Eco. Env. & Cons. 25 (April Suppl. Issue) : 2019; pp.

(S40-S45)
Copyright@ EM International
ISSN 0917–765X

DNA barcoding for identification of commercial fresh

and processed mushroom-based products in
Surabaya
Intan Ayu Pratiwi*, M. Hilman F. Amin and Bambang Irawan

Department of Biology, Faculty of Science and Technology, Airlangga University,

Kampus C Mulyorejo, Surabaya 60115 Indonesia

(Received 2 January, 2019; accepted 28 February, 2019)

ABSTRACT.
The higher of mushroom consumption drives the effective food safety regulation especially in Indonesia.
The challenge of increasing mushroom consumed are species confirmation and identification for fresh
edible mushroom and derivative supplement. Then a fast and appropriate method is needed. One of the
best and fast method is DNA barcoding. DNA barcoding concept has become one of the most important
and significant. Eight types of mushroom products were analyzed in this research. Four samples of fresh
edible mushroom, two samples of dried edible mushroom, one of liquid supplement, and one capsule of
mushroom supplement. The goals of this study was to evaluate the labeled grocery store mushrooms and
content of labeled processed mushroom-based products matched with ITS barcoding. DNA barcoding
used for identification of fresh products and processed mushroom-based products. DNA barcoding
mushroom using ITS can play a very significant role in verification of processed mushroom-based products
and it was proper to used. This result can be used for a food safety and verification partial solution.

Key words : DNA Barcoding, Internal Transcribed Spacer region, Fresh Mushroom, Mushroom-based Products

Introduction ment, in 2002, global market for mushroom in

Refer to National Survey of Socio Economics by BPS
Indonesia, Indonesia Yearly Consumption for Mush-
room already increased to 103% from 0.087 kg/per-
son to 0.177 kg/person for Data comparison 2014
and 2018. If compare to 2011, the number of increas-
ing to be 211%. Jalaku (2011) predict that trend of
mushroom consumption increase due to creativity of
food processing and supplement. Statistic data of
average growth from 2014 to 2018 for utilization of
mushroom is 3.43%. Indonesia. In global data, for
mushroom commodity market from 1999 predicted
approximately 18 Billion Dollar and this number
value higher than coffee commodity in the world
market. Another derivative of mushroom is supple-
*Corresponding author’s email : intan.ayu.pratiwi@fst.unair.ac.id
supplement product approximately 5-6 Billion Dol-
lar.
The higher of mushroom consumption drives the
effective food safety regulation especially in Indone-
sia. Variant control for mushroom product depend on
country, in Indonesia, regulation for mushroom prod-
uct include in food category regulation. Moreover,
the suitability proven that packaging product will be
responsible of industry instead of Government. Fre-
quently product in market has difference contami-
nant and can to be problem for costumer. In business
perspective, for incompatibility product can to be se-
riously tread not only will be negative issue of prod-
uct but also can drive to legal issue. In traditional
market, for fresh mushroom of commercial edible
AYU PRATIWI ET AL
only categorized on macroscopic identification. The
challenge is species confirmation and identification
for fresh mushroom of commercial edible and mush-
room derivative supplement. Hawksworth (2004) re-
ported that another product can be serious problem
due to mycelia sample that contain of mushroom.
General identification process is morphology of
mushroom characteristic. Mushroom morphology
can be used as identification with refer to body of
mushroom, but it characteristic can change after next
processing with drying or milling. Taxonomy identi-
fication with morphology is important but less valid-
ity due to some factors especially uncertain variant,
species hybridization, convergent evolution. One of
the weakness of mushroom morphology identifica-
tion is variant of phenotype characteristic. Morphol-
ogy of mushroom influenced by the environment
and expressive of gen. Revolution to deeply investi-
gation beyond DNA Bar-coding. Sequence DNA of
mushroom resulted for big sub-unit through
nrLSU026S or 28s and n4SSU-18s for small sub-unit.
As well as rRNA 5.8S (include ITS4 and 5) bring to
new era of sequence identification of mushroom mol-
ecule phylogenetic (Huzefa et al., 2017).
The objective of the present study was to identify
the utility of fungal ITS DNA barcoding to the identi-
fication of commercial eligible species in market for
fresh mushroom, liquid supplement and capsule
supplement to address with question, is that true
fungal materials used in commercial mushroom
product. If these techniques could address a ques-
tions, then it was the goal to apply them in the au-
thentication (i.e. verification of species identities) of
fungal materials used in commercial products and
educate stakeholder.
Materials and Methods
Sampling and DNA extractions
Eight types of mushroom products were analyzed in
this study. Four samples of fresh edible mushroom,
two samples of dried edible mushroom, one of liquid
supplement, and one capsule of mushroom supple-
ment. All samples were analyzed using DNA
barcoding taken from grocery stores and company
that sell mushroom supplement. (Table 1). DNA ex-
traction was performed in triplicate (n=3). The mush-
room product from fresh, dried, liquid and capsules
was transferred to DNA lysis buffer (Promega DNA
extraction kit). Subsequently DNA was extracted us-
S41
ing procedures outlined in the Promega DNA extrac-
tion kit. For mushrooms DNA extractions, a small
piece of cap (pili) was removed from the packet and
physic lysed using sonication. Subsequently, ap-
proximately 5 mg of each samples. For liquid and
capsule product used approximately 10 mg of each
sample.
PCR amplification and sequencing
The entire ITS region was PCR-amplified with prim-
ers ITS5 and ITS4 (Gardes, White, Fortin, Bruns, and
Taylor, 1991; White et al., 1990). The total volume
PCR reaction was carried out in 25 ìL containing
Promega PCR mix cocktail 12,5 uL; ddH2O 7,5 uL;
Primer Forward (ITS5) 1,25 ìL; Primer reverse (ITS4)
1,25 ìL; DNA template 2,5 (Promputtha and Miller,
2010). The following thermocycling parameters were
used for the amplification: initial denaturation at 95
_C for 5 min followed by 35 cycles of 95 _C for 30 s,
43–45 _C for 15 s, and 72 _C for 1 min with a final
extension step of 72 _C for 10 min (Schoch et al.,
2012) modifications. The PCR products were on 1%
agarose gel along with a 1 kb DNA ladder (Promega)
to estimate the size of the amplified band. After
checking all the band, the amplicon were send for
sequencing process.
NCBI-BLAST Search for DNA Barcoding
ITS sequence fragment is subjected to an individual
Basic Local Alignment Search Tool (BLAST) in Gen
Bank to verify identity (Hennell et al., 2012). These
locations for submission of molecular data to public
databases for the mushroom identification. We have
used value ranging from 3% to 5% for ITS sequence
divergence to indicate conformity among Kingdom
Fungi. It is reasonable with 80% query coverage
and 97"100% sequence similarity of results from
Gen Bank. We also check the presence of specimen
vouchers and other unpublished collection informa-
tion.
Results
A total of eight samples were analyzed using ITS
barcoding (Table 1). All fresh and dried mushroom
samples were analyzed macroscopic to identify be-
fore extractions. Morphology identifications needed
for knowing common character that they have. And
without morphological identification for processed
mushroom-based products. All the samples were ex-
tracted using kit identification and PCR process. The
S42 Eco. Env. & Cons. 25 (April Suppl. Issue) : 2019

data were collected and analyzed using BLAST Discussion

(Table 2).
Table 1. List of mushroom sample type in this study
for DNA barcoding
No. Sample Code
1 JEF1, JEF2, JEF3
2 JWF1, JWF2, JWF3
3 JKF1, JKF2, JKF3
4 JSF1, JSF2, JSF3
5 JKD1, JKD2, JKD3
6 JSD1, JSD2, JSD3
7 SL1, SL2, SL3
8 KL1, KL2, KL3
Many fresh products and processed mushroom-
based products spread widely. Mushroom is one of
the big commodities food in Indonesia. However
Information
Fresh Enoki mushroom
Fresh White Beech
mushroom
Fresh Ear mushroom
Fresh Shitake mushroom
Dried Ear mushroom
Dried Shitake mushroom
Liquid supplement
Capsule supplement

All data morphological sample shown at Fig. 1.

The samples were list and code before proceed.
Fig. 2. PCR reaction applied to single-species mush-
The PCR products were analyzed using electro-
phoresis with 1% agarose (Fig. 2). There are 87% (7/
8) product were accurately labeled (Table 2). Only liq-
uid supplement product can be detected.
room DNA extracts. Ethidium Bromide fluores-
cence image showing electrophoresis of PCR
product. Gel was run with 1% agarose 10 L of
PCR product was loaded in each well for each of
the templates listed at the top of the gel. From
left to the right; Marker, JEF, JKF, JWF, JSF, JKD.

Fig. 1. Mushroom differences sample were obtained. 1: Fresh Shitake mushroom were labeled Shiang ku. 2: Fresh
Enoki mushroom from market. 3: Fresh Ear mushroom. 4: Fresh White Beech mushroom from market. 5: Liq-
uid supplement were labeled contain of Ganoderma lucidum. 6: Dried Shitake mushroom.

Table 2. List of mushrooms sample.

No Sample Common Name Barcoding ID Accessions Percent
codes Number Similarity (%)
1 JEF Enoki Flammulina velutipes KX058567.1 93
strain S14027
2 JWF White Beech Hypsizygus marmoreus HM561968.1 98
isolate HMB1
3 JKF Ear mushroom Auricularia polytricha HM448467.1 98
cultivar Apw82
4 JSF Shitake Lentinula edodes AB286066.1 94
5 JKD Ear mushroom Auricularia polytricha HM448456.1 97
cultivar Sanyou
6 JSD Shitake Lentinula edodes AB286066.1 98
7 SL Ling zhi ND ND ND
8 KL Shitake Lentinula edodes AB286066.1 93
AYU PRATIWI ET AL
along with the increasing number of mushroom-
based products, it is also expected to increase food
safety protection for consumers. Fresh, dried and
processed mushroom-based products are the biggest
sales in Indonesia (JALAKU, 2011). Mushroom are
utilized routinely for direct consumption and me-
dicinal properties (Wasser, 2002). Unfortunately
there are still many farmers used morphology obser-
vations only in determining the type of mushroom.
Morphological characters can often be misleading
due to hybridization and convergent evolution (Brun
et al. 2010) which caused confusion. Therefore, our
study was to test DNA barcoding for identification
commercial edible mushroom and their product.
DNA barcoding concept has become one of the most
important and significant in the last decade. The
goals of this study was to evaluate the labeled gro-
cery store mushrooms and content of labeled pro-
cessed mushroom-based products matched with ITS
barcoding.
DNA Barcoding is identification process to im-
prove relevant name of species on level intra species.
Stajic et al., (2009) reported that DNA can depict
valid result for identification process of mushroom
with mega-diversity. Huzefa et al., (2017) investi-
gated how Internal Transcription Spacer (ITS)
barcoding can be used for identification of fungal
sample used in dietary supplements, this report can
conclude that manufacturing could demonstrate the
accuracy their labeled ingredients of products that
contain fungi beyond ITS Barcoding. Others research
already presented of DNA product of commercial
mushroom.
All fresh and dried mushroom product were
verified successfully. This result can help to improve
lack morphological characteristics and evaluate their
product label accurately (Table 2). These data de-
scribe the ability ITS DNA barcoding not only for
challenging fresh and dried mushroom, but also for
more observed processed mushroom-based products
(Dentinger and Suz, 2014). The Internal Transcribed
Spacer (ITS) region has been considerable used in
fungal identity at and below the genus level
(Begerow et al., 2010). Challenges of ITS barcoding
with liquid supplement processed mushroom-based
products (Fig. 2; Table 2). We postulate that the appli-
cation of heat processes could be a limitation factor to
acquire the genomic DNA. The liquid product could
not be extracted and detected as well as the labeled
contain of Ganoderma lucidum. Similar research with
Newmaster et al., 2013, postulate that DNA from
S43
highly processed material can often be damaged into
short fragments DNA sizes, which cannot be suc-
cessfully extract and the product may contain DNA
from mixed samples. The ITS sequence analysis re-
vealed JEF to be a species of Flammulina velutipes
strain S14027 (GenBank accession number
KX058567.1); JWF to be Hypsizygus marmoreus isolate
HMB1 (GenBank accession number HM561968.1);
JKF and JKD became same spesies Auricularia
polytricha but different cultivar, one of them detected
as cultivar Apw82 and the other is cultivar Sanyou
(GenBank accession number HM448467.1 and
HM448456.1); JSF, JSD, KL to be Lentinula edodes with
same accession number but have percent similarity
differences respectively 94%, 98%, 93% (Table 2). All
samples Lentinula edodes have same GenBank acces-
sion number i.e. AB286066.1. Interestingly, not deter-
mine data in SL code. This conditions refer to Moshe
et al. (2013) stated the thermal degradation of deox-
yribonucleic acid (DNA), under dry conditions, com-
plete DNA degradation occurs at above 190C and
boiling temperature of water are pressure dependent.
Heat effects on nucleic acids and nucleic acid-associ-
ated processes i.e. heat shock proteins and factors
(HSPs and HSFs), their involvement in the regulation
of transcription, protein homeostasis (Kantidze et al.,
2016). The results obtained from the current study
indicate that the ITS region can be used as a reliable
barcode for identifying fresh products and pro-
cessed mushroom-based products. We firmly believe
that the DNA barcodes generated for each of the
mushroom species in the present investigation, will
help in food safety and commercial eligible species
in market for fresh mushroom, liquid supplement
and capsule supplement.
Conclusions
In this study, we have provided on DNA barcoding
used for identification of fresh products and pro-
cessed mushroom-based products. DNA barcoding
could demonstrate the accuracy of their labeled prod-
ucts that contain of mushroom. These methods can be
to identification and validation of fresh products and
processed mushroom-based products. Before the fi-
nal products are produced, DNA barcoding can be
used for mushroom derived that have not clear mor-
phological characteristics. The processed mushroom-
based products were displayed on the product label
accurately. DNA barcoding mushroom using ITS can
play a very significant role in verification of pro-
S44 Eco. Env. & Cons. 25 (April Suppl. Issue) : 2019

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