Journal of Organic Biochemistry at St. Andrews, Volume 2 (September 2021) by St. Andrew's Episcopal School

VOLUME 2, SEPTEMBER 2021

Journal of Organic Biochemistry at St. Andrew’s Review articles researched and written by Upper School students.

A Note From the Editor

elcome to the second annual issue of the Journal of Organic Biochemistry at St. Andrew’s (JOBSA). Each of the following review articles was researched and written by Upper School students as part of their culminating project in Fundamentals of

Organic Biochemistry, a rigorous upper level course for students interested in learning more about the chemical sciences. During the winter and spring, students learn to read current research articles in biochemistry. In April, students present an in-depth analysis of a recent journal article of their choice. Topics this year ranged from using chemical analysis of pottery to determine the diet of past civilizations in central Africa, to gold exploration using eucalyptus leaves, to how to build an inclusive protein database to better address health needs of diverse communities. The final projects that follow require each student to write their own review style article, synthesized from the information they find in multiple professional publications. Each project is then anonymously reviewed by their peers, simulating the publication process found in science today. A selection of the top articles have been collected here. We hope you enjoy the following works and are similarly inspired to explore the way chemistry impacts our world. Regards, Mr. Ferriby

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A Brief Review of the Health Benefits of Human Milk Oligosaccharides for Human Neonates Bowen Gan

Abstract: Human milk oligosaccharides (HMOs) are a group of structurally complex, unconjugated glycans that are copious in human milk. Initially, it was believed that HMOs’ only function was to shape the intestinal microbiota with health benefits, acting as “food for bugs.” Today, more immunological effects and health benefits of HMOs have been discovered. This review is intended to briefly summarize the current knowledge surrounding the effects HMOs can have on human infants’ health and growth. Introduction of HMOs: The composition and amount of HMOs vary depending on time of lactation and the genetic makeup of each woman. Human breast milk contains an average of 5-15g of oligosaccharides per liter, comparing to 0.05g of oligosaccharides per liter in bovine milk.1 Oligosaccharides are comprised of a lactose backbone at their reducing ends and four possible monosaccharides as constructing blocks, including galactose, Nacetylglucosamine, fucose or sialic acid. Those building blocks are branched and elongated in various ways through various linkages, generating approximately 200 different structures identified to date.2 For HMOs’ compositions and linkages between the monosaccharides, see Figure 1 below:

Figure 1: Human milk oligosaccharide composition blueprint. HMOs can contain five different monosaccharides in different number and linkages, namely glucose (blue circle), galactose (yellow circle), N-acetlylactosamine (blue square), fucose (red triangle), and sialic acid (purple diamond). All HMOs carry lactose at the reducing end.3

Because every HMO carries a lactose at its reducing end, the HMO biosynthesis is possibly an extension of the lactose biosynthesis, which takes place in the Golgi apparatus of mammary epithelial cells by the lactose synthase enzyme complex from two precursors, glucose and UDP-galactose.4 How lactose is extended to form the different HMOs remains poorly understood, as every species has highly differentiated oligosaccharides quantities and compositions, which makes it difficult to study HMO biosynthesis in animal models.1 For ethical reasons, most studies done on human are observational, which is another challenge faced by HMO biosynthesis research. The metabolism of HMOs in human neonates is essential for further studying of their postulated benefits. HMOs are resistant to digestion in the infant upper gastrointestinal tract, and able to survive through the low pH in the infant’s stomach as well as digestion by pancreatic and brush border enzymes and reach the distal small intestine and colon in an intact form.5 Despite being intact, HMOs can cross the

epithelial barrier, as shown in studies in which HMOs have been detected in urine and blood of infants.3 Nevertheless, only approximately 1% of HMOs can be absorbed by the infant. The absorption rate of HMOs may depend on infant age, blood group and feeding regime. For infants in the first stage between birth and two months of life, feces of breast-fed infants contain HMOs that are similar to their original forms in the milk sample, suggesting neither bacteria nor intestine absorb HMOs much; in contrast, in the third stage, when feedings other than human milk are introduced, HMOs entirely disappear from the infant's feces.6 Postulated Beneficial Effects of HMOs: As early as the end of the 19th century, scientists had discovered that breast-fed infants had a much higher chance of survival and had lower incidences of infectious diarrhea and many other diseases than “bottle-fed” infants because of the prebiotic effects of human milk (later identified that it is HMOs in the milk). However, since the early 1990s, more and more evidence indicates that not only can HMOs promote the desired bacteria in the infant’s intestine,

but they also have many other promising benefits summarized in Figure 2:

Figure 2: Postulated HMO effects. HMOs may benefit the breast-fed infant in multiple different ways.1

The long known prebiotic effect of HMOs is due to their capacity to promote the growth of certain strains of Bifidobacterium infantis (B. infantis), a “friendly” genus of bacteria that helps maintain a healthy digestive tract. In 2011, Asakuma et al. collected and purified their sample HMOs from the breast milk of 57 healthy Japanese mothers. They made this HMOs medium to grow strains B. bifidum, B. longum subsp. infantis, B. longum subsp. longum, and B. breve, which are frequently isolated from the feces of breast-fed infants. As a result, there is convincing evidence that B. longum subsp and B. bifidum’s growth can be promoted by HMOs while the other two can not.7 In addition, Sela et al. in 2008 identified that certain sequences in the B. infantis genome revealed entire gene clusters that control the expression of specific glycosidases, sugar transporters and glycan-binding proteins dedicated to HMO utilization, suggesting HMOs as B. infantis’ common feeds.8 With HMOs as a stable trophic source, B. infantis can dominate in the infant’s gut to protect it from many diseases. Empowered B. infantis compete over other potentially harmful bacteria for limited nutrients and produce short-chain fatty acids and other metabolites (post-biotics) that create an environment favoring the growth of commensals over potential pathogen.9 HMOs also serve as antiadhesive antimicrobals to directly reduce microbial infections. Many viral, bacterial, or protozoan pathogen have to adhere to mucosal surface to colonize or invade the host. The mechanism of such adhesion is often initiated by lectin (glycan-binding protein)-glycan interaction. For example, Escherichia coli with type 1 fimbriae bind to mannose-containing glycans, and E. coli with S fimbriae as well as Helicobacter pylori bind to sialylated glycans.10,11 In addition to bacterial pathogens, many deadly viruses also employ lectin-glycan mechanism. Hu et al. discovered that many viruses like

noroviruses or rotaviruses, which are among the most common causes of severe diarrhea in infants and young children and responsible for almost half a million deaths annually, colonize the host through glycanmediated attachment mechanism.12 Since some HMOs resemble mucosal cell surface glycans, they can therefore serve as soluble decoy receptors to prevent pathogen binding.13 HMOs are also able to directly modulate intestinal epithelial cell responses. For example, Angeloni et al. proved that the HMO 3’-sialyllactose, when lining with incubating cultured human intestinal epithelial cells, lowers the gene expression of sialyltransferases ST3Gal1, ST3Gal2 and ST3Gal4 and diminishesα2-3- andα2-6sialylation on cell surface glycans.14 Consequently, the attachment of bacteria that use sialylated cell surface glycans to attach to the host's intestinal epithelial cell is significantly reduced. These oligosaccharides also demonstrate their immunomodulatory properties. Healthy humans should produce a well balanced Th1 and Th2 response (controlled by Th1-type and Th2-type cytokines).15 Some HMOs are capable of regulating the cytokines. Acidic HMOs, for instance, were shown to induce cytokines IFN-g and IL-10 in human cord blood T cells, and could

decrease IL-4 production in allergen-specific T cells.16 IL-4 production is associated with peanut allergy, which led to the conclusion that certain sialylated HMOs may contribute to allergy prevention. Sialic acid has been proven to be an essential nutrient for brain development and cognition for human infants.17 Sialylated HMOs, in the meanwhile, contribute to the majority of Sia in human milk, making breast milk a rich source of Sia. Subsequently, breast-fed preterm infants have superior developmental scores at 18 months of age and higher intelligence quotients at the age of 7, suggesting the beneficial effects of HMOs on human neonates’ brain development.18,19 Conclusions: HMOs contribute to the development of the desired microbiota in the intestine, immune system, and brain of newborn infants. Further research on HMOs may contribute to discover new ways of addressing diseases and body development in the neonates. However, despite many in vitro- and animal experiments, HMOs have not been tested extensively in placebo controlled infant studies. Powered, randomized and controlled intervention studies will be needed to confirm causal relevance for human neonates in the future studies.

Bode, L. (2012). Human milk oligosaccharides: Every baby needs a sugar mama. Glycobiology, 22(9), 1147–1162. https://doi.org/10.1093/glycob/cws074 2 Ninonuevo, M. R., Park, Y., Yin, H., Zhang, J., Ward, Rd Chemistry, 54(20), 7471–7480. https://doi.org/10.1021/jf0615810 3 Triantis, V., Bode, L., & van Neerven, R. J. (2018). Immunological Effects of Human Milk Oligosaccharides. Frontiers in Pediatrics, 6. https://doi.org/10.3389/fped.2018.00190 4 Mardones, L., & Villagrán, M. (2020). Lactose Synthesis. Lactose and Lactose Derivatives. https://doi.org/10.5772/intechopen.91399 5 Engfer, M. B., Stahl, B., Finke, B., Sawatzki, G., & Daniel, H. (2000). Human milk oligosaccharides are resistant to enzymatic hydrolysis in the upper

gastrointestinal tract. The American Journal of Clinical Nutrition, 71(6), 1589–1596. https://doi.org/10.1093/ajcn/71.6.1589 6 Albrecht, S., Schols, H. A., van den Heuvel, E. G. H. M., Voragen, A. G. J., & Gruppen, H. (2011). Occurrence of oligosaccharides in feces of breast-fed babies in their first six months of life and the corresponding breast milk. Carbohydrate Research, 346(16), 2540–2550. https://doi.org/10.1016/j.carres.2011.08.009 7 Asakuma, S., Hatakeyama, E., Urashima, T., Yoshida, E., Katayama, T., Yamamoto, K., Kumagai, H., Ashida, H., Hirose, J., & Kitaoka, M. (2011). Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria. The Journal of biological

chemistry, 286(40), 34583–34592. https://doi.org/10.1074/jbc.M111.248138 8 Sela, D. A., Chapman, J., Adeuya, A., Kim, J. H., Chen, F., Whitehead, T. R., Lapidus, A., Rokhsar, D. S., Lebrilla, C. B., German, J. B., Price, N. P., Richardson, P. M., & Mills, D. A. (2008). The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proceedings of the National Academy of Sciences of the United States of America, 105(48), 18964–18969. https://doi.org/10.1073/pnas.0809584105 9 Gibson, G. R., & Wang, X. (1994). Regulatory effects of bifidobacteria on the growth of other colonic bacteria. The Journal of applied bacteriology, 77(4), 412–420. https://doi.org/10.1111/j.13652672.1994.tb03443.x 10 Firon, N., Ofek, I., & Sharon, N. (1983). Carbohydrate specificity of the surface lectins of Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium. Carbohydrate research, 120, 235–249. https://doi.org/10.1016/0008-6215(83)88019-7 11 Parkkinen, J., Finne, J., Achtman, M., Väisänen, V., & Korhonen, T. K. (1983). Escherichia coli strains binding neuraminyl alpha 2-3 galactosides. Biochemical and biophysical research communications, 111(2), 456–461. https://doi.org/10.1016/0006-291x(83)90328-5 12 Hu, L., Crawford, S. E., Czako, R., CortesPenfield, N. W., Smith, D. F., Le Pendu, J., … Prasad, B. V. (2012). Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen. Nature, 485(7397), 256– 259. https://doi.org/10.1038/nature10996 13 Simon, P. M., Goode, P. L., Mobasseri, A., & Zopf, D. (1997). Inhibition of Helicobacter pylori binding

to gastrointestinal epithelial cells by sialic acidcontaining oligosaccharides. Infection and immunity, 65(2), 750–757. https://doi.org/10.1128/IAI.65.2.750-757.1997 14 Angeloni, S., Ridet, J. L., Kusy, N., Gao, H., Crevoisier, F., Guinchard, S., Kochhar, S., Sigrist, H., & Sprenger, N. (2005). Glycoprofiling with microarrays of glycoconjugates and lectins. Glycobiology, 15(1), 31–41. https://doi.org/10.1093/glycob/cwh143 15 Berger A. (2000). Th1 and Th2 responses: what are they?. BMJ (Clinical research ed.), 321(7258), 424. https://doi.org/10.1136/bmj.321.7258.424 16 Eiwegger, T., Stahl, B., Haidl, P., Schmitt, J., Boehm, G., Dehlink, E., … Szépfalusi, Z. (2010). Prebiotic oligosaccharides: In vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties. Pediatric Allergy and Immunology, 21(8), 1179–1188. https://doi.org/10.1111/j.1399-3038.2010.01062.x 17 Wang B. (2009). Sialic acid is an essential nutrient for brain development and cognition. Annual review of nutrition, 29, 177–222. https://doi.org/10.1146/annurev.nutr.28.061807.15 5515 18 Lucas, A., Morley, R., Cole, T. J., Gore, S. M., Lucas, P. J., Crowle, P., Pearse, R., Boon, A. J., & Powell, R. (1990). Early diet in preterm babies and developmental status at 18 months. Lancet (London, England), 335(8704), 1477–1481. https://doi.org/10.1016/0140-6736(90)93026-l 19 Lucas, A., Morley, R., Cole, T. J., Lister, G., & Leeson-Payne, C. (1992). Breast milk and subsequent intelligence quotient in children born preterm. Lancet (London, England), 339(8788), 261– 264. https://doi.org/10.1016/0140-6736(92)91329-7

Use and Efficacy of Cognitive PerformanceEnhancing Drugs Will Kaine

Abstract: Young adults are using Amphetamine-based prescription stimulants considerably more in recent years both medically to treat ADHD and narcolepsy, and nonmedically as a perceived study drug. These medications alter the chemistry of the brain, temporarily increasing dopamine and noradrenaline levels. Recent findings suggest that while prescription stimulants are effective at providing relief for certain neurological disorders, non-prescribed usage is generally ineffective and can lead to adverse health effects and addiction. This article will give an overview of the chemistry, health effects, and usage of prescription amphetamine stimulants. Introduction Racemic α-methylphenethylamine (amphetamine) was first synthesized in 1927 by chemist G. A. Alles while searching for a substitute for ephedrine, a drug which was used to treat for anesthesia-induced low blood pressure. Pharmaceutical use of the drug has evolved over its lifetime, and it is now commonly used in medication to treat neurological disorders such as ADHD and narcolepsy due to its calming, focusing, and wakefulness-inducing properties.1 These properties have also lead to the misuse of prescription amphetamines as study drugs by young adults.2

Adderall is composed primarily of dextroamphetamine (d-Amphetamine) and levoamphetamine (l-Amphetamine). These molecules possess chemical similarities to the neurotransmitters dopamine and noradrenaline, as shown in the figure below. Dopamine is a neurotransmitter associated with pleasure, movement and attention;3 noradrenaline is a hormone that raises blood pressure and blood sugar. Because of these similarities, the amphetamine molecules are able to bond to noradrenaline and dopamine receptors and reuptake proteins, which inhibits the reuptake of the corresponding molecules.

Figure 1: Comparison of the structural composition of amphetamine molecules with relevant neurotransmitters.1

This inhibition increases the quantity of these chemicals in neuron synapses, which consequentially increases the strength of these signals in the brain. Approximately 3045 minutes after the initial administration of amphetamine stimulants, dopamine levels can reach peaks of 700-1500% their normal amount, and noradrenaline levels can reach 400-450% of their normal amounts.1

Chemistry of Cognitive PerformanceEnhancing Amphetamines The most commonly prescribed amphetamine-based stimulant is Adderall®.

paranoia, hallucinations, anxiety, panic attacks, constipation, weight loss, headaches, tremors, and heart disease.4

Figure 2: The effects of d-Amphetamine and l-Amphetamine on dopamine and noradrenaline levels in the user. The graphs show that both chemicals vastly increase levels of these chemicals compared to their baselines.1

The combination of the effects of heightened levels of these two chemicals in the brain creates a calming and focusing effect in the brain, while simultaneously increasing wakefulness.3 Intentional and Adverse Effects on Human Health Amphetamine stimulants primarily serve to reset chemical imbalances in the brain of an individual with a neurological disorder. In prescribed use, it often has a calming, alerting, and focusing effect, which is why amphetamine stimulants are widely used to combat the symptoms of ADHD and related neurological disorders. Possible acute side effects may include decreased appetite and weight loss, insomnia, nausea and vomiting, abdominal cramps, increase in blood pressure and heart rate, and possible exacerbation of motor tics.2 Over long periods of time, heavy use of prescription stimulants can lead to a wide variety of negative effects. According to the American Addiction Centers, the long-term abuse of Adderall may cause insomnia, difficulty concentrating, lack of motivation, depression, irritability, lethargy, fatigue, aggression, suicidal thoughts, mood swings,

Motivations for Use Many young adults, especially college students, are using prescription stimulants for both medical and nonmedical purposes. In medical applications, prescription stimulants are useful in mitigating symptoms of certain neurological disorders. These include narcolepsy and attention defective disorders, including ADHD and ADD. A 2018 comprehensive analysis conducted by the U.S. Department of Health and Human Services in 2018 found that approximately 6.6% (16 million) of U.S. adults used prescription stimulants in the previous year, 2.1% (5 million) of U.S. adults had misused prescription stimulants at least once, and 0.2% (400 thousand) of U.S. adults had prescription stimulant use disorders (such as addiction).5 On college campuses, the rate of nonmedical use of prescription stimulants (NPS) is much higher. In 2005, a nationwide survey was conducted on 119 private and public college campuses in order to discover the rate of NPS. This survey found that on college campuses, 6.9% of students had misused prescription stimulants at least once, 4.1% had done so in the previous year, and 2.1% had done so in the previous month.6 One main reason for this misuse is typically due to the belief that prescription amphetamines improve cognitive functionality.2 While this can be true when used appropriately by an individual with an attention defective or hyperactive disorder, a study conducted by the University of Maryland Center of Young Adult Health and Development found that the nonmedical use of prescription stimulants may actually decrease cognitive performance.7 During the study, 898 undergraduate students without ADHD

were studied during their second and third year at college. Students who either stopped engaging in NPS in the second year (Desistors) or abstained from NPS entirely (Abstainers) showed significant average GPA improvement between the two years. Students who either initiated NPS in the second year (Initiators) or continues use through both years (Persistors) showed a small overall average decrease in GPA over the study.

Further studies with larger sample sizes are warranted, but the data shows a significant difference between the GPA change of students who did not engage in NPS and those who did. While this data cannot rule out the possibility that NPS prevented a further decrease in GPA, it does conclusively show that nonmedical users of prescription stimulants gained no advantage over their peers who abstained or desisted. This evidence contradicts the common belief that the nonmedical use of prescription stimulants increases cognitive functionality.8 In this area of study, there is an abundance of data with regards to adults in the United States, but there is a lack of data regarding teenagers or people living outside the U.S. Further studies in these data groups would be beneficial in understanding the effects and usage of prescription stimulants.

Figure 3: Change in GPA over two-year period by students who engaged and did not engage in NPS. As shown by the figure, students who engaged in NPS in the second year of the survey (Persistors and Initiators) showed no significant change in GPA.8

Heal, David J, et al. “Amphetamine, Past and Present – a Pharmacological and Clinical Perspective.” Journal of Psychopharmacology, vol. 27, no. 6, 2013, pp. 479–496., doi:10.1177/0269881113482532 2

Abelman, David. “Mitigating Risks of Students Use of Study Drugs through Understanding Motivations for Use and Applying Harm Reduction Theory: a Literature Review.” Harm Reduction Journal, vol. 14, no. 1, 2017, doi:10.1186/s12954-017-0194-6.

“DrugFacts: Stimulant ADHD Medications Methylphenidate and Amphetamines.” Stimulant ADHD Medications - Methylphenidate and Amphetamines | National Institute on Drug Abuse, National Institutes of Health, 12 May 2012, web.archive.org/web/20130312110514/www.drugabuse.g ov/publications/drugfacts/stimulant-adhd-medicationsmethylphenidate-amphetamines.

National Institute on Drug Abuse. “Five Million American Adults Misusing Prescription Stimulants.” National Institute on Drug Abuse, 3 June 2020, www.drugabuse.gov/news-events/newsreleases/2018/04/five-million-american-adults-misusingprescription-stimulants. 6

Smith, M. Elizabeth, and Martha J. Farah. “Are Prescription Stimulants ‘Smart Pills’? The Epidemiology and Cognitive Neuroscience of Prescription Stimulant Use by Normal Healthy Individuals.” Psychological Bulletin, vol. 137, no. 5, 2011, pp. 717–741., doi:10.1037/a0023825.

“Performance-Enhancing Drugs (Adderall, Ritalin, Etc.) at School and Work.” Buckeye Recovery Network, 13 Mar. 2020, buckeyerecoverynetwork.com/performanceenhancing-drugs/. 8

Edited by Amanda Lautieri, B.A. Last Updated: April 12. “Long Term Effects of Adderall on Brain, Personality, and Body.” American Addiction Centers, 12 Apr. 2021, americanaddictioncenters.org/adderall/long-term-effects.

Arria, Amelia M., et al. “Do College Students Improve Their Grades by Using Prescription Stimulants Nonmedically?” Addictive Behaviors, vol. 65, 2017, pp. 245– 249., doi:10.1016/j.addbeh.2016.07.016.

Application of Essential Oils in Active Food Packaging Ruitong Liu

Abstract: Food packaging plays a major role in protecting food from deterioration. This paper evaluates the possibility of utilizing essential oils as active agents in food packaging and provides an overview of its impacts on the packaging material. Introduction Food packaging prevents the product from leaking or breaking and protects food from the environmental influences, such as oxygen, water vapor, pressure, heat, and ultraviolet light. Because of the increasing customer experience expectations and increasing product complexity, researches in food packaging led to the development of innovative packaging with enhanced functionality that is able to accommodate a variety of additional consumer needs. Over the past few years, active packaging and intelligent packaging emerged as new ways of protecting food. Active packaging has been defined as “the incorporation of certain additives into packaging systems … with the aim of maintaining or extending product quality and shelf-life.” 1 By adding additives or freshness enhancers, active packaging enhances the preservation functions. Intelligent packaging refers to “systems which monitor the condition of packaged foods to give information about the quality of the packaged food during transport and storage.” 2 Sometimes, intelligent packaging can be used to test the effectiveness and integrity of the active packaging system. Based on the types of additives, active packaging can be divided into two types, chemoactive and bioactive. Bioactive packaging adds additives that can interact with the organisms like bacterial and

influences its biological process. 3 In chemoactive packaging, the active agents are designed to impact the chemical composition of the product or the gaseous atmosphere inside the pack.4 However, the chemoactive packaging might cause adverse health effects and lead to high waste volume because the resulting packaging might not be sustainable for recycling. Those issues lead researchers to test new alternatives, such as bioactive compounds from natural sources. This article explores the possibility of using essential oils as additives in active food packaging. Essential Oils The International Organization for Standardization defined essential oil as “product obtained from a natural raw material of plant origin, by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation, after separation of the aqueous phase — if any — by physical processes.” 5 Essential oils are named depending on which plant they are extracted from. Different types of essential oils can be identified by their aroma compounds, such as Azadirachta indica (neem), Lavandula angustifolia (lavender), Thymus vulgaris (thyme), Eucalyptus globulus (eucalyptus), Cinnamomum zeylanicum (cinnamon), and others.6 These compounds can preserve food by controlling microbial growth. For example, neem essential oil can enhance the antibacterial activity according to a study conducted by Ali, Sultana, Joshi, and Rajendran.7 Current Application of Essential Oils as Active Packaging: Because essential oils have natural biopreservative effect, essential oils are used widely in the food industry to prolong shelflife of food products. Essential oils normally apply to fruit, vegetables, fish, meat, milk and dairy products. However, since they are volatile compounds combined with other environmental factors, such as light,

oxidation, and heating, essential oils degrade quickly once applied to food products. Therefore, researchers encapsulate essential oils in liposomes, polymeric particles, and solid lipid nanoparticles to improve the stability.8 Essential oils mainly can have two forms when used as active packaging — films and coatings. Normally, films are thin pre-made sheets to be used as covers, wrappers, and layer separation. Coating are refer to the films that can be applied onto the surface of an edible product. 9 For example, chitosan films with Eucalyptus globulus essential oil are designed to package sliced sausages to reduce the antimicrobial activity and limit foodborne contamination in food systems. 10 Another example is chitosan-based coating with lemon essential oil that are able to rely the ripening process of strawberries by reducing the respiratory rate.11

Figure 1: Functions of Edible Films and Coating

Figure (1) that active food packaging with essential oils protects product from gases, vapor, biological, chemical, and physical deterioration. Essential Oils’ Effects on Food Packaging Material’s Microstructure: 1. Tensile properties The interaction between polymer matrix and essential oil components will possibly

influence the tensile properties of food packaging materials. Since essential oils will lead to the reorganization in polymer matrix, the addition of cinnamon oil in the active packaging will increase the packaging’s tensile strength. 12 In addition, essential oils contain several volatile chemicals that are responsible for different functions. One of the most common compounds is phenol, which is able to lead protein to cross-linking and improve the tensile strength of the film.13 2. Barrier properties Because essential oils have non-polar molecule structure, they are hydrophobic. When adding them into hydrophilic polymer matrices will improve the barrier properties and reduce the water vapor permeability. 3. Optical properties: color, transparency, gloss Color, transparency, and gloss of the packaging materials can directly influence the appearance of the product and even consumer’s choices. The color of the packaging materials mainly depends on the concentration and type of the essential oil added into the material. When the concentration of essential oils added increases, the color changes will also increase.14 Moreover, the essential oil might increase the opacity and reduce film transparency, but those changes largely depend on the type of essential oils added. Essential Oil’s Effects on the Antioxidant Properties: Oxidation process normally results in food deterioration. Food with a high amount of fatty acids are more susceptible to lipid oxidation, which often manifested as discoloration, changes in texture, nutrient loos, and production of toxic compounds.15 Figure (2) illustrates recent studies on the use of essential oils as active agents in food packaging.

Figure 2: Recent studies focused on essential oils’ impacts on the in vitro antioxidant properties of film

Limitations of Using Essential Oil in Active Food Packaging: The major drawbacks of using essential oil as active agent in packaging is its low solubility, high volatility, its strong aroma, and the possible of negatively affecting the

organoleptic properties of food. Because of its low solubility and high volatility, the essential oils might be easily lost from food packaging. Moreover, if the level of essential oils is high enough, it will possibly change the taste of the product.

Conclusion Food packaging protects food from environmental factors that might harm the quality and taste of the product, such as UV light, oxygen, water vapor, pressure, and heat. In the meantime, it prolongs the shelflife of food products by preventing chemical or microbiological contaminants. Today, there are several innovative approaches, such

as active packaging, that overtake the traditional packaging methods because they can solve the ecological problems and satisfy consumers’ new demand. Current applications of essential oils can applied onto various types of products and have multiple positive effects on the product. Once they are approved as additives, they are more likely to be used on the food products for the preservation purposes.

J.P. Kerry, M.N. O'Grady, and S.A. Hogan, "Past, Current and Potential Utilisation of Active and Intelligent Packaging Systems for Meat and Musclebased Products: A Review," Meat Science 74, no. 1 (September 2006): 114, https://doi.org/10.1016/j.meatsci.2006.04.024. 2 Ibid. 3 Ibid. 4 John Brockgreitens and Abdennour Abbas, "Responsive Food Packaging: Recent Progress and Technological Prospects," Comprehensive Reviews in Food Science and Food Safety 15, no. 1 (October 21, 2015): 5, https://doi.org/10.1111/1541-4337.12174. 5 International Organization for Standardization, "Aromatic natural raw materials — Vocabulary," International Organization for Standardization, last modified 2013, accessed May 9, 2021, https://www.iso.org/obp/ui/#iso:std:iso:9235:ed2:v1:en. 6 Shubham Sharma et al., "Essential Oils as Additives in Active Food Packaging," Food Chemistry 343 (May 2021): 3, https://doi.org/10.1016/j.foodchem.2020.128403. 7 Ali, W., Sultana, P., Joshi, M., & Rajendran, S. (2016). A solvent induced crystallisation method to imbue bioactive ingredients of neem oil into the compact structure of poly (ethylene terephthalate) polyester. Materials Science and Engineering C, 64, 399– 406. 8 Fern ́andez-Lo ́pez, J., & Viuda-Martos, M. (2018). Introduction to the special issue: Application of essential oils in food systems. Foods, 7(4), 56.

9 Ribeiro-Santos, R., Andrade, M., de Melo, N. R., & Sanches-Silva, A. (2017). Use of essential oils in active food packaging: Recent advances and future trends. Trends in Food Science and Technology, 61, 132–140. 10 Azadbakht, E., Maghsoudlou, Y., Khomiri, M., & Kashiri, M. (2018). Development and structural characterization of chitosan films containing Eucalyptus globulus essential oil: Potential as an antimicrobial carrier for packaging of sliced sausage. Food Packaging and Shelf Life, 17, 65–72. 11 Perdones, A., Escriche, I., Chiralt, A., & Vargas, M. (2016). Effect of chitosan-lemon essential oil coatings on volatile profile of strawberries during storage. Food Chemistry, 197, 979–986. 12 Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122(1), 161–166. 13 Atar ́es, L., & Chiralt, A. (2016). Essential oils as additives in biodegradable films and coatings for active food packaging. Trends in Food Science and Technology, 48, 51–62. 14 Shubham Sharma et al., "Essential Oils as Additives in Active Food Packaging," Food Chemistry 343 (May 2021): 4, https://doi.org/10.1016/j.foodchem.2020.128403. 15 Wang, Z. C., Lu, Y., Yan, Y., Nisar, T., Fang, Z., Xia, N., ... Chen, D. W. (2019). Effective inhibition and simplified detection of lipid oxidation in tilapia (Oreochromis niloticus) fillets during ice storage. Aquaculture, 511(May).

Bio-grout: A Review Meredith Amick

Abstract: Bio-grouting is a term used to describe bio-mediated and bio-inspired methods to stabilize soils. The two best researched and most common methods of bio-grouting are microbially induced carbonate precipitation (MICP) and enzyme induced carbonate precipitation (EICP) MICP relies on bacteria and therefore is biomediated. EICP is bio-inspired, because no living organisms are involved in the process, it uses the enzyme urease which is created by a living organism. This paper aims to review the two processes and their respective strengths and weaknesses. Introduction Traditional use of concrete is not sustainable, as it is extremely energy intensive, costly, and uses materials that have a significant impact on the environment.1 The use of concrete is a source of CO2 emissions, and it releases both nitrogen and sulfur oxides which are major health risks.2 This has pushed researchers to find a better alternative for concrete for soil improvement. The most promising alternatives are biologically based techniques, which use biogeochemical reactions to precipitate calcium carbonate (CaCO3).

Figure 1: Scanning electron microscope image of calcium carbonate crystals between sand gains.1

The calcium carbonate is precipitated as crystals and form bridges between grains of sand, see figure 1.1. In addition, calcium carbonate precipitation strengthens the soil via interparticle binding (as shown in figure 1), but it also fills pores between the particles and roughens the surface of the particles. All of this works together to improve strength, stiffness, and dilatancy of the soil, and reduces soil permeability.3 The most studied mechanism of CaCO3 synthesis is ureolysis. When CaCO3 is produced, bacteria or enzymes catalyze the hydrolysis of urea, producing ammonium and carbonate, see equation 1 below: (1) NH2CONH2 + 2H2O® 2NH4+ + CO32The carbonate produced in equation 1 then reacts with calcium to produce calcium carbonate crystals (calcite), see equation 2 below: (2) CO32- + Ca2+® CaCO3(s) When the CaCO3 is produced it immediately crystalizes between sand particles.1 The two most common techniques of biogrouting are microbially induced carbonate precipitation (MICP) and enzyme induced carbonate precipitation (EICP). The two methods rely on the enzyme urease to catalyze the hydrolysis of urea (ureolysis). These techniques have the potential to be even more effective than the current method of concrete, not only because of the sustainability advantages, but also because the application of bio-grouting is nondisruptive. Bio-grouts could be applied near or under existing buildings, in places where concrete either could not be used or is too costly. Some are also using bio-grouting techniques to mix in bacteria with concrete and bacteria nutrients to fix cracks in concrete or to create a self-healing cementitious material.4 Bio-grout also has the opportunity to prevent landslides,

accelerate tunneling and excavation projects, and prevent desertification.3 Although biogrouting has the great potential and has had encouraging results during the testing phase, it has not been commercialized yet because of a few challenges that need to be solved, which will be discussed further in the following two sections. Microbially Induced Carbonate Precipitation (MICP) Many laboratory studies have shown that MICP is successful in reducing permeability and improving strength, stiffness, and dilatancy in granular soils. MICP has been successful in many different scales. It has been proven successful multiple times in small scale experiments.4 Additionally, in 2011, a study was done on a 1000 m3 area. In the 2011 study, it was found that MICP was successful in achieving borehole stability in gravel on a large scale.5 Most of the studies on MICP use a process called bioaugmentation, which is when researchers use exogenous ureolytic microorganisms (bacteria grown by researchers), which are injected into the soil. The bacteria are sometimes followed by a fixation fluid to help the bacteria attach to soil particles, and then cementation fluid is injected into the soil to begin the bio-cementation process.3 The most commonly used bacteria in bioaugmentation is S.pasteurii, which is used because of its properties of nonpathogenicity, high urease activity, and resistance to high concentrations of ammonium, which is built up in MICP.4 Bioaugmentation does have its drawbacks. Because the bacteria have to be injected before cementation, bio-augmentation cannot be used in soils with smaller pore sizes than the size of bacteria, like in clay or silt.3 To solve this issue, researchers came up with bio-stimulation, which uses indigenous bacteria. Because the bacteria are already distributed in the soil, bio-stimulation only needs an injection of the cementation liquid to begin the bio-grouting process.3

The most researched mechanism of MICP is hydrolysis of urea, or ureolysis. In this mechanism, bacteria have the catalyzing role in ureolysis, as bacteria produce the enzyme urease.4 When bacteria are attached to soil, they also act as nucleation sites for the calcium carbonate, which assists the inter particle connections seen in figure 1. The drawback of ureolysis is that there is an ammonium byproduct, which is a source of groundwater contamination, and current methods of removing it are costly. Another MICP mechanism is denitrification, which uses denitrifying bacteria to oxidize organic matter with nitrate, and produce biomass, nitrogen gas (N2), and inorganic carbon. This increases the pH and the combination of an increase in pH and an increase in inorganic carbon in soil containing calcium ions, produces calcium carbonate.3 This does not produce ammonium like ureolysis, but an incomplete reaction produces N2O, a greenhouse gas. The other downsides of the denitrification process is that it is significantly slower than ureolysis, and has fewer demonstrated research results. However, denitrification can be used in anaerobic conditions unlike ureolysis.3 MICP has some more challenges like cost, environmental impact, dealing with the complexity of natural soils, and Researchers are currently uniformity.3 working to solve these problems before MICP is considered for commercial use. Enzyme Induced Carbonate Precipitation (EICP) EICP uses the ureolysis mechanisms like MICP, but unlike MICP which uses live bacteria in the method, EICP uses free urease enzyme, which is the enzyme used by the bacteria in the MICP process. The free urease is mostly extracted from agricultural sources like soybeans, watermelon seeds, pea plants, and most commonly, jack beans.2 Urease is soluble in water and is significantly smaller than bacteria, so EICP can be used in fine grain soils like silt unlike the bio-

augmentation method of MICP. Another advantage of ECIP is that, because it does not require living organisms, there are fewer complications. EICP is anaerobic, does not need to provide nutrients for the bacteria, and does not need to account for interactions with other microorganisms.2 The downsides of using ECIP are that the enzymes do not have a cell wall so they are faster to decay than bacteria. Enzymes also do not attach to soil particles like bacteria, so there is a lack of nucleation sites. This is a problem because the calcium carbonate is less likely to create bridges between the molecules, which makes ECIP less effective. The lack of nucleation sites also means that the calcium carbonate is more likely to wash away. This problem is solved by slowing down the rate of precipitation and injecting nucleation sites

into the soil. ECIP also has many similar problems as MICP, like cost, environmental impact, complexity of natural soils, and uniformity. Like MICP, researchers are currently looking for solutions to these problems.3 Conclusion Bio-grouting techniques have a great potential to solve problems relating to soil improvement. MICP and EICP are more sustainable than the current cement methods, but they need further research and to solve problems concerning cost, environmental impact, complexity of natural soils, and uniformity. Once these issues are solved biogrouting will be the superior option in soil improvement.

1 Van Paassen, Leon A. "Microbes turning sand into sandstone, using waste as cement." 4th International Young Geotechnical Engineers Conference, Oct. 2009, Alexandria, Egypt. ResearchGate, www.researchgate.net/publication/ 341566470_Microbes_turning_sand_into_sandstone_ using_waste_as_cement. Accessed 9 May 2021.

Almajed, Abdullah, et al. "Enzyme-Induced Carbonate Precipitation (EICP)-Based Methods for Ecofriendly Stabilization of Different Types of Natural Sands." Journal of Cleaner Production, vol. 274, Nov. 2020, p. 122627, doi:10.1016/j.jclepro.2020.122627. Accessed 9 May 2021.

Hamed Khodadadi, T., et al. "Bio-Grout Materials: A Review." Grouting 2017, July 2017, www.researchgate.net/publication/318256473_BioGrout_Materials_A_Review. Accessed 7 May 2021. De Muynck, Willem, et al. "Microbial Carbonate Precipitation in Construction Materials: A Review." Ecological Engineering, vol. 36, no. 2, Feb. 2010, pp. 118-36, doi:10.1016/j.ecoleng.2009.02.006. Accessed 9 May 2021. 4

Van der Star W.R.L., et al. "Stabilization of Gravel Deposits Using Microorganisms." Stand Alone, vol. 0, 2011, pp. 85-90, doi:10.3233/978-1-60750-801-4-85. 5

Hormone Blocks and their Effects on Adolescents: A Comprehensive Review Tom Graeub

Abstract: Gender dysphoria has been brought into the spotlight in the last decade with 0.5% to 1.3% of people in the United States identifying as transgender.1 With the rise in transgender individuals the popularity of hormone blocking medications, also known as puberty blockers, has increased. This article serves as a review of current research surrounding the effects of hormonal therapy in children. Introduction to puberty blockers: Puberty blockers have entered the mainstream market as a safe alternative for children who are unsure of their gender identity. Advertised as delaying unwanted physical changes that don’t match one's perceived gender identity, puberty blockers have some lesser known side effects.2 Understanding how puberty blockers work is crucial to determining their effects on the adolescent body and mind. One such puberty blocker primarily used by female-to-male (FtM) individuals, is leuprorelin. Leuprorelin belongs to a class of drugs called gonadotropin hormonereleasing (GnRH) agonists, which is administered to children with gender dysphoria (GD).3 Leuprorelin acetate (C59H84N16O12) which is branded as Lupron was originally developed to treat prostate cancer but now is being used in children with Central Precocious Puberty (CPP) and GD to delay the onset of puberty4 (Figure 1).Lupron works by cutting off and modifying the hormones that cause pubescent changes, namely testosterone and estrogen.

Figure 1: Structure of Leuprorelin Acetate5

The other most common puberty blocker is histrelin acetate (C68H90N18O14), sold under the brand names Vantas and Supprelin LA6. Histrelin can be used by both FtM and maleto-female (MtF) transgender individuals and was originally used in treating prostate cancer, but it is now the most widely used puberty blocker (Figure 2).Histrelin, which is also a GnRH-agonist, slows the release of gonadotrophins - by which luteinising hormone (LH) and folliclestimulating hormone (FSH), puberty hormones, are produced - by desensitizing the gonadotrophs (GN).7 Over time this slows the production of testosterone and estrogen, bringing puberty to a nearly complete halt.

Figure 2: Structure of Histrelin

Efficacy of Lupron The Journal of Clinical Endocrinology & Metabolism published a widely cited study on the effects of Lupron over a three month period. Of the 30 subjects

enrolled, 24 were girls and 6 were boys, all of which were given 3 cycles of depot leuprolide. Designed to test the effects of different size doses, of which there is little known, the study showed a decrease in LH hormones after an original spike. This decrease would correlate with the suppression of puberty exhibiting hormones.8 Another more recent study demonstrated long term - over two years - effects of depot leuprolide on adolescents. According to the study, “Pubertal progression ceased in all patients, and menses did not occur.”9 The study also showed FSH, and LH levels reaching their minimum levels to the point of being negligible in the onset of puberty. This all bodes very well for the study which proves the great effects of Lupron. However, while these studies prove that Lupron serves well in stopping pubescent development, many ofthe other effects of the drug are overlooked. Over the nine female participants, body fat percent increased anywhere from 2.8% to 24% (Figure 3) compared with the 1.2% national average. Two participants were withdrawn from the relatively small study due to adverse reactions to the medication.

Efficacy of Histrelin: Much like Lupron, Histrelin has been proven effective in treating GD by decreasing production of LHand LSH. LH concentrations decreased from 28.2 ± 20 mIU/ml at baseline to 0.8 ± 0.4 mIU/ml after 12 months10 (Figure 4). Overall this study too was marked with successful treatment of GD; however, bone density did decrease in all of the participants. The study ends in optimism about the future skeletal development and complete use of reproductive system for its participants. Seeing as decreased bone density and irreversible reproductive changes have been subject to heavy criticismin puberty blockers, this optimism is promising.

Figure 4: Amount of LH in study participants over a 12-month period. The swift decrease symbolizes decrease in production of these sex hormones.10

Figure 3: Body fat percent in 9 girls, at the beginning of the study and after one year on Lupron. Darker line represents the beginning and lighter line represents after one year. 7

Finally and most importantly there was a “striking deceleration of bone age advancement.”7 This deceleration of bone development is crucially dangerous in children whose growth will be stunted and could be devastating in adult life with bones that can't support their bodies.

Long term effects of Puberty Blockers: Along with decreased bone density and irreversible change to reproductive organs, behavioral problems have also been reported in significant numbers for adolescents who have started using GnRHa drugs. The University of Louisville study found many instances of acute behavioral problems including, but not limited to, emotional lability and aggression that had never before been seen in these children.11 Two of the children in this study were described as, “uncontrollableand very aggressive, even biting and kicking,” and, “she would kick, scream, cry uncontrollably

and even bite other children without inciting events” while on these puberty blockers.9 According to another study, many on puberty blockers also experience, “ severe joint pain, osteoporosis,compromised immune systems, and (increased) mental health issues such as severe depression.”3 The FDA has gotten over 24,000 reports, deemed serious medical conditions of people on Lupron. One of the most serious effects was found in study M84-042. The study found evidence that, “62.5% of patients [treated with Lupron for endometriosis] had not regained baseline estrogen levels by one year after stopping Lupron.”3 This study suggests that the effects of puberty blockers might not be as reversible as people are told. Some people suffering from GD “desist”, meaning they no longer fall under the diagnostic criteria for GD. For those who may want future use of their reproductive parts, this causes a major problem. Conclusions and Further Research: While there has been extensive research 1 Garg,

conducted on the short-term effects of puberty blocks, research about the long-term effects- 5 or more years - is severely lacking.There are so few cases of people who have been put on GnRHa’s for an extensive amount of time that the potential subject pool is small. The question is do the costs out way the benefits which is a personal, case by case decision. There needs to be more transparency in the media and clinics as to the effects of these blockers, because there is a high chance that some changes are not as reversible as they are made out to be. Glossary: GD - Gender dysphoria FtM - Female-to-Male MtF - Male-to-Female GnRHa - gonadotropin hormone-releasing agonists CPP - Central Precocious Puberty GN - Gonadotrophs FSH - Follicle-stimulating hormone LH - Luteinising hormone

G. (2020, November 29). Gender Dysphoria. StatPearls [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK532313/.

2 Childrens

Hospital. (2020, July 20). Puberty Blockers. Hormone Blockers | St. Louis Childrens Hospital. https://www.stlouischildrens.org/conditions-treatments/transgender-center/puberty-blockers. 3 Robbins, J. (2018, December 18). Why Puberty Blockers Are A Clear Danger To Children's Health. The Federalist. https://thefederalist.com/2018/12/14/puberty-blockers-clear-danger-childrens-health/. 4 Lupron Depot. (n.d.). PROSTATE CANCER. Lupron Depot (leuprolide acetate for depot suspension). http://www.lupron.com/ 5 C;, B. (2021, February 21). High court should not restrict access to puberty blockers for minors. Journal of medical ethics. https://pubmed.ncbi.nlm.nih.gov/33593872/. 6 DrugBank. (2016, May 6). Leuprolide. DrugBank Online. https://go.drugbank.com/drugs/DB00007. 7 Barradell, L.B., McTavish, D. Histrelin. Drugs 45, 570–588 (1993). https://doi.org/10.2165/00003495-199345040-00008 8 Angela Badaru, Darrell M. Wilson, Laura K. Bachrach, Patricia Fechner, Laura M. Gandrud, Eileen Durham, Kupper Wintergerst, Carolyn Chi, Karen O. Klein, E. Kirk Neely, Sequential Comparisons of One-Month and Three-Month Depot Leuprolide Regimens in Central Precocious Puberty, The Journal of Clinical Endocrinology & Metabolism, Volume 91, Issue 5, 1 May 2006, Pages 1862–1867, https://doi.org/10.1210/jc.2005-1500 9 E.K. Neely, R.L. Hintz, B. Parker, L.K. Bachrach, P. Cohen, R. Olney, D.M. Wilson, Two-year results of treatment with depot leuprolide acetate for central precocious puberty, The Journal of Pediatrics, Volume 121, Issue 4, 1992, Pages 634-640, ISSN 00223476, https://doi.org/10.1016/S0022-3476(05)81162-X. 10 Barradell, L.B., McTavish, D. Histrelin. Drugs 45, 570–588 (1993). https://doi.org/10.2165/00003495-199345040-0000 11 K. O. Akintola , A. O. Omoruyi , M. B. Foster , S. E. Kingery , K. A. Wintergerst, (2014, April 25) Behavioral Disorders Associated with GnRH Agonist Therapy. Department of Pediatrics, Division of Endocrinology, University of Louisville Kentucky, USA. https://d-nb.info/1171816359/34

The Impact of Non-sugar Sweeteners on Human Health Lixin Qin

Abstract: Non-sugar Sweeteners (NSSs) have gained increased attention as a popular substitute for sugars. Previous studies about NSSs have not given a comprehensive conclusion of their effects on humans. This paper aims to offer a systematic review of NSSs in order to evaluate the impact of NSSs on human body weight, eating behavior, and the risk of cancer and cardiovascular disease. Introduction to Non-sugar Sweetener: Along with the rising concerns about healthy lifestyle and nutritional diet, people become more aware of their daily sugar intake. With increased consumer interest in reducing energy intake, food products containing non-sugar sweeteners (NSSs) rather than sugars (monosaccharides and disaccharides) have become increasingly popular mainly because NSSs have significantly less calories than nutritive sweeteners.1 However, some studies indicate that NSS could increase the risk of overweight, diabetes, and cancer.2

Therefore, investigation of the health effect of NSSs is meaningful and necessary. Body Weight: Different groups received different results when introducing NSSs into their weight loss journey. In randomized controlled trials, we saw no significant differences in change in body weight between adults receiving NSSs compared with those receiving different sugars or placebo.3 Subgroup analysis by body weight status suggested that NSS use by overweight or obese individuals resulted in reduced body weight of 1.99 kg but no change in individuals of normal weight (0.03 kg, −0.03 to 0.09; two, n=110; fig 2).3 We can conclude that NSSs have a greater effect on weight loss for obese individuals than that of normal weight individuals. The definition of non-sugar sweeteners is confused with artificial sweeteners in some cases. For clarification, we use NSS to represent non-sugar sweeteners, a category including artificial sweeteners (figure 1). The range of NSSs approved in different countries varies. In the United States, for example, the FDA has approved six NSSs for consumption,4 whereas the range of currently approved NSSs in the European Union is wider.5 Currently, the safety of several NSSs have been recognized and support with scientific evidence.

Figure 1. Types of Sweeteners3

Figure 2. Effect of NSSs intake on Weight Change (kg) in Adults3

In one cohort study, 50 researchers assessed different levels of NSS intake and reported that weight gain was 0.09 kg lower in women consuming up to 5.8 g saccharin per day compared with women consuming more than 5.8 g saccharin per day.3 Two randomized controlled trials investigated the effect of NSS intake in overweight populations trying to lose weight, although they did not provide enough data to conduct meta-analysis (standard error or standard deviation not reported): one study6 showed no difference in body weight between the study groups and differences between the study groups with regard to reduction in body weight, body mass index, or body fat. Eating Behavior: One’s eating behavior is influenced by energy intake, satisfaction, and energy intake. According to evidence from a randomized controlled trial, energy intake was lower in the sucralose group than in the sucrose group.3 In one non-randomized controlled trial, mean daily energy intake was reported to be similar between the groups receiving aspartame or saccharin

and significantly increased in the group that received sucrose. Energy intake was 6711, 6640, or 7728 kJ daily with aspartame, saccharin, or sucrose in the preschool group, respectively, and 8100, 8284, and 9293 kJ for school age children, respectively.3 In one randomized controlled trial with overweight children involved in active weight loss, researchers assessed change in appetite as self-reported adverse events, which were reported to be no different between the study groups.3 Therefore, there is no sufficient evidence to illustrate the relationship between NSSs and eating behavior, and further research is needed. Cancer: In one case-control study (n=150), researchers reported no difference in risk for primary brain tumors when looking at aspartame intake from all sources or aspartame intake from diet drinks only.3 Furthermore, no difference in risk of primary brain tumors was seen with different durations or frequencies of aspartame intake.3

Figure 3. Effect of non-sugar sweetener intake on risk (odds ratio) of bladder cancer. Odds ratio of less than 1=increased risk of cancer with non-sugar sweeteners3

Cardiovascular disease: In one randomized controlled trial, total cholesterol concentration decreased strongly in sucrose groups but increased in the aspartame group.3 The change in triglyceride concentration and blood pressure (no numerical data reported, very low certainty of evidence) were similar between the study groups.3 Another randomized controlled trial reported that in overweight children involved in active weight loss, systolic and diastolic blood pressure were similar in those receiving NSSs or placebo (n=55, very low certainty of evidence).3

Conclusion: This comprehensive systematic review covers a broad range of benefits and harms of NSSs in a generally healthy population of adults and children, following rigorous systematic review methods. Overall, studies of adults and children assessed the associations and effects of NSSs on different health outcomes. For most outcomes, there seemed to be no statistically or clinically relevant difference between NSS intake versus no intake, or between different doses of NSSs.3

Institute for Evidence in Medicine (for Cochrane Germany Foundation), Medical Centre of the University of Freiburg, Faculty of Medicine, University of Freiburg, Breisacher Straße 153, 79110 Freiburg, Germany 2 Olivier B, Serge AH, Catherine A, et al. Review of the nutritional benefits and risks related to intense sweeteners [correction in: Arch Public Health 2015;73:49]. Arch Public Health 2015;73:41. 3 Toews I, Lohner S, Kullenberg de Gaudry D, Sommer H, Meerpohl J J. Association between intake of non-sugar sweeteners and health outcomes: systematic review and meta-analyses of randomised and non-randomised controlled trials and observational studies BMJ 2019; 364 :k4718 doi:10.1136/bmj.k4718 4 US Food and Drug Administration. Additional information about high-intensity sweeteners permitted for use in food in the United States: US Food and Drug Administration; 2017. https://www.fda.gov/food/ingredientspackaginglabeling/foodadditivesingredients/ucm397725.htm. 5 Authority EFS. Sweeteners Brussels, Belgium. 2017. http://www.efsa.europa.eu/en/topics/topic/sweeteners. 6 Blackburn GL, Kanders BS, Lavin PT, Keller SD, Whatley J. The effect of aspartame as part of a multidisciplinary weight-control program on short- and long-term control of body weight. Am J Clin Nutr 1997;65:409-18. doi:10.1093/ajcn/65.2.409