Effect of phyllanthus amarus schum et thonn and euphorbia hirta l extracts on the quality of striped catfish (pangasianodon hypophthalmus) fillets during iced storage

VIETNAM NATIONAL UNIVERSITY OF ARICULTURE

HOANG THI DUNG

EFFECT OF PHYLLANTHUS AMARUS SCHUM. ET THONN.
AND EUPHORBIA HIRTA L. EXTRACTS ON THE QUALITY
OF STRIPED CATFISH (PANGASIANODON
HYPOPHTHALMUS) FILLETS DURING ICED STORAGE

Major:

Food technology

Student code:

24180550

Supervisors:

DR. Tran Minh Phu
DR. Pham Kim Dang
PROF. Marie-Louise Scippo

AGRICULTURAL UNIVERSITY PRESS - 2017


DECLARATION
I hereby declare that the data and results of the research in my thesis are honest.
The work contained in this thesis has not been submitted to meet requirements for the
award of any other degrees or diploma in any educational institution. To the best of my
knowledge and belief, the thesis contains no material previously published or written by

another person, except where due reference is made in the text of the thesis.
I hereby declare that, all the help to carry out of my thesis were thanked and the
cited information in this thesis has been written clearly the sources.
Hanoi, May 10th, 2017
Master candidate

Hoang Thi Dung

i


ACKNOWLEDGEMENTS
Foremost, I would like to express my deep gratitude to my supervisor Dr. Tran
Minh Phu, Prof. Marie-Louise Scippo and Dr. Pham Kim Dang for their supervision,
useful advices, continuous guidance and suggestion throughout the study period and
significant contribution to the planning and writing of the thesis also their friendly
encouragement to complete this thesis.
I am grateful to Research and Teaching Higher Education Academy –
Committee on Development Cooperation (ARES – CCD) for awarding the scholarship
grant. I also give my thanks to Dr. Nguyen Thi Thanh Thuy for her guidance and
helping during study.
I also take this opportunity to thank to the Department of Aquatic Nutrition and
Products Processing, College of Aquaculture and Fisheries, Can Tho University that
offered facilities and technical assistances to successful completion of my study.
I would like to offer a big thanks to Mrs. Nguyen Le Anh Dao for her
enthusiasm, good advices, teaching me useful laboratory skills. My sincere thanks also
are sent to my friends, my classmates, especially, special thanks to my juniors Pham
Thanh Son, Huynh Thi Kim Duyen, Truong Minh Khang, Phan Nguyen Tuong Vy for
their assistance in the experimental parts of this thesis.
Last but not least, I am deeply grateful to my parents, my elder sister and my

grandfather for their love, support and inspiration. This successful result could not be
obtained without their encouragement.
Hanoi, May 10th, 2017
Master candidate

Hoang Thi Dung

ii


TABLE OF CONTENTS
Declaration ........................................................................................................................ i
Acknowledgements .......................................................................................................... ii
Table of contents ............................................................................................................. iii
List of abbreviation ...........................................................................................................v
List of tables.................................................................................................................... vi
List of figures ................................................................................................................. vii
Thesis abstract............................................................................................................... viii
Chapter 1. Introduction .................................................................................................1
1.1.

Introduction ........................................................................................................1

1.2.

Objectives ..........................................................................................................2

1.2.1.

General objective ...............................................................................................2

1.2.2.

Specific objectives ..............................................................................................2

Chapter 2. Literature review .........................................................................................3
2.1.

Introduction of striped catfish ............................................................................3

2.1.1.

Biological properties .........................................................................................3

2.1.2.

Nutritional properties ........................................................................................3

2.2.

Changes in fish during cold preservation ..........................................................6

2.2.1.

Biochemical changes during cold preservation.................................................6

2.2.2.

Microbiological changes during cold preservation...........................................9

2.3.

Methods used in striped catfish preservation ...................................................10

2.3.1.

Low temperature storage .................................................................................10

2.3.2.

Packaging technologies combined with low temperature storage ..................11

2.3.3.

The use of chemical preservation ....................................................................12

2.3.4.

The use of chitosan as a natural preservative combined with frozen ..............13

2.3.5.

Smoking process ..............................................................................................13

2.4.

Generality about phyllanthus amarus schum. et thonn. and
euphorbia hirta l. .............................................................................................14

2.4.1.

Phyllanthus amarus Schum. et Thonn..............................................................14

2.4.2.

Euphorbia hirta L. ...........................................................................................17

iii


2.5.

Current research in applicaion of natural antioxidant compounds in
fish presservation. ............................................................................................20

Chapter 3. Materials and methods ..............................................................................22
3.1.

Materials ..........................................................................................................22

3.1.1.

Materials ..........................................................................................................22

3.1.2.

Study period .....................................................................................................22

3.1.3.

Equipment ........................................................................................................22

3.2.

Research contents ............................................................................................23

3.3.

Methods ...........................................................................................................23

3.3.1.

Experimental design ........................................................................................23

3.3.2.

Samplings .........................................................................................................24

3.3.3.

Analytical methods ...........................................................................................24

3.3.4.

Statistical analysis ...........................................................................................31

Chapter 4. Results and discussions .............................................................................32
4.1.

Proximate composition of striped catfish fillets used in the dip experiments .32

4.2.

Temperature .....................................................................................................32

4.3.

Effect of P. amarus and E. hirta extracts on the change of total
viable counts ....................................................................................................33

4.4.

Effect of P. amarus and E. hirta extracts on the change of ph ........................34

4.5.

Effect of P. amarus and E. hirta extracts on the change of total volatile
basic nitrogen ...................................................................................................36

4.6.

Effect of P. amarus and E. hirta extracts on the changes of peroxide value ...37

4.7.

Effect of P. amarus and E. hirta extracts on the changes of thiobarbituric
acid reactive substances ...................................................................................39

4.8.

Effect of P. amarus and E. hirta extracts on the change of texture .................39

4.9.

Effect of P. amarus and E. hirta extracts on the change of water
holding capacity ...............................................................................................41

4.10.

Effect of P. amarus and E. hirta extracts on the change of sensory
properties .........................................................................................................42

Chapter 5. Conclusions and recommendations ..........................................................44
5.1.

Conclusions ......................................................................................................44

5.2.

Recommendations ............................................................................................44

Literature cited ................................................................................................................45
Appendixes .....................................................................................................................53

iv


LIST OF ABBREVIATION
Acronym

Abbreviations

AOAC

Association of Official Analytical Chemists

DHA

Docosahexaenoic acid

DMA

Dimethylamine

DPPH

2,2-diphenyl-2-picrylhydrazyl

EO

Essential oil

EPA

Eicosapentaenoic acid

FA

Formaldehyde

IC50

The half maximal inhibitory concentration

MAP

Modified atmosphere packaging

MIC

Minimum inhibitory concentration

MUFA

Monounsaturated fatty acids

OD

Optical density

PUFA

Polyunsaturated fatty acids

PV

Peroxide value

QIM

Quality index method

SFA

Saturated fatty acids

TBARS

Thiobarbituric acid reactive substances

TMA

Trimethylamine

TMAO

Trimethylamine oxide

TVB-N

Total volatile basic nitrogen

TVC

Total viable counts

WHC

Water holding capacity

v


LIST OF TABLES
Table 2.1.

Proximate compositions of striped catfish fillets ......................................... 3

Table 2.2.

Amino acid compositions of the Tra and the Basa catfish fillets
in the Mekong River Delta (dry matter basis, %) ........................................ 4

Table 2.3.

Fatty acid components (% total fatty acids) of Tra fillets............................ 5

Table 2.4.

Minerals in striped catfish fillets from Vietnam sold in Italy ...................... 6

Table 2.5.

Bacterial spoilage compounds found in Pangasius .................................... 10

Table 2.6.

Phytochemicals presented in Phyllanthus amarus..................................... 15

Table 4.1.

Proximate composition of striped catfish fillets ........................................ 32

Table 4.2.

The temperature of striped catfish fillets during iced storage (oC) ............ 33

Table 4.3.

Changes in pH of striped catfish fillets during iced storage ...................... 35

vi


LIST OF FIGURES
Fig. 2.1. P.amarus (left) and E.hirta (right) plants ........................................................ 14
Fig.4.1. Changes in total viable count of striped catfish fillets during iced storage,
P. amarus extract treatment (left) and E. hirta treatment (right) ..................... 33
Fig.4.2. Changes in TVB-N of striped catfish fillets during iced storage,
P. amarus treatment (left) and E. hirta treatment (right)................................. 36
Fig.4.3. Changes in PV values of striped catfish fillets during iced storage,
P. amarus treatment (left) and E. hirta treatment (right)................................. 38
Fig.4.4. Changes in OD value of striped catfish fillets during iced storage,
P. amarus treatment (left) and E. hirta treatment (right) .................................. 39
Fig.4.5. Changes in texture property of striped catfish fillets during iced storage,
P. amarus treatment (left) and E. hirta treatment (right) .................................. 40
Fig.4.6. Changes in WHC of striped catfish fillets during iced storage,
P. amarus treatment (left) and E. hirta treatment (right) .................................. 41
Fig.4.7. Changes in sensory of striped catfish fillets during iced storage,
P. amarus treatment (left) and E. hirta treatment (right) .................................. 42

vii


THESIS ABSTRACT
Master candidate: Hoang Thi Dung
Thesis title: Effect of Phyllanthus amarus Schum. et Thonn. and Euphorbia hirta L.
extracts on the quality of striped catfish (Pangasianodon hypophthalmus) fillets during
iced storage
Major: Food Technology
Code: 24180550
Educational organization: Vietnam National University of Agriculture (VNUA)
Research Objectives
The objective of this study is to evaluate the changes of striped catfish fillets
quality in iced storage under treatment of Phyllanthus amarus Schum. et Thonn. and
Euphorbia hirta L. extracts through parameters such as total viable counts, biochemical
(peroxide value, thiobarbituric acid reactive substances, total volatile basic nitrogen),
physicochemical parameters (pH, texture, water holding capacity) and sensory
properties.
Materials and Methods
-

Materials:
Striped catfish fillets (80-100 g) were obtained from striped catfish

processing company. Phyllanthus amarus Schum. et Thonn. and Euphorbia hirta L.
extracts were obtained from College of Natural Science, Can Tho University.
-

Methods

The striped catfish fillets were given dip treatments in P. amarus (0.02% and

004%, weight/volume) and E. hirta (0.06% and 0.2%, weight/volume) extract solutions
(4°C) and in tap water as a control, respectively for 30 min. Fish fillets were stored with
fish and ice ratio of 1:1 in weight. The effect of these extract solutions on TVC, PV,
TBARS, TVB-N, pH, texture, WHC and sensory properties of striped catfish fillets
during iced storage was periodically investigated for 16 days.

-

Analytical methods
The sensory quality of striped catfish fillets was evaluated using the quality
index method (QIM) (Sveinsdottir et al., 2003); the taste of cooked fillet
samples was scored using a nine-point scale (Simeonidou, 1997); total viable

viii


counts were determined according to the AOAC Official Method (AOAC,
2002); the texture was determined using a texture analyzer; WHC was
determined on fish fillets using the centrifugation method described by (Ofstad
et al., 1993); pH was measured using a digital pH meter; TVB-N was measured
following the method described by Velho (2001); peroxide values were
determined according to the spectrophotometric ferric thiocyanate method of
Hornero-Méndez et al. (2001); TBARS values were determined according to the
method described by Ke & Woyewoda, 1979; the moisture, ash, protein, lipid
contents were determined according to the AOAC Official Method 942.03
(AOAC, 2016).
Main findings and conclusions
The extracts of P. amarus and E.hirta could be applied and used to enhance the

iced storage of striped catfish fillets. The self life of the fillets could be prolonged until
12 days when the fillets were treated with 0.04% of P. amarus and 0.06% of E. hirta.
-

P. amarus and E. hirta extracts obviously inhibited the formation of primary lipid

oxidation and could retain their good quality characteristics in terms of sensory
assessment in striped catfish fillets after iced storage time.
-

The group of 0.04% P. amarus extract showed the antimicrobial capacity and

reduced the pH values during the initial storage period whereas E. hirta extract did not
present those properties.
-

Both of plant extract dip treatments did not affect the TVB-N, texture, WHC of

striped catfish fillets compared to the control group.

ix


CHAPTER 1. INTRODUCTION

1.1. INTRODUCTION
Striped catfish (Pangasianodon hypophthalmus), a freshwater fish species,
is the main commercial cultured fish in the Mekong delta, Vietnam (Phan et al.,
2009). Striped catfish production reached 1.22 million tons in 2015 (VASEP,
2015). Striped catfish products containing high nutritional quality (Orban et al.,

2008) are mainly exported to more than 140 countries around the world and
around 10% marketed in Vietnam.
However, striped catfish is an easily perishable product because of its
high water activity, nutrient content and presence of autolytic enzymes. The
spoilage of fish during storage is usually caused by biochemical reaction such as
oxidation of lipids, protein degradation, the microbial growth and metabolic
activities, resulting in the short shelf life and the decrease in flesh quality
(Arashisara et al., 2004). Therefore, taking some measures to delay the
deterioration of striped catfish quality and extend its preservation life are
worthwhile.
In recent years, natural antioxidants from herbals have been studied to
improve fish products preservation. Our previous researches have reported the
antibacterial activities of 20 herbal extract samples against two strains of
Aeromonas hydrophila isolated from Red Tilapia (Oreochromis sp.). Among 20
tested plants, Phyllanthus amarus Schum. et Thonn. and Euphorbia hirta L.
presented the highest antimicrobial activity by in vitro test.
Phyllanthus amarus Schum. et Thonn. (P. amarus) is one of the herbs
that show a wide spectrum of pharmacological effects including antioxidant,
antimicrobial, anticancer, anti-inflammatory, antiviral, antidiabetic activities. It
contains various bioactive compounds e.g. lignans, flavonoids, hydrolysable
tannins, triterpenes, alkaloids (Patele et al., 2011). The presence of high amounts
of phenolic compounds in the methanolic extract of P. amarus was found to have
potential antioxidant activity as its lipid peroxidation inhibition capacity and free
radical scavenging ability (Guha et al., 2010). In addition, the extract of P.
amarus also showed significant antimicrobial activity against Shigella spp.,

1


Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas

aeruginosa. The antibacterial action was mainly due to the isolated phyllanthin
(Manzumder et al., 2006).
Similarly, Euphorbia hirta L. (E. hirta) has shown biological activities
such as antioxidant, antibacterial, antifungal and anticancer (Asha et al., 2014).
Perumala et al. (2012) reported that E. hirta hold potential antimicrobial effects
against a wide array of pathogenic microorganisms and therefore can be used as a
safe, reliable, economical and natural antimicrobial source for therapeutics. This
finding may also be useful in food industry as the plant extracts can be used as
food preservatives protected against spoilage bacteria growth and food borne
pathogens. However, there have been few studies on preservative effect of P.
amarus and E. hirta in fish flesh during iced storage.
Therefore, the research entitled “Effect of Phyllanthus amarus Schum. et
Thonn. and Euphorbia hirta L. extracts on the quality of striped catfish
(Pangasianodon hypophthalmus) fillets during iced storage” was conducted to
evaluate the influence of Phyllanthus amarus Schum. et Thonn. and Euphorbia
hirta L. extracts on changes of striped catfish fillets quality during iced storage.
1.2. OBJECTIVES
1.2.1. General objective
The objective of this study is to evaluate the influence of Phyllanthus
amarus Schum. et Thonn. and Euphorbia hirta L. extracts on changes of striped
catfish fillets quality during iced storage in order to provide scientific evidence
for a response to any specific demand on striped catfish fillets quality issues
during storage.
1.2.2. Specific objectives
- To evaluate the changes of striped catfish fillets quality in iced storage
under treatment of Phyllanthus amarus Schum. et Thonn. and Euphorbia hirta L.
extracts through parameters such as total viable counts, biochemical (peroxide
value, thiobarbituric acid reactive substances, total volatile basic
nitrogen), physicochemical parameters (pH, texture, water holding capacity) and
sensory properties.

2


CHAPTER 2. LITERATURE REVIEW

2.1. INTRODUCTION OF STRIPED CATFISH
2.1.1. Biological properties
Striped catfish (Pangasianodon hypophthalmus) is one of the most
dominant freshwater fish of the Pangasiidae family farmed mainly in the
provinces of Dong Thap, Can Tho and An Giang in the Mekong Delta, Vietnam
(Phan et al., 2009). The fish is characterized by a long body and latterly flattened
with no scales, a short dorsal with one or two spines, dark grey or black fins, a
well-developed adipose and a broad mouth with two pairs of barbels (Thuong,
2008). Striped catfish is cultured in earthen pond and floating cage systems using
both artificial feeds and homemade feeds from rice bran, trash fish, and marine
fish and supplemented with vitamins, minerals, and vegetables (Phu & Hien,
2003). In the wild, mature striped catfish can reach a maximum total length of
180 cm and up to 18 kg in weight. Fish farmed in the earthen pond systems reach
1.0-1.5 kg after a year, depending on the size of fingerlings stocked (FAO, 2010).
For production feature, female fish take at least three years to reach sexual
maturity in captivity and grow to over 3 kg in weight while male fish often
mature in their second years (FAO, 2010).
2.1.2. Nutritional properties
The nutritional composition of striped catfish fillets reared in Mekong
delta are presented in Table 2.1.
Table 2.1. Proximate compositions of striped catfish fillets
Value per 100 g wet fillet
Compositions

Value

Moisture (%) (fresh weight)

71.4

Protein (%)

16.5

Total lipid (%)

7.98

Ash (%)

1.21

Total amino acids (%)

14.6
Phu et al. (2014)

3


In the study of Phu et al. (2014), the lipid content is quite high, 7.98% in
wet matter because raw fillets were analyzed without removing the fat and skin.
In other studies of Orban et al. (2008) and Karl et al. (2010), it is reported that
commercial fillets were characterized by high moisture (83.57, 82.1-83.3%), low

protein (13.6%, 13.3-15.7%) and lipid (1.84%, 1.4-3.2%) contents, respectively.
2.2.2.1. Protein
Protein of the striped catfish fillets contains a large amount of essential
amino acids such as isoleucine, leucine, lysine, phenylalanine, histidine,
methionine, threonine and valine. The concentration of these amino acids in
striped catfish on a dry matter basic is higher than in the Basa catfish exception
with threonine and valine (Men et al., 2005).
Table 2.2. Amino acid compositions of the Tra and the Basa catfish fillets in
the Mekong River Delta (dry matter basis, %)
Compositions

Tra-c

Tra-p

Basa

Arginine
Glutamic
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine

3.79

5.75
2.41
2.51
3.35
5.12
4.50
0.79
2.20
2.07
2.82

3.23
5.57
2.66
2.71
3.26
4.87
4.13
1.14
2.11
2.68
2.80

3.24
2.95
3.10
1.07
3.25
4.66
2.37

1.11
2.11
2.92
2.96
Men et al. (2005)

Tra-c: the Tra catfish in cage; Tra-p: the Tra catfish in pond; Basa: the Basa catfish in cage.

Men et al. (2005) also demonstrated that the relative patterns of glutamic
and histidine to lysine were highest in the Tra-p.
2.2.2.2. Lipid
Striped catfish fillets have low cholesterol levels accounted for 2139mg/100g wet weight. The occurring of low cholesterol contents is an
advantageous property for human nutrition (Orban et al., 2008). The fatty acid

4


profile of striped catfish fillets is dominated by saturated fatty acids (42.3% of
total fatty acids) with a high level of palmitic acid (29.33% of total fatty acids).
On the contrary, total polyunsaturated fatty acids (PUFA), which are mainly
illustrated by linoleic acid (8.43%), are contained in low percentage (17.74%) in
these fish fillets. In addition, a low but noteworthy percentage (by fillet wet
weight) of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is
present in striped catfish fillets, 4.74% and 0.31% respectively. These figures are
lower than in salmon (20.2% and 8.27%) and seabass (18.68% and 2.97%).
Nevertheless, the content of DHA and EPA in striped catfish fillets expressed on
a dry matter basis is not dramatically different from Asian seabass (Ho & Paul,
2009). Regarding to nutritional respect, striped catfish fillets are potential to be a
precious source of omega-3 fatty acids and low fat food in human diet (Ho &
Paul, 2009). The fatty acid profile of striped catfish fillets is shown in Table 2.3.

Table 2.3. Fatty acid components (% total fatty acids) of Tra fillets
Fatty acid components

Value

C14:0 (Myristic acid)
C16:0 (Palmitic acid)
C18:0 (Stearic acid)
C20:0 (Arachidonic acid)
Total SFA
C16:1n-7 (Palmioleic acid)

4.97
29.33
7.58
0.28
42.63
1.53

C18:1n-9 (Oleic acid)

30.93

C20:1n-9 (Eicosenoic acid)

1.66

Total MUFA

34.69

C18:2n-6 (Linoleic acid)

8.43

C18:3n-3 (α-Linolenic acid)

1.07

C20:5n-3 (EPA)

0.31

C22:6n-3 (DHA)

4.74

Total PUFA
Σn-3/ Σn-6

17.69
0.72
Ho and Paul (2009)

SFA: saturated fatty acids; PUFA: polyunsaturated fatty acids; MUFA: monounsaturated fatty
acids EPA: Eicosapentaenoic acid; DHA: Docosahexaenoic acid.

5

2.2.2.3. Minerals and vitamins
Striped catfish is also a good source of minerals and vitamins (Orban et
al., 2008).
Table 2.4. Minerals in striped catfish fillets from Vietnam sold in Italy
(Value per 100 g wet fillet, n=4)
Minerals

Mean ± SD

Minimum

Maximum

Na (mg)

387.5 ± 135.9

222.6

594.0

K (mg)

335.6 ± 3.42

330.7

340.2

Mg (mg)

12.08 ± 0.15

11.9

12.3

Ca (mg)

8.03 ± 1.50

5.50

10.10

Hg (μg)

0.03 ± 0.01

0.03

0.04
Orban et al. (2008)

Regarding to minerals, striped catfish fillets have shown a high sodium
content, probably due to the polyphosphates added (Table 2.4). On the contrary,
magnesium levels were found to be lower than in other fish species (Orban et al.,
2007).
2.2. CHANGES IN FISH DURING COLD PRESERVATION
2.2.1. Biochemical changes during cold preservation

2.2.1.1. Trimethylamine oxide (TMAO) reduction
Trimethylamine oxide (TMAO) is found in a large number of fish. The
reduction of TMAO to TMA (trimethylamine) is mainly caused by bacterial
action. In the early stage, the aerobic bacterial on fish use carbohydrates and
lactate as the main substrates generating electrons to concurrently produce CO2
and H2O. In this condition, the TMAO reduction is carried out by the anaerobic
bacterial such as Alteromonas, Shewanella putrifaciens, Aeromonas,
Enterobacteriacceae (FAO, 1995).

6


CH3CHOHCOOH + (CH3)3NO
Lactic acid

CH3COCOOH + (CH3)3N + H2O

TMAO

Pyruvate

CH3COCOOH + (CH3)3NO + H2O
Pyruvate

TMA

CH3COOH + (CH3)3N + CO2 + H2O

TMAO

Acetic acid

TMA

TMA is a volatile amine which is associated with the typical fishy spoiled
odor and is part of spoilage pattern of many fish species. TMA content is used to
measure microbial degeneration leading to fish spoilage during the time storage.
The level of TMA mainly depends on the amount of TMAO in initial fish. The
fresh fish will be definitely rejected when the level of TMA is around 10-15 mg
TMA-N/100 g in aerobically stored fish and about 30 mg TMA-N/100 g in
packed storage (Dalgaard et al., 1993).
In addition, the muscle tissue fish present endogenous enzymes which are
responsible for the transformation of TMAO into dimethylamine (DMA) and
formaldehyde (FA). The enzyme responsible for formation of formaldehyde is
called TMAO-ase or TMAO demethylase. During cold frozen storage,
formaldehyde induces the denaturation of the muscle proteins leading to the
toughening of fish muscle and loss of its water holding capacity. Gill et al.
(1979) reported that the amount of FA is increased by physical maltreatment to
the catch prior to freezing and by temperature fluctuations during the time frozen
storage.
2.2.1.2. Proteolysis
A variety of proteolytic enzymes e.g. cathepsins, calpains and
collagenases in fish muscle become active after catching and storage. Although
several proteolytic enzymes are discovered in the fish tissues, the cathepsins play
a major role in the autolytic degradation of fish tissue. During processing and
storage of fish products, these enzymes are responsible for proteins degradation
related to extensive softening of the tissue (Engvang & Nielsen, 2001). Only a
small level of ammonia is formed by autolytic proteolysis, but the majority
results from the deamination of amino-acids and nucleotide catabolites. In iced
fish fillets, some spoilage bacteria namely Pseudomonas, Achromobacter are

able to produce decarboxylase enzymes which will convert lower molecular
weight compounds of fish muscle such as free amino acids or peptides of fish
muscle into ammonia, dimethyl sulfide and biogenic amines. Some biogenic

7


amines are toxic for the consumer such as histamine or tyramine. Other biogenic
amines are responsible for negative flavours leading to the low commercial value
of fish fillets (Ghaly et al., 2010).

R-CH2-CH(NH2)-COOH

deaminase oxidative

α-ceto-acid Decarboxylase

Decarboxylase

R-CH2-CH2-NH2

RCH2-CO-COOH + NH3

oxydase

RCH2-COOH + NH3

Amine
The combined total amount of TMA, NH3 and other volatile basic
nitrogenous compounds is called total volatile basic nitrogen (TVB-N) and is

widely used as an indicator of fish deterioration. In the spoiled fish, after TMA
has reached its maximum level, TVB-N levels will continue to increase owing to
the formation of NH3 and other volatile amines (Wu & Bechtel, 2008). The
different acceptability levels of TVB-N for human consumption have been
reported by several authors as 25–35 mg/100 g (Ababouch et al., 1996) or 25–30
mg/100 g (Lopez-Caballero et al., 2000).
Additionally, during the time of spoiling iced fish, S. putrefaciens and
some Vibrionaceae can also cause degradation of cysteine and methionine
leading to the forming of volatile sulphur compounds such as hydrogen sulphide
(H2S), methyl mercaptan (CH3SH), dimethyl sulphide ((CH3)2S). Volatile
sulphur compounds are responsible for putrid odor of fish products even at ppb
levels (FAO, 1995).
2.2.1.3. Lipid oxidation
Fish lipids contain polyunsaturated fatty acids which are highly sensitive
to oxidation by an autocatalytic mechanism. The process involves three stages
including initiation, propagation and termination. In the first step, lipid free
radicals are formed through catalysts such as heat, metal ions and irradiation and
then these lipid radicals quickly react with atmospheric oxygen to form peroxyl
radicals. In second stage, the peroxyl radicals react with other lipid molecules
resulting in hydroperoxides and new free radicals. The hydroperoxides can be
determined as peroxide values. During termination, the hydroperoxides are
readily broken down, catalyzed by heavy metal ions, to secondary autoxidation

8


products containing mostly aldehydes, ketones, alcohols, small carboxylic acids
and alkanes. A variety of the produced aldehydes can be determined as
thiobarbituric acid-reactive substances (TBARS) (Ghaly et al., 2010).
In addition to lipid oxidation, fatty fish also undergo hydrolysis due to

endogenous lipolytic enzymes and some psychrotrophic microorganisms during
fish spoilage. These kinds of spoilage usually come in the later stage of spoilage
and happen during chilled and freezing fish preservation. The main enzymes in
lipid hydrolysis are triacyl lipase, phospholipase A2 and phospholipase B. In first
step of fats hydrolysis, enzyme lipases split the triglycerides into glycerol and
free fatty acids leading to negative changes in taste, smell of fish flesh associated
with rancidity and also the changes in texture by binding covalently to fish
muscle proteins (Ghaly et al., 2010).
2.2.2. Microbiological changes during cold preservation
The bacteria on fish will pass through a lag phase from 1 to 2 weeks
during ice storage. Following that, the exponential growth phase will begin and
reach numbers of 108 -109 cfu/g flesh or cm2 skin after 2-3 weeks. However, in
fish stored in ambient temperature, the level of the bacteria reaches 107-108 cfu/g
in 24 hours (Gram et al., 1990). In addition, in fish stored in iced under anaerobic
conditions or in CO2 involving atmosphere, the amount of the psychrotrophic
bacteria such as S. putrefaciens and Pseudomonas spp. is often much lower than
in aerobically stored fish. Nevertheless, the level of typical psychrotrophic
bacteria such as P. phosphoreum reaches a level of 107-108 cfu/g when the fish
spoils (Dalgaard et al., 1993).
During the time of ice storage, microbial growth and metabolism is the
main cause of fish spoilage resulting in a high amount of volatile compounds as
well as biogenic amines associated with negative flavors and odors (Ghaly et al.,
2010). Among various species spoilage bacteria, Pseudomonas spp., Aeromonas
spp. are the specific spoilers of tropical freshwater fish stored aerobically in ice.
S. putrifaciens has been isolated from tropical freshwaters, but does not play an
important role in the spoilage of freshwater fish during ice storage (Gram &
Huss, 1996). In one of the few reports, Noseda et al. (2012) reported that the
dominant spoilage microorganisms of thawed Vietnamese Pangasius fillets
during chill stored modified atmosphere packaged were Serratia, Pseudomonas,

9


Carnobacterium and Brochothrix thermosphacta. In addition, it indicated that
Serratia spp. are present on frozen Vietnamese Pangasius products before
exporting. The compounds formed during spoilage by microbial metabolism are
listed in Table 2.5.
Table 2.5. Bacterial spoilage compounds found in Pangasius
Specific spoilage bacteria

Spoilage compounds

Shewanella putrifaciens

TMA, H2S, CH3SH, (CH3)2S, HX

Photobacterium phosphoreum

TMA, HX

Pseudomonas spp.

Ketones, aldehydes, esters, non –H2S sulphides

Vibrionacaea

TMA, H2S

Aerobic spoilers

NH3, acetic, butyric and propionic acid
Ghaly et al. (2010)

TMA: Trimethylamine; H2S: Hydrogen sulphide; CH3SH: Methylmercarptan;
(CH3)2S: Dimethylsulphide; HX = Hypoxanthine, NH3: Ammonia

2.3. METHODS USED IN STRIPED CATFISH PRESERVATION
There was a wide range of preservation techniques utilized to prevent
spoilage, especially caused by microorganisms, and to extend the shelf life of
fish after postmortem such as icing, chilling, freezing, chemical preservation,
salting and smoking, fermentation, canning. Nevertheless, in the industry today,
low temperature storage and chemical techniques are the most predominant
methods to control the microbial and biochemical changes in freshly caught fish
during distribution and marketing (Ghaly et al., 2010).
2.3.1. Low temperature storage
The low temperature storage is an effective technique for the preservation
of several fish, but they do not enhance the product quality. This method reduces
the physical and biochemical reactions and microbial metabolism leading to fish
spoilage during the time storage (Ghaly et al., 2010).
2.3.1.1. Iced storage
The advantages of using ice as a cooling medium during fish storage
include giving a quick cooling capacity, a cleaning effect during melting,

10


harmlessness and portability (Nalan and Pinar, 2015). Hossain et al. (2005)
reported a shelf life of 20 days for Pangasius fillets stored under ice in an
insulated box. Viji et al. (2014) evaluated the shelf life of suchi catfish steaks
under chilled (4ºC) and iced (0ºC) storage conditions with the ratio of fish to ice

was 1:1 and the melted ice was replaced daily to conserve the ratio to achieve a
temperature 1-2ºC. The result showed that although the chilled and iced steaks
were rejected on the 14 and 17 days respectively by sensory analysis, all the
biochemical quality parameters including pH, total volatile base nitrogen (TVBN), peroxide value (PV) and thiobarbituric acid reactive substances (TBARS)
were within the acceptable limit of human consumption even after the rejection.
2.3.1.2. Freezing storage
Preservation fish for longer periods can be obtained by freezing. Freezing
is the process of removing heat from product to lower product temperature to 18°C or below. This method is efficient to minimize microbial contamination;
however, the enzymatic activity can still continue at a slow rate in frozen fish.
Many parameters can affect the survival of spoilage bacteria during freezing
storage such as microorganisms and fish species, initial fish quality, methods of
catch and the handling and storage processes aboard the fishing vessel (Ghaly et
al., 2010). Akter et al. (2014) investigated the effects of freezing storage method
at -20ºC on the shelf life of Pangasius catfish fillets. This study concluded that
sensory quality of fish fillets was found in acceptable conditions for 120 days of
frozen storage before it becomes inedible.
2.3.2. Packaging technologies combined with low temperature storage
2.3.2.1. Oxygen scavenger packing technology
Oxygen scavengers are able to eliminate oxygen contained in the
packaging headspace and in the product or permeating through the packaging
material during storage. Therefore, packaging containing oxygen scavenger is
advantageous in extending the shelf life of fresh fish products. According to
Mohan et al. (2008), oxygen scavenger was efficient in decreasing oxygen
concentration inside the package by 99.58% within 24h. By using this technique,
the use of a vacuum packing machine can be avoided. Furthermore, the shelf life
of Pangasius fillets was extended up to 20 days, maintaining its chemical,
microbiological and sensory qualities, while the control samples packaged in air

11

and also stored at a temperature between 0ºC and 2ºC were found acceptable only
up to 10 days.
2.3.2.2. Modified atmosphere packaging
The modified atmosphere packaging (MAP) can be combined with cold
storage to considerably extend the freshness and shelf life of fish products. The
effect of four packaging conditions of Pangasius hypophthalmus fillets including
air packaged, vacuum packaged, MAP 1(50% CO2, 50% N2) and MAP 2 (50%
CO2, 50% O2) on microbiological spoilage growth was evaluated during storage
at 4ºC (Noseda et al., 2012). The result showed that the shelf life of the fillets
packaged in air, vacuum, MAP 1 and MAP 2 was 7, 10, 12 and 14 days
respectively. The combination of 50% CO2 with 50% O2 additionally inhibited
on the microbiological growth mainly lactic acid bacteria such as
Carnobacterium maltaromaticum and Carnobacterium divergens. Therefore, this
combination dramatically prolonged the shelf life of fish fillets compared to air
and vacuum packaged fillets. In addition, this study also illustrated that several
volatile compounds such as ethanol, 2,3-butanediol, diacetyl, acetoin, ethyl
acetate, acetic acid and hydrogen sulfide, methylmercaptan, carbon disulfide and
dimethyl disulfide were found in the headspace of Pangasius fillets.
2.3.3. The use of chemical preservation
Many organic acids such as lactic acid, acetic acid, gallic acid, citric acid
are widely used in fish preservation because of their availability, low commercial
price and wide range of permitted concentrations for their use. Organic acids can
be directly added to fish samples or included in aqueous solutions where fish
fillets can be soaked for a certain time before storage. These compounds show
some antimicrobial properties so that the shelf life of fish fillets is enhanced
(Sanjuás-Rey et al., 2011, 2012; García-Soto et al., 2014).
Duy & Ha (2014) investigated the alone and combined effect of acetic
acid and hot water treatment on total bacteria and E.coli of contaminated striped
catfish fillets. The results indicated that a lower bacterial growth was observed in

striped catfish muscle treated with hot water at 75 ºC during 15s and acetic acid
at concentrations 2% during 120s by comparison with control samples. After 7
days storage at 4ºC, the decrease in E. coli and bacterial total levels of treated
samples was 3.65 log cfu/g and 5.65 log cfu/g in comparison with the control

12


fish. This study suggested that the combination of acetic acid and hot water
treatment can be applied to decrease E. coli and bacterial total in order to assure
the safety of catfish fillets, especially for exportation. Another research evaluated
the effect of gallic acid combined with gelatin coating on the quality changes of
refrigerated striped catfish paste by determining microbiological, texture,
peroxide value and sensory parameters for 10 days (Thuy et al., 2015). During
the time of storage, the increase in PV (from 0.15 to 2.32 meq kg-1) of Tra fillets
coated a solution of 2% gelatin with gallic acid at the concentration of 2% was
dramatically lower than control fillets (from 0.25 to 4.07 meq kg-1). The obtained
results indicate that gelatin in the form of coating enriched with gallic acid could
more effectively maintain the good quality and could extend the shelf life of striped
catfish fillets by prevent the lipid oxidation during the refrigerated storage.
2.3.4. The use of chitosan as a natural preservative combined with frozen
Chitosan, the deacetylated form of chitin, has been widely applied to the
preservation of seafood products because of its harmless feature, antimicrobial,
antifungal activities, biodegradability and film forming property (Fan et al.,
2009; Li et al., 2012). Thuy & Thu (2011) compared the preservative capability
of chitosan solution and polyphosphates to striped catfish fillets under frozen
storage. The research results showed that using chitosan concentration of 0.5%
for 25 minutes decreased the change of quality of fish fillets such as weight loss,
protein content and lipid content, and improved sensory quality after 6 months of
storage at -20°C. Moreover, chitosan showed a higher antimicrobial activity than

polyphosphates. Jeyakumari et al. (2016) compared the shelf life and quality of
fish products made with chitosan-corn flour and products prepared without
chitosan. The results shown that sensory acceptability, texture and color
attributes were higher for products incorporated with chitosan. The shelf life of
the product incorporated with chitosan (0.75 %) is extended by 7 days compared
with the control products during chilled storage.
2.3.5. Smoking process
Smoking of fish products is one of the most ancient preservation
technologies. The wood smoke contains amounts of flavorings, antibacterial and
antioxidant substances such as phenol and carbonyl compounds, phenolic
compounds (guaiacol, 4-methylphenol and 2,6-dimethoxyphenol). Therefore, the

13


smoking method can increase the shelf life and contribute to the distinctive
flavor, odor and color of the products (Kostyra & Baryłko-Pikielna, 2006). Luc et
al. (2013) reported that the smoking process of Pangasius fish fillets in smoking
temperature of 34.4 °C and smoking time of 8h55 minutes showed the strongest
effect in inhibiting total aerobic bacteria and improving sensory value. In another
study, the combination of 10% salt and 10% garlic on smoked catfish yielded
best outcome as showed the more useful nutrient property, lower moisture,
higher fat, ash and protein. The study concluded that dipping in a concentration
of salt and garlic before smoking improves overall quality and shelf life of
striped catfish (Begum et al., 2012).
2.4. GENERALITY ABOUT PHYLLANTHUS AMARUS SCHUM. ET
THONN. AND EUPHORBIA HIRTA L.

Fig. 2.1. P.amarus (left) and E.hirta (right) plants
2.4.1. Phyllanthus amarus Schum. et Thonn.

2.4.1.1. Biological properties
Phyllanthus amarus Schum. et Thonn. (P. amarus) belongs to the family
of Euphorbiaceae and is widely spread across the tropical and subtropical regions
of the world including Vietnam. The plant is a branching annual herb which
grows up to 30-60 cm high and has slender stem, yellowish flowers, distichous
leaves. The plant is characterized by typically bitter taste, astringent, cooling,
diuretic, stomachic, febrifuge and antiseptic. In addition, P. amarus mainly
occurrs as a weed in cultivated and waste lands (Patel et al., 2011).
2.4.1.2. Traditional use
There is a wide range of traditional usefulness of P. amarus in health
problems such as dropsy, diarrhea, dysentery, intermittent fevers, jaundice, and

14


diseases of urogenital disorders, scabies ulcers and wounds. It is widely used in
traditional medicine because it has various positive properties including
antiseptic, styptic, carminative, and coolant, febrifugal, stomachic, astringent and
diuretic properties (Joseph & Raj, 2011). The use of medicinal plants for the
treatment of human diseases has been in practice for a very long time. In folk
medicine P. amarus has reportedly been used to treat jaundice, diabetes, otitis,
diarrhea, swelling, skin ulcer, gastrointestinal disturbances and blocks DNA
polymerase of hepatitis B virus during reproduction (Oluwafemi & Debiri, 2008).
2.4.1.3. Phytochemical properties
P. amarus contains various phytocompounds inclunding alkaloids,
flavonoids, hydrolysable tannins (ellagitannins), major lignans, polyphenols,
triterpenes, sterols and volatile oil. Several phytoconstituents isolated from this
plant are listed in Table 2.4
Table 2.6. Phytochemicals presented in Phyllanthus amarus
Secondary metabolites

Lignans
Flavonoids

Ellagitannins

Alkaloids

Triterpenes
Sterols
Volatile oil

Phytoconstituents
Phyllanthin, hypophyllanthin, niranthin, phyltetralin,
nirtetralin,
isonirtetralin,
hinokinin,
Lintetralin,
isolintetralin
Quercetin, quercetrin, rutin, gallocatechin, astragalin,
kaempferol, phyllanthusiin
Geraniin, amariin, furosin, geraniinic acid B, amariinic
acid, amarulone, repandusinic acid A, corilagin,
isocorilagin, elaeocarpusin, ellagic acid, phyllanthusiin
A, B, C and D, gallic acid, 1,6-digalloylglucopyranose,
4-O-galloylquinic acid
Securinine, dihydrosecurinine, tetrahydrosecurinine,
securinol, phyllanthine, allo-securine, nor-securinine, 4methoxy dihydrosecurinine, 4 hydrosecurinine,
4-methoxy-nor-securinine, epibubbialine,
4-methoxytetrahydrosecurinine, isobubbialine
Phenazine, phyllanthenol, phyllanthenone, ursolic acid,

phyllantheol, oleanolic acid
Amarosterol A, amarosterol B
Linalol, phytol
Patel et al. (2011)

15