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

The thesis provides additional scientific data on the nutritional and biological value of grape seaweed. On the other hand, the thesis has also built a process of preliminary processing, preserving sea grape by the method of adjusting MAP gas and assessing the change in quality of grape seaweed by storage time, ... Research results The dissertation is a new scientific data on seaweed, serving teaching and researching activities at Nha Trang University as well as a number of other schools and research institutes.

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

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

pre-processing 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 3215-

79, 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

Table 1.2 Ash content of sea grapes

P mg/100 gram dry weight 1030

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

Table 1.4 Acid amin content (g/100g dry weight) of sea grapes

 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