DESIGN AND PRODUCTION OF GREEN ULTRA LOW-FAT MAYONNAISE AS A MODEL PRODUCT USING MICROFLUIDIC TECHNIQUE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010696
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Food Science

Non Technical Summary
The increase in the consumption of fat and energy-rich foods, accompanied with a lack of physical activity, has resulted in over 60% of the American population being overweight or obese. Because obesity is closely linked to other metabolic disorders, as well as cancers, it has become the second leading cause of death and disease in the United States, with corresponding medical costs of more than $162 billion each year. New York State ranks number two in the nation for both obesity and associated medical costs. One way to overcome Obesity challenge is to formulate and produce reduced-fat products. However, since fat plays key roles in the product properties, reducing and removing 50% of the fat from a product will result in undesirable sensory attributes and adversely affect the product taste, texture, physical appearance and mouthfeel. Therefore, to solve this problem, we need to utilize new techniques which enable us formulate reduced-fat products with desirable sensory attributes and appealing for the consumers. Examples include Mayonnaise, salad dressing, spreads and butter. All of these products are composed of emulsions; emulsions are simply a dispersion of two immiscible liquids in each other.Current techniques to formulate these emulsions lack the required precision and control to provide sufficient data for new formulation of reduced-fat products. To solve this challenge, we will utilize a novel technique "Microfluidics" (developed by the project director at Harvard University three years ago), to form emulsions with identical size and shape. This technique enable us to study the effect of every single ingredients within the product formulation and eventually results in the best combination of the ingredients for the reduced-fat product. We will utilize Microfluidic techniques to produce emulsion-based foods, such as mayonnaise, using natural ingredients. The exquisite precision offered by this novel technique enables us to design, study and develop ultra-low fat mayonnaise with a Green Label without compromising traditional texture, shelf life stability, or taste. We will employ a combination of food-based ingredients and healthy oils to formulate low calorie mayonnaise. Mayonnaise is one example that we will start to formulate, once we develop the platform for formulation of low-fat Mayonnaise, we will customize and tailor this platform for production of other reduced-fat products such as butter, spreads and variety of salad dressing. We will investigate and improve the shelf-life stability and perform sensory evaluation on these products by a systematic approach using microscopy and analytical methods as well as tasting panels.

Animal Health Component

70%

Research Effort Categories

Basic

20%

Applied

70%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	3420	2020	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
3420 - Butter;

Field Of Science
2020 - Engineering;

Keywords

reduced-fat

emulsion

microfluidics

Goals / Objectives
Utilize Microfluidic techniques to produce emulsion-based foods, such as mayonnaise, using natural ingredients. The exquisite precision offered by this novel technique enables us to design, study and develop ultra-low fat mayonnaise with a Green Label without compromising traditional texture, shelf life stability, or taste. To produce low-fat food product, we use mayonnaise as a model food product, once we developed our microfluidic-based technique, we can employ our approach to design other low calorie food product. We will employ a combination of food-based polymers and emulsified healthy oils to formulate low calorie mayonnaise. Investigate the shelf-life stability and perform sensory evaluation on these products by a systematic approach using microscopy and analytical methods as well as tasting panels. Measure the amount of free fatty acid and quantify the fat uptake based on each formulation to result in the best tasting product with low calorie. Examine the effects of fortification with natural antioxidants (i.e. vitamin E and white tea) on sensory attributes and chemical stability of mayonnaise.

Project Methods
We will employ glass-based microfluidic devices, built in our lab, to produce monodisperse emulsions (mayonnaise as a model product) with ultra-low fat contents. Although, to produce emulsion and simulate the physicochemical properties of mayo, it is indispensible to use oil in the formulation, using hydrocolloids and formulating the product with microfluidic will enable production of ultra-low fat product. We will use mayonnaise as an example for reducing fat content, however, once we established the protocol and procedure using Microfluidics, we will be able to tailor our approach towards other food products and customize the approach to reduce the amount of fat within food products without compromising the sensory attributes of the food including taste, flavor, physical appearance and texture. Microfluidics enable us to systematically study emulsions physicochemical properties and create stable products without chemical additives, we exploit the exquisite flow control afforded by microfluidics to control the emulsion stability. To fabricate emulsions with new formulation including natural and beneficial ingredients and minimized fat contents, we will utilize glass capillary devices.We use a glass capillary microfluidic device to prepare mono disperse single anddouble emulsion drops. The device consists of two tapered cylindrical capillaries inserted into the opposite ends of a square capillary, whose inner dimension is larger than the outer diameter of the cylindrical capillaries. The orifices of injection capillaries are in the range of 20 to 250 μm in diameter. We will use glass surface modification methods to render the inner surface area of injection and collection capillaries hydrophobic and hydrophilic respectively. This configuration enables us to accurately align both cylindrical capillaries. We use one cylindrical capillary to inject the innermost aqueous phase. We inject this oil from the other capillary, forcing it to flow in the same direction as the inner aqueous phase, through the interstices between the cylindrical and square capillaries. We will use syringes and injection pumps to inject the oil and water containing surfactant solutions. The average water phase and oil phase flow rates will be in the range of 100 mL/hr to 10,000 mL/hr. The drop generation frequency using microfluidic device is in the range of 1000 to 10,000 Hz, therefore, we will use a fast-camera to record and analyze the emulsion production and investigate the effects of natural and beneficial ingredients, such as antioxidants (carotene and alpha-tocopherol), inulin and Konjacas hydrocolloids, denatured whey proteins or protein-hydrocolloid conjugates asan emulsifier, and vegetableoil with high degree of unsaturated lipids. After formation of emulsions, we will remove and tune the water content of the product to the appropriate oil in water volume fraction and simulate the exact texture and rheology of the mayo. We hypothesize that using microfluidics, we can identify most stable emulsions with desirable physicochemical attributes using low-calorie and health beneficial ingredients. To test our hypothesis, we will produce emulsions with different oil and surfactant and hydrocolloid ratios and investigate their physicochemical stability as a function of time. To simulate the exact rheology of the produced mayo, we will first characterize mayo samples obtained from market using Rheometer and other techniques and utilize these data as our standard. To characterize the stability, structure, and composition of the mayonnaise, we will use confocal and bright field microscopies, rheology, dynamic light scattering, zeta potential, GC-MS, FT-IR, and UV-Vis techniques. In addition, we will perform sensory evaluations to identify the most acceptable formulation. To perform sensory tests and systematic evaluations of low-fat mayo, we will collaborate with Professor Robin Dando at the Department of Food Science at Cornell University. Finally, we will screen the efficacy of different formulations for minimizing calorie intake. We will utilize in vitro digestion models under conditions that simulate the human gasterointestinal (GI) tract. We will subject the produced mayonnaise to simulated GI tract fluids, including oral, gastric, and intestinal phases, and measure the rate of lipid digestion by determining the amount of free fatty acids released from the lipid phase due to lipase activity using analytical methods such as pH-stat and HPLC techniques. To evaluate the absorption of fat components across the intestinal epithelium, we will use physical methods simulating the intestinal fluid or uptake by cell cultures (Caco-2 cells), to mimic the human intestinal epithelium.

Progress 10/01/16 to 09/30/19

Outputs
Target Audience: The scientific society, and the food and agriculture industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided opportunity to train post-doctoral researcher and graduate students in the area of emulsion and emulsion fabrication. How have the results been disseminated to communities of interest? We wrote a manuscript and submitted it for publication in peer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Spreads, fillings and mayonnaise contain high fractions of oil phase. These products usually feature high caloric values but low nutrients.For example, conventional spreadscompose of mostly synthetic emulsifier, flavors and preservatives other than80% fat. To maintain the desired consistency in spreadableproducts, while delivering higher nutritional value, we could take advantage of the technique to creating a high internal phase emulsion (HIPE).HIPE is a highly concentrated emulsion system with internal phase volume fraction exceeding 0.74, the gel-like characteristic of HIPE allows the products to be self-standing and spreadable. However, current food products are limited to forming oil-in-water HIPE, while water-in-oil HIPE with high stability are difficult to fabricate thus are rarely explored.By creating a water-in-oil version of HIPE, we can reduce the amount of fat used, as well as toinclude nutritious milk proteins and vitamins in the water phase.In this research, we propose creating ultra-stable water-in-oil HIPE made with milk-derived ingredients. We will emulsify 80% water phase containing milk protein in 20% milk fat phase using a high shear homogenizer. By applying phase structuring approach within HIPE, we can modulate the rheological properties, stability, enable high loading of milk proteins, as well as providing functionality within a low-calorie yet palatable food matrix. There is also a growing interest in replacement of synthetic surfactants with biopolymers, such as proteins and polysaccharides for food applications.Previously,we have use whey protein microgel (WPM) to fabricated O/W HIPE as low-fat emulsions. We observed a significantly higher stability and more texture consistency in the emulsions stabilized with WPM in comparison with whey protein isolate or tween surfactant. We observed that the physicochemical stability of the HIPE systems prepared using WPM was higher in comparison with the ones stabilized using the synthetic surfactant (Tween). Therefore, our results show that WPM is a promising natural surfactant and can be substituted in different food emulsions to stabilize the system and decrease the chance of creaming and phase separation of the emulsion. Beside using WPM as a stabilizer, we explore creating a low-fat mayonnaise through creating a W/O HIPE.W/O HIPE features the high aqueous phase volume and low oil fractions while maintaining highly viscous nature of the HIPE.The aqueous internal phase provides a large volume for protection of water-soluble deliverables and the continuous external oil phase can be utilized for delivery of lipid-soluble actives. To date, most of the research on HIPE has been focused on studying and formulating water-based continuous phase emulsions O/W and there is lagging effort and much greater need for research on W/O HIPE. Our group has recently made progress in this latter regard with the successful development of an ultra-stable, food-grade W/O HIPE system that is suitable for versatile application.We've applied a simple strategy of generating W/O HIPEs through homogenization using high shear conventional homogenizer, by structuring and modulating the viscoelastic behavior and physical properties of the internal and continuous phases. This W/O HIPE utilizes food-grade materials and the processing require solely the control over temperature and shear rate of the homogenizer. Such W/O HIPEs are very stable and can retain its integrity when stored at 25 °C for 2 months. Our strategy for forming ultra-stable W/O HIPEs involves structuring both oil and aqueous phases, which is different from conventional W/O emulsions consisting of a liquid-in-liquid model with high flowability. Our unique W/O HIPE platform with structured internal and external phases enables the co-loading of both hydrophilic and hydrophobic nutraceuticals which are immobilized in the corresponding phases. To visually and quantitatively demonstrate how hydrophilic components can be protected in our W/O HIPE, we utilized anthocyanin as a model hydrophilic bioactive due to its high sensitivity to pH. Results indicate that our system can protect hydrophilic components from a great range of pHs (pH 1-8). In addition to hydrophilic compounds,β-carotene, are incorporated within our HIPE. Suchβ-carotene incorporated into the structured oil external phase also showed a protective effect. Furthermore, our preliminaryin vitrorelease study at elevated temperatures revealed that both encapsulated hydrophilic and hydrophobic components showed a pH-responsive release profile. In an extension work investigating the application of such W/O HIPEs, we showed the feasibility of incorporation of high concentration of whey protein (20 wt%) into the aqueous phase. We have also developed an optimized W/O HIPE platform where the water phase is saturated with choline chloride (nutrient) and gelled using food-grade hydrocolloids dispersed in the water phase. Structuring the internal water phase using gums and/or proteins enable better control over release profile due to affinity and interaction of amino acids and their entrapment within gum and protein network inside the internal phase. Collectively, we've shown the possibility of developing novel delivery-system featuring simultaneous delivery of hydrophilic and hydrophobic components and releasing these nutrients in a controlled fashion. Based on our current achievement, recently published works and preliminary studies on O/W and W/O HIPEs, we believe there is strong potential for HIPEs to be utilized as a low-fat mayonnaise. Microfluidic emulsification can play a role as it forms highly monodisperse droplets with fully controllable shapes within a narrow size distribution range.We employed a combination of food-based polymers and emulsified healthy oils to formulate low-fat emulsions.We prepared microcapsules with palm oil as middle phase in a water-in oil-in water (W/O/W) emulsion using microfluidic device. A W/O/W double emulsion was producedcomposed of solution of aqueous phase (10% w/w) as the internal aqueous phase,palm oil as the middle phase, and aqueous solution with 5% (w/w) of Pluronic F-108 as the continuous phase. We observed spherical, mononucleated microcapsules, with particle size of 164-190µm. The formulations showed high aqueous core encapsulation efficiency, and stability. This study shows that the microemulsions prepared using a microfluidic device provided aqueous protection and represents a new method to produce low-fat foods. We were able to provide multiple platforms as a low-fat mayonnaise. Such green, low-fat spreadable products can be fabricated through forming HIPEor double emulsions. In our first milestone, we substituted synthetic surfactants with a natural surfactant (WPM) and made a stable, proteinaceous O/W HIPE. In addition to replacing synthetic surfactants, we created aW/O HIPE system using phase structuring technique. This W/O HIPE can be also use to carry both the hydrophilic and hydrophobic nutrients. Lastly, we generated W/O/W microparticles using a microfluidic device. Such results showed that we can fine-tune the structure of each microparticles so it can be suited for low-fat product. Collectively, we were able to control the amount of fat within food products without compromising the sensory attributes of the food including taste, flavor, physical appearance and texture.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lee, M. C.; Tan, C.; Ravanfar, R.; Abbaspourrad, A. Ultrastable Water-in-Oil High Internal Phase Emulsions Featuring Interfacial and Biphasic Network Stabilization. ACS Appl. Mater. Interfaces 2019, 11 (29), 2643326441.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audiences include the scientific society as well as the food and agricultural industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Please see the attached link to view our complete report: Report 03.19.2019.docx? First Approach: We employed a combination of food-based polymers and emulsified healthy oils to formulate low-fat emulsions. We employed glass-based microfluidic devices, built in our lab, to produce monodisperse emulsions with ultra-low fat contents. We prepared microcapsules with palm oil as the middle phase in a water-in-oil-in-water(W/O/W) double emulsion using the microfluidic device.The formulations showed high aqueous core encapsulation efficiency and high stability. This study shows that the microemulsions prepared using a microfluidic device provided protection, and represents a new method to produce low-fat foods. Second Approach:In this work, we demonstratesonochemically-synthesizedhigh internal phase emulsions (HIPEs), which feature high stability due to a permanent layer at the oil-water interface that nearly any material can form as long as it can be cross-linked duringacoustic cavitation. These findings suggestthis sonochemical technique holds promise as a versatile, scalable, and environmentally friendly route for synthesizing ultra-stable and fully natural HIPEs. Additionally, the material performance shown herein provides a proof-of-concept for the design of multifunctional HIPEs in various applications. Third Approach:We synthesized polysaccharides-based high internal phase emulsions (HIPEs) by simply centrifuging ultrasonically prepared oil-filled polysaccharide microspheres without the help of any surfactants and synthetic particles. Key to achieving ultra-stable HIPEs was the cross-linking of polysaccharides at oil-water interface induced by acoustic cavitation.Having determined the feasibility of this strategy, we then further evaluated the influence of ultrasound intensity and polysaccharide concentration on the properties of HIPEs.We found that whatever the ultrasound intensity applied, HIPEs can be produced successfully without any coalescence. Forth Approach:Centrifugation coupled with ultrasonication: a simple universal route for synthesizing ultra-stable "green" high internal phase emulsions.Here we experimentally enable centrifugation as a universal and robust technique for preparing ultra-stable HIPEs by the assistance of ultrasonication.The properties of the centrifugation-produced HIPEs and resultant porous materials are controllable by simply adjusting the ultrasound power.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Formation of shelf stable Pickering high internal phase emulsions (HIPE) through the inclusion of whey protein microgels S Zamani, N Malchione, MJ Selig, A Abbaspourrad Food & function 9 (2), 982-990

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The scientific society. The food and agriculture industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided opportunity to train post-doctoral researcher in the area of emulsion and emulsion fabrication. How have the results been disseminated to communities of interest?We wrote a manuscript and submitted it for publication in peer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?We plan to execute the second phase of the project and also formulate emulsion system with low-caloried value.

Impacts
What was accomplished under these goals? Abstract The increase in the consumption of fat and energy-rich foods, accompanied with a lack of physical activity, has resulted in over 60% of the American population being overweight or obese. Fat is the most energy dense macronutrient and plays a major role in obesity development; hence efficient strategies that tune or decrease lipid content in food and lipid bioavailability are sought enthusiastically. It is, however, noteworthy that decrease in fat content requires redesigning food with new formulation which possesses the similar sensory attributes as regular product but offers the benefit of reduced-fat products. In this work, we propose a novel method to develop stable ultra-low fat emulsions, which can be used in the production of ultra-low fat foods such as mayonnaise. This approach could be potentially applied for production of other low-fat and low-calorie food products. Introduction, results and discussion Engineered oil-in-water emulsions are considered convenient contenders to suppress body energy intake while keeping a normal-fat diet. Interface characteristics and the size and presumably the monodispersity of droplets impact remarkably the digestion fate and behavior of an emulsion. High internal phase emulsions (HIPE), wherein the internal phase volumetric fraction exceeds 0.74, are usually highly viscose, or in gelled form. In this work, we use the unique structural properties of HIPE to produce low-fat emulsions. We prepare the Pickering HIPE by a layer of rigid particles around each droplet rendering more stability against coalescence, and creaming. Using HIPE, we could control the texture and viscosity of our system using different amount of oil and aqueous phases. There is also a growing interest in replacement of synthetic surfactants with biopolymers, such as proteins and polysaccharides for food applications. Thus, we fabricate and use whey protein microgel (WPM) to stabilize our low-fat emulsions. To prepare WPM, solutions of whey protein isolate (WPI) were prepared by dissolving the 4% (w/w) WPI powder in water, adjusting the pH to 5.8-6.0, and heated at 85°C for 45 min; Figure 1 shows WPM prepared and stored within falcon tubes, these WPM samples scatter the light due to their size and that is why they are opaque. We observed a significantly higher stability and more texture consistency in the emulsions stabilized with WPM in comparison with whey protein isolate or tween surfactant (Figure 1). We observed that the physicochemical stability of the HIPE systems prepared using WPM was higher in comparison with the ones stabilized using the synthetic surfactant (Tween). Therefore, our results show that WPM is a promising natural surfactant and can be substituted in different food emulsions to stabilize the system, and decrease the chance of creaming and phase separation of the emulsion. Microfluidic emulsification forms highly monodisperse droplets with fully controllable shapes within a narrow size distribution range. The exquisite precision offered by this novel technique enables us to design, study and develop ultra-low fat emulsions with a Green Label with a high shelf life stability and desired taste. We employed a combination of food-based polymers and emulsified healthy oils to formulate low-fat emulsions. We employed glass-based microfluidic devices, built in our lab, to produce monodisperse emulsions with ultra-low fat contents. We prepared microcapsules with palm oil as middle phase in a water-in oil-in water emulsion using microfluidic device. We established this protocol and procedure using Microfluidics, which can be used to tailor our approach towards other food products and customize the approach to reduce the amount of fat within food products without compromising the sensory attributes of the food including taste, flavor, physical appearance and texture. The encapsulation process was performed using a glass microfluidic device. Cylindrical capillaries (World Precision Instruments, Inc., Sarasota, Florida, USA) were inserted into a square capillary (Harvard Borosilicate Square Tubing), with outer and inner diameters of 1.5 and 1.05 mm, respectively. A water-in-oil-in-water (W/O/W) double emulsion was produced, composed of solution of aqueous phase (10% w/w) as the internal aqueous phase, palm oil as the middle phase, and aqueous solution with 5% (w/w) of Pluronic F-108 as the continuous phase (Figure 2). We observed spherical, mononucleated microcapsules, with particle size of 164-190µm. The formulations showed high aqueous core encapsulation efficiency, and high stability. This study shows that the microemulsions prepared using a microfluidic device provided protection, and represents a new method to produce low-fat foods. Conclusion We substituted the synthetic surfactants with a natural surfactant (WPM) and concluded that WPM stabilized the emulsions even better than the synthetic surfactants. This approach can be used in the production of other food products. We also showed that using WPM, we control the amount of fat within food products without compromising the sensory attributes of the food including taste, flavor, physical appearance and texture. Furthermore, using microfluidic device, we produced microcapsules through a w/o/w emulsion, and showed that we can fine-tune the formulation and preparation parameters of all types of food emulsions.

Publications