Not fish in fish balls: fraud in some processed seafood ...

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



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


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



SB07 Gallus gallus 99 98 0.0

ShB01 Sus scrofa 99 99 0.0

ShB02 Gallus gallus 99 99 0.0

Cytb




SB07 Gallus gallus 100 100 0.0

ShB01 Sus scrofa 100 100 0.0

ShB02 Gallus gallus 99 100 0.0


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.


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).


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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Not fish in fish balls: fraud in some processed seafood ...

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