UUnniivveerrssiittyy ooff AArrkkaannssaass,, FFaayyeetttteevviillllee SScchhoollaarrWWoorrkkss@@UUAARRKK Graduate Theses and Dissertations 5-2014 SSttaabbiilliittyy ooff NNaannoo--eennccaappssuullaatteedd RRiiccee BBrraann DDeerriivveedd BBiiooaaccttiivvee PPeennttaappeeppttiiddee iinn AAppppllee JJuuiiccee Fatima Mohammed Alessa University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/etd Part of the Food Chemistry Commons, and the Food Processing Commons CCiittaattiioonn Alessa, F. M. (2014). Stability of Nano-encapsulated Rice Bran Derived Bioactive Pentapeptide in Apple Juice. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/2320 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. Stability of Nano-Encapsulated Rice Bran Derived Bioactive Pentapeptide in Apple Juice Stability of Nano-Encapsulated Rice Bran Derived Bioactive Pentapeptide in Apple Juice A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science in Food Science By Fatima Alessa King Faisal University Bachelor of Science in Food Science, 2007 May 2014 University of Arkansas This thesis is approved for recommendation to the Graduate Council. ___________________________________ Dr. Navam Hettiarachchy Thesis Director ____________________________________ ____________________________________ Dr. Sun Ok Lee Dr. Suresh Kumar Thallapuranam Committee Member Committee Member ABSTRACT Cereal grains and their components derived Bioactive compounds such as rice bran can promote health and can be derived from Rice bran contains 12-20 % protein and could be a good source for extracting bioactive peptides. A pentapeptide with a secqunce of amino acids Glu- Gln-Arg-Pro-Arg (EQRPR) has been prepared from heat stablized defatted rice bran (HDRB) and has demonstrated anti-cancer proprerties in-vitro. This bioactive pentapeptide can thus be used as a nutraceutical by incorporating it into a suitable food system. Fruit juces can be vehicles to incorporate this pentapeptide. Fruit juices contribute to about 60% of the consumed beverages in the U.S. However, the stability of the pentapeptide in beverages can be a problem due to possible interactions with other components. Nano-encapsulation is a novel and promising technique that can be used to deliver bioactive ingredients into food systems. This study involves the use of a nano-encapsulating technique to protect the bioactive pentapeptide, incorporating the encapsulated pentapeptide into apple juice (model system), and testing for the stability of the peptide. The null hypothesis of the study: The Nano-encapsulated pentapeptide shall degrade over time when incorporated in apple juice and the alternate hypothesis that the Nano- encapsulated pentapeptide incorporated apple juice shall be stable over a storage period of 6 months or more. The specific objectives of this research were to: (1) prepare nanoparticles using polylactic-co-glycolic acid (PLGA) to encapsulate the rice bran pentapeptide, (2) incorporate the encapsulated pentapeptide into apple juice, (3) evaluate the stability of incorporated pentapeptide at 4◦C for 6 months. Nanoparticles that can deliver three different concentrations (200/ 400/and 600 µg/ml) of pentapeptide were prepared, and the particle size were measured using a laser particle size analyzer. Apple juice containing nanoparticles (loaded with pentapeptide) was ultra- centrifuged to separate nanoparticles, and the supernatant was analyzed by high performance liquid chromatography (HPLC) C18 column reverse phase (RP) to test the stability of pentapeptide. Physical properties of the apple juice were studied which included the evaluation of color, microbial count total, acidity (pH), and soluble solid (TSS) during storage period of 60 days. A particle size ranging from 81 to 83 nm was observed, and the results indicated that there were no significant changes in the size over the storage period (0 – 60 days). There was no microbial growth observed in the prepared apple juice samples. Total Soluble Solids content was 11.0 ºBrix for the controls and 31.0 ºBrix for the Nano-encapsulated pentapeptide. The stability of pentapeptide at prepared concentrations: 200, 400 and 600µg/ml in water at pH of 3.7 at 0th day was: 200ug/mL – 87 %, 400ug/mL – 97%, 600ug/mL – 91%) and in apple juice was: 200ug/mL – 96%, 400ug/mL – 98%, 600ug/mL – 94 %. The stability of pentapeptide at 60th day in water was: 200ug/mL – 41%, 400ug/mL – 60%, 600ug/mL – 55%, and in apple juice was: 200ug/mL – 60%, 400ug/mL – 67%, 600ug/mL – 59%. The Nano-encapsulated pentapeptide in water at pH of 3.7 and apple juice was stable over the storage period of 60 days, which implies that the nanoparticles were effective in protecting the bioactive pentapeptide in the acidic environment of apple juice. The PLGA nanoparticles showed a remarkable effect in protecting and stabilizing the bioactive compounds (pentapeptide) during the shelf life at 4ºC. Polylactic- co-glycolic acid nanoparticles can thus be a promising carrier for the bioactive pentapeptide when incorporated into a juice medium. ACKNOWLEDGEMENTS I want to thank God for his guidance. I express my deepest gratitude to my husband, children, and parents who encouraged me with love and supported me through the hardest moments of my life. I am very thankful to my major advisor, Dr. Navam Hettiarachchy, for offering me good learning experience, which will be beneficial for my career and future. She has helped to develop my skills and pushed me to participate in competitions and win. Also, I would like to thank her for the time and patience that she always spent for her students. My deep gratitude also goes to all the faculty and staff of Food Science Department. Three people, Srinivas Rayaprolu, Dr. Eswaranandam Satchithanandam and Madhuram Ravichandran helped me learn the techniques in conducting research. I learned a lot by interacting with those people and enjoyed the friendly environment. I would like to thank my committee members Dr. Suresh Thallapuranam and Dr.Sun-ok Lee for serving in my committee, Dr.Surendra Singh, Dr. Mourad Benamara, and Dr. Denise Greathouse for their efforts in helping me to improve my research. Finally, I would like to thankful my funding agency King Faisal University for offering me the full support and encouragement to attend the University of Arkansas. TABLE OF CONTENTS Chapter 1 ......................................................................................................................................... 1 Introduction ................................................................................................................................. 1 Chapter 2 ......................................................................................................................................... 4 Literature review ......................................................................................................................... 4 A. fruit juice consumption in the U.S. .................................................................................... 4 B. Suitability of vehicles for nutraceuticals and functional foods. ........................................ 5 C. Vehicles to incorporate nutraceuticals, flavors, nutrients, and proteins. ........................... 7 D. Examples of peptides and proteins incorporated into beverages. ..................................... 9 E. Problems associated with incorporation of proteins and peptides into beverages. ......... 10 F. Processes of overcoming the issues associated with incorporation of bioactives. .......... 12 G. Nano-encapsulation of bioactives. .................................................................................. 17 H. Nano-encapsulation of pentapeptide in apple juice. ........................................................ 21 I. Shelf life stability of Nano-encapsulated pentapeptide in apple juice. ........................... 23 Chapter 3 ....................................................................................................................................... 28 Introduction ............................................................................................................................... 28 Materials ................................................................................................................................... 30 METHODS: .............................................................................................................................. 31 Preparation of nanoparticle using plga polymer and incorporation in apple juice ................... 31 Preparation of a standard curve to determine pentapeptide concentrations .......................... 33 Stability of Nano-encapsulated pentapeptide in pasteurized apple juice by HPLC .............. 33 Measurement of the particle size of Nano-encapsulated pentapeptide in pasteurized apple juice ....................................................................................................................................... 34 Scan electron microscopy (SEM) in Nano-encapsulated pentapeptide in pasteurized apple juice ....................................................................................................................................... 34 Testing physical properties of pasteurized apple juice with nanoparticles (contain pentapeptide) ......................................................................................................................... 35 Results and discussion .............................................................................................................. 38 Stability of pentapeptide incorporated nanoparticles in pasteurized apple juice by HPLC .. 38 Particle size of Nano-encapsulated pentapeptide in pasteurized apple juice ........................ 40 Scan electron microscopy (SEM) of Nano-encapsulated pentapeptide in pasteurized apple juice ....................................................................................................................................... 41 Physical properties of Nano-encapsulated pentapeptide in pasteurized apple juice ............. 41 References ..................................................................................................................................... 87 TABLE OF FIGURES Figure 1: The main elements of Scan Elements Microscopy. ...................................................... 37 Figure 2: Standard curve of pentapeptide at increasing concentrations based on peak areas from retention times on an affinity HPLC column .................................................................... 48 Figure 3: HPLC profiles of the pentapeptide (200µg/ml) incorporated in water at pH of 3.7 ..... 50 Figure 4: HPLC profiles of the pentapeptide (400µg/ml) incorporated in water at pH of 3.7 ..... 52 Figure 5(a-f): HPLC profiles of the pentapeptide (600µg/ml) incorporated in water at pH of 3.7. ........................................................................................................................................... 54 Figure 6(a-F): HPLC profiles of apple juice alone. ...................................................................... 56 Figure 7(a-f): HPLC profiles of the pentapeptide (200µg/ml) incorporated in apple juice. ......... 57 Figure 8(a-f): HPLC profiles of the pentapeptide (400µg/ml) incorporated in apple juice. ......... 59 Figure 9(a-f): HPLC profiles of the pentapeptide (600µg/ml) incorporated in apple juice. ......... 61 Figure 10: The stability of varying concentrations of pentapeptide in water at a pH of 3.7 based on the percentage of pentapeptide degraded over the storage period. .............................. 62 Figure 11: The stability of varying concentrations of pentapeptide in apple juice based on the percentage of pentapeptide degraded over the storage period. ......................................... 63 Figure 12(a-f): HPLC profiles of Nano-encapsulated pentapeptide (200µg/ml) incorporated water at pH of 3.7. ...................................................................................................................... 67 Figure 13(a-f): HPLC profiles of Nano-encapsulated pentapeptide (400µg/ml) incorporated water at pH of 3.7. ...................................................................................................................... 69 Figure 14: HPLC profiles of Nano-encapsulated pentapeptide (600µg/ml) incorporated water at pH of 3.7. .......................................................................................................................... 71 Figure 15(a-f): HPLC profiles of Nanoparticles in water at pH of 3.7. ........................................ 73 Figure 16(a-f): HPLC profiles of Nano-encapsulated pentapeptide (200µg/ml) incorporated apple juice. .................................................................................................................................. 75 Figure 17: HPLC profiles of Nano-encapsulated pentapeptide (400µg/ml) incorporated apple juice. .................................................................................................................................. 78 Figure 18(a-f): HPLC profiles of Nano-encapsulated pentapeptide (600µg/ml) incorporated apple juice. ........................................................................................................................ 80 Figure 19(a-f): HPLC profiles of Nanoparticles in apple juice. ................................................... 82 Figure 20: Illustration of electrostatic interactions between nanoparticles and peptide ............... 83 Figure 21: The particle size stability of Nano-encapsulated pentapeptide in apple juice(200/400/600µg/ml) over the storage period (0 to 60 days). .................................... 83 Figure 22: SEM image of Nano-encapsulated pentapeptide in apple juice. ................................. 84 Figure 23: The Chroma changes of the Nano-encapsulated and non-encapsulated pentapeptide incorporated apple juice in storage. .................................................................................. 85 Figure 24: The Hue Change of Nano-encapsulated and non-encapsulated pentapeptide incorporated apple juice in storage. .................................................................................. 86 Figure 25: The color change of Nano-encapsulated pentapeptide incorporated apple juice and control (apple juice) in storage. ........................................................................................ 86 CHAPTER 1 INTRODUCTION The International Markets Bureau (2011) documented in 2010 that fruit and vegetable juices are consumed at per capita consumption of about 30.3 liter/ person, and the retail market in the United States is 8.8 billion dollars. The US Department of Agriculture documented that 49% of Americans consume more than one glass (236.6 mL; 8 fluid oz.) of juice daily (Andon et al., 1996). The International Markets Bureau (2011) further asserts that consumers are increasingly becoming health conscious; therefore, the consumption of fruits and vegetables have increased in popularity with a larger proportion of the American population. Since consumers have become more health conscious and prefer to purchase 100% fruit juices with no additives, the manufacturers have improved the ingredients that are used in fruit juices. Fruit juices can offer health benefits, such as reducing the risk of cancers and cardiovascular disease. Some common ingredients in fruit juices are water, malic acid, sugar, fiber, and minerals. The interactions among native components in fruit juices and bioactive ingredients can be minimized, making juices suitable vehicles to incorporate bioactive compounds (peptides and proteins) (Day, et al., 2009; Tuorila & Cardello, 2002). Vehicles are food systems that can deliver bioactive ingredients so that consumers receive maximum health benefits. Apple juice can be a vehicle to incorporate peptides because it is the second most popular juice consumed in the U.S., making up 12.5% of total juice consumption preceded only by orange juice 60% of total juice consumption. Apple juice is rich in phytochemical compounds, especially flavonoids, and phenolic compounds (Boyer and Lui, 2004). Because of these beneficial components in fruit juices, apple and orange juices are fortified with calcium citrate malate (CCM) and show high calcium absorption from CCM. To mimic the composition of Ca fortified juices, the 1 concentrations of organic acid and carbohydrate in the test solution were manipulated. Apple juice has a high Ca absorption because of high fructose and low organic acid content (Andon et al., 1996). Also, orange juice is fortified with 1000 IU vitamin D3/236.6 mL, and the result shows more than 150 % increase in the serum 25- hydroxyvitamin D3 [25(OH)D] concentrations of adults over a 12-week period. Therefore, vitamin D intake can be increased by orange juice modification with vitamin D3 (Biancuzzo et al., 2011). Fruit juices such as apple and orange juice can be good vehicles for bioactive ingredients such as proteins, peptides, and vitamins due to their efficiency to impart health benefits. Bioactive compounds have positive effects on human physiological health beyond their nutritional values. Examples of bioactives derived from cereal grains include oatmeal, rice bran, wheat bran, and protein rice. Rice bran (RB) is rich in protein; protein makes up approximately 12-20% of rice bran. A pentapeptide is a protein extracted from RB with a sequence of amino acids Glu-Gln-Arg-Pro-Arg (EQRPR), pentapeptide is prepared from heat stablized defatted rice bran (HDRB). EQRPR has demonstrated anti-cancer proprerties, and has the potential of being used as a drug or incorporated into sutitable food products such as orange juice (Kannan et al.,2008). For example, in a study conducted by Khairallah (2011), the stability of RB peptide fractions into orange juice environment was investigated for 6 months. The peptide fractions showed high stability at 4°C and pH 3.0- 4.5, which indicated that fruit juices can be an appropriate food system to incorporate bioactive compounds from rice bran. The pentapeptide EQRPR can be incorprated into several food aplications due to its health benefits, disease prevention, and higher stability. It is likely that the bioactive peptides can interact with the components in juice matrix and influence the stability. The interaction between ingredients in fruit juices can initiate 2