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

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DOCTORAL COURSE THESIS SUMMARY Major: Biotechnology Major code: 62 42 02 01 DOAN THI KIEU TIEN ISOLATION AND SELECTION FOR THERMOTOLERANT YEAST AND STUDY FOR WINE FERMENTATION FROM Cayratia trifolia IN MEKONG DELTA Can Tho, 2020 THIS THESIS WAS COMPLETED AT CAN THO UNIVERSITY Advisor: Prof Dr Ha Thanh Toan Assoc Dr Ngo Thi Phuong Dung This thesis was defended on the meeting of Univeristy doctoral thesis evaluation council Place: Doctoral thesis evaluation hall, 2nd floor – Administration Building, Can Tho University at 14 h 00, 3rd April, 2019 First Reviewer: Assoc Dr Nguyen Duc Hoang Second Reviewer: Assoc Dr Nguyen Minh Thuy This thesis is stored and can be referenced at the Learning Resource Center, Can Tho University and The National Library of Vietnam LIST OF PUBLISHED PAPERS Doan Thi Kieu Tien, Ha Thanh Toan and Ngo Thi Phuong Dung, 2016 Exploration of yeasts applied for wine fermentation from Cayratia trifolia in the Mekong Delta The 12th Young Scientist Seminar; Nov 22nd-23rd, Yamaguchi Prefectural Seminar park, Yamaguchi, Japan, 19 Doan Thi Kieu Tien, Huynh Thi Hoang Anh, Nguyen Ngoc Thanh, Huynh Xuan Phong, Ha Thanh Toan and Ngo Thi Phuong Dung, 2017 Selection of thermotolerant yeasts for production of three-leaf cayratia (Cayratia trifolia L.) wine in Kien Giang Science and technology journal of agriculture and rural development 54-62 Doan Thi Kieu Tien, Vien Thi Hai Yen, Huynh Xuan Phong, Bui Hoang Dang Long, Ha Thanh Toan Ngo Thi Phuong Dung, 2018 Selection of thermotolerant yeasts and application in wine production from three-leaf cayratia (Cayratia trifolia L.) in Hau Giang Can Tho University journal of science 54(4B): 64-71 Doan Thi Kieu Tien, Lu Hang Nghi, Nguyen Ngoc Thanh, Huynh Xuan Phong, Ha Thanh Toan Ngo Thi Phuong Dung, 2018 Isolation and selection of thermotolerant yeasts for wine production from three-leaf cayratia (Cayratia trifolia L.), Journal of agricultural science and technology 55-64 Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Nguyen Duc Do, Ha Thanh Toan Ngo Thi Phuong Dung, 2018 Total polyphenol content and antioxidant capacity of Cayratia trifolia (L) Domin berries before and after fermentation using thermotolerant yeast Saccharomyces cerevisiae HG1.3, 2018 Vietnam journal of science and technology 60-64 Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Huynh Thi Hoang Anh, Huynh Xuan Phong, Nguyen Ngoc Thanh, Bui Hoang Dang Long, Ha Thanh Toan Ngo Thi Phuong Dung, 2018 Bioactive capacity of three-leaf cayratia collected in Kien Giang province and wine fermentation using Saccharomyces cerevisiae AG2.1 National biotechnology conference, 2018 978-983 Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Lu Hang Nghi, Huynh Xuan Phong, Nguyen Ngoc Thanh, Ha Thanh Toan Ngo Thi Phuong Dung Bioactive capacity of three-leaf cayratia collected in Ca Mau province and wine fermentation using Saccharomyces cerevisiae CM3.2, 2018 The conference of biotechnology in the Mekong Delta 104 Doan Thi Kieu Tien, Huynh Xuan Phong, Mamoru Yamada, Ha Thanh Toan and Ngo Thi Phuong Dung, 2019 Characterization of newly isolated thermotolerant yeast and evaluation of their potential for use in Cayratia trifolia wine production, Vietnam Journal of Science,Technology and Engineering 68-73 Doan Thi Kieu Tien, Huynh Thi Ngoc Mi, Lu Hang Nghi, Huynh Xuan Phong, Nguyen Ngoc Thanh, Bui Hoang Dang Long, Ha Thanh Toan Ngo Thi Phuong Dung, 2019 Evaluation of total polyphenol and antioxidant capacity in wine fermentation of three-leaf cayratia from Ca Mau province using Saccharomyces cerevisiae CM3.2, Can Tho University journal of science 55 (2019)(2): 285-291 Chapter INTRODUCTION Rationale of the study Threeleaf cayratia (Cayratia trifolia) is a wild creeper growing in grasslands and glades in several countries like China, Laos, Cambodia, In Vietnam, threeleaf cayratia naturally grows along the fences, bushes in provinces of the central and sourth Vietnam In the Mekong Delta, threeleaf cayratia is found in almost all provinces and cities, especially this plant is abundant in U Minh area The composition of threeleaf cayratia contains highly bioactive compounds (Tsao, 2010), the fruit has a unique color and flavor that were inadequately studied with few researches Wine had a history of thousands of years and were researched thoroughly to diversify in types and to improve in quality During wine fermentation, one of the most important factors is the yeast source Temperature has an important effect on the ethanol convertion from sugar using yeasts Currently, the climate change has gradually increase earth temperature which will definitely affect the fermentability of yeast in the wine production process Therefore, the application of thermotolerant yeast strains can contribute to improve the efficiency of the fermentation process Isolation and selection of thermotolerant yeasts and the evaluation of the ability to ferment threeleaf cayratia wine (Cayratia trifolia) in the Mekong Delta were essential Research objectives To provide references for the wine fermentation from Cayratia trifolia and to propose a solution for the wine fermentation which could improve effectiveness to deal with global warming Thereby, the people in the region can adapt to the climate change of the region and exploit the value of natural sources to improve their income Research contents (1) Collecting and evaluating physical and chemical characteristics and antioxidant properties of Cayratia trifolia in Mekong Delta; (2) Isolation and identification of morphological, physiological and biochemical characteristics of yeast strains from natural Cayratia trifolia; (3) Selection and identification of genetic relationships of thermotolerant, alcohol-resistant yeasts which had good ability of fermenting Cayratia trifolia wine; (4) Study the suitable conditions for the fermentation of Cayratia trifolia using the selected thermotolerant yeast strain New contributions of this thesis - Determining polyphenol content of Cayratia trifolia ranging from 0.47 to 1.54 mg GAE/mL and has antioxidant capacity from 16.60 to 82.86% - Isolation of 151 strains of yeast from natural Cayratia trifolia in the Mekong Delta that were identified as species of five genera Saccharomyces, Candida, Clavispora, Pichia and Hanseniaspora - Thirty (30) strains of natural yeasts were determined that were thermotolerant and could grow on the plate incubated from 37 to 45°C, were capable of growing in an environment with an ethanol content of 912% and could ferment to create over 6% (v / v) ethanol content - Saccharomyces cerevisiae strain HG1.3 was capable of being tolerant to 43°C At 35°C, the wine fermentation with initial pH 4.5, 20 ºBrix and 105 cells/mL After a 6-day fermentation, the content of ethanol reached 11.68% (v/v) The produced wine had polyphenol content of 0.6 mg GAE/mL, antioxidant capacity of 57.3% and had the unique color and flavor Chapter OVERVIEW 2.1 The overview of threeleaf cayratia threeleaf cayratia is a species of vines based on the stem-like tentacle that grows on opposite side of leaves The tentacle is about 2-3 cm long, usually divided into branches (Kumar et al., 2011) and sometimes with 4-5 reddish-brown branches with little of furry and long veins Leaves grow in a double, feathery pattern, with leaves joined in the middle that are usually larger than the sides, with long, slender, ovoid-shaped leaves The trunk has a polygonal cross-section with a green old stem and a reddish-brown young body with many longitudinal veins Flowers grow on axillary leaves and may be tall The lowers were 6-8 mm wide and have a disc shape, green petals (Pham Hoang Ho, 2000) The three leaf Cayratia contains a lot of water with round and flat fruit which can turn purple when ripe and was about 1cm in diameter (Sesagirriravu, 1986) The fruit contains high levels of phenolic, the ethanol extract had high antioxidant capacity (Rabeta and Lin, 2015) 2.2 Overview on yeast Yeasts are defined as single-celled fungi with asexually reproducing by budding Yeast cells are composed of: cell wall, cytoplasmic membrane, cytoplasm and nucleus (Nguyen Lan Dung, 1999) Yeast can be found in many different environments such as water, air, soil, etc Yeast is often able to grow at low pH and anaerobic conditions to reduce or inhibit bacterial growth (Kurtzman et al., 2011) From ancient times, people have applied yeast in science, food, medicine and industries Fermented beverages and foods produced through the activities of yeast have contributed prominently to the sustainable development of human society (Legras et al., 2007, Ulber and Soyez, 2004) 2.3 The wine fermentation and the effects of temperature on the wine fermentation The carbohydrate degradation under anaerobic conditions is called fermentation (Nguyen Lan Dung, 1999) Alcohol fermentation is a complex biochemical process that requires the participation of yeast or other microorganisms During alcohol fermentation, sugar is converted into ethanol and CO2 Alcoholic fermentation is accompanied by the formation of products and energy releasing (Nguyen Thanh Dat, 1986) Conventional yeasts are very sensitive to temperature At 36°C, yeast can be inhibited In a liquid environment, at a temperature of 40-50°C, yeast are almost deactivates and dies after hour 30 minutes; At 60-65°C, the yeast will die after minutes In dry environments (W = 13%), yeasts are more thermotolerant that can to withstand up to 85-105°C The higher the temperature, the lower ethanol content produced by fermentation In contrast, the more sugar supplied in the wine, the higher the sugar concentration can deactivate the fermentation (Bui Ai, 2003) The optimum temperature for yeast growth is 25-28°C (Casellas, 2005) When the fermentation taking place at low temperatures will take longer time However, high temperature can affect the taste of the product and can reduce the activity of the yeast and lead to the contamination of lactate bacteria and wild yeast In addition, high temperature fermentation can create aldehyde ester and reduce alcohol content in associate with CO2 increases (Nguyen Dinh Thuong and Nguyen Thanh Hang, 2005) 2.4 Thermotolerant yeast Temperature is the most important factor affecting yeast activity Arthur and Watson (1976) determined the growth temperature of psychophilic yeast in the range of 2-20ºC; mesophilic yeast were 5-35ºC; thermotolerant yeast were 8-42ºC and thermophilic yeast were 28-45ºC According to Roehr (2001), heat-resistant yeasts had several advantages in ethanol production under high temperature conditions including good metabolic activity and high fermentation rate creating high ethanol contents At high temperature, the solubility of oxygen and other gases in the water decreases that ensure anaerobic conditions for fermentation; The lower viscosity of the fermentation medium at higher temperature can save the energy needed for mixing the environment and minimize chance of contamination Therefore, in order to achieve high temperature fermentation, a highly efficient thermotolerant yeast strain is needed (Limtong et al., 2007) Chapter RESEARCH METHODOLOGY 3.1 Collect and evaluate characteristics of Cayratia trifolia Cayratia trifolia berries were collected from 53 locations in 13 provinces and cities in the Mekong Delta After harvesting, ripe fruits were taken, washed and drained Characteristics including shape, size, color of Cayratia trifolia were evaluated Juice extract of berries was measured for pH, Brix; sugar content, polyphenol content and oxidation resistance by the ability of DPPH free radical capturing (%) 3.2 Isolate the yeast strains from natural Cayratia trifolia for the identification of morphological, physiological and biochemical characteristics The collected fruits were ultilized for yeast isolation using YPD culture medium The enrichment medium was diluted and then spread, transferred by YPD agar medium several times until pure colonies were collected and stored at 4ºC The isolated yeast strains are identified with physiological and biochemical characteristics such as the abilities to use saccharose, maltose and glucose and urea decomposition, gelatin hydrolysis, spore formation by budding These characteristics were investigated for preliminary classification and initial evaluations of ethanol fermentation 3.3 Selection, genetic identification of relationships between thermotolerant and alcohol resistant yeast strains with good ethanol fermentation 3.3.1 Study for the thermotolerant ability of the yeast strains The isolates of yeast were cultured on petri containing YPD agar medium Then, the yeast plates were incubated at different temperatures: 30ºC, 35ºC, 37ºC, 39°C, 41°C, 43°C, 45°C and 47°C for 48 hours Colony observation was taken place to select yeast strains that were capable of growing and developing at high temperatures 3.3.2 Assessment of ethanol tolerance of yeast strains This experiment was applied to select thermotolerant yeast which could grow on plates supplemented with ethanol The thermotolerant yeast strains selected from 3.2.3.1 were spread on YPD agar medium with 3%, 6%, 9%, 12%, 15% v/v absolute ethanol and 0% control samples Yeast plates were incubated at 30°C for 48 hours Observe the formation of colonies of yeast strains on plates under different ethanol concentration Lin (2011) in which the ability of DPPH free radical capturing in fresh fruit juice was determined at 61.47% 4.2 Isolation of yeast strains from natural Cayratia trifolia fruits and identify the morphological, physiological and biochemical characteristics of the isolated yeasts From 53 different sources of Cayratia trifolia in 13 provinces and cities in the Mekong Delta region, 151 yeast strains were isolated This result illustrated the abundance and diversity of natural yeast on the Cayratia trifolia that had the similarity with published studies which isolated 50 yeast strains isolated from yeast starters collected in Mekong Delta (Ngo Thi Phuong Dung, 2009), 40 strains of yeast from an agriculture by-products including cocoa pods, pineapples, bagasse, straw and sawdust, 28 strains of yeast from fruits and citrus leaves (Dung Phong et al., 2016; Nuanpeng et al., 2016; Phong et al., 2016; Techaparin et al., 2017) Morphological characteristics of 151 strains of yeast were divided into groups of shapes: small sphere, large sphere, small oval, large oval, short ellipse, long ellipse and pointed ellipse Spore formation characteristics showed that groups to created round spores, except group Results on the ability to ferment glucose, saccharose and maltose; urea degradatin activity, gelatin hydrolysis of yeast strains showed that there were 140 strains capable of fermenting glucose; 104 strains capable of fermenting saccharose, 108 strains capable of fermenting maltose; 24 strains had urea degradation activity; and 30 strains were capable of degrading gelatine According to the preliminary classification description to the genus level of Kurtzman and Fell (1998), 151 yeast strains could be classified into genera: Saccharomyces, Hanseniaspora, Pichia and Candida 11 4.3 Selection, identification and genetic relationship investigation of thermotolerant and alcoholic resistant yeast strains with good Cayratia trifolia wine fermentation 4.3.1 The thermotolerant ability of yeast strains The colony forming of 151 strains of yeast showed that all of the strains could develop at the temperature from 30°C to 35°C Among 151 strains of yeast, 141 strains were able to grow at 37°C At 39°C, there were 121 strains of yeast capable of growing When the incubation temperature increased to 41°C, there were 89 strains of yeast capable of growing At 43°C, there were 48 strains that could form colonies At a temperature of 45°C, only 10 strains developed All isolates could not grow at 47°C Consequently, the final results selected 141 strains capable of growing at 37°C 4.3.2 The ethanol resistance of thermotolerant yeast strains Most of the selected yeast strains were grown in environments with 3% (v/v) ethanol content, except for strain VL4.4 isolated from the Cayratia trifolia collected in Tra On district, Vinh Long province When the ethanol content increased, yeast growth decreased In specific, there were 128 strains growing in environments with 6% ethanol concentration, 64 strains growing on plates supplemented with 9% ethanol At 12% (v/v) ethanol concentration, there were only 27 strains with colonies When the ethanol concentration increased to 15% (v/v), all strains did not grow According to Casey and Ingledew (1986), the usual ethanol resistance of most alcohol produced yeast strains were at about 5-10% (v/v) ethanol As a result, 64 strains showing resistances to alcohol 12 content ranging from 9% to 12% v / v were selected to assess the ability of ethanol fermentation 4.3.3 Selection of yeast strains with high ethanol fermentation abilities According to Luong Duc Pham (2005), the life cycle of yeast produced a large amount of carbon dioxide This by-product was made up in the anaerobic metabolism of yeast to produce ethylic alcohol, CO2 and other substances The amount of CO2 generated will be accumulated the Durham tube Therefore, the height of carbon dioxide in Durham tube was a criterion for preliminary assess the fermentation ability of the yeast strains Sixty-four strains of yeast were able to convert glucose into ethanol, with the height of the column of gas produced in the Durham tube increased over time Strains KG2.2, KG3.1, DT1.2, CM3.2 and BT1.2 could accumulate 30 mm height of air column in hours Two strains of yeast KG4.1 and AG3.2 had almost no fermentation ability The results were prequalified 57/64 strains capable of ethanol fermentation, gas production filled the 30mm Durham tubes in 48 hours 4.3.4 Cayratia trifolia wine fermentation The wine fermentation of 57 strains of yeast was assessed by the ability to ferment 100 mL of fruit juice After fermentation, the pH and Brix of the Cayratia trifolia fluid were reduced compared to the original pH and Brix (original pH 4,5 and 22 °Brix) yeast uses sugar as a carbon source since during fermentation Therefore, sugars were metabolized through an enzyme reaction sequence until the final products of fermentation including ethanol and CO2 (Le Ngoc Tu et al., 2005) Ethanol content was one of the most important criteria to assess the alcohol fermentation capacity of yeast strains The 13 results showed that the strain HG1.3 was able to ferment and produce 9.9% (v/v) ethanol 4.3.5 Scientific nonclamentures and genetic relationship of thermotolerant and alcoholic resistant From the results of fermentation survey in section 4.3.4, 30/57 strains of yeast capable of fermenting with the ethanol concentration from 6.0% (v/v) or more were selected The identification of 30 strains of yeast were shown in Table 4.2 Table 4.2: Results of identifying 30 yeast strains No Genus Candida Pichia Species Candida tropicalis Candida nivariensis Candida glabrata Pichia kudriavzevii Clavispora lusitaniae Saccharomyces Strains Strain number KG1.1, KG3.2, CM3.3, DT1.2, HG3.3, ST2.1, TG1.1 BT1.2 BL2.1, CT1.1, CT1.3, AG2.3, CT2.3 KG5.1, AG4.2, CM4.4, Clavispora TG4.2 BT2.1, BT3.3, Saccharomyces HG1.3, CM3.2, CT4.2, VL1.1 cerevisiae AG2.1, TV4.2, Total 30 DT3.2, LA1.3, The genetic relationship of 30 yeast strains KG2.1, TG3.1, MEGA integrated with Neighbor-Joining analysis HG2.1 was applied in the establishment of phylogenetic trees based on MEGA software (Neighbor-Joining) The phylogenetic tree of 30 yeast strains was shown in Figure 4.1 The identification results indicated the diversity of thermotolerant yeast strains isolated from Cayratia trifolia The results were also consistent with the research of Nguyen Lan Dung et al (2019) or Basso et al (2008), Saccharomyces cerevisiae was among of traditional species of yeast commonly used in alcohol fermentation 14 Figure 4.1: The phylogenetic tree of 30 yeast strains Note: The number indicated above each branch was the repetition number of same consequence in 1000 time bootstrap test : Genetic distance 4.4 The suitable conditions for the wine fermentation of Cayratia trifolia 4.4.1 The effects of fermentation temperature and pH Results of the change of Brix, pH and ethanol content in the fermentation using S cerevisiae HG1.3 showed that the ability of fermenting wine of HG1.3 strain was different at different temperature and pH (Table 4.3) Treatments of pH 4.5 at room temperature and 35°C resulted high ethanol contents (12.82-12.53% (v/v)), the difference was not statistically significant at 95% confidence Arthur and Watson (1976) identified the growth temperature of thermotolerant yeasts in the range of 8-42°C Thermotolerant yeasts had minimum temperature ranges (Tmin), optimum (Topt), maximum (Tmax), which were at 20-26°C, 26-35°C and 37-45°C, respectively and can even grow at temperatures above 45°C Thus, pH 4.5 at 35°C was selected as the suitable conditions for the fermentation using thermotolerant yeast that was in 15 accordance with published studies This temperature and pH were chosen for further experiments Table 4.3: The effects of temperatures and pH levels to the fermentation No 10 11 12 13 14 15 16 17 18 19 20 Temperature-pH Room temperature – Natural pH Room temperature 4.0 Room temperature 4.5 Room temperature 5.0 35-Natural pH 35-4.0 35-4.5 35-5.0 37-TN 37-4.0 37-4.5 37-5.0 39-TN 39-4.0 39-4.5 39-5.0 41-TN 41-4.0 41-4.5 41-5.0 Postfermentation pH Postfermentation Brix Ethanol content (% v/v) at 20ºC 3.57 8.17 11.68c 3.95 6.33 11.85bc 4.42 7.33 12.82a 4.64 7.50 10.53ef 3.55 3.95 4.24 4.62 3.55 3.93 4.31 4.90 3.46 3.84 4.22 4.07 3.23 3.41 4.04 4.22 9.00 9.00 9.00 9.00 11.17 11.00 10.67 12.00 13.00 12.00 12.00 11.00 14.67 15.00 16.00 16.00 11.55cd 11.68c 12.53ab 9.79fg 9.57g 9.87fg 10.78de 9.14gh 7.98ij 7.99ij 8.45gh 7.21ij 5.98k 5.98k 5.98k 5.19k Note: The figures in the table were the averages of triple repetitions; the same above characters indicated that there were no significant differences at 95% confidence 4.4.2 The effects of yeast density, Brix and fermentation time to Cayratia trifolia wine fermentation In fermentation, the ethanol content is related to the dry matter content in the medium Low dry matter content leads to the lack of nutrition for yeasts that then leads to a decrease in the number of products However, if the sugar content is too high, the concentration of ethanol is reduced since the too high 16 sugar content will increase osmotic pressure, and disorders in yeast physiology (Tahir et al., 2010) Yeast density and fermentation time were factors influencing the fermentation The effects of strain, Brix and fermentation time using S cerevisiae strain HG1.3 were shown in Table 4.4 Table 4.4: The effects of inoculation density, Brix and fermentation times o Brix Ethanol content (%) Days- oBrix-Density No sau lên men ethanol 20 oC (% v/v) - 20 - 103 8.67 11.40bc 20oC (% v/v) 7.33 13.47a - 20 - 10⁵ - 20 - 10⁷ 7.00 12.00bc - 22 - 103 8.67 11.40bc 5 - 22 - 10⁵ 8.33 12.30ab - 22 - 10⁷ 8.67 11.67bc - 24 - 103 11.67 11.40bc - 24 - 10⁵ 10.76 11.07bc – 24 - 107 11.33 10.83cd 10 - 20 - 103 7.67 11.40bc 11 - 20 - 10⁵ 8.33 12.30ab 12 - 20 - 10⁷ 9.33 11.07bc 13 - 22 - 103 9.00 11.40bc 14 - 22 - 10⁵ 9.00 12.30ab 15 - 22- 10⁷ 11.67 10.83cd 16 - 24 - 103 10.33 10.77cd 17 - 24 - 10⁵ 10.33 11.07bc 18 - 24 - 10⁷ 11.67 10.83cd 19 - 20 - 103 8.33 10.83cd 20 - 20 - 10⁵ 8.67 10.77cd 21 - 20 - 10⁷ 9.67 10.77cd 22 - 22 - 103 9.33 10.83cd 23 - 22 - 10⁵ 10.00 10.87c 24 - 22 - 10⁷ 10.67 9.53de 17 No Days- oBrix-Density 25 - 24 - 103 26 - 24 - 10⁵ 27 - 24 - 10⁷ o Brix Ethanol content (%) sau lên men ethanol 20 oC (% v/v) 9.00 8.86ef 20oC (% v/v) 10.33 8.05f 12.67 8.05f Note: The figures in the table were the averages of triple repetitions; the same above characters indicated that there were no significant differences at 95% confidence The data analyzed by Statgraphics Centurion XV software at 95% confidence showed the regression model as follows: H= -27,4471 + 1,58638Z+ 3,66409Y+ 5,10075X 0,019213Z2 - 0,124219ZX - 0,0769965ZY- 0,124352Y2 - 0,181007YX - 0,174352X2 + 0,00619792ZYX (1) Where: H= ethanol content, X= fermentation time (days), Y= density, Z= ºBrix Fixing ºBrix at 20 (Z= 20) would lead to the derrivatives for each variable The solving of equation system H (X ') = and H (Y') = resulted X = 5.8; Y = 5.04 The values were added to the equation to get the predicted optimal ethanol content H = 12.37% (v/v) Therefore, the optimal conditions for the fermentation of Cayratia trifolia wine using S cerevisiae HG1.3 were determined as days of fermentation, 105 cells/mL cell density, 20°Brix, and the initial pH 4.5 at 35ºC In this conditions, the theoretically achieved ethanol content was predicted at H = 12.37% The regression equation was vertified by liter scale fermentation of Cayratia trifolia wine The obtained ethanol content was at 12.0% (v/v) (Table 4.5) Therefore, the verified results showed the compatibility of the regression model and were also similar to the previous studies on the fermentation of watermelon wine (Ngo Thi 18 Phuong Dung et al., 2011), pineapple wine (Huynh Xuan Phong and ctv, 2017) The quality of Cayratia trifolia wine was checked with 15 criteria shown in Table 4.5 The results of testing criteria were compare to the standards of QCVN6-3:2010/BYT on wine quality assessment The results showed that all of the criteria meet the regulations In addition, the Cayratia trifolia wine contains 0.60 mg GAE/mL which showed the ability to capture 57.3% DPPH free radicals Thus, it was concluded that Cayratia trifolia wine had antioxidant capacity to help protect the body Table 4.5: Qualitative checks of Cayratia trifolia wine No Vietnamese Standard 6-3:2010/BYT Remarks Criterion Results Ethanol 12.0% v/v at 20°C ≥ 8% v/v at 20°C Passed Methanol 2.534 g/L alcohol 100° Passed SO2 1.4 mg/L 10g/L alcohol 100° 350 mg/L pH 4.17 - Selfdeclaration º Brix 12 - Selfdeclaration Reducing sugar 0.26 g/100 mL - Selfdeclaration Acid hydrocyanic g/L 0.1 mg/L methyl 2-propanol 2.425 g/L 100° alcohol - Selfdeclaration Aldehyde 0.638 g/L 100° alcohol 100° - Selfdeclaration 10 E coli CFU/ mL products CFU/ mL Passed 11 Coliforms 10 CFU/ mL products ≤ 10 CFU/ mL hẩm Passed 19 Passed Passed Vietnamese Standard 6-3:2010/BYT No Criterion Results 12 Total aerobic bacteria 7.2 x 102 CFU/g 13 Total yeast – mold 14 15 Polyphenol content DPPH free capturing radical 103 CFU/g Remarks Passed 3.6 x 10 CFU/g 10 CFU/g Passed 0.60 mg GAE/mL - Selfdeclaration 57.3a±3.73% - Selfdeclaration Sensory evaluation of Cayratia trifolia wines were established base on 0-5 scale on criteria (clarity and color, smell, taste, interest for the sample) based on the criteria in the grading evaluation method according to Vietnam standard TCVN-3217:79 The Cayratia trifolia wine using the strain HG1.3 had the purity and color of the wine reaching 4.9 points; the smell reached 4.6 points; the taste reached 4.0 points; the liking for wine reached 4.2 points CHAPTER CONCLUSION AND RECOMMENDATION 5.1 Conclusions Cayratia trifolia in the Mekong Delta were classified into two types: round and flat fruits, ripe purple-black fruits, the average diameter of the berries was 1.5 cm The extracted fluid had a very specific purple color, pH value at 3.01-4.75, Brix value at 3.5-10.0, total sugar content at 0.47-2.17 g/100mL and the reducing sugar at 0.22-0.96 g/100mL In particular, the fluid contains polyphenol content at 0.47-1.54 mg GAE/mL and the antioxidant capacity of 16.60-82.86% This was a potential source of raw materials for wine production and could help increase the income for people in the material area 20 The Isolation of natural yeast resulted 151 strains from 53 different sources of Cayratia trifolia in the Mekong Delta Preliminary classification from morphological, physiological and biochemical characteristics showed that 151 yeast strains of genera including Saccharomyces, Hanseniaspora, Pichia and Candida In particular, Saccharomyces was widely distributed and present in large numbers on many Cayratia trifolia samples Identify 64/151 strains capable of growing at 37°C in an environment with an ethanol concentration from to 12% v/v 30/64 strains of thermotolerant yeasts could grow at 37°C to 45°C with the supplemented ethanol concentration from 9-12% and produced 6% (v/v) ethanol The results of genetic sequencing in addition to the morphological and biochemical analysis of 30 yeast strains showed that the strains belonged to the four genera Saccharomyces, Candida, Pichia and Clavispora In particular, Saccharomyces cerevisiae HG1.3, CM3.2, AG2.1, TV4.2, DT3.2, LA1.3, KG2.1, TG3.1 and HG2.1 were the most closely related strains with a 100% bootstrap index The suitable conditions for wine fermentation of Cayratia trifolia juice using thermotolerant yeast strain HG1.3 were determined at 20 oBrix, pH 4,5, 105 cell/mL density in days at 35ºC The obtained wine has unique color and satisfied qualitative requirements according to the regulations In particular, the polyphenol content was determined at 0,6 mg GAE/mL with 57.3% anti-oxidant ability which could help improve human health Figure 4.2 showed the recommended protocol for Cayratia trifolia wine fermentation 21 Cayratia trifolia chín Clean with water Fluid extraction Adjust to 20º Brix and pH 4,5 Sterilize with NaHSO3 (140 mg/L) in hours Inoculate 1% of S cerevisiae HG1.3 Anaeobic incubation at 35ºC in days Filtration Final product Figure 4.2: Protocol for Wine fermentation from Cayratia trifolia 22 5.2 Recommendation Study the pharmacological effects of some other ingredients besides polyphenols Study on characteristics and mechanisms of thermotolerant properties on genome level of thermotolerant yeast strains Assess the characteristics of the isolated yeast strains according to storage time and 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Tahir, A., M Aftab and T Farasat, 2010 Effect of cultural conditions on ethanol production by locally isolated Saccharomyces cerevisiae bio-07 Journal of Applied Pharmaceutical, 3(2): 72-78pp Techaparin, A., P Thanonkeo and P Klanrit, 2017 Hightemperature ethanol using thermotolerant yeast newly isolated from Greater Mekong Subregion Brazilia Journal of Microbiology 48:461-475 Vietnamese standard 3217:1997 (TCVN 3217:1997), (1997) Wine – Sensorial Evaluation – Marking Method Hanoi 25 ... about 1.5 - cm and a dark black color (Figure 4.1) Bảng 4.1: Đặc điểm dạng trái giác vùng ĐBSCL STT Đặc điểm Hình STT Đặc điểm Hình The fruit had round shape with rough surface and a dark black... density, Brix and fermentation time to Cayratia trifolia wine fermentation In fermentation, the ethanol content is related to the dry matter content in the medium Low dry matter content leads to... Huynh Xuan Phong, Nguyen Ngoc Thanh, Bui Hoang Dang Long, Ha Thanh Toan Ngo Thi Phuong Dung, 2019 Evaluation of total polyphenol and antioxidant capacity in wine fermentation of three-leaf cayratia