1167

Journal o f Food Protection, Vol. 75, No. 6, 2012, Pages 1167-1171

doi: 10.4315/0362-028X.JFP-11-361
Copyright © , International Association for Food Protection

Research Note

Antimicrobial Activities of Polyhexamethylene Guanidine

Hydrochloride-Based Disinfectant against Fungi Isolated from
Cocoa Beans and Reference Strains of Bacteria
YAO K. MATHURIN, 12 ROSE KOFFI-NEVRY,1* SIMPEICE T. GUEHI, 1 KABFAN TANO,2 a n d MATHIAS K. OULE3

1Biotechnology and Food Microbiology Laboratory and 2Food Biochemistry and Tropical Products Technology Laboratory, Department o f Food Science
and Technology, University o f Abobo-Adjame, 02 BP 801 Abidjan 02, Cote d ’Ivoire; and 3Department o f Science, University o f St-Boniface, 200 avenue de
la Cathedrale, Winnipeg, Manitoba, Canada R2H 0H7

MS 11-361: Received 25 July 2011/Accepted 11 January 2012

ABSTRACT
This study was conducted to assess the antibacterial and the antifungal activity of a polyhexamethylene guanidine
hydrochloride (PHM GH)-based disinfectant and to determine if it could be used as a disinfectant for the treatment of cocoa
beans. The activity of PHMGH was tested in vitro for efficacy against five reference strains o f pathogenic bacteria and six strains
of fungi isolated from cocoa beans. All the strains tested were sensitive to the disinfectant. The MICs reported were between 0.01
and 1.9 mg/ml and equal to the MBC or minimum fungicidal concentration (MFC) regardless of the strains o f those
microorganisms. The bacteria were more sensitive to PHMGH than were the fungi. Enterobacter cloacae was the most sensitive
bacterium with a MIC and MBC of 0.01 mg/ml, whereas the genus Aspergillus was the least susceptible of the microorganisms
tested, with a MIC and MFC from 1.0 to 1.9 mg/ml. The time required for the activity o f PHMGH varies from 2 min for
Enterobacter cloacae to 12 min for Aspergillus tamarii and generally increases with the MBC or the MFC. Through this in vitro
study, the PHMGH has been proved to be bactericidal and fungicidal on the strains studied. Hence, it could probably serve as a
fungicidal disinfectant for the treatment of cocoa beans after harvesting.

Cocoa is a very important ingredient for several kinds
of foods (cakes, biscuits, ice cream, etc.) It is mainly
produced in Africa and both Central and South America and
is the main raw material for chocolate (7, 25). The export of
raw cocoa beans is an important economic activity for
several tropical countries with intermediate revenues. West
Africa produces two-thirds of the world production with
Cote d’Ivoire and Ghana as the major producers (1, 4).
Unfortunately, this crop is threatened by a multitude of
fungi (Aspergillus, Penicillium, Mucor, Rhizopus, and
Fusarium), which affects the sales price of cocoa beans
and may lead to a loss of revenue (3, 6, 17, 25). In previous
work (8, 9), the fungal profiles of the cocoa produced in
Cote d ’Ivoire were explored, and the presence of several
strains of Aspergillus and Penicillium was revealed. Fungal
activity can lead to a contamination with mycotoxins,
causing deterioration of the quality of the product, and can
pose a health risk to the consumers. Therefore, it is
important for Cote d ’Ivoire to reduce or eliminate the
fungal contamination of the Ivorian cocoa in order to keep
the leading edge of the production.
In Cote d ’Ivoire, apart from diseases such as the ones
caused by the bite or sucking of the pods by insects and the
swollen shoot disease, which is a viral disease, the main
* Author for correspondence. Tel: (225) 07 68 83 34; Fax: (225) 20 30 43
02; E-mail: rosenevry2002@yahoo.fr.
diseases (brown rot of cocoa pods) of cocoa beans are of
filamentous fungal origin (6, 9, 16). Reducing or eliminat­
ing mold contamination could improve the quality of the
cocoa, prevent mycotoxin production, and increase the sales
price of cocoa beans. Most of the methods used to fight
the fungi include chemicals. However, there are constant
increases in microbial infections and in losses of foods and
feeds over time, leading to economic losses as well. In
addition, the resistance of microorganisms to antimicrobial
chemicals (such as antibiotics and food preservatives) is
increasing (19). Thus, in order to reduce food poisoning,
the use of new bioactive molecules is essential (19).
In promoting new products that can help improve food
preservation, the chemical-based polyhexamethylene gua­
nidine hydrochloride (PHMGH) disinfectants, proven to be
powerful bactericides and fungicides (21), draw the
attention of agricultural producers.
Polyhexamethylene biguanide, a member of the
polymeric guanidine family, has a broad-spectrum activity
against microorganisms (12, 18). It is odorless, colorless,
noncorrosive, nonvolatile (13), harmless, and much less
toxic than currently used disinfectants (18) for humans and
animals at a concentration of <1% (10 mg/ml). The
antibacterial activity of the PHMGH disinfectant seems to
be well documented (14, 21, 23). However, its antifungal
activity needs to be studied. The aim of this study was to
verify the antimicrobial properties of the PHMGH disinfec-
1168 MATHURIN ET AL.
tant in order to improve its use in areas such as agriculture,
and also for food preservation, especially vegetables and
fruits such as cocoa beans. In order to reach this goal,
antimicrobial activity tests were performed on the in vitro
growth of five reference strains of pathogenic bacteria and
six strains of fungi isolated from the Ivorian cocoa beans
and that cause the production of mycotoxin. This work
will help compare the antimicrobial activity of this new
disinfectant on both bacteria and fungi in the same study.
MATERIALS AND METHODS
Disinfectant and strains. We used PHMGH, a chemical with
antimicrobial effect developed by the Department of Biological
Sciences, University of Manitoba (Canada). Five species of
bacteria identified by the National Committee for Clinical
Laboratory Standards in 1992 as the most frequent agents of
infection were used for testing: Bacillus subtilis ATCC 6633, a
gram-positive bacterium; and four gram-negative bacteria, Salmo­
nella enterica serovar Typhi 5534 from Pasteur Institute of Paris,
Klebsiella pneumoniae ATCC 13883, Enterobacter cloacae ATCC
13047, and Shigella sonnei ATCC 2571. Stock cultures of these
bacteria were maintained on nutrient agar slants for monthly
transfers. New stocks of tested organisms transferred were
incubated for 2 days at 37°C. The fungal strains Penicillium
chrysogenum, Aspergillus tamarii, Aspergillus tubengensis, As-
pergillus flavus, Absidia corymbifera, and Rhizopus oryzea were
isolated from cocoa beans from Cote d ’Ivoire (9).
Preparation of the different concentrations of PHMGH.
Different concentrations of PHMGH were tested with Mueller-
Hinton broth for bacteria and Sabouraud broth for fungi. A series of
solutions of different concentrations were made by the method
described by Oule et al. (21). Serial dilutions from 0.001 to 2.2 mg of
PHMGH per ml were made with the growth media inside test tubes
(15 by 150 mm). The following concentrations were prepared to
determine the MIC across 14 dilutions: 0.001, 0.004, 0.007, 0.01,
0.04, 0.07, 0.1, 0.4, 0.7, 1.0, 1.3, 1.6, 1.9, and 2.2 mg/ml.
Determination of the PHMGH’s MIC and MBC. The MIC
was assessed by the broth dilution technique reported by Soberon
et al. (24) and Moroh et al. (15). One milliliter of each inoculum of
bacterial or fungal culture 24 or 48 h old was added to each of the
tubes from Ti to T ,4 containing 10 ml of each of the PHMGH
concentrations previously prepared in order to achieve a final
inoculum of 106 CFU/ml (equivalent to a McFarland test tube
number 3 [0.5 McFarland standard]). Ten milliliters of Mueller-
Hinton broth (or Sabouraud broth) without PHMGH was used as
control for bacterial or fungal growth (Tc). The tubes were
subjected to agitation before being incubated. Before incubation, a
first reading of the optical density (initial optical density [dj]) was
measured. The tubes were incubated at 37°C for 24 h for the
bacteria and at 30°C for 72 h for the fungi. Then, a second reading
of the optical density induced by the growth of the fungi studied
was performed, giving a final optical density (df).
Bacterial growth was indicated by turbidity, and the absence
of bacterial growth meant an inhibitory activity. The MIC
was determined by measuring the optical density induced by the
growth of the microorganism studied. The MIC (in milligrams per
milliliter) was the lowest concentration of PHMGH in a broth tube
where there was no growth of the tested organism. That means the
lowest concentration of PHMGH at which the initial optical
density (dj) is equal to the final optical density (df) (15). The
microbial growth in each test tube was indicated by the difference
J. Food Prot., Vol. 75, No. 6
between those two values (df — d; = ADO). The optical density
was obtained by using a spectrophotometer (Milton Roy Spectro-
nic 601) at 600 nm for the bacteria and 550 nm for the fungi. Each
test was performed in duplicate and repeated three times.
In order to determine the MBC or MFC, a sample (0.1 ml)
taken from each test tube used to determine the MIC that did not
show any turbidity was streaked where there was no growth in the
MIC assay onto Mueller-Hinton agar without PHMGH for the
bacteria and onto Sabouraud agar without PHMGH for the fungi
and incubated at 37°C for 24 h for the bacteria and 30°C for 72 h
for the fungi. The controls included aliquots taken from the growth
control tubes. The MBC or MFC was determined as the lowest
concentration of PHMGH at which the tested organism was killed,
i.e., the concentration of PHMGH that did not show any growth on
a new set of agar plates. Each test was performed in duplicate and
repeated three times.
MBC or MFCs MIC. A ratio was established between the
MIC and the MBCs or MFCs in order to show the PHMGH
bactericidal or fungicidal activities for the microorganisms studied
(15, 22). When the ratio of the MFC to MIC of an antimicrobial
agent is below 4 (<4), that agent is considered to be microbicidal.
And when that ratio is above 4 (>4), the agent is microbistatic.
Each test was performed in duplicate and repeated three times.
Determination of the time required for PHMGH bacte­
ricidal or fungicidal activity at MBCs and MFCs. To determine
the time required for antimicrobial activity of PHMGH at MBCs
and MFCs (21), for each organism tested, 1 ml of a 24-h culture for
the bacteria and 72-h culture for the fungi was poured into a tube
containing 10 ml of a PHMGH solution at the corresponding MBC
or MFC (0.01, 0.04, 0.07, and so on up to 0.7 and 1.9 mg/ml, for£.
cloacae, the other bacteria, and the fungi). The tubes were agitated,
after which one loopful of culture and PHMGH mixture was plated
by streaking onto Mueller-Hinton or Sabouraud agar every minute
for a period of 15 min. The plates were incubated at 37°C for 24 h
for the bacteria and 30°C for 72 h for the fungi. The absence of
growth was interpreted as a bactericidal or fungicidal activity. Each
test was performed in duplicate and repeated three times.
RESULTS
The susceptibility test revealed the presence of an
antimicrobial activity of the disinfectant at various concen­
trations. The microbial growth recorded at a given
concentration of PHMGH is presented in Tables 1 and 2.
The turbidity (optical density) related to the growth of the
microorganisms in the test tubes was recorded before (d,)
and after (df) incubation (Table 1). The growth of the strains
tested at a given concentration of PHMGH is shown in
Table 2. Table 3 shows a summary of the MICs, MBCs,
and MFCs of PHMGH against the tested organisms. The
lowest concentration that inhibited Salmonella Typhi PPI
5534, K. pneumoniae ATCC 13883, and B. subtilis ATCC
6633 was 0.04 mg/ml; the one for S. sonnei ATCC 2571
was 0.07 mg/ml; 0.01 mg/ml for E. cloacae ATCC 13047;
O. 4 mg/ml for R. oryzea and A. corymbifera; 0.7 mg/ml for
P. chrysogenum; 1.0 mg/ml for A. tubingensis and A. flavus;
and 1.9 mg/ml for A. tamarii. These concentrations can be
defined as the MICs for PHMGH against the microorgan­
isms tested.
Concentrations lower than 0.01 mg/ml did not have
any effect on the growth of the tested organisms. However,
J. Food Prot., Vol. 75, No. 6 POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE AGAINST FUNGI AND BACTERIA 1169

TABLE 1. MICs obtained through the variation o f the optical density at given concentrations o f PHMGHa
Concn of PHMGH (mg/ml)

H ^
Ti t2 t3 t4 t5 t6 T7 T8 t9 T„ Ti2

oO
T ,3 T l4
Strain tested 0.000 0.001 0.004 0.007 0.01 0.04 0.07 0.1 0.4 0.7 1.3 1.6 1.9 2.2

St 0.66 0.10 0.08 0.07 0.04 0 0 0 0 0 0 0 0 0 0

Kp 0.41 0.04 0.03 0.03 0.01 0 0 0 0 0 0 0 0 0 0
Bs 0.32 0.05 0.04 0.02 0.01 0 0 0 0 0 0 0 0 0 0
Ss 0.46 0.05 0.03 0.03 0.02 0.02 0 0 0 0 0 0 0 0 0
Ec 0.30 0.05 0.04 0.03 0 0 0 0 0 0 0 0 0 0 0
Pc 0.71 0.38 0.31 0.27 0.21 0.13 0.09 0.05 0.02 0 0 0 0 0 0
Ata 0.67 0.51 0.49 0.44 0.40 0.34 0.31 0.23 0.18 0.11 0.09 0.06 0.02 0 0
Ro 0.57 0.31 0.29 0.20 0.12 0.10 0.02 0.01 0 0 0 0 0 0 0
Ac 0.61 0.32 0.27 0.19 0.10 0.08 0.02 0.01 0 0 0 0 0 0 0
Atu 0.58 0.50 0.40 0.33 0.25 0.17 0.11 0.09 0.03 0.01 0 0 0 0 0
Afl 0.65 0.52 0.42 0.32 0.24 0.17 0.11 0.08 0.03 0.01 0 0 0 0 0

a The standard deviation of the optical density varied between 0.001 and 0.004. Tn, test tube numbers. St, Salmonella Typhi; Kp, K.
pneumoniae; Bs, B. subtilis; Ss, S. sonnet, Ec, E. cloacae-, Pc, P. chysogenum; Ata, Aspergillus tamarii; Ro, R. oryzea; Ac, Absidia
corymbifera; Atu, Aspergillus tubingensis; Afl, Aspergillus flavus.

those above 0.04 mg/ml inhibited all the reference bacteria
tested except for S. sonnei. The lowest values were observed
with E. cloacae (MIC = MBC = 0.01 mg/ml), while the
highest values were recorded with A. tamarii (MFC = MIC
= 1.9 mg/ml). Therefore, E. cloacae was the bacterium
most sensitive to the effect of PHMGH, and A. tamarii was
the most resistant strain. The MBC of PHMGH was
therefore 0.04 mg/ml for Salmonella Typhi, K. pneumoniae,
and B. subtilis and 0.07 mg/ml for S. sonnei. The fungi were
the least sensitive to PHMGH with MFCs of 1.0 to 1.9 mg/ml
for Aspergillus spp. and 0.7 mg/ml for P. chrysogenum.
The ratio of MBC to MIC or of MFC to MIC gave the
value of 1 for all the microorganisms studied. This value is
less than 4; Table 4 shows the time required for PHMGH to
effect microbicidal activity according to the MBC or MFC.
As shown in this table, the bactericidal and fungicidal effect
of PHMGH at the MBC (or MFC) was achieved within 2 to
12 min and depended on the type of microorganisms used.
This demonstrates the quick action of PHMGH, even when
applied at low concentrations. The lowest time (2 min)
required for PHMGH bactericidal activity was recorded
with E. cloacae, and the highest (9 min) was with S. sonnei.
The time required for PHMGH fungicidal activity was
10 min for P. chrysogenum and 12 min for A. tamarii.
DISCUSSION
The results obtained in this work indicated that the
effect of PHMGH on the growth varies according to the type
of microorganisms. In fact, no matter what genus of fungi
was tested, the MFC and MIC had the same value. Thakkar
et al. (26) reported that polyhexamethylene biguanide or
other polybiguanide-based compounds might also have
antiviral or vimcidal activity against the human immuno­
deficiency virus type 1.
Because PHMGH is fast acting at low concentrations,
as indicated by the time required for PHMGH bactericidal or
fungicidal activity, it could be a very efficient disinfectant
for the control of fungi and pathogenic bacteria. The MBC
for E. cloacae was the lowest of all the MBCs. The MFC for
P. chrysogenum was about three times lower than the one

TABLE 2. Growth o f the strains tested at a given concentration o f PHMGHa

Concn of PHMGH (mg/ml)

tested 0.001 0.004 0.007 0.01 0.04 0.07 0.1 0.4 0.7 1.0 1.3 1.6 1.9 2.2

Ec + + + — - — — — — — — — — —
St + + + + - - - - - - - - - -
Kp + + + + - - - - - - - - - -
Bs + + + + - - - - - - - - - -
Ss + + + + + - - - - - - - - -
Ro + + + + + + + - - - - - - -
Ac + + + + + + + - - - - - - -
Pc + + + + + + + + - - - - - -
Atu + + + + + + + + + - - - - -
Afl + + + + + + + + + - - - - -
Ata + + + + + + + + + + + + - -

+ , microbial growth in broth after exposure to PHMGH; —, no growth; Ec, E. cloacae-, St, Salmonella Typhi; Kp, K. pneumoniae-, Bs, B.
subtilis; Ss, S. sonnei; Ro, R. oryzea; Ac, Absidia coiymbifera; Pc, P. chrysogenum; Atu, Aspergillus tubingensis; Afl, Aspergillus flavus;
Ata, Aspergillus tamarii.
1170 MATHURIN ET AL. J. Food Prot., Vol. 75, No. 6

TABLE 3. MICs, MBCs, and MFCs for PHMGH against the glucane, and mannose, and the weak resistance of the
tested microorganisms bacteria was due to the fact that theirs are composed of
Tested organism MIC (mg/ml) MBC and MFC (mg/ml) peptidoglycan. The fungal cell wall is a rigid structure that
does not allow any phagocytosis and protects the cell from
E. cloacae 0.01 0.01 mechanical, physical, and chemical aggressions. The chitin
Salmonella Typhi 0.04 0.04 and glucane that are present in the wall protect it and allow
K. pneumoniae 0.04 0.04 it to resist the antimicrobial effects (21).
B. subtilis 0.04 0.04 The highest concentration was recorded with A.
S. sonnei 0.07 0.07
tamarii, for which a concentration of 1.9 mg/ml was needed
R. oryzea 0.4 0.4
to inhibit its growth. This result is in accordance with that
A. corymbifera 0.4 0.4
P. chrysogenum 0.7 0.7 recorded by Koffi-Nevry et al. (10). The results obtained in
A. tubingensis 1.0 1.0 this study on the resistance of Aspergillus agreed with those
A. flavus 1.0 1.0 of Toumas and Katsoudas (27) and Ouattara et al. (20).
A. tamarii 1.9 1.9 These authors indicated that some species of Aspergillus
such as A. fumigatus were particularly resistant to
therapeutic agents. In addition, Aspergillus niger is one of
for A. tamarii, while those for R. oryzea and A. corymbifera
the most resistant microorganisms to antifungal agents (27).
were five times lower. The MBCs for the bacteria tested in Although PHMGH is bactericidal against S. sonnei with an
this study were lower than the MFCs for the fungi, especially MBC equal to 0.07 mg/ml (wt/vol), this bacterium had a
for the strains of Aspergillus. This means that the bacteria higher level of resistance than the other bacteria tested. It is
were more sensitive to PHMGF1 than the fungi, and generally admitted that gram-negative bacteria are more
Aspergillus was the most resistant strain tested. These results resistant to antimicrobial agents than gram-positive ones
go along with those reported by Koffi-Nevry et al. (10), who because of the morphological differences between these
noted the antifungal activity of PHMGH against Mucor spp., microorganisms (11).
Botrytis spp., Penicillium spp., Geotrichum spp., Aspergillus The time required for PHMGH bactericidal activity at
spp., and Colletotrichum spp., common postharvest fungi the MBC was relatively short for all the pathogens studied.
frequently responsible for papaya spoilage. Our work Thus, the time required for PHMGH to inhibit the fungal
confirms the one from Rosin et al. (23), who explained the strains was longer than the time required for the bacteria.
efficiency of PHMGH in medical and food industries. These times were 2 min for E. cloacae, 6 min for
The difference in sensitivity between the bacteria and Salmonella Typhi and K. pneumoniae, 8 min for B. subtilis,
the fungi could be explained by the different compositions 9 min for S. sonnei and R. oryzea, 10 min for A.
of cell wall and cytoplasmic membrane. According to corymbifera, P. chrysogenum, A. tubingensis, and A.flavus,
McDonnell and Russell (14) and also Oule et al. (21), the and 12 min for A. tamarii.
main target of the PHMGH is the cell envelope. The product The results show that the time required increases with
penetrates the cell envelope and attacks the membrane. the MBC or the MFC. Thus, Aspergillus spp. with MFC of
Indeed, the PHMGH provokes the lyses of the cell 1.9 mg/ml has the longest time for PHMGH bactericidal
envelope, the destruction of the cytoplasmic membrane, activity, 12 min. However, E. cloacae (MBC = 0.01
and the release of the cellular contents (2, 5), thereby mg/ml) has the shortest time (2 min). These results confirm
causing the death of the cell. The resistance of the fungal those obtained by Oule et al. (21), where the time required
cells was due to the fact that their walls are made of chitin, for the PHMGH bactericidal activity against Escherichia

TABLE 4. Time required fo r PHMGH bactericidal or fungicidal activity at the MBC or MFCa
Exposure time (min)

Tested organism 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Ec (MBC, 0.01 mg/ml) +

St (MBC, 0.04 mg/ml) + + + + + - - - - - - - - - -
Kp (MBC, 0.04 mg/ml) + + + + + - - - - - - - - - -
Bs (MBC, 0.04 mg/ml) + + + + + + + - - - - - - - -
Ss (MBC, 0.07 mg/ml) + + + + + + + + - - - - - - -
Ro (MFC, 0.4 mg/ml) + + + + + + + + - - - - - - -
Ac (MFC, 0.4 mg/ml) + + + + + + + + + , - - - - - -
Atu (MFC, 1.0 mg/ml) + + + + + + + + + - - - - - -
Afl (MFC, 1.0 mg/ml) + + + + + + + + + - - - - - -
Pc (MFC, 0.7 mg/ml) + + + + + + + + + - - - - - -
Ata (MFC, 1.9 mg/ml) + + + + + + + + + + + - - - -

+ , microbial growth; —, no growth; Ec, E. cloacae; St, Salmonella Typhi; Kp, K. pneumoniae; Bs, B. subtilis; Ss, S. sonnei; Ro, R.
oryzea; Ac, Absidia corymbifera; Atu, Aspergillus tubingensis; Afl, Aspergillus flavus; Pc, P. chrysogenum; Ata, Aspergillus tamarii.
J. Food Prot., Vol. 75, No, 6 POLYHEXAMETHYLENE GUANIDINE HYDROCHLORIDE AGAINST FUNGI AND BACTERIA
coli and methicillin-resistant Staphylococcus aureus was
1.5 min. Since for all the strains of bacteria and fungi
studied, the MBC or MFC is equal to the MIC, the ratio of
MBC to MIC or to MFC is equal to 1. According to Oussou
et al. (22) and also Moroh et al. (15), when the ratio of MBC
to MIC of an antibacterial agent is <4, this agent is
described as a bactericidal substance. On the contrary, when
this ratio is > 4, the agent is not bactericidal. Likewise, the
PHMGH can be characterized as fungicidal or not.
The PHMGH is therefore revealed as an upcoming
disinfectant against microorganisms accustomed to chemi­
cal products used for postharvesting treatment. It appears
from this study that the disinfectant PHMGH has a broad
spectrum of action. The fungi and bacteria tested were
all proven to be susceptible. With its many innovative
properties (odorless, noncorrosive, nontoxic to humans and
animals, etc.), in addition to its significant in vitro
antimicrobial activity, this disinfectant may be recom­
mended as a novel disinfectant for food preservation in
order to prevent or reduce postharvesting losses due to
fungal spoilage and contribute to improving the storage
quality of food, especially cocoa beans from Cote d'Ivoire.
ACKNOWLEDGMENTS
We are thankful to the Food Science Department of the University of
Abobo-Adjame, Abidjan, Cote d'Ivoire, for the laboratory facilities, and to
the Department of Science of the University of St-Boniface, Winnipeg,
Canada, for technical assistance.
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