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Zou_Z.pdf

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1 THE USE AND STABILITY OF MONK FRUIT PLANT DERIVED SWEETENER IN A PROTOTYPE ORANGE JUICE BEVERAGE By ZHOU ZOU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEG REE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 201 9

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2 201 9 Zhou Zou

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3 To my beloved family, Dr. Goodrich, Zihan and Mengzhu for helping me accomplish this education, discover myself and enjoy the journey

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4 ACKNOWLEDGMENTS First of all, I have to thank my family for their unconditional love and support throughout my life. Thank my parents for letting me chase my dreams and giving me the strength to make my own decisions. Thank my grandparents for educating me to be a kind and energetic person. Also, my uncle, aunt and cousins for their love and care while I am growing up. I would like to thank to my major professor, Dr. Rene Goodrich Schneider for supporting me during these past three years. She is someone that you will instantly love once you meet h er She and her husband, Dr. Schne ider, are the most friendly and smartest people I know. I will always remember all those interesting stories and food and culture related discussion s between us. They are my best role model s for food scientist s mentor s and teachers Thank Dr. Goodrich for her scientific advice and knowledge and many insightful and warm suggestions. She is my primary resource for getting my all kinds of questions answered and was instrumental in helping me complete this thesis. I still think fondly of my time as an undergraduate student in her product development class. Since that, I decided to go to pursue a career in R&D department in food industry. I am always very grateful and proud for being her student. I hope that I could be as lively, enthusias tic and professional as Dr. Goodrich and to someday be able to help and influence other people like she encouraged me. I also have to thank the members of my committee, Dr. Lisa House, Dr. Yu Wang have finished this research project without the m. Dr. House has been helpful in providing advice many times during my graduate degree. As an expert in the food economic field, her research projects are extremely interesting and meaningful. Dr. Wang is a great mentor and has helped me a lot with the she lf life study of my project. I

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5 also thank all members from her lab, especially postdoc, Joon and PhD students Jingwen, Yanli, Zoey, who have been helpful in providing guidance during my time in Lake Alfred. Thanks for their time even during weekends. Dr. W ang enthusiasm and love for researching is contagious and respectable. A good and resourceful system is important to everyone in graduate school. I was lucky to be a part of Food Science and Human Nutrition department. I know that I could always ask profe ssional people for their advice and opinions on research or life related issues. Dr. Sims and his lab (Sara, Jessica) are wonderful and generous friends who has been helped me with the sensory evaluation. Dr. Sims is also a great teacher. I really enjoyed his sensory class, and it was one of the reasons why I wanted to do sensory and market related work in the future. I also thank Dr. Macln tosh and Stephen, for their help while the pasteurization. Dr. Mac is a precise but also creative person, he encouraged and expected us to think more independently about our experiments and results. But when you have a problem, he could always come up with a way to solve it. in our depar tment. I remember first meeting Herschel when I tried to decide my course schedule for the first semester. I admire his advice and he shared his experience in China to make me feel associated. All the staffs in our department are very nice and helpful, I w together. Dr. Percival, thanks so much for organizing those activities for us. I also thank all my friends here. They are friendly, hardworking and outstanding with lots of enthusiasm a nd optimism and remind me to keep moving, to be a better person. I am glad to have worked with them and I wish them the best of luck. I value

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6 the friendship will last forever. I will always be willing to help whenever they need me. I also thank my close friends in China, Mengzhu, for providing most significant friendship that I needed. I would like to thank Zihan who has been supportive and caring for pointing out my m istakes and also encouraging me to follow my heart and be myself.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 2 REVIEW OF LITERATURE ................................ ................................ .................... 15 Low Calorie Sweetener Definitions and Applications ................................ .............. 15 Global Food Sw eetener Market Growth and Trends ................................ ............... 17 Drivers of Natural Sweeteners ................................ ................................ ................ 18 Customer Perception on Products with Natural Sweeteners ................................ .. 20 Monk Fruit Extract Introduction ................................ ................................ ............... 22 Traditional Processing a nd Biosynthesis Pathway of Stevia and Monk Fruit .......... 24 Metabolism and Biotransformation of Mogrosides ................................ .................. 26 GRAS Notifications and Manufacturing Process of Monk Fruit Extract ................... 27 Orange Juices and Orange Juice Beverages ................................ ......................... 28 Important Quality Parameters of Orange Juice Beverages ................................ ..... 29 Orange Production and the Orange Juice Market ................................ ................... 30 3 MATERIALS AND METHODS ................................ ................................ ................ 35 Retail Screening of Commercial Low calorie Orange Juice Beverages .................. 35 Beverage Analysis ................................ ................................ ................................ .. 35 Total Solids (Brix) Measurement ................................ ................................ ..... 35 pH Determination ................................ ................................ ............................. 35 Titratable Acidity (TA) ................................ ................................ ....................... 36 Color Determination ................................ ................................ ......................... 36 Viscosity Determination ................................ ................................ .................... 36 Mogroside V Stability in Model Juice Systems ................................ ........................ 36 Model Juice Preparation ................................ ................................ ................... 37 Thermal Processing Treatment on Model Juice ................................ ............... 37 Shelf Life Study and Stability Test of Pure Mogroside V ................................ .. 38 Preparation of Standard Solutions ................................ ................................ .... 38 Sample Preparation ................................ ................................ .......................... 39 LC MS/MS Analysis ................................ ................................ ......................... 39 Sensory Evaluation of Prototype Orange Juice Beverages ................................ .... 40 Preliminary Test on Bench Scale Products ................................ ...................... 40

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8 Pilot Plant Scale Products Manufacture ................................ ........................... 41 Final Sensory Evaluation Customer Preference Test ................................ ..... 42 Statistical Analysis ................................ ................................ ................................ .. 44 4 RESULTS AND DISCUSSION ................................ ................................ ............... 46 Stability Studies on Mogroside V in Model Juices ................................ ................... 46 Standard Curve of Mogroside V ................................ ................................ ....... 4 6 Heat and pH Stability ................................ ................................ ........................ 46 Shelf Life Study/Storage Stability ................................ ................................ ..... 47 Sensory Evaluations on Application of Monk Fruit Extract in Prototype Orange Juice Beverages ................................ ................................ ................................ .. 48 Determination of Optimal Formula Sweetened with Monk Fruit Extract ........... 48 Preliminary Sensory Test: Comparison of Commercial Products and Tested Formulation ................................ ................................ ................................ ... 49 Final Sensor y Evaluation: Customer Preference Test on Sweetener in Low calorie Orange Juice Beverages ................................ ................................ ... 51 5 CONCLUSION ................................ ................................ ................................ ........ 60 APPENDIX A INGREDIENT LIST OF TROP50 ................................ ................................ ........... 62 B FORMULATION DETAILS ................................ ................................ ...................... 63 C FINAL SENSORY EVALUATION BALLOT ................................ ............................. 64 LIST OF REFERENCES ................................ ................................ ............................... 68 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 79

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9 LIST OF TABLES Table page 2 1 USDA standards for Grade A orange juice products from Florida a ..................... 34 3 1 SRM transitions, collision energies and RF lens for LC MS/MS analysis of samples ................................ ................................ ................................ .............. 45 4 1 pH, soluble solids and acidity of three orange juice products ............................. 57 4 2 Descript ive analysis scores for attributes of appearance and overall flavor in preliminary test evaluated by 31 panelists ................................ .......................... 57 4 3 Descriptive analysis on attributes of basic flavor and texture in preliminary test evaluated by 31 panelists ................................ ................................ ............ 57 4 4 Quality evaluation of Trop50 and reformulated samples A and B ..................... 58 4 5 The overall liking and overall appearance liking results for samples in final sensory eval uation with 92 panelists ................................ ................................ .. 58 4 6 Customer preference results for low calorie orange juice beverages in final sensory evaluation with 92 panelists ................................ ................................ .. 58 B 1 The formula information of model juice in stability test ................................ ....... 63 B 2 The formula information of samples in sensory tests ................................ .......... 63

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10 LIST OF FIGURES Figure page 2 1 Structures of main mogrosides isolated from the fruits of monk fruit .................. 32 2 2 Proposed biosynthetic pathway of mogrosides in monk fruit. ............................. 33 4 1 The calibration curve of the dependence of a p eak area on the concentration of mogroside V:MV, mogroside V. ................................ ................................ ...... 55 4 2 Effect of heat treatment1(72C, 15s) and treatment 2 (90C, 30s) on mogroside V in pH 3.5 and pH 5.0 model liquids. ................................ ............... 55 4 3 Effect of storage on mogroside V in pH 3.5 and pH 5.0 model liquids after heat treatment1(72C, 15s) and treatment 2 (90C, 30s). ................................ .. 56 4 4 Results from repeated storage test: effect of storage on mogroside V in heat treated (treatment 1,72C, 15s; treatment 2, 90C, 30s) model systems. ........... 56 4 5 Result of aftertaste evaluations on tested beverages in preliminary test with 31 panelists: TOJ, Tropicana Orange Juice; T50, Trop 50 ; FOJB, Formulated orange juice beverage. ................................ ................................ .... 59

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE USE AND STABILITY OF MONK FRUIT PLANT DERIVED SWEETENER IN A PROTOTYPE ORANGE JUICE BEVERAGE By Zhou Zou August 2019 Chair: Rene Goodrich Schneider Major: Food Science and Human Nutrition Monk fruit ( Siraitia grosvenorii or luo han guo) is a cucurbitaceous edible herb widely planted in China, which produces high potency sweeteners increasingly popular in the food industry as additives in low calorie drinks or foods. The main swe et compound, m ogroside V is a cucurbitane triterpenoid saponin and the major bioactive constituent of monk fruit, which is approximately 400 times sweeter than sucrose. This study aims to clarify its applicability in juice beverages and identify its stabil ity after thermal processing and storage. The stability of pure mogroside V in acidified model systems (pH 3.5 and 5.0) was evaluated chemically after two heat treatment regimes and during shelf life storage over 90 days. Processing methods and storage con ditions were chosen to encompass the typical shelf life of orange juice products. Liquid chromatography tandem mass spectrometry (LC MS/MS) was used to monitor the chemical degradation of th e pure mogroside V compounds. Additionally, in this study, prototy pe orange juice beverages were developed using not from concentrate (NFC) orange juice and commercial monk fruit extract. Pasteurized prototypes and commercial product were compared by sensory evaluation. The result s demonstrated that m ogroside V was stable under all conditions tested, and a prototype formula was highly

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12 acceptable to customers, thus indicating the potential suitability of monk fruit extracts used as a sweetener in juice beverages.

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13 CHAPTER 1 INTRODUCTION The growing concern wit h human health and the greater incidence of overweight, metabolic syndrome and diabetes have resulted in an increased interest in reduced calorie foods and beverages, especially those that use low calorie sweeteners as sucrose substitutes (Dabelea et al., 2007). According to a very recent study, about 25% of children and more than 41% of adults in the United States reported consuming foods and beverages containing low calorie sweeteners (LCS) in a recent nationwide nutritional survey. These numbers represen t a 200% increase in consumption of LCS by children, and a 54% increase among adults from 1999 to 2012 (Sylvetsky et al., 2017). In addition, they also claimed that the LCS market is projected to continue to grow at approximately 5% per year through the ye ar 2020. fully replace added sugar with LCS in many foods and beverages. Interest in the development of food products with LCS, and particularly natural sweeteners, has ma rkedly increased in the last decade (Piernas et al., 2013). As one of the natural sweeteners approved by United States Food and Drug Administration (FDA), monk fruit extract (MFE) has become more and more popular (Li et al., 2015). However, there are few s cientific studies examining how MFE performs in commercial food products. This study aim ed to determine the stability of monk fruit extract after thermal processing, which is one of the more common food processing unit operations, as well as the degradatio n rates of the main functional component over shelf life under different storage conditions. Additionally, to demonstrate a potential application of MFE in a lower calorie juice beverage system, we determine d the

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14 feasibility of using MFE in a model orange juice beverage and develop ed a MFE sweetness equivalence equation with respect to by conducting a series of sensory tests with this beverage /sweetener system. This beverage case study will provide guidance for the use of MFE in lower calorie juice based be verages.

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15 CHAPTER 2 REVIEW OF LITERATURE L ow C alorie S weetener D efinitions and A pplications LCS are a class of high intensity sweeteners which contribute no or few calories to the overall food product They are commonly used in foods and beverages to reduce calorie content while maintaining palatability. They are also referred to as nonnutritive sweeteners, high intensity sweeteners, and non caloric sweeteners (Sylvetsky & Rother, 2016) depending on the specific sweeten ing compound. FDA has approved several of these LCS as food ingredients, including saccharin, aspartame, acesulfame potassium (Ace K), sucralose, neotame, advantame, steviol glycosides, and Siraitia grosvenorii Swingle fruit extract, commonly known as L uo H an G uo or monk fruit extract (FDA, 2014). These LCS were approved for use as table sugar, and in various food categories, such as beverages, snacks, and dairy products. However, different applications processing methods, and food matri ces of those produc ts affect the performance of the sweetener For example, aspartame is commonly used in soda drinks and chewing gu m goods because it loses sweetness when heated. Most of sweeteners that widely used as sugar substitute s or sugar alternatives in commercial food products are generally called artificial sweeteners, since they are chemically synthesized. S ome artificial sweeteners are derived from naturally occurring substances that have been chemically manipulated, for exa mple sucralose from sugar ; Although these sweeteners are considered safe and potentially useful for controlling obesity related health conditions, their use is still questioned by consumers and some research (Rodero et al ., 2009; Bellon et al., 2009;

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16 Ma et al., 2010 ) Therefore, natural sweeteners had gained much attention as substitutes. Studies have shown that people do not prefer artificial nonnutritive sweeteners over sucrose, especially for parents who choose pro ducts for their children drink category globally, leading to a backlash against ingredients perceived to be plant food labeling, FDA has considered that to mean the food products with nothing artificial or synthetic (including all color additives regardless of source) included in the ingredient list (FDA, 2017). Accordingly, some manufactures call their stevia and monk fruit extract plants. Currently, they are only two types of natural plant based sweeteners which approved by FDA : certain steviol glycosides obtained from the leaves of the stevia plant ( Stevia rebaudiana / Bertoni) and extracts obtained from Siraitia grosvenorii Swingle fruit, also known as Luo Han Guo or monk fruit B oth contain no calories and have sweetness related sensory characteristics that can vary based on temperature, acidity, sweetener concentration and the chemical composition of the food product (Cardoso et al., 2004). LCS are playing an increasing significant role for consumers who seek alternatives to sucrose in many product categories (Ng et al., 2012). The multiple characteristics of these sweeteners has also brought unprecedented challenges to new product development process. Conseque ntly, there has been an increase in i ndustry

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17 efforts to reduce sugar are having a profound influence on new product formation or reformulati on by adjusting the sweetener level and type (Williams, 2018). Global Food Sweetener Market Growth and Trends Global market food sweetener sales recorded $84 billion in 2014 and is expected to increase at a compound annual growth rate (CAGR) of 4.5% and reach nearly $111 billion by 2020. Currently, in the sweetener market, sugar (sucrose) still holds the majority share (more than 80%), followed by high intensity sweeteners. Although high intensity sweeteners don't constitute the major share, this is the fastest growing segment. The market for high intensity sweeteners is estimated to reach to $2.2 billion in 2020 at a CAGR of 5.1% (Wood, 2015). This growth reveals the rising health concerns among people and increased awareness of dietary foods. Aspartame and s ucralose are the most common sweeteners in the LCS segment (Wood, 2015). These artificial nonnutritive swe eteners have been widely used in food products and have showed beneficial influence on weight loss for children and adults (Swithers, 2013; Rogers et al., 2016; de Ruyter et al., 2012; and Tate et al., 2012). However, a major trend that must be addressed b y food product developers is the public or personal pressures which are turning consumers away from artificial sugar substitutes to more natural low or zero calorie alternatives (Pawar et al., 2013). Accordingly, the two botanical sweeteners, stevia and M FE have gradually gained popularity in the market and enjoyed a prodigious surge in usage as natural LCS (Li et al., 2015). More specifically, it has been reported that the consumption of products that contain stevia grew by 53% from $123.1 million to $188 .3 million from 2010 to 2011 in the conventional (food, drug, mass market ) channel s (Almendarez, 2012). In the USA, there is increased use of plant extracts known to contain highly sweet terpenoids, which

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18 result in greatly increased interest in MFE (Pawar et al., 2013). Natural LCS is seeing a major demand in the natural sweeteners category and is expected to increase more rapidly than other LCS (Hackett, 2014). T his will become increasingly important as specific constituents of botanical sweeteners such a s stevia and MFE are blended to take advantage of the unique attributes of individual sweet components and improve the sweet quality in the meantime (Azevedo, Schmidt, & Bolini, 2015) Drivers of Natural Sweeteners King (2019) noted that the number one customer driver of natural sweetener is tilizes simple, natural and minimally processed ingredients, and possible production by traditional techniques (Edwards, 2013). Since consumers are nowadays much more interested in information about the production methods and components of the products that they eat (Asioli et al., 2017). For instance, they are concerned about the use of p esticides (Aktar, Sengupta, & Chowdhury, 2009), the use of artificial ingredients, additives or colorants (Lucov Hojerov Pa ourekov & Klimov 2013), and the controversial food technologies like g enetic modification (Grunert, Bredahl, & Scholder er, 2003). These factors have been encouraging consumers to look for products with healthy benefits and that are free from artificial ingredients. Accordingly, the food industry has started to supplying products that are natural and healthy (Katz & Williams, 2011). For example, in 2010 Simply Heinz ketchup was launched after re moving the high fructose corn syrup from the ingredient list and replacing that with sugar (Katz & Williams, 2011). A dditionally beverages could result in an increased use of low calorie sweetener. FDA proposed the

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19 mandatory declaration of added sugars on the nutrition facts label to assist consumers in mainta ining health beneficial dietary practices (FDA 2016). Since then, t he new label has begun appearing on packages on the market. The labeling requirements have been recently clarified (FDA 2019) The final guidance explains that a percent daily value for add ed sugars will be required on the nutrition facts label by July, 2021 to help consumers understand how their consumption will contribute to the daily sugar intake according to dietary guidelines. Interestingly a statement of added sugars content would not be required for products that contain less than 1 gram of added sugars in a serving making high intensity natural sweeteners more attractive for use Other parts of the regulation are very specific and might guide product development. For example allulo se, a monosaccharide that is naturally present in a few foods, such as wheat, figs and raisins. Williamson and others (2014) found that it was eliminated in urine without being used in human body or raising blood sugar. It was exempt from being included as a carbohydrate, sugar, or added sugar in the Nutrition Facts label on foods and beverages by FDA (2019 a ). Therefore, when combined with possible industry reformulations to reduce added sugar content in packaged foods and beverages, the new label policy co uld also drive the industry to replace conventional sugar with LCS (Huang et al., 2019). At the same time, a combination of other factors is also driving the spread and use of natural LCS, including taxation on the sugar sweetened beverages. For instance, Mexico implemented a 1 peso per liter excise tax on sugar sweetened beverages in 2014, and previous studies found a 6 percent and 9.7 percent reduction in purchases of taxed beverages in the following two years, respectively (Colchero, et al., 2016).

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20 Findi ngs from Mexico started a trend, which had been influenc ing other countries to generate fiscal policies in order to take control of sugar consumption and reduce chronic disease (Colchero, et al., 2017). In the USA, Oakland, San Francisco and Seattle are am ong the cities that have soda taxes (Laura and Smith, 2018) As the sweeteners that serve as sugar alternative, the adoption of new technologies that reducing off flavors (Espinoza, et al. 2014), the prevalence and cost reduction of MFE (Feng, et al. 2012) and the awareness of keto friendly, gluten free and hypoglycemic index compounds (Harrington, 2008; Watson, 2019) are also thought to promote the consumption of non nutritive LCS. Customer Perception on Products with Natural Sweeteners Ohmes (2019) clai med that consumers want reduced sugar beverages made with familiar ingredients but not at the expense of great taste. He believes that taste is always the single biggest driver of purchase intent. Technical recommendations from the food ingredient company age groups, gender and ethnicities (Kerry 2019) The results showed that 75% of tested consumers want reduce d sugar products to taste the same. Moreover 71% of American consumers note the sugar content on ingredient labels. When it comes to consumer awareness and preferences, honey, sugar (sucrose) and maple syrup were the t op 3 choices at 64%, 59% and 31%, res pectively. While 58% of consumers were aware of stevia, just 22% preferred it. Despite garnering 63% recognition, high fructose corn syrup was only preferred by 7% of respondents, while aspartame ranked last in consumer preference with 6% of the vote. More over, according to HealthFocus

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21 the top five preferred sweetener s for US consumers are honey, fruit juices, maple syrup, agave are high fructose corn syrup, artificial sweeteners, aspartame, saccharin, and fructose (HealthFocus, 2019). I that consumers are educating themselves around some of these comparatively new sweetener options and understand the benefit of selectin g natural sweetener over sugar or artificial sweeteners as encompassing organic production practices; they typically have a more idealized view of organic farming than what is reality (Baker, 2015). Abrams and others reported preservatives, no additives, no antibiotics, no hormones, and no chemicals Hence, the omers, continues to be perceived as the safer food choice (Abrams et al., 2009). FDA (2015) has been considering clarification In order to investigate customer perception more in detail, several studies have bee n conducted on natural nonnutritive sweeteners in different products. For example, the iso sweetness was determined to indicate the sweet taste intensity as compared to sucrose at different concentrations Zhang and Gruen (2013) reported the iso sweetness of stevia, monk fruit extract erythritol, lactitol, and xylitol to 10.1% sucrose sweetened whey protein beverages and indicated that stevia and monk fruit extract both had sweeter taste than sucrose control (77 and 115 times respectively). A sweetness int ensity perception study on skim chocolate milk sweetened by stevia and monk fruit extract showed that both monk fruit extract and stevia were acceptable by young adults and children ( Li, Lopetcharat, & Drake, 2015). In one study, p arents preferred skim

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22 cho colate milk with natural nonnutritive sweeteners rather than that with sucrose (Li et al., 2015). However, conflicting research does exist For example, in a sensory study conducted on functional dairy food products sen sory acceptance, sucrose sweetened aronia and elderberry kefir products were best accepted ; stevia and MFE were not well accepted in either tests (Du & Myracle, 2018). Few studies dealing with juice products or acidic model systems utilizing stevia and MFE have been found in the literature. The sweetness potency defined as the number of times sweeter a compound, on a weight basis. S tevia potency has been reported to be 202 in water and 216 in peach juice (Parpinello, Versari, Castellari, & Galassi, 2001), and 134 in mango nectar (Cadena & Bolini, 2012). Additionally, Cardello (1999) has reported that the acid pH favored the increase of the potency of stevia. In a customer preference study with sugar reduced orange/pomegranate juice, researchers tried to investigate the effect of information about sugar reduction and the use of sweeteners on consumer perception. Results from their work showed that information about lack of added sugar and the replacement of natural sweetener influenced consumer perception of different dimensions of wellbeing. Specifically, information mainly affected consumers sensory and hedonic perception of the juice sweetened with stevia, but did not have a significant effect on MFE sweetened juice perceptions which had an impact on the results (Reis, Alcaire, Deliza, & Ares, 2017). Monk Fruit Extract Introduction Monk fruit sweetener or extract (MFS or MFE) is the common name for Siraitia grosvenorii Swingle fruit extract, a natural sweetener recently approved as GRAS by FDA (FDA 2010 ). Fruits of S. grosvenorii known as monk fruits have a long history of use in south China as a household remedy for colds and sore throats (Kinghorn, 1986;

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23 Prakash & Chaturvedula, 2014). In the 1970s, Lee first established that a cucurbitane type triterpene glycoside was responsible for imparting the characteristic sweet taste of MFE (Lee, 1970). These compounds possess a triterpene backbone with two to six glucose units attached, forming mogrosides II to VI (Chang, 1996). Five ch emical structure s of mogroside (I, II, III, IV, and V) are recognized by the number of glucose units that are attached to the chemical structure of mogroside unit. Whereas ripe fruits of monk fruit contain primarily mogroside V and have a most sweet taste, unripe fruits, which contain less mogroside V are less sweet even bitter. T hus, t he level of mogrosides sweetness depends on the percentage of mogrodside V in the total mixture of mogrosides derived from fruits (Dianpeng, et al., 2007) However, the matur e status c an S ome methods for quantitating the mogroside V in samples were reported to evaluate the quality of commercial MFE (Xia et al., 2008; Zhang et al,.201 2 ; Sun et al., 2012) MFE works as a swee tener as combination of several different cucurbitane glycosides, mogrosides (Matsumoto, 1990) has been approved for use in dietary supplements in Japan, Australia, New Zealand, and the U.S. nevertheless (Qin et al., 2006). It is noteworthy that none of the monk fruit extract components demonstrate toxic ity to animal or humans ; this is the basis for the GRAS designation by FDA A 90 day oral toxicity study in rats by Qin et al. (2006) assessed a 30% mogroside V product contai ning 7.8 14.1% of unidentified components. It found no toxicity at daily doses of up to 3% of the diet, the highest dose tested. A 28 day oral toxicity study on rats by Marone et al. (2008) also confirmed the safety of MFE, which was determined to be 100,0 00 ppm in the diet, the highest level tested, equivalent to 7.07 and 7.48 g/kg

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24 bw/day for male and female rats, respectively. MFE was well tolerated and produced no significant adverse effects. In addition to their sweetness properties, MFEs were reported to show demonstrated antioxidant activity. A wide range of antioxidant effects of fruit extracts, including MFE, may have anticancer, antiviral, antihyperglycemic, antidiabetic activities and some other benefits (Ukiya, 2002; Lim, 2012). Traditional Proces sing a nd Biosynthesis Pathway of Stevia and Monk Fruit The conventional extraction procedures of stevia leaves and monk fruit were similar. Hot water treatment was used as a classical method (Dacome et al., 2005). And then, HPLC method was used to separate bioactive compounds and other carbohydrates by different mobile phases (Pawar, 2013). However, it should be noted these traditional processing is associated with long extraction time. Also, reproduction of stevia in the wild is mainly by seed, but germina tion and establishment from seed are often poor and sometimes unsuccessful (Shaffert and Chebotar, 1994). Besides, for stevia, plant organs contain different amounts of the sweet glycosides, leaves and flowers have the highest amount (Dwivedi, 1999). But, as a short day plant, stevia flowers from January to March in the southern hemisphere and from September to December in the northern hemisphere. Accordingly, the accessibility of yield of stevia extract was hard to control. Also, for monk fruit, the mogros ides are present at about 1% in the flesh of the fruit (Kinghorn & Soejarto, 2002). But the amount is differed at different growth stages and could be heavily influenced by environmental conditions (Xia, 2008). The success of breeding depends on the choic e of parents, making crosses, raising adequate population and further selections. But selection for plants

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25 producing high amounts of sweet compounds is expensive, time consuming, and relatively inefficient (Yao et al. 1999). There was a need to develop an efficient and low cost method that make these plant based sweeteners utilized commercially. Adari (2015) developed a novel in situ enzymatic transglycosylation of stevioside by pre treating the stevia leaves with cellulaseand adding soluble starch as the glucosyl donor. The results confirmed that the transglycosylation of stevioside led to an enrichment in the rebaudioside A content from amylase from Bacillus amyloliquefaciens in the starch solution to produ ce transglucosylated steviosides with reduced bitter aftertaste was also investigated (Ye et al., 2013). To date, stevia biosynthetic methods has been widely using in the industry, while mogrosides production is still based completely on extraction from fr uit, which result in a high price of this products (Catani et al., 2013) T he biosynthesis pathway of mogrosides has also been extensively studied recent years, and several functional genes have been identified (Dai et al., 2015; Itkin et al., 2016; Zhang et al., 2016). Dai et al. (2015) first reported the ir study of mogrosides synthesized in vivo proposed biosynthetic pathway is shown in figure 2 2. The results also demonstrated that RNA sequencing and digital gene expression profile analysis is a promisi ng approach for identifying genes involved in biosynthesis of mogrosides. Mia n and others (2018) successfully identified CYP87D18 as a key P450 gene involved in the biosynthesis of mogrosides. The P450 supergene family is a large and diverse group of enzym es and plays critical roles in oxidative reactions in the biosynthesis of diverse natural plant metabolites (Nelson et al., 1996). In the yeast BY4741 Z5 expressing SgCbQ/CYP87D18/CPR three

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26 intermediate products were detected between cucurbitadienol and m ogrol. In the future, further characterization of other related genes involved in the biosynthesis of mogrosides may permit engineering of recombinant yeast that produce mogrosides with high yield. M etabolism and Biotransformation of Mogrosides Researchers believed that the glycosidic bonds are not easily broken by either human digestive degradation or the action of intestinal microorganisms, which indicates that the product has neither caloric nor glycemic properties (Suzuki et al., 2005). Mogrosides are therefore non nutritive constitu ents whose sweetness intensity are 100 to 250 times sweeter than sucrose (FDA, 2014). There are a few studies on the metabolism of mogrosides. One important study (for potential use by the food industry) investigated the bio transformation of mogroside III by human intestinal microflora in vitro which suggested that human intestinal bacteria showed potent ability to transform mogroside III to release secondary glycoside mogroside II and the aglycone mogrol (Yang et al. 2007); a recent study illustrated that the MFE was stable in simulated gastric and intestinal juices in vitro as only several intact mogrosides were detected during incubation in model digestion systems (Guisheng et al. 2017). However, Murata (2010) did a resea rch on the in vivo digestion, absorption and metabolism of mogroside V in rats whose results indicated that dehydrogenation, deoxidation, oxidation and isomerization were the major metabolic transformations of mogroside V these results were contrary to p revious reports. Then, t he metabolites were found to be different in the biotransformation of MFE in normal and type 2 diabetic patients in this study. This was consistent with another study in rats, where diabetic model rats produced more metabolites than healthy rats. With respect to distribution studies, there was a study

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27 inves tigating the digestion and absorption of mogroside V in rats (Zhou, Zhang, Li, Wang, & Li, 2018). Researchers found that mogroside V could be degraded by digestive enzymes and inte stinal microflora and was excreted in the feces as mogrol and other metabolites. T he total amount of mogrosides in the feces was about 61% of administered amount ; however no mogroside V was detected in the whole blood or urine and therefore the absorbed am ount of mogroside V and its metabolites was also extremely low. Reseachers claimed that the mogroside V is mostly excreted without absorption in rats. Similar results were presented in another study as well (Xu et al., 2015). After grounded monk fruit admi nistration to rats, mogroside V was the most abundant compound in GI tract of rats. The other metabolisms were mainly excreted by feces, while mogroside V was mainly excreted by urine as the original structure. G RAS Notifications and Manufacturing P rocess of Monk Fruit Extract In July 2009, FDA received a GRAS notice from BioVittoria (Hamilton, New Zealand). The subject of the notice was Siraitia grosvenorii Swingle (Luo Han Guo) fruit extract (SGFE). With mogrosides V constituting more than 30% of their l iquid and in January 2010 (FDA, 2010). This response indicates the ingredient can be used unless there are adverse effects reported by either the marketplace or in the sc ientific literature Since then, m onk fruit extract started to be used as a sweetener and flavor enhancer in foods, as well as use as a tabletop sweetener since then. Based on the primary evidence of safety provided in the first GRAS notice and additional response from the FDA in April 2011. The product they provided was a monk fruit juice concentrate with a soluble solids level of 65 Brix based on the concentration of

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28 mogrosid e V, which was intended for use in conventional foods as well as infant and toddler foods. In January 2018, Nutramax's Luo Han Guo fruit extract powders also received the same response from FDA. Th ose powders were manufactured in a process similar to those described in previous GRNs but with more purification steps the company produced highly purified products (up to 95% mogroside V) using current Good Manufacturing Practices (cGMP). So far, this substance has been used as a sugar substitute in different foods at levels proportional to those specified in five different GRAS notices (GRNs 301, 359, 522, 556, and 706). In general, the commercial preparations (extracts) described in the GRAS notifications discussed above were obtained by mechanically crushing or shredding the fruit, which was then extracted with hot water. Centrifugation and ultrafiltration were then used to remove protein and pectin to produce a liquid fruit concentrate. Activated carbon and/or adsorption/separation polymer resin columns were used to further purify the ingredient(s) of interest by absorbing glycosides onto their surface(s). The desired components were then washed from the resin with ethanol, which could then be removed by evaporation. Extracts as described in the two GRAS noti fications are now commercially available (Pawar et al., 2013). Orange Juices and Orange Juice Beverages In the United States, orange juice is the most popular juice per capita, leading juice consumption at 10.2 liters in 2015 (USDA, ERS. 2015). Meanwhile the interest in developing functional food is rising, driven largely by the market potential for foods and beverages that can improve the health and well being of consumers (Hilliam, 2000).

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2 9 Experiments (da Costa et al., 2017; Luckow & Delahunty, 2004) ha ve shown that a population of consumers significantly preferred the functional orange juices. This indicates that a potential market for functional orange juices does exist (Luckow & Delahunty, 2004). Furthermore, orange juice is a candidate for applicatio n of sweeteners studies since its fresh flavor characteristics are favored by customers (Liu, 2003) but many consumers are seeking that flavor in a lower calorie product. In addition, because these products are generally preserved by thermal processing (Ji menez Sanchez et al., 2017), this is an appropriate application for a potential monk fruit sweetened product. However, it should be clearly defined the differences between r egulations governing fruit juices. Generally, only beverages that are 100% juice can be Accordingly, in the European Union and many other countries, the term orange juice may only be used for juice extracted from sweet oranges, Citrus sinensis In the US, h owever, regulations allow for up to 10% of tangerine or hybrid orange/ tangerine juice to be included in orange juice. Codex standards, a collection of internationally recognized standards relating to foods, permit the inclusion of 10% mandarin juices as w ell. These added mandarin type juices are thought to improve the color and flavor of blended juice. Important Quality Parameters of Orange Juice Beverages USDA (1983) issues the grade standards for fruit juices. Grade standards concern product quality. Th

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30 meeting all the specifications defined for such grades by the USDA. For instance, some of the requirements that orange juice produced in Florida to be labelled as Grade A must meet are shown in Table 2 1 (Fellers, 1990) Analytical parameters can be determined by standard methods of analysis to give reliable results. The quality factors are measured on a 100 point scale. For example, orange juice flavor is evaluated by sensory means; As for color specification, juice manufactures would perform visual comparisons of orange juice in a standard glass tube to set of USDA plastic comparators also in glass tubes (Lee, 2000) The most important properties of orange juice are its sugar content and ratio of sugar to acid (Fellers, 1990) Brix degree for orange juice normally represents the sweetness of products, not only includes the concentration of dissolved sugars but all soluble solids. The level of acid is often measured to indicate the acidity in the juice. Thus, t his ratio is an important indicator for taste. As orange ripen, the ratio increases as sugars are formed and the acid content decreases (Fellers, 1990) Orange Production and the Orange Juice Market Currently, citrus producers in many countr ies are facing serious citrus production problems (Spreen and Zansler, 201 6 producer, Brazilian commercial orange production continued decreasing due to high temperatures and stress from the precious production cycle (USDA 2018). At the same a smaller crop in several provinces (USDA, 2019) Huanglingbing (HLB) or citrus greening was responsible for the decrease in the production of citrus in the United States from 7.98 to 2.22 billion ton s (72.2% reduction) from 2007 08 to 2017 18 (USDA 2018). Additionally, U.S. orange production was decreased due to other reasons

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31 including the damage by Hurricane Irma in 2017 as well as the recent hot weather in California (USDA 2017). However, the fresh fruit market was less impacted than the juice industry, as the juice industry relies on large quantities of fruit for processing efficiencies. Around 90% of the oranges produced in Florida are used for producing it not showing HLB symptoms in this state (Dala Paula et al., 2019) Singerman et al. (2017) reported an increase from $2.89 to $9.34 (3.2 times) of the price of a box of orange since HLB had been detected and widespread in the United States, presumably due to lack of supply. H owever, the control of HLB is still difficult; one of current strategies focus on vector control and management of infected trees (Batool et al., 2018) which are theoretically possible and effective. However, the bacteria species associated with HLB are mainly transmitted by insects, namely psyllids (Bov 2006). Methods developed to prevent contact between the pest and trees are costly and time consuming (Ferrarezi et al., 201 7b). The most effective control strategy has been to remove infected trees in a growing area, but it is uncertain how long a tree can be infected before showing the symptoms of the disease (McCollum and Baldwin, 2017). Florida growers have been using folia r nutritional spray products to compensate for lack of nutrient by the disease. Unfortunately, the beneficial effect of this approach may not manifest unless the vector is thoroughly controlled, since the trees are still infectable (Gottwald et al., 2012; Plotto et al., 2017).

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32 Figure 2 1. Structures of main mogrosides isolated from the fruits of monk fruit

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33 Figure 2 2 Proposed biosynthetic pathway of mogrosides in monk fruit (adapted from Dai et al. 2015) : SE, squalene epoxidase; SgCbQ, cucurbitadienol synthase; P450s, cytochromes P450; UGTs, uridine diphosphate glycosyltransferases. Dark solid arrows represent one step; dark dotted arrows represent multiple steps

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34 Table 2 1. USDA standards for Grade A orange juice products from Florida a Standard Parameters Orange Juice (Not from concentrate)/ Processed Orange Juice Products/ Pasteurized Orange Juice Analytical factors Brix (% w/w) Min. 11.0 Brix to acid ratio 12.5 20.5 Quality factors Appearance Fresh orange juice Color Very good, 36 40 points Flavor Very good, 36 40 points Minimum score b 90 a Adapted from Fellers, 1990 b A limiting rule applies in which the lowest score of any one factor determines the grade

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35 CHAPTER 3 MATERIALS AND METHODS Retail Screening of C ommercial Low calorie O range J uice B everages Low calorie orange juice beverage products found at the retail market were evaluated for formula determination. The commercial low calorie OJB included Trop50 No Pulp (Tropicana Pr oducts, Inc., Chicago, IL), and Minute Maid Pure Squeezed Light No Pulp Orange Juice (The Minute Maid Company, Sugar Land, TX). The se were analyzed for standard quality parameters and sensory sweetness perception. The nutrition facts of these commercial p roducts were also summarized for formula adjustment. Beverage Analysis Total Solids (Brix) Measurement Soluble solids (Brix) were measured using a hand refractometer (0 32% Brix, Fisher Scientific, Pittsburgh, PA, USA). Refractive index was recorded an d converted to Brix. Measurements were performed at 20.0 0.5 C. The refractometer prism was cleaned with distilled water after each analysis. pH Determination The pH of treated beverages and untreated orange juice samples was measured using a digital p H meter ( A ccumet AB150 pH Benchtop Meters, Fisher Scientific, Pittsburgh, PA, USA). The meter was calibrated with commercial buffer solutions at pH 7.0, 4.0 and 2.0. Samples (10 mL) were placed in a 50 mL beaker with a magnetic stirrer and pH electrode ins erted. Samples were measured at 20 0.5 C.

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36 Titratable Acidity (TA) Samples of 20 mL were placed into a 250 mL beaker, and 80 mL of distilled water was added. This solution was then titrated against standardized 0.1 N NaOH (Sigma A ldrich, Dublin, Ireland) to the phenolphthalein end point (pH 8.2 0.1). The volume of NaOH was converted to grams of citric acid per 100 mL of juice, and TA was calculated. Color Determination Samples were cooled to room temperature (20 1.0 C). Color was measured using a Hun ter Laboratory colorimeter ( ColorQuest XE Hunter Associates Laboratory Inc., Reston, VA) based on three color coordinates, namely, L*, a*, and b*. The instrument was calibrated using white and black reference tiles. Color values were measured using total transmission mode. Viscosity Determination The viscometer (RVDV II+P, Brookfield Engineering Laboratories, MA, USA) drives a spindle immersed in 500mL test fluid. When the spindle is rotated, the viscous drag of the fluid against the spindle is measured by the deflection of the calibrated spring. The spindle type and speed combinations will produce satisfactory results when the applied torque is between 10% and 100%; Spindle S01 was used in this experiment. And viscosity of each samples was determined at 10 0 RPM spindle speed. Mogroside V Stability in Model Juice Systems Two citric aci d added model systems (pH 3.5 and 5.0) were prepared into which pure mogroside V was dissolved at 25mg/100mL. The heating experiments were carried out at 72 C for 15s and at 9 0 C for 30s, as commonly used thermal processing methods in juice industry. Samples of the sweetened model juices with and without

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37 heating treatment s were kept in tightly closed glass tubes and stored in the refrigerator at 4C. The samples were chemicall y monitored periodically over shelf life for 90 days. LC MS/MS were used to analyze compound losses and degradation products. Model J uice P reparation A representative model juice was formulated and prepared according to the screening of purchasable produc ts at the retail market. This study mainly focused on the stability of main ingredient, mogroside V (98%, from Siraitia grosvenorii (Swingle), Frontier Scientific, UT, USA) in acidic liquid after thermal processing and over shelf life. Thus, distilled wate r was used as a simple basement in model juice. Sodium citrate (Fisher Scientific, NJ, USA) and citric acid (Fisher Chemical, NJ, USA) were also added to reach the desire pH 5.0 and 3.5, which represented the pH range of many juice products. In addition, 6 .0g sucrose was added to replicate the soluble solids content of commercial products, and mogroside V was added and tasted to achieve the equivalent sweetness in comparison to the commercial products as a reference. Model juice of different pH levels were mixed separately and pooled into 3 groups of 10 mL tubes: untreated juice (Control), Treatment 1, and Treatment 2. Thermal P rocessing T reatment on M odel J uice Pasteurization methods applied on model juice were selected with reference to industrial practice where heating of orange juice is carried out (Braddock, 1999). Treatment 1 represented a conventional mild temperature short time (MTST) thermal processing at 72 C for 15s (Walkling Ribeiro et.al. 2010). Treatment 2 represented a typical high temperature short time (HTST) thermal treatment of orange juice at 90C for 30s (Aguilar Rosas et al 2013). Both methods met the requirements of FDA and could

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38 result in a population reduction of at least 5 log in Salmonella as well as other potential pathogens in or ange juice (Petruzzi et al. 2017). A water bath (Precision GP 05, Thermo Fisher Scientific, Waltham, MA) was used for thermal treatments. After the target temperature reached, 15 mL glass tubes with 10 mL of model juice, closed with plastic caps, were subm erged into heated water bath. The liquid level in the test tubes were below the water level. An additional tube with the rmometer was added to measure and indicate the temperature. Tubes were removed and immediately cooled in an ice bath right after the hol ding time for each treatment to mimic the industrial practices. Shelf L ife S tudy and S tability T est of P ure M ogroside V A 90 day shelf life storage test for the model juice s w as carried out at 4 C. Samples were kept in tightly closed glass tubes during shelf life. The model juices were chemically monitored periodically. LC MS/MS were used to analyze mogroside V losses every 30 days. The effect of heat treatments was evaluated by comparison the mogroside V content of non treated samples and samples right after thermal treatments. Stability of mogroside V at different pH at were also determined. Preparation of S tandard S olutions Stock solution of mogroside V was prepared by dissolv ing the mogroside V powder in water at a concentration of 1.0 mg/ml. Additionally, stevioside was selected and used as an internal standard in this study due to its similar molecular weight to mogroside V. Stock solution of stevioside was also prepared in water at a concentration of 1.0mg/ml. A mixed solution was diluted with water to obtain a series of standard solutions with concentration of 0.25, 0.5, 1, 2, 5, and 10 mg/L. Linearity of response was

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39 determined for six concentrations. The calibration curve was based on the relationship of mogroside V concentration to peak area. Sample P reparation Accurately measured 10 L model juice from each glass tube and 10 L of internal standard, stevioside solution (0.2 mg/mL) were introduced into a 2mL centrifuge t ube and diluted with 1 mL water. The mixture was vortexed for 30 seconds to ensure the ingredients were dissolved. Then, 70 L of the formed sample were transferred into a screw neck vial. A 10 L solution was injected for LC MS/MS analysis. LC MS/MS A nalys is The liquid chromatography (LC) separation was performed using an Ultimate 3000 system (Dionex, Sunnyvale, CA, USA), equipped with a rapid separation (RS) pump, an RS column compartment and an XRS open autosampler. A ACQUITY UPLC BEH C18 column (150 2. 1 mm, 1.7 m particle size) at a column temperature of 35C. The mobile phase consisted of water (solvent C) and acetonitrile (solvent D) with gradient elution as follows: 0 5 min, 90% solvent C; 5 10min, 95% solvent D; 10 15min, 90% solvent C. The flow rate was set to 0.2 mL/min and the injection volume was 10 L. The mass spectrometry (MS) was carried out using a triple quadrupole mass spectrometer (TSQ Quantiva, Thermo Fisher Scientific, San Jose, CA, USA) with selected reaction monitoring (SRM). The instr ument was operated with a heated electrospray ionization (HESI) in positive/negative ion mode. The ion source conditions were set as follows: positive ion, 3500V; negative ion, 2500V; sheath gas, 35 Arb; aux gas, 10 Arb; sweep gas; 0 Arb; ion transfer tube temperature, 325C; and vaporizer temperature, 275C. The MS/MS parameters were optimized as follows: collision gas pressure, 2mTorr; source fragmentation voltage, 0 V; chrom filter, 0 s; and dwell time,

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40 200 ms. The SRM transitions, collision energies and RF lens of the analytes and internal standard are shown in Table 3 1 Xcalibur software (Ver. 3.0) was used for data processing and instrument control. S ensory Evaluation of Prototype Orange Juice Beverages Initial formula was determined using conventional orange juice as a reference. After formulation, b ench scale products were prepared for preliminary test which show ed the perception of MFE and provide some guidance for final formula determination Then, samples were reformulated and a djusted feedback in preliminary test. Prototype beverages were evaluated by panelists in final sensory evaluation. The aim of this phase was to investigate consumer hedonic and sensory perception of prototype orange juice beverages with MFE. Meanwhile, the sensory differences of stevia and MFE as a sweetener used in orange juice beverages were also determined The effects of information about sugar reduction and the use of sweeteners were indicated. Preliminary Test on Bench Scale Produc ts The method of the preliminary test involved in two basic steps: formulation determination and sensory test. In order to prepare low calorie orange juice beverage comparable to commercial Trop50 conventional Tropicana orange juice, purified water, food grade citric acid, and commercial monk fruit extract sweeteners were used for formulation. All formulated samples were prepared by following method: 50% Tropicana orange juice (v/v), 50% water (v/v ), and then monk fruit extract (Monk Fruit in the Row, Cumberland Packing Corp. NY, USA) and citric acid (Now Foods, IL, USA) were added to replicate the taste of conventional orange juice. Tropicana orange juice with no pulp was used as flavor reference t o determine the best formulation. Brix value,

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41 pH and titratable acidity were measured to assist the formula determination and indicate the sweetness, sourness, and balance of sweetness with sourness of tested formulation ( Parish, 1998 ). The dextrose exis ted in the commercial monk fruit extract product was aimed to make the products measurable for customers using, but it could influence the Brix degree. Accordingly, the amount of sweetener and citric acid used in each product was adjusted and determined by tasting Formula details are shown in appendix B. In the preliminary sensory test, 31 panelists were selected randomly on campus, in which there were 12 male panelists, 19 female panelists, to conduct the a single blind taste panel on randomized coded jui ce samples. Tropicana orange juice with no pulp (OJ Original), Trop50 and the formulated orange juice beverage (FOJB) were evaluated with 12 attributes with respect to appearance, texture, flavor, and aftertaste of juice beverages. Overall appearance, c olor, aroma, overall flavor, and overall freshness were evaluated based on 9 hedonic scale. Flavor and mouthfeel attributes including sweetness, sourness, thickness, and tartness were evaluated with 5 anchored scale, in which situation, rating of 3 represe sample was also evaluated to indicate the lingering effect resulting from juice beverages. Ultimately, panelists were asked to rank the samples from 1 to 3 to indicate their preference on the 3 juice beve rages. They were also asked to rate their potential to hedonic scale. Two for data analysis. Pilot Plant S cale Products M anufacture Based results derived from the preliminary test, t wo different formulas of low calorie orange juice beverages were de veloped The formulas of sample A and B were

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42 the same formulation but with different MFE sweetener concentration based on results and comments from the preliminary test. Sample A has the same sweetness level with regular orange juice based on the result of sweetness equivalent test. However, considering reducing the potential bitter taste of sugar substitute and sav ing cost, sample B was prepared with 25% less amount of MFE sweetener added. Additionally, natural and added to the all final samples to create similar appearance to commercial product s. After the addition of all ingredients and mixing, the orange juice mixtures A and B were pasteurized individually in a pilot scale pasteurizer with a tubular heat exchanger (UHT/HTSTLab 25EHVH, MicroThermics, NC, USA ) at 90 C for 30 s. Details of the thermal treatment are described: samples flow rate was maintained at 1L/min, which was shown on the flow meter; products were preheated to 60C, and then continuously heated until the temperature reached 90 C; products were held in the holding tube with the temperature maintained at 90 C for 30 s; the cooling tank equipped in the system was used to make the product temperature was controlled to 40C ; the pasteurized and cooled juice beverages were aseptically collected into aseptic containers with sealed lids and stored at 4 C for 36 hours until final sensory evaluation. Final S ensory E valuation Customer Preference T est Final sensory test was performed in the Taste Panel at the University of Florida, in a separate booth area to enable participants to c onduct the task comfortable and without distractions. Pasteurized sample A, sample B and commercial product Trop50 were evaluated by sensory test. Commercial low calorie orange juice beverage Trop50 contains stevia as sweetener to maintain the sweetness was used as a comparison to

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43 provide reference for the use of MFE in lower calorie juice based beverages. Tests were conducted in the morning, three samples (2oz each) were served in clear plastic cups, received random 3 digit codes and they were presented to the consumers at one time but in a randomized sequence. 92 panelists were recruited based on orange juice consumption and acceptance of low calorie beverages. Demographic information was collected, such as age and gender. Consumer familiarity with juice products and light beverages was assessed. Then, they were asked to rate the overall appearance, overall liking of each sample using 9 point scale (1=Dislike extremely, 5=Neither like nor dislike, 9= Like extremely). The color, sweetness, sourness, bitterness, and thickness of each sample were also asked by using just about right 5 anchored hedonic scale (e.g. 1= Not nearly sweet enough, 2=Somewhat not sweet enough, 3=Just about right, 4=Somewhat too s weet, 5=Much too sweet) to investigate the optimum levels of these attributes in tested products. Check all that apply questions composed of 11 sensory terms: Smooth, Delicious, Lingering, Refreshing, Fresh, Chemical, Fruity, Natural, Artificial, Thin/wate ry, and Other. These sensory terms were selected based on former studies (Reis, Alcaire, Deliza, & Ares, 2017) in which consumers were asked to describe low calorie juice products. Panelists were asked to rank all samples and indi cate their purchase intents on each sample. Consumer attitude toward purchasing products was asked again after product ingredient and nutrition information was given. Data were collected using Compusense software (Compusense Inc., Guelph, Ontario, Canada). Ballots shown in the appendix

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44 Statistical Analysis Data are shown as mean standard error (SE) of triplicate measurements. Sensory data was analyzed with analysis of variance (ANOVA) using Compusense software (Compusense Inc., Guelph, Ontario, Canada). Difference (HSD) test was used for mean comparisons as well. Statistical analysis on instrumental data was carried out using ANOVA by GraphPad software (GraphPad Software, CA, USA).

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45 Table 3 1. SRM transitions, collision energies and RF lens for LC MS/MS analysis of samples Compound Retention Time (min) Precursor ion (m/z) Product ion (m/z) Collision Energy (V) RF lens (V) Mogroside V 4.8 1285.64 1123.56 55 221 Stevioside 5.3 803.34 641.23 19.05 101

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46 CHAPTER 4 RESULTS AND DISCUSSION Stability Studies on Mogroside V in Model Juices Standard Curve of Mogroside V As LC MS/MS was used for compound analysis, l inearity of detector response was determined in the concentration range 0.25 10 mg/L of m ogroside V using 6 standard solu tions. A linear cali bration curve was obtained with a correlation coefficient of 0.9995 (Fig. 4 1). Heat and pH Stability In citric acidified aqueous solution mogroside V was remarkably stable after thermal processing (Fig. 4 2). The mogroside V calculated amounts represent mean values from triplicate analyses. Control values indicate the amount of mogroside V in the model liquid without heat treatment at the same time as other treatments. In each pH group, thermal processing did not significan tly affect the levels of mogroside V. Aqueous solutions containing mogroside V are reported to be stable under boiling conditions (Nabors and Gelardi, 1986). Additionally, different pH level also r thermal treatments. Although there are no literature reports detailing the stability of mogroside V. It is occurring in fruit, which is usually a low pH environment and t he indigenous use of monk fruit involves drying, boiling, indicates that the se bioa ctive compounds are likely to be a heat and pH stable molecule. In addition, s tructurally, Lindley (2012) believed that mogrosides should be stable since they resemble the steviol glycosides that are known to exhibit excellent stability. Early work by Lee (1975) also showed that linkages of glucose units ensure that the mogroside V is a stable compound and intrinsically resistant to

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47 hydrolysis. Overall, in no study was there a significant difference detected under thermal treatments in either pH 3.5 or 5 products, indicating pH and heat stability of the compound of interest, mogroside V. Also, this result demonstrated that there had been no loss of sweetener nor any interaction with citric acid in the acidic model juice system under commonly used ther mal treatment 1 (72C, 15s) and treatment 2 (90C, 30s). Shelf Life Study/Storage Stability The results from the model juice storage trials are documented in Fig. 4 3. The amounts of mogroside V also represent mean values from triplicate analyses. Accordi ng to data analysis, pH 3.5 and pH 5.0 acid conditions did not result in any significant degradation of mogroside V during 90 day storage at 4C. Moreover, the four groups of pasteurized model liquid stored after 90 days did not show any significa nt change in their mogroside V levels. The results from repeated experiments were shown in Fig. 4 4. Thus, the fact that no significant differences was detected for 90 days shelf life for both studies suggest that mogroside V does not deg rade in typical juice beverage applications. The stability of mogrosides has been investigated in baked mince pork slices as natural antioxidants during the storage by Cheng and others (2017) their results showed that after baking process (180C for 3min) more than 75 percent mogrosides could be retrieved for all groups after the storage, thus, they claimed that mogrosides were relatively stable during storage for 21 days at room temperature in pork products. The conclusion was consistent with t he analyti cal results of this study that pH, storage time, and the interaction of pH and storage time do not have any significant degradation regarding the initial mogroside V levels. Concentrations were maintained at the same level indicating that mogroside V in ac idic liquids stored at 4C was stable, even after 90 days storage.

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48 Sensory Evaluations on Application of Monk Fruit Extract in Prototype Orange Juice Beverages D etermination of Optimal Formula Sweetened with Monk Fruit Extract According to the lab scale sensory test by a small group of panelists, the optimal formulation for orange juice beverage was determined details are shown in appendix B T hat was 0.2% citric acid and 1.6% monk fruit extract (w/v), adding to orange juice mix with purified water in 1:1 ratio. Mogroside V has been rated as being in the rages 250 425 sweeter than sucrose by human taste panel, depending on the concentrations of the tested samples (Kinghorn et al., 1998). The optimal formula in this study was selected by comparing the sweetness and sourness with reference original orange juice samples. Formul ated sample tasted most similar to original orange juice with an acceptab le pH level and Brix degree, thus it was brought up to upcoming preliminary sensory analysis to be evaluated. The pH, degrees Brix and acidity of the sensory products is shown in Table 4 1. Based on the result, formulated beverage showed a lower pH degree and acidity, which indicated the taste of formulated OJB might be sourer than other samples. Also, the Brix level was slightly higher than the Trop50 commercial product The pure monk fruit extract had no effect on the Brix level. However, the reason for the increased Brix degree was the dextrose existed in the commercial monk fruit extract product as discussed above. Dextrose, as know as glucose, is a simple sugar derived from corn. The relative sweetness value for dextrose is 74 reported by Biester, Wood and Wahlin (1925). According to the product introduction, dextrose was added to many sugar substitutes that in powder form to make it measurable for consumers. Thus, the main sweetening ingredient in Monk Fruit In the raw is simply monk fruit.

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49 Preliminary Sensory Test: Comparison of Commercial Products and Tested Formulation After preliminary development the formulated sample, conventional orange juice and Trop50 were prepared and evaluated by 31 panelists in the preliminary test. Before tasting, panelis ts were asked to observe the samples only to evaluate the appearance, color and aroma. As shown in Table 4 2 the overall appearance and aroma of original orange juice and Trop50 had no significant difference, but significantly higher rated than that of f ormulated OJB. It was reasonable due to the dilution of color with the addition of water in the formulation. The aroma and overall flavor of original orange juice was significantly higher rated than that of Trop50 and formulated OJB, which indirectly veri fied that the higher concentration and abundance of flavor active compounds in orange juice played an important role for rich flavor and pleasant aroma of orange juice. With reduction of orange juice compensated with water, it was difficult to maintain the similar flavor profile as it before dilution. However, it was notable that rating of overall freshness of FOJB was significantly higher than that of Trop50 which may indicate that compare to Trop50 the overall acceptability of OJB with MFE was better received by participants. The results shown in Table 4 3. was observed that the formulated sample was rated with significantly higher sweetness and significantly lower tartness than the other two products, while the thickness rating was significantly lower than the other two products. The sourness level of formulated OJB and Trop50 was similar, but significantly lower than the sourness of Tropicana Orange Juice. Results of sweetness and sourness complied with the results obtained from chemical analysis as shown in Table 4 1. The formulated OJB with lower acidity but higher Brix value explained the

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50 sourness and sweetness ratings in sensory analysis. According to the ingredient list of Trop50 ( Appendix A ), gellan gum was used as an ingredient, presumably as an emulsifier and thicken agents to increase the mouthfeel of Trop50 making it comparable to real orange juice. Additionally, the lower thickness and tartness ratings of sample were also partially explained b y the same reason for ratings of color and flavor, which was the decrease in characteristic compounds resulting from dilution of original juice formulation. Philipsen and others (1995) also reported that color had specific unique effects on overall accepta nce, flavor quality, and intensity in sensory responses to a flavored cherry beverage. There have been mixed results on how color influences the flavor intensity. Kostyla (1978) reported that lower scores were received when yellow color was added to raspbe rry beverages. However, Dubose et al. (1980) and Johnson and Clydesdale (1982) showed that the flavor ratings of beverages increased as the color intensity increasing. The results of the aftertaste analyses of three samples are shown in Fig. 4 5 Compared with the results from original orange juice, evaluation on aftertaste of Trop50 and formulated OJB were similar but still slightly different Formulated sample had more panelists (23%) rated with favorable aftertaste than Trop50 (13%). Over half of pane lists (52%) believed there was non favorable aftertaste in Trop50 This indicated that the acceptability of the OJB sweeten with MFE in this study might higher than stevia, which also explained that the unfavorable aftertaste of sweetener may be a reason for the low hedonic scores of the Trop50 in the flavor attribute. Guggisberg and others (2010) reported bitter and off flavors in whole milk yogurt sweetened with stevia alone had negative influences on physical and sensory properties. Accordingly, dislik e

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51 of stevia sweetener might also have influenced the preference ratings for formulated OJB, which tended to be higher (p ) than ratings on Trop50 It was a good indicator for the possible success of the final prototype OJB sweetened by MFE in the pilot plant scale sensory evaluation. Final Sensory Evaluation: Customer Preference Test on Sweetener in Low calorie Orange Juice Beverages Sample A and Sample B were formulated based on the results from preliminary test and instrumental analysis. Color additiv appearance, making the color looks like fresh orange juice. The beverage analysis results of final samples shown in Table 4 4. Both product s were pasteurized for panelist food safety and because the commercial products had undergone thermal treatment. After pasteurization at 90C for 30s, t he customer preference test on pilot plant scale products was performed to investigate the suitability o f MFE as a sweetener in orange juice beverages. Comparison between stevia and MFE in terms of sweetness, freshness and aftertaste and other attributors were also determined in orange juice products. A total of 92 panelists evaluated the tested samples. Th e behavioral question suggested that more than 80% of panelists consume light/low calorie beverages (such as diet soda, flavored water, etc.) at least 2 3 times a month. Additionally, more than 70% panelists showed that they consume light/low calorie juice beverages several times a month. Results of o verall liking scores suggested that when tasted three samples in randomize order, panelists preferred samples with MFE sweetener over the commercial

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52 stevia sweetened product (P 0.05; Table 4 5 ). The best overa ll liking was received in sample B made with 25% less MFE regarding to sample A. Trop50 made with stevia, by contrast, received the lowest overall liking score. More specifically, panelists preferred sample B for its appearance and flavor (Table 4 6 ), ind icating that a lower concentration of MFE sweetener in product resulting a proper balance that was more acceptable compared with equivalent sweetness blends. Kamerud and others (2007) means panelists who rated a product with sweetener high in sweetness were not always more likely to rate it high for liking. In contrast, the av erage bitterness rating for a compound was found highly correlated with liking, and the perceived bitterness was obviously increased as the concentration of a given sweetener increasing According to the results of overall appearance rating, Trop50 was ev aluated with the highest score (Table 4 2). However more than 25% of panelists thought the color of Trop50 was consistent with the result of instrumental color analysis. Also, it might e could increase with the darkness. Freely comment result supported the preference test results that orange juice with light color was at least equally acceptable for panelists to samples with dark color. For other major tastes, 60% to 68% of the panelists thought B samples about P 0.05; Table 3), which was significantly higher than the percentages of other tested samples. Trop50 Similar result was obtained by Freitas, Dutra and Bolini (201 6 ), stevia showed a higher perception of bitter taste and the maximum intensity persisted longer than sucrose and other tested sugar

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53 substitutes: sucralose, aspartame, Neotame and saccharin Th is indicates that the addition of color additive made a difference in the acceptability of prototype OJB. Most interestingly, in addition to the influence on taste, the texture was rated no significant difference among samples, even though the analytical v iscosity of sample A and B were nearly half lower than that of Trop50 This evidence confirms that products with proper Koza et al. avor perception by modifying the orthonasal and retronasal odor intensities. Also, the somatosenses of the food and overall multisensory flavor percept could be influenced by color (Spence et al., 2010). Their results were consistent with the result of thi s study as well. The comparison of sample A and preliminary formulated sample indicated that the color plays a significant The results of questions (Fig 4 6 ) showed that 63% of R B. As for Trop50 C ted samples. Descriptor analysis suggested that MFE sweetener has a fresh and natural flavor note in prototype orange juice beverage, compare to stevia which may contribute to some negative and bitter taste. This also confirmed the result of aftertaste eva luation in preliminary test. In the end, consumers indicated their purchase intent of each sample. The result indicated that more than 64% of panelists were willing to buy sample B, which was consistent with the ranking results that sample B was rated the best among samples.

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54 purchase intent. Finally, r esults also indicated that healthier and sugar reduction label erages significantly ( P 0.05 ) The top two boxes rate increased to 73 % when product information Natural Light Orange Juice Beverage, made with 50% less sugar and calories than was given consistent with Krutulyte and others (2011) reported that familiarity of products and functional ingredients leads to higher purchase intent for functional foods. Additionally, results from Reis and others (2017) suggested that the information increased consumer sensory a nd hedonic rating of sugar reduced products, and it could influence consumer perception of physical health and emot ional aspects of wellbeing.

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55 Figure 4 1. The calibration curve of the dependence of a peak area on the concentration of mogroside V:MV, mo groside V. Figure 4 2. Effect of heat treatment1 (72C, 15s) and treatment 2 (90C, 30s) on mogroside V in pH 3.5 and pH 5.0 model liquids.

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56 Figure 4 3. Effect of storage on mogroside V in pH 3.5 and pH 5.0 model liquids after heat treatment1 (72C, 15s) and treatment 2 (90C, 30s) Figure 4 4. Results from repeated storage test: effect of storage on mogroside V in heat treated (treatment 1, 72C, 15s ; treatment 2 90C, 30s) model systems.

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57 Table 4 1. pH, soluble solids and acidity of three or ange juice products Parameters Tropicana Orange Juice a Trop50 Orange Juice Beverage Formulated Orange Juice Beverage pH 3.78 3.88 3.52 B rix (%, w/w) 11.6 6.0 7.8 Acidity ( g/L ) 0.80 0.68 0.63 a Results represent mean values from triplicate analyses. Table 4 2 Descriptive analysis scores for attributes of appearance and overall flavor in preliminary test evaluated by 31 panelists Product s Appearance a,b Color Aroma Flavor Freshness Tropicana Orange Juice 6.8a 6.7a 6.7a 6.6a 6.8a Trop50 Orange Juice Beverage 6.4a 6.2a 5.9b 5b 4.8c Formulated Orange Juice Beverage 5.6b 5.4b 6.1b 5.3b 5.5b a Evaluation of each attribute was based on 9 hedonic scale (1=Dislike extremely, 5=Neither like nor dislike, 9=Like extremely) b Different lowercase letters indicated a significant difference. Table 4 3 Descriptive analysis on attributes of basic flavor and texture in preliminary test evaluated by 31 panelists Product s Sweetness a,b Sourness Thickness Tartness Tropicana Orange Juice 2.8b 3.1a 2.9a 3.2a Trop50 Orange Juice Beverage 3.1b 2.7b 2.8a 2.9a Formulated Orange Juice Beverage 3.7a 2.6b 2.5b 2.5b a Evaluation of each attribute was based on Just about right scale 5 anchored hedonic scale (e.g. 1= Not nearly sweet enough, 2=Somewhat not sweet enough, 3=Just about right, 4=Somewhat too sweet, 5=Much too sweet) b Different lowercase letters indicated a significant difference.

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58 Table 4 4 Quality evaluation of Tro p50 and reformulated samples A and B Parameters Trop50 Sample A Sample B pH 3.88 3.45 3.45 B rix (%, w/w) 6.0 7.6 7.1 Color a L*=24.54 L*=32.24 L*=32.22 a*=13.33 a*=9.73 a*=9.76 b*=41.76 b*=53.24 b*=53.34 Viscosity (cp) b 23.0 13.2 13.1 a Color scores shown in CIELAB color scale b Rotational viscometer: 100RPM, S01spindle Table 4 5 The overall liking and overall appearance liking results for samples in final sensory evaluation with 92 panelists Sampl es Overall Liking a,b Overall Appeara nce Overall Texture A 5.72 ab 6.54 b 6.08a B 6.16 a 6.70 ab 6.22a Trop50 5.63 b 6.92 a 6.27a a Attributes were scored on a 9 point hedonic scale where dislike extremely = 1 and like extremely = 9. b Different letters in rows following means of each attribute indicate significant differences (P 0.05). Table 4 6 Customer preference results for low calorie orange juice beverages in final sensory evaluation with 92 panelists Sample s Color a,b,c Sw eetness Sourness Bitterness A 65.22 % b 48.91 % c 48.91 % b 54.35 % b B 70.65 % a 68.48 % a 59.78 % a 67.39 % a Trop50 70.65 % a 58.70 % b 53.26 % ab 52.17 % b a Just about right scales were scored on a 5 point scale b about c Different letters in rows following means of each attribute indicate significant differences (P 0.05).

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59 Figure 4 5 Result of aftertaste evaluations on tested beverag es in preliminary test with 31 panelists : TOJ, Tropicana Orange Juice; T50, Trop50 ; FOJB, Formulated orange juice beverage. Figure 4 6 Results of descriptive evaluations on Trop50 and prototype s ample s in final sensory evaluation with 92 panelists : *significant difference (P<0.05) ; ** (P<0.1) 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Smooth Delicious Lingering Refreshing Fresh Chemical Fruity Natural Artificial Thin/watery Sample A Sample B Trop50 ** *

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60 CHAPTER 5 CONCLUSION The stability and sweetness intensity perception of MFE in orange juice beverages, and the appropriate sweetness concentration levels in orange juice beverages for target customers were d etermined in this study. Citric acidified aqueous solutions under two pH levels (pH 3.5 and 5.0) with 25mg/100mL mogroside V were stable after different heat treatment regimes and during shelf life storage over 90 days. Results also show that this compound in the samples during storage. The low calorie orange juice beverage sweetened partially by commercially available MFE was acceptable by target panelists. Lowing the sweetness concentration by 25% of MFE in Orange juice beverage samples increased overall liking significantly. People preferred MFE as a sweetener in sugar reduced orange juice beverages over than stevia. In conclusion, MFE can be considered as a potential sugar substitute in the development o f a new low calorie orange juice beverages. Possible limitation of this study would be that all the panelists were recruited from Gainesville, FL area, which may not represent the entire US population. In addition, the orange juice used in final formulati on was commercial pasteurized product, which means these orange juice in final samples had been pasteurized twice. However, that would likely be the case for the commercial product as well, as NFC juice is usually pasteurized before storage and subsequent use in formulated products. Pasteurization times may also influence the freshness or other flavor attributes in beverages. Further research is needed to better understand the impact of heat treatment on orange juice beverages and sweetener. Different flavo ring agents and formulations of Trop50 may

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61 also affect consumer acceptance. Further research could evaluate the different varieties of MFE and the blends of different ingredients in orange juice beverages and other food products.

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62 APPENDIX A INGREDIENT LIST OF TROP50 The ingredients found from the package of Trop50 as follows: Filtered water, not from concentrate pasteurized orange juice, orange juice concentrate, potassium citrate, malic acid, citric acid, magnesium phosphate, ascorbic aci d (Vitamin C), calcium citrate, beta carotene, purified stevia leaf extract, tocopherols (vitamin E), gellan gum, natural flavor, niacinamide (vitamin B3), thiamin hydrochloride (vitamin B2), pyridoxine hydrochloride (vitamin B6), riboflavin (vitamin B2)

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63 APPENDIX B FORMULATION DETAILS Table B 1. The formula information of model juice in stability test Samples Water (mL) Citric Acid (g) Sodium Citrate (g) Sucrose (g) Mogroside V (mg) Model Juice (pH 3.5) 1000 3.022 1.255 60 25 Model Juice (pH 5.0) 1000 1.625 3.393 60 25 Table B 2. The formula information of samples in sensory tests Samples Commercial Orange Juice (mL) Water (mL) Citric Acid (g) Commercial MFE (g) Color Additive (g) Preliminary Sample 500 500 2.25 16 -a Final Formula A 500 500 2.25 16 0.14g Final Formula B 500 500 2.25 12 0.14g a -indicates the ingredient was not used in the formula

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64 APPENDIX C FINAL SENSORY EVALUATION BALLOT Welcome! Today's Samples: Orange Juice Beverages Please indicate your gender: ___Female ___Male Please indicate your age: In which of the following groups would you most likely place yourself? ___ Caucasian ___Hispanic / Latino ___African American ___Native American / American Indian ___Asian ___Other How often do you consume orange juice? ____Daily ____3 4 times a week ____Once a week ____2 3 times a month ____Monthly How often do you consume light/low calorie beverages? For exampl e, diet soda. ____Daily ____2 3 times a week ____Once a week ____2 3 times a month ____Monthly ____Rarely How often do you consume light/low calorie juice beverages ? ____Daily ____2 3 times a week ____Once a week ____2 3 times a month ____Monthly ____ I

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65 The next several questions will be on the APPEARANCE of sample 602 Please do NOT TASTE u ntil you are instructed to do so. Please indicate how much you like the appearance of sample 602. Please rate the color of sample 602. Much too light Somewh at too light Just About Right Somewh at too dark Much too dark 1 2 3 4 5 Take a bite of cracker and a sip of water to rinse your mouth. Remember to do this before you taste each sample. You are now ready to taste sample 602. Please indicate how much you like sample 602 overall Please indicate how much you like the flavor of sample 602. Please indicate how much you like the texture of sample 602. How would you describe the Thickness for sample 602? Thickness

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66 Much too thin Somewh at too thin Just About Right Somewh at too thick Much Too thick 1 2 3 4 5 How would you describe the sweetness for sample 602? Sweetness Not Nearly Sweet Enough Not Quite Sweet Enough Just About Right A Little Too Sweet Much Too Sweet 1 2 3 4 5 How would you describe the sourness for sample 602? Sourness Not Nearly Sour Enough Not Quite Sour Enough Just About Right A Little Too Sour Much Too Sour 1 2 3 4 5 How would you describe the bitterness for sample 602? Bitterness Not Nearly Bitter Enough Not Quite Bitter Enough Just About Right A Little Too Bitter Much Too Bitter 1 2 3 4 5 Which of the following words best describes this light orange juice sample? Smooth Lingering Delicious Chemical Refreshing Fruity Thin/Watery Natural Artificial

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67 Other_____ How else would you describe sample 602? Now that you have tasted sample 602, how likely would you be to PURCHASE it if it were available at a store where you usually shopped? Definitely would not buy Probably would not buy Might or might not buy Probably would Buy Defi nitely would buy 1 2 3 4 5 Beginning with the sample on the left, please taste each sample again and RANK them from the MOST preferred to LEAST preferred. Please carefully read the following product description and then answer the question below. Introducing a NEW, all Natural Light Orange juice, made with 50% less sugar and calories than regular juice, No artificial sweeteners, and only 50 calories per serving. Based on the product description above, how likely would you be to purchase the type of light or low calorie orange juice beverages. If it were available at a store where you usually shop? Definitely would not buy Probably would not buy Might or might not buy Probably would Buy Definitely would buy 1 2 3 4 5 Please feel free to comment on what you liked/dislike about sample 602.

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79 BIOGRAPHICAL SKETCH Zhou Zou came from Changsha, the capital of Hunan province in the south part of China. Her hometown is known for its long history, creative and passionate people, traditional street food and the largest entertainment television media system in China. Changsha g ave her a lot of creativity and imagination, as well as the courage to try new things. Zhou grew up with her grandparents, who has influenced her a lot. Her grandfather is a hardworking and popular doctor who has no judgement of his patients and received m any respects and awards. Zhou developed an early interest in life science during her childhood. At meantime, her grandmother, who is still the best chef in her mind, always has tons of ideas for delicious homemade food. Food and love are tightly tied Her parents have always insisted on giving her the best education and developing her independency and critical thinking ability. Zhou and her family love travelling, these interesting experiences with different cultures inspired her to study aboard. During her undergraduate studies in Nanjing Agricultural University, Zhou completed her course works in the first three years and was admitted as an exchange she Rene Goodrich Schneider with a specialization in low calorie orange juice beverage development. Under her instruction, Zhou gained the experiences in working as a lab assistant, getting food safety training, pre senting at conferences, participating competition and receiving awards. Upon graduation, Zhou plans to pursue a career to apply her skills in food industry.