VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

ISOLATION AND CHARACTERIZATION

OF YEAST STRAINS ISOLATED
FROM FERMENTED FRUITS

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S in Biotechnology

Student name: Nguyen Ngoc Minh Thu – ID no.

BTBTIU15091
Supervisor: Tran Thi My Hanh, PhD

March/2020
 Acknowledgment

Firstly, I want to express my special thanks to Dr. Tran Thi My Hanh for her guild
and support during this project. Her wholehearted help is one of the most
important motivations for me to complete the thesis. Her kindness to give me
time and other resources have been deeply appreciated.

Secondly, I am grateful to all technicians of the laboratory of the Chemical

Technology Institute, especially Dr. Trung for providing the best condition for
sample analyzing. I also want to express a deep sense of gratitude to
technicians of the Marine Lab for offering me facilities and techniques to
complete the thesis. This journey would not be possible without the assistance of
Mr. Anh Kiet, Mr. Anh Quan and Ms. Hoang Tuyen, as well as my beloved lab
mates: Ms. Lai Ngoc Bao Tran, Ms. Tran Ngoc Phuong Uyen, Ms. Truong Nguyen
Ngoc, Mr. Nguyen Quang Truc. I want to send my thanks to Mr. Truong Nhat
Nam, Mr. Hoang Phung and Ms. Nguyen Thi Tu Minh for their generous helping
hand. Besides, I also appreciate the great support of Mr. Huynh Le Quang Du
during the project. Their contribution from the beginning of this project is
sincerely acknowledged.

At last, I would like to thank for my dear family including my dad, mom, and my
brother who always be my side through thick and thin. Their continuous love,
care, help, and support have been a valuable encouragement toward the
completion of my thesis.
ISOLATION AND CHARACTERIZATION OF YEAST STRAINS ISOLATED
FROM FERMENTED FRUITS
Thu N.M. Nguyena, Hanh T.M. Trana,b
a
School of Biotechnology, International University - Vietnam National University
in
HCMC
b
Corresponding author’s email address: ttmhanh@hcmiu.edu.vn
Abstract
Qualities of fermented beverages have long been reported to depend on the Formatted: Font color: Auto
activities of yeasts, not only for alcohol production but also aroma influences
(volatile compound profiles). Recently, there has been an increasing interest in
screening for aroma producing yeasts from diverse habitats. However, there is
only a limited understanding of yeast speciess associated with local fruits in
Vietnam. Thus, the objective of the current research was to isolate yeast strains
from fermented fruits collected from southern Vietnam and evaluated their
volatile compounds and ethanol productions.

11 different yeast strains were isolated from 2 week-old-fermented fruits. Based

on morphological characteristics, strains D1 and D4 from papaya, strain C1 from
ambarella and strain P1 from ambarella were tentatively identified as members
of Saccharomyces genus, strain B10 from banana would be a Rhodotorula sp,
and the rest had no clear and specific morphological features to be properly
classified.

After 7 days of fermentation, 36 volatile compounds were identified from the

individual cultures of the isolated yeasts using GC-MS technique. Among these
volatiles, 15 compounds belong to carbohydrates, 4 belong to alcohols, 12
belong to esters, and 4 would be of other group of organic compounds.
Generally, G12 is the most potential strain for aroma compounds production as
its culture yielded the largest highest content of esters (25.064%), of which,
Isoamyl acetate accounted for 10.77%, 3-Methylthiopropyl acetate was 0.154%
and Phenethyl acetate was 14.14%. In terms of alcohol production, all strains
were found to produce alcohol. The foremost most potential strain for alcohol
production was P1 and G11, which produced 23.54 g /L and 23.39 g/l of ethanol
respectively. Strain B10 could produce red-pink pigment indicating the sign of
carotenoid synthesis. B11 had the ability to produce 24,25-Dihydroxyvitamin D,
which is precursor of vitamin D3.

1
Contents

Abstract ............................................................................................ 1

1. Introduction ............................................................................. 6

2. Materials and methods .............................................................. 8

2.1. Materials ................................................................................. 8

2.2. Fermented fruit preparation ...................................................... 9

2.3. Isolation of yeast strain from fermented fruit ............................ 9

2.4. Morphological identification of isolated yeast strains ................ 10

2.5. Inoculum preparation ............................................................. 10

2.6. Culture preparation for aromatic compounds and ethanol

evaluation .................................................................................... 10

2.7. Sampling method for VOCs detection ....................................... 10

2.8. Qualitative detection of volatile compounds ............................. 13

2.9. Quantitative detection of alcohol ............................................. 13

3. Results and discussion ............................................................ 14

3.1. Isolation and morphological characterization of the isolated yeast

strains from the fermented fruits ................................................1514

3.2. Detection of volatile compounds in the isolated yeasts cultures3619

3.3. Alcohol production of the yeast cultures ...............................4629

4. Conclusions .........................................................................4730

References ......................................................................................... i

2
List of Figures

Figure 1. A jar of fermented dragon fruit after 2 weeks of fermentation........... 9

Figure 2. A nitrogen-assisted headspace solid-phase extraction (NA/HS-SPE)
device: (A) physical map; and (B) schematic diagram (Gao et al., 2019) ........ 11
Figure 3. The system for method testing in the first, second and third trials ... 12
Figure 4. The final modified system. (A) Physical map; and (B) schematic
diagram.................................................................................................. 13
Figure 5. Standard curve for GC analysis of alcohol..................................... 14
Figure 6. Cell morphology under microscope 100x of yeasts isolated from
fermented pineapple. ............................................................................ 3417
Figure 7. Cell morphology under microscope 100x of yeasts isolated from
fermented banana ................................................................................ 3517
Figure 8. Cell morphology under microscope 100x of yeasts isolated from
fermented ambarella. ............................................................................ 3518
Figure 9. Cell morphology under microscope 100x of yeasts isolated from
fermented red dragon fruit..................................................................... 3518
Figure 10. Cell morphology under microscope 100x of yeasts isolated from
fermented papaya................................................................................. 3619
Figure 11. Percentages of esters measured in the yeast cultures ............... 4629
Figure 12. Alcohol produced from the isolated yeast in YEPD Broth ............ 4629

3
List of Tables

Table 1. Standard ethanol concentration and pA ........................................ 14

Table 2. Colony morphologies of yeasts isolated from fermented fruits in Long
An province and Ho Chi Minh city. ........................................................... 3315
Table 3. Cell morphology of yeasts isolated from fermented fruits in Long An
province and Ho Chi Minh city ................................................................ 3416
Table 4. VOCs produced by the yeasts isolated from fermented ambarella in
YEPD Broth .......................................................................................... 3720
Table 5. VOCs produced by the yeasts isolated from fermented red dragon fruit
in YEPD Broth ....................................................................................... 3922
Table 6. VOCs produced by the yeasts isolated from fermented banana in YEPD
Broth .................................................................................................. 4023
Table 7. VOCs produced by the yeasts isolated from fermented papaya in YEPD
Broth .................................................................................................. 4225
Table 8. VOCs produced by the yeasts isolated from fermented papaya in YEPD Formatted: Table of Figures, Left, Right: 0", Line
Broth .................................................................................................. 4428 spacing: 1.5 lines, Widow/Orphan control, Tab stops:
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Table 9. Data analysis of Alcohol Concentration (g/L) produced by 11 yeast
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strains using ANOVA in SPSS 20………………………………………………………………………....iv
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Table 10. Comparison of mean alcohol concentration produced by 11 different Formatted: Font: Not Bold, Font color: Auto
yeast strains using Post-hoc Tukey's HSD test…………………………………………….……..iv Formatted: Font color: Auto
Table 11. Data analysis of Ester percentage produced by 11 yeast strains using Formatted: Font: Not Bold, Font color: Auto
ANOVA in SPSS 20…………………………………………………………………………………………………iv
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Table 12. Comparison of mean alcohol concentration produced by 11 different Formatted: Font: Not Bold, Font color: Auto
yeast strains using Post-hoc Tukey's HSD test……………………………………………….……iv
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Widow/Orphan control
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4
List of abbreviations

VOCs Volatile organic compounds

SPME Solid phase microextraction

SPE Solid phase extraction

YEPD Yeast extract peptone dextrose

GRAS Generally recognized as safe

GC/MS Gas chromatography–mass spectrometry

5
1. Introduction

Yeasts used to be described as fermentative ascomycetous fungi and one of the

most representative ones is Saccharomyces cerevisiae. However, the discovery
of basidiomycetous yeast enlarged the definition of yeast. Consequently, yeasts
were mentioned as fungi with vegetative states that predominantly reproduce by
budding or fission which would mainly develop into a single cell (Kurtzman &
Fell, 1998). Generally, yeasts are divided into four groups: Teleomorphic
Ascomycetes, Anamorphic Ascomycetes, Teleomorphic Basidiomycetes and
Anamorphic Basidiomycetes. In over 1,500,000 species of fungi estimated, there
are only 75,000 species of fungi including yeast have been studied (Hawksworth,
2004). In 1998, there were 700 species of yeast had been researched. However,
the number of described species increased doubly to about 1500 in 2011
(Hawksworth, 2004). Until now, only a dozen of them have been used in
industry and a number of about 70-80 species have been used in laboratory
scale (Deak, 2009).

Yeast is one of the most important elements for the fermentation industry.
Besides sugar, yeast also has the ability to utilize both organic and inorganic
nitrogen sources such as amino acid, amines and urea (Large, 1986). Today in
modern biotechnology, the application of yeast has been significantly broadened.
Traditional yeast fermentation (e.g. beer, wine, soy sauce) had been developed
for a further demand of mankind. In food and feed ingredients, yeast is used as
a potential source for utilizing enzyme, flavor, pigments, amino acid and organic
acid. Besides, the application and development of yeast in biofuel, biocatalyst,
biocontrol, heterozygous protein production and other fields of science research
open a wide range of yeast utilization (Walker, 1998).

Yeast has a significant role in the characteristics of final fermented products.

Their interaction with biochemical components creates other further substances
(Šuranská et al, 2012). One of the most important ones is the aroma
compounds. Since 1923 (Omelianski, 1923), the microbial production of flavors
has been extensively researched. Many publications on this field have been
released (Abbas et al., 2006; Cheon et al., 2014; Dastager et al., 2009; Feron et
al., 1996; Kim et al., 2014). Aroma compounds which are harvested from
fermentation, are also valuable products. But only recently, the increase in
interest in identifying and characterizing the yeast flora associated with diverse
types of habitat isolation has been observed clearly (Grondin et al., 2015).

There has been a growing interest in the production of flavors by using

microorganisms, since they are considered as natural products deserving GRAS

6
(generally recognized as safe). Aroma produced from yeast is perfect
alternatives to extraction from old raw materials like plants due to their
economic feature (Armstrong & Yamazaki, 1986). Besides yeast, bacteria such
as Lactic acid bacteria and Pseudomonas are also well-known for their potential
in flavor production (Janssens et al., 1992). Today, many biotechnological
companies develop various panels of aroma compounds from microbiological
process. The discovery of new strains could open up potential new applications in
this field (Grondin et al., 2015). According to a survey of the US Food Market
Institute, flavors and aromas comprise a yearly overall market of 7 billion U.S.
dollars and 91% of shoppers believe flavors to be the most significant factor
when they pick and buy foods (Arctander, 1969). Generally, the volatile
compounds produced from fermentation process contribute the largest
percentage to the total aroma in wine.

There are a lot of studies on screening for aromatic compounds producing yeasts
from various localities all over the world. In a previous study in 2015, 26
different species were isolated from apple, persimmon fruit, avocado, passion
fruit, rose–apple, pear, pineapple, Cape gooseberry, dragon fruit, peach, and
cocoa in Madagascar and Reunion Island. A total of 52 Volatile Organic
Compounds (VOCs) were detected from the 26 yeast species cultivated on a
glucose-rich medium. Among these VOCs, there were 6 uncommon compounds
identified, as ethyl but-2-enoate, ethyl 2-methylbut- 2-enoate (ethyl tiglate),
ethyl 3-methylbut-2-enoate, 2-methylpropyl 2-methylbut-2-enoate, butyl 2-
methylbut-2-enoate and 3-methylbutyl 2-methylbut-2-enoate (Grondin et al.,
2015). The tropical areas have an advantage of microbial diversity, which
includes yeast. The habitat provides suitable conditions for them to grow and
proliferate (Barriga et al., 2014; Fleet et al., 2003; Nakase et al., 2006).
Vietnam, which is a tropical country, has an advanced dominance for yeast
development due to the favorable climate. Hence, it is of high expectation that
yeasts that are able to produce desired aromatic compounds could be found in
Vietnam. However, due to the insufficiency of facilities and resources, the
number of researches related to the natural aroma from fermentation is very
limited. It is obvious that yeast has the ability to convert sugar into alcohol for
the first fermentation. The high concentration of initial sugar and alcohol also
inhibits the growth of bacteria. Therefore, fermented fruit is a potential source
for isolating new yeast species that could naturally produce alcohol. (Doan Van,
Thuoc & Duyen, Dinh. 2015). Thuoc Doan Van and Duyen Dinh Thi Hong
successfully isolated twenty yeast strains from fermented fruit juice of
Sonneratia caseolaris. A strain was chosen to study alcohol production ability.

7
 The result showed that the yeast belonged to Candida tropicalis and had potency
to produce high alcohol percentage (Doan Van, Thuoc & Duyen, Dinh. 2015).
However, until now, the research on the ability of yeast to produce aromatic
compounds had not been invested domestically yet.

The sampling method for the analysis of potential aromatic compounds from
yeast cultures needs to be carefully designed to restrict the volatile from leaking
during the sample collection process for further analysis. The common ways are
headspace, solid phase microextraction (SPME), and purge and trap. Within
these techniques, purge and trap is the best choice for a preliminary study. The
initial model was based on Rathke & Stratev (2013) and Gao et al. (2019). In
2013, Rathke and Stratev published a procedure for purge and trap the volatile
compounds produced from Dry-Kiln condensate. The target compounds of this
method were esters, alcohols, steroid, and alkane. Then, in 2019, Jing Gao and
his associates established a similar method for analyzing the volatile organic
pollutant in water, honey, sediment and juice. The method successfully captured
4 aromatic hydrocarbons including trichloroethylene, tetrachloroethylene, 1,2-
dichlorobenzene and 1,2,3,5-tetrachlorobenzene.

The objectives of this project were to isolate and morphologically identify yeast
strains from fermented local fruits, then to initially determine the volatile
compounds produced in their cultures. Furthermore, this study also evaluated
the ethanol production and evaluated the potentials of the studied strains.

2. Materials and methods

2.1. Materials
Red dragon fruits were collected from my family member’s garden in Long An
province; ambarella and papaya were purchased from local organic gardens in
Ho Chi Minh city; banana and pineapple were bought from organic sector at Big
C (Ho Chi Minh city).

YEPD Chloramphenicol agar medium including 20 g/l glucose [Xilong], 20 g/l

peptone [ Himedia], 10 g/l yeast extract [ Himedia] ,0.5 g/l Chloramphenicol,
and 15 g/l agar was used for yeast isolation (Grondin et al., 2015).

The nutrient broth was prepared by mixing 5 g yeast extract, 40 g glucose and
10g glycerol with 1L of distilled water. The medium was autoclaved for 1 hour
and store until use.

Solvent used in sampling method for VOCs detection were n-hexane [ Chemso]:
acetone [Xilong] = 10:1

8
2.2. Fermented fruit preparation
Fermented fruits were made by mixing well ripen fruits (either red dragon fruit,
pineapple, banana, papaya or ambarella) with refined sugar and let naturally
fermented in anaerobic conditions within 2 weeks. The method was modified
from Doan Van, Thuoc & Duyen, Dinh (2015)

Figure 1. A jar of fermented dragon fruit after 2 weeks of fermentation

The fruits were peeled off and cut into small pieces, then mixed with sugar with
the ratio of 10:1 and put into a tight jar for 2-week fermentation.

2.3. Isolation of yeast strain from fermented fruit

The fermented liquid from the aforementioned mixture was used for yeast
isolation. A volume of 1 mL aliquot of each mixture was serially diluted with
sterile distilled water with different dilution factors (1:10, 1:100, 1:1000,
1:10000, 1:100000) and 100 µl of each sample was spread onto YEPD
Chloramphenicol agar plates. The purpose of serial dilution is to ensure the
obtainment of distinct single yeast colonies since the numbers of yeasts are
unpredictable in the fermented liquid.

Then the plates were incubated at room temperature in 48 hours. Each type of
fermented fruit was carried out in duplicate (2 replicates per dilution factor per
fruit sample) (Grondin et al., 2015).

When the colonies successfully formed on the surface of the agar plate, the
yeast colony was selected based on the shape of the colony. The double streak
was applied to purify the yeast culture (Kurtzman & Fell, 1998).

9
2.4. Morphological identification of isolated yeast strains
Firstly, a smear slide of each yeast colony was made and left for air dry. After
that, 1 drop of crystal violet solution was dropped onto the smears and
incubated for 1 minute before washing with distilled water. The smears were blot
dried and viewed under a light microscope at the magnification of 1500. Yeast
cells’ morphology and size were recorded. The morphological classification of
yeasts was based on The Yeasts: a taxonomic study by Kurtzman and Fell
(1998).

2.5. Inoculum preparation

A loop of yeast cells was transferred to a 250 mL flask containing 50 mL of broth
medium. Then the flask was placed on a 120-rev/min rotary shaker at room
temperature for 24h. After 24 h, the inoculum was taken out and prepared for
the next steps.

2.6. Culture preparation for aromatic compounds and ethanol evaluation

The culture To prepare for preparation was used for detection of VOCs and Formatted: Font color: Auto
alcohol: .10 ml of the yeast inoculum was then mixed with 60 ml nutrient broth
contained in a 100ml glass serum bottle. The bottle was then closed with a
rubber stopper and covered by an aluminum seal. Let the bottle at room
temperature for 7 days. The sample was used for both qualitative identifications
of VOCs and quantitative detection of alcohol. The method was modified from
Hiralal et al., (2014). In this study, the researchers used wort as a nutrient
source, so to test the ester and alcohol production, the time for fermentation
was 6 days. However, the scale of this research was much larger than my
study’s scale while the fermentation bottle was 10L of volume. Hence, increasing
the fermentation day could enhance a greater chance for ester formation.

2.7. Sampling method for VOCs detection

After 7 days, the culture was then extracted to, capture the VOCs produced Formatted: Font color: Auto
during fermentationfrom the culture was captured. A system was set up to trap Formatted: Font color: Auto
the desired aromatic compounds. The system was modified from Gao et al.
(2019) and Rathke & Stratev (2013) to adapt to our facilities and laboratory
conditions. In the initial method, the diagram below was used.

10
Figure 2. A nitrogen-assisted headspace solid-phase extraction (NA/HS-SPE)
device: (A) physical map; and (B) schematic diagram (Gao et al., 2019)

The sample was diluted to a concentration of 0.1 µg/ml with a total volume of
20mL. Then, a test tube that had a rubber plug with 2 holes was used. One hole
was connected with a stainless-steel needle which allowed a controlled nitrogen
stream to penetrate the sample. A capture system was formed by connecting the
other hole to the solid phase extraction (SPE) cartridge. During this process,
leaking was restricted, nitrogen was continuously introduced to the tube to form
a series of bubble rising to the liquid surface. The bubble was then broken down
and released the volatile compounds to the top. The SPE cartridge then worked
as a compound adsorption due to the interaction between the adsorbent and
compounds. The purging process begins by continuously introducing nitrogen to
the sample at a constant flow rate of 0.2 L/min for 45 mins. Throughout the
process, the temperature was maintained at 40 ± 0.3°C. Finally, 10ml of n-
Hexane: acetone=10:1 was used as an elution solvent for desorption of the
analytes. The eluted targets could be directly analyzed with Gas Chromatography
with Electron Capture Detector (GC-ECD).

Before confirming the final method for sampling, there were several trials had
been done to determine the capability of the purging method in Gao et al
(2019). In the first trial, the below system was used to capture the target
compounds. The test tube in the initial method was replaced by a serum bottle
with a rubber plug and aluminum seal to simplify the transfer from fermentation
bottle to sampling bottle and minimize the leak out of volatile compounds. SPE
cartridge was removed from the system. A glass syringe with stainless steel
needle penetrates one hole of the rubber plug while another hole was connected
with the nitrogen pumping system. Instead of using n-Hexane: acetone=10:1 as
a final step to elute the targets, the solvent was directly added to the syringe.
The balance of pressure between inner gas and outer liquid keeps the solution
stable inside the glass syringe. The flow and time of purging of nitrogen did not

11
change. The system temperature and the sample’s volume were the same.
However, after 5 minutes of purging, all of the solvents vaporized out of the
syringe. For the second trial, the flow rate of nitrogen purging was reduced to
only 30ml/min, 60 ml/min and 100 ml/ min. When the flow rate was 100 ml/
min, the sample totally vaporized after 15 minutes. For the experience with a
flow rate equal to 60ml/min, the result still showed no signal for the volatile
compound, but the solvent vapor to a half. For the experience with a flow rate
equal to 30ml/min, the bubble was very good, the stream was slow and the
vapored solvent was not significant, the peak signal in the result existed but still
was very small. The next time, every criterion was the same as the experience
with flow rate = 30ml/ min except the volume of the sample. The volume of
30ml and 70ml was examined. The result showed that there was an
improvement in the number of volatile compounds present in the sample with 70
ml volume, but the peak signal not yet cleared.

Figure 3. The system for method testing in the first, second and third trials

Finally, 10 ml of the solvent was directly added into the bottle and create a top
layer above 60 ml of the liquid sample. The syringe was removed, a stainless-
steel needle was connected to the system to avoid high pressure. Before
purging, the bottle was shaken carefully for 2 minutes. The purging process was
45 minutes with the flow rate at 30 ml/ min. The diagram below illustrates this
system.

12
Figure 4. The final modified system. (A) Physical map; and (B) schematic
diagram

2.8. Qualitative detection of volatile compounds

After successfully capturing the target with solution n-Hexane: Acetone=10:1, a
GC vial was used to contain a 1mL sample. The analysis of the sample was
conduct by GC- MS instrument SQ 456-GC (SCION Innovative Co., Ltd). The
compounds were separated by gas chromatography on a Rxi-5ms column (30 m
x 0.25 mm × 0.25 µm df, RESTEK Co., Ltd). The carrier gas (Helium) was set at
a flow rate of 1 mL /min. The oven program started with an initial temperature
of 40°C and held for 1 min. The oven was then heated to 200 °C with a rate of
20 °C/min. Then the temperature continuously ramped to 280 °C at 10 °C/min
and finally held isothermally for 3 min. The temperature of the injector was set
at 250 °C.

The detection of captured compounds was based on the comparison of their

spectra and relative abundances with the database from NIST with the NIST MS
Search 2.2 software. The identity was also confirmed by comparing the match
factor (MF) and the probability of difference reference results. The percentage of
each compound was calculated automatically based on the chromatographic
area.

2.9. Quantitative detection of alcohol

The samples were centrifuged at 9000 rpm for 5 mins. Then, 1 mL of the
supernatant was transferred to GC vials for analysis followed Tran et al. (2009).

Different concentrations of ethanol were used to construct a standard curve. The

experimental result was illustrated by below linear regression:

y = 0.0041629218x + 7.2924626656

13
 Where y is the alcohol concentration (g/L) and x is the GC signal magnitude (pA)

After successfully constructing the linear relationship, the signal magnitude of

fermented samples was analyzed using GC system and calculated based on the
above equation.

Table 1. Standard ethanol concentration and pA

Peak area (pA) Ethanol Concentration (g/L)

8063.6 39,5

16753.5 79

36347.3 158

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Figure 5. Standard curve for GC analysis of alcohol
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3.1. Isolation and morphological characterization of the isolated yeast
strains from the fermented fruits
After 2 times of streaking on the YEPD agar plate, the yeasts were totally
purified. After 7 days of incubation at 25°C, yeast colonies were differentiated
from each other based on the differences in morphology including color, texture,
margin, elevation, and size. Generally, in 5 types of fermented fruit including
dragon fruit, pineapple, banana, papaya, and ambarella, there were 11 yeast
strains that had been isolated. The isolated strains were designated as G11, G12
(for dragon fruit sample), C1, C2 (for ambarella sample), D1, D3, D4, D5 (for
papaya sample), P1 (for pineapple sample), and B10, B11 (for banana sample).
Papaya is the sample that had the highest number of strains (4 strains). In
contrast, pineapple had the lowest number (only 1 strain). Most of the yeasts
had various levels of white colony, from white to whitish cream, while only B10
had a red-pink color which indicates the presence of carotenoid pigment
(Kurtzman & Fell, 1998). Moreover, the colonies of B10 and G12 had enlarged
significantly when aging (Table 2)

Table 2. Colony morphologies of yeasts isolated from fermented fruits in Long An

province and Ho Chi Minh city.

Name Fruit Colony Colony description Origin

type
Morpholo Shape & color Margin Elevati
gy on

1 G11 Dragon White, circular, Entire Umbon LA

fruit mucoid and ate
smooth texture

2 G12 Dragon White, circular, Curly Flat LA

fruit smooth texture

3 P1 Pineapple White, circular, Undulate Flat HCMa

membranous
texture

4 B10 Banana Red pink, Entire Raised HCMa

circular,
smooth texture

5 B11 Banana white, curled, Undulate Flat HCMa

butyrous
texture, shiny
surface

15
 6 D1 Papaya white, Entire Raised HCMb
irregular, fluid
texture, shiny
surface

7 D3 Papaya White, Undulate Flat HCMb

membranous
thin texture,
matte surface

8 D4 Papaya white, smooth, Undulate Raised HCMb

fluid texture,
shiny surface

9 D5 Papaya white, circular, Undulate Flat HCMb

membranous
thick texture

10 C1 Ambarell white, circular, Entire Raised HCM

a fluid texture,
shiny surface

11 C2 Ambarell cream white, Entire Flat HCM

a circular, fluid
texture, shiny
surface

LA: yeast isolated from fruit in garden in Long An Province

HCMa: yeast isolated from fruit in Big C organic food sector in Ho Chi Minh city
HCMb: yeast isolated from fruit in garden in Ho Chi Minh city

A loop of yeast colony was stained by crystal violet solution. Observing under the
microscope showed that most of the yeast was in the form of cylindrical with
round end or globose shape. To observe the ascospore, yeast was cultured on
YEPD agar for 7 days at 25°C (Kurtzman et al., 2011). The images of how they
proliferate and distribute were also captured under the microscope. The
characteristic of the cell was recorded based on cell shape, ascospore, and
pellicle formation. By microscopic observation, globose and ellipsoidal ascospores
were noticed with a various number of ascospores per ascus. All isolated strains
did not show any pellicle formation (Table 3, figure 6, figure 7, figure 8, figure 9,
figure 10). The genera of them were initially predicted based on The Yeasts—A
Taxonomic Study by Kurtzman and Fell (1998).

Table 3. Cell morphology of yeasts isolated from fermented fruits in Long An

province and Ho Chi Minh city

N Name Fruit Cell description

type
Cell shape Ascospores Ascospores Pellicle

16
 shape number formation

1 G11 Drago Cylindrical Ellipsoidal 2-4 No pellicle

n fruit with observed
pointed end

2 G12 Drago Cylindrical Ellipsoidal 5-20 No pellicle

n fruit with round observed
end

3 P1 Pineap Cylindrical Globose 2-8 No pellicle

ple with round observed
end

4 B10 Banan Globose Globose 2-15 No pellicle

a observed

5 B11 Banan cylindrical Ellipsoidal 2-20 No pellicle

a with round observed
end

6 D1 Papaya Globose Globose 2 No pellicle

observed

7 D3 Papaya Cylindrical Ellipsoidal 3-6 No pellicle

with round observed
end

8 D4 Papaya Globose Globose 2 No pellicle

observed

9 D5 Papaya Cylindrical Ellipsoidal 2 No pellicle

with round observed
end

10 C1 Ambar Globose Globose 7-28 No pellicle

ella observed

11 C2 Ambar Cylindrical Ellipsoidal 4-5 No pellicle

ella with round observed
end

The figures below illustrated the morphology cell shape of 11 yeast strains under
microscope.

17
 Figures below illustrate the
morphology of each strain including B10, B11, C1, C2, P1, D1, D3, D4, D5, G11
and G12.

Figure 7. Cell morphology under microscope 100x of yeasts isolated from

fermented banana

Figure 8. Cell morphology under microscope 100x of yeasts isolated from

fermented ambarella.

18
Figure 9. Cell morphology under microscope 100x of yeasts isolated from
fermented red dragon fruit

Figure 10. Cell morphology under microscope 100x of yeasts isolated from
fermented papaya

Figure 6. Cell morphology under microscope 100x of yeasts isolated from fermented pineapple.
After 7 days of fermentation, the target compounds in 11 samples were
extracted by using a modified purge and trap technique. Then, these compounds

19
were analyzed using GC-MS. The obtained results are displayed from table 4 to
table 8.

Overall, 11 samples were able to produce VOCs. 15 carbohydrates, 4 alcohols,

12 esters, and 4 other organic compounds were identified. The sample B10
produced the most compounds. However, only 5 over 14 compounds had odor
and only 4 compounds had desired aroma. B10 was also the yeast strain that
had the largest number of carbohydrates in the final product. G12 was the yeast
that had the largest proportion of ester at 25.064 %. Isoamyl acetate and
phenethyl acetate are compounds that contribute the most to the percentage of
total aromatic volatile compound produced by G12. Comparing to other yeast
strains, the proportion of ester in C2 is the least, at only 0.213% (Table 4, figure
11)

All 11 strains had Dodecane and 1-Octanol as their product. Dodecane was also
the compound that holds the largest percentage varied from 66.01 to 85.75%. It
is an alkane with a straight chain that plays an important role in plant
metabolite. The various essential oil of plants including ginger had formed from
this compound. Commercially, Dodecane is also an important chemical used as a
solvent for other chemicals, especially in jet fuel study. 1-Octanol is also a
potential compound that is currently used in food-flavoring, cosmetic and
perfume. The taste of it is known as sweet and slightly herbaceous. Phenylethyl
alcohol and phenethyl acetate are compounds used in food flavor. Phenylethyl
alcohol is also used in other manufacturing including perfumery as rose perfume,
or fragrance in cosmetics. Furthermore, this compound is an antibacterial and
preservative agent (PubChem, n.d.).

In the result of C1 cultured product, there were 8 compounds that had been
detected. Out of these compounds, 4 compounds had desired aroma and 2
compounds were of unpleasant smell. The result showed that carbohydrates and
other compounds, alcohol, and ester occupied 86.065%, 12.915%, and 1.29%,
respectively. (Table 4, figure 11)

For C2, 7 compounds had been detected. There were 4 compounds that had
odor. In which, 2 compounds had desired aroma and the others did not. The
result showed that carbohydrates and other compounds, alcohol, and ester took
87.258%, 12.3%, and 0.213%, correspondingly. (Table 4, figure 11)

Table 4. VOCs produced by the yeasts isolated from fermented ambarella in

YEPD Broth

Name Volatile Formula Odor type MFa RTb %c

20
 compounds

C1 Benzaldehyde,
3-benzyloxy-2-
fluoro-4- C15H13FO 4.41
methoxy 3 586 6 0.75

4.73
e
Nonane C9H20 Gasoline like 862 3 0.285

Bitter Almond, Burnt 6.21

1-Octanol C8H18O Matches, Fat, Floral d 906 4 11.38

Phenethyl Fruit, Honey, Lilac, 6.64

alcohol C8H10O Rose, Wine d 912 1 1.535

7.24
e
Dodecane C12H26 Gaselin like 930 1 85.03

Phenethyl 7.71
d
acetate C10H12O2 Flower, Honey, Rose 910 5 0.613

2-Phenylethyl Sweet, Rose, Fruity, 8.39

propanoate C11H14O2 Berry, Honey e 784 4 0.18

Dodecyl 15.2
Acrylate C15H28O2 792 7 0.659

C2 Isopentyl C7H14O2
acetate 690 4.510 0.213

e
Nonane C9H20 Gasoline like 815 4.73 0.255

Bitter Almond, Burnt 6.21

1-Octanol C8H18O Matches, Fat, Floral d 899 2 10.25

Phenethyl Fruit, Honey, Lilac, 6.63

alcohol C8H10O Rose, Wine d 929 8 1.926

e
Dodecane C12H26 Gaselin like 927 7.24 86.34

9-hexadecen-1- 13.5
ol C16H32O 658 92 0.124

C20H39Cl 15.2
Heptadecyl 3- O2 778 67 0.663
chloropropanoat

21
 e

There were 6 compounds detected from sample G11. In which, 4 compounds

had a good odor and one compound did not have the desired odor. The
proportion of alcohol was 14.367%, ester was 3.913%, carbohydrate and other
compounds were 81.27% (Table 5, figure 11).

G12 yeast cultivated in the liquid medium had 7 compounds detected. Over 6
compounds that had odor, 5 compounds were of pleasant aroma. While alcohol
proportion was at 8.515% and ester was at 25.064%, the percentage of
carbohydrate and other compounds were the highest in total, at 66.213% (Table
5, figure 11).

Table 5. VOCs produced by the yeasts isolated from fermented red dragon fruit
in YEPD Broth

Name Volatile Formula Odor type MFa RTb %c

compounds

G11 2-Methylbutyl Apple, Banana,

acetate C7H14O2 Pear d 742 4.516 0.799

Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 902 6.218 12.19

Phenethyl Fruit, Honey, Lilac,

alcohol C8H10O Rose, Wine d 887 6.639 2.177

e
Dodecane C12H26 Gaselin like 930 7.241 81.27

Phenethyl Flower, Honey,

acetate C10H12O2 Rose d 921 7.719 2.244

Indolyl-3-
ethanol
acetate C12H13NO2 784 12.305 0.87

G12 C7H14O2
Isoamyl Apple, Banana,
acetate Glue, Pear d 935 4.509 10.77

22
 2,4,6- C10H22
Trimethylhept
ane 775 4.736 0.202

Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 895 6.216 8.515

Asparagus,
3- Cabbage, Herb,
Methylthiopro Mushroom, Potato
d
pyl acetate C6H12O2S 691 6.675 0.154

e
Dodecane C12H26 Gaselin like 925 7.244 66.01

Phenethyl Flower, Honey,

acetate C10H12O2 Rose d 945 7.716 14.14

Burnt, Floral,
Indole C8H7N sweet e 815 8.040 0.203

The culture of B10 yeast had the presence of 15 compounds. B10 was also the
yeast that produces the largest number of volatile compounds. Among the total
of 8 compounds had odor, only 4 compounds had desired aroma. As same as
other samples, carbohydrate and other compounds percentage were the highest
in total, at 87.147%, while alcohol was at 12.814%, and ester percentage was
only 0.861% (Table 6, figure 11).

There were 7 compounds detected from B11 yeast culture. Over 3 compounds
that had odor, only 2 had a good aroma. The percentage of carbohydrate and
other compounds from the culture were 83.681%, while alcohol was at 15.957%
and ester was only 0.307. B11 was also the only yeast that was detected with
the production of 24,25-Dihydroxyvitamin D at 0.141% (Table 6, figure 11).

Table 6. VOCs produced by the yeasts isolated from fermented banana in YEPD
Broth

Na Volatile Formula Odor type MFa RTb %c

me compounds

B10 Dodecyl
chloride C12H25Cl 508 4.410 0.91

O-Xylene C8H10 788 4.494 0.27

Strong, sweetish

23
 g
like

e
Nonane C9H20 Gasoline like 803 4.727 0.35

2,2,4,6,6-
Pentamethylhe
ptane C12H26 700 5.587 0.06

Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 905 6.215 12.68

e
Dodecane C12H26 Gaselin like 903 7.242 83.88

Undecane C11H24 617 8.659 0.215

e
Nonadecane C19H40 Sweet, deep-rosy 855 9.966 0.297

e
Hexadecane C16H34 Gaselin like 812 11.376 0.303

e
Octadecane C18H38 Fuel-like 747 12.902 0.24

d
1-Hexadecanol C16H34O Flower, Wax 696 13.588 0.134

9- C23H48
Hexylheptadeca
ne 647 14.480 0.27

Dodecyl
Acrylate C15H28O2 793 15.266 0.861

7-Hexylicosane C26H54 657 16.046 0.196

3-Ethyl-5-(2- C26H54
ethylbutyl)
octadecane 638 17.586 0.156

B11 P-XYLENE C8H10 549 4.503 0.112

Methyl 12,15-
octadecadiynoa
te C19H30O2 525 4.716 0.307

Phenethyl Fruit, Honey, Lilac,

alcohol C8H10O Rose, Wine d 810 6.639 2.347

24
 e
Dodecane C12H26 Gaselin like 930 7.243 82.35

24,25-
Dihydroxyvitam
in D C27H44O3 388 9.981 0.141

Heptadecyl 3-
chloropropanoa
te C20H39ClO2 773 15.266 0.883

Bitter Almond,
Burnt Matches, Fat,
1-Octanol C8H18O Floral d 908 6.21 13.61

The culture of D1 yeast had the presence of 7 compounds. Over 6 compounds

that had odor, 3 compounds had desired aroma. Alcohol percentage was
12.401%, ester was only 0.41%, carbohydrate and other compounds took
86.884% (Table 7, figure 11).

For D3 culture, there was the presence of 7 compounds, in which 6 compounds

had odor but only 4 compounds had a good odor. While alcohol had a
percentage of 13.105%, ester proportion was 2.191%, carbohydrate and other
compounds still took the largest percentage at 85.316% (Table 7, figure 11).

D4 yeast cultivated in the liquid medium had 7 compounds detected. Over 6

compounds that had odor, 3 compounds had a pleasant aroma. While alcohol
proportion was at 14.486% and ester were at 1.65%, the percentage of
carbohydrate and other compounds were the highest in total, at 83.68% (Table
7, figure 11).

There were 8 compounds detected from D5 yeast culture. Over 7 compounds

that had odor, only 5 had a good aroma. The percentage of carbohydrate and
other compounds from the culture were 86.237%, while alcohol was at 12.708%
and ester was only 0.817% (Table 7, figure 11).

Table 7. VOCs produced by the yeasts isolated from fermented papaya in YEPD
Broth

Name Volatile Formula Odor type MFa RTb %c

compounds

e
D1 Nonane C9H20 Gasoline like 810 4.734 0.293

25
 Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 904 6.215 11.33

Fruit, Honey,
Phenethyl Lilac, Rose, Wine
d
alcohol C8H10O 917 6.642 0.9

e
Dodecane C12H26 Gaselin like 921 7.244 85.75

Phenethyl Flower, Honey,

acetate C10H12O2 Rose d 902 7.716 0.41

Heptadecyl 3-
chloropropano
ate C20H39ClO2 801 15.266 0.847

Diacetone pleasant, faint,

alcohol C6H12O2 minty odor f 681 4.21 0.171

D3 C7H14O2
Isoamyl Apple, Banana,
acetate Glue, Pear d 916 4.508 1.296

C9H20 Gasoline like

Nonane odor e 826 4.728 0.31

Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 901 6.215 10.63

Fruit, Honey,
Phenethyl Lilac, Rose, Wine
d
alcohol C8H10O 925 6.635 2.475

e
Dodecane C12H26 Gaselin like 938 7.244 84.34

Fruit, Honey,
Phenethyl Lilac, Rose, Wine
d
acetate C8H10O 881 7.716 0.229

26
 Heptadecyl 3-
chloropropano
ate C20H39ClO2 800 15.266 0.666

D4 Diacetone pleasant, faint,

alcohol C6H12O2 minty odor f 640 4.215 0.19

e
Nonane C9H20 Gasoline like 843 4.735 0.33

Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 906 6.215 13.2

e
Dodecane C12H26 Gaselin like 937 7.246 83.35

Dodecyl
Acrylate C15H28O2 791 15.27 0.965

Fruit, Honey,
Phenethyl Lilac, Rose, Wine
d
alcohol C8H10O 901 6.64 1.096

Phenethyl Flower, Honey,

acetate C10H12O2 Rose d 884 7.72 0.685

D5 C7H14O2
Isoamyl Apple, Banana,
acetate Glue, Pear d 878 4.505 0.707

e
Nonane C9H20 Gasoline like 812 4.726 0.297

Bitter Almond,
Burnt Matches,
1-Octanol C8H18O Fat, Floral d 916 6.213 10.83

Fruit, Honey,
Phenethyl Lilac, Rose, Wine
d
alcohol C8H10O 912 6.641 1.755

2-Ethyl-
decane C12H26 766 7.017 0.72

e
Dodecane C12H26 Gaselin like 929 7.244 85.22

Phenethyl Flower, Honey,

acetate C10H12O2 Rose d 849 7.718 0.11

27
 1-
d
Hexadecanol C16H34O Flower, Wax 741 13.589 0.123

For the final yeast culture, P1 had 5 compounds detected and all of them had
odor. However, only 3 compounds had a pleasant odor. While alcohol had a
percentage of 14.911%, ester proportion was 0.61%, carbohydrate and other
compounds still took the largest percentage at 84.481% (Table 8, figure 11).

Table 8. VOCs produced by the yeasts isolated from fermented papaya in YEPD
Broth

Na Volatile Formula Odor type MFa RTb %c

me compou
nds

e
P1 Dodecan C12H26 Gaselin like 927 7.244 84.2
e

e
Nonane C9H20 Gasoline like 793 4.734 0.281

1- C8H18O Bitter Almond, Burnt 895 6.214 12.24

Octanol Matches, Fat, Floral d

Pheneth C8H10O Fruit, Honey, Lilac, 915 6.642 2.671

yl Rose, Wine d
alcohol

Isoamyl Apple, Banana, Glue, 820 4.514 0.61

acetate C7H14O2 Pear d

a Match factor calculated by NIST MS Search 2.2 software

b Retention time experimentally determined
c Percentage of each compound in the total volatile product collected from the
culture of each strain
d Data were collected from Flavor and Extract Manufacturers Association
e Data were collected from The Good Scents Company

28
f Data were collected from pubchem.ncbi.nlm.nih.gov
g Bullard and Holguin, 1977

VOCs- known as volatile organic compounds are organic compounds that have
low-molecular-weight and easily vapor at room temperature (Pennerman et al.,
2016). Ester is also a VOCs that yeasts produce from their equivalent alcohol
when the acetate group utilized is over the demand of cell for the synthesis of
lipid in membrane (Bamforth, 2003).

The figure below illustrates the percentage of ester produced in each culture.
Assume that the total VOCs produced in each strain are the same, G12 was the
most potential strain for ester production- at 25.064% which is significant higher
than the other strains. The produced esters included isoamyl acetate and
phenethyl acetate. Both of them had the desired aroma and are valuable for
many fields of industry especially food flavor and perfume manufacturing

Figure 11. Percentages of esters measured in the yeast cultures. Duplication was Commented [HT1]: You have duplicates, thus, error bars
should be inserted
used for each strain, and statistically identified differences among them were
Formatted: Font color: Auto
indicated by different letters ( p-value <0.05). Error bars indicate standard
Formatted: Line spacing: 1.5 lines, Don't keep with
errors.
next, Border: Top: (No border), Bottom: (No border),
Left: (No border), Right: (No border), Between : (No
3.3. Alcohol production of the yeast cultures border)
This part of the research investigated the ability of isolated yeast strain in Formatted: Font color: Auto
alcohol production. The experiment was conducted after 7 days of fermentation. Formatted: Font color: Auto
Alcohol concentration was calculated based on the linear equation constructed in Formatted: Font: Verdana

figure 5 (method section). Formatted: Font color: Auto

29
The result showed that all yeast strain was able to create alcohol. Strain P1 and
G11 were identified as producing the highest level of alcohol at 23.54g/L and
23.39g/L respectively, while the least potential strain in alcohol production was
B10 with only 8.3 g/L as the average concentration. Formatted: Font color: Red

Average Alcohol Concentration

25.00 f f
e ef

20.00 d
Concentration(g/L)

15.00 c c c
c

10.00 a b

5.00

0.00
B10 G12 D3 D4 D1 D5 C2 C1 B11 G11 P1
Label

Figure 12. Average alcohol produced from 11 isolated yeast in YEPD Broth. Commented [HT2]: Need to analyze the data to see if
there is any significant differences among the strains?
Duplication was used for each strain, and statistically identified differences
Formatted: Font color: Auto
among them were indicated by different letters ( p-value <0.05). Error bars
Formatted: Font color: Auto
indicate standard errors.
Formatted: Font color: Auto
3.1. Isolation and morphological characterization of the isolated yeast Formatted: Centered
strains from the fermented fruits Formatted: Font: Verdana
Formatted: Font color: Auto

There were 6 compounds detected from sample G11. In which, 4 compounds Formatted: Font: (Default) Verdana, 10 pt

had a good odor and one compound did not have the desired odor. The
proportion of alcohol was 14.367%, ester was 3.913%, carbohydrate and other
compounds were 81.27% (Table 5, figure 11).

a Match factor calculated by NIST MS Search 2.2 software

b Retention time experimentally determined

30
c Percentage of each compound in the total volatile product collected from the culture of each strain
It is possible to isolated yeast from fermented fruits. In general, a total of 11
yeast strains were isolated from red dragon fruit, pineapple, banana, papaya or
Commented [HT3]: You have duplicates, thus, error bars
should be inserted
Commented [HT4]: Need to analyze the data to see if
there is any significant differences among the strains?

ambarella. Based on morphological characteristics C1, D1 and D4 seemed to be Formatted: Font color: Auto

members of Saccharomyces. P1 might also have an opportunity to be

Saccharomyce and B10 might belong to Rhodotorula genus. Molecular technique
should be used in the future for confirmation and identification of all strains.

After 7 days of fermentation, 36 compounds were identified from cultures of the

isolated yeasts. Among these VOCs, there were 15 carbohydrates, 4 alcohols, 12
esters, and 4 other organic compounds. Phenylethyl alcohol and phenethyl
acetate are valuable compound that 10 over 11 strains created. G12 is the most
potential strain for aroma compound production. Over the largest proportion of
ester at 25.064%, Isoamyl acetate took 10.77%, 3-Methylthiopropyl acetate
took 0.154% and Phenethyl acetate took 14.14%. In terms of alcohol
production, all strains produced alcohol. The most potential strain for alcohol
production was P1, which produced 23.54 g /L of Ethanol. Strain B10 could
produce red-pink pigment that shows the ability of carotenoid synthesis .
Carotenoid is an important compound for different industries. The value of Formatted: Font color: Auto
carotenoid is its role as not only a vitamin A precursor but also coloring and
antioxidant sources (Mata-Gómez et al., 2014).

31
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 Appendix 1: Tables for data analysis in SPSS 20 software Formatted: Font: Verdana, 11 pt

Table 9. Data analysis of Alcohol Concentration (g/L) produced by 11 Formatted: Centered

Formatted: Font: 11 pt
yeast strains using ANOVA in SPSS 20.
Formatted: Font: Verdana, 10 pt, Bold
Sum of df Mean F Sig. Formatted: Centered, Right: 0", Space After: 8 pt,
Squares Square Line spacing: At least 20 pt, Widow/Orphan control
Formatted: Font: Bold
Between 1153.77
670.238 10 67.024 .000 Formatted: Font: Verdana, 10 pt, Bold
Groups 6
Formatted: Font: (Default) Verdana, 10 pt
Within Groups .639 11 .058 Formatted: Font: Verdana, 10 pt

Total 670.877 21 Formatted: Font: Verdana, 10 pt

Formatted: Font: Verdana, 10 pt
Formatted: Font: Verdana, 10 pt
Table 10. Comparison of mean alcohol concentration produced by 11 Formatted: Centered
different yeast strains using Post-hoc Tukey's HSD test
Formatted: Font: Bold
Formatted: Font: Verdana, 10 pt
Label N Subset for alpha = 0.05

1 2 3 4 5 6

8.30230
B10 2 Formatted: Font: Verdana, 10 pt
0

9.26575
G12 2 Formatted: Font: Verdana, 10 pt
0

13.01030
D3 2 Formatted: Font: Verdana, 10 pt
0

13.22010
D4 2 Formatted: Font: Verdana, 10 pt
0

13.26250
D1 2 Formatted: Font: Verdana, 10 pt
0

13.91400
D5 2 Formatted: Font: Verdana, 10 pt
0

18.93280
C2 2 Formatted: Font: Verdana, 10 pt
0

22.40435
C1 2 Formatted: Font: Verdana, 10 pt
0

22.63330 22.63330
B11 2 Formatted: Font: Verdana, 10 pt
0 0

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 23.39190
G11 2 Formatted: Font: Verdana, 10 pt
0

23.54240
P1 2 Formatted: Font: Verdana, 10 pt
0

Sig. 1.000 1.000 .069 1.000 .994 .067 Formatted: Font: Verdana, 10 pt

Means for groups in homogeneous subsets are displayed. Formatted: Font: Verdana, 10 pt

a. Uses Harmonic Mean Sample Size = 2.000. Formatted: Font: Verdana, 10 pt

Formatted: Font: Verdana, 10 pt

Table 11. Data analysis of Ester percentage produced by 11 yeast strains
Formatted: Centered, Right: 0", Space After: 8 pt,
using ANOVA in SPSS 20. Line spacing: At least 20 pt, Widow/Orphan control

ANOVA Formatted: Font: (Default) Verdana, 10 pt

Ester percentage

Sum of Squares df Mean Square F Sig.

Between Groups 1020.609 9 113.401 15221.607 .000

Within Groups .074 10 .007

Total 1020.683 19

Table 12. Comparison of mean alcohol concentration produced by 11 Formatted: Font: Verdana, 10 pt
different yeast strains using Post-hoc Tukey's HSD test
Ester percentage

Tukey HSD

Label N Subset for alpha = 0.05

1 2 3 4 5 6 7

D1 2 .3550

D5 2 .7600

B10 2 .7800

C2 2 .9400 .9400

B11 2 1.1450 1.1450

C1 2 1.2950 1.2950

D4 2 1.5750

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 D3 2 2.0950

G11 2 3.8550
25.0300 Formatted: Right
G12 2

Sig. 1.000 .570 .422 .756 .138 1.000 1.000

Means for groups in homogeneous subsets are displayed. Formatted: Font: Verdana, 10 pt

a. Uses Harmonic Mean Sample Size = 2.000. Formatted: Font: Verdana, 10 pt

SUPERVISOR’S APPROVAL

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