BEHAVIOR OF POLYSACCHARIDE STRUCTURES IN THE GUT AND IMPLICATIONS IN CHRONIC DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1015716
• Project Start Date: Jun 18, 2018
• Project End Date: Nov 24, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Food Science & Technology

Non Technical Summary
Obesity and cardiovascular disease (CVD) are major public health concerns worldwide, with coronary heart disease being the leading cause of death for people of most ethnicities in the U.S.Many of these deaths are potentially preventable with appropriate changes to diet and lifestyle. Despite extensive recommendations from public health bodies to improve diet and physical activity habits, only a small minority of the population changes its lifestyle in response to such advice. Strategies focused on reducing the caloric content from the diet do not seem to be working neither, due in part, to a reduction in foods' palatability. An opportunity then exists, to develop strategies that look beyond caloric content in order to produce healthier processed foods with appealing taste, texture and appearance that satisfy the consumer's demands.Certain food polysaccharides and dietary fibers have been recognized as healthy, showing positive effects in reducing blood cholesterol and lipid concentrations as well as promoting feelings of fullness and are, therefore, candidates to combat CVD and obesity. However, despite their clear health benefits, dietary fiber is deficient in the American diet. The U.S. population consumes only half of the fiber recommended intake, with average intakes at around 14-15g/day. Considering that further processing of sources rich in dietary fiber can reduce its efficacy, the daily recommended intake of fiber would need to be highly increased and most consumers would find this difficult or unpalatable. Therefore, approaches are needed to incorporate dietary fibers into commonly consumed foods without compromising their sensorial attributes and making sure that can promote feelings of satiation and hypocholesterolemic effects. Additionally, it is not well understood yet how exactly dietary fibers and polysaccharides achieve the above mentioned healthy effects. Because of the large variety of dietary fibers, more knowledge is needed about the specific biological processes that dietary fibers are involved with, so that their positive physiological effects can be maximized. This project aims to understand 1) how dietary fibers provide controlled lipid digestion, regulation of blood lipids/cholesterol and increased satiety responses and 2) how processing of dietary fibers change their bioactivity and the sensorial attributes of fiber-containing foods. It also aims at using food polysaccharides to design systems for enhancing the delivery of lipid-soluble bioactive micronutrients. In order to achieve these goals, we will follow an approach involving the use of a range of biophysical techniques together with in vitro models of digestion and fermentation to simulate how dietary fibers behave in our gut. Based on these observations, clean-label processing technologies will be applied to understand the impact of food processing. By conducting these experiments, we anticipate that we will identify strategies by which dietary fibers can be processed and added into foods to maintain optimal health benefits without compromising their sensorial quality. Consequently, successful completion of this project would help improve dietary advice and the design of healthier foods, thereby improving the population's quality of life and reducing the prevalence and economic burden of chronic diseases.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	5010	1000	30%
502	5010	2000	25%
502	1410	1000	10%
702	5010	1000	25%
501	1410	1000	10%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components; 501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
1410 - Beans (dry); 5010 - Food;

Field Of Science
1000 - Biochemistry and biophysics; 2000 - Chemistry;

Keywords

food structure

satiety

dietary fiber

polysaccharides

lipid digestion

cholesterol

bile salts

emulsions

delivery system

Goals / Objectives
Thelong-term goalof this researchis to develop strategies to design healthier food products without compromising their sensorial quality and consumer acceptability, in order to address major diet-related challenges such as obesity, CVDs and type 2 diabetes. The overall objective of this projectis to understand how food polysaccharides/dietary fibers influence the behavior of lipids, and how we can design polysaccharide-based renewable structures to improve the delivery of lipid-soluble bioactive molecules. To achieve the main goal, we propose the specific objectives:Elucidate the mechanisms by which dietary fiber decrease blood lipids/cholesterol and promote satiety.Determine how the bile salts (BS) binding ability of dietary fibers influence interactions with BS at the lipid surface.Determine the impact of dietary fibers on lipid digestion in vitro.Evaluate the way that both dietary fiber and BS affect the microbial community in the gut in vitro.Evaluate non-thermal processing methods to retain bioactivity of dietary fibers and to improve sensorial attributes of fiber-containing foods.Determine the effect of high pressure processing (HPP) on the physicochemical and the cholesterol-reducing properties of dietary fibers.Determine the effect of HPP on the sensorial acceptability of fiber-containing foods.Develop a nano-delivery system designed with modified celluloses that improve the oral delivery of lipid-soluble bioactive molecules. Design physically stable nano-delivery systems, using modified celluloses carriers, that maximize dissolution of model lipid-soluble molecules.Evaluate nano-delivery systems for improved bioaccessibility of model lipid-soluble molecules under GI conditions in vitro.

Project Methods
To address the objectives of this proposal we will use a combination of spectroscopic, colloidal and interfacial techniques coupled with in vitro digestion models. Purified food polysaccharides and dietary fibers from different sources (food matrices, agrofood industrial residues) will be tested as potential anti-cholesterol and satiety agents for intestinal sequestration/adsorption of BS and lipids. Structural differences in selected polysaccharides will be studied by NMR, which will allow us to elucidate the key structure-property- performance relationships. Food matrices and residues will be analyzed for their content in dry matter, protein, lipid, starch using standard methods outlined by the AOAC. Soluble (SDF) and insoluble dietary fiber (IDF) contents will be determined by sequential enzymatic digestion and gravimetric filtration according to the AOAC Method 991.43 using a commercial assay kit. The colloidal and physicochemical properties of sample will be determined. Study 1.1. Determine interactions between polysaccharides and BS at the oil/water interface and in the bulk. We will evaluate the interactions between food polysaccharides and BS in two different scenarios found for emulsified lipids on their transit through the duodenum (interactions that take place at the lipid droplet surface and bulk) by using Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). SPR and QCM-D provide real-time monitoring of adsorption and desorption kinetics and give information on the mass, thickness and structural changes of biopolymers assembled at the surface. In order to test the effect of BS micelle formation, the range of BS concentrations include concentrations below and above the critical micelle concentration (CMC). Study 1.2. Impact of dietary fibers on lipid digestion in vitro. Emulsions (20% (w/v) oil) will be stabilized by the non-ionic surfactant Tween-20, mixed with the selected fiber, and then subjected to an in vitro digestion model. In the case that the selected polysaccharide is surface active and has emulsifying properties, emulsions in absence of the non-ionic surfactant will be also prepared. The impact of polysaccharide/fiber type and initial concentration on the emulsion particle size, rheological properties and ζ-potential will be examined. In vitro lipid digestions under duodenal conditions will be performed using commercially available preparations of highly-purified materials, such as lipase, colipase and BS. A preconditioning gastric step will be included in order to consider the change of pH from the gastric to the duodenal environment. Lipid digestions of emulsions will be performed by in situ titration of the digestion mixture as the lipolysis progresses using a pH-stat automatic titration device. Bulk rheology and droplet size will be correlated with lipolysis rate in order to determine the mechanisms responsible for promoting or delaying lipolysis. Before (initial) and after each phase digestion, the droplet size and ζ-potential of the digested emulsions will be measured using the methods already described above. The residues from these replicates will be pooled for subsequent in vitro fermentation in Study 1.3. Study 1.3. Impact of dietary fiber on the microbial community in the gut in vitro. We will investigate the impact of dietary fibers on the gut microbiota composition and the concentrations of SCFAs. Residues from the enzymatic hydrolysis will be subjected to in vitro fermentation. Samples will be collected for microbiota composition and SCFA analyses. Amounts of SCFAs (acetic, propionic, and butyric acids) will be analyzed by GC/MS. BS binding and fiber viscosity will be correlated with production of SCFA. The microbial group of interest will be quantified by real time qPCR. We will pay particular attention to two of the health beneficial bacteria, Bifidobacterium spp. and Lactobacillus spp. Study 2.1. Effect of non-thermal processing on the physicochemical properties and bioactivity of dietary fibers. The effect of High Hydrostatic Pressure (HHP) on selected food matrices and/or agrofood industrial residues will be studied. HPP-treated slurries will be studied as a function of pressure level and slurry concentration. In total, 6 different treatment combinations per sample will be investigated. Samples will be characterized (e.g. physic-chemical, microstructural, colloidal) and their BS binding ability (Study 1.1), behavior during in vitro digestion (Study 1.2), in vitro fermentation (Study 1.3) will be studied before and after processing. BS-binding ability of fibers will be determined using a colorimetric kinetic assay based on enzymatic recycling and compared with cholestyramine. Changes in viscosity will be correlated with BS binding under physiological conditions in order to determine to which extent the BA-binding ability of fiber is related to their viscous and non-viscous components. For this objective, we will use a standardized multistage static in vitro digestion model suitable for food that simulates the mouth, stomach, and small intestine based on that reported previously, with some modifications. Lipolysis rate will be evaluated as in Study 1.2. Study 2.2. Effect of non-thermal processing on the sensorial acceptability of fiber-containing foods. After processing, the best candidates (samples showing the highest ability to bind BS and delay lipid digestion) will be selected for the production for example of baked goods, (e.g. bread or muffins). Instrumental textural measurements and sensorial evaluations will be performed. Study 3.1. Design stable nano-delivery systems, using modified celluloses to maximize dissolution of model polyphenols. Commercial cellulose ethers will be used to synthesize amphiphilic celluloses (AC) using cross-metathesis (CM). Nanoemulsions will be obtained by mixing vigorously an aqueous phase and an oil phase which will be then homogenized by high pressure microfluidization. Oil concentration will be fixed at 20% (w/v) and two concentrations of celluloses will be tested (0.5% and 1% (w/v)). A control emulsifier (commercial Tween 20) will be used for comparison. In order to optimize the fabrication of nanoemulsions we will determine the equilibrium and saturation concentrations of polyphenol within the oil phase to estimate the total amount of polyphenol that could be successfully solubilized within the nanoemulsion at saturation. Polyphenol concentration in the oil phase will be determined using a spectrophotometric method. Nanoemulsions will be prepared in the absence and presence of polyphenol at levels below and above its equilibrium saturation limit in oil. The same total polyphenol concentration will be used for each emulsion. We will then determine the particle size distributions and polydispersity index of nanoemulsions through dynamic light scattering methods. Study 3.2. Evaluate nano-delivery systems for improved bioaccessibility of model polyphenols under GI conditions in vitro. The best candidates (showing the highest stability and polyphenol solubility) from Study 3.1 will be selected for bioaccesibility studies. We will examine how nanoemulsion structure and composition affect the bioaccessibility of encapsulated polyphenols using the in vitro digestion model previously mentioned. We will investigate the effects of varying polymer hydrophobicity and BS concentration (fed and fasted states). The digestion process will be evaluated by evaluating the change in particle size and distribution as well as by measuring the release of FFAs as described in Study 1.2. The bioaccessibility of polyphenols will be quantified at the end of the simulated duodenal phase by measuring the polyphenol concentration within the micelle phase collected by centrifugation followed by UV-visible spectrophotometry.

Progress 06/18/18 to 11/24/20

Outputs
Target Audience: Students and food and colloidal scientists, and in general academic peers involved in research at the interface of food, polymer science, andhealth. Individual consumers and commodity groups, in particular dry bean growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period students (undergraduate, MS and Ph.D.) have received training in technical skills, specifically in 1) the use of surface analysis techniques ([e.g. Quartz Crystal Microbalance with Dissipation monitoring (Q-CMD) and Surface Plasmon Resonance (SPR)] to characterize the adsorption/desorption of cellulose derivatives to surfaces and study the interfacial interactions between them and BS; 2) rheological and colloidal (particle size, zeta potential) characterization of polysaccharide solutions, emulsions and flour dispersions; 3) Differential Scanning calorimetry (DSC) analysis,HPLC and GC analysis; 4) chemical analysis of components and dietary fiber fractions 5)in vitrodigestion methodologies to evaluate the digestion of lipids and bioaccesibility of other nutrients 6) Microscopy techniques (Optical, SEM, and Confocal).Students have also received training in experimental design, data collection and analysis, manuscript preparation and presentation of results to a wide audience. The students have communicated their research findings to scientists of different backgrounds through different research venues and have interacted with members of the IFT Carbohydrate (CHO) division the ACS carbohydrate chemistry, surface science and Agriculture and Food Chemistry divisions. Grad studentshave participated in the FST annual poster competition and were provided feedback by an evaluation committee. The students have been awarded several prizes for their research (i.e. second prize in poster presentation in the 20th Gums & Stabilizers for the Food Industry Conference, second place in the 1st Annual Alpha Epsilon Flash Talk Research Symposium, 2 first and third places in the FST annual poster competition; first prize at the IFT-CAFS video competition, Chinese American Food Society, Annual meeting IFT2020). Two M.S. students and one PhD student completed project and degree requirements.? One PhD student is currently working as a post-doc atCornell University, anda Master student is working as FSQA Associate-Technical Services at Tyson Foods How have the results been disseminated to communities of interest?Results have been disseminated through publications in peer-reviewed journals of high impact factor inthe field of Food Science and Polymer Science including Food Hydrocolloids (I.F=7.053) and Food Chemistry (I.F=6.306), presentations at domestic and international conferences (i.e. 255th ACS National Meeting, 256th ACS National Meeting, 3rd Food Structure and Functionality Symposium, IFT 2018, 32nd EFFost International Conference, the 257th ACS National Meeting, the 20th Gums & Stabilizers for the Food Industry Conference, the 8th International Symposium on Food Rheology and Structure, ISFRS 2019, NUTRITION 2019 Conference, 259th ACS National Meeting & Exposition (Online), 34th EFFoST International Conference (Online)), and an outreach article regarding the health benefits of dry beans and foods containing dry beans Research findings were also presented to an audience of faculty scientists, industry members, graduate students and undergraduate researchers, at the Macromolecules Innovation Institute (MII) Annual Technical conference and Review (Blacksburg, VA) and at the departmental level (i.e.FST Poster competition (Food Science & Technology department, Blacksburg, VA).? What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The purpose of this project was 1) to understand how food polysaccharides/dietary fibers influence the behavior of fats under gastrointestinal conditions, and 2) to design polysaccharide-based encapsulation systems that improve the release of lipophilic bioactive molecules. To address goal 1, two different sources of dietary fiber (DF) were studied: Purified dietary fiber (commercial cellulose derivatives, which are common ingredients used as thickeners and emulsifiers in the food industry) and a whole plant food rich in fiber (common beans (Phaseolus vulgarisL.)). We also tested a chemically modified cellulose (a novel anionic Hydroxypropyl-cellulose (HPC)-derivative with increased hydrophobic character obtained from the commercial HPC).We used an interdisciplinary approach combining structural, physico-chemical and colloidal characterization (rheology, particle size, differential scanning calorimetry,water and oil-holding capacity, emulsifying power and stability)and/or surface adsorption techniques (QCM-D and SPR) coupled within vitrodigestion models simulating the human upper gastrointestinal tract (i.e. mouth, stomach and small intestine) to investigate how cellulose derivatives and major bean components contribute to both, bile salt-binding and the modulation of fat digestion. We also investigated the role played by the bean matrix in these processes. Adsorption and micro-calorimetry studies, together with lipolysis experiments demonstrated thatcellulose derivativesare able to sequester bile salts (BS) (important intestinal components that aid fat digestion) and to delay the digestion of fatsin vitro. We showed that celluloses derivatives are able to interact with BS in the aqueous phase (bulk) of emulsions, and when they are adsorbed at the surface of fat droplets. Specifically, the ability of Hydroxypropyl-cellulose (HPC) to modulate fat digestion is due to a combination of events taking place at these two scenarios, with the binding of BS in the bulk by HPC being the major factor that hinders BS adsorption to the fat droplet surface. We also shown that this effect was dependent on the charge and hydrophobicity of the cellulose, and that the type of BS and their aggregation behavior play a key role in these events. Regardingcommon beans, compositional analysis and anin vitrodigestion model combined with dialysis and HPLC analysis were used to evaluate separately the ability of bean flour and bean components to retain primary BS.We found thatthe ability of beans to sequester/retain BS was not solely based on soluble fiber (SDF) content and its capacity to increase the viscosity of digestive contents, but it was also triggered by other bean components like proteins and insoluble fiber (including resistant starch), and it involves additional factors like molecular interactions. We also evaluated the effect of High Hydrostatic Pressure (HHP), a clean label, non-thermal technology, on the ability of beans to retain BS duringin vitrodigestion, and compared it with traditional cooking (HT).Since not all food products that contain fiber have the same health outcomes, we hypothesized that the bean matrix might influence the positive action of their dietary fiber.Microscopy (SEM) and thermal analysis of beans, together with rheological measurements showedthat the resilience of bean cell walls impacts the ability of bean constituents to retain BS during in vitro digestion, and specifically the degree of disintegration of the cell wall, protein denaturation and starch gelatinization play an important role in BS-binding efficiency. We also demonstrated that the effects of HHP on the physico-chemical properties of beans led to different functional and technological properties, while keeping their nutritional quality and effectively preserving and/or improving their ability to retain BS. Therefore, HHP processing is a promising non-thermal technology for tailoring the functionality of bean flours and therefore for increasing their use as nutritious and hypocholesterolemic in a range of food formulations. The studies on beans highlighted the importance of considering the structure of foods, and not just their nutrient content, when evaluating potential health impacts. By understanding how food processing affects the structural properties of beans, it would be possible to develop new strategies in the formulation of legume-based functional foods. Overall, these studies have provided a deeper insight into how dietary fibers regulate fat digestion, which can be exploited in tailoring novel rich-fiber foods with improved functionality to optimize fat digestion and to reduce blood cholesterol levels. The new knowledge obtained in this project should also help policy-makers distribute and communicate new dietary advice and guidelines from new data on plant foods. This will have a positive impact in the therapeutic treatment of obesity and CVD and will improvewellnessand reduce the health care costs associated with chronic diseasesin the US. To address goal 2, we produced nanofibers from aqueous blends of pullulan (a biodegradable exopolysaccharide) and a cyclodextrin (CD) (functional small molecules with hosting abilities) using electrospinning, and tested their potential to encapsulate and protect quercetin, QE (antioxidant molecule with poor water-solubility, and low stability) from environmental factors. This study showed that the incorporation of hydroxypropyl-β-cyclodextrin (HP-β-CD) improved the electrospinnability of pullulan, promoting the formation of well-formed and bead-free nanofibers at almost half the required polymer in neat pullulan systems. These findings are meaningful not only because HP-β-CD reduces the amount of pullulan to use, but also because it widens the range of applications of pullulan electro spunfibers by increasing their encapsulation functionality without compromising their morphology and diameter. Furthermore, electrospinning in water would avoid the need for post-processing treatments and reduce the degradation of active ingredients to be encapsulated into the spunfibers. In fact, encapsulation of QE in this system increased the water solubility of QE and delayed its photo-degradation when exposed to LED and UV-light. Thus, this encapsulation matrixmight find potential uses in the food industry, in food active packaging applications, to improve the safety, quality, and shelf-life of some packaged foods Overall, this project was a success and has provided an improved understanding of the functional versatility of polysaccharides, which can be of great benefit tothe food and pharmaceutical industry.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lin, T., Okeefe, S, Duncan, S and Fern�ndez-Fraguas, C*. (2020) Manipulation of the dry bean (Phaseolus vulgaris L.) matrix by hydrothermal and high-pressure treatments: Impact on in vitro bile salt-binding ability. Food Chemistry, 310. doi.org/10.1016/j.foodchem.2019.125699
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lin, T. and Cristina Fern�ndez-Fraguas, C*. (2020). Effect of thermal and high-pressure processing on the thermo- rheological and functional properties of common bean (Phaseolus vulgaris L.) flours. LWT - Food Science and Technology, 127, doi.org/10.1016/j.lwt.2020.109325
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Jennifer Zornjak, Jianzhao Liu, Alan Esker, and Cristina Fernandez-Fraguas*. (2020). Bulk and Interfacial Interactions Between Hydroxypropyl-Cellulose and Bile Salts: Impact on the digestion of emulsified lipids. Food Hydrocolloids, 106, 105867. doi.org/10.1016/j.foodhyd.2020.105867
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lin, T., Okeefe, S., Duncan, S. and Cristina Fern�ndez-Fraguas*. (2020). Retention of primary bile salts by common beans during in vitro digestion: Role of bean components and effect of food processing. Food Research International, 109337, https://doi.org/10.1016/j.foodres.2020.109337
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Deepak Poudel, Sarah Swilley-Sanchez, Sean Okeefe, John Matson, Timothy Long and Cristina Fern�ndez- Fraguas*. (2020). Novel Electrospun Pullulan Fibers Incorporating Hydroxypropyl- ?-Cyclodextrin: Morphology and Relation with Rheological Properties. Polymers 2020, 12(11), 2558; doi:10.3390/polym12112558
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Tiantian Lin  PhD Dissertation: Role of dry beans (Phaseolus vulgaris L.) in binding bile salts and modulating lipid digestion: Impact of the bean matrix and high-hydrostatic pressure processing  2020
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Lin, T., Okeefe, S., Duncan, S. and Fern�ndez-Fraguas, C*. Common beans, Dietary Fiber and Lipid digestibility: Impact of Bean processing. 34th EFFoST International Conference (Online), November 2020
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Poudel D. Okeefe, S. and Fern�ndez-Fraguas, C*. Quercetin encapsulation in Cyclodextrin/ Pullulan Fiber Mats produced via electrospinning. 34th EFFoST International Conference (Online), November 2020
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Lin, T.*, Okeefe, S., Duncan, S. and Fern�ndez-Fraguas, C. Role of common beans (Phaseolus vulgaris L) in modulating lipid digestion: Impact of food processing. 259th ACS National Meeting & Exposition, Philadelphia (Online), PA, March 22-26, 2020.
• Type: Other Status: Awaiting Publication Year Published: 2020 Citation: Common Beans: An unexploited and inexpensive super food to fight against CVD in a sustainable way. Research Outreach
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Lin,T.,Okeefe,S.,Duncan,S.and Fern�ndez-Fraguas, C.Dry bean(PhaseolusvulgarisL.)modulate the kinetics of lipid digestion in vitro. Journal of Functional Foods
• Type: Journal Articles Status: Other Year Published: 2021 Citation: JenniferZornjak,JianzhaoLiu,AlanEsker,DianaNovo,KevinEdgarandCristinaFern�ndez-Fraguas.Roleofinterfacial and bulk Interactions Between Cellulose Ethers and Bile Salts in the adsorption of Bile salts to hydrophobic surfaces: a QCMD-study. Journal of Colloid and Interface Science
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Poudel, D. Okeefe, S., Long, T. and Fern�ndez-Fraguas, C. Characterization, photostability and antioxidant activity of pullulan spunfibers embedded with quercetin/hydroxypropyl ?-cyclodextrin inclusion complex. Carbohydrate Polymers
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lin, T. and Fern�ndez-Fraguas, C (2019). Structurally different dry bean matrices modulate in vitro lipid digestion. NUTRITION 2019 Conference, Baltimore, MY, June 8-11, 2019.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: Students and food and colloidal scientists, especially food chemists, and in general academic peers involved in research at the interface of food andhealth. Individual consumers and commodity groups, in particular dry bean growers. Changes/Problems:A change in aim 3 has been made. The initial study within aim 3 was focused on designing delivery systems for polyphenols using modified amphiphilic celluloses carriers. Due to the difficulty in manufacturing these novel polymers, in the past reporting period, we changed the delivery system to hydrogels which needed a lower amount of the modified polymer to be produced. Nevertheless, it was not possible to synthesize enough amount of the modified cellulose for this project. Therefore, we have changed the polymer to cyclodextrins/pullulan, and used electrospinning, which is a simple and cost-effective method, to produce nanofibers as nano-delivery system of polyphenols. Owing to the different encapsulation system developed, the study corresponding to the system design and characterization (Study 3.1) has also changed. Characterization of electrospun nanofibers fromnatural biodegradable polymersincludes rheology and determination of the polymer entanglement concentration, conductivity measurements, analysis of thermal properties by differential scanning calorimetry and structural characterization by scanning electron microscopy. Because of the broad application of this delivery system in the food industry (i.e. functional foods or food active-packaging applications), we consider of interest to evaluate the release of polyphenols from thisencapsulation matrix(Study 3.2) under conditions simulating a food system instead of GI conditions. We will still keep the objective of evaluating the bioaccesibility of polyphenols to address chronic diseases, by investigating the release ofbioactive phenolicsfrom a food matrix (i.e. hibiscus) and an agrofood residue (i.e. hibiscus spent-calyx) rich in dietary fibers. What opportunities for training and professional development has the project provided?Training of graduate students including (MS and Ph.D. students) in technical skills. Specifically, during this reporting period students have received training in the use ofin vitrodigestion methodologies to evaluate the digestion of lipids, Differential Scanning calorimetry analysis and HPLC analysis. Students have also received training in experimental design, data collection and analysis, manuscript preparation and presentation of results to a wide audience. Graduate students have communicated their research findings to scientists of different backgrounds through different research venues, and have interacted with members of the ACS Carbohydrate Chemistry and Agriculture and Food Chemistry divisions. Students also participated in the 7thFST annual poster competition; one of the MS students was awarded the first place. The PhD student was awarded the second prize in poster presentation in the 20th Gums & Stabilizers for the Food Industry Conference. She also was awarded the second place in the 1st Annual Alpha Epsilon Flash Talk Research Symposium, VT, Spring 2019, and selected for the Science Mix event at the 257th ACS National Meeting & Exposition. M.S. student completed project and degree requirement. How have the results been disseminated to communities of interest?Results have been disseminatedthrough publications andpresentations atdomestic and international conferences including the 32nd EFFost International Conference, the 257thACS National Meeting, the20th Gums & Stabilizers for the Food Industry Conferenceand the 8th International Symposium on Food Rheology and Structure, ISFRS 2019. Research findings were also presented at the university level during the 7thFST Poster competition (Food Science & Technology department, Blacksburg, VA). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan toevaluate the capacity of dry beans to bind to individual bile salts differing in conjugation group and the impact that common beans treated by high-hydrostatic pressure and hydrothermal processing has on thein vitrodigestion of lipids.We also plan to conduct this line of work with studies focused onHibiscus, another source ofdietary fiber. We plan to characterize hibiscus calyces andspenthibiscus calyces, considered an agri-food residue, and to evaluate the role of fiber on the bioaccesibility release of polyphenols duringin vitrodigestion. We plan to present the research findings obtained from these studies at an international conference, a national meeting and seminars/symposiums at the university level, and through publications in peer-reviewed journals.

Impacts
What was accomplished under these goals? Objective 1: We have continued investigating the interactions between cellulose derivatives and bile salts (BS) (objective 1.1.) using surface analysis techniques. We have also studied the interactions between hydroxypropyl-cellulose (HPC) and BS in the aqueous phase using DSC, and performed simultaneous adsorption experiments using Q-CMD to evaluate how HPC-BS bulk interactions affect disruption of the lipid droplet surface. We have explored two different BS, NaTC and NaTDC. Also, sequential experiments with a novel modified HPC (anionic and with a more balanced hydrophilic-hydrophobic character) have been performed. Additionally,in vitrodigestions (objective 1.2) of emulsions stabilized with HPC has been carried out under conditions that simulate the gastrointestinal tract.The results of this work showed thatthe ability of cellulose derivatives to modulate lipid digestion is due to a combination of interfacial and bulk events, with the binding of BS in solution by HPC being the major factor affecting displacement behavior of interfacial layers by BS. We have also shown that this effect was dependent on the type of BS and charge and hydrophobicity of the cellulose.This knowledge willprovide a deeper insight into the factors that regulate the digestion of lipids. Objective 2: We have investigated the effect of high-hydrostatic pressure (HHP) and hydrothermal (HT) treatments on the thermo-rheological, functional (protein solubility, water and oil-holding capacity, emulsifying power and stability) and structural properties of dry beans. We have further studied how changes of beans caused by these processing operations impact the BS-binding ability of dry beans underin vitrogastro-intestinal conditions, which is related to the cholesterol-lowering properties of common beans.The results of this workdemonstrate that HHP has the technological potential to manufacture bean flours into food products with improved thermo-rheological and functional properties. Our results also showed that HT and HHP processing had a different impact on BS-binding efficiency, consequence of diverse changes in bean microstructure, rheological properties, and dietary fiber content. This study highlights the importance of considering the structure of foods, and not just their nutrient content, when evaluating potential health impacts. Objective 3: We have studied the feasibility of producing nanofibers from blends of pullulan,awater-soluble, biodegradable exopolysaccharide, and cyclodextrins (CD),functional small molecules,by electrospinning. We have tested different pullulan: cyclodextrin ratios and determined the entanglement concentration, to identify the optimal pullulan/HP-β-CD concentration required for the fabrication of bead-free uniform nanofibers.Our results showed that the CE was found to be ~20% pullulan in water.The incorporation of CD did not significantly affect the CE of pullulan solutions, and electrospinning of these mixtures produced uniform fibers with average diameters of ~800 nm.This study shows that blending pullulan with truncated CDs offers a powerful platform to exploit the inherent benefits of both components.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lin, T.; O'keefe, S.; Duncan, S.; Fern�ndez-Fraguas, C. 2019. Manipulation of the dry bean (Phaseolus vulgaris L.) matrix by hydrothermal and high-pressure treatments: Impact on in vitro bile salt-binding ability. Food Chemistry. 10.1016/j.foodchem.2019.125699
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Bulk and Interfacial Interactions Between Hydroxypropyl-Cellulose and Bile Salts: Impact on the digestion of emulsified lipids. Food Hydrocolloids. FOODHYD_2019_2395
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Effect of thermal and high-pressure processing on the thermo-rheological and functional properties of common bean (Phaseolus vulgaris L.) flours. LWT-Food Science and Technology. LWT-D-19-05002
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Poudel, D., OKeefe, S., Long T and Fern�ndez-Fraguas, C (2018). Encapsulation of natural antimicrobials into electrospun biodegradable nanofibers for active food packaging. 32nd EFFost International Conference, Nantes, France, 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Lin, T., Martinez O. and Fern�ndez-Fraguas, C (2018). Impact of high-pressure and hydrothermal processing of dry beans (Phaseolus Vulgaris L.) on bile salt-binding capacity. 32nd EFFost International Conference, Nantes, France, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Jennifer Zornjak, Jianzhao Liu, Alan Esker and Fern�ndez-Fraguas, C (2019). Understanding the role of interfacial interactions between novel cellulose ethers and NaTDC for a sustained lipid release. 257th ACS National Meeting & Exposition, Orlando, FL, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lin, T., OKeefe, S. and Fern�ndez-Fraguas, C (2019). Analysis of in vitro bile salt-binding ability of structurally modified common bean matrices using high-performance liquid chromatography (HPLC). 257th ACS National Meeting & Exposition, Orlando, FL, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Poudel D., OKeefe, S. and Fern�ndez-Fraguas, C (2019). Optimizing polysaccharides and cyclodextrin blends for electrospinning: Sustainable biomaterials for active packaging. 257th ACS National Meeting & Exposition, Orlando, FL, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lin, T., OKeefe, S. and Fern�ndez-Fraguas, C (2019). Bile acid retention of structurally modified common bean matrices during in vitro digestion. The 20th Gums & Stabilizers for the Food Industry Conference, San Sebastian, Spain, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Poudel D. OKeefe, S. and Fern�ndez-Fraguas, C* (2019). Pullulan/ Hydroxypropyl-B-Cyclodextrin electrospun fibers as encapsulation matrices of Quercetin. The 20th Gums & Stabilizers for the Food Industry Conference, San Sebastian, Spain, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Fern�ndez-Fraguas, C (2019). Adsorption behavior of Hydroxypropyl Cellulose derivatives on hydrophobic surfaces: Impact of Bile salts. The 20th Gums & Stabilizers for the Food Industry Conference, San Sebastian, Spain, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lin, T. and Fern�ndez-Fraguas, C (2019). Effect of high-pressure processing on the microstructure and rheological properties of bean flours. 8th International Symposium on Food Rheology and Structure, ISFRS 2019, Zurich, Switzerland, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Poudel D. OKeefe, S., Long T and Fern�ndez-Fraguas, C* (2019). Rheological aspects in fabricating electrospun pullulan fibers incorporating cyclodextrins. 8th International Symposium on Food Rheology and Structure, ISFRS 2019, Zurich, Switzerland, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Fern�ndez-Fraguas, C (2019). Impact of interfacial and bulk interactions between modified anionic cellulose ethers and two bile salts on the control of lipid digestion. 8th International Symposium on Food Rheology and Structure, ISFRS 2019, Zurich, Switzerland, 2019.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Zornjak, Jennifer. 2018. Characterizing Interfacial and Bulk Interactions Between Cellulose Ethers and Bile Salts: Impact on In Vitro Lipid Digestion
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Poudel, Deepak. 2019. Fabrication and Characterization of Electrospun Pullulan Fiber Mats incorporating Hydroxypropyl-?-Cyclodextrin as Encapsulation Matrices of Quercetin

Progress 06/18/18 to 09/30/18

Outputs
Target Audience:Students and food and colloidal scientists, and in general academic peers involved in research at the interface of food and health. Individual consumers and commodity groups, in particular dry bean growers. Changes/Problems:There are no changes or problems to report for this period. However, a change in the approach of objective3 (which was planned to be started in year 2) will be made next year. The initial study within objective3 planned for next year, focuses on designing delivery systems for polyphenols using modified amphiphilic celluloses carriers. These modified celluloses are difficult to manufacture and do not yet have an industrial scale-up process that would allow them to be commercially produced. Due to its difficulty in manufacturing, only a limited amount of the modified polymers will be synthesized for this project. Consequently, we have changed the delivery system to hydrogels which need a lower amount of the modified polymer to be produced. The delivery target site of polyphenols has been then changed to the colon. Keeping the same objective, this approach is more attractive from the point of view of addressing inflammatory bowel diseases. What opportunities for training and professional development has the project provided?Training of undergraduate and graduate students including MS and Ph.D. students. One M.S. student has been trained in the use of surface analysis techniques ([e.g. Quartz Crystal Microbalance with Dissipation monitoring (Q-CMD) and Surface Plasmon Resonance (SPR)] to characterize the adsorption/desorption of cellulose derivatives to surfaces and study the interfacial interactions between them and BS. One doctoral student has learned how to perform the chemical analysis of components and dietary fiber fractions (soluble and insoluble) of legumes. Additionally, grad and undergraduate students were trained in the rheological characterization of polysaccharide solutions. Graduate students have communicated their research findings to scientists of different backgrounds through different research venues. M.S. and PhD students were able to interact with members of the IFT Carbohydrate (CHO) division, and with members of the ACS carbohydrate chemistry and surface science division respectively. Grad students participated in the FST annual poster competition and were provided feedback by an evaluation committee; they were awarded the first and third place. How have the results been disseminated to communities of interest?The results of this research have been disseminated through presentations at domestic and international conferences including the 255th ACS National Meeting (New Orleans, LA, March 19-20), 3rd Food Structure and Functionality Symposium (Montreal, Canada, June 3-6, 2018) and IFT 2018 (Chicago, IL, USA, July 15 - 18). Research findings were also presented to an audience of faculty scientists, industry members, graduate students and undergraduate researchers, at the Macromolecules Innovation Institute (MII) Annual Technical conference and Review (Blacksburg, VA, April 16-18, 2018), and at the university level during the FST Poster competition (Food Science & Technology department, Blacksburg, VA, April 27, 2018). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will conduct studies related to the proposed objectives 1 and 2. Overall, we plan to further understand how dietary fibers could be used to interfere with lipid digestion and absorption, and hence prevent hyperlipemia and hypercholesterolemia. Objective 1: In particular, we will continue investigating the interactions between cellulose derivatives and BS (objective 1.1.) using surface analysis techniques. We will perform sequential experiments with a novel modified HPC (anionic at physiological pH and with a more balanced hydrophilic-hydrophobic character). Also, simultaneous adsorption experiments using Q-CMD will be performed to evaluate bulk interactions between celluloses and BS and thus determine if cellulose derivatives are more effective ingredients at binding BS in aqueous solutions than at creating strong layers at the surface of fat droplets resistant to BS disruption. We will explore different BS type to investigate how their adsorption behavior influence their functionality. We will compare commercial HPC (non-ionic) and a novel modified HPC for their ability to bind BS in order to understand the role of polymer hydrophobicity and charge on the biological action of celluloses. Additionally, in vitro digestions (objective 1.2) of emulsions stabilized with cellulose derivatives will be carried out under conditions that simulate the gastrointestinal tract. We will examine the potential of HPC to decrease the rate and extent of intestinal lipolysis. Objective 2: We will investigate the effect of food processing operations on the physicochemical/structural properties and BS-binding ability of dry beans. Specifically, studies will be performed to examine how structural and physicochemical changes of beans caused by thermal and non-thermal processing influence different aspects related with lipid digestion. In addition, we will explore to which extent the BA-binding ability of beans is related to their viscous and non- viscous components. The microstructure and viscosity of the different bean matrices generated during food processing will be characterized by SEM and rheology respectively, and their BS-binding capacity will be determined by using a Bile Acid Assay Kit. Graduate students will be involved in these studies and will develop a better understanding of the impacts of this research on the field. We will present the research findings obtained from the studies performed during this period at an international Conference, the ACS national meeting and seminars/symposiums at the university level. We will be submitting two publications during this reporting period.

Impacts
What was accomplished under these goals? We have initiated studies with two different sources of dietary fiber (DF) as potential anti-hypercholesterolemia and anti-hyperlipidemia agents for intestinal sequestration of bile salts (BS) and lipids: Purified dietary fiber (commercial cellulose derivatives) and a food matrix (common beans (Phaseolus vulgaris L.). Cellulose derivatives are common ingredients used as thickeners and emulsifiers in processed foods and were chosen as a model of non-ionic DF, where the hydrophobicity can be tuned by varying the number, type and pattern of substituents. Initially, we have investigated the interactions between cellulose derivatives and BS using surface analysis techniques. In order to better understand how cellulose derivatives and BS behave at the surface of lipid droplets we performed the sequential adsorption of cellulose derivatives and BS on solid surfaces using QCM-D and SPR. We tested different solid surfaces (gold, hydrophobic polystyrene (PS), and various self-assembled monolayers (SAMs) with different degrees of hydrophobicity (SAM-CH3 and SAM-OH)), flow conditions, adsorption times. We compared different commercial cellulose derivatives with different type of substitution, Mw and hydrophobicity (i.e. HPMC, MC, HPC...). This first study demonstrated that the molecular substitution type and hydrophobicity affects the way celluloses adsorb to surfaces. We also found that HPC is able to adsorb irreversibly to a hydrophobic surface and form strong surface layers resistant to complete disruption by BS. These findings show that HPC is a dietary fiber and food emulsifier with potential to hinder the adsorption of BS onto fat droplets, thus controlling lipid digestion and lowering blood lipid levels. Consumption of common beans has shown positive effect on blood lipid and cholesterol levels. This effect is mainly linked to the ability of bean DF to interfere with lipid digestion and metabolism in different and related ways. However, the precise mechanisms involved in the lowering of blood lipids and cholesterol are still not fully understood. Initial studies performed this year with common beans, mainly focused on evaluating their content in dietary fiber fractions (soluble and insoluble) as well as on characterizing bean matrices from a rheological point of view. We also performed a preliminary experiment to set-up a protocol for determining their BS-binding ability. This preliminary study showed that the physical chemistry properties of the bean matrix could influence different aspects related with lipid digestion.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Jennifer Zornjak, Diana Novo, Kevin Edgar and Cristina Fern�ndez Fraguas. (2018) Characterizing interactions between cellulose derivatives and bile salts using Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and Surface Plasmon Resonance (SPR). 255th ACS National Meeting, New Orleans, LA, US.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Tiantian Lin and Cristina Fern�ndez-Fraguas. (2018) Bile salt binding capacity of fiber from dry beans: Impact of the food matrix. IFT 2018, Chicago, IL, USA
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Tiantian Lin and Cristina Fern�ndez-Fraguas. (2018) Effect of high hydrostatic pressure on the bile salt-binding ability of fiber in common beans (Phaseolus vulgaris L.). 3rd Food Structure and Functionality Symposium, Montreal, Canada