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Philippine Science Letters Vol. 11 | No. 01 | 2018 30
Not fish in fish balls: fraud in some
processed seafood products
detected by using DNA barcoding
Katreena P. Sarmiento 1,2, Jacqueline Marjorie R. Pereda1, Minerva Fatimae H. Ventolero 1, and Mudjekeewis D. Santos1,* 1Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute,
101 Mother Ignacia Ave., South Triangle, Quezon City, Metro Manila, Philippines, 1103 2 Department of Biology, School of Science and Engineering, Ateneo de Manila, Metro Manila, Philippines, 1108
ne of the most popular processed seafood products
in the Southeast Asian region, “fishball” is a round,
white food containing fish meat and other
ingredients such as salt, starch, and sugar cooked in
oil and sold as street food. Fish products are among
the leading food categories with reported cases of food fraud—
fish samples purchased at grocery stores, restaurants, and sushi
bars are mislabeled. Fishballs are anecdotally known as being
made up of shark meat. To identify the animal species used in
commercially processed seafood products, we utilized DNA
barcoding by analyzing cytochrome c oxidase I gene, which was
further validated by cytochrome b gene. Twelve seafood
products, including fish balls, fish nuggets, squid balls, and
shrimp balls, were collected from supermarkets, street vendors,
and commercial stalls in Iloilo City, Marikina City, and Quezon
City, Philippines. Results revealed that processed seafood
products manufactured and/or sold by well-established
companies conform to some extent with their respective labels:
fish balls contained meat from Chinese herring, Ilisha elongata,
and broad club cuttlefish, Sepia latimanus; fish nuggets were
composed of blue grenadier, Macruronus novaezelandiae; squid
balls came from Humboldt squid Dosidicus gigas. Products sold
by unknown companies or those with no labels—the products
sold mostly as street food—showed an entirely different
composition. Samples labeled squid and shrimp ball came from
domestic pig, Sus scrofa, and chicken, Gallus gallus, suggesting
that some seafood products being sold are mislabeled and
misdeclared. We highlight the need for increasing quality control
and inspection on lesser-known processed seafood product
sources for consumer welfare and safety. DNA barcoding is an
effective tool in assessing food identity and traceability in
commercial fishery products even for those already processed.
KEYWORDS
processed seafood, mislabeled, misdelcared, traceability,
consumer welfare, DNA barcoding
INTRODUCTION
Value-added commercial fishery or seafood products have been
increasing due to the continued expansion and diversification of
product forms to meet quality expectations and consumer
demand. Thus, technological advancements in food production,
handling, processing, and product distribution need to be
adapted (Filonzi et al. 2010).
One of the most popular products in the Southeast Asian region
is “fish ball” (Kok et al. 2013). It is known in different countries
by various names (“BebolaIkan” in Brunei and Malaysia;
“Ngasoke” in Myanmar; “Bakso” in Indonesia; “Luk chin-pla”
in Thailand; “Yu Yuan” in Singapore; and “Yu Wan” in China)
(Kok et al. 2013). In the Philippines it is called “ola-bola” and
regarded at times as the “Philippine caviar” (Kok et al. 2013;
Golangco 2010). Fish balls are considered to have high
nutritional value because of their high protein and carbohydrate
contents (Huda et al. 2010).
O
ARTICLE
*Corresponding author Email Address: [email protected] Date received: November 10, 2017 Date revised: April 30, 2018 Date accepted: May 17, 2018
Vol. 11 | No. 01 | 2018 Philippine Science Letters
31
Table 1: List of seafood product samples and detailed description
Sample Code Seafood Product Date of Collection Sampling Site Location (PH)
FB01 Fish balls 06-03-2013 Supermarket Iloilo City
FB02 Fish balls 06-03-2013 Supermarket Iloilo City
FN01 Fish nuggets 06-03-2013 Supermarket Iloilo City
SB01 Squid balls 06-03-2013 Supermarket Iloilo City
SB02 Squid balls 06-03-2013 Supermarket Iloilo City
SB03 Squid balls 06-03-2013 Supermarket Iloilo City
SB04 Squid balls 06-21-2016 Commercial stall Quezon City
SB05 Squid ball 06-21-2016 Street vendor Quezon City
SB06 Squid ball 05-13-2015 Street vendor Quezon City
SB07 Squid ball 06-28-2016 Supermarket Marikina City
ShB01 Shrimp balls 06-21-2016 Commercial stall Quezon City
ShB02 Shrimp balls 06-28-2016 Supermarket Marikina City
The wide variation in the types of ingredients used in producing
fish balls, particularly the fish meat source and types of
ingredients, depends on cost, fish ball quality, and fish species
availability (Huda et al. 2010; Kok et al. 2013). Some species
utilized include wolf herring (Chirocentrus dorab), coral fish
(Caesio spp.), Spanish mackerel (Scomberomorus spp.), conger
eel (Congresox spp.), yellow stripe trevally (Selaroides
leptolepis), obtuse barracuda (Sphyraenaobstusata), threadfin
bream (Nemipterus hexodon), monocle bream (Scolopsistae
niopterus), lizard fish (Sauridaundo squamis), and the most
common in Philippine fish balls, yellowtail fusilier
(Caesioteres) (Kok et al. 2013). Other low-cost marine fishes
have been utilized for fish ball production, including jewfish
(Johnius argentatus), sea catfish (Tachysurus thalassinus),
jeweled shad (Ilisha filigera), and horse mackerel (Megalaspis
cordyla) (Hoque et al. 2007). Aside from the fish meat, typical
fish balls also include 3–5% salt, 3% sugar, 3% starch, 1%
monosodium glutamate (MSG), and 40% water (Kok et al.
2013). The increasing growth in the fish ball industry allowed
for the introduction of new product variants using squid and
shrimp meat as replacement for fish meat, called squid ball and
shrimp ball, respectively (World Heritage Encyclopedia 2016).
Fish and seafood were listed as one of the leading food
categories with reported cases of food fraud—various products
purchased at grocery stores, fish markets, restaurants, and sushi
bars were mislabeled (Johnson 2014; Quinto et al. 2015; Willette
et al. 2017).
In the Philippines, mislabeling in fishery products sold as fresh
and/or frozen fillets (Maralit et al. 2013), as well as the detection
of endangered and protected species in dried seafood for export
(Asis et al. 2016), has been documented by using DNA
barcoding. This tool has also been successfully used to identify
fish species in processed products such as from fish paste
(Agmata et al. 2014).
Using DNA barcoding for consumer welfare and safety, the
present study investigated the identity of the popular processed
seafood products labeled as fish balls, fish nuggets, squid balls,
and shrimp balls sold in supermarkets and by street vendors.
MATERIALS AND METHODS
Sample collection
Frozen fishballs (FB), fish nuggets (FN), squidballs (SB), and
shrimp balls (ShB) were obtained from several supermarkets,
street vendors, and commercial stalls in Quezon City, General
Santos City, and Marikina City, Philippines, in 2013–2016.
Twelve (12) samples were obtained and chilled on ice until
laboratory processing (table 1). Among the samples, 8/12 (66%)
were obtained from supermarkets, 2/12 (17%) from commercial
stalls, and 2/12 (17%) from street vendors (table 1). Before DNA
extraction, three pieces (“representative sample”) per sample
were collected and subjected to tissue sampling where
approximately150 mg were taken and stored in absolute ethanol
at -20°C.
DNA extraction and amplification
DNA extraction was carried out by using the modified Cetyl Tri-
methyl Ammonium Bromide (CTAB) technique based on the
protocol of Santos et al. (2010) with minor modifications. The
DNA markers cytochrome c oxidase I(COI) and cytochrome b
(Cytb) were amplified by using the primers in table 2. The 26 µl
polymerase chain reactions (PCR) consisted of ultrapure water,
0.5x PCR buffer, 0.08 mM dNTP’s, 1.4 mM MgCl2, 0.6 µM of
each primer, 0.1 unit Taq polymerase, and 2 µl of template. The
PCR cocktails were subjected to the following conditions: 94°C
for 2 min, 38 cycles of 94°C for 30 sec, 54°C for 40 sec, 72°C
for 60 sec, and a final extension of 72°C for 10 min. Product
amplicons were electrophoresed in 1% agarose gel stained with
ethidium bromide and submerged in 1x TAE buffer, and then
documented with SynGene G:BOX. Standard sequencing and
DNA purification were outsourced to AIT Biotech, Singapore.
Genetic analysis
Only the “representative sample” for each of the 12 samples with
the best sequencing results was used in the final analysis.
Consensus sequences were generated for each sample by using
Geneious (version 6, http://www.geneious.com/). Each
consensus sequence was run in Basic Local Alignment Search
Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and
Philippine Science Letters Vol. 11 | No. 01 | 2018 32
Table 2: Primers used in amplifying genetic markers from samples
Primer name Sequence 5’→3’ Target Reference
VF2_t1 TGTAAAACGACGGCCAGTCAACCAACCACAAA
GACATTGGCAC
Cytochrome
oxidase I
Ivanova et al. 2007
FishF2_t1 TGTAAACGACGGCCAGTCGACTAATCATAAAG
ATATCGGCAC
FishR2_t1 CAGGAAACAGCTATGACACTTCAGGGTGACC
GAAGAATCAGAA
Fr1d_t1 CAGGAAACAGCTATGACACCTCAGGGTGTCC
GAARAAYCARAA
Glud-G TGACTTGAARAACCAYCGTTG Cytochrome b Palumbi 1996
CB2-H CCCTCAGAATGATATTTGTCCTCA
sequences within the GenBank database
(http://www.ncbi.nlm.nih.gov/genbank/) with identity >90%
were used as reference sequences. Model testing was performed
by using Molecular Evolutionary Genetics Analysis (MEGA)
version 6, and phylogenetic tree was inferred by using the
Maximum Likelihood method on the General Time Reversible
model (Nei and Kumar 2000) with 500 bootstrap replications.
The percentage of replicate trees in which the associated taxa
clustered together based on the bootstrap test was shown next to
the branches (Felsenstein 1985), and values >70% were
considered reliable (Hillis and Bull 1993). Genetic distances
between sequences in the units of base substitutions per site were
also analyzed by using the K2P model (Kimura 1980). All
positions containing gaps and missing data were eliminated.
Analyses were conducted in MEGA version 6
(http://www.megasoftware.net/) (Tamura et al. 2013).
RESULTS AND DISCUSSION
Consensus sequences from the fish ball samples were analyzed
to examine each of their fish meat sources. Identity of samples
and corresponding reference sequences for clustering analysis
were based on the BLAST search results for each sample (table
3) that was within the top 10 hits and whose identity is in the
range of 97–100% and E-value at 0.0.
A clustering tree inferred from COI sequences (fig. 1) revealed
that collected fish ball samples used various fish meat sources.
The analysis involved 28 sequences with a total of 306 positions
in the final data set. The reliability of the inferred topologies was
confirmed by the bootstrap values obtained and genetic
distances within the range of 0.000–0.017between samples and
reference sequences (tables 4 and 5).
Samples FB02and FN01, labeled as “fish balls” and “fish
nuggets,” were composed of Chinese herring Ilisha elongate and
Blue grenadier Macruronus novaezelandiae, respectively, while
samplesSB01and SB03, obtained from supermarkets and
labeled as “squid balls,” were from Humboldt squid Dosidicus
gigas, and SB02 labeled as “squid ball” was composed of meat
from Broadclub cuttlefish Sepia latimanus. This suggests
appropriate labels for these samples sold by established
companies. However, FB01 labeled as “fish ball” was found to
be composed of Humboldt squid Dosidicus gigas.
Table 3: Basic Local Alignment Search Tool (BLAST) results showing the top hit for each sample for COI and Cytb markers
Sample BLAST Result (Top hit) % Identity Query Coverage E-value
COI
FB01 Dosidicus gigas 99 99 0.0
FB02 Ilisha elongata 99 100 0.0
FN01 Macruronus novaezalandiae 99 100 0.0
SB01 Dosidicus gigas 99 99 0.0
SB02 Sepia latimanus 99 100 0.0
SB03 Dosidicus gigas 99 100 0.0
SB04 Sus scrofa 99 99 0.0
SB05 Sus scrofa 100 100 0.0
SB06 Sus scrofa 99 100 0.0
SB07 Gallus gallus 99 98 0.0
ShB01 Sus scrofa 99 99 0.0
ShB02 Gallus gallus 99 99 0.0
Cytb
SB04 Sus scrofa 100 100 0.0
SB05 Sus scrofa 100 100 0.0
SB06 Sus scrofa 100 100 0.0
SB07 Gallus gallus 100 100 0.0
ShB01 Sus scrofa 100 100 0.0
ShB02 Gallus gallus 99 100 0.0
Vol. 11 | No. 01 | 2018 Philippine Science Letters
33
Figure 1: Clustering analysis of COI sequences of 12 fish ball samples using Maximum Likelihood tree based on General Time Reversible model. Bootstrap values from 500 bootstrap samples are indicated beside the nodes. The scale bar represents 5% genetic distance, and GenBank accession numbers are enclosed in parentheses.
More surprising, samples SB04, SB05, and SB06, labeled as
“squid balls,” and ShB01, labeled as “shrimp balls,” were found
to be composed of Domestic pig Sus scrofa with a genetic
distance of 0.000–0.017, while SB07, labeled as “squid balls,”
and ShB02, labeled as “shrimp balls,” were composed of
Chicken Gallus gallus with a genetic distance of 0.000–0.003
(table 4).
Verification of the detection of nonfish meat source for samples
SB04, SB05, SB06, ShB01, SB07, and ShB02 was done by
using the marker Cytb consensus sequences. The analysis
involved 13 nucleotide sequences with a total of 421 positions
in the final data set. The results of the clustering analysis (fig. 2)
concur with the results of the analysis of COI sequences showing
supported clustering where “squid balls” SB04, SB05, and SB06
and “shrimp ball” ShB01 grouped with Domestic pig Sus scrofa
and “squid ball” SB07 and “shrimp ball” ShB02 grouped with
Chicken Gallus gallus exhibited computed genetic distances
ranging within 0.000–0.002 (table 5). Although the final data set
used is only 300–400bp long, analysis results show that species
matches are reliable. Studies have shown that lesser COI
fragments of 100–200bp can provide a reliable species-level
identification and that these shorter barcodes are especially
useful for samples that have high chances of DNA degradation
such as the samples in the study that have undergone some form
of processing (Hajibabaei et al. 2006; Ivanova et al. 2009;
Meusnier et al. 2008). Moreover, the primer sets used in the
study, though popularly known to be specific for fish, have been
reported to work with other vertebrate species as well (Ivanova
et al. 2007; Palumbi 1996).
Interestingly, most of the products from established companies,
specifically 5/8 (62.5%) of the samples sold in supermarkets,
namely, FB02, FN01, SB01, SB02, and SB03, revealed correct
labels. By contrast, 4/4 (100%) of the samples from unknown
companies sold through street vendors and commercial stalls
and 2/8 (25%) of the samples sold in supermarkets were found
to have pig and chicken meat. Taken together, of the total
seafood product samples analyzed, only 41.7% were labeled
correctly. Moreover, 50% of the samples used in the study
showed compromised content in that they were found to contain
meat sources other than 100% fish meat as marketed.
Philippine Science Letters Vol. 11 | No. 01 | 2018 34
Table 4: Distance matrix of COI sequences of samples used with representative reference sequence from BLAST search results
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 Dosidicus_gigas_(EU068697.1)
0.000
0.000
0.000
0.078
0.078
0.080
0.068
0.071
0.065
0.065
0.045
0.045
0.057
0.054
0.054
0.057
0.054
2 FB01_COI 0.000
0.000
0.000
0.078
0.078
0.080
0.068
0.071
0.065
0.065
0.045
0.045
0.057
0.054
0.054
0.057
0.054
3 SB01_COI 0.000
0.000
0.000
0.078
0.078
0.080
0.068
0.071
0.065
0.065
0.045
0.045
0.057
0.054
0.054
0.057
0.054
4 SB03_COI 0.000
0.000
0.000
0.078
0.078
0.080
0.068
0.071
0.065
0.065
0.045
0.045
0.057
0.054
0.054
0.057
0.054
5
Gallus_gallus_(KT780168.
1)
0.4
77
0.4
77
0.4
77
0.4
77
0.0
00
0.0
03
0.0
51
0.0
49
0.0
54
0.0
54
0.0
81
0.0
81
0.0
55
0.0
59
0.0
59
0.0
55
0.0
59
6 ShB02_COI 0.477
0.477
0.477
0.477
0.000
0.003
0.051
0.049
0.054
0.054
0.081
0.081
0.055
0.059
0.059
0.055
0.059
7 SB07_COI 0.488
0.488
0.488
0.488
0.003
0.003
0.052
0.050
0.055
0.055
0.083
0.083
0.057
0.060
0.060
0.057
0.060
8 Ilisha_elongata_(HM030768.1)
0.462
0.462
0.462
0.462
0.324
0.324
0.331
0.006
0.044
0.044
0.067
0.067
0.051
0.055
0.055
0.051
0.055
9 FB02_COI 0.481
0.481
0.481
0.481
0.309
0.309
0.316
0.010
0.045
0.045
0.070
0.070
0.054
0.059
0.059
0.054
0.059
1
0
Macruronus_novaezelandi
ae_(KX781872.1)
0.4
28
0.4
28
0.4
28
0.4
28
0.3
40
0.3
40
0.3
48
0.2
75
0.2
82
0.0
00
0.0
69
0.0
69
0.0
57
0.0
57
0.0
57
0.0
57
0.0
57 11 FN01_COI
0.428
0.428
0.428
0.428
0.340
0.340
0.348
0.275
0.282
0.000
0.069
0.069
0.057
0.057
0.057
0.057
0.057
12
Sepia_latimanus_(AP013074.1)
0.286
0.286
0.286
0.286
0.523
0.523
0.534
0.453
0.471
0.459
0.459
0.003
0.054
0.055
0.055
0.054
0.055
13 SB02_COI
0.286
0.286
0.286
0.286
0.523
0.523
0.534
0.453
0.471
0.459
0.459
0.003
0.054
0.055
0.055
0.054
0.055
14 Sus_scrofa_(KX982659.1)
0.375
0.375
0.375
0.375
0.358
0.358
0.366
0.333
0.355
0.361
0.361
0.360
0.360
0.008
0.008
0.000
0.008
1
5 ShB01_COI
0.3
59
0.3
59
0.3
59
0.3
59
0.3
81
0.3
81
0.3
89
0.3
62
0.3
87
0.3
63
0.3
63
0.3
67
0.3
67
0.0
17
0.0
00
0.0
08
0.0
00 16 SB05_COI
0.359
0.359
0.359
0.359
0.381
0.381
0.389
0.362
0.387
0.363
0.363
0.367
0.367
0.017
0.000
0.008
0.000
17 SB04_COI
0.375
0.375
0.375
0.375
0.358
0.358
0.366
0.333
0.355
0.361
0.361
0.360
0.360
0.000
0.017
0.017
0.008
18 SB06_COI
0.359
0.359
0.359
0.359
0.381
0.381
0.389
0.362
0.387
0.363
0.363
0.367
0.367
0.017
0.000
0.000
0.017
Table 5: Distance matrix of Cytochrome B sequences of samples used with representative reference sequence from BLAST search results
1 2 3 4 5 6 7 8 9
1 SB04_CYTB
0.000 0.000 0.000 0.002 0.048 0.048 0.048 0.063
2 SB05_CYTB 0.000
0.000 0.000 0.002 0.048 0.048 0.048 0.063
3 SB06_CYTB 0.000 0.000
0.000 0.002 0.048 0.048 0.048 0.063
4 ShB01_CYTB 0.000 0.000 0.000
0.002 0.048 0.048 0.048 0.063
5 Susscrofa 0.002 0.002 0.002 0.002
0.048 0.048 0.048 0.062
6 SB07_CYTB 0.385 0.385 0.385 0.385 0.385
0.002 0.000 0.058
7 ShB02_CYTB 0.384 0.384 0.384 0.384 0.384 0.002
0.002 0.059
8 Gallus gallus 0.385 0.385 0.385 0.385 0.385 0.000 0.002
0.058
9 Ilishaelongata 0.481 0.481 0.481 0.481 0.476 0.445 0.451 0.445
This finding is not surprising since, in a previous study by
Maralit et al. (2013), frozen products such as fish fillets, choice
cuts, and shrimp sold in Philippine supermarkets were found to
be mislabeled with some products being replaced with lower
market-value species. A more recent study revealed that raw fish
served as sushi in restaurants and sold in high-end grocery stores
also conveyed a high percentage of mislabeling (Willette et al.
2017).Food fraud is evident not only in frozen and fresh forms
but also in processed products such as sausage products sold as
“turkey” sausages but containing chicken meat or “beef”
sausages containing pork meat or “pork” sausages containing
horse meat (Naaum et al. 2017).
Vol. 11 | No. 01 | 2018 Philippine Science Letters
35
Figure 2: Clustering analysis of cytochrome bsequences of 6 fish ball samples using Maximum Likelihood tree based on General Time Reversible
model. Bootstrap values from 500 bootstrap samples are indicated beside the nodes. The scale bar represents 5% genetic distance, and GenBank accession numbers are enclosed in parentheses.
Article 85 (entitled “Mislabeled Food”) of Republic Act 7394,
also known as the Consumer Act of the Philippines, states that
food shall be deemed mislabeled “if its labeling or advertising
is false or misleading in any way” (Philippine Commission on
Women 2018). While the term “fish ball” may not specifically
indicate the species of fish to which it pertains, it does directly
imply that this particular processed product is composed of fish
and seafood meat and expectedly does not pertain to any product
composed of pig or chicken meat. With the results of the study
taken into account, this suggests that the presented case is indeed
an act of mislabeling and highlights the importance of strong
implementation of regulations that would protect consumer
welfare and safety.
The need for stronger regulatory measures is further underscored,
in spite of the increase in studies and awareness, by the
continuing prevalence of mislabeling and fraud, and the possible
extent of negative effects such as on personal beliefs or
preferences and health issues such as allergic reactions, due to
these products’ popularity and consumers’ growing support.
Finally, this study shows that DNA barcoding allows for a fast
and precise method of species identification, thereby providing
reliable data that give support for proper labeling for food
traceability and monitoring and regulation of seafood products,
specifically those that have undergone processing.
CONCLUSION
The results of this study revealed the prevalence of incorrect
species declaration, specifically in processed seafood products
such as fish/shrimp/squid balls as well as fish nuggets, notably
those sold by some street vendors and commercial stalls. This
emphasizes the need to strengthen regulatory measures
particularly on food traceability of processed food products and
frequent assessment of the authenticity of raw materials utilized
for commercial selling. Moreover, this study shows that DNA
barcoding serves as an effective technique in species
identification and food traceability. Thus, this study highly
recommends that local regulatory agencies implement similar
tests in assessing authenticity of commercially sold food
products to avoid similar cases of fraud.
ACKNOWLEDGMENTS
The authors are grateful to the National Fisheries Research and
Development Institute (NFRDI) and Bureau of Fisheries and
Aquatic Resources (BFAR) for funding the study through its
FISHCODES: DNA Barcoding Services Project. We thank the
members of the Genetic Fingerprinting Laboratory (GFL) for
their utmost support.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
CONTRIBUTION OF INDIVIDUAL AUTHORS
KS wrote the manuscript. KS, JMP, and MFV carried out sample
collection, molecular genetic studies, participated in the
sequence analysis, and drafted the manuscript. MS conceived
the study, participated in its design and coordination, and
structured and revised the writing of the manuscript. All authors
read and approved the final manuscript.
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