Nghiên cứu tuyển chọn chủng nấm men để ứng dụng trong sản xuất whisky từ malt đại mạch và ngô tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
HANOI NATIONAL UNIVERSITY OF EDUCATION

NGUYEN VAN QUYEN

RESEARCH AND SELECTION OF YEAST STRAINS FOR
APPLICATION IN WHISKY PRODUCTION FROM
BARLEY MALT AND CORN
Major: Microbiology
Code: 9.42.01.07

SUMMARY OF PhD THESIS IN BIOLOGY

HANOI - 2020


The study is completed at:
HANOI NATIONAL UNIVERSITY OF EDUCATION

Proposed supervisor:

1. PhD. Nguyen Quang Thao
2. Prof. PhD. Nguyen Thanh Dat

Reviewer 1: Prof. PhD. Hoang Dinh Hoa
Reviewer 2: Assoc. Prof. PhD. Dinh Thi Kim Nhung
Reviewer 3: Assoc. Prof. PhD. PhD. Vuong Trong Hao

The thesis is defended before Thesis Examination Board at University level
held at Hanoi National University of Education
at …..hour … date … month… year 2020

The thesis could be found at Library:
National Library of Vietnam, Hanoi
Library of Hanoi National University of Education


1

PREAMBLE
1. Reason to choose the study
Whisky is a strong alcohol (alcohol content from 40 - 50% Vol) produced by distilling
fermented liquor of some cereals (barley, wheat, corn, rice, ...), After distillation, wine will be stored
with oak for a long time to perfect featured flavor, color of the product. Each type of whisky has its
own unique flavor due to the differences in the quality of production materials, yeast strains, climatic
conditions, production processes, storage time and conditions, etc.
According to MarketLine: The market value of strong alcohol will exceed 306 billion
USD in 2015, the growth rate in the nearest five years will reach 17%, sales volume increased
from 19 billion liters (2010) to 22 billion liters (2015), up 10% and market leader is whisky
(accounting for 27% market share). Europe, India and the US are still the number one markets
for this item, accounting for 48% of the world market. The size of the global strong alcohol
market was valued at 336 billion USD in 2016 and is expected to witness a 33.4% annual
growth (CAGR) from 2017 to 2025 in the US market.
In Vietnam, the need to use wines with high alcohol content in general and whisky in
particular is huge and increasing. According to the Vietnam Beer Alcohol Beverage Association, the
consumption of alcohol with high concentrations in Vietnam is about 500-520 million liters/year, of
which mainly imported Whisky, Cognac and Vodka, ... accounting for about 20% of the total alcohol
output in the market (whisky alone accounts for the largest market share of over 30%) means that
Vietnam still has to spend hundreds of millions of United States dollars every year to import alcohol
with high concentrations, significant impact on the sustainable economic development of Vietnam.
Vietnam has the potential of raw materials (microorganisms, cereals, ...) to produce

whisky, to be able to actively produce whisky in Vietnam, we choose the topic: “Research and
Selection of Yeast Strains for Application in Whisky Production from Barley Malt and Corn”
with the desire to contribute a part in scientific and technological knowledge, step by step
perfecting and raising the scale of whisky production in Vietnam, creating good products with
good quality, safety, high economic value, gradually restricting import, contributing to
stabilizing the national economy.
2. Objectives of the study
Selecting 1-2 good quality yeast strains, meeting the technological requirements and
product quality
Identifying the biological, physiological, biochemical, genetic characteristics of selected
yeast strains.
Applying selected yeast strains to study whisky production from barley malt and corn
Developing a technological process for producing whisky from corn and malt barley so
that the product meets quality standards and food safety as prescribed.
3. Main research content
3.1. Researching and selecting yeast strains that meet the production technology
requirements and quality of whisky products;
3.2. Researching basic biological characteristics (morphological, physiological, growth
and development characteristics) and scientific names of selected yeast strains
3.3. Researching kinetics of alcohol fermentation
3.4. Researching and developing technological process for producing whisky from corn
and malt barley
3.5. Applying technological process of product trial production at liquor production
establishments
4. Achievements
4.1. Selecting 01 yeast strain to meet technology and quality requirements of whisky products
4.2. Identifying biological characteristics, genetic characteristics and scientific and
species names for selected yeast strain is Saccharomyces cerevisiae MS42
4.3. Developing a technological process for producing whisky from corn and malt barley.

2
4.4. Producing 10,000 liters of whisky from corn and malt barley at Eresson Beer and
Alcohol One Member Company Limited (including 3 products: Whisky with 100% malt;
Whisky with 100% corn; Whisky with 20% malt and 80% corn), all products meet national
technical standards and Food Safety for spirits as prescribed.
5. Scientific and practical significance
- On the scientific side
The study has selected the yeast strain in Vietnam and the S8 oak that are suitable to
produce whisky from corn and malt barley. The yeast strain has the ability to grow and thrive
on malt and corn environments, the fermentation efficiency reaches 89.51 - 91.40%, at 25 oC,
pH 5.0 - 5.5, capable of fermenting wine yeast reached 12.3 oVol, the product has the typical
flavor and taste of whisky.
Providing a scientific basis for the technological process of producing whisky from corn
and malt barley (especially the process of forming and converting some typical aroma of
whisky in the storage process) in accordance with practical conditions in Vietnam.
- On the practical side
The study applies in manufacturing practices at enterprises and created good quality
products, meeting the prescribed Food Quality and Safety Standards, highly appreciated by
Chinese and Singaporean customers.
The production of whisky in Vietnam will contribute to the effective exploitation of the
available raw materials (corn, yeast strains, etc.) in Vietnam to proactively produce high-class
products of Vietnam, gradually restricting imports in foreign currencies to export typical
products of Vietnam.
6. New contributions of the thesis
6.1. Selection and scientific identification of 01 yeast strain isolated from traditional yeast
source of Mau Son commune, Binh Loc district, Lang Son province (Saccharomyces cerevisiae
MS42) met criteria for producing whisky from corn and barley malt such as: Fermentation reaches a
high alcohol content 12.3%Vol, at 25oC, creating a good and featured flavor and taste on malt and
corn. The M42 strain has good growth ability on malt (M), corn (N) medium after 36 - 48 hours of

culture, reaching the maximum number of cells from 213 x 106 - 215 x 106 cells/ml. Optimal alcohol
fermentation from a fermented liquor with a sugar content:160 – 180g/l ; pHopt: 5,0 – 5,5; Topt = 20 –
30oC; Alcohol content reached: 9,00 – 9,91%Vol. The fermentation efficiency reaches 89.52 91.13%, produces less toxins methanol, acetaldehyde, fufurol.
6.2. Identifying suitable materials to produce whisky including: Malt, corn and oak (symbol
S8) imported from America. Developing technological process for producing whisky from corn
and barley malt that is suitable to the practical conditions in Vietnam.
6.3. Initially studying the formation and transformation of some whisky flavoring agents
in the process of storing wine with oak.
6.4. Providing scientific basis and technological process for producing whisky from
barley malt and corn in Vietnam on an experimental scale with 3 types of whisky products:
100% barley malt; 100% corn and 20% barley malt + 80% corn.
7. Thesis structure
The thesis is structured into 6 parts: preamble, content, conclusion, list of published
scientific works, reference and appendix. The content of the thesis is developed into three
chapters as follows:
Chapter 1: Overview of research issues (38 pages)
Chapter 2: Research materials and methods (20 pages)
Chapter 3: Results and discussion (80 pages)


3

CHAPTER 1. OVERVIEW
1.1. Yeast
1.1.1. Overview of yeast
Yeast is a group of unicellular fungi or single cell aggregation, having a size and a cell
diameter of 3 - 10 μm. Depending on the type of cell yeast, it is spherical, ovoid, oval, elliptical,
felt hat-shaped, Saturn-shaped, sickle-shaped, rhombus, scoreed arc, triangular, lenticular, pear
shaped, hemispherical with narrow margin…
Saccharomyces yeast strain cell wall is composed mainly of mannam and glucan, in

some lines with extra chitin. The cytoplasmic membrane consists of two layers of phospholipid
molecules with ester bonds specific to the eukaryotic membrane. In the cytoplasm, there are
organelles such as: nucleus, mitochondria, riboxoid, vacuole, Golgi body, etc. The cell nucleus
of Saccharomyces cerevisiae has 16 chromosomes (haploid - 1n) or 16 pairs of chromosomes
(diploid - 2n). In addition to the nucleus there are many other genetic factors such as plasmids,
motor genetic factors (Transposon).
Yeast maintains the lineage by two forms of asexual reproduction and sexual reproduction.
However, there is a rotation of asexual and sexual reproduction with different haploid and diploid
stages in the yeast's life cycle or depending on the environment.
Currently, the yeast has been identified: 1110 species/58 lines/17 families/
Saccharomycetales set/Saccharomycetes class / Ascomycota branch
1.1.2. Basic nutritional needs of yeasts
Yeast is capable of growing and developing under aerobic and anaerobic conditions. The
metabolism is different, so the nutritional needs are also different. In culture media, there are
sufficient necessary factors for yeast cells to grow and develop. The ratio of elements in the
biomass of Saccharomyces cerevisiae is C: 1.72; O: 0.44; N: 0.15.
1.1.3. Yeast in alcohol production technology
1.1.3.1. Standards of yeast strains in alcohol production
- Growing strongly in fermented sugar, use many types of sugar (glucose, maltose, maltotriose, ...)
- Having ability to secrete enzyme branch for effective fermentation.
- Having ability to ferment to create high alcohol content during fermentation (12 – 15%Vol)
- Having ability of antibacterial agents (SO2) and fluctuations in fermentation conditions
(pH: 4,5 – 6,5; Temperature: 16 – 35oC)
- Creating good flavor and sensory value, specific to the product, producing less toxins,
do not create strange unwanted flavors.
- Long-term stability in production
- In addition, depending on the technology and product quality requirements, which the
yeast strains used need to have other specific requirements such as the ability to create flavors
in different conditions, using sugars…
1.1.3.2. The yeast strains are used in alcohol production.

In today's alcohol production, 15 typical lines can be used to in alcohol fermentation. In
alcohol production, the widely used species is: Saccharomyces cerevisiae.
1.2. Whisky alcohol
1.2.1. Whisky classification
Whisky is a type of strong alcohol produced by distilling liquor from some cereals
(barley malt, wheat, corn, rice, etc.). after distillation, alcohol is stored with oak for a limited
time. Whisky are classified in many different ways:
* Being classified by raw materials used for production:
- Malt whisky (Single Malt Whisky): is a type of whisky made from 100% malt
- Grain whisky: is a type of whisky made from barley malt that replaces a certain
percentage of cereal grain, usually corn.


4
- Rye: a type of whisky primarily made from at least 51% of rye
- Bourbon: A type of whisky mainly made from at least 51% corn, distilled up to 81% Vol
alcohol and stored up to 61% Vol alcohol.
* Being classified by raw materials and manufacturing techniques:
- Single: is a type of whisky only produced from a specific material to create a pure,
unadulterated product (often used for Scottish whisky: (Single-Malt-Whisky)
- Straight: is also a type of whisky produced only from a specific material (usually used
for whisky from the US).
- Blend: is a type of blended whisky. In the process of producing many different types
of whisky from many different breweries are blended together. In some products there are up to
70 different types of whisky. is a type of blended whisky. In the process of producing many
different types of whisky from many different breweries are blended together. In some products
there are up to 70 different types of whisky.
- Pot Still: is a type of whisky produced using only classic cooking pots (usually used
for some types of Irish whisky)
- Pure Pot Still: is a type of whisky produced using only malt in vintage brewers

(usually used for individual Irish whiskys)
1.2.2.Whisky production technology
1.2.2.1. Main production materials
The main raw material for the whisky production is sprouting barley, although in some cases
a certain percentage of other sprouting grains such as corn, black wheat, barley, etc. are used. Alcohol
produced from barley malt is still considered to be of the highest quality, sensory and economic
efficiency. In the United States and Canada, corn is the main raw material for whisky production
because of its high starch content (about 67-69%).
Oak has the main components including: cellulose (38-52%), hemicellulose (25-30%),
lignin (22-25%) and tannins (5-10%). The cell wall is composed of cellulose, hemicellulose
and lignin molecules, while the intercellular area consists mainly of lignin. The polyphenolic
compound in oak has an important function in alcohol age. Oak polyphenols play an important
role in alcohol maturation, but also have an effect when combined with a strong antioxidant
effect to contribute to the flavor and taste of the alcohol soaked in oak. These compounds are
often classified into three mains: the volatile phenol group; phenolic acids and ellagitannins.
1.2.2.2. Whisky production technology
Raw materials (Matl/barley/corn) are crushed and saccharized (in case of using corn or
barley, amylase enzyme will be added) to collect sugar solution (about 8-12 o Bx), sugar solution
after being adjusted pH, temperature adjustment, etc. are used directly to ferment alcohol. The
fermentation process is carried out in oak barrels or stainless steel tanks (depending on
technology requirements, production scale).
Sacharomyces cerevisiae is added to the fermentation fluid to carry out the fermentation
process to produce alcohol, the fermentation time usually ranges from 5-8 days (depending on
the quality of the liquid and the fermentation conditions). At the end of the fermentation
process, the vinegar solution is distilled twice to reach 63-68%Vol. The alcohol 63-68 %Vol is
soaked in oak casks (traditional technology) or soaked with oak (bar type) in stainless steel
tanks (when produced on a large scale) so that the alcohol extracts aromatic substances. In oak
during storage and creates the unique aroma and flavor of whisky. Oak aging time is not less
than 3 years and usually lasts for tens or even hundreds of years depending on the quality
requirements of each whisky and the degree of alcohol will also decrease with the aging time of

storage. At the end of the incubation process, the expert will decide the option of blending
alcohol (or blending high alcohol with soft water) to create a commercial alcohol of sensory
quality and alcohol content in accordance with the requirements of the firm.


5
1.2.3. Situation of research and production of whisky in Vietnam
The research and production of whisky in Vietnam is still very limited. In 2006, Mr.
Dang Tat Thanh carried out the project "Researching and selecting yeast strain to produce
high- concentration alcohol from barley malt" at an experimental scale.
In 2008, Mr.Nguyen Duc Quang (Research Institute for Beer, Alcohol and Beverage)
carried out the project "Research on the production of high- concentration alcohol from
Vietnam's Barley source", which built a technological process and trial production with scale of
40 liters / batch and 150 liters / batch, recovery efficiency of 88%, alcohol with 30% Vol
alcohol, good product quality
The above topics focused on selecting yeast and fermenting to recover high- alcohol alcohol
from barley malt, the product is not yet whisky because there has not been research on the process of
aging of cozy alcohol such as using oak wood to create the flavor and taste of whisky
In 2016, Ms. Nguyen Minh Thu (Food Industry Institute) carried out the project
"Researching whisky production technology from barley malt and alternative raw materials of
Vietnam" under "Project of technology application and development biology in the field of
processing industry until 2020”, initially introduced the technological process of producing
whisky using corn and rice as alternative materials. In the technology that uses oak wood (in
the form of planets and powders), there is no in-depth research on the process of creating
flavor, the characteristic flavor of whisky, especially scientific knowledge about technology and
sensory quality of the product during storage so it has not been commercialized.
ERESSON One-member Co., Ltd. has invested in a fairly modern equipment system to
test the production of whisky from barley malt and has produced good quality alcohol,
however, the Company has not had any deep and scientific research. Regarding the use of oak
wood as well as the annealing process to create a unique flavor and flavor for whisky, the

product is not eligible for commercialization when produced on a large scale.
Therefore, the research to perfect the whisky production technology in Vietnam ensures
the scientific from the selection of suitable yeasts, the determination of raw materials, oak,
production technology, storage, ... to commercially qualified products are still necessary and
urgent to meet the needs of the market.

CHAPTER 2. RESEARCH MATERIALS AND METHODS
2.1. Material
- Source of yeast for isolation and selection: 107 strains of yeast were isolated and
selected from traditional yeast sources: Hong Mi yeast in Ban Pho, Bac Ha, Lao Cai; Mau Son
leaf yeast in Mau Son, Loc Binh and Lang Son communes; Van village yeast in Van Xa village,
Van Ha commune, Viet Yen, Bac Giang; Saccharomyces cerevisiae strain TCCY belongs to the
seed bank of VNU Hanoi; 04 strains of Saccharomyces cerevisiae of Food Industry Institute:
TN2, EC1118, BDX, R2226.
- Malt: Barley malt we researched and used include: 02 kinds of premium malt, made
from Barly - Sebastian (France) and Buloke (Australia). Products are imported by Malt Sugar
Company (Bac Ninh).
- Corn DK9955: yellow-seed hybrids corns. Supplier: Nguyen Thi Van Anh private
store, address: Quang Minh town, Me Linh district, Hanoi.
- Oak : used for research are 24 commercial oaks for alcohol soaking in USA, France and
Australia, specifically: Australian oak including 5 types.: Arobois HM, Arobois Aroneo, INTENSE,
BALANCED, SAVORY; American oak includes 9 types, sign: BLA42, CSA12, CSA32, CSA02,
CXA02, CXA32, CXA12, MSA42, TXA 82HV; French oak includes 10 types, sign: CXF00F,
CXF02, SLF12, CSF30, CSF02F, CXF32, CXF12, BLF42, MSF42, TXF 82HV.
The oak samples used in the research were coded by us: S1 – S24


6
2.2. Environment
2.2.1. Environment remaining seedlings

2.2.1.1. Hansen environment (sign: M1)
2.2.1.2. Malt-aga extract environment (sign: M2)
2.2.2. YPD yeast biomass Human environment
2.2.3. Basic propagation environment
2.2.4. Environment to determine ability of yeast to thrive, grow and ferment
2.3. Research method
2.3.1. Method of microorganisms and molecular biology
2.3.1.1. Isolating yeast
2.3.1.2. Determining number of cells by red blood cell counting chamber
2.3.1.3. Determining characteristics of yeast cell populations
2.3.1.4. Determining scientific name of the yeast by molecular biology technique
2.3.2. Chemical – physical method
2.3.2.1. Determining reducing sugar content by the DNS method
2.3.2.2. Method of determining the total sugar content (Phenol – axit sunfuric)
2.3.2.3. Determining total acid content by the direct method
2.3.2.4. Determining ester content by TCVN 378-86 allocation method
2.3.2.5. Determining aldehyde content by gas chromatography
2.3.2.6. Determining soluble proteins (Lowry method)
2.3.2.7. Determining content of free amino acids (FAN) following [TCVN 8764:2012]
2.3.2.8. Determining acetaldehyde content (Gas chromatographic method)
2.3.2.9. Determining methanol content (Gas chromatographic method)
2.3.2.10. Determining some basic aromatic substances in alcohol (Gas chromatography method)
2.3.3. Sensory methods
Sensory methods for scores: following TCVN 3217 – 79
2.3.4. Mathematical methods
- Calculating the average overall
- Overall standard deviation
- The variation coefficient of the overall average:
- Processing statistics: Excel software

CHAPTER 3. CONTENT AND RESULTS OF THE STUDY
3.1. Isolating and selecting suitable yeast strains
3.1.1. Criterion of yeast selection
- Growing strongly in fermented sugar and using many type of sugar (glucose, maltose,
maltotriose...)
- Having ability to secrete enzyme system for effective fermentation
- Having ability to ferment malt, corn with high alcohol content (12 – 15%Vol)
-Resisting antibacterial agents (SO2) and wide fluctuations of some fermentation conditions
(pH: 4.5 - 6.5; Temperature: 16 - 35oC)
-Creating flavors, specific characteristics of the product, producing less toxins, do not create
strange unwanted flavors.
-Being stable quality in production, can meet the requirements of large-scale production
3.1.2. Selection of yeast strains
3.1.2.1. Sources of Yeast
From traditional yeast source: Hong Mi yeast in Ban Pho, Bac Ha, Lao Cai; Mau Son
leaf yeast in Mau Son, Loc Binh and Lang Son communes; Van village yeast in Van Xa village,
Van Ha commune, Viet Yen, Bac Giang. The result was 107 yeast strains, of which: Bac Ha
yeast source (BH) isolated 42 types: BH1 - BH42; Mau Son glaze source (MS) isolated 43


7
types: MS1 - MS43; Lang Van bread source (LV) isolated 22 types: LV1 - LV22
3.1.2.2. Evaluating ferment ability and specific favor creation
From the isolated yeast strains, the first fermentation was carried out on 2 types of
environment, 100% malt and 100% corn. After 10 days of fermentation, t = 25oC, extracting the
distilled fermentation and proceeding the 1st sensory evaluation at 40%Vol alcohol
concentration according to the TCVN 3215-79 score method. The result has obtained 15 lines
with good fermentation ability, creating typical sensory products, capable of creating good
flavors, good sensory scores above 15.2 scores, namely:
- Leaf yeast, from Mau Son yeast – Lang Son (03 lines): MS18, MS40, MS42;

- Hong Mi yeast, Bã Ha – Lao Cai (10 lines): BH1, BH2, BH19, BH20, BH24, BH30,
BH33, BH34, BH41, BH42;
- Van village yeast, Viet Yen, Bac Giang ( 02 lines): LV4, LV22.
Detailed results of fermentation capacity of 15 lines on malt (M) environment and corn (N)
environment were obtained (Table 3.1; Table 3.2):
Table 3.1: Results of sensory analysis and evaluation of 15 yeast strains
(on malt environment - M)

No

Yeast
strain

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

LV4

LV22
MS18
MS40
MS42
BH1
BH2
BH19
BH20
BH24
BH30
BH33
BH34
BH41
BH42

Reducin
Residual
g sugar
Actual
sugar
(convert
alcohol
(converted
ed to
percentage
to maltose)
maltose)
(%Vol)
g/l
g/l

170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6
170.6

2.08 ± 0.1
3.26 ± 0.2
3.24 ± 0.2
3.01 ± 0.2
2.05 ± 0.2
3.24 ± 0.2
2.95 ± 0.1
3.25 ± 0.2
2.16 ± 0.1
5.23 ± 0.2
5.62 ± 0.2
3.01 ± 0.2
5.28 ± 0.2

5.28 ± 0.3
2.95 ± 0.2

8.5
9.2
9.2
9.5
9.7
9.0
9.0
9.5
8.8
9.2
9.0
9.5
9.0
8.5
9.2

Alcohol
fermen
tation
perform
ance
(%)

78.40
85.45
86.44
88.11

89.45
83.58
83.44
88.23
81.20
86.47
84.79
88.11
84.62
79.91
85.29

Sensory evaluation (TCVN 3215-79)
Coe
Coe
Coe
Clarity
ffi Sme Ffi
Ffi Total
and
Taste
cient ll cient
cient score
color
(0,8)
(1,2)
(2,0)

3.6
4.6

4.5
4.5
4.5
4.3
4.6
4.5
4.1
3.5
4.6
4.2
4.1
3.9
4.2

0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8

3.9
4.2
4.3
4.1
4.3
3.1
3.9
3.9
3.1
4.1
4.1
3.4
3.2
3.1
3.8

1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2

1.2

3.7
3.8
3.7
3.8
3.9
3.9
3.7
3.9
4.2
3.9
3.8
4.2
4.1
4.3
3.6

2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0

2.0
2.0
2.0

15.0
16.3
16.3
16.1
16.6
15.0
15.8
16.1
15.4
15.5
16.2
15.8
15.3
15.4
15.2

The above results showed that: On a 100% malt fluid environment, the MS42 strain had the
highest score of 16.6 scores, followed by the LV22 strains with 16.3 scores and the BH30 strain
with 16.2 scores. The highest fermentation efficiency: MS42 reaches 89.45%; BH19 reached
88.23%; MS40 reached 88.11%
Table 3.2: Results of sensory analysis and evaluation of 15 yeast strains
(on corn environment - N)

TT

Yeast

strain

Reducing
sugar
(converted to
maltose) g/l

1
2

LV4
LV22

169.3
169.3

Residualsug
ar
(converted
to maltose)
g/l

Actu
al
alcoh
ol
perce
ntage
(%
Vol)

2.58± 0.2
3.26± 0.2

8.50
9.00

Sensory evaluation (TCVN 3215-79)
Alcohol
fermen
tation
perform
ance (%)

79.22
84.22

Clarit
y and
color

Coe
ffi
cient
(0,8)

Sme
ll

Coe

ffi
cient
(1,2)

Tas
te

Coe
ffi
cient
(2,0)

4.5
4.5

0.8
0.8

4.1
3.9

1.2
1.2

3.2
3.5

2
2

Total
Score

14.9
15.3


8
3
4
5
6
7
8
9
10
11
12
13
14
15

MS18
MS40
MS42
BH1
BH2
BH19
BH20
BH24

BH30
BH33
BH34
BH41
BH42

169.3
169.3
169.3
169.3
169.3
169.3
169.3
169.3
169.3
169.3
169.3
169.3
169.3

3.28± 0.2
3.05± 0.2
2.16± 0.1
3.64± 0.2
2.15± 0.1
3.65± 0.2
2.56± 0.2
5.63± 0.3
4.02± 0.2
3.41± 0.2

3.28± 0.2
5.68± 0.3
3.05± 0.2

9.20
9.40
9.50
8.70
9.00
9.00
8.50
8.50
9.00
9.30
9.00
8.50
9.00

86.10
87.85
88.32
81.60
83.66
84.42
79.21
80.70
84.61
87.11
84.23
80.72

84.12

4.5
4.5
4.8
4.2
4.2
4.1
3.6
3.2
4.5
3.9
4.2
3.9
4.3

0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8

4.1
4.1
4.2
4.2
4.1
4.0
4.3
4.1
4.1
4.2
4.1
4.1
3.8

1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2

3.2
3.2

3.5
3.2
3.1
3.5
3.1
3.6
3.5
3.2
3.2
3.5
3.2

2
2
2
2
2
2
2
2
2
2
2
2
2

14.9
14.9
15.9
14.8

14.5
15.1
14.2
14.7
15.5
14.6
14.7
15.0
14.4

On 100% corn fluid environment, the MS42 strain scored the highest at 15.9 scores,
followed by BH30 strains (15.5 scores) and LV22 strains (15.3 scores). Among the 15 strains,
the three strains MS42, BH30 and LV22 all met the criteria and achieved high scores in both M
and N environments, of which the MS42 strain scored the highest in both M and N. 3 lines
LV22, MS42, BH30 continue to use the study for the next criteria.
3.1.2.3. Assessment of the ability to produce toxins and impurities
To determine the ability of fermenting toxins and impurities: methanol, acetandehyde,
Furfurol, higher alcohol (propanol, iso/n-butanol, iso/n-pentanol). Alcohol samples of three yeast
strains: MS42, BH30 and LV22 were analyzed by gas chromatography to determine the
composition and content of toxins and impurities. Results obtained (Table 3.3; Table 3.4):
Table 3.3: Gas chromatographic analysis results from malt solution after distillation
NO

Yeast
strain

Metanol (mg/l) H.HD.QT.196
(GC-FID)

1

2
3

LV22
MS42
BH30

KPH (˂0.5)
KPH (˂0.5)
KPH (˂0.5)

Acetandehyde
(mg/l) H.HD.QT.196
(GC-FID)
35 ± 1
39 ± 1
91 ± 1

High-quality
alcohol (mg/l) H.HD.QT.196
(GC-FID)
218 ± 2
183 ± 2
252 ± 2

Furfurol (mg/l) H.HD.QT.022
KPH (˂0.21)
KPH (˂0.21)
KPH (˂0.21)

Table 3.4: Results of gas chromatographic analysis of corn after distillation
NO

Yeast
strain

1
2
3

LV22
MS42
BH30

Metanol (mg/l) H.HD.QT.196
(GC-FID)
KPH (˂0.5)
KPH (˂0.5)
KPH (˂0.5)

Acetandehyde
(mg/l) H.HD.QT.196
(GC-FID)
131 ± 2
125 ± 2
127 ± 1

High-quality
alcohol (mg/l) H.HD.QT.196
(GC-FID)

229 ± 1
216 ± 1
232 ± 2

Furfurol (mg/l) H.HD.QT.022
0,26 ± 0.01
KPH (˂0.21)
KPH (˂0.21)

In both types of malt and corn, three types of yeast produce less toxin and impurities. In
which MS42 has more advantages: methanol, furfurol undetected, content of acetandehyde in
malt alcohol (39 mg/l), corn alcohol (125 mg/l); higher alcohol in malt alcohol (183 mg/l), corn
alcohol (216 mg/l). These results are much lower than Vietnamese standards TCVN 2043: 2013
on white alcohol: acetandehyde: self-declared; Maximum alcohol level: 2000 mg/l; maximum
methanol: 2000 mg/l; furfurol: not detected.


9
uV(x10,000)

uV(x10,000)
3.5
Chromatogram
n butanol/13.499/501567/15911

3.5 Chromatogram

1.5

n butanol/13.505/438330/14211

1.0

0.5

0.5

iso amyl/15.489/117577/4427

1.0

2.0

iso butyl/11.182/10791/2419

ald/5.331/13561/3952

1.5

iso butyl/11.177/17467/4171

2.0

2.5
ald/5.330/34661/10987

2.5

3.0

iso amyl/15.474/105270/4217

3.0

0.0

0.0
5.0

7.5

10.0

12.5

15.0

min

2.5

5.0

7.5

10.0

12.5

15.0

17.5 min

Hình 3.1: Chromatography of alcohol
Hình 3.2: Chromatography of alcohol
products of MS42 lines on malt (M)
products of MS42 lines on corn (M)
environment
environment
3.1.2.4. Evaluating the fermentation ability to create high- alcohol alcohol
Conducting fermentation of 3 yeast strains on malt environment with the sugar content of
220g / l. Conducting fermentation at 25oC, fermentation time is 10 days. Results (Table 3.5)
Table 3.5: Fermentation ability to create high-alcohol alcohol
Residual
Reducing sugar
Alcohol after
Alcohol
sugar
NO
Yeast strain
(converted to
distillation (%
fermentation
(converted to
maltose) g/l
Vol)
efficiency (%)
maltose) g / l
1
LV22

220
10.79
11.8
87.64
2
MS42
220
8.25
12.3
89.52
3
BH30
220
9.79
12.0
88.70
From the results of Table 3.7 shows: 3 lines MS42, BH30, LV22 have the ability to
ferment to create high alcohol concentration. In particular, the MS42 line is the most advantageous
line: Creating an alcohol concentration of 12.3%Vol, fermentation efficiency of 89.52%, the ability to
ferment thoroughly with low residual sugar content (8, 25g / l). Therefore, the MS42 line was
selected for evaluation with a number of strains currently available.
3.1.3. Evaluation of alcohol fermentation ability of strain MS42
On malt environment of , the content is 220g / l. Making fermentation at 25oC, 10 days time.
Comparing strains MS42 with some control strains of good quality (including: EC1118, R2226,
TN2, BDX) in the yeast seed bank of the Food Industry Institute. Analysis of toxin composition and
impurities created during fermentation, fementation ability to create high alcohol concentration of
strains, assessing organoleptic perception at 40% Vol. Results (Table 3.6):
Table 3.6: Fermentation capacity of strains MS42 with strains
TN2, EC1118, BDX, R2226

Targets
Alcohol content (%Vol)
Residual sugar (g/l)
Fermentation efficiency
Fermentation time(hour)
Methanol (mg/l)
Acetaldehyde (mg/l)
Isopropanol (mg/l)

TN2
11.8
9.16
86.96
204
1.62
6.92
2.29

MS42
12.3
7.25
89.52
192
1.29
3.65
2.15

Line sign
EC1118
12.5

6.12
90.81
192
1.38
3.71
2.21

BDX
11.5
8.34
84.42
204
1.26
5.70
1.30

R2226
11.8
8.98
86.89
192
1.42
3.91
2.03


10
Acetone (mg/l)
n-propanol (mg/l)
Isobutanol (mg/l)

Ethylacetate (mg/l)
n-butanol (mg/l)
Isoamylancol (mg/l)
n-amylancol (mg/l)
Isoamylacetate (mg/l)
Ethylhexanoat (mg/l)
Furfurol (mg/l)
Sensory score

0.55
1.93
0.95
0.79
0.07
2.50
0.34
0.14
˂0.21
15.3

0.23
1.40
0.84
0.93
0.17
5.34
0.12
0.41
0.07
˂0.21

16.6

0.29
1.56
0.90
0.99
0.19
5.70
0.14
0.47
0.07
˂0.21
16.0

0.23
0.55
1.40
0.88
0.13
6.23
0.03
0.71
0.09
˂0.21
15.8

0.40
1.07
0.77
0.79

0.19
2.97
2.94
0.29
0.03
˂0.21
16.2

The results showed that: Methanol, acetaldehyde content of strains was lower than the
permitted level of white alcohol standard (TCVN 7043: 2013), in which two strains had low
toxic and impurity content, MS42 and EC1118. All strains had high fermentation capacity (≥
11.8% Vol), of which EC1118 strain had the highest fermentation capacity of 12.5%Vol, MS42
reached (12.3%Vol). However, the MS42 strain had a better alcohol sensory value of 16.6
scores. The fermentation ability of MS42 strain is fast, in an environment with a sugar content
of 220 g / l, the fermentation time is 192 hours. The amount of toxins and impurities created is
low: methanol (1.29 mg / l), acetaldehyde (3.65 mg / l)
* Conclusion: strain MS42 has characteristics:
- Fermentation reaches a high alcohol content (reaching 12.3%Vol, at 25oC);
- Fermented products with low levels of toxins and impurities, especially the content of
aldehydes, methanol and furfurol produced in the fermentation fluid are very low compared to
TCVN 7043: 2013;
-High molecular alcohols are low in content;
- Fast fermentation time;
- Various fermentation materials, low residual sugar content;
- Alcohol after the second distillation at 40%Vol has a high sensory value of 16.6 scores,
of which:
• Clarity and color: The clear liquid is not cloudy and small objects, colors are typical
for malt, corn products;
• Smell: Harmonious, sweet-aromatic, characteristic for malt and corn products;
• Taste: Harmonious, mellow, sweet aftertaste, typical for malt and corn products.

The MS42 strain satisfies the requirements of technology, product quality, based on the
selection criteria and results (mentioned above), we decided to choose the MS42 strain to
conduct further research.
3.2. Research on some biological characteristics and identification of MS42 strain
3.2.1. Morphological characteristics of yeast strain MS42
Characteristics of colonies on Hansen environment: round, 2 - 4 mm in size, milky
white, shiny surface, smooth colon edge, convex colonies, no concentric circles, no rays
radiation .


11

Figure 3.3: Colon strain MS42
Figure 3.4: Cell shape
on Hansen environment
strain MS42 under electron microscope
3.2.2. Sugar fermentation ability of yeast strains MS42
Conducting the fermentation test of sugars of MS42 strain by: Using API 20C aux and
ID32C test kits. The results showed that MS42 yeast strains were able to use many different types
of sugar, specifically:
Table 3.7: Ability to use some types of sugars of MS42 strain
Type of sugar
D-Glucose
Maltose
Rafinose
Maltotriose
Mannose
Trehalose
D-mannitol

Fermentation ability

Type of sugar
Saccharose
D-Galactose
Fructose
Deoxyribose
Melibiose
Melezitose

Fermentation ability
++
++
++
+
+
+

Note: Having fermentation but not strong: +, Strong fermentation: ++.
3.2.3. Genome sequences series of MS42 strain
Region ITS1, ITS2 of 5.8S and D1, D2 of 28S: The above analysis results show: strain
MS42 belongs to Saccharomyces genus species Saccharomyces cerevisiae. We named it
Saccharomyces cerevisiae MS42.
Taxonomy of Saccharomyces cerevisiae MS42: Territoriality (Domain): Eucalyptus
(Eukayota) / Gender (Kingdom): Mushroom (Fungi) / Sector: Baganus (Ascomycota) / Subbranch: (Ascomycotina) / Class: Bag Mushroom (Ascomycetes) / Set: Endomycetales / Family:
Saccharomycetaceae / Genus: Saccharomyces / Species: Saccharomyces cerevisiae.
3.3. Affecting to the growth and development of Saccharomyces cerevisiae MS42 during
propagation process
From the research results, we have selected the environment and basic propagation
conditions suitable for S. cerevisiae MS42 is the environment (G2) with a sugar level of 100

(g / l), pH = 4.5. Breeding time 24 hours, temperature from 25 - 300C with dissolved oxygen
concentration from 6.5 - 7.5 (mg / l).
The addition of bean sprouts in yeast propagation production applications can use bean sprouts
(100g / l), grated, added to malt then pasteurized to create propagation.
3.4. Researching the effect of several factors on alcohol fermentation
- Sugar content: The amount of sugar from 140 - 160 g / l for whisky production has a
short fermentation time, high fermentation efficiency, the after-fermentation solution reaches an
alcohol content of 7.88 - 9.00%Vol. However, in order to ensure high fermentation efficiency, the
fermentation solution has high alcohol content, limits contamination during fermentation, the
efficiency of alcohol recovery after distillation, we choose the sugar content of the fermentation
solution. at 160 g / l (calculated according to maltose).


12
- PH: 100% malt environment, pH = 4.5 - 5.5 with high alcohol content, fermentation
performance and sensory scores. Meanwhile, the methanol content is lower than the environment
with pH = 4.0. The best result is a fermented solution with a pH = 5.0. When pH increased to 6.0,
the amount of acetaldehyde, alcohols increased sharply and fermentation efficiency decreased.
According to some authors, fermentation solution with pH = 5.0 is not only suitable for the growth
of yeasts but also suitable for the activities of many enzymes to convert sugar into alcohol.
- Temperature: The fermentation temperature from 16°C - 20°C gives better results on the
alcohol content, low content of acetandehyde and methanol. However, fermentation at 16° C 20°C will increase production costs, while the use of appropriate distillation techniques and
equipment will limit many types of toxins and impurities. In our opinion, the fermentation
temperature of 25° C - 30°C is suitable for the production conditions, at this temperature, it still
ensures high fermentation efficiency, low production of toxins and impurities and in accordance
with practical conditions in Vietnam.
- Disinfectant (SO2): The use of K2S2O5 content at 2,0-3,0 g/l is suitable for the fermentation
ability of MS42 strain, and also limits the infection during fermentation. This result is consistent with
the published statements of many authors when using SO2 (maximum SO2 concentration should not
exceed 490 ppm). At the supplemental threshold of K2S2O5 of 0.3 g/l, the fermentation process has a

high alcohol content, although acetaldehyde and methanol content are higher than 0.2 g/l but
maintain better resistance to infection. Therefore, we choose K2S2O5 at 0.3 g/l.
- Number of cells when breeding to alcohol fermentation: To ensure safety of fermentation,
shorten fermentation time, limit bacterial contamination but still ensure product quality, alcohol
fermentation efficiency. In our opinion, it is recommended to select the addition of the original
variety at the rate of 7-10%V (equivalent to 12-18 x106 cells / ml) which is suitable to create the
overwhelming yeast to help support good anti-infection and high fermentation performance.
3.5. Researching the kinetics of alcohol fermentation of Saccharomyces cerevisiae MS42
3.5.1. Researching the kinetics of alcohol fermentation from barley malt
To determine the dynamics of the fermentation process, we used fermented malt fluid
(100%), adjusting the fermentation conditions accordingly according to the results of our studies:
total sugar 160 (g/l), pH: 5,5; K2S2O5: 0,3 (g/l); rate of breeding is 7%V; fermentation
temperature at 25-28oC.Implementing fermentation scale of 10 liters/batch. Conducting analysis
of fermented samples every 12 hours/time. The change of yeast, sugar and alcohol during
fermentation process is shown by graphs (Figure 3.5):

Figure 3.5. Graph of alcohol fermentation in malt solution
* Conclusion: Suitable fermentation conditions for the yeast strain Saccharomyces
cerevisiae MS42 for the production of high-quality liquor from 100% malt fermented liquor,
including: malt with a sugar content of 160 - 170 g / l; pH = 4.5 - 5,0; ToC: 25 - 30oC; K2S2O5: 0.2
– 0.3 (g/l); initial seeding rate: 7 - 10%; fermentation time: 132 - 144 hours.


13
3.5.2. Researching on the kinetics of alcohol fermentation from maize
Determining the dynamics of the fermentation process, we used corn fermentation
solution (100%), adjusting the fermentation conditions appropriately according to the results of
the experiments studied: Total sugar content 160 (g/l), pH = 5.5; rate of breeding is 7%;
fermentation temperature at 25-28oC; fermentation scale with a capacity of 10 liters/batch.
Perform sample collection and analysis every 12 hours/time. The variation of the amount of

yeast, sugar and alcohol during the maize fermentation is shown by the graphs (Figure 3.6):

Figure 3.6: Graph of alcohol fermentation action from corn fluid
* Conclusion: Suitable fermentation conditions for the yeast strain Saccharomyces cerevisiae
MS42 for the production of high alcohol content from 100% maize fermentation include: Corn liquor with
a sugar content of 140-160 g / l; pH = 5.0 - 5.5; ToC = 25-30oC; Additional content of anti-bacterial
K2S2O5: 0.2 - 0.3 (g/l); initial seeding rate: 7 - 10% V; Fermentation time: 156 - 168 hours.
3.6. Research on technology for producing whisky from corn and malt malt (laboratory scale)
3.6.1. Research on whisky production technology from barley malt
3.6.1.1. Processing malt liquid from malt
In order to stabilize and improve the quality of the gastrointestinal fluid from Malt, we added
Termamyl® SC (0.08%) and Dextrozyme® GA (0.3%). The results of malt fluid were as follows:
o
Bx: 18.0; Reducing sugar: 160.5 g / l; Protein: 19.21 g / l; FAN: 264.5 mg / l; pH: 5.4.
3.6.1.2. Fermenting whisky production from malt barley
Conducting fermentation of malt fluid with the scale of 100 liters / batch with the
fermentation conditions identified in the studies: Fermentation temperature: T = 25 oC; sugar
content 160,55 g/l; pH = 5.4; the added content K 2S2O5 0.3 g / l; the same amount as 7% V
(equivalent to 12 to 15 x 106 cells / ml of fermented solution). The alcohol fermentation process
ended after 144 hours after the seeding, we got results (Table 3.8).:
Table 3.8: Changes in alcohol fermentation from malt (M) solution
Component
Time
0h
12 h
24 h
36 h
48 h
60 h
72 h

84 h
96 h
108 h
120 h
132 h
144 h

Sugar content
(converted to maltose
sugar) g / l
160.5 ±0.5
150.2 ±0.5
130.1 ±0.5
106.1 ±0.5
78.4 ±0.3
50.8 ±0.3
31.2 ±0.3
16.6 ±0.2
14.8 ±0.2
11.3 ±0.2
7.2 ±0.2
5.0 ±0.2
4.6 ±0.2

Alcohol content
(%Vol)

No of cells
(x106 tb/ml)

Total acid (converted to
acetic acid) mg / l

0.2±0.05
1.2±0.05
2.1±0.05
3.3±0.1
5.0±0.1
6.6±0.1
7.8±0.1
8.3±0.1
8.6±0.1
8.7±0.1
8.8±0.1
9.2±0.1
9.1±0.1

17 ± 1.0
100 ± 2.0
198 ± 3.0
213 ± 3.0
215 ± 3.0
206 ± 3.0
183 ± 3.0
159 ± 3.0
120 ± 2.0
80 ± 2.0
62 ± 1.0
48 ± 1.0
42 ± 1.0

423.2 ± 2.0
428.5 ± 2.0
432.9 ± 2.0
456.8 ± 2.0
463.8 ± 2.0
465.8 ± 3.0
484.3 ± 3.0
486.2 ± 3.0
509.8 ± 3.0
510.5 ± 3.0
512.8 ± 3.0
518.5 ± 3.0
519.8 ± 3.0


14
The results showed that: At 96 hours after fermentation, the sugar content decreased and
remained almost unchanged after 144 hours. The amount of residual sugar left is 5.1 g/l. The alcohol
content reaches a maximum at 96 hours - 144 hours, after which there is a slight and stable increase
in the final stage of the fermentation, reaching 9.1 ± 0.1 (%Vol). The total acidity in the fermentation
solution increases, so the pH is decreasing but the change is not too large. At the end of the
fermentation, the amount of acid in the fermentation liquid is: 519.8 mg / l.
Analysis results of indicators: Sugar content; alcohol content; The number of cells and pH at
the fermentation scale of 100 liters/batch although there are some slight differences compared to the
experimental scale (10 liters/batch) but not statistically significant, in which the amount of alcohol The
result is 9.1 ± 0.1 (%Vol) which is equivalent to the experimental scale of 9.1 ± 0.1 (%Vol).
The above results show that: MS42 strain has high stability, can be used for larger
scale production.
3.6.1.3. Malt alcohol distillation

a) the first distillation
Liquid after fermentation has an alcohol content: 9.0% Vol is distilled for the first time at a
vacuum pressure of 404.6mmHg, the boiling temperature of alcohol at this pressure is 63,04 oC. The
distillation temperature is from 63 –75oC. For equipment of 100 liters / batch at us, we carry out the
distillation of malt solution at the first stage: 63 – 65oC, middle distillation stage: 68 – 70oC, final
distillation stage: 73 – 75oC.
Distillation recovered all alcohol with a concentration of 20%Vol (evaporation
coefficient of alcohol 3.31).
Components of some toxins and basic impurities in malt distilled alcohol 1 st time:
Acetaldehyde: 56,23 ± 0,1 mg/l; Methanol: 8.32 ± 0.05 mg/l; High alcohol: 238.26 ± 1.0 mg/l;
Fucfurol: 2.28 ± 0.1 mg/l.
b) Second Distillation
2nd distillation at a vacuum pressure of 404.6mmHg, distillation temperature made from
51 – 65oC. Perform the second distillation phase of malt in the first stage: 51 – 54 oC, distillation
stage between 55 – 58oC, final distillation stage: 59 – 65oC.
The 2nd distilled alcohol is separated in stages for analysis to determine the separation of
the first and last liquor to obtain the optimal product. Analyzing the composition, fleet of toxins
and impurities at different stages (beginning - middle - end) to determine the appropriate
distillation and recalling process. Analysis results obtained (Table 3.9):
Table 3.9: Analysis results of the 2nd distilled malt samples
No

Samples

1
2
3
4
5
6

7
8
9

M1 (1%)
M2 (3%)
M3(5%)
M4 (7%)
M5 (giữa)
M6(20%)
M7 (15%)
M8 (10%)
M9 (5%)

Acetaldehyde(mg/l) Metanol (mg/l)
54.53± 0.10
11.52 ± 0.12
47.22± 0.10
9.17 ± 0.10
33.35± 0.10
5.43 ± 0.10
20.58± 0.10
4.21 ± 0.10
18.53± 0.10
3.29 ± 0.08
-

High alcohol
content(mg/l)
2.55 ± 0.05

5.58 ± 0.05
21.63 ± 0.05
50.92 ± 0.08
67.51 ± 1.00
117.63 ± 1.00
329.27 ± 1.00
416.21 ± 1.00
442.29 ± 1.00

Fucfurol
(mg/l)
1.36 ± 0.05
2.11 ± 0.05

For distillation of malt in the first stage, it is from 1-3% content of acetaldehyde,
methanol is quite high, higher alcohol also appears but with very low content, fucfurol is not
available. Continuing the distillation process, acetaldehyde, methanol tended to decrease and
sharply reduced when distilled to 5% of first alcohol. High-grade wines have a gradual increase
in distillation, when distillation to the last 20% of the alcohol content of high-grade alcohol


15
increases significantly (117.63 mg/l) and continues to increase rapidly at the last 15% (329.27
mg/l). At the end of the wine period, 10% - 5% of the higher alcohol content reaches 436.29 –
442.29 mg/l and the appearance of fucfurol begins..
Thus, in order to recover the product, it will eliminate many toxins and impurities , for
malt alcohol, the first and last alcohol separation is necessary and the appropriate amount of
alcohol is, according to our opinion, the beginning amount of alcohol is between 3% and 5%,
the final amount of alcohol is between 10% and 15%
First and last alcohol products are separated to redistribute with the following batches or

can be distilled into a separate batch.
Analysis results of toxins and impurities of alcohol after the second distillation of low
content, including: Acetaldehyde: 18.53 mg/l; Methanol: 3.29 mg/l, High alcohol: 67.51 mg/l,
Fucfurol: not detected
The 2nd malt alcohol product (M5) after 2nd distillation with the alcohol content of 65 68% Vol is stored and flavored with oak to make commercial whisky.
3.6.2. Research on technology for producing whisky from corn
3.6.2.1. Processing liquid fermenting from corn
In the experimental production, the saccharification process of maize juice was
conducted in two stages: gelatinizing the cooked liquid (boiling / 45 minutes); Fluidization: Cool
down to 65oC, adding enzymes of Termamyl® SC 0.35%, Dextrozyme® GA 1.5% and keeping
65oC/60 minutes, to ensure effective saccharification. Continue lowering the temperature to 52 oC,
adding enzyme Neutrase 0.03%, keeping the temperature at 52°C/30 minutes to break down the
protein into a free amino acid (FAN) to help the growth and development of yeast.
The main components of the corn fluid in whisky fermentation are: oBx: 18.0; Reducing
sugar: 160.50 g/l; Protein: 14.23 g/l; FAN 168.5 mg/l; pH: 5.4.
3.6.2.2. Fermenting to produce whisky from corn
Fermentation of corn liquid with a scale of 100 liters / batch with the fermentation
conditions identified in the research (in Section 3.4.2): Fermentation temperature: 25°C; sugar
content 169.3 g/l; pH = 5.6; same amount of 7%Vol (reaching 12 x 106 - 15 x 106 cells/ml
fermented fluid). The alcohol fermentation process ended after 168 hours after the seeding, we
got the results (Table 3.10):
Table 3.10: Changes in alcohol fermentation from corn (C)
Component
Time
0h
12 h
24 h
36 h
48 h
60 h

72 h
84 h
96 h
108 h
120 h
132 h
144 h
156 h
168 h

Sugar content
(converted to
maltose sugar) g/l
160.3±0.5
142.1±0.5
121.5±0.5
110.2±0.5
78.4±0.3
62.8±0.3
40.2±0.3
30.1±0.3
21.8±0.3
14.2±0.3
11.6±0.2
8.2±0.2
6.1±0.2
4.2±0.1
4.1±0.1

Alcohol content

(%Vol)

Number of cell
(x106 cells/ml)

0.2 ± 0.05
1.3 ± 0.05
2.8 ± 0.05
4.6 ± 0.05
6.1 ± 0.1
6.9 ± 0.1
7.2 ± 0.1
7.6 ± 0.1
7.8 ± 0.1
8.0 ± 0.1
8.3 ± 0.1
8.6 ± 0.1
9.1 ± 0.1
9.1 ± 0.1
9.0 ± 0.1

17±1.0
130±3.0
205±3.0
212±3.0
213±3.0
198±3.0
162±2.0
116±2.0
108±2.0

85±2.0
71±2.0
60±2.0
46±2.0
45±1.0
43±1.0

Total acid
(converted to acetic
acid) mg/l
418.5± 1.0
429.1± 1.0
431.5± 2.0
456.9± 2.0
462.8± 2.0
469.8± 2.0
481.3± 2.0
483.3± 2.0
491.9± 2.0
495.5± 3.0
510.8± 3.0
516.8± 3.0
519.7± 3.0
521.6± 3.0
522.3± 3.0

The results showed that: the indicators of sugar content, alcohol content, total acid,
number of cells had similar changes as in the corn liquid fermentation experiments, but the

16
total acid content increased more than the experimental scale (2 liters/batch), this can be
explained by the part of bacteria involved in fermentation.
The research results show that they are consistent with the studies of many previous authors.
The process of fermenting corn fluid in production with the above conditions, the optimal time for
fermentation is from 7 to 8 days.
3.6.2.3. Corn alcohol distillation
a. First distillation
The liquid after fermentation has an alcohol content (9.0%Vol) that is distilled for the first
time at a vacuum pressure of 404.6mmHg, the boiling temperature of alcohol at this pressure is
63.04°C. The first distillation process for corn alcohol is similar to distilling alcohol from barley
malt: First stage: 63 - 65°C, distillation middle stage 68 - 70°C, final distillation stage: 73 - 75°C.
The first distillation, the whole alcohol recovery with a concentration of 20%Vol.
Ingredients of some toxins and basic impurities in the first distilled corn alcohol: Acetaldehyde:
83.62 ± 0.1 (mg/l); Methanol: 10,92 ± 0,05 (mg/l); High alcohol: 295.18 ± 1.0 (mg/l); Fucfurol:
3.42 ± 0.1 (mg/l).
b. Second distillation
The 2nd distillation at a vacuum pressure of 404.6mmHg, distillation temperature made
from 51 - 65°C. Implementing the second distillation stage of corn liquid in the first stage: 51 54°C, distillation middle stage 55 - 58°C, final distillation stage: 59 - 65°C. The 2 nd distilled
alcohol is separated in stages for analysis to determine the separation of the first and last liquid
to obtain the optimal product. Analyzing the composition, content of toxins and impurities at
different stages (beginning - middle - end) to determine the appropriate distillation and
recalling process. Analytical results obtained (Table 3.11):
Table 3.11: Results of analysis of second distilled corn alcohol samples

NO

Sample

1

2
3
4
5
6
7
8
9

M1 (1%)
M2 (3%)
M3(5%)
M4 (7%)
M5 (giữa)
M6(20%)
M7 (15%)
M8 (10%)
M9 (5%)

Acetaldehyde(mg/l)
69.16± 0.10
55.18± 0.10
34.23± 0.10
24.62± 0.10
22.06± 0.10
-

Metanol (mg/l
13.23 ± 0.12
10.26 ± 0.10

6.89 ± 0.10
5.35 ± 0.10
5.02 ± 0.05
-

High quality
alcohol (mg/l)
5.02 ± 0.05
9.15 ± 0.05
13.28 ± 0.05
24.95 ± 0.08
29.83 ± 1.00
129.17 ± 1.00
335.59 ± 1.00
436.92 ± 1.00
465.18 ± 1.00

Fucfurol
(mg/l)
1.32 ± 0.05
4.68 ± 0.05
5.23 ± 0.05

For the vacuum distillation of corn alcohol in the early stages of 1 - 5% of the content of
acetaldehyde, very high content of methanol, higher quality alcohol also starts to appear but with
low content, fucfurol has not been found. Continuing the distillation process, acetaldehyde,
methanol tended to decrease and sharply reduced when distilled to 7% of first alcohol. Highquality alcohol tends to increase gradually during distillation, when distillation up to the last stage
of the last 20%, the higher alcohol content increases significantly (129.17 mg/l) and continues to
increase rapidly at the last 15% ( 335.59 mg/l). At the end of the alcohol period, there will be 10% 5% of high-quality alcohol content, reaching 456.92 - 465.18 mg/l. At the stage of 15% - 10% of
the last alcohol begins to appear fucfurol.

Thus, for corn alcohol, the separation of first and last alcohol liquor to recover high
quality products eliminateing many toxins and impurities, in our opinion, it is necessary to
separate 5-7% of the first and the last content from 15% to 10%.
The analysis results of the toxin composition and the alcohol impurity of the second


17
distillation product in corn alcohol are higher than malt alcohol but both are in low content,
including: Acetaldehyde (22.06 mg/l); Methanol (5.02 mg/l), High alcohol (29.83 mg/l),
Fucfurol: not detected. The second distilled corn alcohol (N5) with the alcohol content of 65 68% Vol is stored and flavored with oak to produce commercial whisky.
3.6.3. Researching the blending ratio of malt alcohol and corn alcohol to make Blend alcohol
In the production, beside alcohol product of 100% malt; 100% corn, to assess malt and
corn blended alcohol, we made blended alcohol of malt and corn at different ratios. Sensory
evaluation of blended products at 40%Vol. Results obtained (Table 3.12):
Table 3.12. Sensory evaluation of the ratio of alcohol blended between malt and corn

Alcohol type /(M:C)
Index
Sensory score

9:1
16.6

8:2
16.6

Malt:corn alcohol ratio (M:C)
7:3
6:4
5:5

4:6
3:7
16.3
16.3
16.1
16.1
15.9

2:8
15.9

1:9
15.2

Thus, to create a blended product, malt and corn can be used according to the malt: corn
ratio of 2: 8 and 3: 8, which is appropriate, while still ensuring good quality and typical flavor
and flavor of the product. This result is also consistent with the recommendations of whisky
experts in the US, whisky made from malt and corn at the rate of no more than 20% corn
alcohol is best rated by consumers. Based on the results of analysis and recommendations of experts,
we choose the ratio of blended alcohol to malt: corn ratio of 2: 8 to research and manufacture
blended whisky products.
The plan for blending malt raw materials: corn is applied by small-scale traditional
production establishments due to the initiative in stabilizing the quality of raw materials and the
fermentation technology. However, the technique of mixing materials is not economical when it
comes to creating many types of products in the blending line. Therefore, in large-scale whisky
production technology, which often produces independently the source of raw materials, experts
often use a variety of alcohol in terms of raw materials and flavors to combine to create high
quality required by the manufacturer.
3.6.4. Researching the process of brewing and flavoring whisky
3.6.4.1. Researching and choosinge the appropriate type of oak

The oak used for the research is 24 commercial oaks soaked in alcohol originating from the
United States, France and Australia. Commercially veneered oak, after weighing to determine the
weight, is washed several times with soft water and soaked in 65%Vol alcohol samples to determine
the type of oak; weight of wood to be used per liter of alcohol, through color perception, taste and
aroma analysis to select a suitable oak for whiskey production.
Among of the 24 oak samples used for the research, after 90 days of incubation, there were 04
samples with aromas, flavors that match the taste, typical flavor of whisky (malt, tannin in oak are
very clear, combined with Vanillin, are oak samples with symbols: S3, S5, S6, S8, in which the
highest sensory score (including flavor, taste and color) is S8 model with 16.4 scores.
Continuing to incubate and keep track of 4 samples of oak flavoring, typical taste of the selected whisky
after the first time extra 90 days (total incubation time is 180 days), continuing the second sensing of samples,
obtained the following results (Table 3.13):

Table 3.13: The second sensory results of 04 oak samples after 180 days of incubation with alcohol
Sensory evaluation (TCVN 3215-79)
Sign of Clarit
Notes (homogeneous
Coeff
Coeff
Coeff Total
NO
oak
y,
alcohol flavor types)
icient Smell icient Taste icient score
types
colo
(0.8)
(1.2)
(2.0)

s
r
1
S3
4.5
0.8
4.3
1.2
3.7
2.0
16.2
chivas whisky
2
S5
4.3
0.8
4.0
1.2
3.9
2.0
16.0
Single malt whisky
3
S6
4.3
0.8
4.0
1.2
3.9
2.0

16.0
Chivas whisky
4
S8
4.5
0.8
4.3
1.2
3.9
2.0
16.6
Single malt whisky


18
The results showed that: Among the selected oak samples, there were 02 oak samples:
S5, S8 with flavor, very typical for high-class whiskey, of which S8 has sensory value (flavor,
taste and color). clarity) suitable for producing 100% malt and 100% malt whisky and also
suitable for corn and malt blending alcohol, so we chose oak with the symbol S8 to conduct
further research.
3.6.4.2. Determining the amount of immersed oak and the appropriate alcohol content
- Determining the amount of appropriate oak immersion: Practising to soak oak S8 with
65°Vol alcohol. Comparing to commercial alcohol (Singleton (Scotchland) 100% malt; Jack
Danies (USA) 100% corn and KNOB CREEK (USA) 80% corn = 20% malt). The above
results show that: After 90 to 120 days of soaking S8 oak (weight 3.0 g in 1 liter of 65% Vol
alcohol) for the best sensory results (color, flavor, taste).
- Determining the appropriate alcohol immersion content (%Vol): When soaking the S8
oak (bar form) with a weight of 3.0 g of wood/1 liter of 65% alcohol, the best color extraction
ability in alcohol samples with a concentration of 65 - 70 (%Vol), color extraction time
achieved equivalent to the control sample from 90 - 120 days. This result is also consistent with

the results of some other authors.
- Comment: From the achieved experimental results we find to produce whisky from
corn and malt barley in Vietnam, the suitable oak is S8 with 3.0 g/liter of alcohol 65 - 70 (% Vol),
typical color extraction time is from 90 to 120 days because after 120 days of storage, the color
change of alcohol is almost negligible, but this time is not enough for the alcohol is "ripe" and still
in the process of changing the flavor and characteristic so it is necessary to continue storing more
time (usually not less than 3 years) in order that the alcohol has a better flavor and taste.
3.6.4.3. Initial researching the changes in aromatic substances during whisky
storage
Within the scope of the thesis, we initially researched the process of creating aroma of
some typical flavorings of whisky by GC-MS chromatography method to determine the
composition and content of the hydrolyzed ellagitannins groups from tannins, which are
typically polyphenols formed primarily from the oxidative bonds of alloyl groups in glucose β1,2,3,4,6-pentagalloyl.
Analytical samples include: 03 alcohol samples used for control include: (Singleton
(Scotchland) 100% malt; Jack Danies (USA) 100% corn and KNOB CREEK (USA) 80% corn =
20% malt) and Whisky samples is researched and produced from research results of this thesis.
a) change of some aromatics in alcohol after 6 months of oak brewing
After 06 months of storage, the composition and flavor impurities have a great change.
Samples of oak-soaked alcohol vary in composition, with significant aroma content.
Typically, such as o-Xylene, n-Propyl acetate, Octanoic acid, ethyl ester, ... Especially
Vanillin (Aldehyd phenolic) has appeared in oak immersion samples. Allene; 2-(4-tert-Butylphenoxymethyl) -4-hydroxy-pyrimidine-5-carboxylic acid ethyl ester; cis-Thujan-3-one 2,4dinitrophenylhydrazone is only found in samples of 100% malt. Benzoic acid trimethylsilyl
ester; iso-Amyl acetate are esters found only in oak immersed corn alcohol samples.
b) Alteration of some aromatics in wine after 12 months of brewing oak
After 12 months of storage, impurities (1,3-Benzodioxole-5-propanol, 4,7dimethoxy-.alpha.-methyl-; 3-Butyn-1-ol; Ethanone, 1- (8-pyridinyl) -, oxime; ...) in flavor has
decreased quite clearly, many impurities have not been detected. The reduction in composition
and concentration of impurities is a very important reason for the increase in sensory value and
flavor in the alcohol product is increased during storage process.
Ingredients n-Propyl acetate; Decanoic acid, ethyl ester; 2- (4-tert-Butylphenoxymethyl) -4-hydroxy-pyrimidine-5-carboxylic acid ethyl ester only appears in malt
alcohol samples. Oxalic acid, cyclohexylmethyl tridecyl ester; Furo [2.3-d] pyrimidine, 4.6dimethyl-2-methylsulfanyl-; iso-Amyl acetate is only found in corn alcohol samples. Vanillin
content in malt alcohol is greater than that in corn alcohol.

19
c) Alteration of some aromatics in alcohol after 18 months of brewing oak
After 18 months of storage showed: n-Propyl acetate; Octanoic acid, ethyl ester; in malt
alcohol samples there are greater concentrations than corn alcohol. o-Xylene; 2- (4-tert-Butylphenoxymethyl) -4-hydroxy-pyrimidine-5-carboxylic acid ethyl ester is high in malt alcohol but
not found in corn alcohol. In contrast to Oxalic acid, cyclohexylmethyl tridecyl ester; iso-Amyl
acetate; 4-methyl-2-pentanone is only found in corn alcohol. Vanillin content in oak soaked alcohol
samples is much higher than samples without oak. The composition of many impurities according
to flavor compared to before storage has been sharply reduced or not detected.
The oak-soaked products have created many unique aroma essence of whisky as a result
of the interaction between the alcohol and the substances in oak, in which the alcohol has
typical ingredients such as Vanillin; n-Propyl acetate; 2- (4-tert-Butyl-phenoxymethyl) -4hydroxy-pyrimidine-5-carboxylic acid ethyl ester; iso-Amyl acetate…
The combination of malt and corn alcohol in stock increases some aroma components in
product alcohol compared to corn alcohol (100%), in which the most obvious is the composition
and content of substances such as: n- Propyl acetate; Octanoic acid, ethyl ester; Decanoic acid,
ethyl ester; Vanillin.
d) Alteration of some aromatics in alcohol after 24 months of brewing oak
After 24 months of storage showed that: in 100% malt alcohol, there were 09 types of
flavoring compounds, including n-Propyl acetate; o-Xylene; Decanoic acid, ethyl ester; Octanoic
acid, ethyl ester; Vanillin in malt alcohol samples has a higher content than corn alcohol. Octanoic
acid, ethyl ester; Decanoic acid, ethyl ester; 2- (4-tert-Butyl-phenoxymethyl) -4-hydroxypyrimidine-5-carboxylic acid ethyl ester has a high content in malt alcohol but not seen on corn
alcohol. In contrast to Oxalic acid, cyclohexylmethyl tridecyl ester; Cyclohexanone; 4-Allyl-1,2diacetoxybenzene; iso-Amyl acetate; 4-methyl-2-pentanone is only found in corn alcohol.
After 24 months of storage, oak-soaked products have created many of the specific
aroma characteristics of whisky as a result of the interaction between the alcohol and the
substances in oak, in which the alcohol product has typical parts such as: n-Propyl acetate;
Vanillin; Octanoic acid, ethyl ester; Decanoic acid, ethyl ester; 2- (4-tert-Butyl-phenoxymethyl)
-4-hydroxy-pyrimidine-5-carboxylic acid ethyl ester; iso-Amyl acetate, ...
The composition and content of flavoring substances in malt and corn alcohol are quite
different. Many compounds are found only in malt alcohol such as: o-Xylene; Octanoic acid, ethyl

ester; 2- (4-tert-Butyl-phenoxymethyl) -4-hydroxy-pyrimidine-5-carboxylic acid ethyl ester. In
contrast, many compounds only found in corn alcohol such as: Ethanol, 2- (vinyloxy) -; 3-Nonen1-ol, (Z) -; Oxalic acid, cyclohexylmethyl tridecyl ester; iso-Amyl acetate; 4-methyl-2-pentanone.
All 3 types of products are similar in color to the imported oversea alcohol lines, with
the typical flavor of whisky (light sweet, soft aftertaste). GC-MS analysis of incense showed
that: incense has many typical similar components: n-Propyl acetate; Vanillin; Octanoic acid,
ethyl ester; Decanoic acid, ethyl ester; 2- (4-tert-Butyl-phenoxymethyl) -4-hydroxy-pyrimidine5-carboxylic acid ethyl ester; iso-Amyl acetate.
e) Sensory evaluation of whisky after 24 months of storage
Practising sensory evaluation on a 20-score scale, Vietnam Standard TCVN 3217 - 79, during
storage process. Results obtained (Table 3.14):

No

Alcohol
sample
symbol

1
2

S8.M
S8.N

3

S8.N8:M2

Table 3.14: Sensory results after 24 months of storage
Sensory evaluation (TCVN 3215-79)
Type of
Clarity Coefficien Smel Coefficien

Coefficien
material
Taste
, color
t (0.8)
l
t (1.2)
t (2.0)
100% malt
100% corn
20% malt,
80% corn

4.5
4.4

0.8
0.8

4.3
4.0

1.2
1.2

4.1
3.8

2.0
2.0

Total
score
s
17.0
15.9

4.4

0.8

4.1

1.2

4.0

2.0

16.4


20
After 24 months of storage with S8 oak, all whiskey products achieved a good sensory score,
in which: whisky from malt (17.0 scores); whisky from corn (15.9 scores); whisky from 8
corn: 2 malt reached (16.4 scores).
The results of the expert's sensory evaluation showed that: The average of sensory scores
with weight of smell and taste of the product after 24 months were higher than 3.8 scores. This
shows that: In addition to whisky products from 100% malt and 100% corn, creating a line of corn
whisky product combined with barley malt still brings high value, characteristic, suitable for the

market and the product price is lower than whisky from 100% malt.
3.6.5. Technological process for producing whisky from barley malt
3.6.5.1. Diagram of technological process for producing whisky from barley malt
Barley malt

Crushing raw materials (d ≤ 0,3mm);
Mixing malt: water = 1:4
Termamyl 0,08%; Dextrozyme 0,3 %
Saccharification (45oC/25' - 52oC/40' - 64oC/60' 75oC/15')

Saccharification fluid of malt
(Cool down on fermentation conditions)

K2S2O5 0,3 g/l
Fermentation fluid
(Sugar content:160 - 165g/l, t = 25 - 28oC pH: 5,2 – 5,6)

Yeast MS42 (7%V) achieved
15 x 106tb/ml

Fermentation
Time: 5–6 days; t = 25–28oC; Alcohol concentration: 9,0–9,1%Vol
1st distillation:
P=404,6mmHg, t = 63–75oC
1st alcohol: 18 – 20%Vol
2nd distillation: P=404,6mmHg,
t=51 – 65oC, Vđ=5%; Vc=10%

2nd alcohol: 65 – 68%Vol
Time ≥ 24 months

t = 20 – 30oC

Pickled oak (S8): 3g/l

Whisky 100% malt
(60 – 63%Vol)

Time ≥ 5 years
t = 20 – 30oC

+ H2O
Whisky 100% malt
(40 – 42%Vol)

Whisky 100% malt
(50 – 53%Vol)

+ H2O


21

Figure 3.7. Whisky production diagram from malt


22
3.6.5.2. Explanation of technological process
a) Crushing and cooking raw materials
Malt (Sebastian - France): being crushed with size (d < 0.3mm), the mixing ratio of malt:
water in the ratio of 1: 4 or 1: 4.2 is suitable for fermentation in production to ensure the sugar

content in the fermented liquid reaches 160 – 170 g/l.
- Process of saccharification fluid:
After being crushed, malt mixed with soft water (RO2 filter) added with Termamyl® SC
(0.08%), Dextrozyme® GA (0.3%) and raised the heat to 45oC/25 minutes, to reach the coefficient of
maximal swelling while activating the enzyme systems found in malt. Being continued to raise to
52oC/40 minutes, which is the condition for the protease enzyme to function optimally, converting
proteins into amino acids. The temperature continues to be raised to 64 oC/60 minutes. At this
temperature, the enzyme amylase will function optimally, breaking down starch molecules into
simple sugars, dextrin, etc. with low molecular weight, easily soluble in water. Finally, the glycemic
solution was raised to 75°C for 15 minutes, under which conditions the amylase enzyme remained
active, thoroughly converting the starch into sugar.
At the end of the saccharification process, the liquid was cooled to 25°C and pumped to
the fermentation tanks, supplemented with K2S2O5 (0,3g/l). Being continue to ferment the seed
to ensure the fermentation process takes place immediately after the amount of fermentation is
generated.
b) Alcohol fermentation
Strain of S. cerevisiae MS42 is activated and propagated on propagation media G1 and
G2. Time for each level is 24 hours. The fermentation temperature is 25 - 28 oC. The same
amount of 7%V (reaching 15 x 10 6 cells / ml of fermented liquid). The fermentation time for
malt is 6-7 days
c) Distillation to collect high-concentration alcohol
The distillation process is carried out twice by vacuum distillation equipment, for the
second distillation, there is the separation of first and last alcohol to reduce unwanted toxins
and impurities in alcohol.
First distillation: vacuum pressure 404.6 mmHg, temperature from 63 - 75 oC. The first
stage: 63 - 65oC, the distillation stage between 68 - 70 oC, the final distillation stage: 73 - 75 oC.
Revoke all alcohol with a concentration of 20%Vol
Second distillation: at a vacuum pressure of 404.6 mmHg, temperature from 51 - 65 oC.
Temperature in the first stage: 51 - 54oC, distillation stage between 55 - 58oC, final distillation
stage: 59 - 65oC. Separate first and last wine products, specifically: Malt alcohol: Vfirst = = 3-5%;

VFinal = 15 - 10%;
d) Storage, flavoring and finishing products
After second distilled alcohol, an alcohol content is 65 – 68%Vol stored with oak (S8)
with content of 3.0g/l. Storage equipment: 50-liter stainless steel tank, in natural conditions,
temperature: 20 - 30oC. Minimum storage time of 2 years.
Ending the process of storage, mixing, coloring with caramelized sugar, flavoring,
filtering wine to finish the product.
3.6.6. Technological process for producing whisky from corn
3.6.6.1. Diagram of technological process for producing whisky from corn


23
Kernel corn

Crushing raw materials (d ≤ 0,5 mm);
Mixing corn: water = 1:4
Saccharification (45oC/20' – boiling/40' - 65oC/60' 52oC/30')

Termamyl® SC (0,3%);
Dextrozyme® GA (1,5%);
Neutrase (0,03%)

Saccharification fluid of corn
(Cool down on fermentation conditions)

Fermentation fluid
(Sugar content:160 - 165g/l, t = 25 - 28oC pH: 5,4 – 5,8)

Yeast MS42 (7%V) achieved
15 x 106tb/ml

Fermentation
Time: 7–8 days; t = 25–28oC; Alcohol concentration: 9,0–9,1%Vol
1st distillation:
P=404,6mmHg, t = 63–75oC
1st alcohol: 18 – 20%Vol
2nd distillation: P=404,6mmHg, t=51 –
65oC, Vđ=5%; Vc=12%
2nd alcohol: 65 – 68%Vol
Time ≥ 24 months
t = 20 – 30oC

Pickled oak (S8): 3g/l

Whisky 100% corn
(60 – 63%Vol)

Time ≥ 5 years
t = 20 – 30oC

+ H2O
Whisky 100% corn
(40 – 42%Vol)

Whisky 100% corn
(50 – 53%Vol)
+ H2O

Figure 3.7. Whisky production diagram from corn
3.6.6.2. Explanation of technological process for alcohol production from corn

a) Crushing and cooking raw materials
Corn (variety DK9955): is crushed with size (d <0.5mm), the ratio of corn mixing:
water in the ratio of 1: 4.0 the sugar content in the fermentation reaches 165 – 170 g/l.
- Process of corn fluid saccharification:
After being crushed and mixed with water (filter RO2), the corn is added to raise
temperature up 45oC/20 minutes to achieve maximum expansion coefficient and activate
enzymes in corn. Cooking the corn fluid to make gelatinization by boiling for 45 minutes, this
is the period of gelatinization of the remaining starch by heat, and pasteurization of
fermentation. At the end of the gelatinization period, lowering the temperature to 65 oC, adding