Advances in Microbiology, 2015, 5, 425-432
Published Online June 2015 in SciRes. http://www.scirp.org/journal/aim
http://dx.doi.org/10.4236/aim.2015.56043
How to cite this paper: Ben Romdhane, I., Saidi, N., Deshaware, S. and Shamekh, S. (2015) Sanitizing Method Effects on
Depending-Culture Microorganisms in Tuber aestivium. Advances in Microbiology, 5, 425-432.
http://dx.doi.org/10.4236/aim.2015.56043
Sanitizing Method Effects on
Depending-Culture Microorganisms in Tuber
aestivium
Ilef Ben Romdhane1, Neila Saidi1*, Shweta Deshaware2, Salem Shamekh3,4
1Laboratory of Treatment and Water Recycling Centre of Research and Water Technologies (CERTE)
Technopark of Borj-Cedria, Soliman, Tunisia
2Food Engineering and Technology Department, Institute of Chemical Technology, Mumbai, India
3Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University,
Aalto, Finland
4Juva Truffle Center, Ju va, Finland
Received 15 May 2015; accep t ed 15 June 2015; published 19 June 2015
Copyright © 2015 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract
Tuber aestivum/uncinatum has been widely used as food, food additives, and traditional medicine.
Truffles are extremely perishable with a short postharvest life quality requiring a special handling
for marketing in order to delay its deterioration. This study aimed to assess the effects of different
sanitizing methods on superficial Tuber aestivium quality ascocarp. The results showed that the
best treatment was obtained by immersing the Truffle ascocarps in boiling water for 1 or 2 min
where counts of total mesophilic microoraganisms (TMM) were respectively 81 and 7 CFU per g of
dry Truffle ascocarps biomass, respectively. However, the highest TMM was obtained after rinsing
Truffle ascocarps in 2% NaOH where recovery was 108 CFU per g of dry Truffle ascocarps biomass.
Treatments applied to disinfect Truffle ascocarps were classified by increasing degree of efficien-
cy as follows to reduce the microbial load expressed in CFU/g: Dipping in boiling water (2 min) 7 ±
3.41; dipping in boiling water (1 min) 81 ± 25.8, rinsing with alcohol 2.102 ± 13; rinsing with tap
water 6.103 ± 36; rinsing with H2O2 6.104 ± 2; brushing 2.105 ± 28 and rinsing with NaOH 108 ± 15.
Keywords
Tuber aestivium Ascocarp s, Total Count Mesophilic Bacteria, Molds and Yeast Decontamination
*
Corresponding author.

I. Ben Romdhane et al.
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1. Introduction
Truffles are the most highly edible valued mycorrhizae fungi in gastronomic and economic terms. Some species
of T r uffl e s uc h a s Tuber magnatum, Tuber melanosporum and Tuber aestivum/uncinatum are highly praised due
to their aroma and organoleptic propreties [1]. Truffles are extremely perishable with a short postharvest shelf
life which req uires specia l han dling for mar ketin g in or der to dela y its deterior ation. T he d eterio ratio n of T ruffle
organolep tic quality is caused by the mold mycelia growth on the surface of Truffle and loss of water, whic h af-
fects their visual appearance and flavours. Tirmenia and Te rfezia species are usually eaten cooked whereas Tu-
ber species are sometimes eaten uncooked.
Several studies reported high microbial loads in the range of 105 cfu/g t o 108 cfu/g i n the gle ba of Tuber bor-
chii, Tuber magnatum, T. melanosporum and T. aestivum species [2]-[4]. High microbial loads and the presence
of potential pathogens such as Listeria and Salmonella species make Truffle spoilage easier [5]. It is essential
for develop ing disinfection techniques to ex te nd Truffle storage and to i mprove sanita ry q uality.
Early decontamination treatment can reduce the initial microbial loads thus increases the storage period. Sev-
eral techniques have been used to reduce microorganisms on whole and cut fresh fruit, and their effectiveness
depends on the product type, mitigation protocol, and other parameters. Contamination prevention seems to be
the best way to eliminate pathogens from products even this is not always possible. The necessity of washing
and sanitizing remains of paramount importance to prevent product spoiling outbreaks. It is well known that
washing and sanitizing methods can’t totally eliminate pathogens. Therefore, sanitizing methods are usually
used to reduce the total flora existing. Some berries are sensitive to washing due to their susceptibility to mold
proliferation. So s imila r fruit are often package d in t he field wit h a light post har vest handling and wa shing.
The increase in demand for T. aestivium leads food technologists to develop appropriate methods for its long
and short term storage in order to retain its quality and reduce the threat of scarcity. After harvesting, T. aesti-
vium is ra pidly altered at room temperature and at 4˚C due to na tural flor a existing in the Tr uffle ascocarps and
to water content (76%). Moreover, Tr uffle ascocarps are sub terranean fruit bodies growing underground, and it
is well known that soil serves as a natural habitat for various microbial communities growing along with asco-
carps [6]. It has been shown that bacteria can promote the establishment of ectomycorrhizal symbiosis [7]-[9].
Several researchers analyzed the microbial load in different Truffle species. These microbial communities
step in growing, physiology and preservation of Truffle ascocarps [10] [11]. The microbial consortium could
have an additive or antagonistic effect on Truffle preservation.
Different decontamination methods such as ionizing radiation, ultrasound, electron beam radiation, gamma
rays and refrigeration at 4˚C have been applied to minimize the microbial load of fresh Truffle [11] [12]. How-
ever, storage at a lower temperature is not an absolutely reliable method since many molds are able to grow at
low temperatures. High concentration of chlorine (500 ppm) has been tried for decontamination but with unsa-
tisfactory results due to high content o f organic matter in Truf fl e and production of potential c arcinogens such as
trehalo-methanes [13]. Chemical fungicides have also been applied for preventing fungal decay; however, con-
sumers demand minimum use of chemical preservatives. As a biological control, Lactic acid bacteria (LAB)
have been reported to produce organic acids, bacteriocins, hydrogen peroxide, and low molecular weight anti-
microbial agents which act as an anti-fungal agent [14]. Sorrentino et al. (2013) [15] reported that strains of
Lactobacillus plantarum can be successful ly used to preve nt the growth of molds belongin g to penicill ium genus
during storage of black Truffle at refrigeration temperature.
The goal of this study is to find an appropriate decontamination method that will reduce the microbial load of
fresh Truffle in order to increase its shelf-life .
2. Material and Methods
2.1. T. aestivum Ascocarps Samples
The ascocarps of T. aestivum were collected in the experimental Truffle field of Juva Truffle Center (JT C), J uva,
Finland, with the help of hunter Truffle dogs. The field soil was sandy with pH 7.2. The main host trees of T.
aestivum were Quercus peduncularis, Quercus robur L and Corylus avellana L. To avoid cross conta mination,
the Truffle ascocarps were individually wrapped in pla stic bags a nd transpo rted to the laborato ry in an insulated
boxes for microbial analysis. The nu mber of Truffle ascocarps used for this study was about 11 weighing 20 - 25
grams.

I. Ben Romdhane et al.
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2.2. Soil Sample
The adhered soil to the Truffle ascoscarps is considered as a separate sample to enumerate the natural associated
flora and it was obtained by ascocarps brushing under laminar flow.
2.3. Quantitative Microbiological Analysis
Eight different decontamination treatments were applied to Truffle ascocarps: Brushing pending wholly extrac-
tion of adhered surface ascocarp s soil, r insing with municip al d rinki ng wate r (prelimina ry ana lysis sho wed tha t
the tap water is exempt of bacteria and was used this water to be near the habits of Finish people when they
clean the T ruffle b y brush or by water), rinsing with ethanol [70%], dipping in boiled water during one or two
min, rinsi ng with NaOH [2% ] and rinsi ng with H2O2 [30%].
2.4. Truffle Ascocarp and Extract Preparation
Bacteria colonize both the external (peridium) and internal part (gleba) of Truffle [16]. For this, we not only
considered the surface ascocarps bacterial extraction but also the total ascocarp body. Briefly, in a laminar hood
and under sterile conditions, each piece of Truffle ascocarp was treated and then cut into 2 - 4-mm length pieces.
Te n g treated T ruffle sampl es wit h moisture content o f 76% (dry weight) was suspende d in 90 ml of sterile sa-
line solution [0.9% w/v NaCl]. The mixture was submitted to a mechanical agitation for 1 hour at 240 rpm to
allow bacteria extraction [17].
2.5. Enumeration of Bacteria
Enumeration of viable aerobic heterotrophic bacteria was performed by standard plate count procedures, in Plate
Count Agar (PCA), Malt Extract Agar (MEA) and Yeast Extract Agar (YEA) (Difco, Germany). Plates were
incubated in Biolog incubator at 25˚C, and the number of Colony Forming Units (CFU) were determined after
48 h and the average number of microorganisms per gram of each sample was then calculated and reported as g
of dried Truffle ascocarp or soil. Plate Count Agar medium were adjusted to a final pH = 7 ± 0.2. This medium
is specific for total count bacteria. Malt Extract Agar medium Sigma-Aldrich 70145 appropriate for molds and
yeast present in gram per liter of purified water Malt extract 30.0; Mycological peptone 5.0 Agar 15.0 fina l pH =
3.5 +/− 0.2 was adjusted by 10% of citric acid instead of 5% of tartric acid.
The typical for mula of Yeast Extract Agar medium Oxoid B O0635M per liter of distilled water included 3 g
of Yeast Extract, 5 g of peptone and 15 g of Agar, final pH ad j usted to 7 ± 0.4. This medium is suitable for both
P. aeruginosa and E. coli.
2.6. Statistical Analysis
Results related to each treatment were repeated three times and analyzed by the SPSS statistical program (SPSS
for Windows, SPSS Inc.). Values mentioned in Table 1 are the average of three replicates and means were se-
parated by the least significant difference according to the StudenNewman-Keuls Test. Pearson correlation de-
terminations were performed using SPSS statistical analysis.
3. Results and Discussion
3.1. Microbial Enumeration in T. aestivium Asc ocarps
The results showed that the more effective treatment was obtained when Truffle ascocarps were immersed in
boiling water for 1 or 2 min with a total mesophilic microorganisms (TMM) count respectively 81 and 7 CFU
per g of dry Truffle ascocarps biomass. However, the highest TMM was obtained after rinsing Truffle ascocarps
in 2% NaOH where the number saved was 108 CFU per g of dry Truffle ascocarps biomass. Applied treatments
were classified with degree efficiency expressed in CFU/g: Dipping in boiled water (2 mi n) 7 ± 3.41; dipping in
boiled water (1min) 81 ± 25.8, rinsing with ethanol (70%) 2. 102 ± 13; rinsing with tap water 6.103 ± 36; rinsing
wit h H2O2 6 104 ± 20, brushing 2. 105 ± 28; rinsing with NaOH 108 ± 1 5. The same order of classification was
obtained for total cou nt bacter ia, molds-yeast and P. aeruginosa-E. coli. H oweve r , 108 of TMM number present
after NaOH and H2O2 was relatively high. In fact, freshly collected Truffles are associated with significant mi-

I. Ben Romdhane et al.
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crobial populations. Studies assessing the total microbial load of entire ascocarps obtained different results.
Nazzaro et al. , (2007) [10] detected a total microbial load in ascocarps of T. aestivium near to107 CFU/g. How-
ever, Reale et al., (2009) [11] reported only 4.0 CFU/g. These differences may be due to different procedures
used to remove ascocarps surrounding soil. The development of superficial mold growth is one of the main
problems in the postharvest phase of ascocarps affecting their vi sual quality, aroma and taste [18].
The significant difference in CFU number per gram of ascocarps between treatments generates four main
group s. T he firs t gro up incl uded boil ing trea tment s for 1 or 2 min. ri nsin g with e thano l tr eatme nt rep resent s the
second group, the third group contains rinsing with tap water and brushing; treatments with H2O2 and NaOH
constitute the last group.
Results showed in Table 1 reveal that the microbial population in T. aestivium is responsive to the type of
treatment applied. This suggests that the treatment application may induce the number of microorganisms ex-
tracted. In fact, actively growing bacteria were effectively expressed by the decontamination method. The high-
est number of micro flora was obtained after NaOH or H2O2 treatment. This resul t pre sumed that NaOH or H 2O2
induced release of microrganisms and positively affected extraction the yield of bacteria. Based on H2O2 effect,
only bacteria producing catalase may survive in presence of H2O2 [19]. The catalyzed H2O2 molecule may pro-
vide o xyge n gas to the med iu m as indicated in Figure 1. This reaction may also induce the aerobic bacteria de-
velopment. The whole and high number of bacteria 108 CFU/g per dry matter expressed following H2O2 treat-
ment may suggest the presence of mainly aerobic bacteria the catalase enzyme is absent i n nearly all anaerobic
bacteria and may thereby be suppressed by H2O2. It is well kno wn that some bacteria are ab le to produce H2O2
and could be used as a substrate for its growing. In fact, related to work of Eschenbach et al., (1989) [20],
Figure 1. Intermediates in the Univalent Pathway of Oxygen
Reduction (Archbald and Fridovichi; 1983) [34].
Table 1. Micro bial load expressed in CFU/g per dry matter of T. aestivium ascocarps in different applied treatments and in
its adhered soil.
Trea tmen ts PCA (1) MEA (2) YEA (3)
Brushing 2. 105 ± 28 (a) 9. 103 ± 71 (a) 2. 104 ± 74 (a)
Rinsing with tap water 6. 103 ± 36 (b) 8. 103 ± 11 (a) 2.103 ± 26 (b)
Rinsing with etha nol (70%) 2.102 ± 13 (c) 2.102 ± 40 (b) 5.102 ± 11 (c)
Dipping in boiled water (1 min) 81 ± 25.8 (d) 3 ± 1.8 (d) 102 ± 17 (c )
Dipping in boiled water (2 min) 7 ± 3.41 (d) 1.4 ± 0.73 (d) 1.2 ± 2 (d)
Rinsing with NaOH 108 ± 15 (e) 6. 107 ± 23 (e) 9. 107 ± 19 (e)
Rinsing with H2O2 6. 104 ± 20 (f) 108 ± 44 (f) 5. 107 ± 19 ( e)
Adhered soil 2.109 ± 16 (g) 5.106 ± 27 (g) 107 ± 10 (e)
(1) P late Count Agar , (2) Malt Extra ct Agar, (3) Ye ast Extra ct Aga r. Eth an ol [7 0 %, v/v], dipp in g in boi led water during one and two minutes, rinsing
with H2O2 [30%, v/v]. (a, b, c, d, e, f, g) means number followed by the same number were not significantly different at p = 0.05 comparison estab-
lished between lines using SPSS statistical program (SPSS for Windows, SPSS Inc.). Values mentioned are the average of three replicates.

I. Ben Romdhane et al.
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some Lactobacillus are kno wn to produc e hydroge n per oxide inducin g an incre ase in the number of Lactobacil-
lus.
Decontamination method using boiled water during one minute keeps 2Ulog of P. aeruginosa and E. coli
higher than total b acteria and fungi (molds and yeast) numb er. It is well known that E. co li and other pathogens
can be removed by a simple pasteurization. The presence of this high load of E. coli may be related to a highly
resistant natural flora of the ascocarps. In fact, during its life cycle, T. aestivium generates a radial stress zone
producing volatile substances able to affect the bacteria community around the ascocarps and likel y ind uce bac-
teria resistance to diverse stresses [21] [22].
The induced alkaline environment is another type of ascocarps stress. In fact, when ascocarps were cleaned
with 2% NaOH, total bacteria, fungi and P. aeruginosa-E. coli count increased. The large number of microor-
ganisms achieved after NaOH application can be a result of specific flora extraction with a shift of the initial pH
of 5 of Truffle ascocarps to neutral pH. Balanced pH may promote the development of some bacteria and inhibi-
tion of fungi growing [23]. Unlike, another study showed that various high pH cleaners containing sodium hy-
droxide, potassium hydroxide, sodium bicarbonate, and/or sodium orthophenylphenate (with or without surfac-
tants) reduced populations of E. coli on orange surfaces [24]. The same study revealed that high pH waxes used
on fresh market citr us pro vided substant ial inacti vatio n of E. co li on the surfaces oranges fruit [25]. The hig h p H
of typical alkaline wash solutions and concerns about environmental discharge of phosphates may be limiting
factors for use of certain alkaline compounds on produce. Furthermore, Somers et al. , (1994) [26] in a lab oratory
study of suspended and attached cells of various foodborne pathogens on non-food surfaces showed that E. coli
O157:H7 populations were reduced by 5 and 6 CFU after a 30-s decontamination method with 1% tri-sodium
phosphate. Besides, H wang and Beuchat, (1995) [27] working in chic ken skin disinfectio n revealed that 0.05%
NaOH significantl y reduce d the Salmonella spp. population but had no effect on L. monocytogenes. The d iffer-
ence in effectiveness degree to remove bacteria with alkaline solution may be the result of many parameters in-
teraction such as pH, water content in Truffle ascocarp and its own composition (hydrocarbons, proteins, fatty
acids contents).
Likewise, in the present study, ethanol may be considered as a potential disinfectant of Truffle ascocarps
since the number of total microflora decreased 3 CFU compared to brushing. It is well known that ethanol is
used i n ma ny fie lds suc h as cl inic al a nd fo od , it possess a large specter effect on bacteria discharge. Ethanol has
pleiotropic effects on bacteria, membra ne damage and inhib ition o f macro molec ular bi osynthe sis being t he ma-
jor mechanisms. However, many bacteria may escaped the ethanol effect when the load of bacteria is very im-
portant or when the bacteria is not present at surface or covered by organic matter. Zhang et al. (2009) [28] sug-
gested dipping in 80% ethanol for 10 seconds combined with mercuric chloride as the most effective surface
disinfec t ion method for inactivating Escherichia coli O157:H7 on lettuce leaves and roots.
As reported by Rivera et al., (2011b) [6], after gamma irradiation treatments, two species of yeasts survived
and became the dominant microbial populations; this selective effect of disinfectant and resistance of one group
rather other s sug gest t he ne cessi ty to c omb ine tr eatme nt in ord er to enhance deco ntamina tio n. For e xampl e, Ri-
vera et al. , (2011b) [6] showed that 70 % ethanol combined with ultrasound has a greater efficacy to reduce mi-
crobial load.
3.2. Microbial Presence in the Adhered Soil of T. aestivium
The layer of soil obtained after brushing Truffle ascocarps may be considered as the source of specific flora
present in T ruffle ascocarps. Wherefore, the adhered soil microflora of Tuber. aestivium ascocarps has been hig-
hlight ed in the present wo rk.
The number of CFU per gram of the adhered soil was respectively about 2.8 × 108 CFU/g of TMM bacteria,
4.7 × 106 CFU/g of both mold and yeast and about 1.8 × 106 CFU/g of both P. aeruginosa and E. c oli. T he high
number of microflora in the adhered soil of T. aestivium substantiates the result of Rougieux (1963) [29]. Re-
sults showed that desert truffle can excrete substances able to stimulate the development of these microorgan-
isms. The abundance of TMM bacteria, mold, yeas t, P. aeruginosa and E. co li has been lo cated in T. borchii soil
[4]. This study showed obviously that TMM bacteria were about 102 CFU/g d r y matt er hi gher t ha n fu n gi o r bo t h
E. coli and P. aeruginosa. A similar study carried out by Dib -Bellahouel and Fortas (2014) [30] showed an es-
timated total count about 8.3 to 8.6 × 10 7 CFU/g of dry soil, while fu ngal ge nera p re sent onl y 8.6 × 104 to 4.5 ×
105 CFU/g. Thus, it is possible to suggest that the number of bacteria and fungi in adhered soil is balanced by

I. Ben Romdhane et al.
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the Truffle presence, this suggestion need to be more demonstrated in other studies.
The soil microflora of Truffle ascocarps has been severally studied; most of them characterized types and ab-
undance of bacteria such as Actinomycetes, yeasts and filamentous fungi [3] [31] [32]. Although, specific bac-
terial communitie s of Tuber species were analyzed, a core micro-biome composed of α-Proteobacteria from the
family of Bradyrhizobiaceae seems to be common to all Tuber species studied [3] [4] [16].
Moreover, many studies have been conducted on bacterial diversity soil horizon containing Truffle. The ho-
rizo n 0 - 20 cm depth sho wed the highest micro bial divers ity [33]. These authors enumerated the number of mi-
croorganisms respectively in s oil adhered to Terfezia boudieri ascocarps and in 0 - 10 cm depth horizon, thus it
was demonstrated that so me microbial communities were higher in soil adjacent to Terfezia boudieri ascocarps
than the horizon of 10 cm depth.
Furt her a nal ysis need to a dvice that an ide al disin fectio n treat ment should b e si mple and easy to use; it will b e
efficient in Truffle for its sensory characteristics preservation such as, aroma, texture and taste. Rivera et al.,
(2011) [6] showed that in the last week of storage, the aroma of non-decontaminated T. aestivium samples was
modified and associated to a strange odor of fungi. This loss of typical aroma influences the overall acceptability
of the no n-decontaminated Truffle out of marketability on day 28. The typical aroma odor and quality in the de-
contaminated summer Truffle samples moderately decreased in the first week of storage.
Chemical disinfectants and chemical residues, depending on their concentrations render Truffles to be unfit
for human consumption and pose a threat for human health.
4. Conclusion
Microbial populations of Truffle were very complex and heterogeneous and were mostly mesophilic bacteria.
Dipping Truffle ascocarps in boiled water for 1 - 2 minutes reduced microbial load and seems to be an efficient
method recommended before T. aestivum storage.
Acknowledgements
This research was supported by Juva Truffle Center Finland. The financial support of Regional Council of
Sout hern Savo , Finla nd is appreciated and thanked. Autho rs are gr ateful for Mr. Antt i kinnune n for ad ministra-
tive service and Mrs. Heli Valtonen for her technical assistance. Authors are grateful to Dr. P. S ivakumar and Dr.
A. M. Ibekwe for their kind comment and improvement of the manuscript. The authors are also grateful to Pr
Kremmer R. for improving English expression and s tyle.
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