Phân lập, tuyển chọn nấm men chịu nhiệt và đánh giá khả năng lên men rượu vang trái giác (cayratia trifolia) ở đồng bằng sông cửu long tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

DOCTORAL COURSE THESIS SUMMARY
Major: Biotechnology
Major code: 62 42 02 01

DOAN THI KIEU TIEN

ISOLATION AND SELECTION FOR
THERMOTOLERANT YEAST AND STUDY
FOR WINE FERMENTATION FROM
Cayratia trifolia IN MEKONG DELTA

Can Tho, 2020


THIS THESIS WAS COMPLETED
AT CAN THO UNIVERSITY

Advisor: Prof. Dr. Ha Thanh Toan
Assoc. Dr. Ngo Thi Phuong Dung

This thesis was defended on the meeting of Univeristy
doctoral thesis evaluation council
Place: Doctoral thesis evaluation hall, 2nd floor –
Administration Building, Can Tho University.
at 14 h 00, 3rd April, 2019

First Reviewer: Assoc. Dr. Nguyen Duc Hoang
Second Reviewer: Assoc. Dr. Nguyen Minh Thuy

This thesis is stored and can be referenced at the
Learning Resource Center, Can Tho University and
The National Library of Vietnam


LIST OF PUBLISHED PAPERS
1. Doan Thi Kieu Tien, Ha Thanh Toan and Ngo Thi
Phuong Dung, 2016. Exploration of yeasts applied for
wine fermentation from Cayratia trifolia in the Mekong
Delta. The 12th Young Scientist Seminar; Nov 22nd-23rd,
Yamaguchi Prefectural Seminar park, Yamaguchi, Japan,
19.
2. Doan Thi Kieu Tien, Huynh Thi Hoang Anh,
Nguyen Ngoc Thanh, Huynh Xuan Phong, Ha Thanh
Toan and Ngo Thi Phuong Dung, 2017. Selection of
thermotolerant yeasts for production of three-leaf cayratia
(Cayratia trifolia L.) wine in Kien Giang. Science and
technology journal of agriculture and rural development.
54-62.
3. Doan Thi Kieu Tien, Vien Thi Hai Yen, Huynh Xuan
Phong, Bui Hoang Dang Long, Ha Thanh Toan và Ngo
Thi Phuong Dung, 2018. Selection of thermotolerant
yeasts and application in wine production from three-leaf
cayratia (Cayratia trifolia L.) in Hau Giang. Can Tho
University journal of science. 54(4B): 64-71.
4. Doan Thi Kieu Tien, Lu Hang Nghi, Nguyen Ngoc
Thanh, Huynh Xuan Phong, Ha Thanh Toan và Ngo
Thi Phuong Dung, 2018. Isolation and selection of
thermotolerant yeasts for wine production from three-leaf

cayratia (Cayratia trifolia L.), Journal of agricultural
science and technology. 55-64.
5. Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Nguyen
Duc Do, Ha Thanh Toan và Ngo Thi Phuong Dung,
2018. Total polyphenol content and antioxidant capacity
of Cayratia trifolia (L) Domin berries before and after
fermentation using thermotolerant yeast Saccharomyces
cerevisiae HG1.3, 2018. Vietnam journal of science and
technology. 60-64


6. Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Huynh
Thi Hoang Anh, Huynh Xuan Phong, Nguyen Ngoc
Thanh, Bui Hoang Dang Long, Ha Thanh Toan và Ngo
Thi Phuong Dung, 2018. Bioactive capacity of three-leaf
cayratia collected in Kien Giang province and wine
fermentation using Saccharomyces cerevisiae AG2.1.
National biotechnology conference, 2018. 978-983
7. Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Lu Hang
Nghi, Huynh Xuan Phong, Nguyen Ngoc Thanh, Ha
Thanh Toan và Ngo Thi Phuong Dung. Bioactive
capacity of three-leaf cayratia collected in Ca Mau
province and wine fermentation using Saccharomyces
cerevisiae CM3.2, 2018. The conference of biotechnology
in the Mekong Delta. 104.
8. Doan Thi Kieu Tien, Huynh Xuan Phong, Mamoru
Yamada, Ha Thanh Toan and Ngo Thi Phuong Dung,
2019. Characterization of newly isolated thermotolerant
yeast and evaluation of their potential for use in Cayratia
trifolia wine production, Vietnam Journal of

Science,Technology and Engineering. 68-73
9. Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Lu Hang
Nghi, Huynh Xuan Phong, Nguyen Ngoc Thanh, Bui
Hoang Dang Long, Ha Thanh Toan và Ngo Thi
Phuong Dung, 2019. Evaluation of total polyphenol and
antioxidant capacity in wine fermentation of three-leaf
cayratia from Ca Mau province using Saccharomyces
cerevisiae CM3.2, Can Tho University journal of science.
55 (2019)(2): 285-291


Chapter 1. INTRODUCTION
Rationale of the study
Threeleaf cayratia (Cayratia trifolia) is a wild
creeper growing in grasslands and glades in several
countries like China, Laos, Cambodia, ... In Vietnam,
threeleaf cayratia naturally grows along the fences, bushes
in provinces of the central and sourth Vietnam. In the
Mekong Delta, threeleaf cayratia is found in almost all
provinces and cities, especially this plant is abundant in U
Minh area. The composition of threeleaf cayratia contains
highly bioactive compounds (Tsao, 2010), the fruit has a
unique color and flavor that were inadequately studied
with few researches
Wine had a history of thousands of years and were
researched thoroughly to diversify in types and to improve
in quality. During wine fermentation, one of the most
important factors is the yeast source. Temperature has an
important effect on the ethanol convertion from sugar
using yeasts. Currently, the climate change has gradually

increase earth temperature which will definitely affect the
fermentability of yeast in the wine production process.
Therefore, the application of thermotolerant yeast strains
can contribute to improve the efficiency of the
fermentation process. Isolation and selection of
thermotolerant yeasts and the evaluation of the ability to
ferment threeleaf cayratia wine (Cayratia trifolia) in the
Mekong Delta were essential.
Research objectives
To provide references for the wine fermentation
from Cayratia trifolia and to propose a solution for the
wine fermentation which could improve effectiveness to
deal with global warming. Thereby, the people in the
region can adapt to the climate change of the region and
1


exploit the value of natural sources to improve their
income.
Research contents
(1) Collecting and evaluating physical and chemical
characteristics and antioxidant properties of Cayratia
trifolia in Mekong Delta; (2) Isolation and identification
of morphological, physiological and biochemical
characteristics of yeast strains from natural Cayratia
trifolia; (3) Selection and identification of genetic
relationships of thermotolerant, alcohol-resistant yeasts
which had good ability of fermenting Cayratia trifolia
wine; (4) Study the suitable conditions for the
fermentation of Cayratia trifolia using the selected

thermotolerant yeast strain.
New contributions of this thesis
- Determining polyphenol content of Cayratia
trifolia ranging from 0.47 to 1.54 mg GAE/mL and has
antioxidant capacity from 16.60 to 82.86%.
- Isolation of 151 strains of yeast from natural
Cayratia trifolia in the Mekong Delta that were identified
as species of five genera Saccharomyces, Candida,
Clavispora, Pichia and Hanseniaspora.
- Thirty (30) strains of natural yeasts were
determined that were thermotolerant and could grow on
the plate incubated from 37 to 45°C, were capable of
growing in an environment with an ethanol content of 912% and could ferment to create over 6% (v / v) ethanol
content.
- Saccharomyces cerevisiae strain HG1.3 was
capable of being tolerant to 43°C. At 35°C, the wine
fermentation with initial pH 4.5, 20 ºBrix and 105
cells/mL. After a 6-day fermentation, the content of
ethanol reached 11.68% (v/v). The produced wine had
2


polyphenol content of 0.6 mg GAE/mL, antioxidant
capacity of 57.3% and had the unique color and flavor.
Chapter 2. OVERVIEW
2.1. The overview of threeleaf cayratia
threeleaf cayratia is a species of vines based on the
stem-like tentacle that grows on opposite side of leaves.
The tentacle is about 2-3 cm long, usually divided into 3
branches (Kumar et al., 2011) and sometimes with 4-5

reddish-brown branches with little of furry and long veins.
Leaves grow in a double, feathery pattern, with 3 leaves
joined in the middle that are usually larger than the sides,
with long, slender, ovoid-shaped leaves. The trunk has a
polygonal cross-section with a green old stem and a
reddish-brown young body with many longitudinal veins.
Flowers grow on axillary leaves and may be tall. The
lowers were 6-8 mm wide and have a disc shape, 4 green
petals (Pham Hoang Ho, 2000). The three leaf Cayratia
contains a lot of water with round and flat fruit which can
turn purple when ripe and was about 1cm in diameter
(Sesagirriravu, 1986). The fruit contains high levels of
phenolic, the ethanol extract had high antioxidant capacity
(Rabeta and Lin, 2015).
2.2. Overview on yeast
Yeasts are defined as single-celled fungi with
asexually reproducing by budding. Yeast cells are
composed of: cell wall, cytoplasmic membrane, cytoplasm
and nucleus (Nguyen Lan Dung, 1999). Yeast can be
found in many different environments such as water, air,
soil, etc. Yeast is often able to grow at low pH and
anaerobic conditions to reduce or inhibit bacterial growth
(Kurtzman et al., 2011). From ancient times, people have
applied yeast in science, food, medicine and industries.
Fermented beverages and foods produced through the
activities of yeast have contributed prominently to the
3


sustainable development of human society (Legras et al.,

2007, Ulber and Soyez, 2004)
2.3 The wine fermentation and the effects of
temperature on the wine fermentation
The carbohydrate degradation under anaerobic
conditions is called fermentation (Nguyen Lan Dung,
1999). Alcohol fermentation is a complex biochemical
process that requires the participation of yeast or other
microorganisms. During alcohol fermentation, sugar is
converted into ethanol and CO2. Alcoholic fermentation is
accompanied by the formation of products and energy
releasing (Nguyen Thanh Dat, 1986).
Conventional yeasts are very sensitive to
temperature. At 36°C, yeast can be inhibited. In a liquid
environment, at a temperature of 40-50°C, yeast are
almost deactivates and dies after 1 hour 30 minutes; At
60-65°C, the yeast will die after 5 minutes. In dry
environments (W = 13%), yeasts are more thermotolerant
that can to withstand up to 85-105°C. The higher the
temperature, the lower ethanol content produced by
fermentation. In contrast, the more sugar supplied in the
wine, the higher the sugar concentration can deactivate the
fermentation (Bui Ai, 2003). The optimum temperature
for yeast growth is 25-28°C (Casellas, 2005).
When the fermentation taking place at low
temperatures will take longer time. However, high
temperature can affect the taste of the product and can
reduce the activity of the yeast and lead to the
contamination of lactate bacteria and wild yeast. In
addition, high temperature fermentation can create
aldehyde ester and reduce alcohol content in associate

with CO2 increases (Nguyen Dinh Thuong and Nguyen
Thanh Hang, 2005).
4


2.4 Thermotolerant yeast
Temperature is the most important factor affecting
yeast activity. Arthur and Watson (1976) determined the
growth temperature of psychophilic yeast in the range of
2-20ºC; mesophilic yeast were 5-35ºC; thermotolerant
yeast were 8-42ºC and thermophilic yeast were 28-45ºC.
According to Roehr (2001), heat-resistant yeasts had
several advantages in ethanol production under high
temperature conditions including good metabolic activity
and high fermentation rate creating high ethanol contents.
At high temperature, the solubility of oxygen and other
gases in the water decreases that ensure anaerobic
conditions for fermentation; The lower viscosity of the
fermentation medium at higher temperature can save the
energy needed for mixing the environment and minimize
chance of contamination. Therefore, in order to achieve
high temperature fermentation, a highly efficient
thermotolerant yeast strain is needed (Limtong et al.,
2007).
Chapter 3. RESEARCH METHODOLOGY
3.1. Collect and evaluate characteristics of
Cayratia trifolia
Cayratia trifolia berries were collected from 53
locations in 13 provinces and cities in the Mekong Delta.
After harvesting, ripe fruits were taken, washed and

drained. Characteristics including shape, size, color of
Cayratia trifolia were evaluated. Juice extract of berries
was measured for pH, Brix; sugar content, polyphenol
content and oxidation resistance by the ability of DPPH
free radical capturing (%).
3.2. Isolate the yeast strains from natural
Cayratia trifolia for the identification of morphological,
physiological and biochemical characteristics
5


The collected fruits were ultilized for yeast
isolation using YPD culture medium. The enrichment
medium was diluted and then spread, transferred by YPD
agar medium several times until pure colonies were
collected and stored at 4ºC. The isolated yeast strains are
identified
with
physiological
and
biochemical
characteristics such as the abilities to use saccharose,
maltose and glucose and urea decomposition, gelatin
hydrolysis, spore formation by budding. These
characteristics were investigated for preliminary
classification and initial evaluations of ethanol
fermentation.
3.3.
Selection,
genetic

identification
of
relationships between thermotolerant and alcohol
resistant yeast strains with good ethanol fermentation
3.3.1. Study for the thermotolerant ability of the
yeast strains
The isolates of yeast were cultured on petri
containing YPD agar medium. Then, the yeast plates were
incubated at different temperatures: 30ºC, 35ºC, 37ºC,
39°C, 41°C, 43°C, 45°C and 47°C for 48 hours. Colony
observation was taken place to select yeast strains that
were capable of growing and developing at high
temperatures.
3.3.2. Assessment of ethanol tolerance of yeast
strains
This experiment was applied to select thermotolerant
yeast which could grow on plates supplemented with
ethanol. The thermotolerant yeast strains selected from
3.2.3.1 were spread on YPD agar medium with 3%, 6%,
9%, 12%, 15% v/v absolute ethanol and 0% control
samples. Yeast plates were incubated at 30°C for 48
hours. Observe the formation of colonies of yeast strains
on plates under different ethanol concentration.
6


3.3.3. Evaluation of ethanol fermentation of
thermotolerant yeast
One milliliter of the enriched medium of yeast
strains were inoculated into tubes, which were inserted

with upside down Durham tubes. The tubes were added
with 9 mL of 2% glucose solution (w/v) and were then
sterilized at 121°C for 20 minutes. Similarly, 1 mL of
yeast enrichment medium was cultured into tubes with an
upside down Durham ring and 9 mL of Cayratia trifolia
extract modified with pH = 4 and 22 ºBrix. The solution in
tubes was shaken thoroughly to fully fill in the durham
tubes with sugar and sample solution. Heigh of gas
column in Durham tubes created by fermentation was
measured after 6, 12, 18, 24, 30, 36, 42 and 48 hours. The
incubation occurred at room temperature (28-30ºC) to
determine ethanol fermentation.
3.3.4. Study for the wine fermentation of
Cayratia trifolia juice using selected yeast strains
One milliliter of yeast enrichment solution selected
in 3.3.3 was cultured to flasks containing 99 mL of
Cayratia trifolia juice. Yeast density was fixed 106
cells/mL. Flasks were incubated in 5-7 days in anaerobic
conditions at a 37ºC. Brix and pH were measured to
during the fermentation time. Distillation for ethanol
collection, measurement of temperature and concentration
of ethanol obtained to determine the ethanol content at
20°C. The experiment was completely randomized with
the yeast factor investigated, performing 3 replicates.
3.3.5. Identify the selected thermotolerant and
alcohol resistant yeast strains with high Cayratia
trifolia wine fermentation
The thermotolerant and ethanol resistant yeast strains
that can ferment Cayratia trifolia and produced at least
6% (v/v) of ethanol were identified by molecular biology

7


with primers NL1 (5 '-GCA TAT CAA TAA GCG GAG
GAA AAG-3') and NL4 (5'-GGT CCG TGT TTC AAG
ACG G-3 ') to amplify the 26S rDNA D1/D2 sequence in
combination with physiological and biological
characteristics. The 26S rDNA sequences of
thermotolerant yeast strains were identified based on
multiple aligment using MEGA 6 program (according to
Neighbor - Joining (NJ)).
3.4. Study for the suitable fermentation
conditions for wine fermentation of Cayratia trifolia
juice
3.4.1. Study for the effects of temperature and pH
levels to the ethanol fermentation
This experiment was completely random and
triplicate with 2 factors: temperature and environmental
pH. The temperature factor consisted of the room
temperature (28-32ºC); 35ºC, 37ºC, 39ºC and 41ºC.
Environmental pH included 4; 4,5; 5 and natural pH in the
left fluid. A total of 4 × 5 × 3 = 60 treatments were
performed. Each experimental unit was carried out at a
volume of 100 mL of Cayratia trifolia juice.
The strains selected through section 3.3 were
cultured in enrichment medium until the cell density of
108 cells/mL. Cayratia trifolia fruit was washed, pressed
and filtered to measure the initial pH and Brix of the fruit
juice. Ninetynine milliliter of fruit juice were added into
the flask and was then adjusted to 22°Brix, with pH levels

at 4; 4,5; 5 and natural pH. The flasks were then
pasteurized with 140mg/L NaHSO3 for 2 hours. One
milliliter of yeast enrichment medium was cultured to
flasks which were then incubated at room temperature
(28-32°C), 35°C, 37°C, 39°C and 41°C for 5-7 days under
anaerobic conditions (covered with waterlock). pH value,
Brix degree were monitored during the fermentation. The
8


ethanol content was tested to choose the appropriate
fermentation temperature and pH for the next experiment.
3.4.2. Study for the effects of yeast density, Brix
and fermentation times
To determine the suitable yeast density, Brix and
fermentation time for the fermentation. The experiment
was designed completely randomly with 3 factors, each
with 3 levels with 3 replications. Strain densities were
analyzed at 103, 105, 107 cells /mL; Brix were designed at
20, 22, 24 ºBrix; the fermentation time was 5, 7, 9 days. A
total of 3 × 3 × 3 × 3 = 81 units were performed. Each
experimental unit was carried out at a volume of 100 mL
of Cayratia trifolia juice.
Proceed similar to section 3.4.1. Adjust the pH of
Cayratia trifolia juice and the fermentation temperature
selected in section 3.4.1. The experiment was monitored
by measuring pH and Brix. Then, distillation were applied
to collect and measure for the content of ethanol at 20ºC.
3.5 Data processing and statistical analysis
Results were processed using Microsoft Office Excel

2010 and the statistical software Statgraphics Centurion
XV.
Chapter 4. RESULTS AND DISCUSSION
4.1. Characteristics of Cayratia trifolia collected in
Mekong delta
The observations on the shape of 53 Cayratia trifolia
samples collected in 13 provinces and cities in the Mekong
Delta showed that there were two main types: round or flat
fruit and slightly flat fruit in some areas. The surface of the
berries was smooth or rough with average size about 1.5 - 2 cm
and a dark black color (Figure 4.1).

Bảng 4.1: Đặc điểm của 2 dạng trái giác ở vùng ĐBSCL
STT

Đặc điểm

Hình
9


STT

Đặc điểm

Hình

1

The fruit had round

shape with rough surface
and a dark black color,
size about 1.5 - 2.0 cm
The leaf surface is
hairless. The leaf edge
was slightly serrated

2

The fruit had a flat shape
and a dark black color,
about 1.5 - 1.7 cm in
size.
Leaf surface was hairy.
The leaf edge was
serrated.

Extracted juice of Cayratia trifolia had different
pH and ºBrix values depending on the collected location;
The difference in natural and climatic conditions of each
samples reflected different pH and °Brix values. The pH
value ranged from 3.01 to 4.75 when the relative °Brix
varied from 3.5 to 10.0. In the berry juice, the total sugar
content ranged from 0.41-2.17 g/100 mL, the reducing
sugar content in the Cayratia trifolia samples ranged from
0.22 to 0.96 g/100 mL. The berry fluid contained
polyphenol content of 0.47 - 1.54 mg GAE/mL and had
the antioxidant to capture 16.6 to 82.86% free radicals.
This result was consistent with the research of Rabeta and
10

Lin (2011) in which the ability of DPPH free radical
capturing in fresh fruit juice was determined at 61.47%.
4.2. Isolation of yeast strains from natural
Cayratia trifolia fruits and identify the morphological,
physiological and biochemical characteristics of the
isolated yeasts
From 53 different sources of Cayratia trifolia in 13
provinces and cities in the Mekong Delta region, 151 yeast
strains were isolated. This result illustrated the abundance
and diversity of natural yeast on the Cayratia trifolia that
had the similarity with published studies which isolated 50
yeast strains isolated from yeast starters collected in
Mekong Delta (Ngo Thi Phuong Dung, 2009), 40 strains
of yeast from an agriculture by-products including cocoa
pods, pineapples, bagasse, straw and sawdust, 28 strains
of yeast from fruits and citrus leaves (Dung Phong et al.,
2016; Nuanpeng et al., 2016; Phong et al., 2016;
Techaparin et al., 2017).
Morphological characteristics of 151 strains of yeast
were divided into 7 groups of shapes: small sphere, large
sphere, small oval, large oval, short ellipse, long ellipse
and pointed ellipse. Spore formation characteristics
showed that groups 1 to 6 created round spores, except
group 7. Results on the ability to ferment glucose,
saccharose and maltose; urea degradatin activity, gelatin
hydrolysis of yeast strains showed that there were 140
strains capable of fermenting glucose; 104 strains capable
of fermenting saccharose, 108 strains capable of

fermenting maltose; 24 strains had urea degradation
activity; and 30 strains were capable of degrading
gelatine. According to the preliminary classification
description to the genus level of Kurtzman and Fell
(1998), 151 yeast strains could be classified into 4 genera:
Saccharomyces, Hanseniaspora, Pichia and Candida.
11


4.3. Selection, identification and genetic
relationship investigation of thermotolerant and
alcoholic resistant yeast strains with good Cayratia
trifolia wine fermentation
4.3.1. The thermotolerant ability of yeast strains
The colony forming of 151 strains of yeast showed
that all of the strains could develop at the temperature
from 30°C to 35°C. Among 151 strains of yeast, 141
strains were able to grow at 37°C. At 39°C, there were
121 strains of yeast capable of growing. When the
incubation temperature increased to 41°C, there were 89
strains of yeast capable of growing. At 43°C, there were
48 strains that could form colonies. At a temperature of
45°C, only 10 strains developed. All isolates could not
grow at 47°C. Consequently, the final results selected 141
strains capable of growing at 37°C.
4.3.2 The ethanol resistance of thermotolerant
yeast strains
Most of the selected yeast strains were grown in
environments with 3% (v/v) ethanol content, except for
strain VL4.4 isolated from the Cayratia trifolia collected

in Tra On district, Vinh Long province. When the ethanol
content increased, yeast growth decreased. In specific,
there were 128 strains growing in environments with 6%
ethanol concentration, 64 strains growing on plates
supplemented with 9% ethanol. At 12% (v/v) ethanol
concentration, there were only 27 strains with colonies.
When the ethanol concentration increased to 15% (v/v),
all strains did not grow. According to Casey and Ingledew
(1986), the usual ethanol resistance of most alcohol
produced yeast strains were at about 5-10% (v/v) ethanol.
As a result, 64 strains showing resistances to alcohol
12


content ranging from 9% to 12% v / v were selected to
assess the ability of ethanol fermentation.
4.3.3 Selection of yeast strains with high ethanol
fermentation abilities
According to Luong Duc Pham (2005), the life cycle
of yeast produced a large amount of carbon dioxide. This
by-product was made up in the anaerobic metabolism of
yeast to produce ethylic alcohol, CO2 and other
substances. The amount of CO2 generated will be
accumulated the Durham tube. Therefore, the height of
carbon dioxide in Durham tube was a criterion for
preliminary assess the fermentation ability of the yeast
strains.
Sixty-four strains of yeast were able to convert
glucose into ethanol, with the height of the column of gas
produced in the Durham tube increased over time. Strains

KG2.2, KG3.1, DT1.2, CM3.2 and BT1.2 could
accumulate 30 mm height of air column in 6 hours. Two
strains of yeast KG4.1 and AG3.2 had almost no
fermentation ability. The results were prequalified 57/64
strains capable of ethanol fermentation, gas production
filled the 30mm Durham tubes in 48 hours.
4.3.4 Cayratia trifolia wine fermentation
The wine fermentation of 57 strains of yeast was
assessed by the ability to ferment 100 mL of fruit juice.
After fermentation, the pH and Brix of the Cayratia
trifolia fluid were reduced compared to the original pH
and Brix (original pH 4,5 and 22 °Brix) yeast uses sugar
as a carbon source since during fermentation. Therefore,
sugars were metabolized through an enzyme reaction
sequence until the final products of fermentation including
ethanol and CO2 (Le Ngoc Tu et al., 2005). Ethanol
content was one of the most important criteria to assess
the alcohol fermentation capacity of yeast strains. The
13


results showed that the strain HG1.3 was able to ferment
and produce 9.9% (v/v) ethanol.
4.3.5 Scientific nonclamentures and genetic
relationship of thermotolerant and alcoholic resistant
From the results of fermentation survey in section
4.3.4, 30/57 strains of yeast capable of fermenting with the
ethanol concentration from 6.0% (v/v) or more were
selected. The identification of 30 strains of yeast were
shown in Table 4.2.

Table 4.2: Results of identifying 30 yeast strains
No.

Genus
Candida

1

2
3
4

Pichia

Species
Candida
tropicalis
Candida
nivariensis
Candida glabrata
Pichia
kudriavzevii
Clavispora
lusitaniae
Saccharomyces

Strains

Strain
number

KG1.1, KG3.2,
CM3.3,
DT1.2, HG3.3,
ST2.1,
TG1.1
BT1.2
BL2.1, CT1.1,
CT1.3, AG2.3,
CT2.3
KG5.1,

5
3
4

8
AG4.2,
CM4.4,
Clavispora
TG4.2
1
BT2.1, BT3.3,
Saccharomyces
HG1.3, CM3.2,
9
CT4.2, VL1.1
cerevisiae
AG2.1, TV4.2,
Total

30
DT3.2, LA1.3,
The genetic relationship of 30 yeast
strains
KG2.1,
TG3.1,
MEGA 6 integrated with Neighbor-Joining
analysis
HG2.1

was applied in the establishment of phylogenetic trees
based on MEGA 6 software (Neighbor-Joining). The
phylogenetic tree of 30 yeast strains was shown in Figure
4.1. The identification results indicated the diversity of
thermotolerant yeast strains isolated from Cayratia
trifolia. The results were also consistent with the research
of Nguyen Lan Dung et al. (2019) or Basso et al. (2008),
Saccharomyces cerevisiae was among of traditional
species of yeast commonly used in alcohol fermentation.
14


Figure 4.1: The phylogenetic tree of 30 yeast strains
Note: The number indicated above each branch was the repetition number
of same consequence in 1000 time bootstrap test.
: Genetic distance

4.4. The suitable conditions for the wine
fermentation of Cayratia trifolia
4.4.1. The effects of fermentation temperature

and pH
Results of the change of Brix, pH and ethanol content in
the fermentation using S. cerevisiae HG1.3 showed that the
ability of fermenting wine of HG1.3 strain was different at
different temperature and pH (Table 4.3). Treatments of pH 4.5
at room temperature and 35°C resulted high ethanol contents
(12.82-12.53% (v/v)), the difference was not statistically
significant at 95% confidence. Arthur and Watson (1976)
identified the growth temperature of thermotolerant yeasts in
the range of 8-42°C. Thermotolerant yeasts had minimum
temperature ranges (Tmin), optimum (Topt), maximum
(Tmax), which were at 20-26°C, 26-35°C and 37-45°C,
respectively and can even grow at temperatures above 45°C.
Thus, pH 4.5 at 35°C was selected as the suitable conditions
for the fermentation using thermotolerant yeast that was in
15


accordance with published studies. This temperature and pH
were chosen for further experiments.
Table 4.3: The effects of temperatures and pH levels to the
fermentation
No

1
2
3
4
5
6

7
8
9
10
11
12
13
14
15
16
17
18
19
20

Temperature-pH
Room temperature –
Natural pH
Room temperature 4.0
Room temperature 4.5
Room temperature 5.0
35-Natural pH
35-4.0
35-4.5
35-5.0
37-TN
37-4.0
37-4.5
37-5.0
39-TN

39-4.0
39-4.5
39-5.0
41-TN
41-4.0
41-4.5
41-5.0

Postfermentation pH

Postfermentation
Brix

Ethanol
content (%
v/v) at 20ºC

3.57

8.17

11.68c

3.95

6.33

11.85bc

4.42

7.33

12.82a

4.64

7.50

10.53ef

3.55
3.95
4.24
4.62
3.55
3.93
4.31
4.90
3.46
3.84
4.22
4.07
3.23
3.41
4.04
4.22

9.00
9.00

9.00
9.00
11.17
11.00
10.67
12.00
13.00
12.00
12.00
11.00
14.67
15.00
16.00
16.00

11.55cd
11.68c
12.53ab
9.79fg
9.57g
9.87fg
10.78de
9.14gh
7.98ij
7.99ij
8.45gh
7.21ij
5.98k
5.98k
5.98k

5.19k

Note: The figures in the table were the averages of triple repetitions; the same above
characters indicated that there were no significant differences at 95% confidence

4.4.2 The effects of yeast density, Brix and
fermentation time to Cayratia trifolia wine
fermentation
In fermentation, the ethanol content is related to the dry
matter content in the medium. Low dry matter content leads to
the lack of nutrition for yeasts that then leads to a decrease in
the number of products. However, if the sugar content is too
high, the concentration of ethanol is reduced since the too high
16


sugar content will increase osmotic pressure, and disorders in
yeast physiology (Tahir et al., 2010). Yeast density and
fermentation time were factors influencing the fermentation.
The effects of strain, Brix and fermentation time using S.
cerevisiae strain HG1.3 were shown in Table 4.4.
Table 4.4: The effects of inoculation density, Brix and fermentation times
o
Brix
Ethanol content (%)
Days- oBrix-Density
No
sau lên men ethanol ở 20 oC (% v/v)
1
5 - 20 - 103

8.67
11.40bc
ở 20oC (% v/v)
2
7.33
13.47a
5 - 20 - 10⁵
3

5 - 20 - 10⁷

7.00

12.00bc

4

5 - 22 - 103

8.67

11.40bc

5

5 - 22 - 10⁵

8.33

12.30ab

6

5 - 22 - 10⁷

8.67

11.67bc

7

5 - 24 - 103

11.67

11.40bc

8

5 - 24 - 10⁵

10.76

11.07bc

9

5 – 24 - 107

11.33

10.83cd

10

7 - 20 - 103

7.67

11.40bc

11

7 - 20 - 10⁵

8.33

12.30ab

12

7 - 20 - 10⁷

9.33

11.07bc

13

7 - 22 - 103

9.00

11.40bc

14

7 - 22 - 10⁵

9.00

12.30ab

15

7 - 22- 10⁷

11.67

10.83cd

16

7 - 24 - 103

10.33

10.77cd

17

7 - 24 - 10⁵

10.33

11.07bc

18

7 - 24 - 10⁷

11.67

10.83cd

19

9 - 20 - 103

8.33

10.83cd

20

9 - 20 - 10⁵

8.67

10.77cd

21

9 - 20 - 10⁷

9.67

10.77cd

22

9 - 22 - 103

9.33

10.83cd

23

9 - 22 - 10⁵

10.00

10.87c

24

9 - 22 - 10⁷

10.67

9.53de

17


No

Days- oBrix-Density

25

9 - 24 - 103

26

9 - 24 - 10⁵

27

9 - 24 - 10⁷

o

Brix
Ethanol content (%)
sau lên men ethanol ở 20 oC (% v/v)
9.00
8.86ef
ở 20oC (% v/v)

10.33
8.05f
12.67

8.05f

Note: The figures in the table were the averages of triple repetitions; the same above
characters indicated that there were no significant differences at 95% confidence

The data analyzed by Statgraphics Centurion XV
software at 95% confidence showed the regression model
as follows:
H= -27,4471 + 1,58638Z+ 3,66409Y+ 5,10075X 0,019213Z2 - 0,124219ZX - 0,0769965ZY- 0,124352Y2
- 0,181007YX - 0,174352X2 + 0,00619792ZYX (1)
Where: H= ethanol content, X= fermentation time
(days), Y= density, Z= ºBrix. Fixing ºBrix at 20 (Z= 20)
would lead to the derrivatives for each variable. The
solving of equation system H (X ') = 0 and H (Y') = 0
resulted X = 5.8; Y = 5.04. The values were added to the
equation to get the predicted optimal ethanol content H =
12.37% (v/v)
Therefore, the optimal conditions for the
fermentation of Cayratia trifolia wine using S. cerevisiae
HG1.3 were determined as 6 days of fermentation, 105
cells/mL cell density, 20°Brix, and the initial pH 4.5 at
35ºC. In this conditions, the theoretically achieved ethanol
content was predicted at H = 12.37%.
The regression equation was vertified by 1 liter scale
fermentation of Cayratia trifolia wine. The obtained
ethanol content was at 12.0% (v/v) (Table 4.5). Therefore,

the verified results showed the compatibility of the
regression model and were also similar to the previous
studies on the fermentation of watermelon wine (Ngo Thi
18


Phuong Dung et al., 2011), pineapple wine (Huynh Xuan
Phong and ctv, 2017). The quality of Cayratia trifolia
wine was checked with 15 criteria shown in Table 4.5.
The results of testing criteria were compare to the
standards of QCVN6-3:2010/BYT on wine quality
assessment. The results showed that all of the criteria meet
the regulations. In addition, the Cayratia trifolia wine
contains 0.60 mg GAE/mL which showed the ability to
capture 57.3% DPPH free radicals. Thus, it was concluded
that Cayratia trifolia wine had antioxidant capacity to
help protect the body.
Table 4.5: Qualitative checks of Cayratia trifolia wine
No

Vietnamese
Standard
6-3:2010/BYT

Remarks

Criterion

Results

1

Ethanol

12.0% v/v at 20°C

≥ 8% v/v at 20°C

Passed

2

Methanol

2.534 g/L alcohol 100°

Passed

3

SO2

1.4 mg/L

10g/L
alcohol
100°
350 mg/L

4

pH

4.17

-

Selfdeclaration

5

º

Brix

12

-

Selfdeclaration

6

Reducing sugar

0.26 g/100 mL

-

Selfdeclaration

7

Acid hydrocyanic

0 g/L

0.1 mg/L

8

methyl 2-propanol

2.425 g/L 100° alcohol

-

Selfdeclaration

9

Aldehyde

0.638 g/L 100° alcohol
100°

-

Selfdeclaration

10

E. coli

0 CFU/ mL products

0 CFU/ mL

Passed

11

Coliforms

10 CFU/ mL products

≤ 10 CFU/ mL
hẩm

Passed

19

Passed

Passed


Vietnamese
Standard

6-3:2010/BYT

No

Criterion

Results

12

Total aerobic bacteria

7.2 x 102 CFU/g

13

Total yeast – mold

14

15

Polyphenol content
DPPH
free
capturing

radical

1

103 CFU/g
2

Remarks

Passed

3.6 x 10 CFU/g

10 CFU/g

Passed

0.60 mg GAE/mL

-

Selfdeclaration

57.3a±3.73%

-

Selfdeclaration

Sensory evaluation of Cayratia trifolia wines were
established base on 0-5 scale on 4 criteria (clarity and
color, smell, taste, interest for the sample) based on the
criteria in the grading evaluation method according to

Vietnam standard TCVN-3217:79. The Cayratia trifolia
wine using the strain HG1.3 had the purity and color of
the wine reaching 4.9 points; the smell reached 4.6 points;
the taste reached 4.0 points; the liking for wine reached
4.2 points.
CHAPTER 5. CONCLUSION AND
RECOMMENDATION
5.1. Conclusions
Cayratia trifolia in the Mekong Delta were classified
into two types: round and flat fruits, ripe purple-black
fruits, the average diameter of the berries was 1.5 cm. The
extracted fluid had a very specific purple color, pH value
at 3.01-4.75, Brix value at 3.5-10.0, total sugar content at
0.47-2.17 g/100mL and the reducing sugar at 0.22-0.96
g/100mL. In particular, the fluid contains polyphenol
content at 0.47-1.54 mg GAE/mL and the antioxidant
capacity of 16.60-82.86%. This was a potential source of
raw materials for wine production and could help increase
the income for people in the material area.
20


The Isolation of natural yeast resulted 151 strains
from 53 different sources of Cayratia trifolia in the
Mekong Delta. Preliminary classification from
morphological,
physiological
and
biochemical
characteristics showed that 151 yeast strains of 4 genera

including Saccharomyces, Hanseniaspora, Pichia and
Candida. In particular, Saccharomyces was widely
distributed and present in large numbers on many
Cayratia trifolia samples.
Identify 64/151 strains capable of growing at 37°C in
an environment with an ethanol concentration from 9 to
12% v/v. 30/64 strains of thermotolerant yeasts could
grow at 37°C to 45°C with the supplemented ethanol
concentration from 9-12% and produced 6% (v/v) ethanol.
The results of genetic sequencing in addition to the
morphological and biochemical analysis of 30 yeast
strains showed that the strains belonged to the four genera
Saccharomyces, Candida, Pichia and Clavispora. In
particular, Saccharomyces cerevisiae HG1.3, CM3.2,
AG2.1, TV4.2, DT3.2, LA1.3, KG2.1, TG3.1 and HG2.1
were the most closely related strains. with a 100%
bootstrap index.
The suitable conditions for wine fermentation of
Cayratia trifolia juice using thermotolerant yeast strain
HG1.3 were determined at 20 oBrix, pH 4,5, 105 cell/mL
density in 6 days at 35ºC. The obtained wine has unique
color and satisfied qualitative requirements according to
the regulations. In particular, the polyphenol content was
determined at 0,6 mg GAE/mL with 57.3% anti-oxidant
ability which could help improve human health. Figure
4.2 showed the recommended protocol for Cayratia
trifolia wine fermentation.

21