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EFFECT OF SOME ORGANIC ACIDS ON SOME FUNGAL GROWTH AND THEIR TOXINS PRODUCTION

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The effect of eight organic acids (propionic, acetic, formic, lactic, tartaric, citric, oxalic and malic acids) as antifungal agents on the growth of four fungi (Aspergillus flavus, Penicillium purpurogenum, Rhizopus nigricans and Fusarium oxysporum) were studied. The high acidity appeared for oxalic acid being 0.14 at the high concentration (10%), while the lowest acidity recorded for propionic acid and acetic acid being 2.71 and 2.56 at the low concentration (5%). It was observed that, there was no relationship between the efficacy of organic acid and its final pH. Acetic acid (10%) has the highest inhibitory effect on A. flavus being 45.21%, but tartaric acid (5%) and citric acid (5%) gave the same lowest inhibition effect (0.42%). The lowest value of mycelium dry weight (MDW) of P. purpurogenum was 5.92 g/l when acetic acid was used (10%), but the highest value was 9.38 g/l when tartaric acid (5%) was used. Formic acid (10%) had a strong effect on the inhibition growth of R. nigricans being 28.65%, similar to propionic acid (10%), acetic acid (10%), lactic acid (10%), tartaric acid (10%) and citric acid (10%) being 26.57%, 26.38%, 26.19%, 23.53% and 24.48%, respectively. But malic acid (5%) and oxalic acid (5%) were having a week effect on R. nigricans being 5.31% and 6.45%, respectively. Lactic acid (10%) has the highest inhibitory effect on F. oxysporum being 34.45% and the lowest value was in the case of tartaric acid (5%) being 1.68%. Four treatments were used to determine aflatoxin B1 production. The highest inhibition (50%) was observed by R. nigricans in the presence of formic acid (10%). Acetic acid in 10% level inhibited the toxic secretion of A. flavus and P. purpurogenum to become 25% and 40%, respectively. Lactic acid (10%) gave 35% inhibition of toxin production in the presence of F. oxysporum.
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International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
1
E
FFECT OF SOME ORGANIC ACIDS ON SOME FUNGAL
GROWTH AND THEIR TOXINS PRODUCTION
Ramadan Hassan
1
, Sherif El-Kadi
2
and Mostafa Sand
1
1
Department of Agricultural Chemistry, Faculty of Agriculture, Mansoura University,
Mansoura City, Egypt.
2
Department of Agricultural Microbiology, Faculty of Agriculture, Damietta University,
Damietta City, Egypt.
A
BSTRACT
The effect of eight organic acids (propionic, acetic, formic, lactic, tartaric, citric, oxalic and malic acids)
as antifungal agents on the growth of four fungi (Aspergillus flavus, Penicillium purpurogenum, Rhizopus
nigricans and Fusarium oxysporum) were studied. The high acidity appeared for oxalic acid being 0.14 at
the high concentration (10%), while the lowest acidity recorded for propionic acid and acetic acid being
2.71 and 2.56 at the low concentration (5%). It was observed that, there was no relationship between the
efficacy of organic acid and its final pH. Acetic acid (10%) has the highest inhibitory effect on A. flavus
being 45.21%, but tartaric acid (5%) and citric acid (5%) gave the same lowest inhibition effect (0.42%).
The lowest value of mycelium dry weight (MDW) of P. purpurogenum was 5.92 g/l when acetic acid was
used (10%), but the highest value was 9.38 g/l when tartaric acid (5%) was used. Formic acid (10%) had a
strong effect on the inhibition growth of R. nigricans being 28.65%, similar to propionic acid (10%), acetic
acid (10%), lactic acid (10%), tartaric acid (10%) and citric acid (10%) being 26.57%, 26.38%, 26.19%,
23.53% and 24.48%, respectively. But malic acid (5%) and oxalic acid (5%) were having a week effect on
R. nigricans being 5.31% and 6.45%, respectively. Lactic acid (10%) has the highest inhibitory effect on F.
oxysporum being 34.45% and the lowest value was in the case of tartaric acid (5%) being 1.68%. Four
treatments were used to determine aflatoxin B
1
production. The highest inhibition (50%) was observed by
R. nigricans in the presence of formic acid (10%). Acetic acid in 10% level inhibited the toxic secretion of
A. flavus and P. purpurogenum to become 25% and 40%, respectively. Lactic acid (10%) gave 35%
inhibition of toxin production in the presence of F. oxysporum.
K
EYWORDS
Antifungal, Organic Acids, Aspergillus flavus, Penicillium purpurogenum, Fusarium oxysporum, Rhizopus
nigricans & Aflatoxin B
1
1.
I
NTRODUCTION
Large amounts of food and feed are lost every year due to spoilage by yeasts and fungi [1]. So
that, preservative agents commonly used include weak organic acids such as acetic, lactic,
benzoic and citric acids, which inhibit the microbial growth in various foods. The effect of
organic acids on the fungal growth, which contaminate food and feed, has been investigated by
several authors [2; 3 and 4].
PH affects on the permeability of the cell membrane and on the enzymes that are active in
degrading the substrate [5, 6 and 7]. Organic acids generally used as safe agents to preserve
foods, these organic acids reduce cytoplasmic pH and stop metabolic activities. On the other
hand, organic acids caused the death by the susceptible organisms act on the plasmic membrane
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
2
by neutralizing its electrochemical potential and increasing its permeability [8]. Organic acids,
which used in food preservation is considered; simple, fast, acid, cheap and efficient. Moreover,
most of them are not limited in the acceptable daily intake for humans. These characteristics favor
their use in food preservation. Many authors have been testing the effect of organic acids on the
microbial growth, and they have taken a consideration in the sensory changes such as color and
flavor. Organic acids are considered weak acids (do not fully dissociate in water) but do so in a
pH dependent manner. The pK
a
is defined as the acid dissociation constant [4]. The decreasing of
pH resulting a greater concentration of protons and increasing the diffusion of acid across the
plasmic membrane and the cytoplasm. However, the substitution of the don able proton with a
monovalent (Na
+
, K
+
) or multivalent (Ca
+2
) cation significantly increases the solubility of organic
acid in aqueous systems. Thus, a balance must be occur between the need to maintain acid
solubility with the need to achieve maximal activity by pH reduction. The inhibitory effect of
organic acids is based in the “weak acid preservative theory”. Lactic and acetic acid have been
used as fungal inhibitors, they are present in the fermented foods and easy to obtain, their
maximum concentration is depending on the sensor parameters. Resistance towards weak acids
dependent on the plasma membrane H
+
-ATPase. This can be explained by the low permeability
of the plasma membrane to undissociated acid. Because of an energy spent during protons
pumping to membrane outside [9 and 10].
On the other hand, the higher acid resistance may be explained by the ability of some microbes to
consume, acetate while growing on fermentable sugar. The observation of higher acid resistance
was explained by two theories, the fungal deacidafication and the formation of alcohols. P.
camembert metabolizes the lactate to CO
2
and H
2
O, which results in deacidafication [4, 11 and
12].
High amount of aflatoxin B
1
was produced even initial spore inoculum levels were low [13]. A.
flavus and A. parasiticus producing aflatoxins were isolated from different Egyptian commodities
[14]. However, high fungi counts (i.e. 10
6
colony forming unite (cfu) or higher) are generally
producing mycotoxins [15]. Fungi can produce their mycotoxins under laboratory conditions or
naturally in various agricultural products [16]. Fungi cause a significant yield reduction and
economic losses because its commonly contaminate crops and foods. In addition, they changes
the appearance, taste, texture and odor of food, and also unsafe for human consumption because
of there mycotoxins. The consumption of foods which contaminated with mycotoxin has been
associated with several cases of human poisoning, sometimes resulting in death [17]. Nowadays,
mycotoxins have been receiving worldwide attention and several groups of mycotoxins are
known such as ergot, aflatoxins, ochratoxins, citrinin, patulin and fumonisines [18]. Fusarium,
Aspergillus and Penicillium were noted to be the major fungal populations in feed and foods. F.
moniliforme present in the feed and food for about a year. The predominant naturally occurring
fungi belonging mainly to Penicillium purpurogenum, Aspergillus glaucus and A. candidus [8].
Aflatoxin (AF) B
1
and mixtures of AFB
1
, AFG
1
and AFM
1
are proven as a human carcinogens
and are classified in the Group 1 carcinogen status [10].
So that, the aim of our research was a trial to inhibit the fungal growth and aflatoxin B
1
production in the presence of some organic acids.
2.
M
ATERIALS AND
M
ETHODS
2.1. Fungal isolates and maintenance
Four local fungal isolates, namely A. flavus, P. purpurogenum, F. oxysporum and R. nigricans
were obtained from Agric. Microbiology Dept., Fac. of Agric., Damietta University, Damietta,
Egypt. The fungal isolates were maintained on potato dextrose agar (PDA) medium slants at 5°C
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
3
till use [19]. Fungal isolates were subcultured on a new slant of PDA and incubated in a digital
incubator (Switc, MPM Instruments s.r.l., Bernareggio/Made in Italy) at 25°C for 10 days before
use.
2.2. Fungal spore suspensions and inoculum size
Spores suspension was prepared as described by [20]. Fungi were grown on a PDA slant at 25°C
for 10 days. 10 ml of sterilized saline solution (0.09%) was added to the slants and the spores
were loosened by gently brushing with a sterile inoculating loop. A vortex mixer (VM-300
power: 220 VAC, 50Hz, 0.16A/Made in Taiwan-Associated with Cannicinc U.S.A.) was used for
one minute to remove all spores from slant [21]. Spores count was performed in a
Hematocytometer slide (model Buerker MOM BUDA pest) and the following equation was used
for fungal spore count: Spore count equation (spores/ml) = mean of spore count in 10 squares x
slid factor (2.5 x 10
6
) x dilution rate. Spores suspension corresponding to give approximately
1x10
6
spores per ml using a micropipette (Micro Volume Pipettor - Accumax A made in China),
thus the count of spores was fixed in the following experiment [2]. The spores suspension stocks
were stored at 4°C.
2.3. Organic acids
Propionic acid (99%, BDH) acetic acid glacial (99.8%, Almasria for Chemicals) formic acid
(85%, BDH) lactic acid (Chemicals Ltd, Poole, England), tartaric acid (99%, ADWIC) citric acid
anhydrous (99%, ADWIC), oxalic acid (99.5%, ADWIC), and malic acid (99%, LOBA Chemie,
India) were used as antifungal agents. The addition of these acids was at the time of inoculation to
reduce the fungal growth and their mycotoxins production. The pH value of each eight organic
acids in water was determined.
2.4. Fungal inhibition
The basal medium was potato dextrose broth (PDB), this medium was used to evaluate the spores
germination and the growth of our fungi by surface culture technique [3]. Fifty ml of PDB was
put into a 250 ml Erlenmeyer flask and autoclaved at 121ºC for 20 min. After cooling, the flasks
were then treated with three levels (0%, 5% and 10%) of organic acids as an antifungal agents
using a Millipore filter (0.2 µ m, Flow Pore D made by Sartorius, W. Germany). All flasks were
inoculated with 1xl0
6
spores from each spores suspension stocks (A. flavus, P. purpurogenum, F.
oxysporum and R. nigricans). After 8 days of incubation in the digital incubator at 25°C, the
inhibition percentage was calculated by the difference between the growth of mycelium dry
weight in the absence or in the presence of the antifungal agent [2]. All experiments were
conducted in three replicates.
2.5. Determination of mycelium dry weight (MDW)
Through a double-layered Whatman filter paper No. 1 the mycelial mat resulting from surface
culture technique was filtered and washed twice with distilled water, dried in an oven (Nemmert,
W. Germany) at 80°C to a constant weight (g/l) [22]. Mycelial mat was determined using an
electronic balance (type BL-32OH Shimadzu Corporation, Japan). The supernatant was collected
and used for mycotoxins determination. The final pH was determined in the culture filtrate also
using a pH meter, (model Hanna pH 211 microprocessor pH meter).
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
4
2.6. Determination of aflatoxin B
1
Culture filtrates were extracted three times with 100 ml volume of chloroform. The residue was
dissolved in 5 ml methanol (70%). ELISA technique was used to determine aflatoxin B
1
[23]
.
Aflatoxin B
1
(standard) was purchased from Sigma Chemical Co. (USA).
3.
R
ESULTS AND
D
ISCUSSION
3.1. The effect of organic acids as antifungal agents
PH values of the eight organic acids when dissolved in water at levels of 5 and 10% were
presented in Table 1. It was clear showed that, when the acid concentration increased the pH
degree decreased. There is an opposite relation between pH degree and the acidity. The high
acidity appeared for oxalic acid being 0.14 at the high concentration (10%), while the lowest
acidity recorded for propionic acid and acetic acid being 2.71 and 2.56 at the low concentration
(5%).
Table 1. PH values of organic acids in water
Organic acids State
The pH values
5% 10%
Propionic acid
(v
/v.)
Liquid
2.71
2.45
Acetic acid (v/v) 2.56 2.28
Formic acid
1.75
1.53
Lactic acid (v/v) 2.09 1.83
Tartaric acid (w/v)
Solid
1.48 1.29
Citric acid (w/v) 1.18 1.12
Oxalic acid (w/v) 0.43 0.14
Malic acid
(w/v)
1.91
1.72
The mechanism of inhibition fungi growth by organic acids is generally not considered a pH
phenomenon. It is known that, growth and morphology of fungi are influenced by the pH of
media [24]. Some mechanisms have been suggested to explain the inhibitory mode of organic
acids. Organic acids resulting a decreasing in pH value, this may influence the growth by
acidifying the cell, which will consume a great amount of energy to maintain the intracellular pH
homeostasis [3]. Other explanations have also been proposed including the membrane disruption,
the interruption of metabolic reactions, and the accumulation of toxic anions. Three of the fungi
(P. roqueforti, P. commune and F. sporotrichoides) and one yeast species (Kluyveromyces
marxianus) did not grow at pH 3 [1]. The inhibition of microbial growth increases by lowering
pH of the media, and most microorganisms are susceptible to antimicrobial effects in the presence
of organic acids. This phenomenon is due to the hydrophobic feature of most organic acids, which
allows free diffusion of the protonized form through cell membrane. This diffusion process takes
place spontaneously due to pH and osmolarity gradients that exist between the inner and outer
sides of the cell. The intracellular pH is higher than the extracellular, and the acid undergoes
dissociation as soon as it enters the cytoplasm and then decreases the intracellular pH by releasing
the proton. In order to counter the decrease of cytoplasmic pH, resulting from the ionization of
the entered acid, the cell allocates the main part of its energy content to eliminate these newly
formed protons which results in slower growth kinetics [10].
Results recorded in Table 2. showed that, the effect of organic acid levels (0, 5 and 10%) on A.
flavus growing in PDB medium. The inhibition percentage was calculated by the difference
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
5
between dried mycelium weight in the absence or presence of the antifungal agent (organic acids)
after 8 days of incubation at 25°C. The final pH and MDW of the control were 3.87 and 9.60 g/l,
respectively. Acetic acid (10%) has the highest inhibitory effect on A. flavus being 45.21% and
the final pH was 3.25, but tartaric acid (5%) and citric acid (5%) gave the same lowest inhibition
effect (0.42%) and final pH was 3.12 and 3.24, respectively. Other authors, [25] studied the
inhibition growth of some species of fungi (A. flavus, A. niger, A. fumigatus, P. glabrum F.
moniliforme and Cladosporium sphaerospermum) using organic acids (lactic, acetic, formic,
oxalic, and propionic) in concentrations 3, 5, 10, 20 and 50 ml/l and they found that, lactic and
oxalic acids did not prove any activity on the chosen concentrations. Propionic acid in
concentration 20 ml/l inhibited the growth of five fungi (A. fumigatus, A. niger, P. glabrum, F.
moniliforme, and C. sphaerospermum). Propionic acid exhibited a fungicidal action; however,
calcium propionate had essentially no effect on A. flavus growth [2]. Our results within the line of
[8] who reported that, acetic acid was more effective than lactic acid and had the best inhibitor of
fungi growth. Different acetic and lactic acid concentrations were studied by [10] for antifungal
activity against different strains of A. flavus: They determined that the increase of acid in the
medium decreases the growth rate and extends the lag phase.
Table 2. Effect of organic acids levels on growth of Aspergilus flavus
Organic acids
5%
10%
Final
pH
MDW
(g/l)
Inhibition
%
Final
pH
MDW
(g/l)
Inhibition
%
Propionic acid 3.96 7.06 26.46% 3.49 5.72 40.42%
Acetic acid 3.76 6.92 27.92% 3.25 5.26 45.21%
Formic acid 3.15 7.50 21.88% 2.56 6.26 34.79%
Lactic acid 3.43 9.16 4.58% 2.62 8.58 10.63%
Tartaric acid 3.12 9.56 0.42% 2.40 8.00 16.67%
Citric acid 3.24 9.56 0.42% 2.60 7.90 17.71%
Oxalic acid
1.96
8.86
7.71%
1.58
8.78
8.54%
Malic acid 2.31 9.46 1.46% 2.62 9.06 5.63%
Table 3. showed the effect of organic acids levels on the growth of P. purpurogenum. The final
pH and MDW of the control experiment were 4.24 and 10.02 g/l, respectively. The lowest value
of MDW was 5.92 g/l when acetic acid (10%) was used, but the inhibition effect and final pH
were 40.92% and 3.31, respectively. The highest value of MDW was 9.38 g/l when tartaric acid
(5%) was used.
P
ropionic acid (20 ml/L) inhibited the growth of P. glabrum [25]. The growth of
two isolates of Penicillium sp. were completely inhibited by the presence of ascorbic acid or
propionic acid. Calcium-propionate and Na-benzoate did not exhibit any inhibitory effects on
these two cultures [2].
Table 3. Effect of organic acids levels on the growth of Penicillium purpurogenum
Organic acids
5%
10%
Final
pH
MDW
(g/l)
Inhibition
%
Final
pH
MDW
(g/l)
Inhibition
%
Propionic acid 4.25 8.10 19.16% 3.49 6.84 31.74%
Acetic acid 4.03 7.66 23.55% 3.31 5.92 40.92%
Formic acid 3.00 9.08 9.38% 2.39 8.32 16.97%
Lactic acid 3.27 9.36 6.59% 2.68 8.22 17.96%
Tartaric acid 3.63 9.38 6.39% 2.44 8.28 17.37%
Citric acid 2.89 9.16 8.58% 2.56 8.00 20.16%
Oxalic acid
2.18
8.50
15.17%
1.75
6.74
32.74%
Malic acid 3.12 8.50 15.17% 2.68 7.94 20.76%
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
6
Similar results were obtained from [24] who published that, the values of final pH of cultivation
broth for lactic acid and acetic acid were 3.35 and 3.81, respectively, these tow acids displayed
little efficacy in controlling fungi growth. Our results were agreeing with that obtained by [1]
who found that, P. roqueforti was the most sensitive to organic acids. Also, they evaluated the
antifungal activity (propionic acid, lactic acid and acetic acid) against A. fumigatus, P. roqueforti,
P. commune, A. nidulans, F. sporotrichoides. The minimal inhibitory concentration values of
propionic, acetic and lactic acid were established for all fungi at pH 3, 5 and 7. Propionic acid,
followed by acetic acid, was the most potent antifungal acid.
The final pH and MDW values in the control flask of R. nigricans were 3.89 and 10.54 g/l,
respectively. Formic acid (10%) had a strong effect on the inhibition growth of R. nigricans being
28.65% (Table 4.), similar to propionic acid (10%), acetic acid (10%), lactic acid (10%), tartaric
acid (10%) and citric acid (10%) being 26.57%, 26.38%, 26.19%, 23.53% and 24.48%,
respectively. But malic acid (5%) and oxalic acid (5%) were a weak effect being 5.31% and
6.45%, respectively. The final pH values were varied between 1.58 and 3.96 in the case of oxalic
acid (10%) and propionic acid (10%), respectively. On the other hand, the values of MDW were
varied between 7.52 and 9.98 g/l when formic acid (10%) and malic acid (5%) was used,
respectively. The mixtures of acetate, lactate and propionate have a synergistic inhibitory effect
on the indicator strains [26].
Table 4. Effect of organic acids levels on the growth of Rhizopus nigricans
Organic acids
5% 10%
Final
pH
MDW
(g/l)
Inhibition
%
Final
pH
MDW
(g/l)
Inhibition
%
Propionic acid 3.65 8.66 17.84% 3.96 7.74 26.57%
Acetic acid 3.47 8.72 17.27% 3.17 7.76 26.38%
Formic acid 2.91 8.54 18.98% 2.59 7.52 28.65%
Lactic acid 2.79 8.94 15.18% 2.40 7.78 26.19%
Tartaric acid
2.80
8.84
16.13%
2.41
8.06
23.53%
Citric acid 2.64 8.64 18.03% 2.58 7.96 24.48%
Oxalic acid 2.03 9.86 6.45% 1.58 9.46 10.25%
Malic acid 2.95 9.98 5.31% 2.81 9.32 11.58%
The effect of organic acid levels on the growth of F. oxysporum was presented in Table 5. The
final pH and MDW of the control experiment were 3.98 and 10.74 g/l, respectively. Lactic acid
(10%) has the highest inhibitory effect on F. oxysporum being 34.45% and the lowest value was
in the case of tartaric acid (5%) being 1.68%.
Table 5. The effect of organic acids levels on the growth of Fusarium oxysporum
Organic acids
5% 10%
Final
pH
MDW
(g/l)
Inhibition
%
Final
pH
MDW
(g/l)
Inhibition
%
Propionic acid 3.85 8.86 17.51% 3.17 8.16 24.02%
Acetic acid 3.16 8.20 23.65% 3.23 9.78 8.94%
Formic acid 3.08 9.76 9.13% 2.45 8.70 18.99%
Lactic acid 3.73 9.14 14.90% 2.49 7.04 34.45%
Tartaric acid 3.89 10.56 1.68% 2.27 10.52 2.05%
Citric acid
3.64
10.16
5.40%
2.71
8.50
20.86%
Oxalic acid 3.45 10.02 6.70% 1.44 9.98 7.08%
Malic acid 3.85 10.22 4.84% 2.56 9.32 13.22%
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
7
Our results were in reserve direction with those [25] who reported that, lactic and oxalic acids did
not prove any activity on the growth of F. moniliforme, but the best organic acid on the fungal
growth inhibition was propionic acid. The growth of Fusarium sp. was completely inhibited by
the presence of propionic acid, sorbic acid or Na-benzoate [2]. 10 inorganic and 12 organic salts
for their inhibitory activity against F. sambucinum were evaluated [27] They found that, several
salts inhibited completely mycelial growth and spore germination of F. sambucinum. Among
these salts, sodium benzoate, sodium metabisulphite, potassium sorbate, trisodium phosphate and
aluminium salts were fungi toxic. Sodium propionate, sodium carbonate and sodium citrate
inhibited the growth of F. sambucinum but to a lesser extent.
3.2. Effect of organic acids on aflatoxin B
1
production:
Four tests were chosen to determent aflatoxin B
1
(AB
1
) production based on the highest inhibitory
effect on fungal growth being: A. flavus, P. purpurogenum, R. nigricans and F. oxysporum treated
with acetic acid (10%), acetic acid (10%), formic acid (10%) and lactic acid (10%), respectively.
All organic acids under study reduced aflatoxin B
1
secretion. The highest one (50%) was
observed for R. nigricans in the presence of formic acid (10%) (Table 6). Acetic acid in 10%
level inhibited the toxic secretion of A. flavus and P. purpurogenum to become 25% and 40%,
respectively. Lactic acid (10%) gave 35% inhibition of toxin production in the presence of F.
oxysporum. Propionic acid and butyric acid were added as sub-lethal doses (1–20%) to a growth
medium of A. flavus for supporting growth and subsequent aflatoxin production [28]. In the same
manner [8] reported that lactic acid bacteria inhibited the fungal growth and biosynthesis of
mycotoxin.
Table 6. The effect of organic acids on aflatoxin B
1
production.
Tested fungi
Aflatoxin B1 production (ppb)
Inhibition (%)
Treated Control
Aspergillus
flavus
8
12
25%
Penicillium purpurogenum 6 10 40%
Rhizopus nigricans 4 8 50%
Fusarium
oxysporum
6.5
10
35%
3.3. Relationship between chemical structure of organic acid and the fungal growth
inhibition:
From Figs. 1, 2 and 3, it was clear that, formic acid (H-CO-OH), acetic acid (CH
3
-CO-OH) and
propionic acid (CH
3
-CH
2
-COOH) was the highest inhibitory effect on A. flavus growth. This
effect may be due to the similarity in their chemical structure and also may be due to their pK
a
,
which are almost the same being 3.77, 4.79 and 4.87, respectively. The effect of propionic acid
concentration (129, 258 and 516 ppm) on the growth of A. parasiticus were studied by [17], the
increasing of propionic acid concentration showed that decreasing in the growth rate. Our results
were similar to those obtained by [3] who measured the residual concentration of dissolved
oxygen in the culture medium after 4 days. The findings of those may explain why acetic acid
showed the strongest inhibition of the fungal growth, where the growth inhibition by this organic
acid was closely related to the inhibition of respiration.
The results in Figs. 4 and 5 appeared that, the lactic acid (CH3-CHOH-COOH) and tartaric acid
(COOH-CHOH-CHOH-COOH) almost had the same effect on the P. purpurogenum and R.
nigricans growth, that may be due to the isomerism in their chemical structure. The minimal
inhibitory concentration (MIC) values for lactic acid concentration are nearly tenfold higher than
MIC values for acetic acid on the A. flavus. Lactic and acetic acid mixtures showed a synergistic
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
8
effect, reducing the concentration necessary of every acid in the mixture for fungal inhibition
compared with the individual MIC values [10].
0
5
10
15
20
25
30
35
40
45
Inhibitio n %
A.fla vus
P.pu rpuro genum
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 1. The effect of propionic acid concentrations on the fungal growth
0
5
10
15
20
25
30
35
40
45
50
Inhibitio n %
A.fla vus
P.p urpuro genum
R.nigric ans
F.ox yspo rium
5% 1 0%
Figure 2. The effect of acetic acid concentrations on the fungal growth
0
5
10
15
20
25
30
35
40
45
50
Inhibitio n %
A.fla vus
P.p urpuro genum
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 3. The effect of formic acid concentrations on the fungal growth
0
5
10
15
20
25
30
35
Inhibitio n %
A.fla vus
P.p urpuro genum
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 4. The effect of lactic acid concentrations on the fungal growth
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
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0
5
10
15
20
25
30
35
Inhibitio n %
A.fla vus
P.p urpuro genu m
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 5. The effect of tartaric acid concentrations on the fungal growth
Citric acid (C
3
H
5
O(COOH)
3
), oxalic acid (COOH-COOH) and malic acid (COOH-CHOH-CH
2
-
COOH) gave highest effect on P. purpurogenum growth (Figs. 6, 7 and 8) that may be due to the
similarity in their chemical structure and containing more than one carboxylic group.
0
5
10
15
20
25
Inhibitio n %
A.fla vus
P.p urpuro genum
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 6. The effect of citric acid concentrations on the fungal growth
0
5
10
15
20
25
30
35
Inhibitio n %
A.fla vus
P.p urpuro genum
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 7. The effect of oxalic acid concentrations on the fungal growth
0
5
10
15
20
25
Inhibitio n %
A.fla vus
P.p urpuro genum
R.nig ricans
F.ox yspo rium
5% 1 0%
Figure 8. The effect of malic acid concentrations on the fungal growth
International Journal of Advances in Biology (IJAB) Vol 2. No .1, February 2015
10
This observation is in agreement with the results of those [29] who reported that, the inhibitory
effect of organic acids on microbial growth has been studied. Organic acids are not members of a
homologous series, but vary in the numbers of carboxy groups, hydroxy groups and carbon–
carbon double bonds in the molecule. Properties correspond to polar groups, the number of
double bonds, molecular size, and solubility in non- polar solvents.
4.
C
ONCLUSIONS
All tested organic acids, which used as antifungal were variations in the effect of fungal growth.
There was a little correlation between the final pH of the organic acids and its efficacy on the
tested fungi. Acetic acid (10%) has the highest inhibitory effect on A. flavus followed by P.
purpurogenum being 45.21% and 40.92, respectively. On the other hand tartaric acid (5%) and
citric acid (5%) gave the same lowest inhibition effect (0.42%) on A. flavus, but tartaric acid (5%)
affected in the growth of F. oxysporum being 1.68%. All organic acids, which used in this study
reduced aflatoxin B
1
production. The treatment of R. nigricans in the presence of formic acid
(10%) was the highest inhibitory effect being 50%.
R
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Authors
Prof. Dr. Ramadan Ahmed Hassan, I am currently working in the Department of
Agricultural Chemistry, Faculty of Agriculture, Mansoura University, Dakahlia,
Egypt. I am interested in general Agricultural Chemistry.
Dr. Sherif Mohamed El-Kadi, I am appointed a demonstrator in Agricultural
Microbiology Department, Faculty of Agriculture, Mansoura University, Egypt since
1999. I have and got the M. Sc. (Studies on the microbial production of citric acid,
2003) and the Ph. D. (Studies on certain microbial polymers, 2008) degrees from the
same department. I am currently working as a lecturer in Agricultural Microbiology
Department, Faculty of Agriculture, Damietta University, Damietta, Egypt. I am
interested in general microbiology, particularly food microbiology. I have specific
interest in environmental microbiology, biotechnology and biofertilization. I am
married and I have four girls. I am living in Domiat El-Jadida city. My favorite game
is chess.
Asst. Prof. Dr. Mostafa Ibrahim Sand, I am currently working in the Department of
Agricultural Chemistry, Faculty of Agriculture, Mansoura University, Dakahlia,
Egypt. I am interested in general Agricultural Chemistry.
... The intracellular pH is higher than the extracellular, and the acid undergoes dissociation as soon as it enters the cytoplasm and then decreases the intracellular pH by releasing the proton. To counter the decrease of cytoplasmic pH, resulting from the ionization of the entered acid, the cell allocates the main part of its energy content to eliminate these newly formed protons which results in slower growth kinetics [21]. According to Hassan et al. (2015) acetic acid shows strongest inhibition of fungal growth among other organic acids [21]. ...
... To counter the decrease of cytoplasmic pH, resulting from the ionization of the entered acid, the cell allocates the main part of its energy content to eliminate these newly formed protons which results in slower growth kinetics [21]. According to Hassan et al. (2015) acetic acid shows strongest inhibition of fungal growth among other organic acids [21]. Earlier reports showed that the major components of organic acids and phenolics in wood vinegar can inhibit pathogenic fungi and bacteria [22][23][24][25]. ...
... To counter the decrease of cytoplasmic pH, resulting from the ionization of the entered acid, the cell allocates the main part of its energy content to eliminate these newly formed protons which results in slower growth kinetics [21]. According to Hassan et al. (2015) acetic acid shows strongest inhibition of fungal growth among other organic acids [21]. Earlier reports showed that the major components of organic acids and phenolics in wood vinegar can inhibit pathogenic fungi and bacteria [22][23][24][25]. ...
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The vinegar produced from different locally grown fruits and industrial produced vinegar was evaluated to determine its antimicrobial properties. Agar well diffusion method was used for this analysis. The antimicrobial activities of both locally and industrially produced vinegar were identified using different microbial isolates which includes Escherichia coli, Staphylococcus aureus and Candida sp using different extracts of the vinegar.The results of the antimicrobial analysis showed that the vinegar exhibit different activities on the clinical isolates. Concentrations of 500mg/ml, 250mg/ml, 125mg/ml and 62.5mg/ml of the vinegar extracts were used. The aqueous and n-butanol extract of Vin A gave the highest zone of inhibition with a diameter of 19mm on Staphylococcus aureus at 500mg/ml concentration.The aqueous extract of Vin A gave diameter range of 15mm-19mm for S. aureus and 11mm-17mm for Candida sp. n-Butanol extract gave the range of 10mm-12mm for E.coli, 12mm-19mm on S. aureus and 7mm-15mm onCandida sp. The ethyl acetate of Vin A gave a diameter of 9mm and 10mm for Candida sp. The n-Butanol extract of Vin B gave the diameter of 10mm for E.coliat 500mg/ml, 6mm and 60mm for S. aureus and 9mm for Candida sp. the aqueous extract of Vin C gave diameter of ranges within 8mm-11mm for E.coli, and 10mm on cCandida sp. The n-Butanol extract gave the range of 7mm-10mm on E.coli, 8mm-12mm on S. aureus and 9-10mm on Candida sp. n-butanol extract of Vin D gave a diameter of 9mm for E.coli, 10mm for staph and a diameter range of 7-11mm for Candida sp. The ethylacetate of the extract gave 7mm and 8mm for E.coli, 9mm and 11mm for Candida sp. The vinegars analysed exhibited bactericidal, bacteriostatic and no activity on the clinical isolates.
... Besides being able to break dormancy, acid, and alkali treatment can also inhibit fungal growth (Turkkan, Ozcan, & Erper, 2017;Hassan, El-kadi, & Sand, 2015). ...
... Carbonate and bicarbonate are known to have an inhibitory effect against fungal plant pathogens (Turkkan, et al., 2017) R. nigricans, is also be able to reduce production fungal toxin such as aflatoxin (Hassan et al., 2015). ...
... Sodium bicarbonate were been able to induce defense mechanism by producing increasing the activity of -1,3-glucanase, peroxidase, and phenylalanine ammonia-lyase (PAL) enzymes (Youssef, Sanzani, Ligorio, Ippolito, & Terry, 2014 (Hassan, et al, 2015). ...
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ABSTRAK Hibiscus macrophyllus merupakan salah satu pohon tropis yang memiliki potensi ekonomi tinggi, namun terdapat permasalahan dalam pembibitannya, yaitu dormansi benih dan patogen terbawa benih. Penelitian ini bertujuan untuk mengevaluasi pengaruh sodium bikarbonat dan asam asetat terhadap kolonisasi cendawan terbawa benih, perkecambahan benih dan pertumbuhan bibit H. macrophyllus. Rancangan acak lengkap digunakan untuk menguji 6 perlakuan, yaitu: (i) benih tanpa perlakuan, (ii) perendaman dalam air mendidih dan dibiarkan selama 24 jam, (iii) perendaman dalam air mendidih dan biarkan selama 24 jam diikuti perendaman dalam asam asetat 1% (15 menit), (iv) perendaman dalam air mendidih dan biarkan selama 24 jam diikuti perendaman dalam sodium bikarbonat 5% (15 menit), (v) perendaman dalam asam asetat 1% (15 menit) diikuti perendaman dalam air mendidih dan biarkan selama 24 jam, dan (vi) perendaman dalam sodium bikarbonat 5% (15 menit) diikuti perendaman dalam air mendidih dan biarkan selama 24 jam. Perendaman dalam sodium bikarbonat 5% (15 menit) diikuti perendaman dalam air mendidih dan biarkan selama 24 jam secara nyata mampu menekan cendawan terbawa benih. Aplikasi sodium bikarbonat 5% dan asam asetat 1% tidak dapat meningkatkan perkecambahan benih. Perlakuan sodium bikarbonat diikuti perendaman dalam air mendidih memberikan pertumbuhan diameter bibit, panjang daun, lebar daun, panjang akar, jumlah daun terbaik. Kata kunci: asam asetat, Hibiscus macrophyllus, benih, bibit, sodium bikarbonat ABSTRACT Hibiscus macrophyllus, an important tropical tree, have high economic potential, however there are the problems in seedling procurement, i.e. seed dormancy and seed-borne pathogen. The purpose of the research was to evaluate the effect of sodium bicarbonate and acetic acid on the fungal colonization, seed germination, and seedling growth of H. macrophyllus. A completely randomized design was used to test the six treatments: (i) untreated seed, (ii) soaking seeds in boiling water and left 24 hours, (iii) soaking in boiling water and left 24 hours followed by soaking in acetic acid 1% (15 minutes), (iv) soaking in boiling water and left 24 hours followed by soaking in sodium bicarbonate 5% (15 minutes), (v) soaking in acetic acid 1% (15 minutes) followed by soaking in boiling water and left 24 hours, and (vi) soaking in sodium bicarbonate 5% (15 minutes) followed by soaking in boiling water and left 24 hours. Soaking in sodium bicarbonate 5% (15 minutes) followed by soaking in boiling water and left 24 hours could significantly decrease the fungal colonization. Sodium bicarbonate 5% and acetic acid 1% treatments could not improve seed germination. The sodium bicarbonate treatment followed by soaking in boiling water increased the seedling diameter, leaf length, leaf wide, root length, and leaf number.jur
... Due to their chemical structures, effects of the acids on the growth of fungi might be different. Formic acid, acetic acid and propionic acid had a similar strong inhibitory effect on Aspergillus flavor as [6] stated. N.V. Narendranath et al. 2001, [7] reported that the inhibitory effect of acetic and lactic acids in a same medium revealed synergism. ...
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... Chemical fungicides are the fastest effective way to control these pathogens, but their negative effects on humans, their environment and on the mushroom product itself owing to the rapid absorption of these pesticides by mushrooms. Studies have tended to find safer alternatives, such as chemicals (nonpesticides) that are degradable, such as some salts and organic acids [13,14]. ...
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... Previous study by Ahn and Shin (1999) reported that different types of organic acids exhibit different antimicrobial effects against different microorganisms. There are many reports on antimicrobial activities of organic acids against pathogenic microbes including Shigella sp., Bacillus sp., Staphylococcus aureus, Escherichia coli, Aspergillus flavus, Penicillium purpurogenum, Rhizopus nigricans, Fusarium oxysporum and other pathogenic microbes (Ye et al., 2013;Feng et al., 2010;Hasan et al., 2015). However the antifungal activity of organic acids against Ganoderma has yet to be reported. ...
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... This can be explained by the lipophilic nature of MCFA that allows them to have a stronger antibacterial activity mainly against Gram-positive species, whereas the presence of lipopolysaccharide (LPS) in the Gram-negative cell wall confers resistance to these species [29]. Propionic acid and butyric acid are strong mold inhibitors, while acetic acid is commonly used as antifungal, also reducing aflatoxins production [30][31][32]. ...
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... As an acidic product with pH ranging from 4.21 to 4.48 [16], YAW may be used as a clean-label alternative or complement to added preservatives, in the case of products in which acid flavors are preferred by consumers [17]. The organic acids contained in YAW (lactic acid 0.65%, citric acid 0.18%, and glutaric acid 0.06%) may have antifungal effects [18], increasing product shelf life. The acidity of YAW may contribute to the flavor of fermented doughs and batters. ...
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The increased production of Greek-style yogurt in the past decade has induced the need for the reintroduction of the nutrients of its byproduct, yogurt acid whey (YAW), into the food system to combat food waste and aid sustainability. However, the processing and treatment of acid whey, which can be environmentally damaging if disposed of incorrectly, can be costly and complex. Upscaling YAW as an ingredient in food products with minimal re-processing is a cost-effective way to bypass the need for further abatement. To span a broad spectrum of baked products (sweet and savory, biologically and chemically leavened, dairy or water based, oven or surface baked, batter or dough, etc.), pilot commercial pizza crust and pancake formulations incorporating acid whey as a functional ingredient were developed. Dimensions and physico-chemical properties of samples were measured at production and over shelf life at room temperature (23 °C). Consumer sensory testing (n = 120 and n = 108, respectively, Just About Right (JAR), nine-point hedonic, purchase intent, and demographics) were conducted for both products. All instrumental trials and analyses (°Brix, aw, color attributes, viscosity, dimension measurements, and texture analysis) were conducted in triplicate for statistical analysis. Cochran’s Q and post-hoc tests on sensory data showed that liking for at least one experimental YAW sample for each of the pizza and pancake formulations were on par with their respective commercial product, despite the reduction of buttermilk, salt and sugar from the YAW formulations. Adding sustainability claims brought the purchase intent on par with the controls. Replacement of water by weight of YAW was more appropriate than by water content of the YAW. Sourness was the main undesirable trait of YAW samples based on penalty analysis. The use of YAW improved the shelf life of baked goods based on their respective failure mechanisms (textural properties and mold growth). YAW is a suitable ingredient in the formulation of sustainable, healthy, safe, and commercially successful baked products that have a tolerance or can benefit from a sour flavor profile.
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This work explores the use of chitosan as an active matrix to develop biofungicides. Two chitosans from marine resources, of 4.8 and 78 kg/mol respectively, were structurally characterized. Both chitosans diluted in acidic conditions (lactic or acetic acid at 1%, v/v) were efficient in reducing the growth of Fusarium graminearum. Ammonium groups of chitosan chains were suggested as key determinants for the polysaccharide's activity. The chitosan of 78 kg/mol was also efficient in decreasing the production of mycotoxins by F. graminearum leading to an inhibition rate of 70%. Moreover, the film forming properties of the 78 kg/mol chitosan were physicochemically characterized and used to mimic how the bioformulation should look when sprayed on the plant. Interestingly, the film was proved to totally inhibit the mycelium growth, contrary to a hydroxypropylmethyl cellulose film, an electrically-neutral analogue to our biopolymeric coating of chitosan.
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