268
Journal of Food Protection, Vol. 59, No.3, Pages 268-275
Copyright ©, International Association of Milk, Food and Environmental
Fungicidal Effect of 15 Disinfectants against 25 Fungal
Contaminants Commonly Found in Bread
and Cheese Manufacturing
KIRSTEN BUNDGAARD-NIELSEN and PER V. NIELSEN*
Department of Biotechnology,
(MS# 95-87: Received 12 April1995; Accepted 22 June 1995)
ABSTRACT
Resistance of 19 mold and 6 yeast species to 15 commercial
disinfectants was investigated by using a suspension method in
which the fungicidal effect and germination time were determined
at 20°C.
Disinfectants containing 0.5% dodecyldiethylentriaminacetic
acid, 10 g of chloramine- T per I, 2.0% formaldehyde, 0.1 %
potassium hydroxide, 3.0% hydrogen peroxide, or 0.3% peracetic
acid were ineffective as fungicides. The fungicidal effect of
quaternary ammonium compounds and chlorine compounds showed
great variability between species and among the six isolates of
Penicillium roqueforti var. roqueforti tested. The isolates of P.
roqueforti var. cameum, P. discolor, Aspergillus versicolor, and
Eurotium repens examined were resistant to different quaternary
ammonium compounds. Conidia and vegetative cells were killed
by alcohols, whereas ascospores were resistant. Resistance of
ascospores to 70% ethanol increased with age. Both P. roqueforti
var. roqueforti and E. repens showed great variability of resistance
within isolates of each species.
Key words: Disinfectants, fungicidal effect, germination
fungi, yeasts, bread, cheese
In several foods the use of preservatives has been
reduced due to consumer demands. This has resulted in
increased incidents of product spoilage. Proper disinfection
of production facilities is important to avoid these problems.
This requires that disinfectants for food industrial hygiene
must contain compounds active against the spoilage fungi.
The most frequent causes of spoilage of bakery products and
cheese are molds and yeasts. The spoilage fungi on both
products are dominated by Penicillium species. Lund et al.
(14) identified 371 fungal contaminants of cheese, of which
91 % were Penicillium species, and Spicher (20) found that
81.3% of the contaminants on rye bread were Penicillium
species. Other frequently occurring species on bread and
cheese are Aspergillus, Eurotium, Cladosporium (8, 14) and
* Author for correspondence. Tel'. +45 4593 3066; Fax: +45 4588 4922
Sanitarians
Technical University of Denmark, DK-2800 Lyngby, Denmark
Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020
yeasts, e.g., Endomyces fibuliger and Hyphopichia burtonii,
which causes "chalky bread" (20).
Data on resistance of molds and yeasts to disinfectant
treatments are limited. Warcup and Baker (23) used 60%
ethanol to separate ascospores from conidia in soil, because
many ascomycetes survive treatment with ethanol. Accord-
ing to Liithi (15) 90% ethanol was the most effective
concentration when 70, 80 and 90% ethanol were tested
against an ethanol-resistant isolate of Penicillium expansum.
Andrews (1) found that 0.37% hypochlorite at a contact time
of 2 min efficiently killed Aspergillus fiavus, A. niger, and P.
chrysogenum. He also showed that ascospores from Euro-
tium repens were resistant to 0.37% hypochlorite, 3%
hydrogen peroxide, and 75% ethanol. To obtain an effective
killing of E. repens with hypochlorite, a 3% concentration
should be used. Beuchen and Marth (3) observed destruction
times between 18.3 and> 120 min in 4% hydrogen peroxide
to obtain a 99.9% reduction of A. parasiticus and A. fiavus
viable counts. They also showed that conidia from 14- and
time,
lO-day-old cultures of two isolates of A. fiavus were more
resistant to 4% hydrogen peroxide than conidia from 7-day-
old cultures. According to Cheng and Levin (5) 4% sodium
hydroxide destroyed A. niger conidia efficiently at 60°C.
In recommended standard disinfection tests such as the
Rideal-Walker test, the improved Kelsey-Sykes test, and the
Chick-Martin test (4, 7, 9) it is recognized that there is
variability in resistance among bacteria species. Data about
variability in disinfectant resistance between mold and yeast
species are limited. As variation in resistance among species
can be expected, this study was designed to investigate the
resistance of common spoilage fungi in bread and cheese
manufacturing to commonly used disinfectants.
MATERIALS AND METHODS
Test microorganisms
The fungi used in this study are listed in Table 1. They were all
obtained from the IBT culture collection at the Department of
Biotechnology, Technical University of Denmark.
 FUNGICIDAL EFFECT OF 15 DISINFECTANTS 269

TABLE 1. Microorganisms examined peptonized water (0.1 % Bacto-peptone [Difco] in distilled water)
and pipetting the suspension into a sterile test tube. This procedure
Organisms Isolates Source
was repeated. Ascosporogenic fungi were cultured for 21 days at
Molds 25 C. Cultures used for testing the resistance of ascospores to 70%
D

ethanol due to aging were cultured from 17 to 110 days. Ascospore

Asperg illus flavus IBT 6919 bread suspensions were prepared as described by Conner et al. (6) where
A. niger IBT6292 bread ascospores are separated from cleistothecia by ultrasound treatment.
A. versicolor IBT 10128 cheese
IBT 12384 cheese
Disinfectants
Cladosporium spp. IBT 13731 bread
The experiments were carried out with commercial disinfec-
Eurotium rep ens IBT6928 bread factory 1
tants representing products used in food-processing facilities. For
IBT 11765 pectin
comparative testing of fungicidal activity, each disinfectant was
IBT 11773 rye bread
mixed in distilled water according to manufacturers instructions.
IBT 11774 malt
The disinfectants are listed in Table 2.
IBT 14048 bread factory 2
Monascus ruber IBT 10951 bread
Neosartorya pseudo- Test procedures
fischeri IBT6472 cherry filling One milliliter of the mold or yeast suspension was transferred
Penicillium commune IBT 10253 cheese to 9.0 ml of the disinfectant solution. Mter a contact time of exactly

Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020

IBT 10727 cheese 10 min at 20 C, 1.0 ml ofthe mixture was transferred into 9.0 ml of
D

IBT 13994 bread peptonized water and mixed. Tenfold dilutions of the mixture were
P. caseifulvum IBT 14760 cheese
P. corylophilum IBT 14040 bread
P. chrysogenum IBT 12572 cheese TABLE 2. Disinfectants
P. crustosum IBT 14747 cheese
Class Components Product
P. discolor IBT 14765 cheese
P. nalgiovense IBT 10252 cheese Quaternary ammo- Benzalkonium Sirafana
IBT 13039 cheese nium chloride Desinfektionsmidletb
P. roqueforti var. N-alkyl(C9-C12) P3-Sterila
roqueforti IBT 4176 cheese manufacturing dimethylam-
IBT 5575 cheese manufacturing monium chloride
IBT 11460 beer Dodecyl- + didecyl-
IBT 11524 bread benzyldimethyl-
IBT 14088 bread ammonium chlo-
P. roqueforti var. car- ride
neum IBT 14042 bread Quaternary ammo- Benzalkonium chlo- Tegodor
P. solitum IBT15l70 cheese nium + aldehydes ride + cetalkoni-
P. verrucosum IBT 13036 cheese umchloride +
Scopulariopsis brevi- formaldehyde +
caulis IBT 12870 cheese glutaraldehyde
Yeasts Alcohols Ethanol
Isopropanol
Debaryomyces han-
Chlorine compounds Chlorine dioxide Jettadam-A100 C

senii IBT 17 cheese

Hypochlorite Stafilex SU 34()d
Hyphopichia burtonii IBT249 bread
Sodium dichloroiso- Klortabs C

Moniliella suaveolens IBT680 bread

cyanuric acid Trefa
Pichia norvegensis IBT47 cheese
Pichia anomala Chloramine- T
IBT 283 bread
(sodium toluene-4-
Torulaspora del-
sulfone-chlo-
brueckii IBT6l4 cheese
ramine)
Ampholyte Dodecy ldiethy lene- Tego 5lc
triaminacetic acid
Media
Aldehyde Formaldehyde Rivonita
All Penicillium, Cladosporium and Scopulariopsis isolates
Alkaline Potassium hydroxide Deconex lIe
were cultured on Czapek yeast autolysate (CYA) agar (18). The
Peroxides Hydrogen peroxide Oxidanc
Aspergillus and Eurotium isolates were cultured on 18% dichloran
Peracetic acid Oxidan Ekstra C

glycerol (DG-18) agar (11). Oatmeal (OAT) agar (19) was used for
the growth of Neosartorya pseudofischeri and Monascus ruber. a Henkel Hygiejne AlS, Carl Jabobsensvej 27-29, 2500 Valby,
The yeasts were cultured on yeast malt extract (YM) agar (21 g of Denmark.
YM broth [Difco], 20.0 g of agar, and 1,000 ml of distilled water). b Cleansolve International Aps, Rugardsvej 224, 5210 Odense,
Denmark.
Preparations of suspensions of test microorganisms C Novadan Kemi AlS, Platinvej 29,6000 Kolding, Denmark.
Conidia and yeast cell suspensions were prepared from d Lever-Otares AlS, Petersmindevej 30, 5100 Odense, Denmark.
cultures incubated for 7 days at 2YC by flooding colonies with e Borer Chemie AG, Solo thurn, Switzerland.
270 BUNDGAARD-NIELSEN AND NIELSEN

TABLE 3. Fungicidal effect (FE) and detection time (Dt) of disinfectants against different fungi and yeasts after an exposure time of 10 min

Benzalkonium N-alkyIdimethylamm. Dode- + didecyI

chloride chloride ammochloride
1.5% 2.0% 2.0%

Organisms Isolate FE Dt FE Dt FE Dt

Conidia
A.jlavus IBT 6919 2.0 ND" 2.2 ND >5.2 ND
A. niger IBT 6292 >5.2 ND 2.7 ND >5.2 ND
A. versicolor IBTlO128 4.0 ND 3.0 1.5 3.0 ND
IBT12384 1.2 1.8 2.3 ND 1.5 1.8
Cladosporium spp. IBTl373 1 >4.1 ND 2.9 ND >4.1 ND
P. commune IBTl0253 2.7 ND 4.3 ND 2.7 ND
IBTlO727 4.0 ND 3.2 ND 4.0 ND
IBTl3994 4.0 ND 4.3 ND 4.3 ND
P. caseifulvum IBTl4760 2.7 ND 2.7 ND >4.9 ND
P. corylophilum IBTl4040 >4.8 ND 3.7 ND >4.8 ND

Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020

P. chrysogenum IBTl2572 >5.2 ND 4.0 ND 4.0 ND
P. crustosum IBT14747 4.3 ND 4.0 ND >5.2 ND
P. discolor IBTl4765 3.0 2.3 3.0 ND 3.0 ND
P. nalgiovense IBTl0252 >4.2 ND 3.0 ND >4.2 ND
IBTl3039 2.3 ND 2.3 ND 2.0 ND
P. roqueforti var. roqueforti IBTl1524 1.0 1.5 2.9 1.6 3.0 1.5
P. roqueforti var. carneum IBTl4042 1.2 1.2 4.0 ND 4.0 ND
P. solitum IBTl5170 >4.8 ND 3.7 ND 3.7 ND
P. verrucosum IBTl3036 3.1 ND 2.1 ND >4.2 ND
S. brevicaulis IBTl2870 >4.2 ND >4.2 ND >4.2 ND
Ascospores
E. repens IBT 6928 >4.5 ND 3.3 1.4 3.3 1.5
M. ruber IBTl095 1 >4.1 ND 2.9 ND >4.1 ND
N. pseudofischeri IBT 6472 >4.5 ND 3.3 ND 3.3 ND
Yeasts
D. hansenii IBT 17 >4.5 ND >4.5 ND >4.5 ND
H. burtonii IBT 249 >5.2 ND 4.0 ND >5.2 ND
M. suaveolens IBT 680 >4.5 ND >4.5 ND >4.5 ND
P. norvegensis IBT 47 >5.9 ND 4.7 ND >5.9 ND
P. anomala IBT 283 3.0 ND 4.0 ND 4.0 ND
T. delbrueckii IBT 614 >4.8 ND >4.8 ND >4.8 ND

a No detection of growth within 14 days.

made to inactivate the disinfectant. Four O.l-ml aliquots of the
mixture were inoculated into microtiter well plates containing 0.1
ml CYA per welL The mean numbers of colony-forming units
(CPU) per ml were determined by most probable number methods
(17) after 3, 7, and 14 days. Two 0.1 ml mixtures were dispensed
from a 100-fold dilution into Bactometer modules. Modules were
incubated in a Bactometer® M123-3 (bioMerieux ltd., UK) at 25°C
for 100 h. Microtiter plates were incubated at 25°C and examined
after 3, 7, and 14 days.
extract (Difco), 30.0 g of glucose (anhydrous), 10.0 g of KH2P04
(Merck), 20.0 g of agar, and 1,000 ml of distilled water. Autoclaved
medium (0.75 ml) was added to each well in the Bactometer
modules.
Data analysis
The fungicidal effect (FE) of the disinfectant at the given
concentration was expressed by the formula (13)

Detection time
After treatment with disinfectants, the microorganisms were
incubated in the Bactometer. The system consists of three Bacto-
matic Processing Units each having two accurately temperature-
controlled incubators. Each incubator holds four disposable Bactom-
eter modules with 16 wells each. Detection of fungal growth was
based on measurements of impedance change in the substrate
caused by metabolic activity. Determination of the detection time
was based on the rate and magnitude of change in capacitance.
Detection time was measured with the Bactometer by cultur-
ing the test microorganisms on a media containing 7.5 g of yeast
where ND is the number of CFU/ml of the test microorganism with
disinfectant, and Nc is the number of CFU/ml of the test microor-
ganism without disinfectant. The relative detection time of the
microorganisms after treatment with the disinfectant was expressed
as
where DtD is the detection time of the test microorganism with
disinfectant, and Dtc is the detection time of the test microorganism
without disinfectant.
 FUNGICIDAL EFFECT OF 15 DISINFECTANTS 271

The microorganisms tested were considered sensitive to a
treatment if growth could not be detected by Bactometer or visually
within 14 days. Dt values for these microorganisms were therefore
not detected. Thus, all fungi with real Dt values are considered
resistant to the applied treatment.
An estimate of the standard deviation of the fungicidal effect
was determined by repeating three disinfection tests (1.5% benzal-
konium chloride, 70% ethanol, and 0.3% hypochlorite) for two
isolates (P. roqueforti var. roqueforti IBT 11524 and E. repens IBT
6928). The standard deviation calculated as the average of these six
estimations was 0.6 FE.
RESULTS AND DISCUSSION
Effect of disinfectants on fungal spores and yeasts
Quaternary ammonium compounds. Considerable vari-
ability in fungicidal effect among species was observed after
exposing conidia and ascospores to four disinfectants contain-
roqueforti var. roqueforti lET 11524 to more than 5.2 for A.
niger lET 6292. Results for P. commune, P. nalgiovense, and
A. versicolor show that variability in resistance within
strains of the same species exists. The majority of species
did not survive treatment with quaternary ammonium com-
pounds, because FE were high and growth could not be
detected by the Bactometer within 100 h. However six
isolates were much more resistant. P. roqueforti var. roque-
forti lET 11524 was resistant to all four disinfectants. It had
a low FE and only slightly prolonged detection times,
indicating that surviving spores were unaffected by the
disinfectants. P. roqueforti var. carneum lET 14042, P.
discolor lET 14765, A. versicolor lET 12384 and lET
10128, and E. repens lET 6928 were resistant to at least one
of the quaternary ammonium components, as growth of
,these molds could be detected by the Bactometer within
100 h. The higher tolerance against different quaternary

Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020

ing quaternary ammonium compounds (Tables 3 and 4). In ammonium compounds of these six molds may be due to a
benzalkonium chloride the FE varied from 0.8 for P. positively charged or hydrophobic spore surface which

TABLE 4. Fungicidal effect (FE) and detection time (Dt) of disinfectants against different fungi and yeasts after an exposure time of 10 min

Benzalkonium chloride
+ formaldehyde
+ glutaraldehyde Ethanol Isopropanol
+ cetalkonium chloride 2.0% 70% 70%

Organisms Isolate FE Dt FE Dt FE Dt

Conidia
A.fiavus IBT 6919 >5.2 NDa 4.0 ND 4.0 ND
A. niger IBT6292 >5.2 ND >5.2 ND >5.2 ND
A. versicolor IBT 10128 3.0 ND >5.2 ND >5.2 ND
IBT 12384 2.0 1.8 3.3 ND 3.3 ND
Cladosporium spp. IBT 13731 >4.1 ND >4.1 ND >4.1 ND
P. commune IBT 10253 2.7 ND 2.9 ND 2.7 ND
IBT 10727 3.2 ND 4.0 ND 4.0 ND
IBT 13994 4.3 ND 4.0 ND 4.0 ND
P. caseifulvum IBT 14760 2.7 ND 2.7 ND 2.7 ND
P. corylophilum IBT 14040 >4.8 ND 4.6 ND >4.8 ND
P. chrysogenum IBT 12572 4.0 ND >5.2 ND >5.2 ND
P. crustosum IBT 14747 >4.2 ND 4.0 ND 4.0 ND
P. discolor IBT 14765 3.0 ND 3.0 ND 3.0 ND
P. nalgiovense IBT 10252 >4.2 ND 3.0 ND 3.0 ND
IBT 13039 2.3 ND 2.3 ND 2.3 ND
P. roqueforti var. roqueforti IBT 11524 1.7 1.3 3.6 ND 3;5 1.7
P. roqueforti var. carneum IBT 14042 4.0 ND 3.4 ND >5.2 ND
P. solitum IBT 15170 3.7 ND 3.2 ND 3.7 ND
P. verrucosum IBT 13036 3.1 ND 3.1 ND 3.1 ND
S. brevicaulis IBT 12870 >4.2 ND >4.2 ND >4.2 ND
Ascospores
E. repens IBT6928 3.3 ND 3.0 ND 2.3 1.5
M. ruber IBT 10951 >4.1 ND 1.3 ND 0.9 1.2
N. pseudofischeri IBT6472 3.3 ND 4.0 ND 3.3 ND
Yeasts
D. hansenii IBT 17 >4.5 ND >4.5 ND >4.5 ND
H. burtonii IBT249 >5.2 ND >5.2 ND >5.2 ND
M. suaveolens IBT680 >4.5 ND >4.5 ND >4.5 ND
P. norvegensis IBT 47 >5.9 ND >5.9 ND >5.9 ND
P. anomala IBT283 3.0 ND >5.2 ND >5.2 ND
T. delbrueckii IBT 614 >4.8 ND >4.8 ND >4.8 ND

a No detection of growth within 14 days.

272 BUNDGAARD-NIELSEN AND NIELSEN

could prevent the quaternary ammonium compound from
getting in contact with the surface of conidia and ascospores.
P. anomala was the most resistant yeast, which may be
due to the formation of ascospores (detected by microscopic
inspection). Jones et al. (12) and Neumayr et aL (16) also
found yeast ascospores to be more resistant to disinfectants
and biocides than vegetative cells.
Alcohols. Vegetative cells and conidia could not survive
treatment with 70% ethanoL However, P. roqueforti var.
roqueforti could withstand contact with 70% isopropanol for
10 min (Table 4). Most resistant to both alcohols was the
ascospore-forming mold M. ruber, followed by E. repens.
The relative detection time for M. ruber (1.2) indicates that
the surviving ascospores were affected by the ethanol
treatment. The delay in detection may be due to slow
evaporation of alcohols from the Bactometer wells, which
were closed with plastic caps (the ethanol concentration in a
niger and B. pumilus was reversibly inhibited by 0.5%
ethanoL Inspection of Bactometer wells after 7 and 14 days
showed growth of M. ruber and E. repens when the number
of surviving ascospores was more than 5 . 102 (two wells
each inoculated with 0.1 ml of a 100-fold dilution results in a
detection limit of 5· 102 cfu/well). This indicates that
germination of survived spores was inhibited until most of
the ethanol had evaporated. Ascospores of the heat-resistant
species N. pseudofischeri were not resistant to alcohoL
Ethanol treatment resulted in higher FE and longer
detection times for P. roqueforti var. roqueforti, E. repens,
and M. ruber as compared to isopropanoL In contrast,
reports have shown that isopropanol has a slightly greater
bactericidal effect and stronger inhibition of detection times
than ethanol (4, 22).
Chlorine-containing compounds. Disinfectants contain-
ing chlorine dioxide and hypochlorite (Table 5) performed

Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020

Bactometer well is approximately 0.7%). Trujillo and Laible similarly against molds and yeasts. Growth was not detected
(22) found that spore germination of Bacillus subtilis var. within 100 h. in the Bactometer. However, chlorine dioxide

TABLE 5. Fungicidal effect (FE) and detection time (Dt) of disinfectants against different fungi and yeasts after an exposure time of 10 min

Sodium
dichloroisocyanuric Chlorine dioxide Hypochlorite
acid 0.37 gil 5.0% 3.0%

Organisms Isolate FE Dt FE Dt FE Dt

Conidia
A·fiavus IBT 6919 4.3 2.5 4.0 ND 4.3 ND
A. niger IBT6292 2.0 NDa >5.2 ND >5.2 ND
A. versicolor IBT 10128 >5.2 ND 3.0 ND >5.2 ND
IBT 12384 >4.5 ND 3.3 ND >4.5 ND
Cladosporium spp. IBT 13731 2.9 ND >4.1 ND >4.1 ND
P. commune IBT 10253 1.7 2.3 >4.9 ND 1.7 ND
IBT 10727 3.7 2.9 >5.2 ND 4.0 ND
IBT 13994 4.0 ND 4.3 ND >5.2 ND
P. caseifulvum IBT 14760 1.9 2.3 2.7 ND >4.9 2.3
P. corylophilum IBT 14040 4.4 ND >4.8 ND >4.8 ND
P. chrysogenum IBT 12572 3.7 ND >5.2 ND >5.2 ND
P. crustosum IBT 14747 2.3 ND 4.0 ND >5.2 ND
P. discolor IBT 14765 1.9 3.2 3.0 ND >5.2 ND
P. nalgiovense IBT 10252 >4.2 ND >4.2 ND >4.2 ND
IBT 13039 1.7 2.6 3.3 ND >4.5 ND
P. roqueforti var. roqueforti IBT 11524 0.9 1.6 3.5 ND >5.2 ND
P. roqueforti var. carneum IBT 14042 2.7 1.1 4.0 ND >5.2 ND
P. solitum IBT 15170 3.7 ND 3.7 ND >4.8 ND
P. verrucosum IBT 13036 2.4 ND 3.1 ND >4.2 ND
S. brevicaulis IBT 12870 >4.2 ND 3.0 ND >4.2 ND
Ascospores
E. repens IBT 6928 0.3 1.4 >4.5 ND >4.5 ND
M. ruber IBT 10951 1.6 ND >4.1 ND 2.9 ND
N. pseudofischeri IBT6472 3.6 ND 3.3 ND 4.0 ND
Yeasts
D. hansenii IBT 17 >4.5 ND 3.3 ND >4.5 ND
H. burtonii IBT249 >5.2 ND 4.0 ND >5.2 ND
M. suaveolens IBT 680 >4.5 ND >4.5 ND >4.5 ND
P. norvegensis IBT47 >5.9 ND >5.9 ND >5.9 ND
P. anomala IBT283 3.0 ND 3.0 ND >5.2 ND
T. delbrueckii IBT 614 >4.8 ND >4.8 ND >4.8 ND

a No detection of growth within 14 days.

 FUNGICIDAL EFFECT OF 15 DISINFECTANTS 273

TABLE 6. Fungicidal effect (FE) and detection time (Dt) of disinfectants with poor killing effect against different fungi and yeasts after an
exposure time of 10 min

Chloramine-T Ampholyte Formaldehyde

10 gil 0.5% 2.0%

Organisms Isolate FE Dt FE Dt FE Dt

Conidia
Penicillium roqueforti var. roqueforti IBT 11524 0.0 1.1 0.4 1.2 0.0 1.2
Ascospore
Eurotium repens IBT6928 0.0 1.0 2.0 1.5 0.0 1.0
Yeast
Pichia anomala IBT283 0.0 1.0 3.0 NDa 0.0 1.1

a No detection of growth within 14 days.

was generally less efficient than hypochlorite. Diluted niger conidia in 0.5% formaldehyde after an exposure time
chlorine dioxide (1:10) generally gave larger FE and longer of 2 h. However, this is an unrealistic contact time in most

Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020

detection times than diluted hypochlorite (1: 10), indicating a
better oxidation capacity even under diluted conditions. D.
hansen ii, H. burtonii, and the ascospore-forming yeast P.
anomala were slightly more resistant to chlorine dioxide
than other yeasts.
Sodium dichloroisocyanuric acid was effective against
all yeasts, except the ascoporogenous P. anomala, but did
not show consistent killing of molds.
Chloramine-Twas inactive as a disinfectant against
molds and yeasts (Table 6). Hegna and Clausen (10) also
found that A. fumigatus and Candida albicans were resistant
to chloramine- T under "clean" conditions, whereas chlora-
mine proved to have a strong fungicidal effect when 5%
autoclaved bakers yeast was added to the test tube in order to
simulate "dirty" conditions.
Ampholyte. The least efficient surface active disinfec-
tant was an ampholyte containing 0.5% dodecyldiethyltriami-
nacetic acid (Table 6). P. anomala was the only microorgan-
ism sensitive to the disinfectant. The ampholyte lost its
effect completely upon dilution (1: 10).
Formaldehyde. A disinfectant containing 2% formalde-
hyde (Table 6) showed no fungicidal activity or effect on
detection times of P. roqueforti var. roqueforti, E. repens,
and P. anomala. Longer contact times may result in better
inhibition: Spicher and Peters (21) found a FE of 4.5 for A.
production facilities, unless it is used for soaking.
Potassium hydroxide. A solution of 0.1 % potassium
hydroxide (Table 7) showed no fungicidal effect on P.
roqueforti var. roqueforti, E. repens, and P. anomala and
only the surviving conidia of P. roqueforti var. roqueforti
were slightly affected, because the relative detection time
was 1.3. The poor effect of this disinfectant is probably due
to a combination of too Iowa concentration and tempera-
ture; Cheng and Levin (5) used 4% sodium hydroxide at
60°C to destroy A. niger conidia efficiently.
Peroxides. Table 7 shows that treatment with 3.0%
hydrogen peroxide and 0.3% peracetic acid resulted in
comparable FE and detection times for P. roqueforti var.
roqueforti, E. repens, and P. anomala. Peroxide showed no
fungicidal effect on E. repens and P. anomala and only a
minority of P. roqueforti var. roqueforti conidia were killed,
i.e., the FE were 0.3 in both disinfectants. A 3.0% hydrogen
peroxide solution showed low fungicidal effect against 18
molds (data not shown), i.e., the FE were in the range of 0 to
1.0. However, against 7 molds the detection times were
significantly prolonged (almost doubled) indicating that the
conidia were damaged by hydrogen peroxide. The poor
killing effects of hydrogen peroxide and peracetic acid are
probably caused by too Iowa concentration or too short a
contact time (10 min). Beuchen and Marth (3) thus observed

TABLE 7. Fungicidal effect (FE) and detection time (Dt) of disinfectants with poor killing effect against different fungi and yeasts after an
exposure time of 10 min

Potassium Hydrogen
hydroxide peroxide Peracetic acid
0.1% 3.0% 0.3%

Organisms Isolate FE Dt FE Dt FE Dt

Conidia
P. roqueforti var. roqueforti IBT 11524 0.0 1.3 0.3 1.2 0.3 1.4
Ascospore
E. repens IBT6928 0.0 1.0 0.0 1.0 0.0 1.0
Yeast
P. anomala IBT283 0.0 1.0 0.0 1.0 0.0 1.1
274
6 resistant to treatment with hypochlorite, and the one isolate
2 for 10 min were strongly increased by age, as illustrated in
..........
e .A. .
o observed after 35 days for E. repens and 110 days for N.
1.5% Benzalkonium 70% thanol
chloride
FIGURE 1. Fungicidal effect (FE) on five isolates ofP. roqueforti
var. roqueforti in three disinfectants after an exposure time of 10
min. Isolates: 0, IBT 1/524; e, IBT 14088; 6, IBT 1/460; A, IBT
5575; \7, IBT4176.
times between 18.3 and > 120 min in 4% hydrogen peroxide
to obtain destruction of A. parasiticus and A. jlavus conidia.
Variation in resistance within isolates of one species
The previous results for the three species P. commune,
P. nalgiovense, and A. versicolor indicate differences in
resistance within isolates from the same species. Five
isolates each of P. roqueforti var. roqueforti and E. repens
were tested against three different active components. Fig-
ures 1 and 2 shows that the FE vary between isolates of the
same species. The differences between the largest and
smallest FE are greater than the method standard deviation
of :±:0.6 FE. The largest variation in resistance within
isolates was found for the five isolates of P. roqueforti var.
roqueforti (Figure 1). Resistance of these isolates in benzal-
konium chloride indicate that resistance depended upon the
origin of the species. Two isolates from bread (lET 11524
and lET 14088) were resistant to 1.5% benzalkonium
chloride whereas isolates from cheese manufacturing (lET
5575 and lET 4176) or beer (lET 11460) were not. Similar
grouping was not observed with 70% ethanol and 0.3%
hypochlorite nor for E. repens isolates. Comparing the
results of 70% ethanol and 0.3% hypochlorite shows that P.
roqueforti var. roqueforti isolates killed by ethanol were
5
4 3
.A.
3 6············
~ ~ ~
2 ···············9·································\7····
o
0.15% Benzalkonium 70% thanol
chloride
FIGURE 2. Fungicidal effect (FE) on five isolates ofE. repens in
three disinfectants after an exposure time of 10 min. Isolates: 0,
IBT 1/765; e, IBT 1/773; 6, IBT 6829; A, IBT 14089; \7, IBT
1/774.
BUNDGAARD-NIELSEN AND NIELSEN
resistant to ethanol was killed by hypochlorite. A similar
effect was not observed for E. repens isolates.
Resistance of ascospores to 70% ethanol due to aging
Resistance of ascospores to treatment in 70% ethanol
Figure 3. More than 1/1,000 M. ruber ascospores survived
after 21 days; however, the same resistance was first
0.3% Hypochlorite
pseudofischeri.
Increased resistance with age for ascospores suggests
that they undergo structural and/or biochemical changes
with time that may explain the change in ethanol resistance.
Conner et al. (6) investigated whether heat resistance due to
aging of Neosartorya fischeri var. glaber was due to
biochemical changes. They found that heat-resistant asco-
Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020
spores contained higher levels of glycerol, mannitol, and
trehalose than heat-sensitive ascospores, suggesting that the
lowering of internal water activity or membrane stabilization
may account for increased heat resistance. Banner et al. (2)
found that ascospores of Byssochlamus fulva differed from
other spores by a greater quantity of fatty acids longer than
C20 in the more heat-resistant ascospores. The presence of
water is essential for alcohol to be effective to denature
proteins. The increased resistance of ascospores against
ethanol might therefore be caused by a lower internal water
activity or a more stable membrane.
Age did not affect the resistance of conidia to 70%
ethanol (Figure 4). Decreases in FE were observed after 19
to 24 days in four of five mold species; whether this was
caused by biochemical changes or method standard devia-
tion (:±:0.6 FE) cannot be determined from these results.
Luthi (15) investigated the resistance of P. expansum conidia
in 70% ethanol during 870 days and found that the ethanol
resistance began to increase rapidly after 30 days; it was
suggested that the increase was caused by a lowering of
water activity in the spore. Our results for other Penicillia
spp. did not confirm these findings, but as P. expansum was
5
~-
w
u.
- ---.,0.
2
.
0.3% H
•
ochlorite
o
o 20 40 60 80 100 120
Age of ascospores (days)
FIGURE 3. Fungicidal effect (FE) on ascospore-forming molds at
different age when exposed to 70% ethanol for 10 minutes. Molds:
0, M. ruber IBT 10951; e, E. repens IBT6928; 6, N. pseudofisch-
eri IBT 6472.
 FUNGICIDAL EFFECT OF 15 DISINFECTANTS
5 2.
-4
rr--
~ 44:545-548.
~
4 peroxide on conidia from toxigenic strains of Aspergillus jlavus and
w 3rd ed. Lea & Febiger, Philadelphia, PA.
u..
3 5.
2 changes in heat resistance of Neosartorya fischeri ascospores. Trans.
o 20 40 60 80 100 120
Age of conidia (days)
FIGURE 4. Fungicidal effect (FE) on conidia at different ages
when exposed to 70% ethanol for 10 min. Organisms: 0, P.
roqueforti var. roqueforti 1BT 11524; e, P. roqueforti var. cameum
1BT 14042; t-" P. corylophilum 1BT 14040; .•., P. commune 1BT
10253; \1, P. solitum1BT 15170.
not included in our study a different behavior of P. expansum
cannot be disputed.
Conclusion
Resistance of molds and yeasts strongly depends on the
strain and species of the isolate and the active component of
the disinfectant. Different isolates of one species may show
different responses to the same disinfectant, resulting in an
effective kill in one case and almost no effect in others. To
obtain proper manufacturing hygiene it is, therefore, impor-
tant to know the resistance of the spoilage fungi in the
process and product and from this knowledge to select
proper disinfectants. The use of one disinfectant is usually
not enough. It is necessary to make a disinfection plan where
at least two different disinfectants containing different active
components are used in rotation during the week to keep the
contamination of molds and yeasts in control and to avoid
selection of superresistant isolates.
REFERENCES
1. Andrews, S. 1986. Optimization of conditions for the surface
disinfection of sorghum and sultanas using sodium hypochlorite
solutions, p. 28-30. In A. D. King, Jr., J. I. Pitt, L. R. Beuchat, and J.
E. L. Corry (ed.), Methods for the mycological examination of food.
Plenum Press, New York.
275
Banner, M. J., L. R. Mattick, and D. F. Splittstoesser. 1979. Chemical
composition of the ascospores of Byssochlamus fulva. J. Food Sci.
3. Beuchen, S. Y., and E. H. Marth. 1977. Sporicidal action of hydrogen
Aspergillus parasiticus. J. Food Prot. 40:617-621.
4. Block, S. S. (ed.). 1983. Desinfection, sterilization and preservation,
Cheng, K. c., and R. F. Levin. 1970. Chemical destruction of
Aspergillus niger conidiospores. J. Food Sci. 35:62-66.
6. Conner, D. E., L. R. Beuchat, and C. J. Chang. 1987. Age-related
changes in ultrastructure and chemical composition associated with
Br. MycoL Soc. 89:539-550.
7. Croshaw, B. 1981. In C. H. Collins, M. C. Allwood, S. F. Bloomfield,
and A. Fox (ed.), Disinfectants: their use and evaluation of effective-
ness, p. 1--48. The Society for Applied Bacteriology technical ser. no.
16. Academic Press, London.
8. Frisvad, J. c., and O. Filtenborg. 1989. Terverticillate Penicillia:
Chemotaxonomy and mycotoxin production. Mycologia 81:837-861.
9. Hayes, P. R. 1985. Food microbiology and hygiene, p. 268-295.
Elsevier Applied Science Publishers, London.
Downloaded from http://jfoodprotection.org/doi/pdf/10.4315/0362-028X-59.3.268 by guest on 05 March 2020
10. Hegna, I. K., and O. G. Clausen. 1988. An investigation of the
bactericidal and fungicidal effects of certain disinfectants by use of an
capacity test. Ann. Inst. PasteurlMicrobioL (Paris) 139:473--483.
11. Hocking, A. D., and J. I. Pitt. 1980. Dichloran glycerol medium for
enumeration of xerophilic fungi from low-moisture foods. AppL
Environ. MicrobioL 37:488--492.
12. Jones, M. V., M. D. Johnson, and T. M. Herd. 1991. Sensitivity of
yeast vegetative cells and ascospores to biocides and environmental
stress. Lett. AppL MicrobioL 12:254-257.
13. Lensing, H. H., and H. L. Oei. 1985. Investigation on the sporicidal
and fungicidal activity of disinfectants. ZbL Bakt. Hyg., I. Abt. Orig.
B 181:487,--495.
14. Lund, F., O. Filtenborg, and J. C. Frisvad. 1995. Associated mycoflora
of cheese. Food MicrobioL 12: 173-180.
15. Ltithi, H. 1954. Uber die alkoholresistens der konidien von Penicil-
lium expansum. Mitt. Geb. Lebensmittelunters. Hyg. 45:26-33.
16. Neumayr, L., G. Heyderhoff, and J. Kramer. 1989. Differences in the
resistance between vegetative cells and ascospores of selected yeast
strains against disinfectants on the basis of biguanoides, peracetic acid
and quaternary ammonium compounds. Yeasts 5: 17-22.
17. Nimelli, S. 1983. Statistical evaluation of results from quantitative
microbial examination. Nordisk Metodikkommitte fUr Livsmedel
Report no. 1, 2nd ed. NMKL, Helsinki, Finland.
18. Pitt, J. I. 1979. The genus Penicillium and its teleomorphic states
Eupenicillium and Talaromyces. Academic Press, London.
19. Samson, R. A., and E. S. van Reenen-Hoekstra. 1988. Introduction to
food-borne fungi, 3rd ed. Centraalbureau voor Schimmelcultures,
Baarn, The Netherlands.
20. Spicher, G. 1985. Die erreger der schimmelbildung bei backwaren. 4.
Mitteilung: Weitere Untersuchungen tiber die auf verpackten schnitt-
broten auftretenden schimmelpilze. Dtsch. Lebensm. Rdsch. 81: 16-20.
21. Spicher, G., andJ. Peters. 1976. Microbial resistance to formaldehyde
in comparative studies in some selected species of vegetative bacteria,
bacterial spores, fungi, bacteriophages and viruses. ZbL Bakt. Hyg., I.
Abt. Orig. B 163:486-508.
22. Trujillo, R., and N. Laible. 1970. Reversible inhibition of spore
germination by alcohols. App!. MicrobioL 20:620-623.
23. Warcup, J. H., and K. F. Baker. 1963. Occurrence of dormant
ascospores in soil. Nature 197:1317-1318.