INFORM October 2024

inform October 2024 Volume 35 (9)

OLEOSOMES for the FOOD INDUSTRY

ALSO INSIDE: Membrane targeted treatments Soybean quality by regions Assessing risk

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October 2024 inform

New food chemistries with oleosomes Oleosomes are oil containing spheres encased in a shell of phospholipids dotted with proteins. Their natural physical properties mean they can be used as emulsions. Read how food scientists are using oleosomes to save on manufacturing resources and reduce ingredient lists. 8 FEATURES

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The quest for new antibiotics finds a new frontier: membrane lipids The arsenal for treating life threatening diseases becomes less and less effective each year as bacteria outsmart therapeutic defenses. Now, some researchers and pharmaceutical companies are focusing on the lipids in bacteria cell membranes. They have identified potential weaknesses that could lead to treatments to overcome rampant antibacterial resistance. Assessing health risks when faced with contradictory research Years of studies on every aspect of human health provide an ever-growing pile of results. This article discusses how experts and non-experts should approach assessing new findings to make better decisions about health risks. Soybean meal and oil quality by production region Increased demand for soybean products world-wide prompted researchers to evaluate how the quality of soybean oil and meal differs depending on the growing region. Read about their findings in this article that is based on their review paper published in the Journal of the American Oil Chemists’ Society . Can fungi turn food waste into the next culinary sensation? Chef-turned-chemist Vayu Hill-Maini contributed to a recent Nature Microbiology paper about using fungi to turn food waste into culinary treats. This story describes his inspiration and the delicious outcome of his research.

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CONTENTS

4 Index to Advertisers 19 AOCS Meeting Watch

5 Editor’s Letter 6 Division Update

28 Regulatory Review 30 Extracts & Distillates

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AOCS MISSION STATEMENT AOCS advances the science and technology of oils, fats, proteins, surfactants, and related materials, enriching the lives of people everywhere.

inform International News on Fats, Oils, and Related Materials ISSN: 1528-9303 IFRMEC 35 (9) Copyright © 2013 AOCS Press

EDITORIAL ADVISORY COMMITTEE

Julian Barnes Etienne Guillocheau Jerry King

Gary List Thais L. T. da Silva Warren Schmidt Raj Shah

Ryan Stoklosa Ignacio Vieitez Bryan Yeh

AOCS OFFICERS PRESIDENT: Tony O’Lenick, SurfaTech, Lawrenceville, Georgia, USA VICE PRESIDENT: Gerard Baillely, Procter & Gamble, Mason, Ohio, USA TREASURER: Greg Hatfield, Bunge Limited, Oakville, Ontario, Canada SECRETARY: Fabiola Dionisi, Societe’ Des Produits Nestlé - Nestlé Research, Lausanne, Vaud, Switzerland PAST PRESIDENT: Grant Mitchell, Salas O’Brien, Cincinnati, Ohio, USA CHIEF EXECUTIVE OFFICER: Patrick Donnelly

AOCS STAFF EDITOR-IN-CHIEF: Rebecca Guenard MEMBERSHIP DIRECTOR: Travis Skodack

PAGE LAYOUT: Moon Design

The views expressed in contributed and reprinted articles are those of the expert authors and are not official positions of AOCS.

INDEX TO ADVERTISERS *CPM Crown. ......................................................................................................... C4 *Desmet USA, Inc................................................................................................... C2

*Corporate member of AOCS who supports the Society through corporate membership dues.

EDITOR’S LETTER

inform October 2024, Vol. 35 (9) • 5

New perspectives

As fall brings changes to the landscape, the October edition brings you stories with a fresh point of view. The theme of this month’s issue is phospholipids and the first two stories describe scientists taking a new perspective on cell membranes. In the first story the goal is to keep them intact. In the second they are identi fied as a weakness. Our cover story is about the potential of oleosomes—tiny oil-containing spheres with a natural shell of phospholipids and proteins—in the food industry. Food scientists are investigating ways to maintain these structures and revolutionize the indus try by simplifying ingredient lists and reducing manufacturing resource requirements. This feature explores how they believe oleosomes may be the key to a more efficient and sustainable food production future. In the realm of medicine, the fight against antibiotic resis tance takes a groundbreaking turn as researchers explore bac terial membrane lipids. With traditional antibiotics losing their efficacy, the research community is turning to the lipid struc tures in bacterial cell membranes as a potential Achilles’ heel.

This story uncovers the latest research that could lead to new, life-saving treatments in the ongoing battle against resistant bacteria. The next feature navigates the daunting maze of health research and when to take notice of cautionary study results, especially when they contradict previous findings. Our article on assessing health risks amid contradictory research offers guid ance for both experts and laypeople alike. It provides a thought ful examination of how to interpret and weigh new findings. And how health research agencies can help the public make informed decisions in an era of constant healthcare updates. Lastly, we explore the global stage with a feature on the quality of soybean meal and oil. As the demand for soybean products surges, researchers have taken a closer look at how production regions impact the quality of these essential com modities. Their findings shed light on important factors that could influence the future of the soybean industry.

Yours in science,

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Phospholipid Division members research food and nutrition applications

The Phospholipid Division promotes professional interest, communication, and competence of experts in the phospho lipid and lecithin fields. This year’s division leaders include Sugasini Dhavamani, Chair; Andrea Stange, Vice Chair; Gulustan Ozturk, Secretary of Education and Events; and Prasuna Desam, Secretary of Membership Communications. To join the Phospholipid Division, contact Steph Adams, steph. adams@aocs.org . SPOTLIGHT ON MIKAEL FABRITIUS Mikael Fabritius is a postdoctoral researcher in the Food Sciences unit at the University of Turku, Finland. His main expertise is method development in liquid chromatographic and mass spectrometric applications. Throughout his academic career, his work has focused on deep structural characteriza tion of lipid molecules, particularly triglycerides and phospho lipids found in natural fats and oils. INFORM: What part of your research or work involves phospholipids? Fabritius: I am currently working on developing a tandem mass spectrometric analysis method for characterizing the positioning of individual fatty acids in phospholipid molecules. Previous work showed that the position of fatty acids in tri glycerides influences their absorption and nutritional proper ties, especially in infant nutrition. For phospholipids, we are still mostly in uncharted territory as there have not been suit able protocols for this type of analysis. Our method, initially developed for the phosphatidylcholine class, is now being expanded to other classes, such as phosphatidylethanolamine and phosphatidylserine. INFORM: What advancements do you find most promis ing in your field of research? Fabritius: As an academic researcher doing mostly analytical method development, I am always very interested in the lat est developments in instrumentation, particularly mass spec trometry. New features such as alternative fragmentation mechanisms allow us to take the molecules, fragment them in a different way compared to traditional mass spectrome ters, and get additional structural information about the mol ecules. Machine learning applications have also helped us to

Mikael Fabritius

decipher extremely complex data, something that was not previously possible. INFORM: How does attending the AOCS Annual Meeting support your research and interests? Fabritius: Conferences are always very important for meeting peers and sharing ideas. The AOCS Annual Meeting especially is one of the largest gatherings in this field, which allows me to network with some of the best experts in this field globally. I have now attended the Annual Meeting twice, and both times the conference program has been extensive, featuring a nice balance of fundamental research and industrial applications. INFORM: What future research directions are you excited about concerning phospholipids? Fabritius: The characterization of phospholipids in natural fats and oils is currently done on mostly the molecular spe cies level. The identity of the attached fatty acid is known, but

YOUR AOCS COMMUNITY

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not their positioning on the glycerol backbone. I am excited that with the methodologies we developed, we can reach a new layer of depth in the structural analysis of lipids, possibly revealing something that has previously been overlooked. This can potentially lead to revelations in our understanding of the nutritional and biological significance of individual phospho lipid molecules. SPOTLIGHT ON CAMRYNN SIMON Camrynn Simon is a masters’ student in food science in the food colloids lab of professor Supratim Ghosh at the University of Saskatchewan, Canada. She was born and raised in Saskatchewan, and her love for food and cooking developed at a young age when her grandparents introduced her to the Food Network. So, it was only fitting that when she discovered food science at the beginning of post-secondary education, it sparked a passion in her that is ever-growing and changing, just like the field of food science. INFORM: What part of your research or work involves phospholipids? Simon: My research involves emulsion and emulsion dynamics. Specifically, I am making plant protein and phospholipid-based, oil-in-water emulsions. They contain a high percentage of oil and a major focus of my research is on the dynamics at the oil-water interface of the dispersed droplets. Analyzing and understanding these interfacial interactions are extremely important and this is where phospholipids come in. I am using phospholipids in my high oil-containing emulsions to help with stability and to attain different functional characteristics. In addition, I am working with phospholipids that come from plants, as with the rest of my materials. This provides an extra layer of difficulty but it is a very interesting and informa tive research experience. INFORM: What new research or advancements are you looking for to move your industry forward? Alternatively, what advancements do you find most promising? Simon: Coming from a lab that works with a wide variety of plant proteins, I find the sourcing of phospholipids from plants quite interesting, as well as the advancement of the refining process of those phospholipids. Additionally, I find that there is a lot of research on harnessing phospholipids and their capa bilities in the biomedical field, but they are not as well studied in food science and in food products. So, seeing how we can take this knowledge from other fields and apply it in different ways in the food sector is something that interests me. INFORM: How does attending the AOCS Annual Meeting support your research and interests? Simon: Attending the AOCS annual meeting furthers my pas sion for food science but also increases my love for being a part of this research community. I find that, to no fault of its own, when you are part of a lab that all have similar projects by nature it will sometimes lack diversity in ideas. Do not get me wrong, it creates an incredibly supportive environment

Camrynn Simon

with an endless amount of background knowledge but can sometimes lack the diversity that is needed when it comes to the problem-solving side of research. So, being introduced to a wide range of topics and researchers helps me gather new ideas and understand new concepts that I may not have found on my own. Also, I find that being surrounded by pas sionate people, whatever the topic, is very invigorating for myself and my research. I’ve been lucky enough to make it to two AOCS annual meetings, and the first one was at the very beginning of my master’s degree. I was fresh out of my undergraduate degree and not exactly sure what an aca demic research landscape was like. I would say that going to AOCS not only helped me see what a passion for one’s research is all about but also to see the faces behind these papers that I’ve been reading. INFORM: What future research directions are you excited about concerning phospholipids? Due to my work with Dr. Ghosh and my experience with emul sions in his lab, I’m looking forward to how phospholipids can be used further in emulsions and emulsion-based food prod ucts. I am also interested in the further advancements in iso lating and analyzing specific phospholipids from plant sources. I think phospholipids are under-researched ingredients when it comes to the food science sector, where there may be more in-depth research in other areas, and seeing how previous knowledge, even in different contexts, can be applied to food products is very exciting. Additionally, coming from a lab that uses a large number of plant-based products, I am always look ing forward to materials that can be sourced from plants in sustainable ways.

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Across large swaths of the Canadian prairies and the United States’ Dakotas yellow canola flowers blanket the landscape. It is one of the most common oil-producing agricultural plants on the planet. When processing an oilseed plant like canola, the oil has historically been the primary product. As AOCS members know, producing clean oil in high yields can require high temperatures and harsh solvents. The leftover seed cake has historically been relegated to animal feed, unsuitable for human consumption, because of moisture and fiber that complex and denature the protein. New food chemistries with oleosomes Dan Samorodnitsky

However, the food industry seeks to identify alternatives to using highly purified ingredients and limit the copious resources their process ing requires. Subcellular structures within oilseeds, called oleosomes, are a recent curiosity for food scientists. Researchers in industry and aca demia are considering how to incorporate oleosomes as ingredients in food formulations while navigating some of their less preferred. “Oilbodies, like oleosomes, are the way that seeds store oil,” says David Dzisiak, the COO of Botaneco, an ingredient manufacturer based in Calgary, Alberta, Canada. They are to plants what fat deposits and lipo somes are to mammals. The essential organelles provide energy storage during seed dormancy and protect against environmental stress. They consist of a stable complex of triacylglycerols encased in a membrane composed of a continuous phospholipid monolayer embedded with hydrophobic proteins. Oleosomes range in size from less than a microme ter to 2.0 μm, depending on the source; they are found in both seeds and nuts. They are a core part of plant biology that, with further investiga tion, could provide the food industry with an ingredient that meets global sustainability goals. MAINTAINING STRUCTURE In conventional processing the oil bodies are crushed and the oil is squeezed out. The proteins holding oleosomes together—known as oleosins—collapse and the structure falls apart. Dzisiak says Botaneco extracts oleosomes intact using an aqueous process. When combined

• Oleosomes are natural emulsions inherent to oilseeds. • With a consumer focus on clean labels could this be the time for the food industry to commercialize these lipid filled vessels? • Hear from one ingredient producer who has proven the potential of oleosomes in multiple applications. • What hurdles remain for large-scale manufacturing of oleosomes?

are added. This avoids the mechanical emulsifying step often needed for other plant-based thickeners, or the need for addi tional emulsifiers, like lecithins, to combine ingredients in a product. Take plant-based milk as an example. “Plant-based milk is often thin and watery,” said Dzisiak, but titrating in oleosomes creates a stable emulsion that gives the drink body, mouthfeel, and texture, as well as preventing separation.” Physically, oleo somes act like soap bubbles when they bump into each other and resist merging with neighboring structures. “Oil bodies have great promise in foods that do not oth erwise have a natural fat system,” said Dzisiak. Botaneco has been producing oleosomes for about 10 years, but that has been mostly for the personal care industry. Oleosomes give such products a “natural feel” without the use of an animal-based ingredient to achieve a thicker or creamier consistency. The oleosomes are produced as pre-packaged emulsions and are good for more reasons than just viscosity and tex ture. Oilbodies could replace tropical oils in plant-based food formulations. They also provide a “cleaner label,” an ingredi NATURAL INGREDIENTS BECAUSE OF THEIR INHERENT PROTEIN-AND-FAT COMPOSITION, OLEOSOMES HAVE A BUILT-IN ASSET OF NATURAL EMULSIFICATION.

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Oleosome

TAGs Proteins Phospholipids

Oleosome structure. Source: Nikiforidis, JCIS , 274, 102039, 2019.

with twin screw pressing, ultrasonication or enzyme treat ments this extraction method can produce higher yields. Because of their inherent protein-and-fat composition, oleosomes have a built-in asset of natural emulsification. They do not need to be stirred, whipped, or otherwise agitated to create a smooth, thick, creamy consistency wherever they

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ent list with fewer items listed, and better nutrition panels due to lower saturated fat content. Many consumers, particularly younger generations, perceive shorter ingredient lists as being more natural and, therefore, better (https://doi.org/10.1016/j. foodqual.2020.104062). MARKET POTENTIAL Plant scientists have known about oleosomes for decades, but only recently has the food industry turned its attention toward them. Part of the reason for that is a renewed interest in a cir cular economy and agricultural efficiency—in the past oleosins were often simply a waste by-product when canola seeds were crushed to extract and purify their oils. Burcu Guldiken, lead scientist at Botaneco, says oleo somes were not targeted by the food industry because demand was low. “Demand defines products that will enter the market,” he says. “In the food market, development of plant-based milks in the last decade has been the largest tar get where oleosomes could be involved.” In 2023, the milk-al ternative market was a billion-dollar industry and 41 percent of US households purchased a plant-based dairy replacement, with almond and oat milk leading the way (https://tinyurl.com/ bdh3wm4a). Producing oleosomes for plant-based dairy will not require new machinery or logistics changes, according to Lorenz Plankensteiner, a food chemistry doctoral student at Wageningen University in The Netherlands. He says the gen eral process for acquiring oleosomes is simple—soaking, blending, then gravitational separation. “A lot of dairy equip ment can be reused for oleosomes, but the roadblocks are in fine-tuning and scaling the processes,” he said. Research teams are investigating how extraction condi tions affect the colloidal state and the size distribution of oleo somes to achieve a method for consistent retrieval of a stable oil body that could be used in a range of food applications, like flavor delivery. Since they can store hydrophobic compounds,

they have already been considered in applications to house and deliver drugs. This is an active area of research, particu larly for delivering chemotherapeutics in cancer treatments ( https://doi.org/10.1021/acsami.7b19255 ). ALTERING INDUSTRY PERSPECTIVES Plankensteiner noted that market research shows consum ers want sustainable oils that do not require the widespread habitat destruction that has occurred in Asia, Africa, and Latin America for edible oil production. Oleosomes align with this current naturalist paradigm shift. The food industry is focused on making products as clean as possible, says Jason Hargreaves, Botaneco’s head of research and development. “With oil, beyond the crush you then have to refine, you have to bleach, and you have to deodorize in order to make that oil suitable for human con sumption, and it is similar with a protein.” Creating plant-based food products requires multiple protein ultra-filtrations—to get rid of ash, carbohydrates, and various micro-compounds that give plant products off-flavors. Oleosomes function because they are a natural mixture. “Instead of highly functional protein concentrates, we pro pose to the industry that mixed ingredients are okay,” says Hargreaves. It is not necessary to process and use all that energy, he says. A mixture of highly functional protein and fat, in the form of an oleosome, can be used to make delicious food. You can produce a mayonnaise with only two ingredi ents: oleosomes and vinegar. Oleosome-interested parties are focusing on a few key plants. Sunflower, canola, and hemp are the big three oleo some-producing plants so far. Companies like Botaneco can dip into existing, reliable supply chains that have decades of scal ability behind them. These plants also produce oleosomes with the best physical properties for food applications, with about 40 percent fat and 20 percent protein. Soy is generally avoided for oleosome extraction, since it contains 20 percent fat to 40

Rapeseed

– Pectinase

+ Pectinase

oleosome oleosin cruciferin napin pectin

Pectinase – +

wet milling

pH 5.7

pH 5.7

Enhanced bridging floccula ti on

Bridging floccula ti on

pH 8.5

pH 8.5

Depletion floccula ti on Coalescence

Depletion floccula ti on Limited coalescence

Changes in canola oleosome properties with changing extraction conditions. Source: Alpiger and Corredig, FRI , 175, 113736, 2024.

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THE LARGEST CHALLENGE LOOMING ABOVE ALL FOR OLEOSOMES IN THE FOOD INDUSTRY: THE TASTE. KEEPING THEIR TASTES CONSISTENT IS A PROBLEM SINCE DIFFERENT SOURCES OFFER DIFFERENT TASTES.

percent protein, non-ideal for current oleosome-based food applications. Safflower makes a higher fat product but tastes bad and is best left for personal care applications. With canola Botaneco says they are working on process improvements to optimize co-extraction of oilbodies and protein isolates. The sunflower process is well developed at Botaneco and is the best overall oilbody source for both neutral taste, cost effec tiveness, and application benefits, says Hargreaves. LOOMING OBSTACLES There are still a variety of technical challenges to overcome for oleosomes to gain prominence in the food ingredient mar ket. To start, aqueous extractions tend to produce unwanted interactions that can destabilize intact oleosomes, according to Simone Bleibach Alpiger, a food science researcher assistant at Aarhus University in Demark, who studies oleosomes. Extracting intact oleosomes is a balancing act, says Alpiger. “They are colloidally unstable, that is the main chal lenge,” she said. That is to say that while the oleosomes them selves may remain intact, under certain chemical conditions they might form large flocculates with other coextracted components, changing their colloidal stability. At lower pHs, towards the isoelectric point of oleosins, the proteins start to aggregate. Adding a pectinase, an enzyme that breaks down co-extracted soluble fibers, intensifies the aggregation. “If you have mixed flocculates, it could potentially be suitable for a spread or other food product where an emul sion-filled gel structure is desired,” said Alpiger. But, she adds, this is generally not preferable. At higher pH there is less aggregation, but then producers need to lower the pH back down to make it palatable for food, otherwise the product would be bitter. Previous research has shown that depending on the extraction conditions, phospho lipids will migrate from the oleosome membrane. Given this observation, when used in food applications they could frac tionate and break an emulsion, forming a cream that rises to the top. Alpiger says this phospholipid behavior needs to be studied further. Another challenge is the natural paradox. “Oleosomes are natural and you do not want to manipulate them too much,” Plankensteiner says. But sometimes manipulation is necessary. Oleosomes with a diameter above 1µm have a greater tendency to form a cream layer. Oleosome diameters vary from one plant to another and even within each plant— canola oleosomes range from 0.2µm to 20µm (https://doi. org/10.1016/j.foodhyd.2021.107078). This can give an advan tage to certain sources, says Plankensteiner. Soybeans, for instance, tend to produce small diameter oleosomes of around 0.5μm. “You can change the size of oleosomes—like through homogenization—but that is an additional processing step making the production more energy intensive,” he says. Then there is the challenge of economics. Traditional oil extraction from a plant like canola is well-established technol ogy that yields upwards of 95 percent. For the past few years, extracting oleosomes from the same plants has only achieved yields around 60 percent (https://doi.org/10.1016/j.jfood -

eng.2019.109890). This might be changing soon, however, with other plant sources. Scientists have reported yields of nearly 90 percent (https://doi.org/10.1016/j.foodhyd.2022.108419). Still, the largest challenge looming above all for oleosomes in the food industry: the taste. Keeping their tastes consis tent is a problem since different sources offer different tastes. Soybean oleosomes reportedly have a bean-y flavor, where canola is a bit nutty. Since canola is a Brassica species, it tends to taste sulfurous and mustard-y, but further processing tones down the flavor if desired. “Some oleosomes have a lingering mouthfeel and can be a bit astringent,” said Plankensteiner. This could be due to phe nolic compounds, not present in oleosomes but in the seeds that co-extract from the plants. However, he is hopeful that Plankensteiner, is using them to help reframe dairy-free prod ucts for consumers. “We need to convince the consumer to not just want a replicate for milk, but to start enjoying the specific flavors from oleosome-containing products,” he said. Considering everything that we make from milk, like cheese or cream, replicating all those products would be challenging. Oleosome-containing alternatives would simply be a different product from what they imitate. Plankensteiner says that is the advantage of oleosomes and dairy-free products generally, the opportunity to cre ate something entirely new. Instead of producing a neutral flavored product that imitates dairy milk, he says, we should explore options like oleosomes from roasted hazelnuts. There are many possibilities. “It is not just a replacement for milk but an additional product, an attractive product due to its own properties,” says Plankensteiner. “That is what moves us forward.” Dan Samorodnitsky is a science journalist based in Minneapolis. He is the co-founder of Sequencer Magazine , where he covers biology, health, genetics, and other science stories. flavor issues can be resolved. THE PATH FORWARD The real opportunity for oleosomes, according to

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The quest for new

antibiotics finds membrane lipids

Christiana Nunez

a new frontier,

Since they arrived on the market about four decades ago, carbapenems have been a go-to anti biotic for tough, life-threatening infections such as pneumonia or meningitis. When other drugs do not work, carbapenems often do. So, when the powerhouse of last resort began to fail against certain illnesses over the past couple of decades, it was one more worrying example of an ongoing global health threat, antimicrobial resistance.

Antimicrobial resistance is when bacteria or other pathogens stop responding to medicines. In 2019 alone, it contributed to nearly 5 mil lion deaths worldwide, directly causing more than 1 million of them. In the United States, treating six of the worst types of antimicrobial-re sistant infections cost an estimated $4.6 billion in 2017, according to a 2021 study. However, innovations in antibiotics have been few and far between, partly because of a lack of investment. A new avenue of research that targets membrane lipids offers hope for disabling some of the most dangerous bacteria. Researchers from the pharmaceutical company Roche have pinpointed a drug candidate that blocks the transport of a key glycolipid in carbapenem-resistant Acinetobacter baumannii (CRAB). Their paper, “A novel antibiotic class targeting the lipopolysaccharide transporter,” appeared in Nature in January (https://doi.org/10.1038/s41586-023-06799-7). “Antimicrobial resistance is often talked about as a silent pandemic. COVID-19 has brought this sharply into light, demonstrating the conse quences of a pandemic on the whole of society,” said Kenneth Bradley, global head of infectious disease discovery at Roche Pharma Research & Early Development, in an email interview. “The difference between anti microbial resistance and COVID-19 is that we have known about antimi crobial resistance for years.” THE RISE OF RESISTANT PATHOGENS Before the debut of penicillin in the 1940s and the other antibiotics that followed, an everyday wound could easily become deadly if it became infected. Patients who received penicillin early in its development included a woman who fell ill after a miscarriage and a man who had scratched himself working in his rose garden. In the era of commercial antibiotics, infectious diseases receded as a leading cause of death and life expectancies rose.

• Antimicrobial resistance is reducing the effectiveness of existing antibiotics, prompting a search for new strategies. • A drug candidate from the pharmaceutical company Roche blocks the transport of a key glycolipid in carbapenem-resistant Acinetobacter baumannii (CRAB). • Research, led by Uppsala University in Sweden, intends to disrupt the synthesis of membrane lipids in E. coli . • The studies demonstrate the potential of membrane lipids as an underexploited target in antibiotic treatments.

LIPID-CENTRIC THERAPIES

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THE GRAM-NEGATIVE CHALLENGE CRAB, E. coli, and many other drug-resistant species fall within a category called gram-negative bacteria, which have a mul tilayered cell envelope that makes them difficult to defeat. (The name refers not to the unit of weight but to the scientist, Christian Gram, who developed a test to help classify them in the late 1800s.) “It has been more than 50 years since the last distinct class of antibiotic was launched that is capable of treating infections by gram-negative bacteria,” Bradley said. “At the same time, drug resistance to all existing classes of antibiotics has been on the rise in various gram-negative bacteria for sev eral decades.” Penicillin disrupts the peptidoglycan cell wall, which is present in both gram-negative and gram-positive bacteria. Other types of antibiotics block synthesis of the proteins bac teria need to grow. But the bacteria’s membrane layers have been relatively underexploited as targets. The drug candidate from Roche targets the outer membrane that sits on top of the cell wall in gram-negative bacteria and gives them an extra layer of protection against antimicrobials. Roche’s approach relies on a class of molecule called tethered macrocyclic peptides (MCP). Merck recently called macrocyclic peptides “the next wave of drug discovery” in a corporate article, and they are being explored not only for antimicrobials but cholesterol medicines, cancer therapies, and other applications. The peptides often prompt references to Goldilocks because of their ability to achieve “just-right” func tionality, penetrating cells like small molecules can but retain ing the target specificity of larger molecules. It was the peptide that led Bradley and colleagues to focus on the bacterial membrane, not the other way around. They screened a collection of approximately 45,000 macrocyclic peptides for their ability to kill bacteria, rather than filtering for a specific component of the cell.

While these medicines have been transformative in treating staph, tuberculosis, strep, and a host of other bac terial illnesses, they do not do a thing for others—but doc tors prescribe them anyway. They were used as a last resort in the absence of a diagnosis; sometimes, because patients demanded them. The US Centers for Disease Control and Prevention estimates that one in three antibiotics prescriptions is unnecessary. Overprescribing, combined with excessive use of antibiotics in agriculture and an anemic pipeline of new drugs, provided the opportunity for bacteria to evolve more defenses. “We have virtually exhausted traditional drug discovery resources, such as chemical libraries and finding bacteria- and fungi-derived antibiotics in the environment that can be grown easily in the laboratory,” said Anthony Fauci, then director of the National Institute of Allergy and Infectious Diseases, in a 2018 interview. CRAB is among the most costly and stubborn superbugs that haunt hospitals and nursing homes. It can make peo ple very sick, infecting the lungs, urinary tract, skin, and other parts of the body, but it can also colonize on skin or in the body without causing symptoms, creating another transmis sion pathway. It primarily strikes immunocompromised people, persists on surfaces for days to weeks, and is impervious to most antibiotics. In other words, it is a nightmare in a health care setting. The World Health Organization put CRAB in the top “crit ical” tier of its 2024 bacterial priority pathogens list, noting that critical pathogens earned the label “because of their abil ity to transfer resistance genes, the severity of the infections and disease they cause and/or their significant global burden, particularly in low- and middle-income countries.” Overall, the list covers 24 culprits, including forms of Escherichia coli, Salmonella , Shigella , Neisseria gonorrhoeae , Pseudomonas aeruginosa , and Staphylococcus aureus .

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a

b

“This phenotypic screening approach overcomes well-doc umented technical hurdles encountered in target-based screens, specifically by selecting for ‘hits’ that have the ability to cross the bacterial envelope and inhibit essential targets, thereby killing the bacteria,” Bradley said. Optimization of an initial hit from the phenotypic screen led to a potent antibacterial compound, zosurabalpin, but the Roche team did not know how it worked. They collaborated with researchers at Harvard to establish the mechanism of action, demonstrating that MCP block the trafficking of lipo polysaccharides (LPS). LPS is part of the gram-negative bacteria’s armor. Chains of sugars anchored by Lipid A, a hydrophobic phos phoglycolipid, line the outer membrane, helping “create a dense electrolyte mesh that prevents entry of hydro phobic molecules including most antibiotics,” as Harvard companion paper author Daniel Kahne described it on his lab’s website. The bacteria’s inner membrane synthesizes LPS, moving it through other interior layers to the outer membrane. “Surprisingly, this new class of antibiotics binds both to the transport complex in the inner membrane, as well as the LPS itself, preventing its transport to the outer membrane,” Bradley explained. “Consequently, the LPS remains trapped in the inner membrane complex. Without the ability to transport LPS, the bacteria die.” Roche’s zosurabalpin is currently in human clinical tri als, with the goal to assess the safety, tolerability, and phar macology of the molecule. “Zosurabalpin has many features that position it to be a medical breakthrough,” Bradley said. “Future human clinical trials will inform whether it has the potential to address a major gap in the fight against antimicro bial resistance.” EXPANDING THE TOOLBOX FOR ANTIBIOTICS Roche is not alone in exploring membrane lipids as a possi ble venue for new antibiotics approaches, but the field is not exactly crowded. Based on both the science and expectations in the market, an antibiotic must be cheap and effective, but used as sparingly as possible to prevent resistance. Some vari eties may target a limited set of ailments, further constraining potential revenue. “Antibiotics are not particularly lucrative, and so pharma ceutical companies have left the industry. There is almost no one left,” said Doug Huseby, a researcher in the department of medical biochemistry and microbiology at Uppsala University in Sweden. Bradley echoed Huseby’s comments, saying, “Companies working in this space have faced severe financial challenges, because there is not a viable market for new antibiotics based on traditional incentives or reimbursement models.” Besides zosurabalpin, Roche has one other novel antibi otic in the works, Genentech research and early development’s LepB inhibitor (RG6319), which also is focused on the treat ment of carbapenem-resistant gram-negative infections. Both Bradley and Huseby noted a need to fix the economics of anti

biotics through incentives or different commercial models that could stimulate investment. Huseby published research in April that also targets LPS in gram-negative bacteria, albeit in a different way and in a different organism, E. coli . His paper, “Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria,” was published in the Proceedings of the National Academy of Sciences (https://doi.org/10.1073/ pnas.2317274121). The outer leaflet of gram-negative bacteria is “something that is difficult to overcome, but it also represents a weakness,” Huseby said, because human cells do not have it. “If you can find something to stop the synthesis of this outer membrane, it means it is unlikely to specifically interact with any human sort of pathways.” His team’s research, which is also based on phenotypic screens, inhibits one of nine enzymes needed to make Lipid A, the anchor in LPS. “The importance of LPS, and Lipid A in par ticular, for the viability and virulence of gram-negative bacteria makes the inhibition of Lipid A biosynthesis a promising target for the development of antibiotics,” Huseby and coauthors wrote. Researchers continue to tweak existing antimicrobial mechanisms and use new technologies to search for untapped natural sources of antibiotics. Iterating on current strategies and exploring new ones, like targeting membrane lipids, are both required against an adversary that has billions of years of evolution in its favor, Huseby said. “Bacteria are always trying new things. They have huge pop ulations and a lot of variation, and so there is always pressure for them to come up with solutions to the problems that they are facing,” he said. “In that sense, resistance is almost inevitable.” Christina Nunez is a writer and editor based near Washington, D.C. She writes about science, technology, and innovation for a variety of organizations, including National Geographic and the U.S. Department of Energy. A schematic (a) of lipopolysaccharide (LPS) transport between inner and outer membranes of a bacterium cell. Researchers at Roche discovered that macrocyclic peptides (b) block the transport killing Acinetobacter strains. Source: Pahil, K.S., et al. , Nature , 625, 572-577, 2024.

INTERPRETING DATA

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Assessing

health risks when faced with contradictory research George Hale

A new study showing increased risk of atrial fibrillation among people taking omega-3 supplements has drawn attention from researchers and shows the importance of viewing new findings with a critical eye when assessing health risks. The past few decades have seen the rise and fall of heroes and villains in the world of health and wellness. One such hero is omega-3 fatty acids, which have been found to improve health outcomes ranging from cardiovascular disease to diabetes to various types of cancer to perhaps even mental health. Could the findings from a controversial new study tarnish omega-3’s heroic reputation? Along with critics’ questions about the study and its findings, it also raises an important question: How should experts and non-experts assess health risks? FATTY FRIEND OR FOE? As AOCS members know, the omega-3 fatty acids eicosapen taenoic acid (EPA) and docosahexaenoic acid (DHA) carry the ‘essential’ designation since they are required for normal metabolism, but the body cannot produce them on its own.

• Despite an existing body of research, recent media coverage shined a spotlight on one negative aspect of an omega-3 study. • Experts and non-experts alike can routinely use a refresher on how to report or understand health risks from the scientific literature. • For scientists, integrating the data from an ever-growing pool gets complicated and requires a healthy understanding of statistics to properly weigh new evidence. • For the general public, the United States National Institutes of Health (NIH) has established a training program for how to evaluate health risks.

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These fatty acids support healthy heart, brain, and vision func tion. Fish oil is a rich source of EPA. Whether through natu ral sources such as fatty fish or through supplements, experts have recommended that people take in an adequate amount of omega-3 each day. The Global Organization for EPA and DHA Omega-3s (GOED), an organization representing the worldwide EPA and DHA omega-3 industry, recommends a daily dosage of 500 mg of omega-3 fatty acids from some combination of food and supplements. Multiple studies have found decreased risks of cardio vascular disease and improved outcomes associated with increased omega-3 intake. However, a new study published in the journal BMJ Medicine reports that people without car diovascular disease who regularly take omega-3 supplements may have a higher risk of atrial fibrillation (a-fib), a type of abnormal heartbeat that can lead to blood clots and stroke (https://www.bmj.com/content/368/bmj.m456). This finding captured widespread media attention despite other results in the study showing several improved health outcomes among people who already have cardiovascular disease (nytimes. com/2024/06/24/well/eat/omega-3-fish-oil-heart-health. html). At the same time, the study has also drawn the inter est of other researchers who have issue with media cover age of these findings and with the study itself (https://tinyurl. com/3u7x399j). For instance, GOED notes that only eight out of the more than 90 long-term clinical trials involving omega-3 supplementation and cardiovascular disease out comes reported on a-fib. Similarly, the reported 13 percent increase in relative risk of a-fib reflects a small difference in a-fib incidence between people with and without cardiovascu lar disease. RISKY BUSINESS Researchers have questions about the study and its find ings. Because of this, GOED is planning to publish a paper using a health economics perspective to provide a risk-ben efit assessment and will commission a research project that takes a look at patient data from other studies to bet ter understand the implications of this study’s findings. But what do these findings mean for assessing health risks? Regulators set policies intended to keep the public safe from health risks. At the same time, the public makes their own decisions about health risks every day. However, while these two groups aim to assess health risks, their methods can differ drastically. Regulators have an important and challenging role in assessing health risks. Not only do they have to assess a large volume of research, they have to weigh risks and ben efits for a wide range of people. Regulatory agencies fol low guidelines on properly evaluating research, which is sometimes inconsistent or, like the recent omega-3 study, contradicts other scientific knowledge. Some assessment approaches focus solely on data, with some guidelines hav ing rules in place to handle conflicting evidence, while oth ers rely more heavily on expert judgment.

However, the growing volume of research and increased demand for transparency has raised the need for updated risk assessment approaches. One approach is outlined in a 2015 paper published in the journal Risk Analysis (https://doi.org/10.1111/risa.12206 ). In that paper, author Lorenz Rhomberg, describes the challenges of integrating dif ferent sources of data when assessing health risks and pro poses a hypothesis-based, weight-of-evidence assessment method. This approach builds on existing methods to evalu ate contradictory evidence and make use of expert judgment that is informed by research. The intent is a flexible approach that transparently shows how evidence supports expert conclusions. SAFE AT HOME While regulatory agencies play an important role, the pub lic also needs to make informed decisions about health risks. However, this can be challenging because many lack the in-depth knowledge needed to evaluate complex research. Complicating matters further is the way popular media often overemphasizes findings that are new or show the potential for harm. The new omega-3 study may have found an elevated risk of a-fib in some people, but that was only part of the study’s findings. The researchers found that omega-3 sup plements were associated with improved outcomes in other areas. For instance, people who were already diagnosed with a-fib showed a lower chance of having a stroke or other adverse event. However, the negative outcome is the one that attracted the most media attention. Bill Harris, president of the Fatty Acid Research Institute and professor of medicine at the University of South Dakota, said that this tendency is one thing to consider when evaluat ing health risks. “You have to be even-handed about it,” said Harris. KNOWING THE SCIENCE The fact that the study contradicts the majority of other research on omega-3 and health outcomes is something that should make the public take notice. It could be pointing to something important, but it also could indicate a problem with the study or with its media coverage. However, without a good understanding of what to look for, it can be hard to find accu rate health information. The National Institutes of Health (NIH) created an initiative called Know the Science to assist the pub lic in these cases (tinyurl.com/3kn4dhfj). Through this initiative, NIH is giving the public tools to make better decisions about health risks, determine the quality of a health news story, and make sense of scientific publications. One such tool is a list of questions to guide the public when consuming health news media. These questions include whether a study was done on animals or people, if enough people were included in the research, whether the study pop ulation represents the whole population or different sub groups, how well the story discussed the study’s limitations,