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HUE UNIVERSITY

UNIVERSITY OF SCIENCE

LE NHAT TAM

INVESTIGATE THE VARIATION OF THE

FACTORS INFLUENCING THE POST-HARVEST

QUALITY OF BLACK TIGER SHRIMP DURING

STORAGE AT 0

O

C

Summary of PhD Dissertation

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HUE UNIVERSITY

UNIVERSITY OF SCIENCE

LE NHAT TAM

INVESTIGATE THE VARIATION OF THE FACTORS

INFLUENCING THE POST-HARVEST QUALITY OF

BLACK TIGER SHRIMP DURING STORAGE AT 0

C

Major : Organic chemistry

Code: 62. 44. 01. 14

PhD DISSERTATION

Supervisors:

1. Associate professor. Tran Thi Van Thi

2. Associate professor. Do Thi Bich Thuy

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INTRODUCTION

Aquatic products are important sources of nutrients containing high
levels of amino acids, peptides, proteins and other essential nutrients for
daily meals. The decomposition of aquatic species occurs during
preservation by microbial activity and chemical reactions.

The method used to evaluate the quality of aquatic products depends
on their characteristics, and the preservation conditions. Each method
only assessed one stage of change, or one aspect of the preservation
process. Thus, a combination of different methods is very neccessary to
evaluate all changes of aquatic products during storage giving more
accurate, and reliable results.

Therefore, we carried out the research entitled "Investigate the 
variation of the factors influencing the post-harvest quality of black 
tiger shrimp during storage at 0 oC "

In this work, we conducted a study to find out the correlation among

sensory, chemical and microbial changes during preservation of shrimp
(Penaeus monodon). Basing on the obtained results, the method using to 
classify the quality of tiger shrimp is suggested

2. Research objectives
3. Material

Black tiger shrimp (Penaeus monodon), phylum: Arthropoda, 
subphylum: Crustacea, order: Decapoda, family: Penaeidae, genus:
Penaeus, species: Penaeus monodon.

4. Methodology

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total aerobic microorganism (TVC), and QIM

- Quantitative methods of chemical indices: TVB-N, TMA-N,
histamine, hypoxanthine and pH values.

- Statistical analysis: using Statgraphics centurion XVI software, linear
model by MS. Excel (2010).

5. New scientific results

The results indicated some important findings as follows:

+ It is a very first time that histamine and hypoxanthine indices were
used to evaluate the shrimp quality.

+ Changes in the sensory and chemical qualities of black tiger shrimp
were observed during storage at 0 oC.

+ The linear regression models between QI score, TVB-N, TMA-N,
histamine or hypoanthine with storage time were found.

+ The combination of sensory evaluation and chemical methods could
be appled to classify shrimp quality.

6. Conclusion

The results of this work provided primary infomation in evaluating the
quality of aquatic products after harvest.

CHAPTER 1. LITERATURE REVIEW
1.1 Introduction

Characteristics of black tiger shrimp

Chemical composition of some shrimp species

1.2 Type of spoilage and sensory changes during storage: spoilage is
caused by microorganisms, enzymes or chemical reactions.

1.3 Quality assessing method: sensory evaluation, chemistry, physics,
and microbiology measurement.

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1.5 Quantitative method: high performance liquid chromatography

(HPLC); ultraviolet-spectral method

1.6 Some models of quality assessment are based on a combination of
different methods

Chapter 2. RESEARCH OBJECTIVES AND METHODS
2.1. Research objectives

- Investigating the correlation between sensory, chemical, and
microbiological changes of tiger shrimp preservation (Penaeus

monodon) during storage at 0 ° C basing on quality indices.

- Developing the quality classification scale of tiger shrimp basing on
quality indices.

2.2. Research content

The research content of the study consisted 5 parts:

- Developing method for assessing the quality of shrimp (Penaeus
monodon) by QIM.

- Developing quantitative method of hypoxanthine on shrimp by HPLC.
- Developing linear regression equations between TVB-N, TMA-N,
histamine and hypoxanthine.

- Investigating the sensory and chemical changes of black tiger shrimp
during storage at 0oC and the correlation between sensory quality and
chemical quality.

- Developing the quality classification scale basing on sensory and
chemical indices.

2.3. MATERIALS, CHEMICALS AND EQUIPMENTS
2.3.1.Materials

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Fig 2.1 Experimental design
2.3.2. Chemicals and equipments

The chemicals used in this research were good quality.
2.4. MEASUREMENT

2.4.1 Determining TVC method (Leboffe et al.) [140].
2.4.2. QIM method

2.4.2.1. Sample preparation (according to 2.3.1)
2.4.2.2. Selecting and training the panel

The panel was selected and trained carefully for the sensory evaluation
[226].

Steps for organizing the panel:

Step 1: Primary recruitment for the panel

Fig. A schematic overview of the experimental study. QIM refers to quality index method;
TVB-N refers to total volatile basic nitrogen; TMA-TVB-N refers to trimethylamine nitrogen; His refers to

histamine; Hx refers to hypoxanthine, TVC refer to total visible counts

Shrimp was stored at 0

QIM
n =3

Farm 1 Farm 2 Farm 3

TVB-N
n = 3

Shrimp harvested from farm (10 kg per farm)

Washed using filtered clean flowing water

Packaged in polyethylene bags

TMA-N
n = 3
Stored in ice in polyethylene boxes

Transported to the laboratory

His
n = 3

Hx
n = 3

Analysis

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Step 2: Recruiting according to the requirement
Step 3: Training the testers

Step 4: Final recruitment the testers for official assessment
- Primary recruitment for the panel [110]

20 people participated in the recruitment, and selecting 12 members for
the panel.

-Training the testers (TCVN 11045: 2015) [239].

During this process, 12 members attended the training sessions and
recruitment. During this period, six members were considered for the
official panel Selectng testers in the official sensory evaluation
-Selecting testers for the official panel (TCVN 11045: 2015)

Six testers having the ability, health and time for the sensory evaluation
process were selected.: Guidance on sensory evaluation in laboratory for
fish and shellfish [5].

2.4.2.3. Assessing quality

Assessing quality of shrimp according to QIM includes the following
steps:

+ Describing the attributes of black tiger shrimp
+ Developing QIM

+ Applying QIM to determine the QI for each piece of shrimp every day
during storage

+ Assessment QIM method

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Sample preparation was conducted according to 2.3.1 section
2.5.2. TVB-N

The concentration of TVB-N was determined according to TCVN 9215:
2012 [7].

2.5.3. TMA-N

The TMA concentration was measured according to AOAC 971-14 [8].
2.5.4. Histamine

Histamine was determined by the HPLC accoding to the earlier report of
Gouygou et al. (1987).

2.5.5. Hypoxanthine

Hypoxanthine in shrimp was extracted as described by Ryder (1985)

[217] and was measured according to Kock et al. [127] and Nacalai's
method [267]

2.5.6. pH

The pH value was performed according to the report of Özogul et al.
[193].

2.6. EXPERIMENTAL DESIGN

2.6.1. Determining aerobic microorganisms

Experiment 1: Investigating the amount of TVC in black tiger shrimp
during storage

2.6.2. Developing QIM and evaluating quality of shrimp according QIM
Experiment 2: Developing desriptors

Experiment3: Verificating desriptors by Cata method

Experiment 4: Evaluating shrimp quality every day during storage days,
establishing the correlation between QI and storage time.

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2.6.3. Investigating the change of chemical indices during storage
2.6.3.1. Sample preparation

2.6.3.2. Measuring the changes in quality indices

Experiment 6: Selecting the method to determine the hypoxanthine
content of black tiger shrimp

Experiment 7: Developing the standard curve, recovery efficiency,
LOD, LOQ and RSD values

Experiment 8: Evaluating the TVB-N content in black tiger shrimp
samples during storage

Experiment 9: Evaluating the TMA-N content in black tiger shrimp
samples during storage

Experiment 10: Evaluating the histamine content in black tiger shrimp
during storage

Experiment 11: Evaluating the hypoxanthine content in black tiger
shrimp during storage.

Experiment 12: Determining the pH value of black tiger shrimp during
storage.

2.7. Statistical analysis

All experiments were carried out 3 times. All data were processed by
Statgraphics centurion XVI software (P<0.05), MS. Excel (2010).

CHAPTER 3. RESULTS AND DISCUSSION
3.1 Results of TVC in shrimp during storage.

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10 was 5,84; 6,39; 6,37, respectively. As a result, the number of TVC
increased rapidly from the 8th of storage.

Fig 3.1 The changes of TVC in shrimp during 10 days of storage at 0°C
According to International Commission on Microbiological
Specifications for Foods (ICMSF), the maximum log cfu / g of frozen
shrimp was 6. This is also in agreement with the regulation of Health
Ministry (No. 46/2007 / QD-BYT, 19 December 2007). Thus, tiger
shrimp stored at 0 ° C had a shelf life up to 8 days basing on
microorganism aspect.

3.2. Developing QIM and evaluating shrimp quality according to QIM
3.2.1. QIM model for quality evaluating of black tiger shrimp.

The QIM (Table 3.1) was developed during training panel basing on
TCVN 3726-89 [2] and previous reports [4], [22], [91], [291].

Table 3.1 QIM for black tiger shrimp 
Attribute

s Descriptors

Scor
e

Color Head

Light pink, no black spot 0

Light blue, no black spot 1

Light blue turns to light black 2

Big black mark 3

0
2
4
6
8

1 2 3 4 5 6 7 8 9 10
TVC

(logcfu/g)

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Body

Blue, no black spots, shiny 0

Grey blue, slightly yellow, no black

spot 1

Reddish brown, black spots 2

Black body 3

Tail

Light pink, no black spot 0

Light blue 1

black spots not more than two tail at the

bottom 2

Visible turbidity at the tail 3

Flesh

Fresh, trasparent, with no black spot 0

Turning to turbidity 1

Pale, the position near the head was

light blue 2

The position near the head was light

yellow or green 3

Structure

Structu

Intact 0

Head was slightly loose to the body 1
Head was separated with the body 2
Head was separated with the body, soft

shell 3

Flesh

Firm, elastic 0

Less elastic 1

Soft 2

Very soft 3

Odor Odor Fresh shrimp characteristic odor 0

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No odor, slight ammoniac odor 2
Unpleasant odor, strong ammoniac

odor 3

3.2.2 Quality changes of black tiger shrimp during storage

Fig. 3.2 presented the quality changes of shrimp during storage according
to QIM

Fig. 3.2 Linear Regression model between storage time and QI of 
shrimp

The results indicated that QI had linear correlation with storage
days. This finding was in accordance with earlier reports (Martinsdöttir
et al., 2001) and [107], [171]. According to this obtained equation, we
can estimate the shelf life of shrimp.

3.2.3. Evaluating the accuracy of QIM

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Fig. 3.3 Testing the accuracy of QIM method according to estimated 
storage time and real storage time

3.2.4. Quality classification of shrimp by QI
According to Hanpong et al. [91], the evaluation scale for color, taste and
structure of black tiger shrimp stored at 0 ° C was from 1 to 9, where 1=
extremly like and 9= extremly dislike. The results showed that shrimp
preserved from 1 to 2 days was perfect quality, from 3 to 5 days was good
quality, from day 6 to day 7 was moderate quality, at day 8 was still
acceptable quality. After the 8th day, the quality of shrimp was 
unacceptable. Jayaweera and Subasinghe et al. [113] found that type 1
shrimp (Penaeus indicus) chilled storage had a shelf life from 1 to 3 days, 
type 1 shrimp had shelf life until 7 days, accepted quality till 10 days.
After 10 days, shrimp was rejected due to poor quality. Cann [38] also

reported that black tiger shrimp preserved at 0 ° C decreased the quality
of taste from 2 to 4 days and appeared black spots from 6 to 9 days. Relly
et al. [209] indicated that the quality of shrimp reduced after 2 days of

storage at 0°C.

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protein, amine, lipid and fatty acid occurred. Therefore, the changes in
color, odor and structure were detected clearly. The spoilage symptom
appeared at the 8th day. According to the obtained results, the shelf-life of 
black tiger shrimp stored at 0°C was 8 days and the quality classification
of shrimp was shown in Table 3.2.

Table 3.2 Quality classification of shrimp by QI

Classification QI

Perfect ≤ 3,27

Good 3,67 ÷ 7,47

Moderate 7,47 ÷ 11,37

Acceptable 11,37 ÷ 14,50

3.3. DEVELOPING METHOD FOR HYPOXANTHINE
DETERMINATION IN SHRIMP

3.3.1. Result selection method

Determining hypoxanthine concentration was conducted as previous
report of Kok et al. (1993).

3.3.2. Standard curve

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Fig 3.4 Linear regression model between peak area and concentration
The results of HPLC showed the correlation between the peak area
and the concentration. The equation of standard curve was y= 126,35x +
10,285 (R2 = 0,9977).

3.3.2.1 Determine LOD and LOQ values

0.01 ppm hypoxanthine standard was used (7 times) to evaluate LOD.
The value of LOD = 3Cm / T [206], the empirical data was LOD = 3.
0.01 / 13.13 = 0.002 ppm, LOQ = 3LOD = 0.006 ppm.

3.3.2.2. Determining the relative standard deviation (RSD)
RSD values are determined on 10 standard samples with 1ppp
concentration. The RSD value was 5.32%

3.3.2.3. Recovery efficiency (H)

Experiment 7 was carried out to determine the recovery efficiency H,
and the average H = 90.10%.

3.4. QUALITY CHANGES OF SHRIMP BASING ON QUALITY
INDICES

3.4.1. TVB-N

TVB-N concentration was measured in experiment 7. The changes of
TVB-N in shrimp during storage were shown in Fig. 3.5. The TVB-N
value consisted of TMA-N, DMA-N, NH3 and volatile amines.

y = 126.35x + 10.285
R² = 0.9977

0
100
200
300
400
500

0 0.5 1 1.5 2 2.5 3 3.5

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Fig. 3.5 TVB-N concentration during storage

In general, the amount of TVB-N in shrimp was significant difference
among storage days. TVB-N values increased slowly from day 1 to 4
(6.47 to 11.39 mg/100g, respectively) and leveled up rapidly from day 5.
Figure 3.5 showed that the angular coefficients of two linear regression
equations representing the correlation between TVB-N content and 2

stages of storage (day 1-4 and day 5-10) were different.

3.4.2. TMA-N

TMA-N concentration was measured in experiment 8. The changes of
TMA-N in shrimp during storage were shown in Fig. 3.6.

y = 4.5243x - 8.5678
R² = 0.9907
0

10
20
30
40
50

0 5 10 15

TVB-N

Ngày
y = 1.6003x + 4.6748

R² = 0.9785

0
2
4
6

8
10
12

0 2 4 6

TVB-N

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Fig. 3.6 TMA-N concentration during storage 
The TMA-N concentration in shrimp was significant difference
among storage days (p < 0,05). The change of TMA-N had the similar
trend to TVB-N. TMA-N increased slowly from day 1 to 4 (0,66 to 1,51
mg/100 g, respectively) and leveled up rapidly from day 5. The TMA-N
content reached the maximum value at day 10 with 10,45 mg/100 g.
3.4.3. Histamin

Histamin concentration was measured in experiment 9. The changes of
histamin in shrimp during storage were shown in Fig. 3.7. In general,
histamine content in shrimp was significant difference among storage
days (p < 0,05).

Fig 3.7 Histamin concentration during storage

y = 0,16x + 0,31
R² = 0,994

0
0.5

1
1.5

0 2 4 6

Histamine

Days

y = 0,44x - 1,18
R² = 0,991

0
1
2
3
4

0 5 10 15

Histamine

Days
y = 0.2778x + 0.3467

R² = 0.9577
0

0.5
1

1.5
2

0 1 2 3 4 5

TMA-N

Days

y = 1.7587x - 6.9313
R² = 0.994
0
2
4
6
8
10
12

0 5 10 15

TMA-N

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The histamine content in shrimp related to the metabolism of
microorganisms, the rate of formation varied from the first stage (day 1
to 4) to the second stage (day 5 to 10). In the first stage of storage, the
histamine level increased slowly (from 0.46 mg / 100 g to 0.95 mg / 100
g). In the second stage, histamine content increased rapidly from 1.13 mg

/ 100 g to 3.23 mg / 100g.

3.4.4. Hypoxanthine

Fig. 3.8 showed the hypoxanthine content in shrimp during storage.

Fig. 3.8 Hypoxanthine concentration during storage

Overall, hypoxanthine content in shrimp increased significantly
during storage (p <0.05). The linear regression equation describing the
correlation between hypoxanthine content and storage time was y = 0.25x
- 0.23 (R2 = 0.976) where y: the hypoxanthine content, x: the storage time. 
In comparison with the TVB-N, TMA-N, and histamine, hypoxanthine
content increased linearly and there was no difference between the two
periods of storage from day 1 to 4 and day 5 to 10.

3.4.5. pH

The pH change was shown in Fig. 3.9. The pH value on the 1st day 
was 7.07 and decreased to 6.93 on the 3rd day. Then the pH started to rise 
on day 4 until day 10. The reduction in pH might be due to the
decomposition of glycogen at the initial time. The increase of pH was
because of the breakdown of the protein producing biological amines.

y = 0.2487x + 0.2282
R² = 0.9709

0
1
2

3
4

0 1 2 3 4 5 6 7 8 9 10 11

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Fig. 3.9. pH value of shrimp during storage

3.5. Correlation equation between chemical quality indices and QI 
sensory

Table 3.3 summarized the changes in quality of black tiger shrimp
during storage. The three indices including TVB-N, TMA-N and
histamine related to bacterial counts, therefore, the concentration of
TVB-N, TMA-N and histamine was significant difference in the two
periods of storage (day 1 to day 4 and day 5 to day 10). The results
indicated that chemical quality indices linearly correlated with the
storage time. Table 3.4 presented the linear regression equations of
quality indices.

6.6
6.8
7
7.2
7.4
7.6
7.8

0 1 2 3 4 5 6 7 8 9 10 11

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Storage time QI TVB-N

(mg/100g) 
TMA-N 
(mg/100g) 
Histamine 
(mg/100g) 
Hypoxanthine

(µM/g) pH

Day 1 1,53b ± 0,06 6,47a± 0,09 0,67b ± 0,02 0,46b ± 0,02 0,68b ± 0,03 7,07a ± 0,02 
Day 2 3,67c ± 0,055 7,77b ± 0,08 0,89c ± 0,02 0,66c ± 0,03 0,78c ± 0,04 6,75b ± 0,03 
Day 3 5,60d ± 0,10 9,09c ± 0,04 1,08d ± 0,02 0,79d ± 0,01 0,95d ± 0,04 6,93c ± 0,03 
Day 4 7,47d ± 0,06 11,39d ± 0,07 1,51e ± 0,02 0,95e ± 0,02 1,02d ± 0,01 7,09a ± 0,01 
Day 5 9,53e ± 0,06 14,57e ± 0,09 2,09f ± 0,03 1,13f ± 0,02 1,38e ± 0,02 7,20d ± 0,01 
Day 6 11,37f ± 0,12 18,89f ± 0,10 3,34g ± 0,03 1,35g ± 0,01 1,59f ± 0,03 7,29e ± 0,03 
Day 7 12,93g ± 0,12 22,73g ± 0,06 5,15h ± 0,03 1,88h ± 0,02 2,07g ± 0,02 7,36f ± 0,04 
Day 8 14,33h ± 0,12 28,15h ± 0,06 7,36i ± 0,04 2,41i ± 0,01 2,13g ± 0,02 7,43g ± 0,04 
Day 9 15,93i ± 0,25 32,30i ± 0,03 9,14k ± 0,05 2,83k ± 0,01 2,49h ± 0.02 7,59h ± 0.04 
Day 10 16,93k ± 0,12 37,52k ± 0,07 1,48l ± 0,07 3,23l ± 0,02 2,87i ± 0.19 7,71i ± 0.02

Linear

regression
equation (x:
storage time)

QI = 1,72 x + 0,55
R2 = 0,993

(x: 1 10)

TVB-N = 1,60x +
4,68
R2 = 0,978

(x: 14)
TVB-N = 4,52x

+8,57

TMA-N = 0,28x + 0,35
R2 = 0,958

(x: 1 4)
TMA-N = 1,76x +6,93

R2 = 0,994

(x: 5 10)

His = 0,16x + 0,31
R2 = 0,994

(x: 1 4)
His = 0,44x -1,18

R2 = 0,991

(x: 5 10)

Hx = 0,25x + 0,23
R2 = 0,976

(x: 1 10)

Giá trị pH khơng
tuyến tính với thời

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Table 3.3. QI, TVB-N, TMA-N, histamine, hypoxanthine and pH during storage

Table 3.4. Linear regression equation between chemical quality indices of shrimp

Linear regression equation between chemical quality indices of shrimp (day 1 to 4)

TMA-N Histamine Hypoxanthine

TVB-N

TVB-N = 5,69(TMA-N) +
2,76

R2 = 0,996

TVB-N = 9,89His – 1,61
R2 = 0,966

TVB-N = 15,81Hx – 2,32
R2 = 0,930

TMA-N TMA-N = 1,72His – 0,19

R2 = 0,947

TMA-N = 0,33Hx + 0,35
R2 = 0,894

Histamine

His = 1,60Hx + 0,4
R2 = 0,968

Linear regression equation between chemical quality indices of shrimp (day 5 to 10) 
TVB-N

TVB-N = 2,55(TMA-N) + 9,40
R2 = 0,980

TVB-N = 10,12His + 3,75
R2 = 0,978

TVB-N= 18,96Hx + 6,88

R2 = 0,978

R2 = 0,991

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20
TMA-N

TMA-N = 3,97His – 2,21
R2 = 0,998

TMA-N = 7,30Hx – 6,16
R2 = 0,963

Histamine

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3.6. The proposal model to classify the quality of black tiger shrimp 
basing on the combination of QIM method and chemical quality 
indices

This work suggested that the classification of shrimp quality was
divided into four categories: Perfect, good, moderate and acceptable.
Table 3.5 showed the sensory evaluation scores (QI) and the values of
the chemical quality indices for each category.

Table 3.5. Classification of shrimp (Penaeus monodon) basing on 
the combination of QIM method and chemical quality indices
Classification Perfect Good Moderate Acceptable 
QI ≤ 3,67 3,67 ÷ 7,47 7,47 ÷ 11,50 11,50 ÷ 14,50
TVB-N ≤ 7,77 7,77 ÷ 11,37 11,37 ÷ 18,89 18,89 ÷ 28,15
TMA-N ≤ 0,89 0,89 ÷1,51 1,51 ÷ 3,34 3,34 ÷ 7,36

His ≤ 0,66 0,66 ÷ 0,95 0,95 ÷ 1,35 1,35 ÷ 2,41

Hx ≤ 0,78 0,78 ÷ 1,02 1,02 ÷ 1,59 1,59 ÷ 2,13

CONCLUSION

The results of this thesis “Evaluating the effects of factors on quality
of shrimp (Penaeus monodon) during storage at 0°C after harvest” 
indicated some important findings as follows.

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g, Hx = 2.13 μM / g, QI = 14.50.

3. Developing method to measure the hypoxanthine content in shrimp
during storage by HPLC.

4. R2 value in the linear regression equation containing chemical 
indices showed a strong correlation between TVB-N, TMA-N,

histamine and hypoxanthine in tiger shrimp preserved at 0°C. This is the
new scientific finding, the values of the indices can be found from the
regression equation.

5. The spoilage process of black tiger shrimp was divided into two
stages: autolysis (from day 1 to 4) and decomposition (from day 5 to day
10). The correlation between TVB-N, TMA- N and histamine was
described by two linear regression equations at these two stages. This is
also the new scientific finding.

6. It is the first time, two chemical indices of His and Hx were used to
assess shrimp quality and proved to be strong correlation with shrimp

quality. This point is new as well.

7. Developing the method to examine the quality of black tiger shrimp
basing on the combination of sensory evaluation and chemical indices.
This method proved to be efficient in assessing the shrimp quality. It is
new scientific result.

PUBLICATIONS

International journal

1. Le, N. T., Doan, N. K., Ba, T. N., & Tran, T. V. T. (2017). Towards
improved quality benchmarking and shelf-life evaluation of black tiger
shrimp (Penaeus monodon). Food Chemistry, 235, 220-226.

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1. Le Nhat Tam, Tran Thi Van Thi (2015), Preliminary studies
assessing the quality balack tiger shrimp by sensory evaluation and
combined with the quantitative indicators trimethylamin, total volatile
base, histamine, Journal chemistry, 53, 190-196.

2. Le Nhat Tam, Doan Nhu Khue, Tran Thi Van Thi (2016),
Chemical quality indices for freshness evaluation of black tiger shrimp
(Penaeus monodon), Journal science & technology, 54(2B), 162-169.

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