Journal of Fisheries science and Technology – No 3/2016

Journal of Fisheries science and Technology – No 3/2016 present content ace-inhibitory activity of protein hydrolysate from the skin of striped catfish (Pangasius hypophthalmus); potential impacts of climate change on fisheries and aquaculture in Sri Lanka (Roaming through the climate change management stories of Sri Lanka); effect of cation concentrations (K+, Ca2+) and hormonal stimulation on sperm motility of common carp cyprinus carpio...

ACE-INHIBITORY ACTIVITY OF PROTEIN HYDROLYSATE

FROM THE SKIN OF STRIPED CATFISH (Pangasius hypophthalmus)

Hue Quoc Hoa 1,2 , Nguyen Xuan Duy 3

Received: 21/7/2016; Revised: 09/8/2016; Accepted: 26/9 /2016

ABSTRACT

There has recently been an increasing demand to produce protein hydrolysates containing peptides with specifi c biological properties, which could be marketed as functional food ingredients The objective of this study was to evaluate the in vitro angiotensin converting enzyme inhibitory activity of striped catfi sh skin hydrolysates and its corresponding fractionates The striped catfi sh skin from fi llet processing was extracted

in an autoclave at 121 0 C for 30 minutes to obtain an extracted protein Then it was further hydrolysed with Alcalase with the enzyme to substrate ratio of 20 units/gram protein at 50 o C, pH 8 for 7h to obtain protein hydrolysate The degree of hydrolysis (DH) increased with the increase of hydrolysis time and reached the highest DH of 91.9% after 7h hydrolysis The 5-h hydrolysate (DH= 60.8%) exhibited the highest ACE-inhibitory activity (IC 50 = 831 µg/ml) Therefore, the 5-h hydrolysate sample was used as material for studying enrichment of ACE-inhibitory peptides by ultrafi ltration using three different molecular weight cut-off membranes (10, 5, and 1 kDa) Six sample fractions obtained during ultrafi ltration process (permeate and retentate) were tested for angiotensin converting enzyme inhibition activity Permeate of 1 kDa membrane showed the highest activity The obtained hydrolysates were fractioned using Sephadex M G-15 Based on gel

fi ltration chromatography results, angiotensin converting enzyme inhibitory peptides had molecular weight ranging of 307 Da to 429 Da Our fi ndings revealed the potential of using catfi sh skin as a promising material for retrieving angiotensin converting enzyme inhibitory substances

Keywords: Alcalase, ACE-inhibitory activity, hydrolysate, ultrafi ltration, Pangasius hypophthalmus

1 Nutraceutical and Functional Food R&D Center, Prince of Songkla University, Thailand

2 Department of Technology, Dong Thap Community College, Vietnam

3 Faculty of Food Technology, Nha Trang University, Vietnam

* Correcponding email: hqhoa@dtcc.edu.vn

I INTRODUCTION

High blood pressure is a major risk factor

associated with cardiovascular disease, the

biggest cause of casualty Hypertension

is commonly treated with antihypertensive

or blood pressure lowering drugs, such as

captopril, benazepril, enalapril These

drugs are angiotensin I converting enzyme

(ACE) inhibitors ACE (EC 3.4.15.1) is a

zinc-metallopeptidase that needs zinc and

chloride ions for its activity In the renin-angiotensin

system (RAS), ACE plays a crucial role in

the regulation of blood pressure as well as

cardiovascular function (Li et al., 2004)

Within the enzyme cascade of the RAS, ACE

converts the inactive angiotensin I by cleaving

dipeptide from the C-terminus into the potent vasoconstricting angiotensin II This potent vasoconstrictor is also involved in the release

of a sodium-retaining steroid, aldosterone, from the adrenal cortex, which has a tendency

to increase blood pressure As many synthetic drugs like ACE inhibitors have side effects, peptides from food sources provide an attractive alternative (Howell and Kasase, 2010) Recent researches have reported discoveries of peptides, which are isolated and characterized from a number of fi sh skin by-products such

as Nile tilapia skin (Vo et al., 2011), Pacifi c cod skin (Ngo et al., 2011), Atlantic salmon skin (Gu et al., 2011), Skate skin (Lee et al., 2011), Pangasius catfi sh (Mahmoodani et al., 2014)

that inhibited ACE and can be used as

nutraceuticals and functional food ingredients

A group of peptides from sardine (Fujita, 2001)

could decrease blood pressure and approved

products containing these components can

claim that the product is suitable for individuals

with slightly elevated blood pressure

A commercial product from sardine peptides

that lowers blood pressure was approved by

food for specifi ed health uses (FOSHU), an

offi cial functional food approved by the

consumer affairs agency of Japan (Shimizu,

T, 2003) Striped catfi sh (Pangasius

hypophthalmus) is a large freshwater fi sh It is

an important species in freshwater aquaculture

in Vietnam, Thailand, Malaysia, Indonesia and

China The fi llet processing generates

considerable quantities of by-products,

including abdominal organs, head, bone and

skin, that in total represent about 65% of the

fi sh by weight (Thuy et al., 2007) The objective

of this study was to investigate ACE inhibitory

activity of protein hydrolysate from striped

catfi sh skin by-products by enzymatic

hydrolysis using Alcalase

II MATERIALS AND METHODS

1 Materials

Catfi sh skins were obtained from a striped

catfi sh processing plant (Dong Thap, Vietnam),

the skins were frozen and stored at -20oC

before use Alcalase from Bacillus licheniformis

2.4 L, o-phthalaldehyde, DL-dithiothreitol,

ACE from rabbit lung and other chemicals

were purchased from Sigma-Aldrich Chemical

Company Polysulphone hollow fi ber membranes

with 10, 5, and 1 kDa MWCOs (diameter = 1, 1,

and 0.5 mm; area = 0.01, 0.01, and 0.014 m2)

were purchased from GE Healthcare

Bio-Science Ltd (Bangkok, Thailand)

2 Methods

2.1 Extraction of protein from striped catfi sh skin

The clean skins were added with distilled

water (1:2, w/v) and the protein was extracted

using an autoclave at 121oC for 30 min After extraction, the extracted protein solution was

fi ltered through a metal sieve to remove skin residues Extracted protein solution was centrifuged at 3,000g for 20 min at 25oC to remove insoluble residues and used as a substrate for enzyme hydrolysis Protein content in the skin and the extracted protein solution were determined by Kjeldahl method (AOAC, 1999)

2.2 Enzymatic hydrolysis of extracted protein solution

The extracted protein solution was diluted

to obtain a protein concentration of 1% (w/v)

by 0.1 M sodium phosphate buffer, pH 8.0 The protein solution was hydrolysed by 20 units/g protein of Alcalase 2.4 L at pH 8.0 and 50oC in

a 4-L reactor for 6h The pH of the mixture was measured by a pH meter (Eutech, Cyber Scan

pH 110, Singapore) and manually adjusted to

pH 8.0 during the hydrolysis by 6N NaOH and 6N HCl Aliquots of hydrolysate were collected every 60 mins during the hydrolysis The sample aliquots were heated in boiling water (950C) for 10 mins to inactivate Alcalase They were kept in plastic bottles at - 20oC for analyses

The degree of hydrolysis (DH) of the sample was determined by measure the available cleaved peptides bonds upon hydrolysis, using the o-phthalaldehyde (OPA)

method as described by Hue et al (2013) 2.3 Enrichment of ACE-inhibitory peptides derived from hydrolysate of striped catfi sh skin

2.4 Angiotensin-I converting enzyme inhibitory

activity of protein hydrolysates from striped

catfi sh skin

The inhibition of ACE activity was

determined by the method of Cushman

and Cheung (1971) described by Lee et al

(2010) with some modifi cations The reaction

mixture contained 8.3 mM

Hippuryl-L-Histidyl-L-Leucine (Hip-His-Leu) in 0.5M NaCl and

5 mU ACE in 50 mM sodium borate buffer

(pH 8.3) A sample (50 μl) was added to above

reaction mixture (50 μl) and mixed with 8.3 mM

HHL (150 μl) containing 0.5 M NaCl After

incubation at 37oC for 1 h, the further reaction

was stopped by the addition of 0.1M HCl (250 μl)

The resulting hippuric acid was extracted by

the addition of 1.5 ml of ethyl acetate After

centrifugation (800 x g, 15 mins), 1 ml of the

upper layer was transferred into a glass tube

and evaporated at room temperature for 2 h in

a vacuum The hippuric acid was redissolved

in 3 ml of distilled water, and absorbance was

measured at 228 nm using a spectrophotometer

(GENESYS 10S UV-VIS Thermo Scientifi c,

Tokyo, Japan) The control and blank were

prepared in the same manner, except that 50 μl

of buffer was used instead of the sample The

ACE inhibitory activity was expressed as IC50

value (μg/ml) The IC50 value was defi ned as

the concentration of inhibitor required to inhibit

50% of the ACE activity The percentage of

inhibition level was calculated as follows:

Inhibition level (%) = AControl - ASample

x 100

AControl - ABlankWhere AControl is the absorbance of control

ASample is the absorbance of the sample

ABlank is the absorbance of the blank

2.5 Fractionation of ACE-inhibitory peptides

from hydrolysate

The obtained hydrolysate from UF with the

highest ACE-inhibitory activity was used for

fractionation It was dried using freeze dryer (Flexi Dry, Dura Dry, NY, USA) The hydrolysate was fractioned using SephadexM G-15 The elution was carried out with 50 mM sodium phosphate buffer pH 7.0 at a fl ow rate of 0.3 ml/min The 3 ml fractions were collected and their absorbance was read at 220 and 280

nm A standard distribution was determined by chromatographing independently using the following standards: Reduced glutathione (429 Da), Hip-His-Leu (307 Da), and Tyrosine (181.91 Da) The fractions of SephadexM G-15 column were determined for their ACE inhibitory activity All fractions were determined soluble protein content by Lowry method

(Lowry et al., 1951).

2.6 Statistical analysis

All experiments were carried out in triplicate Analysis of variance was performed Mean comparisons were run by Duncan’s multiple range tests Analysis was performed using an SPSS package

III RESULTS AND DISCUSSION

1 Effect of hydrolysis time on degree of hydrolysis (DH)

The DH is generally used as a proteolysis monitoring parameter, and it is the most widely used indicator for comparison among different

protein hydrolysates (Guérard et al., 2002)

There was a sharp increase of DH in the fi rst

30 min (DH = 28%) and it increased slightly during 30 to 180 min hydrolysis stage From

180 min onwards, the DH rose dramatically and reached a peak of 91.9% at the end of the period (Figure 1) High value of DH resulted from the increase of short peptides These results indicated that rapid cleavage of peptides from the extracted protein solution by Alcalase occurred after 3 h

Journal of Fisheries science and Technology No.3 - 2016

2 Effect of hydrolysis time on ACE inhibitory activity of hydrolysate

ACE inhibitory activity of hydrolysate with different hydrolysis time expressed as IC50 is shown in Figure 2 IC50 value of hydrolysate decreased as hydrolysis time increased (p < 0.05) ACE inhibitory activity of extracted protein (IC50 value of 1,556 ± 16.61 µg/ml) increased after hydrolysis (IC50 value ranging from 1,233 ± 29.31 µg/ml to 831 ± 33.39 µg/ml)

It was suggested that peptides with ACE inhibitory activity could be generated during hydrolysis The ACE inhibitory activity appeared to increase as hydrolysis time increase because the lengths of peptides were shortened and increased ACE inhibitory

activity (Je et al., 2004) The highest ACE

inhibitory activity of striped catfi sh skin protein hydrolysate (IC50 value of 831 ± 33.39 µg/ml) was found at hydrolysis time of 5 h The highest ACE inhibitory activity of skin hydrolysate in the present study was almost

similar with that of blacktip shark gelatin (0.94 -

1.77mg/ml) (Kittiphattanabawon et al., 2013), salmon skin gelatin (1.17 mg/ml) (Gu et al.,

2011), and skate skin gelatin (1.89 mg/ml)

(Lee et al., 2011) Enzyme hydrolysis was

performed in order to achieve the desired degree of hydrolysis to obtain biologically active peptides From previous studies, ACE inhibitory activity of peptides increased with prolonged incubation with enzyme However, longer hydrolysis time led to the peptides lost

their ability to inhibit ACE (Wu et al., 2008; Xu

et al., 2014) The structure of amino acid for

interactions between the substrate and the active site of ACE affected ACE inhibitory

activity (Ondetti et al., 1977) Cushman

and Cheung (1971) reported that peptides containing aromatic at the C-terminal end and the branch-chain aliphatic amino acid at the N-terminal were effective for high ACE inhibitory activity because of the interaction between these amino acids at the active site of ACE

Figure 1 Degree of hydrolysis of protein hydrolysate during hydrolysis with Alcalase

Figure 2 ACE inhibitory activity of striped catfi sh hydrolysate at various hydrolysis times

Different letters on the bars indicate signifi cant differences (p < 0.05) The lower IC 50 value represents the higher ACE inhibitory activity

3 Effect of different MWCO membranes on

ACE-inhibitory activity of peptides

Permeate of MWCO 1 kDa membrane

showed the highest ACE inhibitory activity

The results indicated that molecular weight

of most ACE inhibitory peptides, which was

produced and separated from the hydrolysate, was smaller than 1 kDa This result was in

accordance with Je et al (2004), who reported

that Alaska pollack frame protein hydrolysate that having a molecular mass below 1 kDa showed the highest ACE inhibitory activity

Figure 3 ACE inhibitory activity of peptides in permeate and retentate during ultrafi ltration

of 5-h hydrolysate in batch mode (TMP = 1.5 bars, CFV = 1.5 m/s, temperature = 50 o C)

10 kDa MWCO (A), 5 kDa MWCO (B), and 1 kDa MWCO (C) membranes The lower IC 50 value represents

the higher ACE inhibitory activity

Figure 3 shows fi ltration time versus ACE

inhibitory activity of peptides in permeation and

retentiveness during ultrafi ltration of protein

hydrolysate In general, the ACE inhibitory

activity of peptides in permeance and retention

fell steadily when the operating time increase

(IC50 value increased steadily) The ACE

inhibitory activity of peptides in permeates

was always higher than that in the retentate because low molecular weight of peptides

in permeates exhibited high ACE inhibitory activity The ACE inhibitory activity (IC50 average value) of permeates of MWCO 10, 5, and 1 kDa membranes were 159.7, 125.0, and 8.3 µg/ml,respectively

4 Fractionation of ACE-inhibitory peptides

from hydrolysate

The chromatogram of hydrolysate subjected

to SephadexM G-15 column is shown in Figure 4

Amarowicz and Shahidi (1997) reported that

the optical density at 220 nm (A220) indicates

the peptide bonds and the optical density at

280 nm (A280) represents peptides, proteins

or amino acids with aromatic rings Figure 4

shows the chromatogram of the hydrolysate

from permeates of UF 1 kDa MWCO membrane

which was fractionated using SephadexM G-15 gel

fi ltration chromatography A peak of A220

was observed in fraction 4, refl ecting the

presence of peptides bonds and a distinct peak

of A280 was found in the same fraction indicated

the presence of peptides containing aromatic

amino acids The highest ACE inhibitory

activity was obtained at fractions 15 to 18 that having molecular weights 307 Da to 429 Da Similar fi ndings were also observed from

previous works by Je et al (2004); Mahmoodani

et al (2014); Raghavan and Kristinsson (2009),

who reported that peptides with molecular masses below 1 kDa showed the highest ACE inhibitory activity The peaked fractions showed the highest ACE inhibitory activity (IC50 value ranging from 1.22 to 5.88 µg/ml) (Table 1), which ranged from 141.45 to 681.72 fold higher than hydrolysate (IC50 value 831.7 µg/ml) Fractions 15-18 showing the highest ACE inhibitory activity The result suggests that peptides without or low ACE inhibitory activity was removed during fractionation while peptides with high ACE inhibitory activity were concentrated

Figure 4 Elution profi le of striped catfi sh skin hydrolysate (from UF 1 kDa MWCO membrane)

separated by size exclusion chromatography on Sephadex M G-15

Reduced glutathione (MW = 429 Da), Hip-His-Leu (MW = 307 Da), Tyrosine (MW = 181.91 Da),

were used to calibrate the standard molecular weights

Table 1 ACE inhibitory activity of peaked fractions from Sephadex M G-15 column

1 Amarowicz, R and Shahidi, F., 1997 Antioxidant activity of peptide fractions of capelin protein hydrolysates Food Chem, 58 (4): 355-359

2 AOAC, 1999 Offi cial Methods of Analysis, 16th ed Arlington, VA: Association of Offi cial Analytical Chemists

3 Cushman, D W and Cheung, H S., 1971 Spectrophotometric assay and properties of the angiotensin I-converting enzyme of rabbit lung Biochem Pharmacol, 20: 1637-1648

4 Fujita, H 2001 Human study of sardine peptides on blood pressure Nutrition Reseach, 21: 1149

5 Gu, R Z., Li, C Y., Liu, W Y., Yi, W X and Cai, M Y., 2011 Angiotensin I converting enzyme inhibitory activity of low molecular weight peptides from Atlantic salmon (Salmo salar L.) skin Food Res Int, 44: 1536-1540

6 Guérard, F., Guimas, L., Binet, A., 2002 Production of tuna waste hydrolysates by a commercial neutral protease preparation Journal of Molecular Catalysis B: Enzymology, 19: 489-498

7 Howell, N K and Kasase, C., 2010 Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Blackwell Publishing Ltd John Wiley & Sons, Inc Iowa: 203-219

8 Hue, Q H., Youravong, W., Sirinupong, N., 2013 Antioxidant activities of protein hydrolysate from the skin

of striped catfi sh (Pangasius hypophthalmus) fi llet processing waste J Fish Sci Technol Special issue: 70-77

9 Je, J Y., Park, P J., Kwon, J Y and Kim, S K., 2004 A novel angiotensin I converting enzyme inhibitory peptide from Alaska Pollack (Theragra chalcogramma) frame protein hydrolysate J Agric Food Chem, 52 (26): 7842-7845

10 Kim, S K and Byun, H G., 2001 Purifi cation and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin Process Biochem, 36: 1155-1162

11 Kittiphattanabawon, P., Benjakul, S., Visassanguan, W and Shahidi, F., 2013 Inhibition of angiotensin converting enzyme, human LDL cholesterol and DNA oxidation by hydrolysates from blacktip shark gelatin LWT Food Sci Technol, 51: 177-182

12 Li, G H., Le, G W., Shi, Y H and Shrestha, S 2004 Angiotensin-converting enzyme inhibitory peptides from food proteins and their physiological and pharmacological effects Nutr Res 24: 469-486

13 Lee, J K., Jeon, J K and Byun, H G., 2011 Effect of angiotensin-I converting enzyme inhibitory peptide purifi ed from skate skin hydrolysate Food Chem, 125: 495-499

14 Lee, S H., Qian, Z J and Kim, S K., 2010 A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats Food Chem, 118: 96-102

IV CONCLUSION

This study found that the protein hydrolysate

from striped catfi sh skin exhibited strong

ACE-inhibitory activity The ultrafi ltration usage

of 1 kDa was successful for separation ACE

inhibitory activity peptides since ultrafi ltration

of the hydrolysate resulted in a signifi cant

increase its ACE inhibitory activity in the

permeate fractions (IC50 = 8.3 µg/ml) It

was concluded that peptides receiving from

alcalase hydrolysis of striped catfi sh skin

could be utilized as a part of functional food or ingredients of a formulated drug in order to control high blood pressure

ACKNOWLEDGMENTS

The authors would like to express their sincere thanks to the Mekong 1,000 Project - The People’s Committee of Dong Thap Province - Vietnam, and the Faculty of Agro-Industry, Prince of Songkla University - Thailand

15 Lowry, O H., Rosebrough, N J., Farr, A L and Randall, R J., 1951 Protein measurement with the folin phenol reagent J Bio Chem, 193: 265-275.

16 Mahmmdani, M., Ghassem, M., Babji, A S., Yusop, S M., 2014 ACE inhibitory activity of pangasius catfi sh (Pangasius sutchi) skin and bone gelatin hydrolysate/ J Food Sci Technol 51 (9): 1847-1856

17 Ngo, D H., Ryu, B., Vo, T S., Himaya, S W A., Wijesekara, I and Kim, S K., 2011 Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacifi c cod (Gadus macrocephalus) skin gelatin Int J Biol Macromol, 49: 1110-1116

18 Ondetti, M.A., Rubin, B and Cushman, D W., 1977 Design of specifi c inhibitors of angiotensin-converting

enzyme: new class of orally active antihypertensive agents Science, 196: 441–444

19 Raghavan, S and Kristinsson, H G., 2009 ACE inhibitory activity of tilapia protein hydrolysate Food Chem, 117: 582-588

20 Shimizu, T 2003 Health claims on functional foods: the Japanese regulations and an international comparison Nutrition Research Reviews, 16: 241-252

21 Thuy, N T., N T Loc, J E Lindberg., Ogle B., 2007 Survey of the production, processing and nutritive value of catfi sh by-product meals in the Mekong Delta of Vietnam Livestock Research for Rural Development, 19: 124-103

22 Vo, T S., Ngo, D H., Kim, J A., Ryu, B and Kim, S K., 2011 An antihypertensive peptide from Tilapia gelatin diminishes free radical formation in murine microglial cells J Agric Food Chem, 59: 12193-12197

23 Wu, H., He, H L., Chen, X L., Sun, C Y., Zhang, Y Z and Zhou, B C., 2008 Purifi cation and identifi cation

of novel angiotensin-I converting enzyme inhibitory peptides from shark meat hydrolysate Process Biochem, 43: 457-461

24 Xu, W., Kong, B H and Zhao, X H., 2014 Optimization of some conditions of Neutrase-catalyzed plastein reaction to mediate ACE-inhibitory activity in vitro of casein hydrolysate prepared by Neutrase J Food Sci Technol, 51 (2): 276-284

POTENTIAL IMPACTS OF CLIMATE CHANGE ON FISHERIES

AND AQUACULTURE IN SRI LANKA (ROAMING THROUGH

THE CLIMATE CHANGE MANAGEMENT STORIES OF SRI LANKA)

Jayasinghe A.D 1 , Niroshana K.H.H 2

Received: 07/6/2016; Revised: 29/7/2016; Accepted: 26/9/2016

ABSTRACT

There is an increasing concern over the effects of climate change on aquaculture worldwide Given

a mounting evidence of the impacts of climate variability and change on aquatic ecosystems, the resulting impacts on fi sheries and aquaculture are likely to be substantial in Sri Lanka This paper reviews potential impacts of climate change on Sri Lankan Fisheries and Aquaculture together with certain possible measures that the nation can adapt to cope with the impacts The sea level rise has been identifi ed as the mostly affecting factor on the Sri Lankan Fisheries and Aquaculture Through ArcGIS techniques, the study has found that in Puttalam district 32.8%, 33.7%, 35.8% and 37.6% of aquaculture farm areas will be irreversibly affected by the future sea level rising in 2025, 2050, 2075 and 2100, respectively Insights to the possible coping strategies were also provided in the study for the government, NGOs and the private sector to cooperate collectively in search of most suitable solutions to deal with climate change.

Keywords: Climate change, impacts, aquaculture

1 Institute of Aquaculture, Nha Trang University, Vietnam

2 Department of Oceanography and Marine Geology, University of Ruhuna

I INTRODUCTION

It is a well-known fact that Sri Lanka earns

a high amount of income from its fi sheries and

moreover the sector provides about 540,000

direct and indirect employment opportunities

people island wide (The Ministry of Fisheries

& Aquatic Resources Development, 2016)

Fishery constitutes the major economic activity

in the coastal region which contains 25% of

the population The fi shery sector has received

much attention in the national development

agenda as shown by its recognition (Climate

Change Secretariat and Ministry of Environment

Sri Lanka, 2010) Although Sri Lanka is endowed

with large fresh and brackish water resources

it does not have a tradition of aquaculture and

only shrimp aquaculture and ornamental fi sh

culture have been developed to any extent

(FAO, 2016)

Capture fi shery of Sri Lanka was given

more weight in the history of the climate change

impacts on Sri Lanka Being touched thoroughly the SL stories of climate change and the fi sheries this paper provides insights on one of the most critical issue of climate change impact on Sri Lanka: the sea level rise Literature is available

in supporting to get predictions on the sea level rise in the Puttalam District of Sri Lanka Yet,

no evidence is available in favor of predicting the possible consequences of such impacts This paper consists of two main parts: (i) the holistic nature of the CC and then the CC impact on local level specifi cally Sri Lanka and (ii) the ArcGIS technique was used to make predictions about scenarios of sea level rise in Puttalam District This paper gives more weight

to the impact of sea level rise on shrimp farming

in the Puttalam district of Sri Lanka as it was the blood vein of the Sri Lankan export fi shery sector

Least developed countries in tropical regions have already been identifi ed as particularly

vulnerable to climate change because of their

greater economic and nutritional dependence

on fi sh and fewer available resources to invest

in climate adaptation (Barange et al., 2014)

Human population growth is faster in least

developed countries where fi sh provides a

larger contribution to non-grain protein needs

South Asian region stands out as a place

that is not only projected to face decreasing

catches, but also has a high dependency on

fi sheries and a sizeable, rapidly growing

population whose consumption of fi sh is likely to

increase with its rapid economic development

(Delgado et al., 2003) Climate change affects

people and nature in countless ways, and it

often increases the prevailing threats that

have already put pressure on the environment

According to the statistics presented by the

National Aquaculture Development Authority

of Sri Lanka the fi sh production by inland and

aquaculture sector is much lower than the

production by the marine fi sh catch For example,

in the year 2014 total fi sh catch from fi sheries

counted as 459300 million tons (MT) while

the fi sh catch from aquaculture accounted as

75750 MT Therefore, studies should prioritize

to investigate the impacts of climate change on

aquaculture in terms Nevertheless it is also

necessary to highlight that we should revise

the aquaculture impacts on accelerating the

climate change

II METHODOLOGY

1 Study area

Though Sri Lanka is bestowed with a great

potential of aquaculture there was no virtually

history on aquaculture until the beginning of

1980s along the coastal border of the Puttalam

district of the North Western region of Sri

Lanka Afterwards there was a rapid expansion

of the shrimp production along the coastal

boarder of the Puttalam district mainly due to

three reasons Firstly, Puttalam is endowed

with number of abundant natural resources

such as mangrove swamps, coastal lagoons,

tidal fl ats and estuaries well-suited to shrimp farming Secondly, this region is close to the Katunayake International airport and Colombo harbor together with good road facilities which allowed for swift access to infrastructure needs for the export of special fresh products And the third reason is that “industry development

in the Puttalam district coincide with a heavy demand for shrimp in international markets”

(Munasignhe et al, 2010) Depending on the

aforementioned facts, this study was carried out in Puttalam District based on mainly IPCC reports and other secondary data on Aquaculture industry in Sri Sri Lanka and Climate Change

2 Assessing sea level rise impacts on aquaculture farming industry in Puttalam district

In order to assess the sea level rise impacts on aquaculture industry in Puttalam district, information was required on the distribution of aquaculture farms in Puttalam district and the areas of inundation due to predicted sea level rise Hence, the distribution

of the aquaculture farms in the Puttalam district were extracted from Google Satellite Images

Figure 1 Distribution of aquaculture farms in

Puttalam district, Sri Lanka

Inserted fi gure is the map of Sri Lanka with the location of Puttalam district This map was constructed by using ArcGIS®

software by Esri

(Google earth, 2016) and GPS locations were

obtained by Garmin 72 Handheld GPS device

for ground verifi cation Areas of inundation due

to predicted sea level rise for 2025, 2050, 2075

and 2100 were extracted (by using manual

digitizing method) from predicted sea level rise

maps for 2025, 2050, 2075 and 2100, which

were published in IPCC report in 2007

Subsequently, the distribution of aquaculture farms in the study area and inundations layers for 2025, 2050, 2075 and 2100 were then overplayed and then potential areas

of aquaculture industry to be affected from predicted sea level rise were determined All these maps were constructed by using ArcGIS 10.1 software by Esri

III RESULTS AND DISCUSSSIONS

Figure 2 Affected aquaculture farms from inundation due to predicted sea level rise in Puttalam district in (a)

2025, (b) 2050 (c) 2075 and (d) 2100

The distribution of the aquaculture farms

(both operating and abounded) in Puttalam

district was determined by using area

calculation technique in ArcGIS 10.1 software

and the total area of aquaculture farms in this

district was recorded to be 6766 ha by the

middle of 2016

The total areas of aquaculture farms to be

affected from predicted sea level rise in Puttalam

district were determined for the years of 2025,

2050, 2075 and 2100, after overlaying the

layer of the distribution of aquaculture farms in

the study area with inundations layers for the

years of 2025, 2050, 2075, and 2100 by using

ArcGIS 10.1 software The obtained prediction

values revealed that 2216 ha, 2280 ha, 2426

ha and 2547 ha of aquaculture farm areas in

this district will be affected by inundations due

to the predicted sea level rise in 2025, 2050,

2075 and 2100, respectively (Figure 2a, 2b,

2c, 2d) Therefore, in Puttalam district 32.8%,

33.7%, 35.8% and 37.6% of aquaculture farm

areas will be irreversibly affected by the future

sea level rising by 2025, 2050, 2075 and 2100,

respectively

1 Predicted impacts of sea level rise

According to the Intergovernmental Panel

on Climate Change (IPCC) report in 2007,

for the tropical Asia the most obvious climate

related impact is the sea “Level Rise”

Furthermore it was also stated that this

densely settled area will be more vulnerable to

coastal erosion and land loss, inundation and

sea fl ooding and upstream movement the

saline/fresh waterfront, and etc The national

climate change adaptation strategy, introduced

in December 2010, recognizes the importance

of sea level rise and changes in the oceanic

environment under the areas of fi sheries,

urban development, and human settlements

(Rodrigo and Senaratne, 2014)

There are two main causes for sea level

rise: melting of ice and expansion of ocean

water from warmer ocean temperatures It was

estimated approximately 15 - 20 cm sea level rise worldwide during the last century, out of which 2 - 5 cm has resulted from melting ice and 2 - 7 from the expansion of water The forecasts for global sea level rise in this century vary considerably, but the Inter-governmental Panel on Climate Change (IPCC, 2007) has provided a central estimate

of 0.2 m and 0.5 m rise by the years 2050 and

2100, respectively According to Sri Lanka Disaster Knowledge Network (2009), the mean rate of current sea level rise of SL is 1.8 mm per year for the past century More recently it was found during the satellite altimetry era of sea level measurement, at rates in the range

of 2.9 - 3.4 ± 0.4 - 0.6 mm per year from 1993 -

2010 All in all it is obvious that global warming

is the main cause of this sea level rise which results due to anthropogenic activities such as burning coal and oil as well as deforestation which ultimately leads to increase the atmospheric concentration of heat trapping gases (Union of Concerned Scientists, 2013)

2 Impacts of sea level rise on shrimp farming in Sri Lanka

Out of all the fi sheries products in the export market shrimp comprises the highest value Production of shrimp has declined during recent years mainly due to civil war confl icts as well as due to disease outbreaks and unsustainable farming practices When considering the coastal resources for the shrimp culture in Sri Lanka thirteen of the twenty four administrative districts in Sri Lanka have maritime boundaries and the development

of the coastal aquaculture is limited to these districts (Wijegoonawardena and Siriwardena, 1996) Furthermore it was also highlighted that majority of areas suitable for shrimp farming is located in the northwestern areas with a signifi cant potential in the southern coastal areas also (NARA, 1989) In the year 2007, farmed shrimp export accounts for nearly 50%

of the total export earnings from the Sri Lankan

fi sheries (FAO, 2004) Japan is the leading

shrimp exporter from Sri Lanka and the black

tiger shrimp, Penaeus monodon is the main

species cultured (Munasignhe et al, 2010)

With sea level rise an additional issue is

the sinking shoreline causing loss of mangrove

forest protection and increases the chances of

coastal subsidence erosion and storm

damage goes up (Huxham, 2015) Location

of the river estuaries would be triggered by

sea level rise causing a great change in

fi sh habitat and their breeding grounds For

example, Penaeid prawns are bred and

developed in brackish water (where salt and

fresh water mixed up) Yet due to the sea level

rise it causes this interface backward changing

habitat of prawn When considering a different

sea level rise phenomenon for instance

fl ooding; it causes massive harm to the sector

by overfl owing shrimp pond and let the shrimps

to set free in open water (Sarwar, 2005)

IV CONCLUSION

For Sri Lanka climate change has a

signifi cant impact on many vital livelihood

opportunities and precious ecosystems

Fisheries and aquaculture are also two of the

most impacted fi elds Sea level rise has found

to be the most hazardous impact on the Sri Lankan fi sheries Since we cannot alter the nature, there is only one rational way to tackle with this phenomenon that is adapting with the upcoming challengers by trying to mitigate the causes of the impacts of the climate change Sri Lankan government together with certain other parties are running at its maximal capacity to fi nd adaptation measures towards these impacts of climate change As a concluding remark it needs to be said that the best approach to cope with the impacts of climate change on the fi sheries and aquaculture

in Sri Lanka is to enhance the adaptive capacities of the livelihoods who engage in that

fi eld and as well as to enhance the research possibilities in search of fi nding technically vital mitigation methods for both the humans and marine inland ecosystems, to cope up with theses impacts Concerning more on the adaptive measures, responsible parties should make sure that the upcoming aquaculture farms in the areas such as Puttalam are built after taking into account the predicted inundation levels due to Sea Level Rise

REFERENCES

1 Athulathmudali S, Balasuriya A, Fernando K., 2011 An Exploratory Study on Adapting to Climate Change

in Coastal Areas of Sri Lanka Working Paper no 02/2011, Published by NTNU Globalization Research Programme, Faculty of Humanities Norwegian University of Science and Technology N 7491 Trondheim

2 Basnayake, S., 2004 Impacts & Adaptation to Climate Change - A Sri Lankan Perspective Paper presented in SICCIA 28th June - 2nd July 2004, Grainau, Germany

3 CCD, 2006 Revised Coastal Zone Management Plan Coast Conservation Department and the Ministry of Fisheries and Aquatic Resources

4 Climate Change Secretariat and Ministry of Environment Sri Lanka, 2010 Sector vulnerability profi le: Agriculture and Fisheries: 37-44, Accessed on; 1st of October 2015, Available at: www.climatechange.lk

5 Delgado, C L., Wada, N., Rosegrant, M W., Meijer, S & Ahmed, M Fish to 2020: Supply and demand in changing global markets International Food Policy Research Institute and Worldfi sh Center, 2003

6 FAO, National Aquaculture Sector Overview National aquaculture sector overview - Sri Lanka National Aquaculture Sector Overview Fact Sheets Text by Siriwardena, P P G S N., in FAO Fisheries and Aquaculture Department [online], Rome, Italy, 2004, http://www.fao.org/fi shery/countrysector/naso_sri-lanka/en#tcNA0132

7 Huxham M, 2015 How Shrimp Farming Wreaked Havoc on Sri Lanka’s Coasts, ELSEVIER SciTech Connect, IPCC, 2007 Sea Level Rise Hazard Profi les of Sri Lanka [PDF], available at ; http://www.dmc.gov.lk/hazard/hazard/Report/UNDP%20BOOK%20CHAP%2007_%20SEA%20LEVEL%20RISE.pdf

8 Mawilmada N, et al., 2010 Sector Vulnerability Profi le: Agriculture and Fisheries, Prepared with assistance

from ADB TA 7326 (SRI) Strengthening Capacity for Climate Change daptation Implemented by: Climate Change Secretariat Ministry of Environment Sri Lanka, Accessed on; 10th of October 2015 Available at; www.climatechange.lk

9 Michel, D and Pandya, A., 2010 Coastal Zones and Climate Change, ISBN: 978-0-9821935-5-6 [pdf]

10 Munasinghe M.N, Stephen C, Abeynayake P,Abeygunawardena I.S., 2010 Shrimp Farming Practices in the Puttallam District of Sri Lanka: Implications for Disease Control, Industry Sustainability, and Rural Development,

Veterinary Medicine International, Volume 2010 (2010), Available at; http://dx.doi.org/10.4061/2010/679130

11 NARA, 1989 Identifi cation of suitable sites for shrimp farming: Phase I North western coastal belt

A consultancy report for Export Development Board and National Development Bank National Aquatic Resources Agency (NARA), Colombo, Sri Lanka, 291 p

12 Rodrigo C, Senaratne A 2014 Adapting Sri Lanka’s coasts and ocean resources to a changing climate - Accessed on; 4th of October 2015, See more at: http://www.ft.lk/2014/06/05/adapting-sri-lankas-coasts-and-ocean-resources-to-a-changing-climate/#sthash.pvSyoq6e.dpuf

13 Sarwar G.M, 2005 Impacts of Sea Level Rise on the Coastal Zone of Bangladesh, Lund University, pdf

14 The Ministry of Fisheries & Aquatic Resources Development, 2016 Ministry reports about the Ministry, Available at; http://www.fi sheries.gov.lk/content.php?cnid=abt_mnstry

15 Union of Concerned Scientists, 2013 Causes of Sea Level Rise, Fact Sheet [PDF], Available at; http://www.ucsusa.org/sites/default/fi les/legacy/assets/documents/global_warming/Causes-of-Sea-Level-Rise.pdf

16 Weerakoon, D.E.M., and P.P.G.S.N Siriwardena, 1988 Final report on cage and pen culture roject to the International Development Research Centre, Canada Inland Fisheries Division, Ministery of Fisheries, Colombo, Sri Lanka, 80 p

17 Wijegoonawardena, P.K.M., and P.P.G.S.N Siriwardena, 1996 Shrimp farming in Sri Lanka: Health management and environmental considerations In Health Management in Asian Aquaculture Proceedings of the Regional Expert Consultation on Aquaculture Health Management in Asia and the Pacifi c R.P Subasinghe, J.R Arthur & M Shariff (eds.):127-139 FAO Fisheries Technical Paper No 360, Rome, FAO 142p

EFFECT OF CATION CONCENTRATIONS (K+, Ca2+) AND HORMONAL STIMULATION ON SPERM MOTILITY OF COMMON CARP

Cyprinus carpio

Le Minh Hoang 1 , Pham Phuong Linh 1

Received: 25/1/2016; Revised: 08/7/2016; Accepted: 26/9/2016

ABTRACT

Semen quality assessment through sperm motility plays an important role in the production of artifi cial

fi sh and contributes to the preservation of fi sh sperm more effectively This study aims to evaluate the effect of cation concentrations and hormonal stimulation on sperm motility of common carp Experiments on the effect

of cation concentrations use two types of cations in the following concentrations: Ca 2+ (0.5; 2.5; 5 mM) and

K + (5; 25; 50 mM), then use the best cation concentration to check the effects of the injection of the hormone

on sperm motility The results showed that the best cation concentration was 50 mM (K + ) and 2.5 mM (Ca 2+ ) These parameters before and after hormonal stimulation showed no difference The results of this experiment can help us to create an environment for carp sperm activity based on the above parameters.

Keywords: Carp, Cyprinus carpio, cation, hormone stimulation

1 Institute of Aquaculture, Nha Trang University (NTU), Vietnam

I INTRODUCTION

Spermatozoa of both marine and freshwater

fi sh species are immotile in the testis and

seminal plasma Motility of spermatozoa

occurs after they are released into surrounding

aqueous environment during natural

reproduction or into a diluents artifi cial

reproduction (Islam and Akhter 2011)

Spermatozoa motility is a prerequisite

parameter in determining fi sh semen quality

and fertilizing capacity (Le et al 2011)

However, spermatozoa motility is also

infl uenced by several factors such as:

temperature, dilution ratio, different diluents,

ion concentration or osmolality (Le et al 2011)

and time after hormonal stimulation (Kowalski

et al 2012)

Some studies have carried out with

Persian surgeon - Acipenser persicus (Alavi

et al 2004), European perch - Perca fl uviatilis

(Alavi et al 2007), yellow croaker - Larimichthys

polyactis (Le et al 2011), tiger grouper -

Epinephelus fuscoguttatus (Hoang and Le,

2014) Le and Hoang (2015) reported the

effects of diluent ratio, diluents and osmolality

on sperm motility of common carp Up to now, there are no studies those related to the effect of cation concentration and hormonal stimulation

on sperm motility have been done in common carp spermatozoa

Common carp is traditional species of Vietnam It distributes in ponds, lakes, from Northern to Southern Besides, it has high commercial value on the market Nowadays, the indiscriminate exploitation of the wild with the introduction of new multi-line carp gradually causes the loss of pure carp seed The determination of the optimal environmental factors for sperm motility of common carp has an important role in enhancing the rate of fertilization as well as helps preserve carp sperm more effectively so that we will preserve pure carp seed varieties to cater for the breeding and protection of genentic resources However, there is no research or publication on the effect of cation concentration and hormonal stimulation

on sperm motility of common carp in Vietnam Therefore, this study aims to evaluate the

effect of cation concentrations and hormonal

stimulation on sperm motility of common carp

II MATERIALS AND METHODS

All experiments were carried out at the

Institute of Aquaculture in Nha Trang University

Male carp were collected from the wild and

then semen were stripped in 1.5 ml eppendofe

and held on ice and immediately brought to the

laboratory for observation and analysis

Spermatozoa motility (SM) assessment:

SM was determined immedicately after semen

collection The percentage of sperm exhibiting

rapid, vigorous and forward movement were

classifi ed under a microscope by diluting the

sperm into distilled water at ratio 1:100 (mixing

1µl semen and 99 µl distilled water)

* Effect of cation concentrations on SM:

To assess the effect of cation concentrations

on motility, semen was diluted at ratio 1:25 in

cation solution concentrantions: KCl (5 mM,

25 mM or 50 mM) and CaCl2 (0.5 mM, 2.5

mM, 5mM) Then, check SM and analyse this

results to fi nd the optimal cation concentration

* Effect of injection hormone on SM: All

treatments of effect of injection hormone

on motility were conducted similar to the

above experiments Males were injected with

hormone LHRHa Then semen was stripped after 6 hours and 12 hours These sperm were tested by the best dilution ratio 1:25, the best diluents as 0.3% NaCl, the best osmolality at

100 mOsm/kg (Le and Hoang, 2015) and the best ion Ca2+ and ion K+ (in this study)

Data representing infl uence of dilution ratio, diluents and cation concentration on motility were analyzed by one-way ANOVA using SPSS 16.0 Results are presented

as mean±standard error Difference with a probability value (P) of 0.05 (P<0.05) were considered signifi cant

III RESULT AND DISCUSSION

1 Effect of K + and Ca 2+ concentrations on sperm motility

1.1 Ion K +

Effect of the change of ions and osmotic pressure level of ambient environment on SM

has demonstrated by Morisawa (Seifi et al

2011) and they have considered as two factor of infl uence SM In family carp, ion K+may increase velocity and motile sperm so that channel K+ has capacity inhibited movement

of sperm fl agellum (Islam and Akhter 2011) Effect of K+ concentration was described in Figure 1

Figure 1 Effect of K + concentrations on sperm motility of common carp

Values with different alphabetic letters on each bar indicate signifi cant difference between concentrations of K + (P<0.05)

The results showed in Figure 1 indicated

that the best concentration of K+ is 50 mM with

time and percentage motility is 835.5±292.2 s

and 92.5±1.1%, respectively No signifi cant

difference between concentrations of 5, 25 or

50 mM, however, time motility at 5 and 25 mM

is lower than 50 mM Alavi et al (Alavi and

Cosson 2006) also indicated the same result

that solution containing 50-200 mM KCl is

able to stimulate SM effectively Increasing the

concentration of K+ of solution also increase

velocity of SM in European perch - Perca

fl uviatilis and the best SM has observed at

50 mM K+ but ion K+ has no signifi cant effect

on the proportion SM In general, the highest

percentage motility has observed at concentration of K+ above 20 mM when

measured within 15 s after activation (Alavi et

al 2007) For yellow croaker - Larimichthys polyactis, the best SM at concentration of 0.4 M (Le et al 2011) Therefore, the concentration of

K+ is different on each fi sh species

1.2 Ion Ca 2+

Sperm motility can be initiated by alteration

of the concentration of Ca2+ ions in many species, such as in cyprinids (Islam and Akhter 2011) The presence of extracellular Ca 2+

is necessary for the initiation of SM in carp

(Krasznai et al 2000) Effect of concentrations

Ca2+ on SM in carp was described in Figure 2

Figure 2 Effect of Ca 2+ concentrations on sperm motility of common carp.

Values with different alphabetic letters on each bar indicate signifi cant difference between concentrations of Ca 2+ (P<0.05)

The result showed that the optimal

concentration of Ca2+ in carp sperm is 2.5 mM

with time and percentage motility is 51±0.5 s

and 86.6±2.5%, respectively At concentrations

of 0.5 or 5 mM, motile time is 46.3±1.7 s and

40.8±2.4 s No signifi cant difference between

concentrations of Ca2+ Some studies have

indicated that the role of concentration Ca2+ in

increasing sperm motility patameters includes

time total motility, percentage motility and

velocity of sperm Studying on European

perch-Perca fl uviatilis indicated that the

concentration of Ca2+ at 2.5 mM increased

motile sperm percentage (Alavi et al 2007)

For yellow croaker-Larimichthys polyactis,

the highest motile sperm obtained in solution containing 0.2 M CaCl2 (Le et al 2011)

When extracellular Ca2+ enters into the cell membrane of sperm, it will increase the concentration of intracellular Ca2+ and stimulate sperm motility Therefore, Ca2+ is an important ion to stimulate motile sperm

2 Effect of hormonal stimulation on sperm motility

Using hormone to stimulate maturation of gonad fi sh is a popular factor in aquaculture (Zohar and Mylonas 2001) Wild carp stimulation to produce of milt in captivily

requires hormonal (Seifi et al 2011) Comparing

effect of injecting hormone on SM was described in Figure 3

The activation medium as NaCl 0.3%

was used to analyse the motile sperm before

and after hormone injection After analyzing,

the result showed that there is no signifi cant

difference (P>0.05) At concentration of 100

mOsm/kg, 24 hours after injection, motile

sperm increased slightly but not with signifi cant

difference (P>0.05) for both K+ and Ca2+ ions

Studying on other species, Krol et al (Krol et al

2009) observed time of motile sperm in

European smelt-Osmerus eperlanus increase

when stimulating by ovaprim+domperidone

For European eel-Anguilla Anguilla, injection

of HCG does not effect SM (Austriano et

al 2006) Frequently, using hormone to

stimulate releasion of sperm in carp does not

effect sperm motility

IV CONCLUSION AND RECOMMENDATION

- The optimal ion K+ concentration was

50 mM with time and percentage motility 835.5±292.2 s and 92.5±1.1%, respectively

- The optimal ion Ca2+ concentration was 2.5 mM with motile time at 51±0.5 s and percentage of motile sperm at 86.6±2.5%

- Be able to use NaCl 0.3%, medium 100 mOsm/kg, medium KCl 50 mM and CaCl2 2.5 mM for evaluating sperm motility but these parameters before and after hormonal stimulation show no difference

- Future research should use another hormones and fertility test for evaluating sperm motility

Figure 3 Sperm motility in relation time of hormonal stimulation

Different alphabetic letters on each bar indicate signifi cant difference (P<0.05)

REFERENCES

1 Alavi S.M.H and C osson J., 2006 Sperm motility in fi shes (II) Effects of ions and osmolality: A review Cell

Biology International, 30:1-14

2 Alavi S.M.H., Coss on J., Karami M., Amiri M.B and Akhoundzadeh M.A., 2004 Spermatozoa motility

in the Persian sturgeon, Acipenser persicus: effects of pH, dilution rate, ions and osmolality Reproduction, 128:819-828

3 Alavi S.M.H., Rodi na M., Policar T., Kozak P., Psenicka M and Linhart O., 2007 Semen of Perca fl uviatilis L.: Sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility Theriogenology, 68: 276-283.

4 Austriano J.F., Mr co-Jimenez F., Perez L., Balasch S., Garzon Penaranda D.L., Vicente D.S., Viudes-de-Castro J.S and Jover M., 2006 Effect of HCG as spermiation inducer on European eel semen quality Theriogenology, 66:1012-1020

5 Bastami K.D., Iman pour M.R and Hoseinifar S.H., 2010 Sperm of feral carp Cyprinus carpio: optimization of activation solution Aquaculture International, 18: 771-776

6 Cabrita E., Robles V and Herráez P., 2009 Sperm quality assessment In: E Cabrit, V Robles, and P Herráe (eds) Methods in Reproductive Aquaculture Marine and Freshwater Species: CRC Press Taylor & Francis Group:93-149

7 Cosson J., Groison L A., Suquet M., Fauvel C., Dreanno C and Billard R., 2008 Marine fi sh spermatozoa: racing ephemeral swimmers Reproduction September 1, 136:277-294

8 Islam S.M and Akhte r T., 2011 Tale of fi sh sperm and factors affecting Sperm Motility: A Review Advances

in Life Sciences, 1:11-19

9 Krasznai Z., Marian T., Izumi H., Damjanovich S., Balkay L., Tron J and Morisawa M., 2000 Membrane hyperpolarization removes inactivation of Ca2+ channels, leading to Ca2+ infl ux and subsequent initiation of sperm motility in the common carp Proc Natl Acad Sci USA, 97

10 Krol J., Kowalski R K., Hliwa A., Dietrich G.J., Stabinski R and Ciereszko A., 2009 The effects of commercial preparations containing two different GnRH analogues and dopamine antagonists on spermiation and sperm characteristics in the European smelt Osmerus eperlanus Aquaculture International, 286:328-331

11 Kowalski R.K., Hliwa P., Cejko B.I., Krol J., Stabinski R and Ciereszko A., 2012 Quality and quantity of smelt (Osmerus eperlanus) sperm in relation to time after hormonal stimulation Biology of Reproduction, 12: 231-246

12 Lahnsteiner F., Berg er B., Weismann T and Patzner A.R., 1997 Sperm structure and motility of the freshwater teleost Cottus gobio Journal of Fish Biology, 50: 564-574

13 Le M.H., Lim H.K., M in B.H., Park M.S., Son M.H., Lee J.U and Chang Y.J., 2011 Effects of varying dilutions,

pH, temperature and cations on spermatozoa motility in fi sh Larimichthys polyactis Environmental Biology, 32:271-276

14 Le M.H and Hoang H.G., 2015 Effect of dilutions ratio, diluents and osmolality on sperm motility in common carp Cyprinus carpio Journal of Fisheries Science and Technology, 4: 34-38

15 Seifi T., Imanpoor R M and Golpour A., 2011 The Effect of Different Hormonal Treatments on Semen Quality Parameters in Cultured and Wild Carp Turkish Journal of Fisheries and Aquatic Sciences, 11:595-602

16 Zohar Y and Mylonas C.C., 2001 Endocrine manipulation of spawning induction in cultured fi sh from hormone

to gene Aquaculture International, 197:99-139

EFFECT OF PACKAGING TO QUALITY AND SHELF-LIFE OF FRESH

SEA GRAPES (CAULERPA LENTILLIFERA J.AGARDH, 1837)

Le Thi Tuong 1 , Nguyen Thi My Trang 1 , Vu Ngoc Boi 1 , Nguyen Huu Dai 2

Received: 23/8/2016; Revised: 20/9/2016; Accepted: 26/9/2016

ABSTRACT

Fresh sea grapes are succulent, soft, loose and easily perishable by environmental factors The purpose

of this study was to determine the type of packaging which is suitable for preserving fresh sea grapes Three types of packaging, namely Polyamide (PA), Polypropylene (PP) and polyvinyl chloride (PVC) were used to research The results showed that while the shelf-life of sea grapes preserved in PVC was only 2 days, that in

PA, PP were up to 10 days Moreover, the weight loss, the rate of damage and total aerobic microorganisms of grape seaweeds preserved in PA were lower than that in PP This means that suitable packaging will help to maintain the quality and extend the shelf life of fresh sea grapes.

Keywords: Sea grapes, shelf-life

1 Faculty of Food Technology, Nha Trang University, Vietnam

2 Dai Phat B Plus Company, Cam Ranh city, Khanh Hoa province

I INTRODUCTION

Sea grapes (Caulerpa lentillifera, J.Agardh,

1837) is a seaweed belonging to species of

Caulerpa They wereinternationally documented

since the 70s of the 16th century They fully

contain essential nutrients, including fi ber,

vitamins, amino acids, minerals and bioactive

compounds which can be seen as a potential

food Sea grapes, nowadays, are signifi cantly

cultured, growth and processed in many

countries such as Japan, China, Korea, India

and the Philippines [8, 9, 10, 11, 12, 13]

In Vietnam, sea grapes were known in

the early years of the 20th century There had

been certain successful aquaculture research

in coastal areas in Khanh Hoa, Binh Thuan and

Phu Yen provinces [3] The estimated capacity

reached up to 100 tons of fresh seaweed per

year in 2002 However, the characteristics of

sea grapes are succulent, soft-loose so immuno

ability and stability are low Sea grapes are

easily perishable under room temperature [4]

Therefore, it is crucial to study suitable

containers to prolong self-life of sea grapes

and maintain its quality

II MATERIALS AND METHODS

1 Materials

Sea grapes: sea grapes were purchased

at Dai Phat B Plus Company, Cam Ranh city, Khanh Hoa province Then, the sea grapes are immediately transported to a Nha Trang University’s laboratory

PA (Polyamide) containers were 20x30cm in size and 0,9µm in thickness These containers were transparent and high-gloss surface Besides, they were high gas permeability resistance, particularly resistance to oxygen but low water vapor permeability PP (Polypropylene) containers were 20x30cm in size and 0,6µm in thickness PVC (Polyvinyl chloride) containers were not plasticized and 20x30cm in size, 14µm in thickness They were transparent, high mechanical strength and surface gloss PVC containers were better than PA and PP containers in water vapor and gas permeability

PA, PP and PVC containers were supplied

by the A Chau plastic packaging Co-operation company Tan Binh District, Ho Chi Minh City

The containers were produced following food safety standards, particularly QCVN 12-1:2011/BYT standard regarding plastic packaging containers that directly contact food [5]

2 Methods

2.1 Sampling and sample preparation

Sea grapes were harvested in the early morning, then transported to the laboratory Next, they were washed and re-growth before being storage The number of each collection were around 6kg All experiments were run in triplicate

2.2 General process

2.3 Experimental design

Washing: Washing removes impurities and

reduce the risk of damage during storage Sea

grapes are washed with 15 liters sea water/1kg

in 7 minutes/time and washing times are 3 With

the above washing conditions, sea grapes are

clean and their quality is not affected

Re-growth: To restore the health of harvested

sea grapes They are re-grown with 1kg/40

liter of water for 3 days and the oxygen

concentration in the water is saturated With

such conditions, the texture and color of sea

grapes are improved the best

Centrifugation: In order to remove water

on sea grape surfaces after re-growth process

and reduce damage during storage, the sea

grapes are centrifuged at the speed of 120 rpm

for 2 minutes With these conditions, water is

removed signifi cantly and the quality of the sea

grapes are not affected

Determination on the rate of sea grapes

that are damaged during storage time: Putting

exact 250g preliminarily treated sea grapes

in PA, PP and PVC containers, then securely

closing the lid All samples were stored at room

temperature (290C±1) They were checked for

every two days, damaged sea grapes were

collected, then weighted to determine the rate

of sea grapes damage during storage time

Similar experimental designs were established

to determine the rate of weight loss, aerobic bacteria total, and average sensory score of sea grapes

2.4 Analytical methods

The rate of weight loss and sea grapes damage were determined by weighting using a Germany electronic balance branded QUINTIX SARTORIUS224-1S, scales 220g, accuracy 10-4g

Damage features of sea grapes: sea grapes’ trunks were soft, thrombocytes were broken off and slimy The color of sea grapes turns to white or yellow or dark blue There stenches of rotting sea grapes

Aerobic bacteria total was determined by NMKL86:2006 [7] method

Sensory quality assessment was conducted following TCVN 3215- 79 [6] There were 5 members in assessment board All members were equipped and trained with assessing method before doing experiments

2.5 Data analysis

All experiments were run in triplicate Analysis of variance (ANOVA) was performed

to compare difference with means at the

α = 0,05% signifi cance level Then SPSS soft ware was applied to determine statistical

variance between means.

The results in fi gure 1 showed that the

packaging containers could strongly affect

the average sensory-score total regarding

storage time

In term of PVC containers, after 2 days,

there was a signifi cant decrease in sensory

quality of sea grapes, particularly average

sensory-score total was 20 at 0 storage day

then reduced to 4.2 after 2 days stored This

means that the sea grapes were damaged

after 2 days There was a similar trend

witnessed to control samples (without packaging)

In opposite, regarding PP containers, samples were good at quality after 6 stored days (scored at 16) However, after 8 days, the sensory quality was at medium (11,2 points) while PA containers showed the sensory quality was still at good (15,2 points) after 10 days stored Thus, the results indicated that using PA containers provided better sensory quality and longer shelf-life than that using PP and PVC containers

2 Effect of packaging containers and storage time on weight loss

III RESULTS

1 Effect of packaging containers and storage time on total sensory scores

Figure 1 Effect of packaging containers and storage time on total sensory scores

a,b,c,d,e: Presenting statistical variance between pairs of means according to storage time; x, y: present statistical

variance between pairs of means according to packaging containers.

Figure 2 Effect of packaging containers and storage time on weight loss

a,b,c,d,e: Presenting statistical variance between pairs of means according to storage time; x, y: present statistical variance

between pairs of means according to packaging containers

The results in fi gure 2 showed that the

packaging containers could affect the weight

loss regarding storage time PVC containers

and the control samples (without packaging)

were seen to be signifi cantly infl uenced

For examples, after 2 stored days, PVC

containers and the control samples showed the

weight loss reached 35% in comparison to 0

stored day, while the weight loss was remarkably

lower (under 10%) in term of PA and PP

containers In addition, there was no statistical

variance between two forms of containers in the fi rst 6 stored days However, the difference appeared after 6 stored days For examples, the weight loss after 10 stored days packed by

PA was 11.9% while it was 15.5% in that by

PP containers This means that PA containers were less effect to weight loss than that of PP and PVC containers

3 Effect of packaging containers and storage time to sea grapes damage

Figure 3 Effect of packaging containers and storage time on sea grapes damage

a,b,c,d: Presenting statistical variance between pairs of means according to storage time; x, y: present statistical

variance between pairs of means according to packaging containers

As can be seen in Figure 3, the spoilage

rate of sea grapes increased in accordance

with storage time in almost samples However,

the increase depended on forms of containers

For example, sea grapes which were packed

with PVC showed a remarkable increase in

the rate of damage After 2 stored days, this

percentage was 39.4% in comparison to the

day of zero

There was also difference in the spoilage

rates of sea grapes packed by PA and PP

containers after 4 stored days While sea grapes which packed by PA containers had not appeared damage after 4 days, the damage was witnessed to samples packed by PP containers, particularly low percentage (smaller than 5%) After 10 days of storage, the damage rate in sea grapes packed by PA and PP were 15.1% and 21.1%, respectively This showed that PA containers was better to be selected than that of PP containers

Bacteria were important criteria which

directly effects on sea grapes damage and food

safety Therefore, it was crucial to study the

variance of sea grapes related to storage time

The results showed that the microbial population

signifi cantly increased in samples packed

by PVC containers after 2 days of storage

This number was 6 times more than of 0 day

of storage Regarding control samples (without

packaging), the microbial population raised 5

times more than that of 0 days of storage

Besides, there were moderate increases in

samples packed by PP and PA containers

during storage time

Thus, the results in Figure 1; 2; 3 and 4

showed that PA were the best containers which

should be used to preserve fresh sea grapes

PVC containers could preserve fresh sea

grapes in 2 days but it was 10 days to PA and

PP containers However, the weight loss, the

rate of damage and aerobic bacteria total of

samples packed by PA containers were lower

than those of PP containers, and sea grapes

packed by PA containers also provided better

sensory quality than that of PP containers This

couldexplain that PVC containers were able to

be high gas and water vapor impermeability (higher than those of PP containers and PA containers) [1] Therefore, during storage time, respiratory speed increased leading to increasing temperature and produced water vapor so the sea grapes damaged quickly

IV CONCLUSION AND PETITION

PA containers showed that the sensory quality was maintained better than that of PP containers This means that, the selection of appropriate packaging containers could maintain the quality and prolong the shelf-life of fresh sea grapes

2 Recommendation

It is necessary to continue to study the effects of temperature and storage conditions

to maintain the quality of fresh sea grapes

4 Effect of packaging containers and storage time on total aerobic bacteria

Figure 4 Effect of packaging containers and storage time on total aerobic bacteria

a,b,c,d,e,f: Presenting statistical variance between pairs of means according to storage time; x, y, z: present statistical

variance between pairs of means according to packaging containers

In Vietnamese

1 Đống Thị Anh Đào, 2005 Kỹ thuật bao bì thực phẩm NXB Đại học quốc gia Thành phố Hồ Chí Minh: 188-204

2 Hà Văn Thuyết, Trần Quang Bình, 2000 Bảo quản rau quả tươi và bán phế phẩm NXB Nông Nghiệp

3 Nguyễn Hữu Đại, 2009 Di nhập và trồng rong nho biển (Caulerpa lntillifera) ở Khánh Hòa Hội nghị khoa học

toàn quốc về sinh thái và tài nguyên sinh vật lần III Hà Nội 22/10/2009: 942-949

4 Nguyễn Xuân Hòa, Nguyễn Hữu Đại, Nguyễn Thị Lĩnh, Phạm Hữu Trí, 2004 Nghiên cứu các đặc điểm sinh

lý, sinh thái của loài rong Nho biển (Caulerpa lentillifera) nhập nội có nguồn gốc từ Nhật Bản làm cơ sở nuôi

trồng Báo cáo tổng kết đề tài cơ sở năm 2004, Phòng Thực vật biển, Viện Hải dương học Nha Trang, Nha Trang

5 Quy chuẩn quốc gia QCVN 12-1:2011/BYT về an toàn vệ sinh đối với bao bì, dụng cụ bằng nhựa tổng hợp tiếp

8 Darcy-Vrillon B., 1993 Nutritional aspects of the developing use of marine macroalgae for the human food

industry Int J Food Sc Nutr 44:23-35.

9 Fleurence J., 1999 Seaweed proteins: biochemical, nutritional aspects and potential uses Trends in Food

Science and Technology 10: 25-28

10 Fu L., X.-R Xu, R.-Y Gan, Y Zhang, E.-Q Xia & H.- B Li, 2011 Antioxidant capacities and total phenolic contents of 62 fruits Food Chemistry 129(2): 345-350

11 Matanjun P, Mohamed S, Mustapha NM, Muhammad K, Ming CH, 2008 Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo J Appl Phycol DOI 10.1007/s10811-007-9264-6

12 Nisizawa K., H Noda, R Kikuchi and T Watanabe, 1987 The main seaweed food in Japan Hydrobilologia 151/152: 5-29

13 Trono G C & Jr (1988), Manual on seaweed culture: Pond culture of Caulerpa, Manual No.3 ASEAN/ SF/88.

EVALUATION OF THE QUANTITIES, VALUES AND PROPOSAL

OF UTILIZATION OF FISH BYPRODUCT FROM FISH PROCESSING

INDUSTRY IN VIETNAM

Luu Hong Phuc 1

Received: 03/6/2016; Revised: 30/8/2016; Accepted: 26/9/2016

of catfi sh byproduct and general fi sh byproduct for Vietnam.

Keywords: fi sh byproduct, byproduct utilization, fraction of byproducts

1 Faculty of Food Technology, Nha Trang University, Vietnam

I INTRODUCTION

Production of fi sheries equates to

more than 6 million tonnes each year in

Vietnam, with increase in both catch and

aquaculture, and this trend is expected to

continue (VASEP, 2016) The number of

fi sheries enterprise is also growing every

year, based on statistics provided by the

National Bureau of Agro - Forestry - Fisheries,

there were 700 fi sheries factories in 2015 in

Vietnam The main fi sheries products from

these factories are shrimp, catfi sh, tuna, and

other fi sh and mollusk Finfi sh account for

more than 45% of fi sheries production (VASEP,

2015) During this process of fi sh utilization,

fi sh processors annually create a great amount

of byproducts and wastes It has been

documented that in particular frozen catfi sh

or Pagasius catfi sh monopolizes a high

proportion of the total fi sh products, with a

share of 44 % of fi sh exported product in 2014

(VASEP, 2016) As a result a vast amount of

processing leftovers from various processing

operations have been generated, including trimmings, fi ns, scale, frames, heads, skin and viscera At present, these byproducts (frames, head, and fi lleting residuals) are used or sold cheaply to livestock owners and used for stock feeding Moreover, there is currently limited information available on fi sh byproduct weight and composition analysis within the fi sheries industry

In order to utilize more than just fi sh fi llet for human consumption and get more profi t from the raw material in the fi sh industry in Vietnam, knowledge about quantity and weight composition of byproducts is required Therefore, a study about quantities and values of

fi sh byproduct is necessary for the development

of processing techniques that convert fi sh waste and byproduct into other high value material in addition to assisting the sustainability

of fi shery itself Subsequently, the data from this study will provide the base information on the potential to use fi sh byproduct for human consumption in Vietnam

II MATERIALS AND METHODS

A survey was carried out within eight fi sh

processing factories in Vietnam including

three factories in An Giang province, three

factories in Ba Ria Vung Tau province and two

factories in Khanh Hoa province These

factories are mainly joint stock companies and

private companies The products from Shutchi

catfi sh (Pangasius hypophthlmus), Skipjack

tuna, Yellowfi n tuna and Black Marlin were

fi lleted Other kinds of fi sh were produced as

frozen product, dried whole fi sh product, and

dried fi llet products The survey focused on

every-day processing activities by workers at

the nine selected sites of each processor All

input of raw materials and all output of fl esh

and waste were counted and classifi ed into

specifi c fractions of by-product

1 Determination of waste and fl esh yield

At the random selected sites of each fi sh

processor, all raw materials (about 9 - 15kg

per each site) were weighed After processing

activities, all fl esh and waste were weighed

and classifi ed for type distribution analysis,

typical species were also recorded

Shutchi catfi sh (Pangasius hypophthlmus),

Skipjack tuna, Yellowfi n tuna and Black Marlin

were selected to determine the fraction of

byproduct, due to the fact that these fi sh

species are presently the main fi sh processing

product in Vietnamese processors The

fractions of byproduct include head, bone,

skin, viscera, fat and crushed fl esh with these

byproducts being separated into six main

anatomical parts at each site The weight of

each separated part was then determined as

a percentages of the whole weight input (raw

material) of the species

In addition to the main fi sh species, some

fi sh processors in Vietnam also process a

variety of less common fi sh species The

percentages of byproduct of these fi sh species

are presented as approximate calculations

of mixed head, bone, fi n, scale and viscera

These fi sh species include Wahoo, Lizardfi sh, Maigre, Mandarinfi sh, Yellow strip trevally, Bartail Flathead, Speck Tongue sole, Silver silago, Frigate mackerel and Cuttle-fi sh The fi sh species were divided into groups characterized by the size of raw fi sh materials and the characteristics of the byproduct Proposal of utilization of fi sh byproducts was presented based on the divided groups

2 Preservation and analysis of catfi sh byproduct samples

Due to the fact that catfi sh are presently a large proportion of fi sh production

in Vietnam, the chemical composition of head and bone byproduct from catfi sh was analyzed in order to determinate the potential utilization The catfi sh byproduct that includes head and skeletal frames were analyzed for protein, lipid and mineral Shutchi catfi sh byproduct samples were selected randomly from some fi sh processors with the samples including head and skeletal frames They were vacuum-packed and preserved in crushed ice in an insulated container and sent to the laboratory The samples were ground and homogenized before analyzed for protein, lipid and minerals

The moisture content of the samples was determined by oven-drying approximately 10g

of the homogenized samples at 1020 C to a constant weight (AOAC International 1995) Crude fat was extracted and determined by continuous extraction with petroleum ether and dried in hot air oven at 600C until it reached

a constant weight (AOAC International 1995) The ash content of the samples was determined by their mineralization at 5500C (AOAC International 1995) The total protein (crude protein, N × 6.25) content was analyzed by using Kjeldahl method (AOAC International 1995)

3 Statistical analysis

The result of anatomical fractions is expressed as the mean of percentage ± standard

error for each kind of fi sh including: Shutchi

catfi sh, Skipjack tuna, Yellowfi n tuna, Black

marlin Differences between selected sites of

Shutchi catfi sh, Skipjack tuna, Yellowfi n tuna,

Black marlin were evaluated with two-way

analysis of variance (ANOVA) Values were

considered to be signifi cantly different at

probability level of P< 0.05

The results of other kinds of fi sh may not

be evaluated by statistical analysis because of

small numbers of selected sites of signifi cant

difference between the sites, therefore, the

results were shown in approximation

III RESULTS AND DISCUSSION

1 Anatomical weight composition of

byproduct of some main fi sh species

product in Vietnam

This study reported the weight composition

in catfi sh, tuna and black marlin in the period

of survey; however the percent of byproduct

and fl esh may be dependant on seasonal

difference

The importance of the anatomical (weight)

composition analysis lies in its prediction of

the meatiness and amount of waste that can

be generated through processing As shown in

Table 1, the percentage of byproduct of

Shutchi catfi sh (67.4%), Skipjack tuna

(62.4%), Yellowfi n tuna (56.6%) and Black

marlin (36.8%) has the potential to generate

a minimum of approximately 674 kg, 624 kg,

566 kg and 368 kg respectively of byproduct

for every 1 metric ton of fi sh dressed during the

processing operation This indicates the great

potential for fi shing industries to utilize these

byproducts

Black marlin generated the lowest

byproduct yield as it has the highest fl esh yield

The low proportion of Black Marlin byproduct is

relative to the size of this species (1500 - 2000 mm)

There appeared to be a decrease in the

proportion of the byproduct with a corresponding

increase in the size of the processed fi sh In

addition to the size, tuna and Black Marlin are

meaty fi sh and therefore offer more fl esh on their fi llets

The percentage of the viscera to the whole body weight of tuna is approximately 8.8% - 9.0%, the highest among the 3 fi sh species This was in accordance with previous fi ndings highlighting the percentage of viscera to body weight in Skipjack tuna during November reported by Balogun and Talabi (1985) This larger percentage provided a possibility for enzyme or fi sh oil extraction however in order

to utilize the viscera of tuna it is necessary to have further knowledge about the possible variation in content and chemical composition Catfi sh generated the highest volume

of byproduct (Table 1) with the proportion of catfi sh byproduct (67.4 %) being signifi cantly larger than that of the Black Marlin (36.8 %) The factors that contribute to the high percentage

of catfi sh byproduct involve the high fat content that mainly arises from the pieces of fat in the abdomen; this fat is separated easily from the viscera and this valuable faction in catfi sh has been attributed towards its commercial value Many studies have been carried out to identify the potential to convert this fat to a higher value product or to add this product as an ingredient

in food or bio-diesel (Nguyen, 2003, Nguyen

et al., 1997) Another fraction of catfi sh with a high percentage of weight in byproduct is bone and head

Results from the statistical analysis regarding difference between selected sites for each kind of fi sh are shown in Table 1 Most of percentages of proximate composition

of the anatomical fractions for each kind of

fi sh had no statistical signifi cant difference between selected sites (Table 1) The difference between one or two selected sites and others may depend on the size of fi sh, worker practices However, in general, it would be possible that the result of the average percentage of byproducts in this study can

be used as approximate percentages The approximate percentages can be a special

reference to plan the recovery and utilization of each kind of byproducts by researchers and authorities The results also provide the overview of byproduct status in fi sheries industry in Vietnam.

Table 1 The average percentage proximate composition of the anatomical fractions of some

Black marlin d (Makaira

Table 2 The percentage of byproduct of some fi sh species

150mm (from 4 selected sites) Frozen skinless fi sh 60 - 62.9 skin, crushed proteinHead, fi n, viscera, Silver silago (from 4 selected

Frigate mackerel (PS:

250-260mm) (from 4 selected sites) Frozen, canned, and smoked product, 64 - 67 Head, bone, fi n, visceraCuttle-fi sh (from 3 selected sites) Frozen product 66.6 Teeth, eyes, viscera, skin

As can be seen from Table 2, the percentage

of byproduct of Yellow Strip Trevally is the

highest with 79 -82% and the lowest proportion

of byproducts is Wahoo with less than 40%

Generally, the percentages of byproduct are

dependant on the size of the processed fi sh

Therefore, the size of processed fi sh can be

used to estimate the percentage of byproduct

and divided into groups as easy reference to

establish the protocols of fi sh byproduct from

the fi sh processor

2 Chemical composition of head and bone

of catfi sh byproduct

The chemical composition of this head

and bone of catfi sh is given in Table 3 and the

amount of protein of head and bone byproduct

indicates the high potential for conversion

of this protein to protein biomass Moreover,

the high fat content in the head and bone of

catfi sh need to be considered for extraction

The chemical composition of catfi sh byproduct

in this study is comparable to that found in other studies Nguyen (2012) reported that crude protein in bone and head of catfi sh were 17,18% This result is higher than that found in this study Explanation for the higher percentage

of crude protein from the study of Nguyen (2012) may be due to the samples collected from different areas and seasonal harvest

or the errors of experiments Comparison of moisture percentage between the result from Nguyen (2009) (moisture – 62.77%) and this study showed that the percentage of moisture was lower than that from this study The difference between two studies may be due

to different methods of sample collection and errors of experiments However, in general, all studies concluded the signifi cant potential

of utilization of catfi sh byproducts for human consumption or producing added value products in the future

Table 3 Chemical composition (%) of head and bone byproduct from catfi sh

f, g Values are mean values of 3 replicates, ± standard deviation

3 Proposal of fi sh byproduct utilization

Many studies have been conducted on the

different possible modes of utilization of fi sh

processing byproduct Fish and parts of fi sh

can have a variety of potential applications

The suggestions below show some of the uses

and potential utilizations of different parts of

fi sh byproduct

Fish frame (head, bone and fi n) and cutoff

Fish frames and cutoffs are a result of fi sh

processing and can be hydrolyzed to recover

protein biomass Kim and Mendis (2006)

reviewed that several hydrolytic enzymes from

microbes, plants and animal are employed for

hydrolysis for various kinds of fi sh byproducts

such tuna, wahoo and mackerel byproducts

Guerard et al (2002) and Benkajul & Morrissey,

(1997) showed the possibility of obtaining

biologically hydrolysis and active peptides from

tuna byproduct and whiting fi sh byproducts respectively Other products that are derived from the bones, fi ns, and fi sh heads include collagen, gelatin and fi sh oil Nagai and Suzuki, (2000) investigated to extract collagens from bone and fi n of skipjack tuna, horse mackerel and Japanese sea-bass Beside collagen and gelatin, Chantachum et al (2000) also reported the separation and quality of fi sh oil from precooked and non-precooked tuna head In addition, fi sh bone, which is separated after removal of muscle protein on the frame, is another potential source to obtain calcium Many studies have concluded that inorganic substances in fi sh bone were not different among species of fi sh, They are mainly composed of calcium phosphate and hydroxyapatile (Chantachum et al., 2000; Hamada & Nagai, 1995; Larsen et al., 2000;

Luu & Nguyen, 2009; Ozawa and Suzuki,

2002) Therefore, fi sh bone should be

considered a sources of calcium to provide

regular diets for Vietnamese Fish frame and

cutoff of fi sh species above are also common

byproducts from Vietnamese fi sh processor,

therefore development of technologies for

marine processing byproduct from Vietnamese

processor will bring more value out of what is

today used as fi sh meal

Fish skin

Fish skin could be used as potential source

to isolate collagen and gelatin Many studies

have been conducted to extract collagen and

gelatin from fi sh skin that are similar with fi sh

byproducts from Vietnamese fi sh processors

(Blanco et al., 2007; Cheow, Norizah et al

2007; Nagashima et al., 2003; Mendis et al.,

2005) For example, Gomez-Gillen et al.,

(2002) extracted gelatin from several marine

species including fl at-fi sh species (sole and

megrim) Nagai and Suzuki (2000) isolated

collagen from fi sh skin of mackerel, tuna, and

sea bream

Viscera

A large amount of offal generated from

fi sh processors would be a potential source to

produce good quality fi sh oil for human

consumption It has been shown that one

method of improving the utilization of fi sh

processing byproduct is to extract fi sh oil from

fat or livers (in the case of sardine and tuna

species) (Bakes, Nichols et al 1995; Dumay et

al., 2006; Nguyen et al., 1997; Nguyen, 2003)

In addition, the internal organs of fi sh were also

examined for the potential to extract enzymes

including pepsin, trypsin, chymotrypsin and

collagenases (Haard 1992; Kim, Jeon et al

1997; Kim, Park et al 2002; Park, Lee et al

2002; Byun, Park et al 2003) There are

enzymes that may be commercially extracted

from marine fi sh viscera of common species

in large scale in Vietnam Furthermore,

intestines, stomachs, and gills of some of fi sh

species can be used as new biologically active

compounds such as the antifungal and antibacterial properties of the epidermal mucous of various fi sh species (Aspmo et al., 2005; Batista and Pires, 2002) Moreover, some researchers have identifi ed lectins from

fi sh eggs of tuna and perch and showed great potential exists to development of extraction methods (Bazil & Entlicher, 1999; Jung et

al 2003)

Crushed protein, mix component byproduct and stale raw materials

Crushed protein can be used to produce

fi sh mince and restructured products (Uresti, Te’llez-Luis et al 2004) In addition, fi sh meal

is mostly used as an ingredient in food for fi sh and crustaceans is one main product obtained from mix component byproduct and stale raw materials (Aksnes and Mundheim 1997; HevrØy, Sandnes et al 2004; Falch, Rustad et

al 2006; Toppe, Aksnes et al 2006) Beside

of fi sh meal, mixed byproduct can be used to obtain fi sh protein hydrolysates (Benkajul & Morrissey, 1997; Guerard et al 2002)

In Vietnam, the present and potential utilization for the use of fi sh byproduct in Vietnam have been suggested above, however

in contemporary Vietnam there are some major problems affecting the potential to utilize fi sh byproduct They are related to the infrastructure and the technology needed to manage the fi sh, both on board of ships and ashore To date a limited investment in new technology has been applied to byproduct processes and therefore,

it has been diffi cult to obtain a higher added-value product Furthermore, the applicability of byproduct as a bioactive compound and the nutraceutical values have not been adequately researched for commercial application In turn this leads to the current low price of byproduct

In the near future, the implementation

of the practice, procedures and machinery

to manage fi sh byproduct is necessary It is necessary to establish and implement protocols

of byproduct separation, classifi cation and

storage, as well as to examine conservation

or pre-processing alternatives when possible,

both concerned with on board and on

land management, in turn this would

assist in the maintenance of the fi sh and fi sh

product and would ensure appropriate

processing conditions

4 Proposal of utilization of catfi sh byproduct

in Vietnam

The discussion and proposal of utilization

of catfi sh byproducts was presented based on

current situation and future trend for catfi sh

industry in Vietnam One advantage of catfi sh

byproduct is in regard to the fact that the

byproduct is fresh due to the raw material in

catfi sh factories being processed as live fi sh

(all dead fi sh are eliminated) The processing

of live catfi sh is primarily due to the majority

of the catfi sh factories being located close to

the catfi sh farms, in turn catfi sh is harvested

and transferred directly to fi sh processors and

the processing time is short, resulting in the

byproduct of catfi sh being raw material suitable

for human consumption

The largest fraction of catfi sh byproduct is

the head and bones (Table 1), furthermore in

Vietnam, the fi sh head and bone can be used

as ingredients for popular dishes Moreover,

the head and bones of catfi sh have high

contents of protein and lipid (Table 3), which

may be a potential additional source of protein

for daily diet The head and bone byproduct

can be supplied directly to markets and

restaurants However, the suitability of

consuming this byproduct can be restricted

by the location of the factory due to the

preservation of the fi sh and related transportation

expenses, thus the proportion of byproduct

consumed is currently limited and a sustainable

use of this byproduct requires the

development of new methods The frame and

cutoff of catfi sh can be hydrolyzed to recover

protein biomass and extracted gelatine from

residual soft connective tissues Furthermore,

the residual bone from the extraction procedure can be used as a source of calcium The skin of catfi sh can be processed into food products suitable for human consumption In some Vietnamese factories, the catfi sh skin

is spiced and fried until it becomes crispy, the crispy fried skin is consumed in Vietnam and internationally Due to the fact that the fi sh skin mainly comprises of gelatin and collagen (Morimura et al., 2002, Kim and Mendis, 2006), the skin of catfi sh can be used to produce gelatin and collagen and has potential for a variety of applications (Doan, 2003) Despite this potential, it still limits to apply it in large industrial gelatin production

Another important category of catfi sh byproduct is viscera, found in the stomach, bladder, liver and intestine The stomach, which is separated from the internal organs,

is treated in order to convert it into an edible form for human consumption The catfi sh stomach can be fried, steamed, cooked, marinated, cooked in soup, stuffed with pig meat or fi sh meat (Vietnamese Fisheries, 2014; Vo, 2010) The bladder of the catfi sh is processed to achieve a higher value product, such as frozen breaded catfi sh bladder mixed with a spice paste, breading and frozen or dried or canned (Vietnamese Fisheries, 2014) These processed stomach and bladder products are currently produced by all catfi sh factories in Vietnam and becoming more popular in Vietnam and overseas However currently there are no studies identifying the potential application of catfi sh’ liver and intestines Alternatively it has been well documented that fi sh liver can be extracted for fi sh oil (Blanco et al., 2007), therefore liver

of catfi sh should be considered as a potential source of fi sh oil

Recently much attention has been given to catfi sh fat in Vietnam; some catfi sh factories in Vietnam have successfully produced biodiesel using catfi sh fat This product features superior characteristics to the diesel produced from

petroleum as it has a non-toxic status

and a lower rate of exhaust (Vietnamese

Fisheries, 2014)

IV CONCLUSION

The characteristics of the fi sh byproducts

from some fi sh processors in Vietnam were

analyzed and the weight compositions of main

fi sh species, currently processed in large

quantities by Vietnamese fi sh processors were

collected from a physical survey The mean

percentage yields of byproduct of catfi sh,

skipjack tuna, yellow tuna and black marlin

that includes crushed fl esh, head, bone, skin, viscera and fat were found to be about respectively 67.5%, 62.4%, 56.6% and 36.8%

of raw material input This study also presented the byproduct of some fi sh species that were processed a variety of less common fi sh species in Vietnamese fi sh processors The general utilization of each fraction of byproduct was suggested for future utilization This study confi rmed that fi sh byproduct in Vietnam was a high potential resource of nutrition and

nutraceuticals for human health

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41 Ozawa, M & Suzuki, S., 2002 Microstructural development of natural hydroxyapatile originated from

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44 Uresti, R M., Te’llez-luis, S J & Rami’rez, J A., 2004 Use of dairy proteins and microbial transglutaminase

to obtain low-salt fi sh products from fi lleting waste from silver carp (Hypophthalmichthys molitrix) Food Chemistry, 86, 257-262

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TRAFFICKING EXPLORATION AND CONTAMINATION MECHANISM

IN MAJOR DOMESTIC FISH DISTRIBUTION CHAINS IN VIETNAM

Luu Hong Phuc 1

Received: 23/6/2016; Revised: 31/8/2016; Accepted: 26/9/2016

ABSTRACT

This study described the operations of the distribution chains, as well as assembling and analysed the contaminant factor for raw fi sh of domestic fi sh distribution chains (DFDCs) in Vietnam The research collected secondary data from management boards of fi shing ports and fi sh markets, authorities’ offi ces and institutes The primary data were collected by semi-structured interviews with authorities, managers, and fi sh distributors The research also observed and monitored time/temperature of fi sh batches to cross check data obtained from interviews at points along distribution chains The result showed that fi sh raw materials have to proceed through many stages Temperature abuse occurred at all of the stages along the distribution chain The potential contaminant sources and factors affecting fi sh safety within the DFDCs were identifi ed and discussed

It can be concluded that the food hygiene and safety of the DFDCs was unsafe and unhygienic handling practices Therefore, more attention should be paid to these issues by both the government and seafood industry in Vietnam.

Keywords: Seafood safety, distribution chain, seafood handling, fi sh contamination

1 Faculty of Food Technology, Nha Trang University, Vietnam

I INTRODUCTION

Seafood had the second highest proportion

of poisonous food occurrences during 2004 -

2009 in Vietnam (VFA, 2013) The issues of

seafood-borne diseases are considered as

comparatively serious because of some

characteristics of Vietnamese seafood

distribution chain and food consumption habits

Fish is an important food source in Vietnam

The number of domestic fi sh markets is growing

every year, with increasing affl uence, the diet of

Vietnam’s people is diversifying to include fi sh

and increasing in quality (Nguyen, 2012) The

domestic fi sh distribution chains (DFDCs) in

Vietnam constitute a complex process involving

many stages and traders They are affected by

numerous factors, such as the large range of

geographical areas served, the assortment of

products offered, the seasonality of production,

and the stakeholders who participate along

the chain (Nguyen, 2008; Pham, 2008) The

government and Vietnamese people are

demanding fi sh with guaranteed food safety and higher quality standards, and these demands are being passed along each stage

of the fi sh distribution chain (Le & Nguyen, 2012; Lem, Tietze, Ruckes, & Anrooy, 2004; MARD, 2008; Vo, 2003) This poses new challenges for the seafood industry and government, because only little information has been published so far on the domestic distribution chain in terms of operations and processing issues related to seafood safety.There is little information available on the DFDCs and utilization of raw fi sh and fi sh products in the domestic markets However, information on production is available on topics such as export markets, and the seafood value chain for exports Several studies have described the distribution chains and value chains from farms or sea exploitation to the

export markets, including those of (Duijin et al

(2012); Nguyen and Nguyen (2011); Nguyen (2005); Nguyen (2011); Nguyen (2009)); and

(Vo et al , 2010, Lem et at., 2004) These

studies mentioned stakeholders and

productions involving export markets and gave

limited information about stakeholders and

operations of the domestic markets Some

studies were specifi c to seafood products such

as tuna or shrimp supply to markets in order

to analyse information for both export and

domestic supply chains (Lewis, 2005; Pham,

2008; Vo, 2006) Several studies regarding

local seafood markets and fi shing ports,

including those of (Le and Nguyen (2012);

Nguyen (2011); Pham (2008)), focused on

evaluating production and consumptions In

general, there is only minimal information

available on the DFDCs and the domestic

seafood markets The same is true for the

related stakeholders in the DFDCs This lack

of information hampers the evaluation of the

levels of food safety throughout the chains

The lack of understanding of the process

and operations within the DFDCs has

affected efforts to improve quality (Lem et al.,

2004; Pham, 2008) What is needed is a clear

understanding of the industry as a whole, and

therefore it is important to have a study that

describes the background, the principles, and

the operations of the distribution chains as well

as assembling and analysing the contaminant

factors of raw fi sh at every step of fi sh

distribution chains in Vietnam Therefore, a

key aim of the present study is to describe

the DFDCs by semi-structured interviews and

observation It is necessary to commit to

addressing in a direct way

This study provides the required information

about the distribution chains by presenting

process fl ow charts, describing the process,

and monitoring time/temperatures of fi sh

batches of raw fi sh at points along distribution

chains, and analysing contamination

mechanisms to raw fi sh within the DFDCs in

selected areas in Vietnam

II MATERIALS AND METHODS

This study was carried out from March to May 2014, at three target provinces, including: Khanh Hoa, Ba Ria Vung Tau, and Ben Tre, these provinces are generally representative

of provinces in the coastal region and have a large volume of fi sh captured in Vietnam The study of DFDCs was based on both primary and secondary data The research process was as follows:

Step 1: Collected secondary data from

management boards of fi shing ports and fi sh markets, authorities’ offi ces and institutes

Step 2: Conducted semi-structured interviews

with authorities, managers, and fi sh distributors

Step 3: Observed fi sh batches to

cross check data obtained from step 2 and monitor time/temperature of fi sh batches at points along distribution chains (Steps 2 and 3 provide primary data)

The secondary data analysis includes several earlier research reports, and annual reports from management boards of fi shing ports and fi sh markets, authorities’ offi ces and institutes The main purpose of the secondary data collection was to search for documents and reports related to the DFDCs, including those related to: operations of chains; stakeholders in distribution chains; amount of

fi sh consumption in domestic markets; and fi sh safety status at each visit place In Vietnam, most documents relating to the DFDCs are not published through the Internet, journals, or magazines Therefore, searching for the documents or reports in the departments required the researcher to make direct contact with those agencies and businesses

The primary data were collected by semi-structured interviews with authorities of provincial departments of agriculture and rural development in three provinces; managers of

fi shing ports and fi sh markets Fish batches

were then observed and informal conversations

were conducted with fi sh distributors to cross

check primary data collection obtained from

the semi-structured interviews

The semi-structured interviews were

conducted with 53 fi sh traders (41 middlemen

and 12 retailers who bought the fi sh in fi shing

ports or establishments and then sold fi sh in

retail markets) using guided questions about

numbers of workers in the distribution-line,

places and times of supply events, fi sh species

for trading, fl ow diagrams of the DFDCs,

methods of preservation, fi sh handling activities,

whether or not their business was registered

with the local government, and their relationships

with other traders

The fi sh traders were interviewed for the

purpose of describing in order to describe the

operations of fi sh distributors in the distribution

chains The interviews were held at six fi shing

ports, 29 establishments and 12 fi sh markets

All interviews ranged from thirty minutes to two

hours, the fi rst 10 to 15 minutes of which were

used to make the acquaintance of interviewees

and build rapport between the researcher and

respondents The hand written record of the

interviews was typed out on the same day or

the next day and interviewees’ answers were

collated to construct fl ow charts of DFDCs

In addition to undertaking interviews, the

fi sh traders were asked to allow the researchers

to observe their fi sh batches and to accompany

their fi sh batches from fi shing ports to retail

markets After receiving information about

places, and times of fi sh batch transportations

from the fi sh traders, the researchers randomly

selected fi sh batches at each of the 6 fi shing

ports on each day of visiting fi shing port A total

of 75 random fi sh batches from six fi shing ports

were observed with respect to the handling

procedures they underwent and researchers accompanied them in their journey to their local retail markets During the observation, all activities that involved the handling of the fi sh batches were recorded in order to confi rm the operations as described by the traders who were interviewed The proportion of fi sh batches that might be divided into smaller amounts to supply each chain was also recorded during observation

The structures of the DFDCs as described

by traders who were interviewed were confi rmed

by the researcher accompanying each fi sh batch from fi shing ports to retail markets

Time and temperature measurement

Duration of fi sh batches (time) was recorded

at intervals throughout and along the process

of domestic distribution chains Temperatures

of fi sh samples in each fi sh batch were measured along each chain with a stem digital thermometer (IP67 Testo, Australia) Time-temperature (t/T) of fi sh was measured after fi nalization of each stage In the retail markets, the temperatures of fi sh were measured twice - at times one hour and fi ve hours after fi sh reception by retailers

Fifteen batches of fi sh in each distribution chain were observed for time and temperature Therefore, a total of 60 batches of fi sh were measured for temperatures and the durations

of their travel, storage, and display at each stage along the chains were recorded 3,600 samples in 60 fi sh batches were measured for temperature (Tables 1) The temperature measurement was taken as the internal temperature of each fi sh It took about 2 minutes for each fi sh sample to be measured for temperature A random sampling strategy was applied for choosing fi sh samples for the measuring of the internal temperatures of fi sh

III RESULTS AND DISCUSSION

1 Characteristics of the domestic fi sh

distribution chains

The interviews with managers and

authorities identifi ed no exact number of fi sh

traders or number of fi sh distribution lines

involving the DFDCs in their local areas They

pointed out that there were many fi sh traders

who were not registered with the authorities

Therefore, reliable information on numbers of

traders, and on the production of the DFDCs,

could not be compiled However, the

interviewer elicited from the interviewees, the

main distributing places and stakeholders in

the DFDCs

The Vietnamese fi sh distribution chain

was examined from ship offl oads to retail local

markets This included fi shing ports or water’s

edge, middle trading places (establishments),

wholesale markets, and retail markets The

stakeholders in the distribution chain in this study

were “distributors”, embracing fi sh handlers,

middlemen, and retailers Fish handlers were

defi ned as those who handle the fi sh in their

work The main activities of the fi sh handlers

include sorting, transporting, unloading and

loading Middle traders or fi sh traders played a

role as owners or fi sh handlers, depending on

the size of their business They might organise

their business at a family household level or

as an enterprise or as a group of traders Their

jobs were trading and moving fi sh from the ship

offl oad areas to the fi nal stages of the marketing chains They did not sell the fi sh directly to the fi nal consumers The fi sh traders might operate at various levels Retailers were the last in the distribution chains They worked at local retail markets and sold the fi sh directly to consumers Their activities in local retail markets might include:

fi sh handling, fi sh scaling, and gutting

Based on semi-structured interviews with managers and fi sh traders and on the observation data, the process of domestic distribution chains can be described as follows:

Processing steps

The structure of the fi sh distribution namely:

fi shing ports, trader’s establishment, wholesale markets, and retail markets refl ects the four functional stages of fi sh distribution The relationships between each stage can be shown in terms of chains – 1, 2, 3 or 4 as in the following fi gure (see Figure 1) In this section, each centre of operation (element) was described

in terms of its own activities and internal governance, and the relationships between the stages/elements were explored

Chain (1) and (2), from fi shing ports to local retail markets or wholesale markets: fi sh may

be transported directly from ports or the water’s edge to wholesale markets or local retail markets

by middlemen traders or retailers, respectively Chain (3), fi shing port to traders’ establishments

to local retail markets: At the traders’ establishments, fi sh may be resold to the

Table 1 Number of fi sh batches and fi sh samples measured temperature

and places of measurement

Chains Total of fi sh batches

fi sh samples

6 fi shing ports establishments 10 1 wholesale markets 11 fi sh retail markets Transpo- rtation

second level traders and transported to

wholesale markets by small van In the wholesale

markets, fi sh may be sold to third level traders

or retailers Chain (4), from fi shing ports to

traders’ establishments to wholesale markets:

fi sh are gathered by the fi rst level traders (Nau

vua - Vietnamese) at fi shing ports or water’s

edge and transported to traders’ establishments

Fish may be resold to retailers or the second

level traders at traders’ establishments Then

fi sh are transported to retail markets or other

traders’ establishments before reaching local

retail markets by motorbike or small van Based

on data from the semi-structured interviews with fi sh traders and observation of 75 fi sh batches, Table 2 shows percentage amount of

fi sh and percentage of traders involved in each chain of the DFDCs Interestingly, while over 60% of fi sh was transported directly to retail and wholesale markets, the majority of middle traders transferred fi sh from the fi shing ports to their establishment before transporting them to wholesale or retail markets

Figure 1 Marine fi sh distribution chains from fi shing ports to retail markets

Table 2 Percentage of fi sh and traders in different distribution chains

Percent-batches (%)

Percentage

of traders (%)

Fishing port → Traders’ establishments → local retail markets 32

54.7Fishing port → Traders’ establishments → wholesale markets → retail markets 6.7

The distribution chain for processing and sale

of raw fi sh involves various levels of handling

from fi sh landing at fi shing ports to middle

traders to retailers Once fi sh are unloaded

from ships, they are sold to the fi rst level traders

and then proceed through a number of middle

trading levels before reaching retailers The

fi rst level traders in turn sell their fi sh to the

second level traders and so on, with each

level of trader handling regressively smaller

volumes In some cases, the retailers buy the

fi sh from different middle traders and mix the

fi sh together to sell Based on interviews with

fi sh traders and authorities, the longest middle

trading chain includes up to fi ve middlemen

before reaching the retailers at local markets

A wholesale agent who is the fi rst level trader (“nau vua” in Vietnamese) is usually registered with the provincial government Transactions between the fi rst level traders and the second level traders or retailers or between the second level traders and the third level traders are almost always based on verbal agreements without formal contracts; however, they bind

by virtue of the conventional socio-cultural norms based on friendship and family-based networks

Some of the fi rst level traders or the retailers can buy fi sh without brand name of the fi sh companies or the fi sh suppliers with no way of knowing from which fi sh company or supplier they come from, let alone from which or ship: