The separatory funnel was
shaken vigorously for about 1 minute. The layers were
allowed to separate, and the lower aqueous-acetone layer
was drained into a second 500 ml separatory funnel. The
petroleum ether layer was then discarded. Then 50 ml of
chloroform were added to the aqueous-acetone solution and
the separatory funnel was shaken vigorously as before.
After the layers separated, the lower chloroform layer was
collected in a 500 ml flask. Aflatoxin extraction from
the aqueous-acetone layer was repeated one more time using
50 m1 of chloroformzacetone (50:30, v/v). The aqueous
layer remaining after the chloroform extraction was discar-
ded. The chloroform-acetone extract was then evaporated to
dryness in a Rotavapor R (Buchi, Switzerland), using a water
bath setting at 35°C.

Silica Gel Column Chromatogtgpgy. A 250x25 mm glass

 

column (Fisher & Porter) was filled using about 50 ml of
chloroform. A slurry of 10 g of silica gel 60 (70-230 mesh
ASTM-EM Laboratories Inc.) in 60 ml of chloroform was added
to the column and the chloroform was then drained to about
10 cm above the top of the silica gel. Another 3 cm layer
of anhydrous NaZSO4 was added on top of silica gel. The

excess of chloroform was drained by gravity to the top of

31

the upper Na2504 layer.

The aflatoxin extract was dissolved in about 5 ml of
chloroform-hexane (1:1, v/v) and then transferred to the
column with a disposable glass pipet. The sides of the flask
were washed three more times with chloroform-acetone (1:1,
v/v) and the washings were added to the column. After each
addition of the chloroform extract, the column was drained
to the top of the packing and the eluate was discarded.
Interfering substances were eluted from the column in 100 ml
of the ether-hexane (3:1). The eluate was then discarded.

The aflatoxins were eluted from silica gel column
with 150 ml of chloroform-methanol (97:3, v/v). The
eluate was collected in a 250 ml flask and evaporated to
near dryness in a rotary evaporator as described earlier
herein. The sample extract was dissolved in acetone and
quantitatively transferred to 2 dram vials using a disposable
glass pipet. The acetone solution was evaporated to dryness
(N-Evap evaporator, Model 106, Organomation Assoc.) under
a steam of nitrogen using a water bath setting of 35°C.
Special care was taken to avoid overheating of the dry
extract. The aflatoxin extract was then dissolved in a
100 pl of benzene-acetonitrile (9:1, v/v), and the vial
was sealed with a teflon lines screw cap. The vial con-
taining the aflatoxins was shaken vigorously for about 1
minute on a vortex shaker before removing the samples for

analysis.

32

Thin Layer Chromatography. Precoated 20x20 cm silica
gel plates (Sil-G-HR-25, Brinkman Instrument Inc.) were
scored and spotted as shown in Figure 3. A 20 ul sample of
aflatoxin extract was applied to the plate with a 25 ul
syringe (Hamilton Co.). Standards of 0.5131, 0.1668,

0.540 and 0.1791 pg of aflatoxin B B

G and 62’ respec-

1’ 2’ l
tively, were spotted on TLC plates about 1 cm from the
edge (Figure 3).

The plates were developed in the first direction with
chloroform:acetone (85:15, v/v) in a sealed and unequili-
brated tank. After the development in the first dimension
was completed, the plates were removed from the tank and
dried under a hood for about 2 minutes, the drying was
finished by placing in a forced-draft oven for 1 minute and
immediately developed in a second dimension with anhydrous
ethyl ether-methanol-water (95:4:1, v/v). The plate was
removed from the tank and dried as before to remove the
ether. After drying the plate was analyzed visually and

prepared for densitometric analysis.

Densitometric Analysis of Aflatoxins. Aflatoxins were

 

quantified with a double beam scanning recording-integrating
spectrodensimeter SD 3000-4 (Shoeffel Instruments). The
plates were scored prior to spotting as shown in Figure 3
with a Schoeffer Scoring Device SDA 303, which provides

10 mm strips. Three aflatoxins standards were spotted within

three strips parallel to the second dimension of development.

33

2nd direction

 

 

 

3 cm’{

 

 

 

standard
spots

11 cm
lst direction

sample spot

1 l \,

h—+ .J,.__..

2 cm 12 cm 6 cm

 

 

 

 

 

 

 

Spotting and scoring pattern for 2-dimensiona1

Figure 3.
TLC plate.

34

The average of the three readings of the aflatoxin stan-
dards was used to compare and calculate the concentration
of the sample. For analysis of the standard, the scanning
head was placed within each of the three strips and parallel
scanning took place. For the sample spot, the plate was
viewed under uv light and each two spots were localized
within an imaginary strip identified through four pencil
marks made on the silica gel plate. Then the plate was
placed on the plate carrier in such a way as to be driven
and scanned parallel to the direction of the imaginary
lines.

Aflatoxin concentrations were calculated according to
the following formula:

pg/Kg=(B x Y x S x V)/(Z x X x W)

where:

B = Area of aflatoxin peak in the sample

Y = Concentration of aflatoxin standard in pg/ml

S = pl of the aflatoxin standard

V = Dilution of sample extract in pl

2 = Area of aflatoxin standard peak (Average of three

replications)
X = pl of sample extract spotted on the plate

w = Grams of sample in the final extract

General Confirmatory_1est for Aflatoxin. After devel-

 

opment of the TLC plates and identification of the spots

under uv light, the plate was sprayed with 25% sulfuric

35

acid. The plate was then dried in a chromatographic oven
at 45°C under a stream of nitrogen. Fluorescence change of
the aflatoxins from blue or green to yellow after the treat-
ment with H2504 was used as additional confirmation for the
presence of aflatoxin in the sample (Przybylski, 1975).
Preparation of Aflatoxin Reference Standards. Afla-
toxin reference standards were prepared according to the
AOAC method (1980) and contained 0.5 pg/ml of aflatoxin B1
and G1 and 0.1 pg/ml of aflatoxins 82 and B2. A solvent
mixture of benzenezacetonitrile (98:2, v/v) was used as the
solvent for all aflatoxins. The aflatoxin standard solu-

tions were stored at —16°C.

Fat and Moisture Analysis

Moisture Content. The A.0.A.C. (1980) procedure for

 

determining moisture was used. About 5 g of sample were
accurately weighed to four decimal places into a previously
dried aluminum dish. The sample plus the dish were then
dried for 18-24 hours in an oven at 1oo-1os°c. The dried
sample was cooled in a desiccator and weighed to four decimal
places. The loss in weight was calculated as percentage
moisture.

Fat Content. The fat content was determined using the

 

Goldfisch extraction method of the A.0.A.C. (1980). The
sample used for moisture analysis was utilized. The aluminum
dish containing the dried meat sample was carefully folded

into a porous thimble and clipped into the Goldfisch

36

extractor. The fat was extracted with anhydrous diethylether
for approximately 8 hours into a previously dried and tared
beaker. The extract was then dried for 1 hour at 100°C in

an air convection oven, cooled in a desiccator and weighed

as before. The percent fat was calculated as grams of fat
extracted per hundred grams of sausage.

Safety Procedures. All material, glassware and vials

 

in contact with aflatoxins were soaked either with 5-6%
Na0C1 (household bleach) or with sulfuric acid-dichromate
solution (120 9 Na Cr 0

-2H20 1600 ml conc. H250 and diluted

2 2 7 4
to a volume of 3 liters with water)to destroy any residual
aflatoxins. Plastic disposable gloves were worn routinely
during all work with aflatoxins. Respirator masks were

worn when handling culture of A. flavus, when making the
spore suspension, during sausage inoculation and when pre—
paring sausages for aflatoxins analysis. After the holding
experiment, all surfaces of the containers were treated by
spraying them with a solution of disinfectant solution (3-5%
Lysol) and then with NaOCl solution. All TLC plates used

for aflatoxin analysis were thoroughly soaked in NaOC solu-
tion before discarding. The waste material, after treat-
ment with ammonia, was collected in plastic bags and placed
inside tightly closed containers and labeled properly until
removed by the MSU Animal Waste Disposal Unit. The discarded

solvents involved in the experiment were removed by the MSU

Chemical and Biological Safety Unit. All work involving

37

aflatoxins was done under a fume hood, this included prepara-
tion of silica gel columns, spotting, development and drying
of the TLC plates. Similarly, any work involving the use

of toxic solvents, such as ammonia, chloroform, benzene,

methanol and acetonitrile, was also carried out under the

fume hood.

RESULTS AND DISCUSSION

Testing of Two Strains of A. flavus for Production of Afla-
toxins

Preliminary work testing two different strains of At
flavus on suitable media showed that only one of the strains
produced aflatoxins. The two molds tested included At
f1avus-NRRL-6549 and A. flavuseNRRL-6550. Only NRRL-6549
produced measurable amounts of aflatoxin 8]. Results are
in agreement with the conclusions of Heathcote and Hibbert
(1978), who showed that many strains of A. flavus do not
produce aflatoxins.

Using the basal YES medium containing 2% yeast extract
and 20% sucrose, the fungus produced from 1.71 to 3.80 09
of aflatoxin per liter of media. Davis gt gt. (1969)
reported that some other strains of A. flavus produced
aflatoxins at levels of 6.2 and 6.4 mg/100 g of mycelia.
The amount of aflatoxin B1 produced by the mold in the
preliminary test was adequate, since the purpose of this
experiment was to find out if aflatoxins could be produced
under simulated conditions of manufacture and storage of

summer sausage.

38

39

Visual Estimation of Mold Growth

The three lots of sausages consisted of: (a) lot 1-
control, smoked and uninoculated, (b) lot 2-unsmoked and
inoculated, and (c) lot 3-smoked and inoculated. They were
aged for either 3 or 6 weeks at either 75-79% or 87-89%
relative humidity at temperatures of either 10 or 30°C.
After incubation, the sausages were analyzed visually for
mold growth.

Incubation for 3 Weeks. After incubation of the samples
at 10°C and a relative humidity of 75%, only slight mold
growth was found on the surface of the sausages. Mold
growth was only noticeable on the uninoculated-smoked
control sausages, which showed about 4 small colonies per
sausage. At the same temperature but at high relative
humidity (87%), slight growth was observed on both the
smoked uninoculated control sausages and on the smoked
inoculated ones. However, the unsmoked inoculated sausages
contained about 5 large colonies per sausage, with the
colonies being considerably larger than those of the smoked
uninoculated control.

On holding the sausages at high temperature and low
humidity (30°C, 79% r.h.), considerable difference in the
amount of mold growth was evident. All sausages in all
treatments were covered by heavy mold growth. However,
visible differences were evident between colonies for the

uninoculated smoked control and for those from the other

40

samples which were inoculated with A. flavus. The uninocu-
lated smoked control sausages exhibited some yellow-green,
some white and some brown colonies. 0n the other hand,
both lots of inoculated sausages (smoked and unsmoked) were
entirely covered by mold, which exhibited the characteristic
yellow-green color of A. flavus colonies. Shrinkage was
observed on the surface of all sausages, and was probably
due to the loss of surface moisture. This resulted in some
surface drying and wrinkling, but was not confirmed by
moisture analysis of the entire sausage (Table 3), indica-
ting that the apparent shrinkage was a surface phenomenon.
At optimum conditions of temperature and humidity for
mold growth (30°C, 89% r.h.), very heavy mold growth was
present on all samples. As previously observed, the uninocu-
lated smoked control showed a mixture of colonies that
exhibited green, yellow-green, brown, black and white
colonies. Both inoculated lots (smoked and unsmoked) had
a heavy mold covering on all exterior surfaces. Under
these conditions, no differences could be observed between
the mold growth for the unsmoked and smoked sausages. No
shrinkage was apparent on any sample under these conditions
of incubation.

Incubation for 6 Weeks. All sausages held for 6 weeks

 

at 10°C and 75% relative humidity showed slightly more mold
growth than those held for 3 weeks. The colonies were

larger in all cases. Slight mold growth was evident on

41

Table 3. Production of aflatoxin B] by A. flavus NRRL-6549
on summer sausages, after 3 weeks of incubation
at 10°C and 30°C. Each value is the average of
three samples expressed in pg/kg.

 

 

 

 

Temperature 10°C 30°C

Relative Humidityi 75% 87% 79% 89%
Controla ___c ---C . ---° ---°
Smokedb ___c ___c ---C ...C
Unsmokedb ---C ---C ---C 0.25

 

aUninoculated smoked sausages.
bSausages inoculated with A. flavus at 10
CNo aflatoxin was detected at >0.02 ppb.

6 spores/sausage.

42

both the smoked and unsmoked inoculated sausages, but more
growth was apparent on the smoked uninoculated controls.
Increasing relative humidity and keeping temperature
the same (87% r.h. and 10°C, respectively) caused only
slight changes in mold growth. The number of colonies was
almost the same for the smoked uninoculated control and the
inoculated smoked sausages, but 60-75% of the surface of
the unsmoked inoculated sausages was covered with mold in
comparison to less than 20% in the case of inoculated smoked
sausages. In all cases the color of all colonies was blue-
green, which is characteristic of A. flavus. Although some
shrinkage was observed on all sausages at 10°C and 75%
relative humidity, at 10°C and 87% relative humidity no
shrinkage was apparent on visual examination, probably
because the high relative humidity prevented moisture losses.
At 30°C and 79% relative humidity, a great deal of
surface shrinkage was evident for all treatments. The
surface of the smoked uninoculated controls was completely
covered with brown, green and white colonies. The brown
colored mold appeared to exude a dark brown fluid on the
surface of the sausages. The smoked inoculated sausages
were completely covered with a brownish—gold mold, which
also exuded a similar dark fluid. In contrast to the
uninoculated smoked controls, the inoculated smoked sausages
showed only the brownish—gold mold colonies. The unsmoked

sausages were somewhat different than the smoked inoculated

43

ones, in that they were completely covered with a brownish-
gold mold, but in addition, also contained some greenish
white secondary contaminations on each sausage.

At 30°C and 89% relative humidity, which are the
optimum conditions for the growth of A. flavus, very little
surface shrinkage was apparent. This was confirmed by
moisture analysis of the sausages (Table 4). The uninocu-
lated smoked control was completely covered with a mixture
of colonies. Brownish-gold mold with brown fluid soaked
areas and several white and green colonies of secondary
contaminations were observed on each sausage. Under these
conditions, both the inoculated smoked and the inoculated
unsmoked sausages were similar in appearance. Their sur-
faces were covered with brownish-gold growth accompanied
by fluid soaked areas similar to those observed on the
controls. As in the case of the uninoculated smoked control,
but to a lesser degree, the inoculated smoked sausages and
inoculated unsmoked sausages both exhibited several small
white colonies of secondary contaminations. Under these
conditions all the sausages were soft, watery and lacked

firmness upon slicing.

Factors Influencing Aflatoxin Production

 

Aflatoxin Production During 3 Weeks Storage. Results

 

show that temperature and time are important factors influ-

encing the ability of the mold to produce aflatoxins. After

44

Table 4. Moisture content in summer sausages incubated for
3 weeks at 10°C and 30°C. Each value is the
average of three samples expressed as percent

 

 

 

moisture.
Temperature __. 10°C 30°C
Relative Humidity 75% 87% 79% 89%
Controla 49.65 47.74 57.84 46.65
Smokedb 49.67 45.52 58.06 50.38
Unsmokedb 49.44 47.39 59.04 49.00

 

aUninoculated smoked sausages.

Sausages inoculated with A. flavus at 10°

spores/sausage.

45

incubation for 3 weeks at 10°C, n0 aflatoxins were detected
in any of the three lots at either 75% or 87% relative
humidity (Table 3). These results agree with those reported
by Bullerman gt _1. (1969), who concluded that there is a
relationship between time, temperature and aflatoxin produc-
tion by A. flavus on fresh beef, bacon and ham. Results of
the present study confirmed those of Oldham §£._l- (1971),
who reported that A. flavus did not produce detectable afla-
toxin in cheddar cheese or luncheon-meat when kept at refri-
geration temperatures.

Data in Table 3 also show that even after the temperature
was increased (30°C) and all samples showed visible mold
growth, only the unsmoked inoculated lot held at high relative
humidity (80%) contained any measurable amount (0.26 pg/kg)
of aflatoxin B]. The data suggested that the mold requires
both time and favorable conditions of humidity to produce
aflatoxins.

The data in Table 3 also indicate that the combination
of low temperature (10°C) at either low relative humidity
(75-79%) or high relative humidity (87-89%), and smoking
retarded mold growth and aflatoxin production on the sausages.
Changes in relative humidity also affected the moisture con-
tent in the samples as shown in Table 4. High relative

humidities tended to increase the moisture content, with the

sausages apparently taking up moisture from the environment.

46

Austwick and Ayerst (1963) found that the most important
factors in growth and aflatoxin production by A. flavus are
the moisture content of the products and the relative humi-
dity in the environment surrounding the natural substrate.
Results of this study (Table 3) show that time and tempera-
ture also are important factors for aflatoxin production,
since neither visible mold growth nor aflatoxin production
were detected during 3 weeks of storage at low temperature
(10°C). 0n the other hand, 0.26 pg/kg of aflatoxins were
produced during storage at high temperature (30°C) and high
relative humidity (89%).

Aflatoxin Production During 6 Weeks Storagg. After 6

 

weeks incubation at 10°C and either 75 or 87% relative
humidity, there were no detectable levels of aflatoxins
in any of the samples except for the unsmoked inoculated
sausages held at high relative humidity (89%). These
sausages that had previously shown visible mycelial growth
produced measurable levels of aflatoxin (1.40 pg/kg, Table
5). In the rest of sausages held under the same conditions,
no aflatoxins were detected. The data indicate that for
periods of up to 6 weeks duration, low temperature, low
relative humidity, and smoking prevented growth mold and
aflatoxin production (Table 5).

Data in Table 5 also demonstrate that aflatoxins were
produced by A. flavus during 6 weeks of incubation. In

the smoked inoculated sausages, the levels of aflatoxins were

47

Table 5. Production of aflatoxin B] by A. flavus NRRL-6549
on summer sausages after 6 weeks of incubation at
10°C and at 30°C. Each value is the average of
three samples expressed in uQ/kg.

 

 

 

 

Temperature 10°C 30°C

Relative Humidity 75% 87% 75% 89%
Controla ___c ---c I ___c ---C
Smokedb ---c ---° 4.04 2.30
Unsmokedb ---c 1.40 1.64 6.60

aUninoculated smoked sausages.
bSausages inoculated with A. flavus at 10
cAflatoxins not detectable at >0.02 ppb.

spores/sausage.

48

4.04 pg/kg and 2.30 pg/kg at 79 and 89% relative humidity,
respectively. The unsmoked inoculated sausages contained
1.64 pg/kg at low relative humidity (75%) and 6.60 pg/kg at
high relative humidity (89%). These results demonstrated
that A. flavus can produce aflatoxins during 6 weeks storage
at 30°C, even at low relative humidity and after smoking.
The optimal conditions for aflatoxin production were Shown
to be 30°C and 89% relative humidity.

The results of this study agree with the report of
Austwick and Ayerst (1963) showing that the relative humidity
surrounding a natural substrate is one of the most important
factors in growth and aflatoxin production. The visual
estimation of growth, previously described, and the data in
Tables 3 and 5 demonstrate that mold growth and aflatoxin
production were only detected when relative humidity was
79% or more. Thus, results indicate that an increase in
relative humidity during storage favored increased levels of
aflatoxin production.

Increasing the hold time resulted in a noticeable
change in the moisture content of the sausages. Comparing
the results in Table 4 with those in Table 6 shows that all
samples increased in moisture content when they were held
in a relative humidity of 75% at 10°C for 6 weeks. Similar
changes were also observed during storage for 6 weeks at

relative humidities of 79 and 89% at 30°C.

49

 

 

 

 

 

Table 6. Moisture content in summer sausages incubated for
6 weeks at 10°C and at 30°C. Each value is the
average of three samples expressed as percent.

Temperature 10°C 30°C

Relative Humidity 7 % 87% 79% 39%

Controia 57.87 51.08 73.00 62.84

Smokedb 59.17 44.90 68.36 58.83

Unsmokedb 60.77 48.47 68.31 60.38

aUninoculated smoked sausages.

bSausages inoculated with A.f1avus at 10° spores/sausage.

50

Other Factors Related to Aflatoxin Production

Effects of pH. The pH values are given in Tables 7 and
8 and showed that the samples stored at low temperature (10°C)
did not change in pH at either 75 or 87% relative humidity.
At high temperature (30°C), on the other hand, the pH read-
ings obtained after 6 weeks were higher than those after 3
weeks, regardless of relative humidity (79 or 89%). On
studying the effects of pH on mold growth and aflatoxin pro-
duction, Davis _t _l. (1966) concluded that pH did not
influence either aflatoxin production or growth, so long as
the pH was higher than 4.0. Based on their results and in
light of the fact that yeasts and molds are more acid-
tolerant than other microorganisms (Frazier and Westhoff,
1978), it is probable that the absence of aflatoxins in the
present study was due to either low temperature or low
relative humidity instead of pH.

Effects of Smoking. Data in Tables 3 and 5 demonstrated
that smoke inhibits mold growth and aflatoxin formation.
Smoking of inoculated sausages delayed mold growth. Thus,
aflatoxins were not detected or were present at lower levels
than in the unsmoked inoculated sausages. When A. flavus
did grow, however, aflatoxins were present fn both smoked
and unsmoked inoculated sausages. Growth of mold and afla-
toxin production thus required favorable conditions of time,

temperature and relative humidity.

51

Table 7. Th8 pH of summer sausages incubated for 3 weeks at
10 C and 30°C. Each value is the average of three

 

 

 

 

samples.
Temperature 10°C 30°C
Relative Humidity 75% 87% 79% 89%
Controla 4.8 4.8 5.3 5.0
Smokedb 4 8 4.9 5 1 6 3
Unsmokedb 4.8 4.9 6.9 6.7

 

aUninoculated smoked sausages.
bSausages inoculated with A. flavus at 106 spores/sausage.

52

Table 8. The pH of summer sausages incubated for 6 weeks at
10°C and 30°C. Each value is the average of 3

 

 

 

samples.
Temperature 10°C 30°C
Relative Humidity 75% 87% 79% 89%
Controla 4.9 4.9 6.99 6.97.
Smokedb 4.9 4.9 6.70 '7.24
Unsmokedb 4.9 5.5 7.23 7.40

 

aUninoculated smoked sausages.

bSausages inoculated with A. flavus at 106

spores/sausage.

53

Effect of Curing Ingredients. There is controversy
regarding the influence of curing ingredients on aflatoxin
production. Strzelecki (1973) reported that potassium
nitrate inhibited aflatoxin production, but that sodium
nitrate stimulated production. He further concluded that the
complete curing mixture stimulated aflatoxin formation to the
greatest extent. These data are opposite to the results of
Bullerman gt gt. (1969), who reported that the curing salts
inhibited aflatoxin production. In this study the effect of
curing ingredients was not investigated since the purposes
of the experiment were to determine the production of afla-
toxins by a known strain of A. flavus on sausages produced
under simulated conditions of manufacture. Nevertheless,
results of this experiment suggest that the curing ingredients
do not inhibit aflatoxin formation, since aflatoxins were
produced whenever environmental conditions were favorable.
Further work will be needed to clarify the effects of curing

salts on production of aflatoxins.

CONCLUSIONS

A preliminary screening test showed that Agpergillus

 

flavus NRRL-6550 did not produce any aflatoxins but NRRL-6549
produced large amounts of aflatoxin B1. Thus, the latter
strain was used in all subsequent studies.

Aflatoxins were not produced during the first 3 weeks
of storage at 10°C, on any of the sausages at either 75 or
87% relative humidity. The growth of mold on the smoked
inoculated sausages and the unsmoked inoculated sausages was
greatly enhanced by increasing the temperature (30°C). How-
ever, high relative humidity (87%) was also required in
order to produce aflatoxin B1 during 3 weeks of storage at
30°C.

In periods of up to 6 weeks, low temperature (10°C),
low relative humidity (75%) and smoking retarded mold growth
and aflatoxin production. At 10°C and high relative humidity
(87%), only the unsmoked inoculated sausages produced
detectable levels of aflatoxins (1.40 ug/kg). However,
during 6 weeks storage at 30°C, aflatoxin B1 was produced in
amounts from 2.30 to 6.60 ug/kg, even at low relative humi-
dity and after smoking.

The presence or absence of aflatoxins was due to the

effects of temperature and relative humidity rather than pH.

54

55

Smoking inhibited mold growth and aflatoxin formation. When
A. flavus did grow, however, aflatoxins were detected in
both the smoked and unsmoked inoculated sausages regardless
of whether or not they were smoked.

Results demonstrate that aflatoxins can be produced on
summer sausages under favorable conditions for the growth
of A. flavus, which include time, temperature and relative
humidity. Although low levels of aflatoxins may occur in
summer sausages under refrigeration temperatures (10°C), the
amount of mold growth found on most samples held at high
temperatures (30°C) and high relative humidity (89%) would

make the sausages unacceptable for human consumption.

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