Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.Sử dụng chế phẩm protease trong xử lý cám gạo dùng cho chế biến thức uống dinh dưỡng.MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY THE ABSTRACT OF THE DOCTORAL THESIS Major Food Technology Code 62 54 01 01 LE HOANG PHUONG PROTEASE PREPARATIONS IN RICE BRAN TREATMENT AND THE AP.
MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY THE ABSTRACT OF THE DOCTORAL THESIS Major: Food Technology Code: 62.54.01.01 LE HOANG PHUONG PROTEASE PREPARATIONS IN RICE BRAN TREATMENT AND THE APPLICATION IN PRODUCING THE NUTRITIONAL DRINK Can Tho, 2023 THE PhD THESIS WAS COMPLETED AT CAN THO UNIVERSITY Supervisor: Assoc Prof Dr Ly Nguyen Binh The doctoral thesis was evaluated by The Board of Examiners at the basic level Meeting at: Meeting room 3, 2rd floor, Campus II – Control Hall, Can Tho University At 14:00 ICT, July 3rd 2022 Reviewer 1: Assoc Prof Ho Thanh Binh Reviewer 2: Assoc Prof Phan Thi Thanh Que The thesis can be found in the library: Learning Resource Center, Can Tho University The National Library LIST OF PUBLISHED PAPERS OF THE AUTHOR RELATING TO THE THESIS 1.Hoang Phuong Le; Diep Thanh Nghi Hong; Thi Thao Loan Nguyen; Thi My Hanh Le; Shige Koseki; Thanh Binh Ho; Binh LyNguyen 2022 Thermal stability of fructooligosaccharides extracted from defatted rice bran: a kinetic study using liquid chromatographytandem mass spectrometry Foods, 11, Issue 14, 2054 Le Hoang Phuong, Nguyen Van Thanh, Do Thanh Xuan, Ly Nguyen Binh, 2020 Some Factors Affect The Rice Bran Extraction Industry and Trade Magazine, ISSN: 0866-7756, no 28,11/2020 Le Hoang Phuong, Ngo Thi Cam Tu, Ly Nguyen Binh, 2021 The Influence of Protease Preparations on The Hydrolyzed Rice Bran Industry and Trade Magazine, ISSN: 0866-7756, no 10, 5/2021, page 6368 Le Hoang Phuong, Nguyen Van Thanh, Nguyen Van Thuan, Ngo Thi Cam Tu, Vo Tan Thanh, Phu Thi Thanh Khiet, Do Thanh Xuan, Ly Nguyen Binh, 2021 The Influence of Extracting Conditions on Lipase Activity in Rice Bran Science and Technology Journal of Agriculture and Rural Development, ISSN: 1859-4581, no 5, 2021, page 53-58 Le Hoang Phuong, Nguyen Van Thanh, Vo Tan Thanh, Ly Nguyen Binh, 2022 Optimization of Lipase Enzyme Extraction from Rice Bran Science and Technology Journal of Agriculture and Rural Development, ISSN: 1859-4581, no 4, 2022, page 67-72 Le Hoang Phuong, Vo Tan Thanh, Ngo Thi Cam Tu, Ly Nguyen Binh, 2022 Effects of Protamex and Flavouzyme combination on the hydrolysis of ricebran, applications in yogurt processing Proceedings of Vietnam national conference on biotechnology 2022 ISBN: 978-804357-052-6, November 2022, page 571-577 Chapter INTRODUCTION 1.1 The necessity of the thesis In five years, from 2016-2020, Vietnam produces nearly 43 million tons of rice/year, and the Mekong River Delta is the country's granary providing 23.8 million tons, accounting for 56% of the rice production (FAOSTAT) The Mekong River Delta is an area that exports approximately 90% of the country's total rice exports About 2.4 million tons of bran are produced annually from rice milling in the Mekong Delta The by-products of the rice processing industry are abundant, with great potential for exploitation In particular, rice bran is a source of raw materials containing many nutrients for many fields such as food, pharmaceuticals and cosmetics (Khalid et al., 2015) Rice bran is about 10% of the total weight of raw rice, including pericarp, seed coat, the cuticle of the kernel, aleuron layer and germ However, rice bran is often used for bran oil production and animal feed Improving the quality of rice bran for human food has not been studied and applied Moreover, during the processing and storage of bran, the fats from the bran are easily hydrolyzed and oxidized Lipase-inactivating protease and some other enzymes have both scientific and practical significance Protease hydrolyze lipase at a relatively low temperature (~55oC) while preserving the sensory values (color, taste) and nutritional values (vitamins, protein digestibility) Moreover, in the process of hydrolysis with protease, some peptides and amino acids are formed These products both increase absorption capacity and biological activity to support health Rice bran has a high nutritional content, including 14÷16% protein, 12÷23% fat, 8÷10% fiber, and is rich in vitamins, especially vitamin B, minerals and other sterols with appropriate content and high digestible fiber can be considered very good for human consumption (Saunders & R.M., 1985) Moreover, the publication of Han et al., (2015) showed that rice bran protein has more superior features than soy protein and animal protein (casein) in terms of digestibility and other biological values According to published scientific research, rice bran is a source of essential fatty acids Therefore, rice bran is not only a feed source for livestock but can be used to create new products from this nutrient-rich source However, this superiority makes it challenging to preserve because of its high nutritional content, especially unsaturated fat The most considerable damage and concern are about the rancidity of the oil due to the action of lipase and lipoxygenase enzymes that oxidize unsaturated fatty acids such as linoleic and linolenic acids to form hydroperoxides and release carbonyl group, which has a rancid odor and changes the natural taste of rice bran and can form carcinogens In order to eliminate harmful factors to food products, many methods have been applied and using enzymes is a superior method due to the outstanding properties of enzyme-catalyzed reactions Enzymes are biological catalysts that are not only significant for the growth and reproduction of plants but also play a crucial role in food processing, medicine, analytical engineering, genetic technology and environmental conservation (Nguyen Duc Luong, 2004) With outstanding advantages, enzymes are increasingly being studied and widely applied Therefore, the author carried out the research "Using protease preparations in rice bran treatment for nutritional drink processing" to combine abundant raw materials processed by advanced biological methods to create a variety of safe food products for consumers' health 1.2 Research purpose Using commercial enzymes with optimal conditions has been established for lipase enzyme processing and its application in producing rice bran nutritional drinks 1.3 Research objectives The research was divided into four main objectives: - The first objective: Study the influence of some factors on inactive enzyme lipase in rice bran treatment by using commercial Protamex and Flavorzyme preparations - The second objective: Study the effect of sterilization modes and storage time on the volatile compounds of rice bran beverage - The third objective: Study on thermolysis kinetics of fructooligosaccharides (FOS) [1-kestose (GF2), nystose (GF3), and 1fructosyl-nystose (GF4)] were extracted from rice bran - The fourth objective: Application of processed rice bran in producing the nutritional drink 1.4 The benefits of the thesis Currently, there are few published studies on agricultural byproducts in our country Research shows that the combined use of protease preparations is effective in rice bran treatment, and applying this raw material to process nutritional products is a potential new direction In addition, the study initially investigated the presence and processing conditions affecting the heat stability of FOS, an essential prebiotic for human health Moreover, the topic is also modern because it uses fingerprinting and headspace techniques to trace and quantify flavour compounds during the heat processing of drinking water products from rice bran Therefore, the topic has high scientific, practical and topical value 1.5 The new findings of the thesis The research results contribute to the scientific basis for the research problem about rice bran that still has left behind The combined use of protease preparations shortens the time and increases the lipase enzyme's efficiency That effect helps recover the hydrolytic fluid, which contains outstanding nutrients and increases the value of the applied product, including yoghurt and nutritional powder These are two feasible new products that were transferred to reality production At the same time, studying the fructooligosaccharide (FOS) composition in rice bran is an outstanding content, but no research has been published yet In addition, the "metabolomics" method has been used to evaluate the quality, processing and safety of the raw materials and final products used in the study From here, it can be seen that this study is novel in terms of both objects and methods Chapter OVERVIEW 2.1 Rice bran overview Rice bran is a by-product of the rice milling process Rice bran is a cheap fibre source and a good source of fat to use as cooking oil (rice bran oil) Because rice bran is only considered animal feed, it is not used much in human nutrition and health promotion The high nutritional value of rice bran has made it more attractive in public health According to research by Professor Elizabeth (2011) from the University of Colorado, a serving of rice bran (equivalent to 28 g according to USDA standards) provides more than half of the need for vitamins such as thiamine, niacin and vitamin B6 during the day The chemical composition and nutritional value of rice bran vary greatly, depending on the rice milling technique (Trần Thị Thu Trà, 2010) Rice bran is usually powdery, soft and fine Rice bran contains many valuable nutrients With 14÷16% protein, 12÷23% fat, and 8÷10% fiber, rice bran is also a rich source of vitamin B and minerals such as iron, potassium, magnesium, manganese, and chlorine (Saunder, 1985) The rice bran compositions have many vitamins, good fats, and a lot of easily digestible fiber, which can be considered very good for human consumption 2.2 Protease preparations overview Enzyme production technology has brought great benefits to many countries; production and sales of enzyme products on the world market increase by 20-30% per year (Murado et al., 2009) Among enzymes used in food processing, hydrolytic enzymes account for the largest proportion (Rao et al., 1998), about 97% of global industrial enzyme sales (Haard & Simpson, 2000) Protease is a hydrolytic enzyme with great commercial value, accounting for about 60% of the total commercial value of industrial enzymes supplied on the world market with broad applications in many fields such as industry, agriculture, cosmetics, especially in modern medicine (Tunga et al., 2003; Rao, 2010) Hydrotic enzymes as a catalyst have many advantages over chemicals, including high specificity, catalytic efficiency at moderate temperatures and environmental friendliness 2.3 Fructooligosaccharides overview Fructooligosaccharides (FOS) are oligosaccharides that are naturally present in plants such as onions, chicory, garlic, asparagus, bananas, artichokes, and among many others FOS has critical physiological effects as a prebiotic, increasing mineral absorption and reducing cholesterol levels, triacylglycerols and phospholipids FOS consists of linear chains of fructose units linked by β (2-1) bonds The number of fructose units ranges from to 60 and usually terminates in a glucose unit The general formula of FOS sugar is GFn, where n = 2, 3, (G is the glucose base, F is the fructose base), including three main representatives, the sugars GF2 (1-kestose), GF3 (nystose), GF4 (fructofuranosylnystose) FOS is not hydrolyzed by glycosidases in the small intestine and reaches the large intestine with an unchanged structure; intestinal bacteria metabolize them to form short carboxylic acids, L-lactate, CO2, hydrogen and other metabolites They not provide energy, not cause cancer and are considered soluble fiber, which is good for health 2.4 Volatile organic compounds and determining method Volatile organic compounds (VOCs) are organic compounds containing one or more carbon atoms that have a high vapor pressure that readily evaporates in the air They are formed by a complex group of chemicals such as esters, alcohols, aldehydes, ketones, lactones, ketones, and terpenoids Volatile compounds can be produced from fats, amino acids, terpenoids and carotenoids (El Hadi & Zhang, 2013; Goff & Klee, 2006) In addition, some sulfur compounds such as S-methyl thiobutanoate, 3-(methylthio) propanal, 2-(methylthio) ethyl acetate, 3(methylthio) ethyl propanoate, and 3-(methylthio) propyl acetate also contribute to the fruit flavor (Song & Forney, 2007) Depending on the type of food and the intended use, we will have appropriate methods of determining volatile compounds One or a combination of techniques can achieve the research goal Gas chromatography (GC) and mass spectrometry (GC/MS) are the most commonly used instruments 2.5 Thermal decomposition kinetics of food properties Heat processing aims to inactivate microorganisms (pathogenic and spoilage microorganisms) and inactivate spoilage enzymes However, heat treatment causes undesirable deterioration of nutrients, color, texture and other properties, along with the inactivation of microorganisms and enzymes 2.6 Metabolomics method in food research The term metabolism has emerged as an essential tool in many fields, such as healing and nutrition In food science, metabolomics has recently been seen as a tool for assessing raw materials and end products' quality, processing, and safety Metabolomics, the study of possible small metabolites in a system, has been applied broadly in many research fields Studies and perspectives on human diseases, pharmaceutical discoveries, botanical analysis, and human nutrition… have shown metabolism's broad impact and rapid development (Juan, 2009) Targeted analyzes are important for evaluating the activity of a specific group of compounds in a sample under defined conditions Targeted metabolites typically require a higher degree of purity and selective extraction In contrast, non-targeted (also called holistic) metabolites focus on detecting as many groups of metabolites as possible to obtain patterns or fingerprints without necessarily identifying or quantifying them Therefore, based on the purpose of analysis and use, most metabolic studies are classified as separative, informative or predictive studies Chapter RESEARCH METHODOLOGY 3.1 Research methodology 3.1.1 Place and time of the research The experimenting, data collecting and processing were conducted at the Department of Food Technology, Faculty of Agriculture, Can Tho University; Experimental Practical Center, Kien Giang University The duration of the project was from November 2016 to June 2021 3.1.2 Equipment and tools GCMS gas chromatography-mass spectrometry (7890B GC System, USA), UV 1800 visible spectrophotometer (Germany), colorimetric spectrophotometer (ZE-6000/Nippondenshoku, Japan), Metrohm potentiometer titrator, heated ultrasonic washing bath (S100H/Elma, Germany), centrifuge (EBA 21/Hettich, Germany), liquid chromatography-tandem mass spectrometry (LC-MS/MS) with software Masslynx calculations include MS mass spectrometer with XEVO TQSmicro ESI ionization source (Waters Corporation, USA) and UPLC super performance liquid chromatography system (Waters Corporation, USA) Luna amino - NH2 type chromatographic column (150 mm × mm × µm) of Phenomenex - USA 3.1.3 Materials and chemicals Raw rice bran was purchased at the Co-operative in Can Cu Hamlet, Vinh Phong Commune, Vinh Thuan District, Kien Giang Province The rice bran used in the study was a by-product of the rice variety ST24, grown in shrimp fields Rice bran used in the study of fructooligosaccharide composition is defatted rice bran provided by Wilmar Agro Vietnam Co., Ltd., located at Hung Phu Industrial Park, Tan Phu Ward, Cai Rang District, Can Tho City, Vietnam The chemicals used in the research were pure and manufactured by Merck and Sigma companies a) Protamex: Protamex was purchased from Novozymes (Denmark), provided by Trung Son Technology Co., Ltd., No 403 Nguyen Thai Binh, Ward 12, Tan Binh District, Ho Chi Minh City Protamex belongs to the endopeptidases group derived from Bacillus and approved for use by FAO/WHO Protamex has a labelled activity of 1.5 AU/g, suitable for pH 5.5÷7.5 and temperature 45÷65oC Protamex can be inactivated in 30 minutes at 55oC (122oF) at pH and in 10 minutes at 85oC (185oF) when pH is The best storage temperature of Protamex is 0÷5oC b) Flavourzyme: Flavorzyme 500 MG was purchased at Novozymes company (Denmark), provided by Trung Son Technology Co., Ltd, No 403 Nguyen Thai Binh, Ward 12, Tan Binh District, Ho Chi Minh City, manufactured from Aspergillus oryzae by submerged fermentation Flavorzyme belongs to the group of exopeptidases Flavorzyme works well in neutral or slightly acidic media when hydrolyzing proteins Flavorzyme has a labelled activity of 500 LAPU (Leucine Aminopeptidase Units)/g, suitable pH in the range of 5÷7 and suitable temperature in the range of 50÷55oC The best storage temperature of Flavorzyme is 0÷4oC c) Cellulase: Cellulase preparations were provided by Novozymes (Bagsvaerd, Denmark), Trung Son Technology Co., Ltd., 403 Nguyen Thai Binh, Ward 12, Tan Binh District, Ho Chi Minh City, Vietnam The cellulase is brown with an activity of 700 EGU/g, and the best storage temperature is 0÷4oC d) Commercial Yeast (Probiotics Yogurt Starter) (Prance): Product of Yo'gourmet - France Composition of active probiotic strains for the fermentation: L casei, B longum, L bulgaricus, S thermophilus, L acidophilus e) Standard FOS kit: Standard fructooligosaccharide kit consisting of 1-kestose (GF2), nystose (GF3) and 1-fructosyl-nystose (GF4) with 99% purity was purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan): 1-Kestose, 99.0+% (HPLC) 250 mg × vial CAS No 470-69-9, C18H32O16 = 504,44 Nystose, 99.0+% (HPLC) 250 mg × vial CAS No 13133-07-8, C24H42O21 = 666,58 1F-Fructofranosylnystose, 80.0+ % (HPLC) 250 mg × vial CAS No 59432-60-9, C30H52O26 = 828,72 3.2 Research methodology 3.2.1 Analytical methods The basic indicators were analyzed by standard methods, which were summarized in Table 3.1 Table 3.1: Some methods of analyzing basic physicochemical properties Indicators Analytical methods Moisture content Dried the sample to constant weight at 105oC according to TCVN 3700-90 Viscosity Determined by viscometer Brookfield DV1MLTJ0 Protein content Used Lowry method (1951) Lipase activity, Used Whyte's (1964) spectrophotometric through quantification of method at 635 nm glycerol Lipase activity, Used continuous titration with 0.05 N NaOH through quantification of according to the Cherry Crandall method fatty acids 1-kestose (GF2), Used Parallel Mass Spectrometry (UPLC-ESInystose (GF3), and 1MS/MS) (Prošek et al., 2003) fructosyl-nystose (GF4) determinations 3.2.2 Data collection and processing methods The experiments were carried out using a completely randomized design with three replications The data were collected and analyzed using SAS 9.1, Statgraphics Centurion 15.1, Excel and XLSTAT software 3.3 Experimental setup methodology The general research process is shown through the diagram in Figure 3.2 Figure 3.2: Diagram of the research process The general research objectives of the study are presented in Figure 3.3 Figure 3.3: Diagram of the general experimental setup 3.3.1 Study the influence of some factors on inactive enzyme lipase in rice bran treatment by using commercial Protamex and Flavorzyme preparations The raw rice bran's initial chemical compositions (protein, fat, carbohydrates and ash) were determined Then, the rice bran is used as a raw material in the hydrolysis process to inactivate the lipase enzyme with Protamex and Flavorzyme preparations Purpose: Study the lipase extraction conditions in rice bran and factors that affect the hydrolysis capacity of Protamex and Flavorzyme Research parameters: Type of solvent used: distilled water, brine, ethanol Solvent concentration (M): 0.5; 1,0; 1.5; 2,0 The ratio of solvent and raw materials: 5:1; 7:1; 9:1 Temperature of the extraction (oC): 29; 32; 35; 38; 41 Hydrolysis temperature (oC): 40; 45; 50; 55; 60; 65 Extraction time (minutes): 120; 180; 240; 300; 360 Hydrolysis time (minutes): 60; 120; 180; 240; 300; 360 Enzyme concentration (%): 0.1; 0.2; 0.3; 0.4; 0.5 Indicators of the evaluation: Content of initial chemical compositions (protein, fat, carbohydrates and ash) (% of dry matter), lipase activity (UI/mL), glycerol content (%), total protein content (mg/mL), amino acid content (%) 3.3.2 Study the effect of sterilization modes and storage time on the volatile compounds of rice bran beverage Purpose: Determine the appropriate thermal setup program for sterilization and the sampling and storage conditions that affect the volatile compounds in the rice bran beverage that have inactivated the lipase enzyme Research parameters: Determine the appropriate thermal setup program: X1; X2; X3; X4 Equilibrium sampling temperature (oC): 40; 50; 60 Equilibrium sampling time (minutes): 20; 30; 40 Ratio of saturated NaCl solution (%): 10; 20; 30 Indicators of the evaluation: the number of substances appearing in the chromatogram 10 3.3.3 Study on thermolysis kinetics of fructooligosaccharides (FOS) [1-kestose (GF2), nystose (GF3), and 1-fructosyl-nystose (GF4)] were extracted from rice bran Purpose: Study the transformation rules of FOS (including 1kestose (GF2), nystose (GF3) and 1-fructosyl-nystose (GF4)) in rice bran under the effects of temperature and pH Research parameters: Treatment temperature (oC): 90; 100; 110 pH of FOS solution: 5; 6; Treatment time (minutes): 5; 10; 20; 30; 40; 50; 60 Indicators of the evaluation: residual FOS content (including 1kestose (GF2), nystose (GF3) and 1-fructosyl-nystose (GF4)) of the samples corresponding to experimental setup data Thermal decomposition kinetics analysis of FOS based on suitable kinetic equations 3.3.4 Application of processed rice bran in producing the nutritional drink Purpose: Apply lipase-inactivated rice bran fluid in processing nutritional drink Research parameters: Study on the processing of making yoghurt product with lipase enzyme-treated rice bran liquid (%): 15; 25; 35 Indicators of the evaluation: pH value, viscosity, water separation and sensory test Investigate processing conditions and monitor the storage time of mixed rice bran powder: nutritious cocoa-flavoured rice bran powder and green tea-flavoured rice bran powder Yeasts, moulds and total aerobic microorganisms 11 Chapter RESULTS AND DISCUSSION 4.1 Study the influence of some factors on inactive enzyme lipase in rice bran treatment by using commercial Protamex and Flavorzyme preparations 4.1.1 Basic chemical compositions The chemical composition and nutritional value of rice bran depend significantly on the rice milling technique at the level of husk separation before rice milling In this study, the rice bran material sample was sent for analysis at the Center for Agro-Forestry-Fisheries Quality Region 6; the results are shown in Table 4.1 Table 4.1: Results of elemental chemical compositions in rice bran No Analytical indicators Results Protein Lipid Carbohydrate Ash 12,22 16,03 50,74 9,35 Unit % dry matter % dry matter % dry matter % dry matter 4.1.2 Effect of extracting conditions on the ability to obtain lipase enzyme from rice bran Extraction of lipase enzyme in rice bran material will facilitate the inactivation process more efficiently Experiments to investigate the influence of solvent type, solvent concentration, temperature and time for the extraction showed that: using a salt solution at a concentration of 1M, extracting for 240 minutes at 38oC for the best results The optimal values of this process, with three factors: solvent concentration, solvent/material ratio and extraction time, are shown in Table 4.2 Table 4.2: Optimal values of NaCl solution concentration, extraction solution/material ratio and extraction time during lipase extraction from rice bran Factors Optimal value NaCl solution concentration 1M Solution/material ratio 7:1 Extraction time hours 12 4.1.3 Effects of some factors on the ability to inactivate lipase enzyme in rice bran by Protamex and Flavorzyme preparations The parameters of hydrolysis temperature, enzyme using rate and processing time of Protamex and Flavouzyme preparations are determined at the same temperature, 50oC Protamex (endopeptidase) concentration for rice bran lipase substrate is 0.4%, and catalytic time is hours With Flavourzyme (exopeptidase) preparation, the optimal enzyme concentration is 0.4%, and the optimal processing time is hours However, the two preparations have different substrate specificities, giving excellent results when combined in enzyme treatment Specifically, hours is the optimal value in lipase inactivation treatment, corresponding to the amount of glycerol producing (by remaining lipase activity) is 2.62%, and the degree of lipase inactivation is 76.6% so that the processing time is shortened by ~50% At the same time, the combination results in a nutrient-rich hydrolytic fluid containing more essential amino acids (Figures 4.1, 4.2 and 4.3) Hình 4.1 Amino acids content in rice bran fluid after lipase inactivated process by protease preparation The correlation between the method of using two preparations, Flavorzyme and Protamex, and the distribution positions of amino acids on the graphs of Figure 4.2 and Figure 4.3 through principal component analysis (PCA) showed that 11 amino acids produced with the highest concentration distribute nearly with the combined use of Protamex and Flavorzyme (first quadrant) In contrast, the remaining five amino acids 13 produced with the highest concentration circulate near the individual use of the preparation Flavorzyme Therefore, the combination of Protamex and Flavorzyme not only inactivates lipase enzyme but also gives more nutritious hydrolytic fluid than using the preparation solely Figure 4.2 Bi-plot shows the correlation between amino acid compositions of rice bran fluid hydrolyzed by Flavorzyme and Protamex preparations The connection between the method of enzyme usage and the group of amino acids formed with the highest concentration Figure 4.3 The bi-plot shows the correlation between essential and non-essential amino acids of rice bran fluid hydrolyzed by Flavorzyme and Protamex preparations 14 4.2 Effect of sterilization modes and storage time on the volatile compounds of rice bran fluid For the analysis of the volatile compounds present in the sample by gas chromatography-mass spectrometry (GC-MS), the thermal program has a decisive influence on the analysis The temperature of the program can change the analysis time and change the ability to detect components (presence of peaks in the chromatogram) The study identified an appropriate heat program with a total running time of 33 minutes This study's rice bran fluid has a pH of 6.0 The sterilization process requires the sterilization value F (also known as F-value) at the reference temperature of 121.1oC in minutes The z value of Clostridium botulinum is 10oC In this experiment, the volatile compounds present in the pasteurized rice bran fluid sample and the control (non-sterilized) sample are determined by gas chromatography-mass spectrometry (GCMS) The results of this study show that the heat treatment significantly impacts the volatile compounds present in the sample The factors of the ratio of saturated NaCl solution (compared to the sample solution, v/v), temperature and equilibration time in static headspace sampling significantly affect the amount of volatile compounds recorded when performing the gas chromatographic-mass spectrometry analysis The research has determined: the optimal sample incubation temperature is 50oC, the sample incubation time is 30 minutes, and the ratio of saturated NaCl solution used (compared to the sample solution, v/v) is 20% During the storage time, volatile compounds present in bran water are changed For the heat-treated and unpreserved samples and the heat-treated and preserved samples for the first month, the volatile compounds in rice bran water did not change much, and these samples distribute in the same quadrant I However, volatile compounds were present in the rice bran fluid The samples that had been heat-treated and stored for months and months have a marked change, these samples distribute in the second quadrant, and the cumulative variance reaches 97.43%, a very high value 4.3 Thermolysis kinetics of fructooligosaccharides (FOS) [1kestose (GF2), nystose (GF3), and 1-fructosyl-nystose (GF4)] were extracted from rice bran 4.3.1 Making calibration curves and determining initial FOS content after extraction 15 From the initial FOS standard (consisting of 1-kestose (GF2), nystose (GF3) and 1-fructosyl-nystose (GF4)), chromatography analysis of the standard solutions is conducted to make three calibration curves using Eq regression between signal peak area and analytic compound concentration The results showed that: applying the extraction procedure according to the method of Patindol and Wang (2007) with modifications shows the presence of FOS (including 1-kestose (GF2), nystose (GF3) and -fructosyl-nystose (GF4)) in the initial material sample 4.3.2 Thermolysis kinetics of fructooligosaccharides (FOS) [1kestose (GF2), nystose (GF3), and 1-fructosyl-nystose (GF4)] were extracted from rice bran in different pH values The effect of the combination between temperature and pH on the thermal degradation of 1-kestose (GF2), nystose (GF3) and fructofuranosylnystose (GF4) extracted from rice bran dissolved in buffer solution is investigated at 90, 100 and 110oC and pH values of 5.0, 6.0 and 7.0 The decrease in temperature-pH value affects the GF2, GF3 and GF4 in rice bran samples can be fully described by the first-order model in the temperature range of 90 -110oC The decomposition rate constants, k values, estimated using linear regression analysis of ln(A/A0) versus t, are reported in Table 4.3 Table 4.3 Estimated rate constants, k values (min−1), of first-order degradation of GF2, GF3, and GF4 extracted from rice bran pH GF2 GF3 GF4 90oC 100oC 110oC Ea (kJ.mol-1) R2 5.0 0.0112±0.0012a 0.0174±0.0020 0.0430±0.0056 77.7 0.9565 6.0 0.0109±0.0011 0.0141±0.0015 0.0318±0.0031 61.8 0.9090 7.0 0.0052±0.0005 0.0090±0.0008 0.0159±0.0012 64.6 0.9995 5.0 0.0147±0.0011 0.0236±0.0020 0.0473±0.0052 67.5 0.9848 6.0 0.0081±0.0009 0.0192±0.0016 0.0401±0.0030 92.3 0.9993 7.0 0.0061±0.0005 0.0136±0.0014 0.0246±0.0026 81.0 0.9949 5.0 0.0102±0.0009 0.0186±0.0020 0.0353±0.0041 72.1 0.9990 6.0 0.0084±0.0006 0.0157±0.0011 0.0270±0.0031 67.6 0.9992 7.0 0.0072±0.0007 0.0123±0.0011 0.0169±0.0014 49.2 0.9824 a Standard error of the regression 16 Samples are dissolved in 0.2 M Na2HPO4/0.1 M citric acid buffers (at pH 5.0, 6.0 and 7.0) at different combinations of temperature and pH The results show that the degradation rate constant increases with the increasing temperature at different pH values However, the degradation rate constant decreases with increasing pH value These findings are comparable to the data that L'Homme et al (2003) used for studying the pH-temperature hydrolysis of standard fructooligosaccharides dissolved in the buffer at different pH and temperature values The rate constants predict the first-order degradation of rice bran GF2, GF3 and GF4 in the combinations of temperature and pH The thermal degradation of GF2, GF3 and GF4 in rice bran fluid occurs more readily at acidic pH than at neutral pH (L'homme et al., 2003) For each combination of temperature and pH, GF3 showed faster degradation than GF2 and GF4 Table 4.4 Estimated half-life time (min) of GF2, GF3, and GF4 extracted from rice bran at different temperatures and pH values GF2 GF3 GF4 pH 5.0 6.0 7.0 5.0 6.0 7.0 5.0 6.0 7.0 90 oC 62.1 63.9 133.7 47.2 85.2 113.9 68.3 82.7 96.2 100 oC 39.8 49.3 76.9 29.4 36.2 51.0 37.3 44.1 56.4 110 oC 16.1 21.8 43.7 14.7 17.3 28.1 19.6 25.7 41.1 Samples are dissolved in 0.2 M Na2HPO4/0.1 M citric acid buffers (at pH 5.0, 6.0 and 7.0) 4.3.3 Modelling of combined temperature and pH dependence of degradation rate constants of fructooligosaccharides [1-kestose (GF2), nystose (GF3), 1-fructosyl-nystose (GF4)] extracted from rice bran With X1 as the temperature variable and X2 as the pH variable on the experimental data, the model parameters were estimated using nonlinear regression analysis (proc NLIN, SAS) Based on the model parameters estimated, however, it was shown that the terms β0 and β22 were redundant, as indicated by the significant standard error (~100%) Model parameters estimated based on Equation (4.1) are shown in Table 4.5 17 ... are easily hydrolyzed and oxidized Lipase-inactivating protease and some other enzymes have both scientific and practical significance Protease hydrolyze lipase at a relatively low temperature... garlic, asparagus, bananas, artichokes, and among many others FOS has critical physiological effects as a prebiotic, increasing mineral absorption and reducing cholesterol levels, triacylglycerols... nutritional values (vitamins, protein digestibility) Moreover, in the process of hydrolysis with protease, some peptides and amino acids are formed These products both increase absorption capacity