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: PhD Nguyen Quang Thao 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 PREAMBLE 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 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 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 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 4.4 Producing 10,000 liters of whisky from corn and malt barley at Eresson Beer and Alcohol One Member Company Limited (including 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 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 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 types of whisky products: 100% barley malt; 100% corn and 20% barley malt + 80% corn Thesis structure The thesis is structured into 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) CHAPTER 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 - 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, 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 - 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 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 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 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 types.: Arobois HM, Arobois Aroneo, INTENSE, BALANCED, SAVORY; American oak includes 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 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 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, 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 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 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 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 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 Total Score 14.9 15.3 8 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 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 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) LV22 MS42 BH30 KPH (˂0.5) KPH (˂0.5) KPH (˂0.5) Acetandehyde (mg/l) H.HD.QT.196 (GC-FID) 35 ± 39 ± 91 ± High-quality alcohol (mg/l) H.HD.QT.196 (GC-FID) 218 ± 183 ± 252 ± 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 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 ± 125 ± 127 ± High-quality alcohol (mg/l) H.HD.QT.196 (GC-FID) 229 ± 216 ± 232 ± 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 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: - 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: - 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 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 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 to 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 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 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 - 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: 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: 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 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 S3 4.5 0.8 4.3 1.2 3.7 2.0 16.2 chivas whisky S5 4.3 0.8 4.0 1.2 3.9 2.0 16.0 Single malt whisky S6 4.3 0.8 4.0 1.2 3.9 2.0 16.0 Chivas whisky 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 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 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 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 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 S8.M S8.N 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 corn: 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 ≥ 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: 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 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 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 ≥ 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