Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Synergism between hydrogen peroxide and seventeen

acids against five agri-food-borne fungi and one yeast
strain
H. Martin and P. Maris
Anses, Fougères Laboratory, Fougères Cedex, France

Keywords
acid, combination, disinfection, fungus,
hydrogen peroxide, synergism, yeast.
Correspondence
Hélène Martin, Anses, Fougères Laboratory,
La Haute Marche, Javené, BP 90203, 35302
Fougères Cedex, France. E-mail: helene.
martin-lesaout@anses.fr
Research paper on improvement of fungicidal
efficacy of hydrogen peroxide in terms of
synergy when associated with various acids.
2012/1262: received 12 July 2012, revised 31
August 2012 and accepted 10 September
2012
doi:10.1111/jam.12016
Abstract
Aims: The objective of this study was to evaluate fungicidal efficacy of
hydrogen peroxide administered in combination with 17 mineral and organic
acids authorized for use in the food industry.
Methods and Results: The assays were performed on a 96-well microplate
using a microdilution technique based on the checkerboard titration method.
The six selected strains (one yeast and five fungi) were reference strains and
strains representative of contaminating fungi found in the food industry. Each
synergistic hydrogen peroxide/acid combination found after fifteen minutes
contact time at 20°C in distilled water was then tested in conditions simulating
four different use conditions. Twelve combinations were synergistic in distilled
water, eleven of these remained synergistic with one or more of the four
mineral and organic interfering substances selected. Hydrogen peroxide/formic
acid combination remained effective against four strains and was never
antagonistic against the other two fungi. Combinations with propionic acid
and acetic acid stayed synergistic against two strains. Those with oxalic acid
and lactic acid kept their synergism only against Candida albicans. No
synergism was detected against Penicillium cyclopium.
Conclusions: Synergistic combinations of disinfectants were revealed, among
them the promising hydrogen peroxide/formic acid combination.
Significance and Impact of the Study: A rapid screening method developed in
our laboratory for bacteria was adapted to fungi and used to reveal the
synergistic potential of disinfectants and/or sanitizers combinations.

synergistic interaction of hydrogen peroxide with acids

Introduction
Moulds and yeasts are ubiquitous in water, soils, plants
and animals and consequently in a range of fresh and
processed food, in raw materials and in numerous other
products (Wirtanen and Juvonen 2002). Spoilage fungi
also proliferate on process surfaces as complexes of yeast
cells and excretory substances that protect them from
sanitizing agents. Food industries present all the condi-
tions necessary (temperature, hygrometry and nutritive
conditions) to favour development of moulds and yeasts
brought mostly by air, water or contact. At least 200 spe-
cies of fungi exist in food (Deak 1991; Kurtzman and Fell
1998; Barnett et al. 2000). We previously showed the
on bacterial contaminants (Martin and Maris 2012). Few
reports have investigated the susceptibility of food-borne
fungi to chemicals as disinfectants used in the food
industry, and they focused on only a few spoilage species,
food processes and sanitizing agents. Two papers tried to
complete our knowledge in this field. The first one using
a suspension method studied the resistance to 15 com-
mercial disinfectants in 19 moulds and six yeast species
found in bread and cheese factories (Bundgaard-Nielsen
and Nielsen 1996). In that work, a 3
0% hydrogen perox-
ide solution with peracetic acid showed low fungicidal
effects (0 to 1-log10 reduction) against 18 strains after
10-min contact time at 20°C. However, hydrogen

Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology 1451
Synergism between hydrogen peroxide and seventeen acids
peroxide damaged the conidia in seven of the moulds.
According to the authors, the poor killing effects proba-
bly resulted from the low solution concentration and the
short contact time used. The second work evaluated the
efficacy of 17 disinfectants and foam cleaners on 25 yeast
strains isolated from dairy, fermented functional food,
bakery, praline, jam and sugar processes; all strains were
tested in suspension tests, and 16 strains were tested by
disinfection tests based on yeasts biofilms (Salo and
Wirtanen 2005). A disinfectant with peracetic acid and
hydrogen peroxide was effective at 1% on vegetative cells,
except for some Candida spp. In fact, the efficacy of
hydrogen peroxide against yeasts, including Candida albi-
cans and some specific fungi, is already established in
ophthalmology and dentistry (Rosenthal et al. 1999;
Szymanska 2006). These results and the synergistic com-
binations between hydrogen peroxide and some acids that
we reported against bacteria in a previous work (Martin
and Maris 2012) prompted us to test the disinfectant effi-
cacy of hydrogen peroxide with different acids (four min-
eral and 13 organic acids) towards a panel of fungal
strains including reference strains selected in the French
standards as model for yeasts and fungi representative of
contaminants found in the food industry (Splittstoesser
et al. 1980; Anon 1987a,b; Moreau 1988; Hamdy et al.
1990).
Materials and methods
Chemicals
Hydrogen peroxide and the 17 mineral and organic acids
were purchased from three suppliers. Hydrogen peroxide
(50% m/v), phosphoric acid (purity 84%), nitric acid
(purity 65–69%), formic acid (purity 100%), acetic acid
(purity 100%) and oxalic acid (purity 75%) were pro-
vided by Prolabo (VWR, Fontenay-sous-Bois, France).
Boric acid (purity 99
8%), sulfuric acid (purity 95–97%),
citric acid (purity 91
4%), benzoic acid (purity 91
4%),
adipic acid (purity 100%), glutaric acid (purity >99%),
propionic acid (purity 99%), succinic acid (purity
99
5%), tartaric acid (purity 99
5%) and mandelic acid
(purity 99%) were provided by Merck (VWR). Sulfamic
acid (purity 100%) and lactic acid (purity 85%) were
provided by Sigma (Saint-Quentin-Fallavier, France).
Stock solutions were prepared in sterile distilled water at
1% (m/v) and 2% (m/v) for adipic and boric acids, at
2% (m/v) for benzoic acid, at 5% (m/v) for oxalic and
succinic acids, at 10% (m/v) for mandelic and sulfamic
acids and at 20% (m/v) for citric, glutaric, tartaric acids
or sulfuric and nitric acids. Stock solutions of the other
acids (formic, acetic, phosphoric, propionic and lactic
acids) were prepared at 20, 40 or 80% (v/v).
1452 Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
H. Martin et al.
Yeast and fungal strains
We selected six fungal strains (one yeast and five fungi)
including two reference strains and four strains represen-
tative of different contaminant species found in the agri-
food industry. Candida albicans ATCC 2091 and Absidia
corymbifera CIP 1129-75 were reference strains in the
French A.F.NOR standards recommended by French reg-
ulation, before the edition of the European standards, to
determine the fungicidal efficacy of commercial disinfec-
tants. These fungi are cosmopolitan and ubiquitous fungi.
Candida albicans is a yeast often found in fruit and vege-
table products, and A. corymbifera is well-known for its
resistance to products and grows easily in humid and
warm environments. The four other fungi selected, Geo-
trichum candidum CIP 285-54, Scopulariopsis brevicaulis
CIP 210-53, Aspergillus versicolor CIP 1187-79 and Peni-
cillium cyclopium CIP 1231-80, were strains representative
of meat, milk and cheese industries contaminants and/or
strains found in fruit, vegetable and cereal transformation
or storage processes.
Assay conditions
Combinations between hydrogen peroxide and acids were
tested first using sterile distilled water. Then, synergistic
combinations were tested using interfering substances.
Four interfering substances were chosen among those rec-
ommended in the French NF T 72-170 standard (Anon
1988). Two hard water levels (300 and 600 mg l 1 CaCO3)
were tested to evaluate the impact of water hardness on
fungicidal activity of combinations. Two organic sub-
stances were chosen: one to simulate clean conditions
(0
3% bovine albumin with hard water at 300 mg l 1
CaCO3), the other to simulate dirty conditions (1% bovine
albumin + 1% yeast extract) according to the conditions
of use of products in food industry area. The concentra-
tions indicated were the final concentrations in the assays.
Test procedure
The ND/1500 checkerboard titration method was used
also to study the efficacy of each component of the com-
bination against each strain. The minimal fungicidal con-
centrations (MFCs) were thus obtained under basic
conditions (with distilled water) or conditions simulating
the practical use (with interfering substances). The ND/
1500 method was based on the checkerboard titration
method that has long been used for antibiotics (Krogstad
and Moellering 1980). The ND/1500 method was used to
prevent insufficient neutralization of the products. This
method combined the use of a 1 : 1500 dilution with the
use of a chemical neutralizer to ensure sufficient
H. Martin et al.
neutralization of the products. This method was devel-
oped in our laboratory to test efficacy of disinfectant
combinations against bacteria (Martin and Maris 1993)
and was already applied for bacteria in a similar study
(see descriptions of the ND/1500 test procedure, the
neutralization control and the results validation methods
in Martin and Maris 2012). In this work, ND/1500
method was adapted for yeast and fungi following the
recommendations of the French A.F.NOR NF T 72-200
(Anon 1987b) and NF T 72-170 (Anon 1988). These
modifications concerned the level of inocula in contact
with products (1 or 39107 CFU ml 1 instead of 1 or
39108 CFU ml 1 for bacteria), the contact time between
combinations and yeast or fungus (15 min at 20°C
instead of 5 min at 20°C), the composition of the liquid
for preparing spore suspensions of fungi (0
05% (v/v)
aqueous solution of polysorbate 80) and the culture med-
ium used to measure the survival of yeast and fungi
(yeast extract 5 g, glucose 20 g, Bacto agar 15 g and dis-
tilled water 1 l). The following temperatures and incuba-
tion times for agar plates were selected: 30°C during 72 h
for C. albicans, 30°C during 40–70 h for A. corymbifera
and 25°C during 4 days for the other strains. As for bac-
teria, MFC value of a product was the lowest product
concentration for which no vegetative fungal cell was
detected on agar plates. Synergistic combinations were
detected and their strength calculated (see below) when
no fungal growth was detected on agar plates, corre-
sponding to the combinations of products at the lowest
concentrations. Neutralization of products was considered
sufficient if results of numerations collected on agar
plates for neutralized product concentrations were higher
or equal to the half of the control numeration. To defi-
nitely validate our results, the consistency of MFC values
was checked for each product as described in the study
by Martin and Maris (2012) for bacteria using MFC val-
ues previously collected with a home-made test (Maris
et al. 1982) adapted and routinely used in our laboratory
to control the efficacy of disinfectants against fungi. A ±1
dilution difference was accepted between results of ND/
1500 method and the home-made test, when a MFC
value was reached with the two methods (for hydrogen
peroxide and some acids). For acids that were not effec-
tive on a fungal strain (MFC not reached, even with the
highest product concentration), ND/1500 assay was
performed using serial dilutions made from the highest
concentration of acid that it is possible to prepare
(depending on the acid dissolution capacity in distilled
water). Acid serial dilutions were then mixed in the
microplate with hydrogen peroxide serial dilutions as
described in the study by Martin and Maris (2012). For
each MFC and each synergistic combination, pH value in
assay conditions was measured.
Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
Synergism between hydrogen peroxide and seventeen acids
Exploitation of results
Minimal Fungicidal Concentration (MFC): for the micro-
plate row or column corresponding to each product
alone, fungal growth or total microbial destruction was
noted. First dilutions without fungal growth were consid-
ered to be the minimal fungicidal concentrations
(MFCs). At these concentrations, a 3-log10 reduction in
viable count was theoretically reached. Results were
expressed in acid or hydrogen peroxide percentages (v/v
or m/v).
Fractional Fungicidal Concentration index (FFC): each
result was expressed as a fractional fungicidal concentra-
tion index (FFC), representing the degree of interaction
of products when used in combination. FFC values were
expressed as a fraction of the MFC (FFC = MFC product
in combination/MFC product alone). We then calculated
the sum of the FFC values (Σ FFC = MFC product A in
association/MFC product A + MFC product B in associa-
tion/MFC product B) for interpretation of the results
based on the following criteria:
1 Σ FFC  0
50: synergism
2 0
50 < Σ FFC < 2: addition
3 Σ FFC  2: antagonism
Interference index: to facilitate interpretation of the
interference data on the effect of hard water and organic
substances on the efficacy of the combinations, MFC val-
ues representing optimal synergy were converted into
interference indices. These interference indices corre-
sponded to the ratio of the MFC value of each product
when tested in combination in the presence of an inter-
fering substance (hard water or organic substance) to the
MFC value of the same product tested in combination
in the absence of interfering substance. We used the
interference classification system previously described
(Guiraud-Dauriac and Crémieux 1984; Martin and Maris
2012). The intervals, chosen arbitrarily, were as
follows:
1 Class 1: no effect, interference index  1.
2 Class 2: weak effect, interference index between 1
1
and 5.
3 Class 3: intermediate effect, interference index between
5
1 and 10.
4 Class 4: strong effect, interference index between 10
1
and 25.
5 Class 5: very strong effect, interference index >25.
Results
Fungicidal efficacy of hydrogen peroxide in distilled
water
1453
Synergism between hydrogen peroxide and seventeen acids
Median MFC values (± 1 dilution) obtained in basic
assay conditions using the ND/1500 micromethod
showed that hydrogen peroxide was fungicidal at concen-
trations varying from 0
39% (m/v) for the more sensitive
fungus (G. candidum) to 12
5% for the less sensitive fun-
gus (A. corymbifera). The means and the standard errors
calculated from the 16 or 18 results of MFC collected for
each strain in distilled water gave a more precise result
for these MFC values and confirmed the results furnished
by median values (Table 1). The hydrogen peroxide MFC
values collected for each synergistic combination detected
in basic assay conditions during each ND/1500 checker-
board titration assay are indicated in Table 2.
Fungicidal efficacy of the 17 mineral and organic acids
MFC values obtained for the 17 acids were already pub-
lished (Martin and Maris 2005). To facilitate comparisons
between acids, MFC values were expressed in acid per-
centages (v/v or m/v) and in molar concentrations
(mol l 1). For each MFC, the pH was measured and the
ratio between undissociated and dissociated acid was cal-
culated. A MFC value was reached for nine of the 17
acids in water. The number of effective acids varied from
5 (for A. versicolor) to 8 (for C. albicans and G. candi-
dum). Four acids (nitric, formic, propionic and acetic
acids) were fungicidal on all six strains tested, two acids
(mandelic and lactic acids) on five strains, one acid (sul-
furic acid) on four strains and two acids (oxalic and
phosphoric acids) on only one strain (either C. albicans
or S. brevicaulis). As seen for hydrogen peroxide, A. cor-
ymbifera was the most resistant strain and G. candidum
was the most sensitive. The MFC values of acids collected
for each synergistic combination detected in basic assay
Table 1 MFC values of hydrogen peroxide in distilled water with ND/
1500 method (median and mean values)
MFC*% median MFC*% mean
value (m/v or v/v) value ± standard
Strains (± 1 dilution) error (m/v or v/v)
Absidia corymbifera 12
5 12
50 ± 0
CIP 1129-75
Candida albicans 3
12 3
90 ± 1
54
ATCC 2091
Scopulariopsis brevicaulis 3
12 5
51 ± 3
03
CIP 210-53
Aspergillus versicolor 1
56 1
71 ± 0
59
CIP 1187-79
Penicillium cyclopium 1
56 2
34 ± 1
28
CIP 1231-80
Geotrichum candidum 0
39 0
50 ± 0
34
CIP 285-54
*Minimum number of assays per result: 16–18.
1454 Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
H. Martin et al.
conditions during each ND/1500 checkerboard titration
assay are indicated in Table 2.
Characteristics of the products combinations: detection
of synergies
Among the 102 assays realized in distilled water, 12 pairs
of product combination/fungal strain had a synergistic
effect with Σ FFC between 0
09 and 0
50 (Table 3). In
most cases (92%), Σ FFC was  0
37, a sign of strong
synergy, indicating that concentrations <25% of com-
pound MFCs alone were fungicidal. Six synergistic com-
binations were detected for C. albicans, three for
A. corymbifera, one for S. brevicaulis, A. versicolor and
G. candidum and none for P. cyclopium. A synergy
between hydrogen peroxide and formic acid was noted
for four of the six strains, and combinations with propi-
onic acid, acetic acid, or oxalic acid were synergistic for
three or two strains, including the resistant strain, A. cor-
ymbifera. All the four acids cited plus lactic and sulfuric
acids were synergistic in combination against C. albicans
in distilled water conditions. Oxalic, lactic and sulfuric
acids were effective in combination only against
C. albicans. Concerning P. cyclopium and G. candidum,
no synergistic combination was detected in distilled water
conditions.
Maintenance of synergism between hydrogen peroxide
and acids in the presence of interfering substances
The 12 synergistic combinations (Σ FBC  0
50) were
then tested in the presence of four interfering substances.
Synergism was maintained for 11 combinations (Table 2).
The respective concentration of each component of the
hydrogen peroxide/acid combination and pH value at the
optimal point of synergy (OPS) are given in Table 2. Of
these 11 pairs (product combination/fungal strain), only
seven stayed synergistic with the four interfering sub-
stances: three couples with formic acid, two with acetic
acid, one with propionic acid and one with oxalic acid.
One couple with formic acid and one with lactic acid
maintained synergy with three interfering substances. In
Table 4, pH values reached at OPS measured for all the
synergistic combinations found for C. albicans were gath-
ered as example of pH values evolution in the presence
of various assay conditions simulating use conditions.
Interference indices: the effect of mineral or organic
substances on MFC values of acid and hydrogen
peroxide in a synergistic combination
Interference indices calculated for hydrogen peroxide (see
Table 5) in the presence of hard water and organic
 H. Martin et al.

Table 2 Values of acid and hydrogen peroxide concentrations (%/%) and pH measured in combinations staying synergistic with interfering substances

MFC of
acid* alone MFC of H2O2†
Acid Combination Acid/H2O2 Acid/H2O2 Acid/H2O2 Acid/H2O2 Acid/H2O2 (%) alone (%)

Bovine albumin Bovine albumin Distilled

1 1
Assay conditions Distilled water 300 mg l CaCO3 600 mg l CaCO3 (0
3%) yeast extract (1%) water Distilled water
In combination
with H2O2 Strain %/% PH %/% PH %/% PH %/% PH %/% PH % PH % PH

Formic Absidia sp. 0
31/1
56 2
80 0
31/0
78 2
75 0
31/0
78 2
65 0
31/0
78 2
95 0
31/1
56 3
36 2
5 2
05 12
5 3
35§
Candida sp. 0
039/0
195 3
22 0
15/0
19 2
76 0
078/0
39 2
88 0
15/0
39 3
11 0
078/0
78 4
19 1
25 2
19 3
12 6
14¶
0
078/0
39 2
91 0
078/0
78 3
37
Scopulariopsis sp. 0
078/0
39 3
02 0
15/0
78 2
82 0
15/0
195 2
81 0
156/0
19 2
95 ‡ – 0
31 2
50 3
12 4
29
0
078/1
56 2
91 0
078/1
56 3
42
Geotrichum sp. 0
019/0
095 3
35 0
039/0
19 3
13 0
039/0
095 3
10 0
078/0
19 3
43 0
078/0
78 4
08 0
15 >2
50 0
39 4
91
Propionic Absidia sp. 2
5/3
12 2
87 2
5/3
12 2
84 2
5/1
56 2
87 2
5/3
12 3
23 2
5/3
12 3
64 20 2
54 12
5 3
35
Aspergillus sp. 0
62/0
39 3
22 ‡ – ‡ – ‡ – 1
25/0
195 3
89 5 2
79 1
56 4
69
Acetic Absidia sp. 2
5/0
78 2
90 1
25/0
78 2
95 2
5/0
78 2
76 2
5/1
56 3
09 5/0
78 3
25 20 2
30 12
5 3
35

Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
Candida sp. 1
25/0
195 3
00 2
5/0
78 2
74 1
25/0
39 2
85 1
25/0
39 3
25 1
25/0
39 3
69 10 2
36 3
12 6
14
0
31/0
78 3
19 1
25/1
56 2
84
Oxalic Candida sp. 0
62/0
39 1
75 0
62/1
56 1
64 1
25/1
56 1
40 1
25/1
56 1
42 0
62/1
56 2
15 2
5 1
17 3
12 6
14
Lactic Candida sp. 2
5/0
39 2
26 5/1
56 2
08 ‡ – 5/0
78 2
22 5/3
12 2
52 10 2
03 3
12 6
14
2
5/1
56 2
40
Sulfuric Candida sp. 0
78/0
78 0
99 ‡ – 2
5/3
12 ND ‡ – ‡ – 5 0
60 3
12 6
14

ND, not determined.

*Value of MFC (%) measured for acid alone during ND/1500 assay realized in distilled water conditions.
†Value of MFC (%) measured for H2O2 alone during ND/1500 assay realized in distilled water conditions.
‡Combination not found synergistic in the assay(s) condition(s) (FFC >0
50).
§MFC value was determined in assay conditions using liquid for preparing spore suspensions.
¶MFC value determined in assay conditions using liquid for preparing yeast suspensions.
Synergism between hydrogen peroxide and seventeen acids

1455
Synergism between hydrogen peroxide and seventeen acids H. Martin et al.

Table 3 Recapitulative table of the synergistic combinations detected in distilled water. The synergy is expressed as the sum of the fractional
fungicidal concentrations (Σ FFC)

Nature and characteristics of the acid in combination

with hydrogen peroxide Σ FFC per fungus

Number of synergistic
1
Acid MWg mol pK1;2;3 1 2 3 4 5 6 combinations per acid

Formic 46
02 3
74 0
25 0
09 0
37 * * 0
37 4

Propionic 74
08 4
87 0
37 0
50 * 0
37 * * 3
Acetic 60
05 4
74 0
18 0
19 * * * * 2
Oxalic 90
04 1
27; 4
27 * 0
37 * * * * 1
Lactic 90
08 3
83 * 0
37 * * * * 1
Sulfuric 98
08 –; 1
92 * 0
37 * * * * 1
Nitric 63
02 – * * * * * * 0
Succinic 118
09 4
16; 5
61 * * * * * * 0
Benzoic 122
1 4
2 * * * * * * 0
Tartaric 150
09 2
95; 4
25 * * * * * * 0
Citric 192
12 3
14; 4
77; 6
39 * * * * * * 0
Sulfamic 97 1 * * * * * * 0
Phosphoric 98 2
15; 7
21; 12
67 * * * * * * 0
Mandelic 152
14 3
40 * * * * * * 0
Adipic 146
14 4
42; 5
42 * * * * * * 0
Glutaric 132
11 4
34 * * * * * * 0
Boric 61
83 9
24 * * * * * * 0
Total 12

1: Absidia corymbifera; 2: Candida albicans; 3: Scopulariopsis brevicaulis; 4: Aspergillus versicolor; 5: Penicillium cyclopium; 6: Geotrichum
candidum.
MW: molecular weight.
pK1;2;3: pK values found coming from http://sakura.cpe.fr/pka.html.
–Strong acid.
*Combination not synergistic (ΣFBC > 0
50).

Table 4 PH values measured in fungicidal mixtures (acid or combination solutions + yeast suspension + distilled water or interfering substances)
at CMF of acid or at the optimal point of synergy (OPS) found in the different assay conditions tested for Candida albicans ATCC 2091

pH
Acid in association
with hydrogen MFC of OPS of combination OPS of combination OPS of combination OPS of OPS of
peroxide acid alone distilled water 300 mg l 1 CaCO3 600 mg l 1 CaCO3 combination OM1‡ combination OM2§

Formic 2
19 3
22 2
75; 2
91 2
88 3
11; 3
37 4
19

Acetic 2
36 3
00; 3
19 2
74; 2
84 2
85 3
25 3
69
Oxalic 1
17 1
75 1
64 1
40 1
42 2
15
Lactic 2
03 2
26 2
08 * 2
22; 2
40 2
52
Sulfuric 0
60 0
99 * ND * *

*Combination not found synergistic.

1
‡0
3% bovine albumin (in hard water at 300 mg l CaCO3).
§1% bovine albumin + 1% yeast extract.
ND, Not determined.

interfering substances were class 1 interference indices
(interference index values equal to 0
25, 0
50 and 1). One
exception was for G. candidum and hydrogen peroxide/
formic acid combination. In this case, we found interfer-
ence indices of class 2 (interference index equal to 2) in
hard water conditions and class 3 with a high organic
load (interference index equal to 8). Candida albicans
showed class 2 (with interference indices between 2 and
4) or class 3 interference indices (interference index equal
to 8) with lactic acid and bovine albumin plus yeast
extract. Acids generally showed similar interference indi-
ces (see Table 5) to hydrogen peroxide (class 1 and 2,
with interference indices usually between 1 and 2, and
not more than 4). No class 3 interference was detected.

1456 Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
H. Martin et al. Synergism between hydrogen peroxide and seventeen acids

Table 5 Interference indices (FFC) calculated for acid and hydrogen peroxide for each combination synergistic in the presence of interfering
substances

Acid Hydrogen peroxide

Nature of acid
1 1 1 1
in combination 300 mg l 600 mg l 300 mg l 600 mg l
Strain with H2O2 CaCO3* CaCO3† OM1 ‡
OM2 §
CaCO3 CaCO3 OM1 OM2

Absidia corymbifera Formic 1 1 1 1 0
50 0
50 0
50 1

CIP 1129-75 Acetic 0
50 1 1 2 1 1 2 1
Propionic 1 1 1 1 1 0
50 1 1
Candida albicans Formic 4-2 2 4-2 2 1-2 2 2-4 4
ATCC 2091 Acetic 2-1; 8-4 1-4 1-4 1-4 4-8; 1-2 2-0
50 2-0
50 2-0
50
Oxalic 1 1 2 1 4 4 4 4
Lactic 2 2 2-1 2 4 ¶ 2-4 8
Sulfuric ¶ 3 ¶ ¶ ¶ 4 ¶ ¶
Scopulariopsis Formic 2 2 2 * 2 0
50 0
50 ¶
brevicaulis
CIP 210-53
Aspergillus versicolor Propionic ¶ ¶ ¶ 1 ¶ ¶ ¶ 0
50
CIP 1187-789
Geotrichum Formic 2 2 4 4 2 1 2 8
candidum
CIP 285-54

*hard water at 300 mg l 1 CaCO3.

†hard water at 600 mg l 1 CaCO3.
1
‡0
3% bovine albumin (in hard water at 300 mg l CaCO3).
§1% bovine albumin + 1% yeast extract.
¶combination not synergistic (ΣFBC > 0
50).

These interference indices were stable under all assay con-
ditions. Absidia corymbifera showed interference values of
1 or less.
Discussion
The sensitivity of A. corymbifera, C. albicans and S. brevi-
caulis to hydrogen peroxide was similar to Gram-positive
bacteria (Martin and Maris 2012), with MFCs between
3
12 and 12
5%. Hydrogen peroxide MFCs for A. versicol-
or and P. cyclopium were identical to Gram-negative bac-
teria and equal to 1
56%. Geotrichum candidum was the
most sensitive fungus to hydrogen peroxide with a MFC
of 0
39%, 32-fold lower than the MFC for A. corymbifera.
Bundgaard-Nielsen and Nielsen (1996) found that
10-min contact with a 3% hydrogen peroxide solution at
20°C had lower fungicidal effects (0 to 1-log10 reduction)
on Penicillium, Cladosporium, Scopulariopsis, Aspergillus
and Eurotium, but conidia damage occurred in seven
moulds. We found a 3-log10 fungicidal reduction after
15 min at 20°C with 1
56 or 3
12% hydrogen peroxide
solutions against S. brevicaulis, A. versicolor and P. cyclo-
pium (Table 1). Similarly, Buchen and Marth (1977)
noted times between 18
3 min and >120 min were neces-
sary to destroy Aspergillus parasiticus and Aspergillus
flavus conidia in 4% hydrogen peroxide. A 3% hydrogen
peroxide system showed an average 0
4 to 4-log10 reduc-
tion for C. albicans, Fusarium solari and Aspergillus
fumigatus after 2–6 h of contact lens disinfecting time
(Rosenthal et al. 1999). In a study on dental unit water-
line disinfection (DUWL), Szymanska (2006) confirmed
the antimycotic effectiveness of a disinfecting procedure
constituted of the use of a 0
25% hydrogen peroxide
solution enhanced with silver ions for 30 min followed
by the constant presence of 0
02% hydrogen peroxide in
DUWL. This disinfecting method used on yeast-like and
mould fungi caused a significant decrease both in the
number of total fungi and in individual species as
C. albicans (constituting from 31
2 to 85
7% of the total
fungi). Application of hydrogen peroxide caused a reduc-
tion of total flora from 410 to 5
6 CFU ml 1 in reservoir,
578
5–6
4 CFU ml 1 in hand piece and 43
43 CFU
mm 2–0
22 CFU mm 2 in biofilm. However, different
isolates of one species may show different responses to
the same disinfectant, resulting in an effective killing one
case and almost no effect in others (Bundgaard-Nielsen
and Nielsen 1996).
Generally, yeast and fungi grow better in acidic media,
whereas bacteria grow better in neutral or slightly alkaline
pH (Genigeorgis 1981). The MFCs for the 17 acids
against the six fungi were previously presented (Martin
and Maris 2005). In this last paper, a relatively low

Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology 1457
Synergism between hydrogen peroxide and seventeen acids
variability was shown between pH values reached at
MFCs of acids determined for the six fungal strains
tested. An example was the case of formic acid: a pH
level of 2
86 was necessary to kill G. candidum when pH
values between 2
05 and 2
50 were necessary against the
five other fungal strains. The mineral acids were fungi-
cidal at low pH (between 0
77 and 1
02 for nitric acid,
0
40 and 0
85 for sulfuric acid and phosphoric acids),
and the organic acids were fungicidal at higher pH
(between 1
75 and 2
92). Sensitivity gradients for formic
acid, acetic acid, or propionic acid showed A. corymbifera
as the most resistant fungus and G. candidum as the least
resistant fungus. Strong acidity was always necessary to
destroy fungal contamination. Indeed, Salo and Wirtanen
(2005) confirmed that sulfamic acid was ineffective
against strains growing in biofilm, even at a concentra-
tion fourfold higher than recommended by the manufac-
turer (one tablet of 15–30% maleic acid + 5–15%
sulfamic acid in 2
5 and 1
25 l). Propionic, benzoic and
sorbic acids require long treatment periods to be effective
against acidophilic fungi, but can inhibit the growth of
moulds and bacteria (Collins 1971; Thabib et al. 1982).
Only six acids were found synergistic with hydrogen
peroxide against fungi, but these synergies were mostly
stable regardless of the assay conditions (Table 2). Formic
acid was the most effective pairing against A. corymbifera,
C. albicans, S. brevicaulis and G. candidum, as well as for
bacteria (Martin and Maris 2012). Fungicidal activity of
these combinations is rarer than bactericidal activity, but
the combination of hydrogen peroxide with formic acid
keeps all its interest against fungi. Propionic acid also
showed interesting efficacy in combination against A. cor-
ymbifera, but not against C. albicans and A. versicolor
when interfering substances were added. Acetic acid
showed stable synergy on A. corymbifera and C. albicans.
Candida albicans was the most sensitive strain to syner-
gistic interactions (six synergistic acids in water and five
with interfering substances). In the work presented here,
the six combinations of hydrogen peroxide with an acid
were synergistic at pH conditions less acid than when the
molecule of acid was used alone. So, in distilled water
conditions, variations of pH values between +0
23 and
+1
03 were measured at the optimal point of synergy of
the combination comparing to pH value reached at MFC
of acid (see Table 2). As for bacteria (Martin and Maris
2012), when tested in hard water conditions, the remain-
ing synergistic combinations were effective at similar or
slightly lower pH values than in distilled water condi-
tions, related to protective effect on fungicidal cells of
calcium ions. Organic interfering substances interfered
with products, and fungicidal efficacy was maintained
with higher concentrations of each pH and less acidic pH
conditions (Tables 2 and 4). Assay conditions tested in
1458 Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
H. Martin et al.
our work were hard water and/or organic conditions
imposed by the French standard institution (A.F.NOR) to
simulate practical utilization planned for disinfectants.
These interfering reference substances were used in
France until European regulation came into force to
determine the fungicidal efficacy of commercial disinfec-
tants used in agriculture and food industry. However, the
conditions of use of these combinations may be different,
mainly if these combinations are used as fresh vegetables,
meat or cereals sanitizers and they do not always reflect
all the possibilities of application of the products. The
impact of food pH, composition and texture and the
impact of the temperature of storage are parameters that
could influence favourably or not the performance of
acid and hydrogen peroxide combinations. Each active
substance constituting our synergistic combinations has
yet been investigated individually for its capacity of
decontamination, most often against bacteria, and its
impact on organoleptic quality of produce has been
sometimes investigated (Cai et al. 1995; Wagenaar and
Snijders 2004; Rico et al. 2007). As the organoleptic con-
sequences of the utilization of sanitizers are related to
their level of concentration and as lower concentrations
were necessary for each active substance in synergistic
combinations, the negative organoleptic effect of each
active substance on produce may be reduced. However,
complementary studies are necessary to confirm this
assumption.
As the objective of our work was only to screen and to
place in a prominent position synergistic combinations,
we were not interested in dealing about the additive or
antagonistic combinations. However, it is important to
notice that, contrarily to bacteria for which no antagonis-
tic combination was determined (Martin and Maris
2012), we noticed such combinations for two of the six
selected fungi, P. cyclopium and G. candidum. So, con-
cerning P. cyclopium, the Σ FFC values were >2 or
 2
25 (results not shown) for the combination of
hydrogen peroxide with each of the seven following acids
(acetic acid, nitric acid, propionic acid, sulfamic acid,
succinic acid, benzoic acid and mandelic acid). For
G. candidum, 11 antagonistic combinations were found,
with Σ FFC values between 2
03 and 2
50 (acetic acid,
boric acid, propionic acid, oxalic acid, sulfamic acid,
orthophosphoric acid, sulfamic acid, succinic acid, glu-
taric acid and citric acid) or  4
12 (nitric acid) (results
not shown). However, these antagonisms were weak, per-
haps related to the composition of the fungal membranes
that may contain degradation enzymes.
In conclusion, synergies between hydrogen peroxide
and 17 different acids were found against one yeast and
five fungi, and their stability in conditions similar to use
conditions was confirmed. Only five acids, formic acid,
H. Martin et al.
acetic acid, propionic acid, oxalic acid and lactic acid, had
a synergistic effect in the presence of mineral or organic
matter. The combination of hydrogen peroxide with for-
mic acid was synergistic against four strains (A. corymbif-
era, C. albicans, S. brevicaulis and G. candidum), those
with acetic acid and propionic acid against two strains
(A. corymbifera and C. albicans or A. corymbifera and
A. versicolor). For oxalic acid or lactic acid, synergies were
maintained with interfering substances for only one strain
(C. albicans). Candida albicans was sensitive to more
numerous combinations of hydrogen peroxide and acid
than fungi. Less synergistic combinations were found for
fungi than for bacteria. However, using the rapid screen-
ing micromethod described for bacteria and adapted in
this work for yeast and fungi, we confirmed the interest of
the hydrogen peroxide and formic acid combination. Its
synergy was demonstrated in distilled water against fungal
suspensions and was maintained with various interfering
substances. Such combination could be promising to
destroy acid-resistant food industry contaminants as disin-
fectant or sanitizer and may have interesting applications
in ophthalmology, dentistry, food industry, and hospital
or veterinary medicine. However, the maintenance of such
synergy using surface tests simulating practical conditions
of application of products would be demonstrated. The
efficacy of such combinations against other microbes as
viruses will be investigated in a next future.
Acknowledgements
I wish to thank the excellent technical assistance of
Mrs G. Jehannin, A. Rault and R. Fresnel and the aid of
Mrs MH Moreau for pH measures. We also thank Mrs
Garnier for her help with the English translation.
References
Anon. (1987a) L’aérocontamination d’origine fongique. LCB
INFO 3, 1–3.
Anon. (1987b) Antiseptiques et désinfectants utilisés à l’état
liquide miscibles à l’eau et neutralisables. Détermination de
l’activité fongicide. Méthode par dilution - neutralisation. N.
F.T. 72-200. Paris: Association Française de Normalisation,
A.F.NOR.
Anon. (1988) Antiseptiques et désinfectants utilisés à l’état liquide
miscibles à l’eau et neutralisables. Détermination de l’activité
bactéricide en présence de substances interférentes définies.
Méthode par dilution - neutralisation NF T 72-170, Paris:
Association Française de Normalisation (A.F.NOR).
Barnett, J.A., Payne, R.W. and Yarrow, D. (2000) Yeasts:
Characteristics and Identification, 3rd edn. Cambridge, UK:
Cambridge University Press.
Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
Synergism between hydrogen peroxide and seventeen acids
Buchen, S.Y. and Marth, E.H. (1977) Sporicidal action of
hydrogen peroxide on conidia from toxigenic strains of
Aspergillus flavus and Aspergillus parasiticus. J Food Prot
40, 617–621.
Bundgaard-Nielsen, K. and Nielsen, P.V. (1996) Fungicidal
effect of 15 disinfectants against 25 fungal contaminants
commonly found in bread and cheese manufacturing. J
Food Prot 59, 268–275.
Cai, T., Pancorbo, O.C., Merka, W.C., Sander, J.E. and
Barnhardt, H.M. (1995) Stabilization of poultry processing
by-products and poultry carcasses through direct chemical
acidification. Bioresour Technol 52, 69–77.
Collins, E.B. (1971) Preservatives in dairy foods. J Dairy Sci
54, 148–152.
Deak, T. (1991) Food borne yeasts. Adv Appl Microbiol 36,
179–278.
Genigeorgis, C.A. (1981) Factors affecting the probability of
growth of pathogenic microorganisms in foods. J Am Vet
Med Assoc 179, 1410–1417.
Guiraud-Dauriac, H. and Crémieux, A. (1984) Inactivation par
les protéines et les ions calcium des désinfectants en
fonction de leur nature chimique et des espèces
bactériennes. Pathol Biol 32, 611–614.
Hamdy, M., Yassien, N. and Mansour, N. (1990) Mycological
investigations of air in camel and cattle slaughter halls.
Fleischwirtsch. 70, 429–430.
Krogstad, D.J. and Moellering, R.C. (1980) Combinations of
antibiotics, mechanisms of interaction against bacteria. In
Antibiotics in Laboratory Medicine ed. Lorian, V. pp. 298–
341. Baltimore: Williams and Wilkins.
Kurtzman, C.P. and Fell, J.W. (1998) The yeasts, a Taxonomic
Study, 4th edn, Amsterdam, The Netherlands: Elsevier.
Maris, P., Ribouchon, J.L. and Perrin, G. (1982) Etude
comparative de l’activité bactéricide des antiseptiques et
désinfectants par microméthode et par méthode de
dilution - neutralization (NF T 72-150 et T 72-170). Rev
Inst Past (Lyon) 15, 253–261.
Martin, H. and Maris, P. (1993) Etude des associations
d’agents antimicrobiens. Mise au point de méthode. Ann
Pharm Fr, 51, 175–185.
Martin, H. and Maris, P. (2005) Bactericidal and fungicidal
efficacy of organic and mineral acids. Sci Aliments 25,
105–127.
Martin, H. and Maris, P. (2012) Synergism between
hydrogen peroxide and seventeen mineral and
organic acids on six bacterial strains. J Appl Microbiol
113, 578–590.
Moreau, C. (1988) Les mycotoxines: Chapitre 7. In
Microbiologie Alimentaire. 1 Aspect microbiologique de la
sécurité et de la qualité alimentaires. Collection Sciences
et techniques agro-alimentaires. Paris, France: Technique
et Documentation, Lavoisier.
Rico, D., Martin-Diana, A.B., Barat, J.M. and Barry-Ryan,
C. (2007) Extending and measuring the quality of fresh-
1459
Synergism between hydrogen peroxide and seventeen acids
cut fruit vegetables: a review. Trends Food Sci Technol 18,
373–386.
Rosenthal, R.A., Bell, W.M. and Abshire, R. (1999) Disinfecting
action of a new multi-purpose disinfection solution for
contact lenses. Cont Lens Anterior Eye, 22, 104–109.
Salo, S. and Wirtanen, G. (2005) Disinfectant efficacy on
foodborne spoilage yeast strains. Institution of chemical
Engineers. Trans IchemE, Part C, Food and Bioproducts
Proc 83, 288–296.
Splittstoesser, D.F., Bowers, J., Kerschner, L. and Wilkison, M.
(1980) Detection and incidence of Geotrichum candidum
in frozen blanched vegetables. J Food Sci 45, 511–513.
Szymanska, J. (2006) Antifungal efficacy of hydrogen peroxide
in dental unit waterline disinfection. Ann Agric Environ
Med 13, 313–317.
1460 Journal of Applied Microbiology 113, 1451--1460 © 2012 The Society for Applied Microbiology
H. Martin et al.
Thabib, Z., Hagler, W.M. and Hamilton, P.B. (1982)
Comparison of physiological parameters useful for
assessment of activity of antifungal agents in feed and
ingredients. J Food Prot 45, 1030–1037.
Wagenaar, C.L. and Snijders, J.M.A. (2004) Decontamination
of broilers with hydrogen peroxide stabilized with
glycerol during processing. Int J Food Microbiol 91,
205–208.
Wirtanen, G. and Juvonen, R. (2002) Disinfectant efficacy on
spoilage yeasts isolated from various food processes. In
Proceedings of a Conference: Fouling, Cleaning and
Disinfection in Food Processing 3–5 April 2002,
pp. 189–196. Cambridge, UK: Dept. Chemical
Engineering, Cambridge University.