Ứng dụng các kỹ thuật lên men, xử lý nhiệt và nano trong chế biến và nâng cao giá trị các chất có hoạt tính sinh học trong tỏi tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

SUMMARY OF DOCTORAL DISSERTATION
Major: Food Technology
Code: 62540101

NGUYEN AI THACH

APPLICATION OF FERMENTATION,
HEATING TREATMENT TECHNIQUES
AND NANOTECHNOLOGY IN
PROCESSING AND ENHANCING THE
VALUE OF BIOACTIVE COMPOUNDS IN
GARLIC

Can Tho, 2020


THE DISSERTATION WAS COMPLETED AT
CAN THO UNIVERSITY

Supervisor: Assoc. Prof. Dr. Nguyen Minh Thuy
Co-supervisor: Dr. Ha Phuong Thu

The doctoral dissertation was evaluated by the Committees at
the basic level
Meeting at: …………………………………, Can Tho University
At:

Reviewer 1:

Reviewer 2:

You can find a copy of this dissertation at the library:
Learning Resource Center, Can Tho University.
National Library of Viet Nam.


LIST OF PUBLISHED WORKS
1. Nguyen Ai Thach, Nguyen Minh Thuy, Nguyen Thi My
Tuyen and Vo Thi Dieu. 2016. Changes in physico-chemical
properties of garlic bulbs (Allium sativum L.) during maturation
and storage. Can Tho University Journal of Science. 45b: 1624. ISSN 1859-2333.
2. Nguyen Ai Thach, Le Thai Anh Thu and Nguyen Minh
Thuy. 2016. The effect of blanching, Lactobacillus plantarum
density and salt concentration on bioactive compounds in
pickled garlic. Vietnam Journal of Agriculture and Rural
Development. Green Agriculture, November - 2016: 161-166.
ISSN 1859–4581.
3. Nguyen Ai Thach, Nguyen Minh Thuy. 2017. Effects of
aging temperature on the physicochemical characteristics of
black garlic. Vietnam Journal of Chemistry. 55(4E23): 130134. ISSN: 0866-7144.
4. Nguyen Ai Thach, Nguyen Minh Thuy. 2017. The effects of
aging temperature on the contents of bioactive compounds in
black garlic. Vietnam Journal of Chemistry. 55(4E23): 135139. ISSN: 0866-7144.
5. Nguyen Ai Thach and Nguyen Minh Thuy. 2017. Effect of
extraction conditions on polyphenols, flavonoids, S-Allyl
cysteine content and antioxidant activity of black garlic extract.
Vietnam Journal of Science and Technology, 55(5A), 18-25.
ISSN: 0866-708X.
6. Nguyen Ai Thach and Nguyen Minh Thuy. 2018. Effects of

drying temperature and time on bioactive compounds and
antioxidant activity of black garlic product. Journal of Vietnam


Agricultural Science and Technology, 1(86): 59-63. ISSN:
1859-1558.
7. Nguyen Ai Thach and Nguyen Minh Thuy. 2019. Study of
synthesis and characteristics of total polyphenols, total
flavonoids and S-allyl cysteine-loaded alginate nanoparticles
with various black garlic extracts and alginate ratios.
International Journal of Engineering Sciences and Reasearch
Technology, 8(6): 261-272. ISSN: 2277-9655.
8. Nguyen Ai Thach, Ha Phuong Thu and Nguyen Minh Thuy.
2019. In-vitro evaluation of cytotoxicity, antimicrobial, and
enzyme inhibition activity of black garlic and its nanoparticles.
International Journal of Engineering Sciences and Reasearch
Technology, 8(10): 59-63. ISSN: 2277-9655.


Chapter 1: INTRODUCTION
1.1 The rationale of the study
In Vietnam, garlic is grown in areas such as Hai
Duong, Ha Noi, Bac Giang, Phan Rang, Ly Son, Da Lat,...
Phan Rang garlic is characterized by small bulbs, white skin,
fragrant essential oils. Many farmers in Phan Rang-Thap
Cham city who applied the VIETGAP (Vietnamese Good
Agricultural Practices) to production, providing consumers
with safe and quality garlic products.
Storage of garlic is very important to provide products
for the market of fresh vegetables and for food processing.

Garlic bulbs can be stored about 1-2 months under good
conditions at ambient temperatures with low relative
humidity (RH<75%). However, in these conditions, the
garlic bulbs will gradually become soft and wrinkly due to
dehydration. Investigation of maturity and storage methods
helps to maintain a stable source of raw materials for food
processing and trading.
The consumption of unprocessed fresh garlic was
limited due to its characteristics such as pungent smell, spicy
taste that tend to cause uncomfortable stomach. In recent
years, various processing methods such as heating treatment,
aging and fermentation were used to eliminate unpleasant
odors and improve the taste of garlic (Bae et al., 2014).
Black garlic has a sweet taste whose soft texture like dried
fruit, but it does not cause unpleasant taste the same as fresh
garlic (Wang et al., 2010). Furthermore, the processing of
black garlic is involved in the formation of phenolic, S-allylL-cysteine (SAC) and water-soluble compounds with strong
antioxidant effects (Corzo-Martinez et al., 2007; Imai et al.,
1994). So, this is an ideal functional food for the prevention
and treatment of heart disease, Alzheimer's, arthritis and
chronic diseases like diabetes, especially cancer disease. In
1


addition, the fermentation process, it is not only to being a
method for preserving fresh ingredients for a short time
before further processing but also bring some advantages
(improving odor and enriching transfer substances). It was
reported in fermentation products of other plants
(Buckenhuskes et al., 1990). In-depth research on the

processing and quality changes of black garlic and lactic
acid bacteria fermentation garlic products has started to be
interested in Vietnam. However, studies have only stoped at
creating new products (black garlic), not paying much
attention to the bioactive compounds content in the these
products. Therefore, the research, establishment and control
of processing specifications are essential and urgent in order
to create products of high biological value which prevent
disease of in Viet Nam.
The ability to absorb the precious substances of garlic
into the body was still limited, but it can be affirmed that
when nanoizing of bioactive ingredients, the absorption
capacity will increase dozens of times (Shaikh et al., 2009).
The process of synthesis black garlic nanoparticles is
complicated because they only form this shape-size when
they meet the criteria of specific physicochemical properties.
Black garlic nanoparticles have also not been synthesized
and published in domestic and international studies.
Consequently, research processing of garlic products with
high content of bioactive substances, together with effective
extraction of these ingredients and application of
nanotechnology to enhance their activity are very important.
1.2 Objective of the research
Enhance and improve the bioavailability of the
bioactive compounds in garlic through the processing of
garlic products (black garlic and lactic acid bacteria
fermentation garlic) and nanotechnology.
2

1.3 Research contents
The first content: Investigation the change of physical
properties and bioactive compounds content of garlic (Allium
sativum L.) during maturing and storage.
The second content: Improving of the bioactive
compounds content in garlic bulbs through the black garlic
processing and lactic acid bacteria fermentation garlic.
The third content: Application of nanotechnology to
improve the bioavailability of bioactive compounds in black
garlic products.
1.4 Subject and scope of the study
Raw materials of fresh garlic with soft necks and local
varieties which were harvested and selected in the 2014,
2015, 2016 and 2017 seasons at Van Hai ward, Phan Rang Thap Cham city, Ninh Thuan province.
Black garlic and lactic fermented garlic have been
processed and analyzed at the Department of Food
Technology, College of Agriculture, Can Tho University.
Black garlic nanoparticles were synthesized by using
short-chain alginate polymers and tested for antibiotic
activity, carcinogenic toxicity, inhibition of -glucosidase
and AChE enzymes, ability to lower blood sugar levels in
white diabetic mice at the Vietnam Academy of Science and
Technology.
1.5 New contributions
1.5.1 Science contributions
The thesis has provided many research results on
maturity, harvest time and storage conditions for fresh garlic
bulbs. In addition, the mechanism of formation of black and
lactic acid fermented garlic during processing was also
clarified. The proposed models and settings related to

kinematic parameter values that were evaluated to gain an
accurately understanding of the change of bioactive
3


compounds content in food products during processing.
Black garlic nanoparticles were successfully synthesized and
in-vitro and in-vivo evaluation. They have contributed to
diversifying the medical database to support and treat
diseases.
1.5.2 Life contributions
Through processing of black garlic and lactic acid
fermented garlic will increase garlic consumption for
farmers. Simultaneous study of the production process of
these products also aims to create new food sources of high
biological value that is a work of scientific and practical
meaning. Black garlic and lactic-fermented garlic were
processing with a higher content of bioactive compounds (Sallyl cysteine, polyphenols, flavonoids,...) and higher
antioxidant activity than their fresh garlic. In particular, these
compounds have high applicability to health protection and
are a cancer prevention therapy for whose consume them.
Black garlic and lactic-fermented garlic also
contributed to the diversity of garlic products on the market
and they provide a rich source of "medicinal value" in daily
meals.
Nanotechnology was applied which will increase the
biological value of these products. It will open new ways for
the using of Vietnamese garlic products at a higher and more
effective level. Beside that, the appearing of black garlic
nanoparticles system will be a new hope for cancer, diabetes

patients and some other diseases.
1.6 New points
The thesis provides some information about the
maturity time and storage conditions of fresh garlic in
relation to the physical properties and bioactive compounds
during storage.
4


Processing of black garlic and lactic acid fermented
garlic to produce garlic products which are high-quality
superior to fresh garlic (to improve bioactive compounds
content and antioxidant capacity in black garlic). In addition,
the study identified the appropriate storage conditions for
two types of black garlic and lactic acid-fermented garlic.
Developing a process for extraction of bioactive
compounds in black garlic and applying nanotechnology to
create black garlic nanoparticles containing bioactive
compounds with dimensions is less than or equal to 100 nm
which is close to the sphere and high stability. Some of
efficiency obtained from the in-vitro and in-vivo testing of
black garlic nanoparticles were also determined.
1.7 Outline of the dissertation
The dissertation consists of 137 pages with five
chapters: Chapter 1: Introduction (pages: 1-5); Chapter 2:
Literature review (pages: 6-30); Chapter 3: Materials and
methods (pages: 31-47); Chapter 4: Results and discussion
(pages: 48-135) and Chapter 5: Conclusions and suggestions
(page 136-137). The primary content has 36 tables and 84
figures. The dissertation consists of 282 references (263

English references and 19 Vietnamese references).
Chapter 2: LITERATURE REVIEW
2.1 Overview of garlic bulbs
Garlic (Allium sativum L.) bulbs, one of the oldest
plants was used in medicine, have been an important part of
human life over the centuries. Garlic bulb is used as a food
spice, boosting the health of soldiers during the war, curing
colds, infections and treating a variety of diseases, from
cardiovascular disease to cancer and even plague (Amagase,
2006). The composition of garlic is complexible with over
200 different compounds which were contributed to the
beneficial effects of garlic. The most important and unique
5


feature of garlic is high content of organosulfur compounds.
Garlic bulb contains more sulfur (at least 4 times) content
than many other high sulfur vegetables: onion, broccoli and
cauliflower. Garlic also contains many carbohydrates such as
fructose, protein, fiber, saponins, phosphorus, potassium,
zinc, moderate amounts of selenium and vitamin C, steroid
glycosides, lectin, prostaglandin, essential oils, adenosine,
vitamins B1, B2, B6, and E, biotin, nicotinic acid, fatty acids,
glycolipids, phospholipids, anthocyanins, flavonoids,
phenols, and essential amino acids (Borek, 2001; Imai et al.,
1994; Amagase et al., 2001; Ide et al., 1999; Borek, 2006;
Rahman, 2003).
2.2 Researches which related to harvest and storage
conditions of garlic bulbs
In Turkey, garlic bulbs could be stored at 0-5oC, 6070% RH at least 3-4 weeks in stock. From 2012, no

treatment has been carried out before and after harvesting
garlic bulb in Turkey. Garlic was stored under controlled
atmosphere conditions and atmosphere modification which
were commonly used with various methods of irradiation,
chemicals and heating abroad (Selen and Nilgün, 2012).
Geraldine et al. (2008) assessed the physical
properties of agar-agar (1%) based on a coating combined
with 0.2% chitosan and 0.2% acetic acid, as well as their
effect on the garlic coating process. The average moisture
loss of the membrane-coated garlic was three times lower
than that of the control (uncoated) sample. There was a
significantly increase in the color difference (DE*) of the
control sample compared to other treatments. Coated garlic
bulb has a respiration intensity (30 mg CO2/kg.hours) that is
halved compared to the control garlic.
Currently, in Vietnam, garlic bulbs after being
harvested from fields that will be handled by farmers, sun
6


dried for many days and stored. This simple process could
make the garlic more decay, dehydration and lead to reduced
economic value. In addition, post-harvest garlic research has
not been published much in our country.
2.3 Researches to improve bioactive compounds content
in garlic that related to black garlic and lactic fermented
garlic processing
In Vietnam, black garlic has only studied at Military
Medical University on a state code number KC10.TN05/1115. According to the owner of this project’s name was
"Research on fermentation of black garlic from Ly Son garlic

bulbs and assessing the biological impact of this product",
the biological activity of Ly Son garlic increased
significantly, in that total sugar content increased about 13
times, fructose increased about 52 times and SAC content
increased about six times.
Black garlic was made from freshly harvested garlic
in Quang Hoa commune, Quang Trach district, Quang Binh
province which was incubated and fermented during 35-55
days. Equipment was vented with a thermometer to monitor
the temperature in the pot (Nguyen Duc Vuong et al., 2015).
Besides that, Nguyen Duc Vuong et al. (2017) also used the
natural fermentation process by heating to turn white garlic
into black garlic. Conducting fermentation under 70oC and
fermentation time about 35-45 days.
Increasing S-allyl cysteine (SAC) content was also an
important change in the processing of black garlic. Fresh
garlic contains 20-30 g/g SAC (Kodera et al., 2002) and the
amount of SAC in black garlic was 5-6 times that higher than
fresh garlic (Bae et al., 2012; Wang et al., 2010). SAC
content was formed by the hydrolysis of -glutamyl-S-allyl
cysteine (GSAC) by -glutamyl transpeptidase (-GTP, EC
2.3.2.2) (Kodera et al., 2002). SAC is one of the main sulfur7


containing amino acid compounds and it is the most
beneficial substance of garlic bulbs such as antioxidant, anticancer, liver and neurological diseases (Kodera et al., 2002).
Pickled garlic from five companies in Spain was
analyzed for physical and chemical properties (approximate
composition) such as ascorbic acid, thiamin, riboflavin, tocopherol, and amino acid content. Acid titration ranged
from 0.70 to 2.66%, while the salt content ranged from 2.39

to 7.40%. A significant difference in vitamin content, as well
as physicochemical characteristics. The levels of analytical
vitamins were thiamine (0-0,055 mg/kg), riboflavin (0.0130,032 mg/kg), to-tocopherol (0.36-2.53 mg/kg), and ascorbic
acid (0-47,9 mg/100 g) (Casado et al., 2004).
Garlic cloves were peeled, processed (blanched and
unblanched) and fermented in salt solution with the
participation of the bacterium L. plantarum. Fermentation
taked place about seven days at a temperature that was
maintained at 30oC. Monitor the physical and chemical
properties of garlic cloves during fermentation and after
three months of storage. The growth of yeasts or molds was
not detected in the blanched or unblanched garlic cloves
during the seven days of the fermentation process. For
blanched garlic cloves, the pH of the salt solution decreased,
reaching 3.77 at 7th day. For unblanched garlic cloves, there
was a beginning to reduce the pH of the medium (4,00),
because the growth of L. Plantarum did not take place, the
pH increased (5,65), probably due to the diffusion of acid
formation into garlic in salt solution (de Castro et al., 1998).
Fresh (peeled) garlic cloves were immersed in 2%
acetic acid and NaCl solution which was stored at 20°C for
sixty days. The pH of pickled garlic drops to about 4,0 on the
20th day of storage. The amount of allicin (the main
pungency ingredient) of pickled garlic cloves that analyzed
8


by HPLC decreased by 5.9% on the 40th day of storage
compared to allicin content in fresh garlic (Kim et al., 1993).
Domestic researches on garlic bulbs were also limited.

In particular, in-depth studies on the effects of the
processing, storage and absorption capacity of bioactive
compounds in garlic products are still very limited in
national and international scientific literature.
2.4 Researches of synthesis of nanoparticles loaded
bioactive compounds from garlic or garlic products
According to Lu et al. (2014), allicin which the main
active ingredient derived from a traditional odorant agent in
garlic, created a wide range of biological effects.
Unfortunately, the great potential of garlic in its processing
was limited by its sensitivity to the heating and alkal
conditions accompanied by the decreasing pungent odor of
garlic. In this study, nanoliposome allicin was prepared using
reverse-phase evaporation to overcome the stated limitations.
Vu Binh Duong and Ho Anh Son (2015) evaluated the
effect of inhibiting the cell lines of non-small cell lung
cancer A549 of dried black garlic by trypan blue and MTT
methods. The results showed that at 24 hours, 48 hours and
72 hours after incubation with black garlic concentration of
2.5-10 mg/mL, black garlic strongly inhibited cell
proliferation A549 with IC50 in 24 hours was 10.57 mg/mL.
Up to this time, there have not been any researches
inside and outside Vietnam successfully produce
nanoparticles that smaller than 100 nm loaded bioactive
compounds from black garlic accompanied by in-vitro and
in-vivo assays.
Chapter 3: MATERIALS AND METHODS
3.1 Materials
3.1.1 Research location and time
9

Experiments were conducted through sampling,
experimenting, analyzing and processing data at:
Garlic fields in season 2014, 2015, 2016 and 2017 in
Van Hai ward, Phan Rang - Thap Cham city, Ninh Thuan
province.
Department of Food Technology, College of
Agriculture; Institute of Biotechnology Research and
Development; Intensive Laboratory - Can Tho University.
Laboratory of Biomedical Nanomaterials, Institute of
Materials Science; Laboratory of Applied Biochemistry,
Laboratory of Biologically active, Laboratory of Natural
Products Research, Institute of Chemistry; Laboratory of
Biotechnology Enzyme, Institute of Biotechnology; Division
of Shared Equipment, Institute for Tropical Technology;
Institute of Natural Products Chemistry - Vietnam Academy
of Science and Technology.
Implementation time was from June 2014 to March
2018.
3.1.2 Equipment, tools and chemicals
3.1.2.1 Equipment and tools
Some basic equipment and tools used in the experiments
were as follows:
- Ohaus Analytical balance, - Sanyo Freezer, model SFmodel PX224/E (USA)
CR27K (Japan)
- Rheotex Texture analyser, - Amerex Dryer, model
model SD700 (Japan)
CV250 (USA)
- Spectrophotometer, model - HPLC-UVD system

722N (China)
(Shimadzu,
Shimadzu
Corporation, Japan)
- Alaska Refrigerator, model - Colunm LiChroCART
SL-8C (China)
(Merck, Germany)
- Aged garlic equipment (80cm - Hitachi Field Emission
x 71cm x 110cm, 50-100oC, Scanning
Electron
10


maximum RH at the respective
temperatures) (Viet Nam)
- Malvern-Zetasizer (DLS)
Zetasizer Nano, model 7.03
(UK)
- Ika Rotary evaporator, model
RV10 digital V (Germany)
- Fourier-transform infrared
spectroscopy Perkin Elmer
(Waltham,
Massachusetts,
USA)

Microscope, model S-4800
(Singapore)
- Moisture balance, model
MX50 (Godex, USA)

- Elisa reader (Biotek,
ELx800, USA)
Tecan
Genios
Fluorescence
Luminescence Microplate
Reader, model A-5082
(Switzerland)
- Omron Blood glucose - IKA Magnetic stirrer with
monitor, model HGM-111 heating,
model
C(Japan)
MAGHS7 (Germany)
- Ultrasonic cleaner, model
Elma P300H (Germany)
3.1.2.2 Chemicals
Some basic chemicals used in experiments were as follows:
- Folin-Ciocalteu reagent S-allyl-Lcysteine
(purity  99%) (Merck, standard ( 98%, Sigma,
Germany)
USA)
- 1,1-diphenyl-2-picryl- Acetonitrile (purity 99.8%)
(Merck, Germany)
hydrazil (purity  99%)
(Merck, Germany)
- Quercetin standard (purity  - Enzyme -glucosidase
98% Sigma, USA)
(CAS No 9001-42-7, Sigma,
USA)

- Dimethyl sulfoxide (CAS - Alloxan monohydrate
No 67-68-5, Sigma, USA)
(Sigma, USA)
- Acetylcholinesterase (CAS -Nitrophenyl--DNo 9002-53-8, Sigma, USA)
glucopyranoside (CAS No
3767-28-0, Sigma, USA)
11


- 4-Notrophenol (CAS No - Ellman's (DTNB) reagent
100-02-7, Sigma, USA)
(Sigma, USA)
- MRS broth (Hemidia, India) - Natri alginate (Sigma,
USA)
Other chemicals meet the standards for use in analysis.
3.1.3 Raw materials
The fresh garlic materials (soft neck and local variety)
were harvested in the early morning, sunny weather. The
outer shape showed that the neck of garlic tree had fallen to
the side, its leafy head was dark-yellow and within 130 to
140 days (after planting) in Van Hai ward, Phan Rang - Thap
Cham city, Ninh Thuan province. Choose garlic bulbs that
were not pest, rot or fungal infections. Garlic was put into a
carton and transported by car to the laboratory within 24
hours.
3.2 Contents and methods of research
3.2.1 First content: Surveying the changes in physical
properties and bioactive compounds content of garlic
(Allium sativum L.) in maturing and storage
3.2.1.1 Objective

Determine the effects of maturation and storage
conditions on physical properties and bioactive compounds
during storage. Choose the harvest time with the best quality
garlic bulbs and appropriate storage conditions to apply in
processing garlic products.
3.2.1.2 Experimental designs
a The quality of garlic bulbs at the time of harvest
Garlic bulbs were harvested in many garlic fields in
Van Hai ward, Phan Rang - Thap Cham city, Ninh Thuan
province. At each age would harvest five kg of fresh garlic.
Garlic bulbs were stored in mesh bags (size of hole about 0.9
mm). Garlic was analyzed in the laboratory at room
12


temperature 28-30°C and RH 68-71%. Experiment was
designed a factor.
Factor A: maturity (days after planting): 120, 125, 130, 135
and 140
b The effects of storage temperature on garlic quality.
The postharvest garlic bulbs were pretreated and
separated a outer layer of garlic skin to remove the sandy,
soil. Then garlic bulbs were stored in mesh bags at different
temperatures. Experiment was designed a factor.
Factor B: storage temperature (oC): 0, 5, 20 and room
temperature (28-30)
c The effects of packaging types which used on the loss of
mass
Choose garlic bulbs at the appropriate age and storage
temperature from previous experiments. The amount of

garlic in each package was 500-550 g. Types of packaging
used in the experiment: PE (24 x 34 cm and 38 m thick),
carton (30 x 22.5 x 12.5 cm) with a perforation rate of 0.5%
(5 mm holes diameter, 20 holes on both sides of the package
at even intervals) and mesh bags (24 x 34 cm and 0.21 mm
thick, holes size about 0.9 mm). The samples were stored at
the appropriate temperature. Experiment was designed a
factor.
Factor C: packaging types: PE, carton and mesh bag
Criteria analysis: Weight loss (%), total polyphenols
content (TPC) (mg gallic acid equivalent (GAE)/g d.w), total
flavonoids content (TFC) (mg quercetin equivalent (QE/g
d.w), total thiosulfinate content (mol/g d.w), antioxidant
activity (%), internal sprouting index, color, moisture (%),
respiratory intensity.
3.2.2 Second content: Enhancing the bioactive compounds
content in garlic bulbs through black garlic and lactic
fermented garlic products processing
13


3.2.2.1 Investigation the effects of preheat treatment methods
on the production process of black garlic products
a Objective: Identify optimal parameters for the production
of black garlic in order to create black garlic products with
high quality (increasing bioactive compounds content and
antioxidant capacity of black garlic).
b Experimental designs
Experiment 1: Investigation of the effects of garlic
material heat treatment on the quality of fresh garlic

Selection garlic cloves intact with skins about 3.60.2 cm in
diameter to conduct the experiment
- Method 1: Blanching with steam (100oC) for 4, 6, 8 and 10
minutes.
- Method 2: Freezing at -18oC for 12, 24, 36 and 48 hours.
Control sample: heating untreatment.
Criteria analysis: TPC (mg GAE/g d.w), TFC (mg
QE/g d.w), thiosulfinate content (mol/g d.w), DPPH free
radical scavenging capacity (%) and sensory evaluation
according to the Quantitative Descriptive Analysis (QDA)
method.
Experiment 2: Investigation of the effects of aging
temperature and time on the quality of black garlic product
Fresh garlic bulbs after being heating treated would
continue to age in order to become black garlic. The quality
of black garlic was analyzed every five or seven days during
aging until the end of 45 days of processing. The experiment
was randomly designed with:
Factor D: aging temperature (oC): 60, 70, 80 and 90
Criteria analysis: Moisture (%), pH, oBrix, reducing
sugar content (% d.w), total polyphenols content (TPC), total
flavonoids content (TFC), total thiosulfinate content (mol/g
d.w), antioxidant activity (%), SAC content (mg/kg d.w) by
HPLC method.
14


Experiment 3: Investigation of the effects of drying
temperature and time on the quality of black garlic products
Garlic after aging was conducted to dry its outer skin.

The study was designed according to Box-Behnken model
with two factors:
Factor F: drying temperature (oC): 50, 60 and 70
Factor G: time (hours): 8, 12 and 16
Criteria analysis: total polyphenols content (TPC),
total flavonoids content (TFC) and antioxidant activity (%).
Sensory evaluation by using logistic regression method.
Experiment 4: Investigation of the effects of the
storage process on the ability to maintain bioactive
compounds in black garlic product
Garlic bulbs (after finishing the drying process) were
stored at ambient (282oC) and cool temperature (5oC) with
two types of preservation packaging: PA and composite
packaging (aluminum-coated). Each package contains 300
grams black garlic. The purpose of this experiment was to
determine the appropriate temperature and type of packaging
to maintain value of black garlic product with the best
quality. Experiments were designed completely random.
Storage temperature (oC): ambient (282) and cool
temperature (5)
Packaging types: polyamide (PA) and composite
packaging (aluminum-coated)
Criteria analysis: Total polyphenols content (mg
GAE/g d.w), total flavonoids content (mg QE/g d.w),
antioxidant activity (%).
3.2.2.2 Investigation of the effects of factors (pH, salt
concentration, lactic acid bacteria density) on the quality of
lactic fermented garlic products.
a Objective: To identify optimal parameters for the
fermentation processing to create high-quality pickled garlic

15


product (enhancing bioactive compounds content and
antioxidant capacity of this product).
b Experimental designs
Experiment 1: Investigation of effects of blanching on
product quality
Choose peeled garlic cloves between 20–30 mm long,
between 5–10 mm wide, between 5–10 mm thick.
Experiments were designed completely random. The garlic
cloves were peeled and they were placed in a blanched bag
with 2 L of water that has been heated to the setting time and
temperature. After blanching, the garlic samples were
quickly cooled in an ice bath for 3 minutes. Garlic was
drained in a cool place, after that they placed in PA bags and
stored at 5oC until analysis.
Factor H: blanching temperature (oC): 70, 80 and 90
Factor I: blanching time (seconds): 60, 90, 120, 150 and 180
- Control HoIo: untreatment
Criteria analysis: Thiosulfinate content (µmol/g d.w),
total flavonoids (mg QE/g d.w), total polyphenols (mg
GAE/g d.w), antioxidant activity (%) , hardness (g force),
color.
Experiment 2: Surveying the effects of the
concentration of salt solution and Lactobacillus plantarum
density on the quality of lactic acid bacteria fermentation
garlic
The purified Lactobacillus plantarum bacterium used
which was derived from the collection of microorganisms of

The Institute of Biotechnology Research and Development,
Can Tho University. Lactobacillus plantarum strains used
include XK 1.4 strain that isolated from pickled cucumber
(traditional formulation) and S 1.2 strain that isolated from
pickled “bon bon” (Typha orientalis G.A.). Each batch of
fermented garlic had 0.5 kg peeled garlic cloves, blanched at
16


the appropriate temperature and time from the previous
experiment. Then add 0.5 L NaCl 0.5, 1.0, 1.5 and 2.0%
solution and sucrose to five oBrix and this solution was
pasteurized at 100oC for 5 minutes. Starter strain of
Lactobacillus plantarum that was developed about 24 hours
at room temperature in a MRS Broth medium with a density
of 108 CFU/mL. Then dilute to 100 mL of Lactobacillus
plantarum bacterial solution with a density of 105, 106 and
107 CFU/mL to proceed with fermentation which took place
at ambient temperature and was monitored regularly.
Experiments were designed completely random.
Factor J: Salt concentration (w/v,%): 0.5, 1, 1.5 and 2
Factor K: bacterial density (CFU/mL): 105, 106 and 107
The control sample did not add L. plantarum bacteria.
Criteria analysis: Thiosulfinate content (µmol/g d.w),
total flavonoids (mg QE/g d.w), total polyphenols (mg
GAE/g d.w), antioxidant activity (%), pH, sensory
evaluation.
Experiment 3: Surveying the effects of the percentage
and supplement vegetables on quality of fermented garlic
The garlic peeled cloves was blanched at the

appropriate temperature and time which selected from the
previous experiment. Addition shallots, onions, green onions
with different weight percent (2.5; 5; 7.5; 10%) to the
blanched garlic cloves. Subsequently, fermentation was
carried out with the optimum Lactobacillus plantarum
density in the salt solution with the suitable concentration
selected from the above experimental results. After finishing
the fermentation process, the auxiliary materials were
removed, the remaining garlic after fermentation was
conducted to analyze the criteria. Experiments were designed
completely random.
Factor L: Vegetable types: shallots, onions, green onions
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Factor M: Ratio (%): 2,5, 5, 7,5 and 10 (compared to amount
of raw garlic peeled cloves)
Criteria analysis: Thiosulfinate content (µmol/g d.w),
total flavonoids (mg QE/g d.w), total polyphenols (mg
GAE/g d.w), antioxidant activity (%) .
Experiment 4: Surveying the effects of the storage
conditions on the ability to maintain bioactive compounds in
fermented garlic product
After the appropriate fermentation period, the
auxiliary material would be removed, the fermented solution
pasteurized at 100°C for 5 minutes, then hot pouring at 70°C
into garlic after fermentation. The samples were stored at
two different temperature conditions (30±2 and 5±1°C). The
experiment was designed completely randomly with one
factor.

Temperature (oC): Room (30±2) and cool temperature (5±1)
Criteria analysis: Thiosulfinate content (µmol/g d.w),
total flavonoids (mg QE/g d.w), total polyphenols (mg
GAE/g d.w), antioxidant activity (%) , hardness (g force),
color, sensory evaluation.
3.2.3 Third content: Application of nanotechnology to
improve the bioavailability of bioactive compounds from
processed garlic products
3.2.3.1 Investigation of the effects of extraction conditions on
bioactive compounds centent in black garlic
a Objective: To identify optimal extraction conditions for
bioactive compounds in black garlic
b Experimental designs
Experiment 1: Surveying the effects of solvent type
and ratio of material/solvent which extracted to content of
bioactive compounds
Factor R: Solvent types: water and ethanol solution (50% and
100%)
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Factor S: Material/solvent ratio: 1/5, 1/10 and 1/15
Experiment 2: Investigate the effects of extraction
temperature and time on the bioactive compounds content
Factor T: Temperature (oC): 40, 50, 60, 70 and 80
Factor U: Time (minutes): 30, 60, 90 and 120
Criteria analysis: S-allyl cysteine content (mg/kg),
polyphenols (mg/g d.w), flavonoids content (mg/g d.w).
3.2.3.2 Application of nanotechnology which contributes to
improving the biological value of black garlic product

a Objective: synthesizing black garlic nanoparticles loaded
bioactive compounds with size  100 nm, its shape close to
the sphere, high stability.
b Experimental design: Black garlic was extracted with a
ratio of material/solvent was 1/10 in 50% ethanol at 60°C for
90 minutes. Drop slowly black garlic extract into an alginate
solution (1 mg/mL) that took place an ultrasonic vibrator
with a frequency of 40 KHz according to the alginate/extract
solution ratio was 1/1, 2/1, 3/1 , 1/2, and 1/3. The mixture
was tightly closed and stirred at room temperature for 48
hours. Then proceed to evaporate ethanol in a vacuum
evaporator at 35°C for 1 hour and 50 rpm. After the ethanol
has been completely removed, the mixture was centrifuged at
a rate of 5000 rpm for 5 minutes, collection the supernatant
and store at 5°C.
Determine properties and characteristics of materials:
Entrap efficiency (EE%), morphology, size and zeta
potential, Fourier transform infrared spectroscopy (FT-IR).
3.2.3.3 In-vitro and in-vivo assays of absorption capacity
and biological activity of black garlic nanoparticles
a Objective: To identify and compare the effectiveness of
black garlic and black garlic nanoparticles in-vitro and invivo evaluation
b In-vitro evaluation
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Experiment 1: Evaluation of the cytotoxic effects
Cancer cell lines derived from American Type Culture
Collection (ATCC), including: human mouth epidermal
carcinoma KB (CCL-17TM), liver cancer Hep G2 (HB 8065TM), lung cancer LU-1 (HTB- 57TM), breast cancer

MCF-7 (HTB - 22TM), Vero monkey kidney carcinoma and
skin cancer SK-Mel 2 (HTB - 68TM). The cytotoxic activity
was performed based on the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium) method which was described
and modified by Scudiero et al. (2006). Experimental results
were determined by the absorbance values/optical density
(OD) that were measured at 540 nm on the Genios Tecan
spectrophotometer. The experiment was repeated three times.
The value of IC50 was determined by the value of inhibition
of cell growth and the Rawdata software.
% inhibition = (ODcontrol (+) – ODsample)/( ODcontrol (+)–
ODcontrol (-)) x 100
IC 50  Highconc 

HighInh%  50x Highconc  Lowconc 
HighInh%  LowInh %

(In there, HighConc/LowConc: test substance at high
concentration/test substance at low concentration;
HighInh%/LowInh%: percent of inhibition at high
concentration/percent of inhibition at low concentration).
Experiment 2: Evaluation of antimicrobial activity
Representative strains of bacteria and mold that cause
disease in human of the Institute of Chemistry - Vietnam
Academy of Science and Technology, including gramnegative bacteria: Pseudomonas aeruginosa (Pa) ATCC
15442, E. Coli (Ec) ATCC 25922; gram-positive bacteria:
Staphylococcus aureus (Sa) ATCC 13709, Bacillus subtilis
(Bs) ATCC 6633, Lactobacillus fermentum N4, Enterococus
faecium B650 and mold: Candida albicans (Ca) ATCC
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10231. Assays of antibiotic activity according to the method
which was modified by Paul et al. (2005).
Experiment 3: Evaluation of inhibitory activity of
enzymes which causing diabetes (-glucosidase) and
Alzheimer's disease (acetylcholinesterase)
Enzyme -glucosidase: The inhibitory activity of enzyme glucosidase was conducted by two methods:
First method (1) (Yamaki and Mori, 2006): Each well
consists: 20 µL of sample (control well replaced with 20 µL
of 0.5 M potasium phosphate buffer, pH 6.7), 50 µL of 25
mg/mL emzyme α-glucosidase concentration, 50 µL of 3
mM p-NPG substrate ( -nitrophenyl -D-glucopyranoside)
and 120 µL of 0.5 M potasium phosphate buffer, pH 6.7. The
mixture was incubated at 37°C for 45 minutes, then added 50
µL of 0.67 M Na2CO3 to each well to stop the reaction.
Reaction mixture was measured at 415 nm on Elisa reader
(Biotek, ELx800, USA). The degree of α-glucosidase
inhibition was calculated by the formulation: % Inhibition =
[(A415control - A415sample)/A415control]x 100. In there: A415control
and A415sample are OD values of control samples and testing
samples which measured at 415 nm .
Second method (2) modified by Hakamata et al.
(2009): The absorbance of the reaction was determined by
Tecan GENios equipment with a wavelength of 405 nm (A).
The inhibition ability of -glucosidase of samples was
determined by formulation: Inhibition (%) = [A(negative control) A(test sample)]/A(negative control) x 100% and IC50 values were
calculated by Tablecurve software.
Enzyme acetylcholinesterase (AChE): The inhibitory activity
of AChE that causes Alzheimer's disease was tested by using
a test method that modified by Karthikeyan et al. (2015). The

reaction well includes: 10 µL of test compound diluted at
concentrations; 15 µL Phosphate buffered saline (PBS
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