Science highlights from St. Louis

July/August 2019

• The 2019 AOCS Annual Meeting & Expo, held in St. Louis, Missouri, USA, on May 5–8, offered many interesting and informative oral presentations.
• Those who attended could choose from more than 400 oral and 150 poster presentations given during 57 technical sessions and 10 poster sessions.
• Although complete coverage is not possible, this article highlights several talks that exemplify the high-quality science presented at the meeting.

Despite a forecast calling for storms, attendees of the AOCS Annual Meeting May 5–8, reveled in sunny days in St. Louis, Missouri, USA. Blue skies and warm temperatures made for picturesque selfies near the St. Louis Arch during the day and comfortable viewing of a Cardinals’ game in Busch Stadium at night. While inside the conference hall, over 1,300 attendees experienced educational short courses, lively division meetings and receptions, and a bustling Expo. They took home metal straws and memories of energizing comradery and a multi-block city blackout.

Making new connections and gaining a fresh perspective on your career is what the annual meeting is all about. Even our established members got a jump start from attending. Kaustuv Bhattacharya, edible applications technology division vice chair, said he was grateful for the opportunity to meet Filip Van Bockstaele, food science professor at Ghent University in Belgium, Netherlands. “He has a great mind, and I plan to communication with him to discuss our division activities,” Bhattacharya said. For governing board member Phillip Kerr, the highlight was the AOCS Member and Volunteer Appreciation Luncheon, where volunteers for the society were recognized before a broad audience of their peers and fellow members. “The luncheon provided an opportunity to recognize the extraordinarily impact of the careers of many long-term AOCS members, while highlighting those mid-career members who are on track to make a significant contribution to the industries they serve,” Kerr said. Bhattacharya and Kerr both said they look forward to new experiences at the 2020 AOCS Annual Meeting & Expo Montréal, Québec, Canada on April 26–29.

The following is a sliver of the impactful science that was shared at the 2019 meeting. Unfortunately, there are not enough pages in Inform for a complete review of all the stimulating presentations that were given.

Use of epigenetics to identify the role of fatty acids in personalized nutrition
Presented by Iwona Rudkowska, Department of Kinesiology, Laval University, Canada
Featured Session

Dietary fat intake affects our cardiometabolic health benefits by influencing the amounts of low-density lipoprotein (LDL), high-density lipoprotein (HDL), insulin resistance, and stiffness of our arteries. However, there is no simple formula determining what food we should eat to stay healthy. We may believe that the lipid-rich plaque in our arteries will decrease if we simply change our diet or decrease our intake of saturated fats or cholesterol. That is often not the case.

One of the first studies to show the variability of LDL, HDL, and triglycerides after a low-fat diet was published in 1997, by Schaefer et al. While 55% of men on the low-fat diet decreased their LDL, 3% of them showed an increase. For women, only about 39% decreased their LDL, with 13% increasing it. Given the age of the study, it is apparent that we have known for a long time that few people respond the same way to a diet.

The question remains: What causes these differences in response? Our gender, bodyweight, health status, and environment factors, such as physical activity, smoking, and medications, all play a role, but so do our genetics, epigenetics, and microbiome. Due to this myriad of factors, Rudkowska stated that we cannot determine that any one food will be beneficial to all people. Any formula for determining if a food has cardiometabolic health benefits for a person should evaluate the food’s dietary fat content against that person’s individual variations, considering the bioavailability of the food and the individual’s biological responsiveness.

Aside from about 1% of the genome, all humans are genetically identical. That 1% could account for our different responses to various foods. In addition, our interaction with our diet modifies our gene expression, determining our phenotype. To explain, Rudkowska relayed results of experiments on omega-3s she performed as a post doc at Laval University. A range of results on the effect of triglyceride lowering by omega-3s have been previously reported in the literature. This could be due to multiple factors, like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) doses or differences in background diets. Rudkowska was curious if it was due to genetics. She conducted a study of the French-Canadian population, giving participants three grams of omega-3s (EPA and DHA) for six weeks. Most of the population was identified as responders, meaning they showed a decrease in triglycerides. However, about a third showed no change. She performed a genome-wide association study (GWAS), looking at all the genotypes of the individuals, and compared the genotypes of those who responded to those who did not respond to omega-3s (https://doi:10.1194/jlr.M045898). Her team identified 13 polymorphisms that were significantly different between the groups (Fig. 1). This indicates that there is a genetic component that could determine whether an individual responds to omega-3s. Using these results, the team created a genetic risk model that predicted who would respond and who would not respond to the omega-3s. The model successfully predicted responders for a different set of French-Canadian participants. They tried the same study on a different population with a different genetic background and found that the model was unable to predict who would be a responder. This finding reiterates that fact that dietary response has a genetic component.

FIG. 1. GWAS Manhattan plot showing differences in allele frequency between responders and nonresponders to n-3 PUFA supplementation (Iwona Rudkowska et al. J. Lipid Res. 2014)

Further, Rudkowska reviewed a study by Corella et Ordovas (https://doi.org/10.1002/bies.201300180) on the Mediterranean diet. This diet incorporates more monounsaturated fatty acids (MUFAs), lower saturated fat (SFAs), and moderate polyunsaturated fatty acid (PUFAs) compared to a western diet. This study examined if there was a genetic component to the Mediterranean diet’s success. The TCF7L2 gene regulates blood sugar and individuals who have the gene are prone for diabetes. One polymorphism (rs7903146) of this gene has the C-allele or T-allele carriers. People with this T-allele usually have higher risk of hyperglycemia, hypertriglyceridemia, hypercholesteremia, type-2 diabetes, and cardiovascular disease compared to the C-allele carriers. Would the added fat component of the Mediterranean diet still improve the health of people with this polymorphism? These researchers found that people with the T-allele experience a normalization of their plasma glucose, cholesterol, and triglycerides on the Mediterranean diet.

Rudkowska went on to explain that in addition to genetics, an individual’s response to dietary fats is determined by their epigenetics and microbiome. Nutritional genomics is a field of study that identifies the dietary patterns for individual variations that determine a phenotype and can predict nutritional profiles that will benefit that individual. They evaluate if a Mediterranean diet, a low-fat diet, or a high-fat diet, is best, and recommend specific foods and nutrients, such as MUFAs, PUFAs, and SFAs that may benefit certain individuals. She acknowledged that more studies are needed to confirm the findings of many of the reported results and that the field is too new to have conducted long-term viability studies.

New insight into molecular origins of cocoa butter polymorphism
Presented by Saeed M. Ghazani
Edible Applications Technology

Cocoa butter is fat extracted from the seeds of the cocoa bean after it is dried, fermented, and roasted. The physical properties of chocolate (sharp melting profile, gloss, snap, etc.) are related to the physical properties and polymorphisms of the main three triacylglycerols (TAGs) in cocoa butter: 1,3-dipalmitoyl, 2-oleoyl glycerol (POP); 1-palmitoyl, 2-oleoyl; 3-stearoyl glycerol (POS); and 1,3-distearoyl-2-oleoyl glycerol (SOS). Understanding the interactions of these three TAGs may expedite the development of a product that mimics cocoa butter. A full replacement of natural cocoa butter may eventually be needed since cocoa supplies are diminishing. Cocoa trees are under threat for various reasons depending on the region where they are grown. Demand for chocolate currently outweighs supply with reports of an outright shortage by 2020.

To find a fat that mimics the same chemical and physical properties as cocoa butter, Ghazani and his colleague first looked at the triacylglycerol composition of cocoa butter to figure out the polymorphism and physical properties of the three major TAGs present. They also studied binary and ternary interactions between POP, POS, and SOS in their triclinic solid-state structure. They accomplished this using cocoa butter crystallized from both an organic solvent and directly from the melt. Ghazani started isolating pure TAG components from Kpangnan butter and Chinese tallow butter to obtain high-purity POP and SOS. He developed crystallization procedures to obtain the most stable crystal polymorphic form of POS (β1), then crystalized the samples for about two years at room temperature to allow for the most stable crystal form to be achieved and avoid transients. He then synthesized cocoa butter equivalents using enzymatic interesterification to observe the physical property effect on manipulating the POP, POS, and SOS ratios.

Two new triclinic polymorphs, β3, and β2, were obtained for POS. The small and wide-angle powder X-ray diffraction pattern of β3 POS was identical to that of form V in cocoa butter (Fig. 2). The melting point of POS in β3, β2, and β1 crystal polymorphic forms was 32.9°C, 33.8° C, and 38.7° C, respectively. Next, Ghazani evaluated the binary interaction of POP, POS, and SOS in both the meta and the most stable crystal polymorphic forms using static tempering (AOCS protocol) or shear tempering. He found that the static tempering method was not sufficient to transform the crystal polymorphic form of POP and POS individually or in their binary mixtures (POP:POS and POS:SOS) from pseudo-β’ to β forms. By contrast, solvent crystallization yielded the form β2, similar to results obtained with cocoa butter. Temperature cycling (30° C to 18° C) was found to influence the 1:1 binary interaction between POP:POS, POP:SOS, and POS:SOS. In ternary interactions between POP, POS, and SOS, he found a strong eutectic region between POP:POS mixtures. The researchers conclude that POS dominates crystal forms IV and V of cocoa butter and modulates their transformations from form IV to form V. This study suggests a relationship between POS polymorphic transitions and the fat bloom defect formation in chocolate. Since POS showed a critical functionality in the crystal polymorphism and melting behavior of ternary mixtures of POP, POS, and SOS, they recommend the inclusion of POS in the design of cocoa butter equivalents.

FIG. 2. X-ray diffraction data and microscopic image of 1-palmitoyl, 2-oleoyl, 3-stearoyl glycerol (POS) in β3 crystal polymorphic form which dominates cocoa butter’s triclinic polymorphism (form V). (Ghazani and Marangoni, Crystal Growth and Design, 2019).

Dishwashing appliance trends’ impact on detergent formulation
Presented by Monica Ochoa Ruiz, Home and Personal Care, DuPont, The Netherlands
Surfactants and Detergents

The surfactants and detergents industry obviously must keep pace with changing trends in automatic dishwashers. However, Ochoa Ruiz explained that these trends are determined by multiple consumer preferences, not just established by the machines themselves.

Three factors affect automatic dishwashing: the detergent, the appliance, and the food. No factor can be considered independently as a focus of customer satisfaction. For example, consider the relationship between the food and the appliance. If manufacturers only focus on running the appliance at lower temperature, customer satisfaction will not be met because the appliance will not be able to remove the fat from the food we eat. The relationship between the food and the detergent requires detergent formulations that are specific to the foods consumers routinely consume. The detergent must contain active ingredients and enzymes that are effective against foods popular with consumers. Lastly, there is a potential for auto-dosing to become popular in appliances. Homecare professionals need to think about how such appliance technology will affect detergent formulas. It is important for formulators to consider all these factors when looking to the future.

The current appliance trend in North America is a focus on sustainability. Consumers want to have a clear conscience about the products they use, and they want brands that embrace similar values, reports Ochoa Ruiz. Today, appliance manufacturers are investing in reducing the impact of dishwashing on the environment. According to the Consumer Electronics Show held in Las Vegas, USA, in January 2019, two popular manufacturing trends influenced by sustainability were half-load options and more efficient drying. Half-load only runs a wash in half the machine, saving time, water, and energy. Drying efficiency is achieved by building machines with stainless steel interiors which are standard in Europe and becoming more so in North America. Aside from sustainability, manufacturers seek to provide customers with high-performance, convenient appliances. They have added a steam option to machines as a prewash for stain removal, and redesigned wash arms that operate in dual zones, offering variable water force where needed. Machines are now connected to smart devices so they can be started at any time and from any place. The ultimate combination of all these trends is a product by Heatworks based in Mt. Pleasant, South Carolina, USA. They have created a dishwasher the size of a toaster oven that sits on the counter and does not require a water supply connection (Fig 3.). Some of these trends will improve the cleaning performance of dishwashing appliances, but they will likely create new challenges for detergent formulators.

FIG. 3. The latest trend in dishwashers by Heatworks in Mt. Pleasant, South Carolina, USA. It sits on your kitchen counter and requires no water supply hook-up. (Credit: Heatworks)

Just as a focus on sustainability is popular among consumers, so is a greater focus on eating healthfully. The population of people who are eating vegan or vegetarian has increased over the years. Globalization has also influenced consumer food choices, since food from all over the world can easily be found in North America. To understand how dietary choices will influence detergent formulations, Ochoa Ruiz’s group performed a consumer study. They surveyed North American consumers about their most-consumed food, how often the food stains, and how difficult it is to remove the stains. Tea, coffee, and curry were the most commonly reported and difficult-to-remove stains. The survey respondents also reported difficulty removing dried-on and baked-on food.

After gathering all this information about trends in consumer preferences. Ochoa Ruiz sought to determine what these trends mean for detergent formulators. She tested standardized stains, of mixed starches, to see how detergents performed at different cycle lengths. She found that performance of both amylase and protease enzymes were reduced when wash cycles were shortened from one hour to 30 minutes. The Sinner’s cycle states that temperature, chemistry, time, and mechanical action must all be in harmony to achieve optimal cleaning. If any aspect is decreased, another must compensate. Ochoa Ruiz stated that if less or cooler water is going to be used to satisfy consumer trends toward sustainability, detergents need to be improved. Enzymes are key in this capacity, she said. After adding more enzymes to the detergent formulations used in the cycle length comparison study there was no difference in cleaning performance despite a shorter cleaning time.

Restructuring lipids for functionality and health
Casmir C. Akoh, University of Georgia, USA
Biotechnology

Akoh presented an overview of the research that takes place in his lab, where they focus on structuring lipids with the intention of creating molecules that will provide a health benefit on consumption. Either chemical catalysts or enzymes are used to modify these lipids. However, enzymes provide more mild conditions than chemical catalysts and are, therefore, used more often in the Akoh lab.

One food material Akoh is studying is a replacement for human milk fat. Human milk fat analogs primarily contain oleic acid, palmitic acid, and linoleic acid, in stereospecific arrangements, for example oleic-palmitic-oleic (OPO) or oleic-palmitic-linoleic (OPL). In addition, they contain substances like docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6). Akoh’s lab is working on infant formulas that would duplicate this stereospecific structure of the fats found in human breast milk.

They begin with the vegetable oil tripalmitin and replace the outermost palmitic acid groups with oleic acid groups in one- or two-step reactions using a sn-1,3-specific lipase enzyme (Fig. 4). However, the OPO produced by either method does not have many of the free fatty acids that an infant will need and must be mixed with a preformed DHA and arachidonic acid in infant formulas. More recently, the Akoh group attempted another synthesis method by starting with two triacylglycerols and then adding two different enzymes along with the desired free fatty acids. They allowed these reactants to compete, and the result was a mixture of molecular species which included OPO, as well as some fraction of triacylglycerols with DHA or ARA attached in place of the oleic acid groups. This synthesis was then improved by a two-step method that better controlled the fatty acid placement. By forming an intermediate with a higher concentration of palmitic acid on position 2 of the triacylglycerol backbone, the researchers had the ability to selectively add DHA, ARA, or oleic acid.

These experiments used vegetable oils as a starting point for the structured lipids, however, human milk fat has a unique triacylglycerol structure. In milk produced by humans, the glycerol has unsaturated fatty acids at the 1 and 3 positions. Research shows that unsaturated fatty acids in these positions allow for palmitic acid to reside at the 2 position, which is critical for calcium absorption by the infant. The Akoh group has started to develop triacylglycerols with unsaturated chains to take advantage of this health benefit.

Triacylglycerols with different degrees of saturation, in addition to a mixture of chain lengths, have a benefit not just to infants, but also for endurance athletes. Akoh’s group is working on products that will provide both immediate and sustained energy for active consumers. They produced medium- and long-chain oils by starting with coconut oil and removing the undesirable fatty acids. They were able to replace the 50% lauric acid concentration in the oil with high oleic sunflower oil, resulting in a product containing only 20% lauric acid.

Before concluding his talk, Akoh described the other lipid restructuring efforts in his lab, which include menhaden oil structured lipids, and the synthesis of 1-o-galloylglycerol and cocoa butter substitutes. Throughout the talk, he emphasized his group’s reliance on enzymes to make these structural changes possible.

FIG. 4. One- and two-step synthesis of human milk fat. (Pande et al. & Akoh, J. Agric. Food Chem., 2013)

MCPD and glycidyl esters—present and future EU legislation, implementation in German risk management
Martin Kaminski, Federal Office of Consumer Protection and Safety, Germany Analytical

Maximum levels for contaminants in foods were set by the European Union (EU) in 2006. These regulations cover contaminants such as, nitrates, mycotoxins, heavy metals, dioxins, and 3-MCPD. The regulation was amended several times to include other contaminates, the latest amendment made in 2018, that set maximum limits (ML) for glycidyl esters. EU regulation 290 determined that the maximum level of glycidyl fatty acid ester in vegetable oils is 1,000 µg/kg. If the oil is being used to make baby food, the limit is 500 µg/kg. If the vegetable oil-based infant food or formula is intended for medical purposes, the acceptable limit drops to 75 µg/kg for powders and 50 µg/kg for liquids (these values will drop to 50 µg/kg and 6 µg/kg, respectively starting in July of 2019).

Kaminski reported that Germany’s Federal Office of Consumer Protection and Food Safety is currently discussing their maximum limits for these contaminants. For oils like coconut, rapeseed, and palm kernel, they are considering a maximum limit of 1,250 µg/kg and a limit of 2,500 µg/kg for marine-based oils. Baby food and formula limits will likely be set just above the EU standards. These potential maximum limits were issued for glycidyl esters, but not for 3-MCPD because of discrepancies in the limits set by the European Food Safety Authority (EFSA) and the Japanese Food Safety Authority (JFSA). EFSA set the tolerable daily limit of 3-MCPD at 0.8 µg/kg of body weight, but JFSA established a limit of 4 µg/kg. Due to the discrepancy the EFSA revaluated their data and in 2018 reset the TDI to 2 µg/kg.

When considering these proposed maximum limits for 3-MCPD, Kaminski first questioned whether they were achievable. Looking at 2012 data from the EFSA on 3-MCPD in vegetable oils, he found that for most oils on the market the ML was less than those under consideration by the German government. This is true even in the case of infant products, which require stricter food contaminant MLs given high-quality raw materials are used. However, the palm oil and olive pomace oil industries may have difficulty meeting proposed limits since both oils exceed them in the 2012 data.

Since most MLs can be met, Kaminski stated that regulating oil blends is likely to pose the most difficulty. An oil mixture will have to comply with the MLs for all the components of the mixture. For example, the resulting limit for a mixture of 50% coconut oil with an ML of 1,250 µg/kg and 50% palm oil with an ML of 2,500 µg/kg, would be 1,875 µg/kg for glycidyl esters. If the composition of the mixture is unknown, the ML is 2,500 µg/kg. Kaminski stated this puts an undue burden on official control laboratories to determine the composition of the oils, which are often unknown or proprietary. Even when compositions are reported, the laboratories cannot be certain they are accurate. This puts them in the position of always adhering to the higher of the two limits, which may not be safe. Kaminski stated the lower limit would be preferable. The regulation of blended oil becomes increasing complicated when having to consider both the EFSA and the JFSA 3-MCPD standards.

Kaminski reported that his lab is responsible for Germany’s risk management of 3-MCPD and glycidyl esters by organizing the proficiency tests and coordinating method standardization. Once established, these tests are implemented in German control laboratories. The last test, to be released in early 2020, will be the determination of 3-MCPD and glycidyl esters in fish oil. The current 3-MCPD and glycidyl esters standard methods for various food matrix categories are limited to oils, and oil-based emulsions, but Kaminski said methods are needed for matrixes like food supplements, fried potato products, instant noodles, and fish meal products. In January of 2019, his lab established a standardization working group to address these missing methods.

Rebecca Guenard is the associate editor of Inform at AOCS. She can be contacted at rebecca.guenard@aocs.org.

References

Ghazani, S.M. and A.G. Marangoni, Cryst. Growth Des. 19: 90–97, 2019.

Zou, L., G. Pande, and C.C. Akoh, Infant formula fat analogs and human milk fat: new focus on infant developmental needs, Annu. Rev. Food Sci. Technol. 7: 139–165, 2016.

Pande, G., J.S.M. Sabir., N.A. Baeshen, and C.C. Akoh, Enzymatic synthesis of extra virgin olive oil-based infant formula fat analogues containing ARA and DHA: one-stage and two-stage syntheses, J. Agric. Food Chem., 2013.

Li, J., X. Zeng, and A. Stevels, Ecodesign in consumer electronics: past, present, and future, Crit. Rev. Env. Sci. Technol. 45: 8, 840–860, 2015.

Da Silva, M.S., D. Chartrand, M.C. Vohl, O. Barbier, and I. Rudkowska, Dairy product consumption interacts with glucokinase (GCK) gene polymorphisms associated with insulin resistance, J. Pers. Med. 7: 8, 2017.

Guenard F., A. Bouchard-Mercier, I. Rudkowska, S. Lemieux, P. Couture, and M.C. Vohl, Genome-wide association study of dietary pattern scores, Nutrients 9: 649, 2017.

Jędrkiewicz, R., M. Kupska, A. Głowacz, J. Gromadzka, and J. Namieśnik, 3-MCPD: a worldwide problem of food chemistry, Crit. Rev. Food Sci. Nutr. 56: 2268–2277, 2016.

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