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Abstract— Chanterelle (Cantharellus cibarius) mushroom can be cultured from its fruit body on agar medium. The present study showed that the growth rate of chanterelle mycelia in agar medium is slow whereas the pigment of the cultured mycelia was medium dependent. Different mycelia colors were detected in this study: from orange to pink and brown.This study also revealed bacterial growth near mycelia fragments, which appeared only at the initial phase of mycelia growth after which the mycelia continued to grow, blocking bacterial growth in the center of the agar plates. Therefore, we presumed that these bacteria were able to transfer the color to the chanterelle mycelia and may serve as fungal growth helper bacteria. The first step was to isolate these accompanying bacteria in pure culture and relate its phenotypical aspect to the mycelia aspect. The second step consisted of chemically treating the mycelia to suppress bacteria around and verify the mycelia’s ability to enhance or decrease color production. As a third step, the Chanterelle mycelia were treated separately with different chemical reagents [Sodium nitrate, Potassium phosphate monobasic, Ammonium nitrate, Citric acid, Acetic acid, Boric acid (0.05 g/ml), 1% NaOH, 1% KOH and 0.5%.HCl] followed by incubation in different agar plates. We demonstrated that some treatments killed all bacteria after which the mycelia lost its growth capacity. As a final step, agar plates showing no development of mycelia were inoculated with bacteria. After this inoculation, mycelia growth resumed and obtained the color of the inoculated bacteria. The results clearly showed that endogenous bacteria present in Chanterelle mycelia serve to initiate mycelial growth and impart color to the Chanterelle mycelia. The isolated bacteria produced aromas, lecithinase, amylase and laccase as well. However, these bacteria were unable to produce oxidase, catalase or protease. Index Terms— Chanterelle, endogenous bacteria, color, pink, brown, orange, perfume, enzymes.
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International Journal of Engineering and Applied Sciences (IJEAS)
ISSN: 2394-3661, Volume-3, Issue-9, September 2016
58 www.ijeas.org
Abstract Chanterelle (Cantharellus cibarius) mushroom can
be cultured from its fruit body on agar medium. The present
study showed that the growth rate of chanterelle mycelia in agar
medium is slow whereas the pigment of the cultured mycelia was
medium dependent. Different mycelia colors were detected in
this study: from orange to pink and brown.This study also
revealed bacterial growth near mycelia fragments, which
appeared only at the initial phase of mycelia growth after which
the mycelia continued to grow, blocking bacterial growth in the
center of the agar plates. Therefore, we presumed that these
bacteria were able to transfer the color to the chanterelle
mycelia and may serve as fungal growth helper bacteria.
The first step was to isolate these accompanying bacteria in
pure culture and relate its phenotypical aspect to the mycelia
aspect. The second step consisted of chemically treating the
mycelia to suppress bacteria around and verify the mycelia’s
ability to enhance or decrease color production. As a third step,
the Chanterelle mycelia were treated separately with different
chemical reagents [Sodium nitrate, Potassium phosphate
monobasic, Ammonium nitrate, Citric acid, Acetic acid, Boric
acid (0.05 g/ml), 1% NaOH, 1% KOH and 0.5%.HCl] followed
by incubation in different agar plates. We demonstrated that
some treatments killed all bacteria after which the mycelia lost
its growth capacity. As a final step, agar plates showing no
development of mycelia were inoculated with bacteria. After this
inoculation, mycelia growth resumed and obtained the color of
the inoculated bacteria.
The results clearly showed that endogenous bacteria present
in Chanterelle mycelia serve to initiate mycelial growth and
impart color to the Chanterelle mycelia. The isolated bacteria
produced aromas, lecithinase, amylase and laccase as well.
However, these bacteria were unable to produce oxidase,
catalase or protease.
Index Terms Chanterelle, endogenous bacteria, color, pink,
brown, orange, perfume, enzymes.
I. INTRODUCTION
Chanterelles are ectomycorrhizal fungi growing in a
Neila Saidi, CERTE - LEMBE Technoparck Borj Cedria Tunis Tunisia,
21655641366 .
Shweta Deshaware, research fellow, pursuing Ph.D (Food
Biotechnology) from Institute of Chemical Technology, Mumbai. India. Ph:-
022-25841881 Mob:- 09892727197.
Ilef Ben Romdhane Senior research fellow, pursuing Ph.D in
microbiology CERTE, LTEU Technoparck Borj Cedria Tunis Tunisia.,
Matab Nadim, Juva truffle center, 51901, Juva, Finland.
Marwa Laarib High Institute of Food Tunis Tunisia ESIAT
Abdelkader Ltifi CERTE - LEMBE Technoparck Borj Cedria Tunis
Tunisia,
Robert Kremer University of Missouri 302 Natural Resources Bldg
Columbia, MO 65211
Salem Shamekh Juva truffle center, 51901, Juva, Finland.
mutually and beneficial association with certain trees.
Ectomycorrhizal fungi in general are a subset of mycorrhizal
fungi that form sheaths over the root tips of their host trees.
Chanterelles are highly prized for their flavor and can be
safely collected and consumed because they are easily
identified [1]. For the propagation of fungal mycelia in
laboratory an optimum medium is required, the composition
of which provides all the essential nutrients offered via
symbiosis naturally by host trees. Routinely used media such
as Malt Extract Agar (MEA), Potato Dextrose Agar (PDA)
and Potato Dextrose Yeast Agar (PDYA) may lack certain
essential growth components required for the growth of
Chanterelle mushrooms. Other commonly observed factors
inhibiting its growth include unknown specific nutritional
requirements, difficulty in using complex carbohydrate
sources, and high natural contamination of sporocarps by
moulds and bacteria [2, 3].
However, several attempts have been made for aseptic
cultivation of pure mycelium and fruiting bodies of
Chanterelle mushrooms. Nylund (1982) [4] supplemented the
agar medium used for chanterelle mycelium production with
several antibiotics. Antibiotics kept the co-inhabiting bacteria
and fungi sequestered for 17-53 days following which a pure
mycelium was obtained which was confirmed by genetic
sequencing.
Danell & Camacho (1997) [5] produced the first cultivated
Cantharellus cibarius in a potted 16-month-old Pinus
sylvestris with mycelium grown for only one year in culture.
Chanterelle mushrooms may serve as an important source for
natural pigments since fungi can be grown in higher yields
using biotechnological techniques, compared with higher
plants whose growth rates are slow. Different species of
Cantharellus produce carotenoid pigments, which produce
yellow, orange and red colors. The natural pigment of orange
yellow chanterelle mushroom is canthaxanthin. Major
applications of this pigment are in the cosmetic and food
industries including dairy products, confectionary, fish meat
and poultry products, beer and wine [6].
Chanterelle mushrooms also produce some enzymes such as
polyphenol oxidases [7], superoxide dismutase (SOD),
catalase (CAT) and peroxidase (POX) [8].
Due to high nutritional and gourmet values, wide applications
in food and cosmetic industries, as well as production of
several biologically important metabolites, the demand for
chanterelle mushrooms is on the rise.
Based on the background provided, this study aims to
demonstrate the role of endogenous bacteria associated with
Endogenous Starter Bacteria Associated to
Chanterelle mycelia Enhance Aroma, color and
growth of mycelia
Neila Saidi, Shweta Deshaware, Ilef Ben Romdhane, Matab Nadim, Marwa Laaribi, Abdelkader
Ltifi, Robert Kremer, Salem Shamekh
Endogenous Starter Bacteria Associated to Chanterelle mycelia Enhance Aroma, color and growth of mycelia
59 www.ijeas.org
chanterelle in color production and growth of the Chanterelle
mycelia. In addition, the study tested the ability of isolated
strains for enzyme production.
II. MATERIAL AND METHODS
A. Fragment of Chanterelle body inoculation in solid
medium
Chanterelle fruits were harvested from Juva forest located
close to Juva Truffle Center, Finland. They were brushed and
washed several times with tap water. One piece of 0.5 x 0.5
cm was cut under sterile conditions and placed on the surface
of four different solid agar media.The following agar media
were used: Potato Dextrose Agar (Potato extract, 4 g; Glucose,
20 g; Agar, 20 g; Distilled water, 1 L), Yeast extract Agar
(Yeast extract, 20 g; Sucrose, 150 g; Agar, 20 g; Distilled
water, 1 L), Malt Extract Agar (Malt extract, 20 g; Peptone, 1
g; Glucose, 20 g; Agar, 20 g; Distilled water, 1 L), and
Nutrient Agar.
B. Mycelia and associated bacteria observation
Three observation methods were performed to investigate the
existing of other microorganisms than Chanterelle mycelia
growing on the agar media. The first method was based on
visual observation of bacterial colonies developing around
the chanterelle mycelia. The second observation method was
performed by placing the agar cultures containing the
chanterelle mycelia under the microscope to check the
existence of bacterial colonies around the mycelia. The third
observation technique was based on gram staining method.
After microscopic observation, Actinomycete cultures were
inoculated in Actinomycete Isolation Agar: sulfate, 0.001 g/L,
magnesium sulfate, 0.1 g/L. Serratia spp. were identified
using API 20E; and Staphylococcus spp. were confirmed
using API Staph.
C. Chemical treatment of Chanterelle fragments to destroy
associated bacteria
Different compounds were used such as sodium nitrate,
potassium phosphate monobasic, ammonium nitrate, citric
acid, acetic acid and boric acid (0, 05 g/ ml) as chemical
treatments in order to destroy the existing bacteria. Other
treatments with 1% NaOH, 1% KOH and 0.5% HCl were
performed. The treatment protocol involved immersing 10
chanterelle fragments (0.5 x 0.5 cm) in a 9 ml assay tube
containing 1 ml of the respective chemical for 2 hours at room
temperature. The fragments were then washed several times
with sterilized water followed by re-streaking on PDA,
YEAST, and NA medium.
D. Detection of mycelial growth in absence of bacteria
The treatment showing inhibition of all bacteria around the
mycelia was considered for artificial inoculation by arbitrarily
choosing strain 4 (red in color), after which mycelia were
grown by incubation for 1 month.
E. Enzymatic assays
The qualitative investigation of enzymes was carried out
according to methods based on [9, 10]. A 5-microliter volume
of bacterial suspension was spotted in the centre of agar plates
containing substrates for specific enzymes dissolved in
growth media. After 2-3 days of incubation at room
temperature, the plates were flooded with the suitable
indicator. Formation of clear zones or zones with different
colors around the bacteria colony indicated the presence of
the enzyme activities.
Amylase
Amylase activity was assessed by culturing bacterial
suspensions on TSA (MPA, Sigma-Aldrich, Germany)
medium supplemented with 0.2% soluble potato starch
(Sigma-Aldrich, Germany) at pH 6. After incubation for 2
days, the plates were flooded with 1% iodine in 2% potassium
iodide solution. Formation of a clear zone surrounding the
colony was considered as positive result for amylase
production.
Protease
For proteolytic activity, TSA medium amended with 0.4%
gelatine was used for growing corresponding bacteria.
Degradation of gelatine was seen as clear zone around the
colonies. Plates when flooded with aqueous ammonium
sulphate resulted in the formation of a precipitate making the
agar opaque and enhancing the clear zone.
Lipase
For lipase activity measurement, the bacteria were grown on
Peptone Agar Medium (Peptone 10g, NaCl 5g, Agar 16g,
Distilled water added to make 1L) supplemented with 1% w/v
tween 20 (separately sterilized). Positive lipase activity was
indicated by the formation of a visible precipitate of calcium
salts of lauric acid.
Peroxidase
For peroxidase activity, TSA Medium was used to culture
fungal isolates. After 4 days of incubation the presence of
peroxidase was evaluated by flooding the plates with a freshly
prepared mixture of 0.4 % H2O2 and 1 % pyrogallol
dissolved in water. Plates were checked 3 and 24 hours after
applying the indicator agent. The formation of a dark yellow
brown color around the mycelium indicated peroxidase
activity.
Laccase
Laccase activity was determined by culturing fungal isolates
on TSA medium amended with 0.05g/L 1-napthol. As the
mycelia grows, the colorless medium changes to blue due to
oxidation of 1-naphthol by laccase enzyme.
Catalase
For catalase activity, TSA medium was used for bacteria and
Potato Dextrose Agar used for fungi essay. After 4 days of
incubation the presence of catalase was evaluated by flooding
the plates with a freshly prepared 0.4 % H2O2. The presence
of bubbles indicated the presence of catalase activity.
Data Presentation
Mycelial and bacterial growth associated with the chemical
treatments were scored for presence or absence of growth in
culture and detection of aromas were noted and recorded.
Reactions and aroma production in enzyme assays were
scored on a scale from (-) to (++) with (-) indicating absence
and (++) indicating excessive production.
International Journal of Engineering and Applied Sciences (IJEAS)
ISSN: 2394-3661, Volume-3, Issue-9, September 2016
60 www.ijeas.org
III. RESULTS
A. Effect of medium agar composition on the color of
Chanterelle mycelia incubated in Petri dishes
Chanterelle fragments incubated at room temperature in PDA,
YEA and NA, showed a change in mycelia color related to the
change of culture media used (Figure 1). This change in color
may differ according to the biochemical pathway used by the
mycelia for nutrient consumption. For example, YEA medium
has high content in nitrogen and in this medium the mycelia
appeared brown. It is important to mention that the brown
color was only found in YEA. However, Chanterelle
fragments incubated in PDA showed the development of two
colors (white and taupe). When incubated in Malt extract
agar, chanterelle mycelia developed two colors: orange or
pink. This change in mycelia color compared across different
growth media may result from differences in specific nutrients
available for bacterial development and secondary
metabolites produced relative to the specific culture medium.
This is the first study reporting results on the description
Chanterelle mycelia color relative to associated bacteria in
different culture media.
Danell (2001) [11] found that the initial white color of
chanterelle mycelium changed to yellow when growing in the
dark on MFM (Modified Fries Medium), although carotenoid
concentrations within the mycelium varied from time to time,
depending on carbon dioxide levels and incubation
temperature. However, different aspects of some fungus
species related to their medium culture were assumed. In fact,
the studies of Zain et al. (2009) [12] showed that Aspergillus
terreus, Penicillium janthinellum and Penicillium duclauxii
cultivated on different growth media including yeast extract,
malt extract, yeast-malt extract, and potato dextrose,
demonstrated that the growth and the secondary metabolites
of these three fungal strains were greatly affected by the
culture medium types.
These authors showed that the color of the culture and
secondary metabolites were noticeably altered and changed
according to the growth medium used. Alam et al. (2001) [13]
used different types of media for the study of mycelia
pigments and pycnidia of the fungus Botryodiplodia
theobromae Pat, and revealed that pigmentation of the fungus
increased with the increase in glucose, however growth rate
gradually decreased.
Figure 1. Phenotypic aspect of fragments of Chanterelle after
growing in different culture media A, B, C: PDA medium; D,
E, F: Nutrient broth; G and H: Malt extract agar; I: yeast
extract agar.
In this study, almost similar cultural characteristics were
observed in all the media with slight variation in PCM; the
color of the mycelia was white to light grey at the beginning
and became darker with the age; maximum pigmentation was
observed in PDA (Potato Dextrose Agar) (Black 75% of the
total mycelia surface) and moderate (15-25%) in PSA (Potato
Sucrose Agar). Additionally, pink color development was low
(5-10%) in Richard’s agar and Czapek’s agar. According to
Shresta et al. (2006) [14] , various degrees of colony
pigmentation were produced by incubating all isolates of
Cordyceps militaris in all media exposed to light, except for
poor or nutrient-deficient media ( water agar :WA), which
produced no pigmentation.
Results of this study revealed that nutrition source was the
main factor responsible for the degree of pigmentation in C.
militaris under incubation in light.
However, the exact nutrient factor responsible for the
induction of pigmentation in C. militaris was not known,
although observations showed that peptone and yeast extract
were the main components in inducing pigmentation. Media
without peptone or yeast extract or other organic nitrogen
sources produced lighter pigmentations than the media
prepared with standard concentrations of these substances.
The specific role of yeast extract on pigmentation of C.
militaris was evaluated by observing the difference in
pigmentation between CDA (Czapek-Dox agar) and CZYA
(Czapek Yeast Extract agar). Besides, a single amino acid,
DL-asparagine, was found to be as efficient as peptone or
yeast extract for inducing pigmentation, as revealed by
production of similar types of pigmentations on MM
(Schizophyllum (Mushroom) Genetics Minimal Medium) and
MCM (Schizophyllum (Mushroom) Genetics Complete
Medium plus Yeast Extract). The authors suggested that
incubation period was also an important factor for the
development of pigmentation in C. militaris under light
incubation. In most of the media, pigmentation was most
pronounced after three weeks of incubation when compared
to early periods, but tended to fade after four weeks in some
media. Deep pigmentation during late incubation periods of
C. militaris could be due to different phases of photo-induced
carotenogenesis such as light reaction, protein synthesis and
accumulation of carotenoid pigments [15].
Friederichsen & Engel (1958) [16] reported carotenoid as the
compound, which produces orange color of C. militaris.
Boonyapranai et al. (2008) [17] reported that PDA is one of
the most commonly used culture media, because of its simple
formulation and its ability to support mycelial growth and
pigment production for a wide range of fungi.
Maheshwari et al. (1999) [18] stated that PDA and PDB could
be the best culture media for mycelial growth and pigment
production. While UKNCC (1998) [19] suggested that most
fungi thrive on PDA, but this can be too rich in nutrients, thus
encouraging the mycelia growth with ultimate loss of
sporulation. Tseng et al. (2000) [20], investigating the growth
and pigment production of the edible mushroom Monascus
purpureus, suggested that pigment production and mycelia
growth ran in parallel in all cultures.
Endogenous Starter Bacteria Associated to Chanterelle mycelia Enhance Aroma, color and growth of mycelia
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B. Presence of bacteria around Chanterelle
After plating the Chanterelle fragment at the surface of the
three considered Agar medium (PDA, YEA and NA) , and
after 2 days of incubation at room temperature, approximately
25°C, we noticed that some bacteria grow around the
Chanterelle fragment at the center of the Petri dishes (Figure
2).
Figure 2. Photo illustrating the presence of bacteria around a
fragment of Chanterelle located in the centre of Petri dishes,
and the presence of mycelia only from the center to the
periphery of the Petri dishes.
AB: Associated bacteria, CF: Chantarelle Fragment, CM:
Chantarelle mycila
When the incubation time was extended to one month, it was
noticed that the bacteria preserved its place in the center of the
Petri dishes. However, the mycelia continued growing alone
to reach the peripheral agar surface without the associated
bacteria (Figure 2).
After two nights incubation at room temperature,
approximately 25°C, every bacteria isolated from the area
around the mycelia revealed the same color that occurred in
the mycelia (Figure 3).
Figure 3. Photo of bacteria associated with the Chanterelle
mycelia and as purified isolates.
Each photo A, B, C, D, E and F present two Petri dishes the
left one or the right one shows the inoculated plates with the
fragment of the mycelia, the second Petri dish presents the
purified corresponding bacteria.
A: Strain 1, B: Strain 2, C: Strain3, D: Strain 4, E: Strain 5, F:
Strain 6
This experiment showed clearly that when the fragment of
Chanterelle was plated at the surface of the Petri dishes, some
endogenous bacteria associated to Chanterelle may grow in
symbiosis with the mycelia (Figure 4).
Different aspects of bacteria associated to Chanterelle
mycelia were observed by microscopy (Figure 5).
bacteria were incorporated in fungal tissues during primordial
formation, thus growing actively between the cells without
harming the mushroom. In addition, Rangel- Castro (2001)
[22] showed that amino acids, organic acids, and sugars
released by chanterelles serve as nutrient source for the
bacteria. Bacterial contamination presents a potential
problem that has plagued the culturing of chanterelles [23,
24].
Pilz et al. (2003)[21] showed that Chanterelle tissue may
serve as nutrient media more favorable for bacteria
originating from the chanterelles than fungal hyphae, thus
precluding subsequent isolation of uncontaminated
chanterelle hyphae. Recently, Dutch scientists used an
antibacterial nutrient media formulation [25] to grow and
isolate pure chanterelle mycelium from chanterelle tissues
[26, 27].
Figure 4. Depiction of bacteria around mycelia, Microscopic
observations conducted in Petri dishes including agar
medium.
A : Strain 3,B: Strain 2, C: Strain 4, D: Strain 1
International Journal of Engineering and Applied Sciences (IJEAS)
ISSN: 2394-3661, Volume-3, Issue-9, September 2016
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Figure 5. Depiction of bacteria developed around the
mycelia, (Microscopic observation conducted in Petri dishes
including agar medium).
In fact, the bacteria first appeared around the mycelia was
isolated and purified. In total, six bacterial isolates were
obtained, noted as S1, S2, S3, S4, S5 and S6.
Danell et al. (1993) [3] found that the presence of bacteria
and other microorganisms within the sporocarp tissues is one
reason chanterelles have been so difficult to cultivate.
Bacteria are present in millions per gram of fresh weight).
Pilz et al. (2003) [21] suggested that these associated
C. Bacteria Gram staining and identification
Four Bacteria S1, S2, S3 and S5 were Gram positive.
According to the Gram reaction, it seems that these bacteria
belong to the Actinomyces group (Figure 6). However, two
other bacteria S4 and S6 were Gram negative. These two
bacteria, when streaked in Asparagine and Acetamid
mediums, produced a fluorescent color therefore they belong
to the fluorescent Pseudomonads. Strain S4 produced red
pigment and was identified as Serratia maresens using API
20 E and the strain S6 was identified as Staphylococcus using
API Staph.
Figure 6. Gram staining of bacterial strains 1, 4, 5 and 6
objectives, Scale 1 cm line 25 µm
Danell et al. (1993) [3] isolated and identified aerobic
bacteria from C. cibarius (fruit body) FB and found that most
of them belong to the fluorescent Pseudomonas group.
Bacillus spp., Xanthomonas spp., and Streptomyces spp. were
also found, though in significantly lower amounts, they also
observed that the proportion of fluorescent Pseudomonas in
soil samples of FB growing sites was only 12%, while inside
the FB these bacteria represented an average of 78% of the
total culturable community. Utilization of mannitol and
trehalose, which are carbohydrates exuded by C. cibarius
mycelia, was a common trait among Pseudomonas spp. from
all environments studied.
Trehalose degradation is not a common trait among
bacteria, and exhibited by only a limited number of
fluorescent Pseudomonas spp [28, 29].
A helper effect of the bacteria on fungal growth was
examined by Danell (1994) [30] in C. cibarius but no helper
effect was found. Danell et al. (1993) [3] suggested that the
fungus probably produced trehalose, and this was involved in
the selection of bacteria associated with the fruiting body. It
has been observed that bacteria located in different
environments have different capacities to utilize diverse
substrates [31]. Rangel-Castro et al. (2001a) [32] found that
mannitol and trehalose are exuded by C. cibarius mycelia in
vitro cultures. This may support the suggestion that bacteria
growing inside FB have a greater capacity to utilize these
exudates than bacteria from other environments. However,
the association between C. cibarius and bacteria is not well
understood (Figure 6).
D. Chemical treatment of Chanterelle fragments
The Chanterelle fragment treated with different chemical
reagents did not show the same microbial biomass behavior
(Table 1).
In fact, the treatments by sodium nitrate, potassium
hydroxide, ammonium nitrate suppressed the mycelia’s
growth, and allow bacteria to grow (Figure 7).
In addition, some bad odors were detected following these
treatments. In PDA, only black fungi possessing prompt
radial grew and filled all the Petri dishes. Additionally, after
boric acid, acid acetic and potassium hydroxide treatments, it
was noticed that only fungi development enabled growth of
both the Chanterelle mycelia and the endogenous bacteria.
Figure 7. Example of treatments showing bacterial or fungal
development with bacteria enabling the development of
Chanterelle mycelia.
Only the treatment of the Chanterelle fragments with
hydrochloric and acetic acids and sodium hydroxide
prevented the development of both bacteria and fungi. These
plates were inoculated with the red bacteria strain 4 (Figure
8).
Endogenous Starter Bacteria Associated to Chanterelle mycelia Enhance Aroma, color and growth of mycelia
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Incubation of mycelia without bacteria at room temperature,
revealed no development of the Chanterelle mycelia growth
(Figure 9A). However, after one week incubation at room
temperature, approximately at 25°C, the inhibited mycelia
associated with strain 4 showed regrowth (Figure 9B). The
growth kinetics of the mycelia when associated with strain4
was enhanced over time after two and three weeks (Figure
9C 9D, respectively).
Effective use of hydrogen peroxide as a chemical sterilant in
mushroom production and selection of cultivable mushroom
strains for tropical conditions was tested on Pleurotus
mycelium [33]. when eight strains were cultured aseptically
on agar at six hydrogen peroxide concentrations (00.1%,
v/v) at 27 °C and another fast growing strain P. sajor-caju
strain 1, was cultured non-aseptically at six hydrogen
peroxide concentrations (00.1%, v/v) at 27 °C. Results
showed that the mycelial growth rate in all strains increased
when hydrogen peroxide was increased from 0 to 0.001%
(v/v), and then decreased with further increments in hydrogen
peroxide concentration. The hydrogen peroxide
concentration associated with 50% reduction in maximum
mycelial growth rate due to toxicity (EC50) ranged from
0.009 to 0.045% (v/v); in non-aseptic culture of P. sajor-caju
strain 1, bacterial growth was observed at concentrations
≤0.016%, whilst the upper hydrogen peroxide concentration
limit for fungal growth was 0.025% (v/v).
The highest hydrogen peroxide concentrations 0.016% (v/v)
and 0.025% (v/v) in which bacteria and fungi, respectively,
were observed to grow were within the concentration range
0.0090.028% (v/v).
Wong & Preece (1985) [34] revealed the effect of
N-cetylpyridinium chloride, benzalkonium chloride,
Cetrimide, bronopol (2-bromo-2-nitropropane-1,3-diol),
Panacide and Chloramine T tested as possible disinfectants
for use in growing mushrooms (Agaricus bisporus) where
Pseudomonas tolaasii blotch is prevalent. The most effective
materials in vitro against P. tolaasii were quaternary
ammonium compounds and bronopol.
In 8 min ‘clean’ and ‘dirty’ tests incorporating yeast cells
showed that only bronopol failed to kill the pathogen. If
mushroom casing (peat plus limestone) was added to these
short duration tests the pathogen survived all six disinfectants.
When tests with added casing were extended to 20 h,
bronopol was very effective (biocidal value 100 µg/ml) and
the pathogen was not killed by the other five disinfectants. In
experiments on agar plates, bronopol and chloramine T were
stimulating to the growth of A. bisporus. Growing mushroom
caps treated with bronopol remained white, whereas caps
treated with the other five disinfectants turned brown within
30 min. It is thus likely that bronopol could be used to control
the source of bacterial blotch epidemics in mushroom
growing.
(Tseng el al., 2000)[20] noted the effect of different levels of
sodium chloride, sodium nitrite, polyphosphate on growth
and pigment production of the edible mushroom Monascus
purpureus.
The addition of sodium chloride (>50•0 g/l) and
polyphosphate (>3•0 g/l) to broth medium decreased mycelial
growth andpigment production, whereas low concentrations
of sodium nitrite (<0•2 g/l) promoted mycelial growth and
pigment production. The fungus showed more tolerance to
salt and polyphosphate in ground meat than in broth medium
and used sucrose as a carbon source as well as glucose for
growth and pigment production.
Acetic acid and sodium hydroxide added to cultures on PDA,
MYE and NA inhibited Chanterelle mycelial growth in the
absence of associated bacteria may serve for further studies
considering effects of artificial contamination by bacteria.
Figure 8. Photo presenting a collection of Chanterelle ycelia
treated by hydrogen chloride.
E. Bacterial enzymes
Results gathered in Table 2 show that bacteria isolated from
Chanterelle were also able to produce perfume, lecithinase,
amylase and laccase. However, these bacteria were unable to
produce oxidase, catalase and protease.
According to Toljander et al., (2007) [35] the mycelial
exudates not only increased bacterial growth and vitality but
also influenced the bacterial community composition. This
suggested that some bacteria preferentially utilized different
compounds available in exudates. To capture and utilize
resources from the fungal partner, particular enzyme
complexes may be necessary for the fungal-associated
bacteria [36]. Thus, the efficient use of such enzyme systems
to obtain essential energy and carbon sources from the fungal
partner emerges as a key mechanism involved in the bacterial
interaction with soil fungi.
Figure 9. Example of treated Chanterelle fragment with
chemical substances in order to suppress all microorganisms’
development which have received artificial contaminant
bacteria (strain 4).
International Journal of Engineering and Applied Sciences (IJEAS)
ISSN: 2394-3661, Volume-3, Issue-9, September 2016
64 www.ijeas.org
(A): mycelia which did not receive the bacteria serving as a
control (B): After one week of growing at room temperature
approximately at 25°C. (C): After two weeks. (D): After three
weeks.
It has been demonstrated by Rangel-Castro et al. (2001b) [29]
that C. cibarius has a limited capacity to utilize organic N. To
compensate for this, the fungus may access recalcitrant
sources of N by taking advantage of the bacterial enzymatic
capacity. However, the ability of bacteria to secrete lytic
enzymes, such as proteases, might be very important for the
vegetative soil mycelium.
IV. CONCLUSION
This study is the first one showing the contribution of
endogenous bacteria of chanterelle in Chanterelle mycelial
growth and pigmentation. Is it possible that the orange color
of the Chanterelle is related to soil culture? Furthermore,
under hydroponic conditions and considering different
substrates, is it possible that these different colored bacteria
have the ability to yield different Chanterelle fruits? To
answer these questions, more in-depth studies must be
considered. In addition, the present study provides six strains
of bacteria, which are able to produce color, perfume and
enzymes suggesting possible exploitation in further
biotechnology studies.
Table 1: Effects of Chemical reagents in fragmented body fruits of Chanterelle and behavior of associated mycelia and bacteria.
Hydrogen Acetic Citric Boric Sodium Sodium Phosphate Hydroxyde Nitrate
chloride acid acid acid Nitrate Hydroxyde Potassium Potassium Ammonium
Chemical Formula HCl C2H4O2 C6H8O7 H3BO3 NaNO3 NaOH K3PO4 KOH (NH4)(NO3)
PDA My - Bact - My - Bact - Fungi + Fungi +
My - bact + Bad odor My - Ba ct - Fungi + My - bact + Bad odor My - ba ct + Bad odor
YEA My - Bact - My - Bact - Fungi + Fungi +
My - bact + Bad odor My - Ba ct - Fungi + My - bact + Bad odor My - ba ct + Bad odor
NA My - Bact - My - Bact - Fungi + Fungi +
My - bact + Bad odor My - Ba ct - Fungi + My - bact + Bad odor My - ba ct + Bad odor
Different treatments were applied (Sigma) (Sodium nitrate,
Potassium phosphate monobasic, Ammonium nitrate, Citric
acid, Acetic acid, Boric acid) (0.05 g/ml). Also, other
treatments with 1% NaOH, 1% KOH and 0.5%.HCl were
considered. The experiment consisted on putting 10
fragments chanterelle bodies having 0.5 x 0.5 cm in size in 1
ml of each treatment in 9 ml liquid essay tube. After storage
during 2 hours at room temperature, several washings with
sterilized water were performed and fragments were
restreaked in PDA, YEAST, NA. My-: inability of
Chanterelle Mycelia to grow. Bact-: inability of associated
bacteria to grow, Bact +; ability of associated bacteria to
grow. Fungi+: The specific treatments enhance the
development of some fungi. Bad odor: The associated
treatment induce bad odor.
Table 2: Ability of selected strains for enzyme and perfume production in three different media (PDA, YEA, NA)
Perfume
Lecithinase
Amylase
Laccase
Oxydase
Catalase
Protease
PDA
YEA
NA
+++
++
-
++
++
++
-
-
-
+++
++
-
++
++
++
-
-
-
+++
+
-
++
++
++
-
-
-
++
+
-
+
+
+
-
-
-
++
+
-
+
+
+
-
-
-
++
+
-
+
+
+
-
-
-
For perfume: +++: Refers to high production; ++: average production; +: very low level smelling; -: Absence of smell perfume
[no quantifying test was performed but the detection of the smell is only the method adopted in the present study in order to
verify whether the bacteria is producer or not of perfume].
For Enzymes production: +: Refers to the presence of the activity; −: Refers to absence of the activity; ++: Refers to an
excessive presence of the activity.
ACKNOWLEDGMENT
The experimental study was conducted at Juva Truffle
Center Finland. The financial support of Regional Council of
Southern Savo Finland and Ministry of Higher Education and
Scientific Research of Tunisia are appreciated. Authors are
grateful to Mr. Antti kinnunen for administrative service and
Mrs. Heli Valtonen for her technical assistance.
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Neila Saidi, I am involved in Centre of Research and water
technologies CERTE since 1984 Actually I am Lecturer in Microbiology
applied to Soil ad Water,
I have more than 50 articles published in international journal indexed
impacted factor. Key Skills Biofilm and bacteria virulence, phage use in
waste water treatment, antagonisms and substance bioactive (to limit some
plant disease), composting and soil amendment, UV disinfection, bacteria
exploitation in biotechnology colorant production, plastic, perfume
production, bioremediation from heavy metals, decolouration, waste
watertreatment by ecological method (Wetlands) and Biosurfactant.
Second Author Shweta Rakesh Deshaware, senior
research fellow, pursuing Ph.D (Food Biotechnology) from Institute of
Chemical Technology, Mumbai. My research area includes, polymorphism
in bitter taste receptors, studying gene diet interaction and looking for
approaches for debittering of foods.
Ilef Ben Romane, engineer in agronomy and a senior
research fellow, pursuing PhD (Microbiology) from the Faculty of sciences
of Tunis and the Centre of Research and water technologies CERTE
(Tunisia). My research topic is ‘Truffles Biotechnologies’ including
Biology, Microbiology and Genetics of Truffles.
ResearchGate has not been able to resolve any citations for this publication.
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