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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: Food Technology Code: 62.54.01.01 TRAN QUOC BINH RESEARCH ON TECHNOLOGY OF SYNTHESIZING PUERARIN-MALTOSE COMPLEX BY MALTOGENIC AMYLASE ENZYME AND APPLICATION OF FERMENTED DRINK PRODUCTION FROM KUDZU ROOTS STARCH AND PINEAPPLE JUICE Can Tho, 2021 THE PhD THESIS WAS COMPLETED AT CAN THO UNIVERSITY Advisor: Assoc Prof Dr Ly Nguyen Binh Dr Le Quang Tri The doctoral thesis was evaluated by The Board of Examiners at the basic level Meeting at: Doctoral Thesis Defense Room, 2nd Floor, Executive Building, Can Tho University University At: 14 p.m., date 17/11/2018 Reviewer 1: Reviewer 2: Reviewer 3: You can find a copy of this thesis at the library: - Learning Resource Center, Can Tho University - National Library of Vietnam LIST OF PUBLISHED PAPERS (1) Le Quang Tri and Tran Quoc Binh, 2014 Study on the production process of functional alcohol from kudzu roots Journal of Science and Education, ISSN: 2354 – 0567, 01/2014, 175 – 183 (2) Le Quang Tri and Tran Quoc Binh, 2014 Research on production of slow-digesting rice starch by Maltogenic Amylase enzyme Paper presented at the 2014 ICSAF conference in Bangkok Thailand, July 16-18, 2014 (3) Tran Quoc Binh, Le Quang Tri, Doan Nguyen Thuy Quynh and Pham Thi Minh Hoang, 2017 Modifying tapioca starch by αamylase and Maltogenic amylase Food Science and Technology: integration for ASEAN Economic Community Sustainable Development Proceedings of the 15th ASEAN Conference on Food Science and Technology November 14-17, 2017 Ho Chi Minh City, Viet Nam Volume 2: 10-17 ISBN: 978 604 67 1006-6 (4) Tran Quoc Binh, Pham Thi Minh Hoang, Ly Nguyen Binh, Le Quang Tri, 2020 Optimization of the process of denaturing the functional compound puerarin in kudzu by maltogenic amylase enzyme Journal of Agriculture and Rural Development, 2, June 2020, ISSN 1859-4581 (5) Tran Quoc Binh, Pham Thi Minh Hoang, Ly Nguyen Binh, Le Quang Tri, Nguyen Phu Thuong Nhan, 2021 Optimizing the fermentation process of drinking kudzu-pineapple juice supplemented with puerarin-mlatose complex Vietnam Trade and Industry review, 2, 01/2021, ISSN 0866-7756 (6) Tran Quoc Binh, Pham Thi Minh Hoang, Le Quang Tri, Ly Nguyen Binh, 2021 Identifying consumers’ drivers for acceptance of fermented misture of pineapple and kudzu Vietnam Trade and Industry review, 2, 01/2021, ISSN 0866-7756 CHAPTER INTRODUCTION 1.1 The rationale of the study Pineapple is the main export and domestic fruit of Tien Giang province To enhance added value for this type of product, it is necessary to further investigate and intensify the processing stage, thus setting it as the main product of the province Pineapple provides vitamins A, C, calcium, manganese to help tissues avoid the harmful effects of oxidation Pineapple juice also helps reduce stress and work more efficiently In particular, pineapple contains abundant bromelains, conferring it the effect of hydrolyzing protein into amino acids and aiding digestion and improved resistance Kudzu root contains starch content, which could hydrolyze and ferment alcohol, and valuable compounds such as puerarin However, kudzu is still a raw material that has not been extensively studied and thus find limited applications Puerarin is effective in preventing cancer and diseases related to cardiovascular such as high blood pressure, disorders heart rate, arterial spasm, atherosclerosis However, low water solubility has prevented its application in pharmaceutical products and functional foods (Liu et al., 2012) An important application of puerarin in the treatment of diabetes is that it enhances the absorption of glucose into insulin (Kato & Kawabata, 2010) To improve the solubility and applicability of cassava, this study, titled “Research on technology of synthesizing puerarinmaltose complex by Maltogenic amylase enzyme and application of fermented drink production from kudzu roots starch and pineapple juice” was conducted to produce puerarin-enriched pineapple-fermented drink as a valuable product Successful implication of the process is consistent with the province's orientation, and contributes to the formation of the value chain of agricultural products 1.2 Objective of the study To improve the value and explore more effective use of abundant and available pineapple raw materials of Tien Giang and kudzu and to incorporate puerarin compounds in kudzu into pineapple juice that has undergone metabolic processes by fungi and enzymes 1.3 Research contents Part 1: Optimization of the hydrolysis of kudzu starch under the action of the β-amylase enzyme by investigating the influence of substrate concentration, pH, temperature, time and concentration of β-amylase enzyme Part 2: Optimization of the synthesis puerarin-maltose complex under the catalysis of BSMA enzyme through investigating the effects of pH, temperature, time, and concentration of BSMA enzyme Part 3: Optimization of mixed pineapple juice puerarinmaltose fermentation through investigating the effects of pH, soluble content, yeast counts under the fermentation at ambient temperature 1.4 Scientific and practical significance - Scientific significance: Kudzu roots is a raw material of low economic value, but contains a functional compound puerarin - which is very valuable for human health Having undergone denaturation processes by combining two enzymes β-amylase and maltogenic amylase, the puerarin structure from water-insoluble to water-soluble form through the formation of a puerarin-maltose complex, from which it can be applied to create many new directions in scientific research on puerarin present in kudzu in particular and in plants in general - Practical significance: The success of this research significantly contributes to agricultural production industry by increasing the quality of fruit processing, contributing to improving the difficulties for farmers involved in production The result of the project is a new process in fruit processing, creating valuable products, contributing to the competitive strengths of fruit products in the province, region, and in the country 1.5 New contributions of the thesis Proposing a research method and successfully building a process to produce fermented drink from pineapple enriched with puerarin with an active and stable source of puerarin through enzymatic synthesis of puerarin-maltose Determining the optimization parameters of the puerarin denaturation process was done through two steps (1) hydrolyzing kudzu starch under the action of β-amylase enzyme to produce hydrolysate rich in puerarin; (2) process of synthesizing puerarin-maltose complex under the action of BSMA enzyme The technological process of pineapple juice fermentation with the addition of puerarin-maltose complex from kudzu has been established at a laboratory scale with the quality criteria meeting the Vietnamese standards (TCVN) for alcoholic beverages From there, it serves as a scientific basis for the development and diversification of nutritious products from agricultural products of the Mekong Delta (pineapple) and Vietnam (kudzu) in general 1.6 The structure of the thesis The thesis consists of chapters with 103 pages: Chapter 1: Introduction (pages: 1÷3); Chapter 2: Literature review (pages: 4÷44); Chapter 3: Research Methods (pages: 45÷63 with experiments); Chapter 4: Results and discussion (pages: 64÷101) and Chapter 5: Conclusions and suggestions (page 102÷103) The primary content has 36 tables and 59 figures The thesis consists of 184 references (171 English references and 13 Vietnamese references) CHAPTER LITERATURE REVIEW 2.1 Introduction of Kudzu roots Kudzu roots, genus Pueraria DC, belongs to the Fabaceae family which is widely grown in Asia, Australia, America, and some other countries including Vietnam In particular, kudzu roots have a lot of nutrients that are beneficial for human health Kudzu roots (Radix puerariae, RP) contain bioactive isoflavones such as daidzein, daidzin and puerarin (Chen et al., 2001; Hirakura et al., 1997; Jiang et al., 2005; Miyazawa et al., 2001) These phytochemicals have important activities such as anticancer and have been used in the treatment of alcoholism safely and effectively 2.2 Puerarin compounds Puerarin compound is a C-glucoside isoflavone, which is considered as a major component in the isoflavones of kudzu Puerarin has low solubility in water (11 mM at 25°C) (Wang & Cheng, 2005), which is the main limitation of puerarin compound in pharmaceutical and food applications The solution to improve the solubility of puerarin includes chemical and enzymatic applications (Bertrand et al., 2006; Jiang et al., 2008); microincapsulation from natural or synthetic polymers (Sansone et al., 2001; Oidtmann et al., 2012 ); emulsification (Quan et al., 2007); preparation of derivatives (Li et al., 2004; Ye et al., 2007, Jiang et al., 2008) 2.3 Pineapple Pineapple (Vietnamese is “Khóm”), scientific name is Ananas comosus In Vietnam, pineapple is grown in many provinces such as Tien Giang, Long An, Kien Giang, Bac Lieu, Ca Mau, Quang Nam, Thanh Hoa, The total production is 337,500 tons of fresh pineapple, accounting for about 2% of the total production worldwide, ranking 11th in terms of pineapple production in the world (FAO, 2004) They have a digestive effect because they contain enzymes, typically the bromelain enzyme 2.4 Bacillus stearothermophilus maltogenic amylase (BSMA) Maltogenic amylase (EC.3.2.1.133) (MA), the other names: 1,4-alpha-D-glucan alpha-maltohydrolase; Maltogenic α-amylase; Glucan 1,4-α-maltohydrolase MA is an enzyme that binds to cell membranes and is not produced outside the cell (Tang et al., 2006) MA showed the possibility in hydrolyzing both α–1,4 and α–1,6 glycosidic bond followed by converting into α–1,3 glycosidic 1,4 glycosidic bond or other glycosidic bonds (Bae et al., 2002) MA has the ability to hydrolyze acarbose (a pseudotetrasaccharide capable of strongly inhibiting a number of carbohydrase enzymes such as: α–amylase, α–glucosidase and cyclodextrin glycosyltransferases (CGTase)); transfer acarviosine - glucose (hydrolysis product) to the receiving carbohydrate molecules MA transfers mono and disaccharides to types of receptor molecules (Bae et al., 2002); MA is more active on cyclodextrin (CD) substrates than on starch or pullulan; MA decomposes βCDs (7 glucose units) 100 times faster than starch or pullulan In contrast, other amylases with a hydrolytic property cannot degrade CDs or pullulans MA hydrolyzed the α-1,4 bond more efficiently than α-1,6 bond, with low substrate concentrations and maltose is dominant the final product 2.5 Related studies BSMA preferentially converts the maltose unit that is released from hydrolysis to the acceptor molecule by forming an α-(1,6)-glycoside bond between the donor and acceptor (Cha et al., 1998) This enzyme also attacks and hydrolyzes the donor (maltotriose) to form an intermediate complex between the hydrolytic substrate (maltose or glucose) and the enzyme Water or acceptor molecule (puerarin or maltooligosaccharide) attacks the intermediate complex in the reaction mixture Three types of products are formed: products of hydrolysis, products of maltooligosyl-transglycosylation, and products of puerarin metabolism Li et al (2004) used soluble starch as a substrate for the metabolism of puerarin, the yield was 40.9% when using 0.5% starch, 1% puerarin; when using 5% maltotriose as substrate under the same reaction conditions of 55°C, 55 minutes, BSMA concentration U/mg, the yield was 66.6% At starch concentration of 5%, the efficiency improved to 70% According to Li et al., (2004), the metabolism of BSMA (EC 3.2.1.133), T maritima maltosyl-transferase (TMMT; EC 3.2.1.25), T scotoductus 4-α-glucanotransferase (TS4αGTase; EC 3.2.1.25) and Bacillus sp I-5 cyclodextrin glucanotransferase (BSCGTase; EC 3.2.1.19) enzymes was used to test acceptability specificity for puerarin The reaction was performed with 1% puerarin and 5% maltotriose Results showed that TMMT and TS4αGTase form very few metabolites of puerarin even though these enzymes hydrolyze and metabolize maltotriose TMMT and TS4αGTase have no acceptor specificity for puerarin because they not form an Oglucoside bond between D-glucose and 7-OH-daidzein BSMA and BSCGTase form a large number of metabolites of puerarin Park Kwan Hwa et al 2012 studied the biotransformation of isoflavones by the conversion activity of MAase enzyme MAases catalyze substrate hydrolysis; perform conversion reactions to different acceptor molecules by forming α-(1,6)glycoside bonds BSMA is the most efficient enzyme among metabolic enzymes such as maltosyltransferase (MTase), 4-αglucanotransferase (4-α-GTase) and other metabolic enzymes MTase and 4-α-Gtase are not specific for puerarin because no O-glycoside bond is formed between D-glucose and 7-OH- daidzein The two main products are glucosyl-α-(1,6)-puerarin and maltosyl-α-(1,6)-puerarin Zhong et al (2012) studied "Physicochemical, microstructural, and antibacterial properties of β‐chitosan and kudzu starch composite films" The concentration of kudzu starch varied from 0%, 20%, 60% and 100% in 1% chitosan solution It was found that 60% starch kudzu will reduce water vapor permeability and solubility compared with pure βchitosan film by 15% and 20%, respectively While the mechanical strength is about 50% and the flexibility is 25% of the β-chitosan film According to Wu et al (2013), following optimal conditions for extraction of puerarin from Radix Pueraria by microwave technique were found Ethanols were used for extraction: solvent concentration was 52.36%, microwave irradiation time was 60 seconds, microwave energy 184.8 W, solvent: material ratio 25:1 (mL/ g) corresponding to the puerarin content 11.97 mg/g CHAPTER MATERIALS AND METHODS 3.1 Research facilities - Research period: 6/2015 - 6/2019 - Research location: Laboratories Faculty of Agriculture and Food Technology, Tien Giang University; Center for Engineering and Biotechnology, Tien Giang Department of Science and Technology; Center for consultancy and quality assurance of agroforestry-fishery products, Tien Giang Department of Agriculture and Rural Development ; Tien Giang Fruit and Vegetable Company, Long Thuan Private Enterprise (Tien Giang) 3.2 Research methods 3.2.1 Materials Kudzu (Pueraria thomsonii Benth) is grown in Hoanh Son area, Kinh Mon district, Hai Duong province, and was harvested one year after planting after each sampling period Kudzu is sliced and dried to constant weight at 70oC, stored in - a refrigerator (5oC), used up to year, periodically each sampling period, analyzing and measuring for homogenization sample quality Pineapple (Ananas Comosus) belongs to the Queen pineapple variety, grown in Thanh My area, Tan Phuoc district, Tien Giang province The pineapple used in the study is a ripe pineapple, with an average weight of 0.5 kg/fruit Industrial maltodextrin powder is supplied by Hoa Nam chemical company, 239/4 Ly Thuong Kiet, Ward 15, District 11, Ho Chi Minh City Enzyme β-amylase is provided by Novozymes company, liquid form, the optimum temperature is 50oC and pH BSMA enzyme is provided by DMS company (Singapore), solid form, the temperature is 60oC and optimal pH 5.5 Saccharomyces oviformis yeast was obtained from Biotechnology Laboratory, Saigon University of Technology, 180 Cao Lo, Ward 4, District 8, Ho Chi Minh City 3.2.2 Analytical methods Total dissolved solids content: Using an Atago refractometer 0-32 Humidity (%): determined according to AOAC 934.06 pH: determined by pH meter Total sugar content (%): determined according to TCVN 4594-88 Reducing sugar content (mg/g): According to Miller's method, 1959 Amylose content: According to the method of Juliano, 1971 Amylopectin content: According to the method of Juliano, 1971 Starch content (%): TCVN 4594-88 Ethanol content: TCVN 5562-2009 Yeast cell count (CFU/mL): Count on the red blood cell counter Puerarin content: HPLC analysis 3.3 Experimental Experimental process: The kudzu juice after hydrolysis by β-amylase enzyme and BSMA was added pineapple juice with variable ratios at five levels and filtered, adjusting the appropriate values for the propagation process: pH ,5-5,5; Peptone: 1%; Bx: 15 After balancing the nutrients as above, sterilize the medium at 121oC for 30 minutes and cool to room temperature Using a culture rod, remove the yeast S oviformis from the inclined agar tube into Erlen 500 mL containing the above medium Carry out propagation at a temperature of 28-32oC in a thermostatic shaker During the propagation process, take a sample every 24 hours to check the number of yeast cells in mL of culture solution Evaluation criteria: colony-forming unit/mL (CFU/mL) of culture solution over time Determined by direct counting on the red cell cout 3.5.2 Experiment 7: Investigate the effects of mixing ratio of pineapple juice - kudzu juice on the sensory value of products Purpose: Determine the appropriate mixing ratio to create products with appropriate alcohol content as well as high sensory value Experimental design: Factor L: ratio of pineapple juice to kudzu, changing at five levels: L1: 2:8 ; L2: 4:6 ; L3: 5:5 ; L4: 6:4 ; L5: 8:2 The experiment was performed three times Total number of experimental units: x = 15 Experimental process: The kudzu juice after hydrolysis by β-amylase and BSMA enzymes was heated at 100oC for 15 minutes to stop the hydrolysis reactions by the above enzymes and kill harmful microorganisms that that may be harmful affect the fermentation process After heating, the kudzu juice was cooled to room temperature and mixed with pineapple juice according to the ratios investigated so that the final volume was 500 mL Addition of yeast cultured with appropriate time in the pineapple-kudzu juice medium (experiment 5) at the rate of 12 10% Adjust Brix, pH with corresponding values of 20, 4.5, and carry out fermentation at normal temperature After one day, the results are recorded once, until the desired alcohol content (56o) is reached, then stop the fermentation process Evaluation criteria: The alcohol content is produced overtime Brix, pH changes during fermentation The change of total yeast cells in fermentation fluid Sensory evaluation of products 3.5.3 Experiment 8: Optimization of pineapple juice-kudzu fermentation (investigation of the influence of pH, Bx, yeast ratio) under normal temperature fermentation conditions Purpose: To find the optimal parameters for yeast growth to obtain the most appropriate alcohol content as well as the best sensory quality of the product Experimental design: Table 0.3: Factors and levels of survey Symbol Factors Unit Levels M pH 3,5 4,5 5,5 N Bx 20 22 24 O Yeast percentage % 10 15 Experimental process: The experiment was randomly arranged with three survey factors (pH, Brix, yeast percentage), the remaining factors were fixed The experiment was carried out in the same way as Experiment The experiment was performed three times Total number of experimental units: 30 Evaluation criteria: The alcohol content is produced overtime Brix, pH change during fermentation The change in the total number of yeast cells in the fermentation solution Sensory evaluation of products Evaluation of the quality of fermented drink products from kudzu and pineapple Determination of puerarin content in fermented beverage products 13 Analysis of parameters of fermented drink products (according to TCVN) Sensory evaluation of fermented beverage product quality CHAPTER RESULTS AND DISCUSSION 4.1 Chemical composition of raw kudzu roots The results are shown in Table 0.4 Fresh kudzu roots have high humidity content (52.15%) Amylose content in kudzu powder was 30.19% The HPLC analysis results showed that the puerarin content accounted for 0.96% (9.6 mg/g) Table 0.4: Analytical results of the basic physical-chemical composition of kudzu roots powder No Parameters Method Results (%) Humidity AOAC 934.06 6.5 Total sugar content TCVN 4594-88 10.77 Starch content TCVN 4594-88 56.46 Amylose content TCVN 5716 30.19 Amylopectin TCVN 5716 69.81 content 4.2 Optimization of the hydrolysis of kudzu starch under the action of β-amylase 4.2.1 Effect of substrate concentration on the hydrolysis of kudzu starch under the action of enzyme β-amylase The highest concentration of reducing sugars (141.68 mg/g) corresponds to the substrate concentration of 10% and the lowest (94.79 mg/g) corresponds to the substrate concentration of 16% Kudzu starch samples at concentrations 8, 10, 12, 14% produced significantly different reducing sugars compared with samples at 16% concentration When the substrate concentration increased from to 10%, the reducing sugar content increased and decreased with increasing substrate concentration to 16% 14 Table 0.5: Effect of substrate concentration on reducing sugar content Sample Substrate concentration (%) Reducing sugar (mg/g) 119.56±6.4c 10 141.68±4.44d 12 116.99±7.86bc 14 108.04±3.98b 16 94.79±4.89a Ratio F 27.4 P-value 0.0000 4.2.2 Effect of pH on the hydrolysis of kudzu starch under the action of β-amylase Table 0.6: Effect of pH on the amount of reducing sugars formed STT pH Reducing sugar content (mg/g) 131.33±3.01b 5.5 149.24±5.88c 160.01±1.86c 6.5 137.74±5.81b 104.76±10.54a Analytical results showed that β-amylase enzyme activity was best shown in the pH range from to and reached the highest at pH 6, which is suitable for enzyme activity The hydrolyzed sample at pH 5.5 had a reducing sugar yield of 149.24 mg/g which was not significantly different from the sample at pH with a reducing sugar yield of 160.01 mg/g 4.2.3 Effect of temperature, time, and concentration of βamylase enzyme on the hydrolysis of kudzu starch The efficiency of starch hydrolysis by enzymes depends on many conditions, especially temperature, concentration, and hydrolysis time with the objective function of reducing sugar content The correlation model has high statistical significance, the value for the coefficient R2 = 0.988 The F and p values of the model are 288.72 and 0.0000, respectively, which means the model has been accepted 15 (A ) (B) (C) Figure 0.1: Response surface graph showing the influence of some factors on reducing sugar content formed when hydrolyzing kudzu starch by β-amylase (mg/g) A: Effect of time and temperature (at concentration 40U/g powder) B: Effect of time and concentration (at 55oC) C: Effect of temperature and concentration (at h) The equation to predict the amount of reducing sugar obtained is as follows (1): Y = - 520,813 + 21,5708X1 + 16,9202X2 + 2,8513X3 – 0,205667X12 + 0,00129995X1X2 + 0,0187132X1X3 – 1,74789X22 – 0,052651X2X3 – 0,0439538X32 (1) 16 The coefficients in the model including first order coefficient, quadratic coefficient, interaction coefficient are all significant (p0.05), so this coefficient can be omitted to simplify the equation, equation (1) becomes (2): Y = - 521,099 + 21,576X1 + 2,8513X3 + 16,9917X2 – 0,205667X12 – 0,0439538X32 – 1,74789X22 + 0,0187132X1X3 – 0,052651X2X3 (2) Equation (3) describes the compatibility between experimental and model reducing sugar content The coefficient R2 = 0.9989 confirms that the predictive data has high compatibility y = 0,9989 x + 0,1478 (3) The obtained data show that the p = 0.4278>0.05 of Lack of fit is not statistically significant, so the fit of the model is very high Optimal hydrolysis conditions: temperature 54.34 oC, time 4.24 hours and enzyme concentration 41.46 U/g starch Experimental value: content of reducing sugar 161.07±2.96 mg/g, which is not significantly different from predicted results from model 160.225 mg/g at 95% confidence 4.3 Optimization of the synthesis of puerarin-maltose complex under the effect of enzyme BSMA 4.3.1 Effect of pH on denaturation of puerarin compounds under the action of enzyme BSMA Table 0.7: Changes in puerarin content under the influence of pH STT pH Puerarin content (mg/g) 4.5 6.84±0.22b 7.75±0.16c 5.5 7.10±0.22b 6.31±0.29a 6.5 6.31±0.27a The analysis results showed that when the pH changed from 4.5 to 6.5, the puerarin content changed At pH 4.5 values; and 5.5, the synthesized puerarin content was significantly different from that at pH and 6.5 Of which, the sample 17 corresponding to pH exhibited the highest puerarin content (7.75 mg/g) that is significantly different from the puerarin content of the remaining samples The activity of BSMA was in the weak acidic pH range (from 4.5 to 5.5) and the highest at pH The pH value was chosen as an appropriate parameter for the process of denaturing puerarin compounds under the action of enzymes BSMA 4.3.2 Effect of temperature, time, and concentration of enzyme BSMA on the denaturation of puerarin The value for the coefficient R2 is 0.96 The F and p values of the model are 90.54 and 0.00, respectively; that means the model has been accepted Correlation model between puerarin content and factors was built as equation (4) Z = 1,59074 + 0,129549X4 + 0,32109X5 + 0,197767X6 – 0,00116001X42 – 0,000426667X4X5 + 0,000357167X4X6 – 0,0483066X52 + 0,00614X5X6 – 0,00786672X62 (4) The coefficients in the model including the first order coefficient, the quadratic coefficient, the interaction coefficient are all significant (p
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