Summary of the Doctoral thesis: Study on pre-processing and preservation of post-harvested sea grapes (Caulerpa lentillifera J. Agardh 1837)

MINISTRY OF EDUCATION AND TRAINING
THE UNIVERSITY OF NHA TRANG

LE THI TUONG

STUDY ON PRE-PROCESSING AND PRESERVATION OF
POST-HARVESTED SEA GRAPES
(CAULERPA LENTILLIFERA J. AGARDH 1837)

SUMMARY OF THE DOCTORAL THESIS

Major

: Aquatic Product Processing Technology

Code

: 9540105

KHÁNH HÒA - 2019


The work has been completed at Nha Trang University

Science instructor:

1. Vu Ngoc Boi, Associate Professor
2. Nguyen Huu Dai, Associate Professor

Reviewer 1: Ngo Đang Nghia, Associate Professor
Reviewer 2: Nguyen Duy Nhut, Dr

Reviewer 3: Nguyen Duy Thinh, Associate Professor

The thesis is protected at the Thesis Evaluation Board, held at Nha Trang
University at............, on.....................................................

This thesis can be found at: The National Library
The Library of Nha Trang University


SUMMARY OF NEW CONTRIBUTIONS OF THE THESIS
Thesis topic: Research on pre-processing and preservation of post-harvested sea
grapes (Caulerpa lentillifera J. Agardh 1837)
Major : Aquatic Product Processing Technology

Code: 9540105

PhD student: Le Thi Tuong

Course: 2012

Science instructor: 1. Vu Ngoc Boi, Associate Professor
2. Nuyen Huu Đai, Associate Professor
Place of education: Nha Trang University
Contents: The thesis has gained some new results added to the researches on preprocessing and preservation sea grapes:
1) Proper conditions have been identified for the pre-processing of sea grapes
(including the process of washing, recovery of sea grapes) as follows: the suitable
conditions for the washing process of sea grapes: amount of water: 15 liters/kg of sea
grapes, washing time: 8 minutes and number of times: 3. The suitable conditions for
recovery of sea grapes: the rate of sea grapes in water is 2%; growing time is 2 days; the
dissolved oxygen in water is 8 mg/l; the light intensity is from 10-15klux, the

temperature of medium is 280C. Sea grapes planted in these conditions can recover
95.7% with the green intensity of 43.7%, the total aerobic bacteria remain on sea grapes
of 323 cfu/g and the sensory qualities of sea grapes meet TCVN 3215-79.
2) The thesis has designed and manufactured the circulating planting equipment
used in the preliminary processing of sea grapes at pilot scale, the yield of 300kg/batch.
The equipment works on the principles: the static tank works with the dynamic process
and carries out by the circulation of the circulating water and aeration. The rectangular
equipment is made of the stainless steel 304, size of 6000mm x 1080mm, curved bottom.
It is equipped with sensors to adjust the temperature in the range of 200C ÷ 300C, the
salt concentration from 0 ÷ 10%, the dissolved oxygen concentration from 0 ÷
20mg/litter. Sea grapes pre-processed in 2 days on this equipment have a recovery rate
more than 80% and sea grapes meet the quality standards for fresh food according to the
current regulations of the Ministry of Health, Vietnam. .


3) The thesis has identified the suitable conditions for fresh sea grapes
preservation procedure by MAP method. Fresh sea grapes after pre-processed in the
suitables conditions are wrapped in PA packagings, in nitrogen 90% medium, the
preservation temperature of from 230C to 290C. With the above conditions, fresh sea
grapes preserved in 18 days still have good sensory qualities according to TCVN 321579, the rate of damaged sea grapes is about 7%, the weight loss rate is 14.8%, the vitamin
content C remains 0.0106 mg/g, the polyphenol content remains 0.077 mgGAE/gFW,
DPPH free radical reduction effect is 39.8% compared with that of sea grapes before
storage.
4) The thesis evaluated the variation in quality of sea grapes after 18 preservation
days of MAP method. Preservation results shows that after 18 preservation days, the
total polyphenol content decreases by nearly 50%, the vitamin C content decreased by
35%, DPPH-free radical reduction decreases by nearly 30%, the protein content
decreases by 15% , the lipid content decreases by 18%, the damage rate increases by
6.6%, the weight loss rate increases by 15%. The ash and fiber content are quite stable
during preservation time.

5) The thesis proposes the preliminary processing and preservation of sea grapes
by MAP method: collect sea grapes from 35-40 days old, then remove the horizontal
stems, collect stems without being damaged, with the same size, green color and length
more than 6cm. Wash with clean water at a rate of 15 liters/kg, washing time: 8
minutes/times, number of washing: 3 times. Then sea grapes are recovered in the
continuous circulation equipment with a rate of 2% in the culture medium; the planting
time: 2 days; the dissolved oxygen in water: 8 mg/l; the light intensity from 10 to 15klux,
the temperature of the culture medium is 280C. The recovery period ends, sea grapes are
removed, centrifuged to separate 10% of water grasping on the sea grapes. After that,
pack the sea grapes in PA packagings, pump 90% nitrogen and store them at a suitable
temperature of 260C ± 30C. After 18 preservation days in the above conditions, the
quality of sea grapes is not significantly reduced.


OPENING
1. Urgency of thesis
Sea grape (Caulerpa lentillifera J. Agardh 1837) is a species of the genus
Caulerpa, which is common in the tropical and subtropical regions. Genus Caulerpa is
very diverse, Caulerpa lentillifera is the most valuable species.
In the world, sea grades are known in the 1970s of the 16th century, nowadays
they are planted in some countries, especially in Japan, South Korea, India and the
Philippines. In Vietnam, the sea grapes are known in the early twentieth century.
Vietnamese scientists have discovered the species of Caulerpa dramatically developing
in coastal tidal areas, on densely populated islands, such as Ly Son (Quang Ngai), Phu
Quy (Binh Thuan), Phu Quoc (Kien Giang). However, sea grapes discovered in Vietnam
have small sizes, they haven’t been planted. Sea grapes (Caulerpa lentillifera J. Agardh,
1837) was taken from Nhat Ban by Associate Professor Nguyen Huu Dai which are first
planted in Khanh Hoa. At present, sea grapes are grown in Khanh Hoa, Ninh Thuan,
Binh Thuan and Phu Yen provinces.
The biologic and nutritious values of sea grapes contain many vitamin group A,

B, C, polyphenol, chlorophyll and some micro minerals (such as iron, iodine, calcium,
etc.) that are necessary for human body. Although the protein and lipid content in sea
grapes are low, they contain the essential amino acids and fatty acids that human body
cannot synthesize itself. Especially, sea grapes contain caulerpin which reduces total
cholesterol in serum, blood pressure control, anticancer, anticoagulant, antivirus,
antioxidant….
Consuming sea grapes in Vietnam and in the world is more and more increasing,
their price at the Japanese market is about $10/kg of fresh sea grapes. Vietnamese sea
grapes imported to Japan is about $5/kg of fresh sea grapes. Sea grapes in some Asian
countries are considered as a high quality vegetable and they are as precious as green
salmon caviar. Sea grapes contain a lot of water, their water content is up to 94%, their
tissue structure is loose and their pigment is easily destroyed by the physical agents.
Therefore, the fresh sea grapes are quickly spoiled, which reduces their economic
values. As a consequence, the potential for commercial development of fresh sea grapes
products is limited. Therefore, the research to propose solutions to prolong the


preservation time of fresh sea grapes is necessary. The author conducts the thesis:
“Research on pre-processing and preservation of post-harvested sea grapes
(Caulerpa lentillifera J. Agardh 1837)”
2. The purpose of thesis: Build the pre-process and preservation procedure for fresh
sea grapes over ten days
3. The objects and scope of research
3.1. The objects of the research
Sea grapes (Caulerpa lentillifera) are bought at the farm of Dai Phat B Plus
Company, Cam Ranh, Khanh Hoa.
3.2. The scope of research
1) Study on pre-process before preservation
2) Study on sea grape preservation by MAP method
3) Study on evaluating the quality by preservation time.

4) Propose the pre-process and prservation of sea grapes by MAP method.
4. Research methods
Using high precisely analytical methods such as atomic absorption spectroscopy
- graphite furnace - chemical modifier (CM-GF-AAS) to determine the content of heavy
metals Cd, Pb, ...; high performance liquid chromatography to determine vitamin A, C,
B1 content; gas chromatography to determine the compositions of fatty acids, etc. The
thesis also uses some Nordic food analysis methods to determine the total number of
aerobic bacteria, Escherichia coli and Salmonella. Besides, the thesis also used TCVN
analyzes to determine the raw protein, the fiber content or the sensory quality. At the
same time, the thesis also uses mathematical methods in optimizing the experimental
process and processing data collection statistics in order to ensure high reliability test
results.
5. The structure of the thesis
The thesis includes 160 pages: 39 pages of overview, 23 pages of research
methods, 83 pages of research results, 2 pages of conclusion, 13 pages of references, 39


tables, 83 images, 119 references (51 Vietnamese documents, 3 websites and 65 English
documents), 01 page of lists of published works of thesis and 53 pages of annex.


CHAPTER 1. DOCUMENTAL OVERVIEW
1.1. RESEARCH SITUATION OF SEA GRAPES
 Introduction of sea grapes
Sea grapes (Caulerpa lentillifera, J. Agardh, 1837), which were first described
by J. Agardh as a species of the Bryopsidale Green algae, Caulerpa, are a common and
diverse species living in the tropical and warm temperate regions. This species was
described by Lamouroux in 1809 with dark green features, cylindrical horizontal stems
with many branches, diameter of 1 – 2mm. Many vertical stems are grown from
horizontal stems. Many small branches are grown from vertical stems. The end of the

branch is spherical bead-like shapes (the grapes) arranged densely on a vertical stem, its
diameter of 1.5-3mm. This is the valuable part for use, many “unreal roots” on the
horizontal stems branch into bunches like fuzz which can cling to mud. The
characteristics of horizontal stems: they have long horizontal stems, branch into the clam
by the root system. From horizontal stems, they will grow the vertical stems.

Figure 1.1. Picture of sea grapes (Caulerpa lentillifera, J. Agardh, 1837)

 Nutritional values of sea grapes
Sea grapes are a kind of seaweed, so they have the typical biological and
nutritional values of sea grapes such as low energy, high nutritional and biological
values. They also have many applications in food processing and pharmaceutical field.


Sea grapes contain a lot of minerals, fiber, vitamins. Especially, they contain almost
essential amino acids for the human body, including Asparagine, Glutamate, Serine,
Glycine, Histidine, Arginine, Threonine, Alanine, Prolin, Tyrosine, Valine, Methionine,
Isoleucine, Leucine, Phenylalanine, Lysine. Compared with the genus brown sea grapes,
red sea grapes, green sea grapes, sea grapes have higher nutritional values.
Table 1.1. Compositions of Caulerpa Lentillifera and Ultra Reticulate
Compositions

Caulerpa lentillifera

Ulva reticulate

Protein (N=6,25), %

12,49±0,3

21,06±0,42

Fat, %

0,86±0,10

0,75±0,05

Fiber, %

3,17±0,21

4,84±0,33

Ash, %

24,21±1,7

17,58±2,0

Carbohydrate

59,27

55,77

Moisture, %

25,31±1,15

22,52±0,97

Table 1.2. Ash content of sea grapes
Ash

Unit

Content

P

mg/100 gram dry weight

1030

Ca

780

Mg

630

Zn

2,6

Mn

7,9

Fe

9,3

Cu
I

g/100 gram dry weight

2200
1424


Table 1.3. Vitamin content (mg/100g) of sea grapes
Vitamin

Content (mg/100g)

Total

170

Vitamin E

2,22

Vitamin C

1,00

Thiamin

0,05

Riboflavin

0,02

Niacin

1,09
Table 1.4. Acid amin content (g/100g dry weight) of sea grapes
Fatty acid

Content (mg/g dry weight)

Threonine

0,79

Valine

0,87

Lysine

0,82

Isoleucine

0,62

Leucine

0,99

Phenylalanine

0,61

Aspartic axit

1,40

Serine

0,76

Glutamic axit

1,78

Glycine

0,85

Arginine

0,87

Histidine

0,08

Alanine

0,85

Tyrosine

0,48

Proline

0,57


 Pre-processing and preservation technology of sea grapes
In the world, some countries such as Japan, the Philippines, India... preservation
and pre-processing technology of sea grapes are done by the following ways. Sea grapes
grown in the seabed are harvested by pulling up both horizontal and vertical stems; The
ones grown in hanging nets, the harvest process is relatively easier, The nets are
collected first, sea grapes are collected then. In those nets, we will choose to cut the
vertical stems which meet standards, the rests including the horizontal stems and the
young stems are retained in the nets and replanted for next exploiting. The harvested sea
grapes will then be thoroughly washed in sea water to remove mud and other dirt. They
are classified, the unqualified sea grapes and other sea grapes species are removed after
that. The clean sea grapes are placed in the bamboo baskets, wrapped into the plastic
bags which are sealed. The baskets of sea grapes are put in shade and transported to the

market.
When preserving fresh sea grapes, the temperature factor is very important, this
factor greatly affects the quality of sea grapes. When the temperature is high, the quality
of the sea grapes drops rapidly, they do not retain their original color and taste. Some
studies in Japan on the effect of temperature on the quality of fresh sea grapes showed
that fresh sea grapes were not suitable for storage under 150C. In addition, some
Philippine researchers pointed out that, in temperate climates, the average temperature
from 180C to 270C is better than those are stored in higher climates. Fresh sea grapes are
quickly damaged and wilthered when they are stored under the normal temperature
conditions and this is a major concern in the export of sea grapes. The studies show that
at the normal temperature, fresh grapes are withered and quickly deteriorated. On the
other hand, sea grapes also can not be stored in the cold temperature, so we can not use
cold temperatures to store sea grapes. The appropriate temperature extending the storage
time for fresh sea grapes to export is between 180C and 270C.
Exported sea grapes are either fresh or embalmed in sea water or salt water. They
are washed in sea water several times, then people pick good quality ones up, draining
them, package them in the spongy boxes with vents above or around them. The fresh
sea grapes can be preserved from 5 to 7 days, the salty ones can be preserved from 2 to
3 months.


In Vietnam, there are three types of sea grapes products: fresh, dried and
salty ones.
Fresh sea grapes products: After being cleaned, raw sea grapes are centrifuged
to separate water on their surface. This process can reduce sea grapes’ weight about 10%
before being packaged. We use transparent PP plastic boxes so that the sunlight can
shine inside and keep sea grapes fresh for a long time. This technology can extend the
preservation time about 7-8 days. Producers can evaluate the quality of sea grapes
through the practical experiences (basing on color, firmness, densely beads on the stems,
size, etc.).

Dried sea grapes products: Sea grapes are cleaned and centrifuged to separate
water on their surface, then cool-dried at the temperature from 400C to 450C. Thanks to
this separation process, the drying step is to gain the preservation moisture. The rate of
products collected per 1 kg of raw sea grapes can be processed 25-30 grams of dried
products; dried products, after being dried, are packaged with a silica gel package inside,
the preservation time is around 3 – 4 months. However, there are no published data on the
quality of dried sea grapes.
Salty sea grapes products: Sea grapes, after being cleaned, are soaked in salt
saturated water for 5 - 10 minutes. Before eating, we should pick up and soak the salty
sea grapes in water for 3-5 minutes, they will rise, then, pouring out the first salt water,
soaking the sea grapes into the water for the second time in 5 - 10 minutes, picking out
and processing as fresh sea grapes.
From the documental overview, it can be shown that sea grapes are very
nutritious and suitable for human. Sea grapes can be eaten when they are fresh, or they
are processed into ready-to-eat products. However, the researches on post-harvest
preservation of sea grapes is still limited, they do not commensurate with their values
and potential. Up to now, there have not been any scientific researches that completely
announce about the pre-processing and preservation of fresh sea grapes. In addition,
among all preservation methods of fresh sea grapes, the fresh preservation is the most
important one because fresh sea grapes are popularly chosen by consumers and their
nutritions haven’t almost reduced.


1.2. PRESERVATION METHODS OF FRESH VEGETABLES BY MODIFIED
ATMOSPHERE PACKAGING (MAP)
This is a preservation method in a modified atmosphere environment, which the
compositions and proportions of atmosphere change in favor of preservation. This
method uses a special package called “smart package” which automatically adjusts to
appropriate atmosphere compositions so as to maintain the respiratory process for each
type of vegetables and extend the preservation time. The packages such as PE, LDPE,

HDPE, PP, PA, PVC, etc. help prevent water vapor and selective diffusion of oxygen
and carbon dioxide, reduce the respiratory intensity and metabolic actions; therefore, the
quality of products are improved and the preservation time of post-harvest vegetables
are also extended. Thus, the applications for preservating vegetables, flowers, fresh
fruits by MAP method mainly depends on packaging materials.
The advantages of MAP method help significantly increase the preservation time
because the respiratory process, metabolic actions and variation of subtances are
limitted. Hence, post-harvest losses reduce, the quality and value of products still remain
without using any preservative chemicals. Products preservated by MAP are "clean"
products without using any preservative chemicals, so they are absolutely safe for
consumers’ health and the surrounding environment. Products preserved by MAP
technology helps fruits and vegetables maintain their firmness and stiffness. Chlorophil
decreases slowly, carotenoids and anthocian don’t increase. Therefore, MAP has
become a popular method to meet the requirements of preservation technology,
transportation and retailing of vegetables such as mushrooms, bananas, rambutans,
cherries, beef, fish and fish products. ...
With this method, raw materials are packed in the semi-permeable membranes
such as: PE, LDPE, HDPE, PP, PA, PVC, .. Sea grapes preserved by these membranes
can prevent evaporation, oxygen and carbon dioxide selective diffusion, reduce the
respiratory intensity and metabolic activities. Therefore, the quality of products is
improved and prolong the preservation time of post-harvest materials.
MAP preservation method has two types of packaging: a vacuum packaging and
a modified atmosphere packaging. The vacuum packaging is commonly used for


products with low permeability. The modified atmosphere packaging is commonly used
for products with high permeability and strong respiratory intensity.
The modified atmosphere packaging: This method allows adjusting the
proportion of suitable components in the atmosphere for each type of vegetables so as
to limit their respiratory intensity. Normally, we reduce O2 rate and increase N2 rate or

decrease O2 rate, increase CO2 rate. According to ISO 6946-1988 about principles and
techniques of vegetables preservation in the controlled environment, there are two types
of preservation environment . Type 1 adjusts 79% of N2, 8% of CO2; 13% of O2 content
v/v. This type is used for preservating apples and can be useful for the tropical regions
as preservating fruits such as bananas. Type 2 adjusts the O2 content of 2% to 4% v/v
(average content is 3% v/v) and the CO2 content of 3 to 5% v/v or significantly reducing
the O2 content (from 3% to 10% v/v) and the CO2 content from 1% to 2% v/v, the rest
is N2. For example: 1% of CO2; 5% of O2; 94% of N2 v/v. Other studies have also shown
that using N2 in preserving fresh vegetables can delay oxidation and inhibit the growth
of aerobic microorganisms (Farber, 1991; Carol A. Phillips, 1996).
The vacuum packaging: This method creates an environment where the
pressure is lower than the atmospheric pressure, thus the unwanted effects which
atmospheric pressure can cause are limitted. At the normal temperature, food is quickly
gone off as oxygen reacts with organic ingredients in food, which loses their values and
makes them spoil. Sucking the air out of food bags can prevent oxidation, food is
preserved fresher and longer than normal preservation methods. This is an advanced
food preservation technology, which saves time, money, ensures the quality and extends
the preservation time of vegetables and fruits. Research results of Geetha Padmanaban
et al., (2014) showed that the vacuum-preservation method for papayas lasts one week
at the room temperature and four weeks at the cooler temperature in comparison with
the samples (under normal conditions, no vacuum). In addition, the vacuum packaging
reduces the weight loss of vegetables and fruits, restricts the growth of aerobic
microorganisms and fungi, limits the water vapour. However, the amount of the sucked
air depends on the mechanical strength of vegetables. The sea grapes have very poor
mechanical strength, the amount of the sucked air is 65% (the rest of air in the packaging


is 35%) considered as the most suitable one (Pham Thi Khanh Quynh and Le Thi Tuong,
2015).
From the combination of documents and the the actual surveys show that fresh

sea grapes can not suffer the cold temperature below 200C. The characteristic of fresh
sea grapes is juicy, loose and soft structure, they are easily damaged by the external
factors, especially the temperature. Therefore, in order to prolong the storage time of
fresh sea grapes, the MAP control method is selected for the study. This method is
considered to be highly effective preservation with much lower investment cost than
airconditioning – CA. MAP method has two types of packagings: a vacuum packaging
and a modified atmosphere packaging. The modified atmosphere packaging can use N2
with a content greater than 79% but less than 95%, the O2 content is not less than 5% to
maintain the aerobic respiration. The vacuum packaging can suck the air (65%) because
mechanical strength of sea grapes is very poor. These packagings are considered to
inhibit the growth of sea grapes’ respiratory intensity, inhibit microbial activity and
inhibit the production of ethylene, thereby they can extend the preservation time of sea
grapes. The types of packagings used for packing are based on the respiratory intensity
of the sea grapes. However, most common types of packagings used for food are PE,
LDPE, HDPE, PP, PA, PVC.
1.3. MAJOR CHANGES IN PRESERVATING FRESH VEGETABLES
In the preservation process of fresh fruits and vegetables, depending on the
preservation conditions, the changing speed of vegetables and fruits is different. These
changes include: physical changes such as water vapor, weight loss, and heat. These
changes make vegetables quickly wither due to dehydration and decrease their weight,
even spoil due to heat. Biochemical, chemical changes, biochemical changes are mainly
due to the respiratory. Chemical changes are mainly loss of nutrients due to the
participation in respiration or enzymatic activity. The higher the respiratory intensity is,
the greater the loss of nutrients gets. Therefore, during the preservation process, it is
necessary to limit the respiratory intensity of vegetables and also limit the impact of
preservation environment on vegetables and fruits to extend their preservation time.


1.4. MAJOR FACTORS AFFECT THE PRESERVATION TIME OF
VEGETABLES

The major factors affecting the preservation time of sea grapes are the initial
quality of sea grapes, the air temperature, relative humidity of the air, the atmospheric
compositions and the environmental lights. All these factors greatly affect the
preservation time of sea grapes, they determine the quality and the preservation time of
sea grapes. If the initial quality of sea grapes is not fresh and intact, their resistance is
poor, the effects of microorganisms and environmental factors will make them spoil
quickly. The environmental preservation factors have a strong impact on physical
changes and biochemical, chemical changes that rapidly reduce weight and nutrients of
sea grapes. However, each type of sea grapes adapts to each environmental preservation
condition. Therefore, in order to extend the preservation time of sea grapes and fruits, it
is necessary to study their appropriate preservation conditions.


CHAPTER 2. MATERIALS AND METHODS
2.1. MATERIALS
2.1.1. Raw sea grapes
Sea grapes (Caulerpa lentillifera) are bought at Dai Phat B Plus Company, Cam
Ranh, Khanh Hoa. Sea grapes have the vertical length more than 6centimeters. They
have specific green color and they are not damaged. Sea grape beads are not broken and
have the same size.

Figure 2.1. Raw materials (Caulerpa lentillifera)
2.1.2 Packagings, nitrogen
PA, PP and PVC packagings are provided by Asia Plastic Packaging Joint Stock
Company in Tan Binh District, Ho Chi Minh City. The packagings meet hygiene and
food safety standards in accordance with QCVN 12-1: 2011 / BYT about hygiene and
safety for the synthetic plastic bags and utensils contacting directly with food.
Nitrogen has no odor, no taste, no color, purity 99.5%. It is manufactured
according to TCVN 3286-79. It is allowed to use in food technology, provided by
Sovigaz Nha Trang Company, Khanh Hoa.

2.2. RESEARCH METHODS
2.2.1. Approaches to the research content


Based on the assessment of domestic and international researches related to the
processing of sea grapes and based on the preliminary results, the thesis approaches the
contents presented in Figure 2.2.
Raw sea grapes

Quality evaluation

Pre-process
- Growing time
- The rate of sea
grapes in the culture
medium.
- Soluble oxygent
content.

- Washing time
- Times of washing
- The amount of
washing water

Recovery

Separation of water

- PA packaging
- PP packaging

- PVC packaging

Packaging

Preservation

Evaluating the changes in the quality of
sea grapes by the preservation time

Physical changes

Chemical changes

Microorganism changes

Indentify the preservation
method

Preliminary processing and
preservation of sea grapes

Figure 2.2. Schematic approach to the research content of the thesis


2.2.2. Analytical methods:
+ Determination of moisture: The moisture of the samples is determined by
drying method to the constant weight according to TCVN 1867: 2001.
+ Determination of the protein content: the protein content is determined
according to the standard TCVN 4328: 1-2007.
+ Determination of the lipid content: the lipid content is determined by Folch et

al., (1956).
+ Determination of of the total fiber content: The total fiber content is determined
according to TCVN 5714: 2007.
+ Determination of the ash content: The ash content is determined according to
AOAC 923.03-1995.
+ Determination of the carbonhydrate content: the carbohydrate content is
determined according to AOAC: 1990.
+ Determination of the vitamin C content: the vitamin C content is determined
by the iodine titration.
+ Determination of the vitamin B1 content by the fluorescence method based on
the principles: When oxidizing vitamin B1 (thiamin) with potassium citrate in alkaline
medium will form a green fluorescent thiocromium compound under the ultraviolet
light, Thiamin will form a thiocrom molecule. To determine the thiamine content, a pure
standard solution of thiamin compares with the reference solution. Prior to the reaction,
thiamin must be released from the sample by enzyme phosphatase, papayolin or
takadiatase.
+ Determination of vitamin A contents by the method of high performance liquid
chromatography.
+ Determination of fatty acids: Fatty acids are determined by gas chromatography.
+ Determination of Amino Acids: The amino acids are determined by high
pressure liquid chromatography.
+ Determination of heavy metal elements: Heavy metals Cd, Pb, .. are determined
by atomic absorption spectrometry (CM-GF-AAS).


+ Determination of free radical scavenging activity (DPPH): Determination of free
radical scavenging activity (DPPH) is determined according to Fu et al (2002).
+ Determination of total polyphenol content: Determination of total polyphenol
content is determined by the method of Fu et al (2011).
+ Determine the total number of aerobic bacteria according to NMKL86: 2006.

+ Determination of Escherichia coli by NMKL 96: 2003.
+ Determination of Salmonella spp according to NMKL 71: 1999.
+ Determination of the color of sea grapes by analyzing them through the image
processing software - Image J. The color of sea grapes is captured by the digital camera
Nikon coolpix s3300, 16 Megapixel, Nikon STYLE Series (S), Japan. The samples of
sea grapes are photographed in a black box to prevent the interference from the outside
light because the light can make the number wrong. The shooting distance is kept fixed
for all samples. Images in JPEG format are analyzed for red (R), green (G), blue (B) by
Image J software, distributed by Natl, Inst. Of Healt Bethesda, Md, USA
+ Evaluation of the sensory quality of sea grapes by the grading method according
to the standard TCVN 3215-79.
+ Determination of the weight loss rate and the damage rate of sea grapes by
weighing method with the accuracy of 10-4g.
2.3. DATA PROCESSING METHODS
Using SPSS 18.0 software for analyzing ANOVA in order to determine
statistically significant differences among mean values with  = 0.05% and Post Hoc
Test after ANOVA to understand the statistically specific differences among pairs of
mean value; Using Design Expert 8.0 software to provide regression equations,
mathematical models and relating charts; Using Excel 2013 software to calculate mean
values, standard deviation and to draw graph.


CHAPTER 3. RESULTS AND DISCUSSIONS
3.1. DETERMINATION OF THE QUALITY OF RAW SEA GRAPES
After being harvested, sea grapes are transported to the laboratory for removing
the vertical stems, damaged sea grapes and unqualified ones, then they are washed,
analyzed the chemical compositions, microbiological criteria and some heavy metals.
The study results show that the ash content accounts for 25.32%, the raw protein content
accounts for 12.07%, the raw lipid content accounts for 2.72%, the fiber content
accounts for 3.47%, the carbohydrate content (including the fiber) accounts for 59.89%,

the vitamin C content accounts for 0.21mg/g, the total polyphenol content accounts for
2.28mg GAE/g and some substances with antioxidant activity (DPPH) account for
47.83%. Besides, there is no presence of E.coli and Salmonella in the raw sea grapes.
These two types of bacteria cause the intestinal diseases that have been prescribed for
raw vegetables by the Ministry of Health. Cadmium and Pb content of sea grapes are
below the level permitted by Ministry of Health according to QCVN regulation 8-2:
2011/BYT.
3.2. STUDY ON PRE-PROCESSING RAW SEA GRAPES
3.2.1. The process of washing raw materials
The purpose of washing is to remove most microorganisms and some impurities
on the surface of the sea grapes, and to minimize the risk of damage.
The research shows that the appropriate amount of water to wash is 15 liters/kg
sea grapes; washing time: 8 minutes/ times and number of washing times: 3 times. With
these good washing conditions, the microbiological criteria and the quality meet
standards.
3.2.2. The stage for re-planting raw materials
When being harvested, the vertical stems of sea grapes are cut from the horizontal
roots. The re-planting period helps sea grapes heal the cuts, grow roots and live
normally, increase the resistance of sea grapes, prolong the preservation time.
The study shows that the suitable conditions for re-planting sea grapes are the
rate of sea grapes in the culture medium: 2%; the culture time: 2 days; the dissolved
oxygen in water: 8 mg/l; the light intensity from 10-15klux, the temperature of culture


medium is 280C. The sea grapes grown in the above conditions can heal with the rate of
95.7%, green intensity is 43.7%, the total aerobic bacteria remains on the sea grapes is
323 cfu/g and the sensory quality of fresh sea grapes is good.
3.2.3. Centrifuging to separate water on the surface of sea grapes
At the end of the re-planting stage, the water content on the sea grapes is quite
high. If they are immediately packaged and stored, they will be quickly spoiled.

Therefore, centrifuging is necessary.
The research results show that the percentage of water removed from the surface
of fresh sea grapes corresponding to a centrifuge speed 300 rpm in 30 seconds is 10%.
After being extracted water, sea grapes have enough conditions for preservation.
3.2.4. Design of quipments used for the preliminary processing before
preservation at the pilot scale, the yield of 100 kg of material / batch
The thesis studies the design, produces the circulatory equipment for preliminary
processing, the recovery growing of sea grapes is at the pilot scale, the yield is 100kg of
material/batch. The equipment works on the principles: the static tank working with the
dynamic process carried out by the circulation of circulating water and aeration. The
rectangular equipment is made of the stainless steel of 304, size of 6000mm x 1080mm,
the curved bottom. The equipment has full range of sensors which can adjust the
temperature in the range of +20 ÷ + 300C, the salt concentration in the range of 0 - 10%,
the concentration of dissolved oxygen in the range 0-20mg/litter. Sea grapes planted
within 2 days in this equipment has the recovery rate of more than 80%, even they can
recover completely and meet the quality standards of fresh food according to the current
regulations of Vietnam.


Parts for seawater cooling

Parts for handling seagrapes

Overcover parts

Tank 1

Pneumatic

Tank 6

Tank 2

Water supply

Figure 3.1. The principles of preliminary processing system
Notes:
MN:air compressor

DN: Outdoor unit air

RV: Bottom exhaust valve

conditioner cooling (Gree)
F2: water filter

Drying filter

P: Pump

HS: Heat resistance

DL: The hose device with BTL: Throttle
the horizontal cover.

* The operation principles of the system:
- The cooling system will cool, control and mantain the temperature of sea water
at a temperature of 280C ± 20C. At the cooler, the vertical twist hose is insulated with
PU (poly urethane). At this device, a heat resistance is installed in case the
environmental temperature is below 28°C, the cooler system will stop and the resistance

is supplied the electricity to produce heat and make sea water hot before entering the
tank for pre-processing.
- Sea water after being cleaned is pumped through the cooling system to maintain
the proper temperature for the pre-processing of sea grapes (28  2)°C, and then it is
pumped into the pre-processing tank. In the preliminary tank, the aeration system will
provide the gas in order to dissolve the oxygen in water and helps sea grapes, water
circulate from the bottom to the top. As a result, sea grapes at the bottom will be moved
upward and expose to light to increase the photosynthesis, they can recover their
"health". This process makes the dirt on the sea grapes be removed, which helps sea
grapes clean. When the dirt is removed from the sea grapes, water will be dirty, then the


user will turn on the water recirculation pump system to pump dirty sea water out of the
tank, and refill the filtered water back to the tank. Thus, sea water in the tank is always
circulated in the tank and it is sometimes pumped out of the tank when it is dirty.
Thus, during operation, the temperature of the sea water in the tank, the dissolved
oxygen content, the salt concentration of water in the tank and the illumination intensity
of the lighting system are controlled to ensure the proper conditions for photosynthesis,
growth and recovery of sea grapes.

Figure 3.2. The equipment fot recovery of sea grapes, the yield of 300kg
material/batch

Figure 3.3. Sea grapes are damaged
after harvest

Figure 3.4. Sea grapes have a recovery
after being pre-processed

Figure 3.5. The color of the sea
grapes before preliminary
processing

Figure 3.6. The color of the sea
grapes after preliminary
processing

3.3. SEA GRAPES PRESERVATION AFTER PRE-PROCESSING
3.3.1. Determining the respiratory intensity of sea grapes
For MAP method, choosing the suitable packagings for packaging the fresh sea
grapes is a successful factor because the semi-permeable membrane of the packaging is
directly related to the respiratory intensity of fresh plants. For plants with low respiratory
intensity, the packageings with low permeability should be chosen and vice versa
(acording to Lisa Kitinoja et al, 2004). Therefore, measurement of the respiratory
intensity of the sea grapes is the basis for selecting the appropriate packagings in order
to prolong the preservation time.
The study shows that sea grapes have low respiratory intensity. The mg of CO2
producing per kilogram of sea grapes in an hour maximizes only 0.008 on the 6th day
(0.008 mgCO2/kg.h). Therefore, to prolong the preservation time of sea grapes, they
should be wrapped in low permeability packagings (Lisa Kitinoja et al., 2004). PP, PA,
PVC packagings are low permeability packagings commonly used in food packaging.
3.3.2. Determining type of packagings to pack sea grapes
The study of the thesis shows that PA packaging is more suitable than PP and
PVC packagings for preserving sea grapes. If sea grapes are packed in PA packaging,
the sensory quality, chemical compositions and microbial criteria of sea grapes are less
variable than those of PP and PVC.