INNOVATIVE STRUCTURING-LAYERING TECHNOLOGY FOR LARGE-SCALE PRODUCTION OF FISH ANALOGS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: EXTENDED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028038
• Grant No.: 2022-67018-37101
• Project No.: MASW-2021-09401
• Proposal No.: 2021-09401
• Program Code: A1364
• Project Start Date: May 1, 2022
• Project End Date: Apr 30, 2025
• Grant Year: 2022

Project Director
Lu, J.

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Dept: Food Sciences

Non Technical Summary
Overfishing, environmental pollution, and short shelf-life pose challenges for increasing theconsumption of fish meat. Developing plant-based fish analogs provides a sustainable and environmentally friendly alternative solution. Current commercial fish analogs do not mimic thetexture of the fish meat, this is mostly because structure forming processing technologies appliedfor land animals cannot mimic the unique texture of fish meat characterized by short muscle fibers and flaky muscle alignment. This project evaluates the feasibility of an innovative structuring-layering process based on coating technology that uses a type of bio-ink made of a thermodynamically incompatible biopolymer mixture. The proposed technology will be able to create a single layer of texture with a thickness on the order of a fish muscle fiber and fabricate the fish analog from the bottom up. In addition, the coating technology can potentially be applied for large-scale production. Thisproject will use a multidisciplinary approach to develop and test this technology. Various plant-based ingredients will be investigated using a soft matter physics approach to identify a proper bio-ink. The appropriate processing parameter region will be identified with the help of high-fidelity numerical simulations. And finally, process design will be iteratively improved by comparing the fabricated product to the texture profile of a benchmarked Atlantic salmon. The result of this project will allow the fabrication of high-quality plant-based fish analogs and would lead to a new generation of high-quality, affordable, safe, nutritious, and accessible fish analogs. Moreover, it would create new economic opportunities by creating the knowledge and technologies to stimulate the development of plant-based food companies.

Animal Health Component

0%

Research Effort Categories

Basic

60%

Applied

20%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	7410	2020	70%
502	5010	2000	30%

Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
7410 - General technology; 5010 - Food;

Field Of Science
2020 - Engineering; 2000 - Chemistry;

Keywords

coating

fish analog

layering

plant protein

processing

Goals / Objectives
The overal goalof this projectis to improve the sustainability and healthiness of the food supply by advancing the knowledge required to createhigh-quality plant-based food.The overal objective of this project is todevelop an innovative structuring-layering process based on the coating technology that can be used to create next-generation fish analogs that closely resemble the major characteristics of real fish muscle texture.The following specific aim will be addressed:Aim 1: Identify and characterization of functional bio-inks. In this aim, we will test and characterize physicochemical properties of various potential functional plant ingredients including solubility, structure-forming, gelation, rheology, and the phase separation regions between the mixture of bi-polymer system.Aim 2: Identify feasibility for design parameters and processing regions for laminating protein fibers embedded biopolymer sheets To facilitate the process development, computational aided engineering (CAE) will be used to identify feasible design parameters of the die as well as processing regions based on critical relations between process and product that are difficult to access experimentally. High-fidelity numerical simulations will be conducted to estimate internal structure formation and orientation, multi-layer lamination process. The determined processing region and design parameters will be used to guide experiments in Aim 3 for fabricating fish analogs.Aim 3: Fish analog fabrication and texture characterizationIn this aim, we will build a bench-top coating rig that combines a slot die coating and an adjustable die slip. Following the feasibility region obtained by the simulation in Aim 2, we will create fish analogs using bio-inks identified in Aim 1. We will evaluate the internal structure of the fish analogs, as well as their their texture profile.A structure to texture relation will be established. In addition, the obtained texture profiles will be compared against our previously determined texture profile of real fish meat and iteratively improve the bio-ink formula and process design.

Project Methods
Aim 1: Identity and characterization of functional bio inksIn this aim, we will first characterize physicochemical properties of various plant-based ingredients, such as pulse proteins (e.g., pea protein) and polysaccharides (e.g., pectin). We will then identify suitable bio inks to be used in Aim 3. In addition, some of the properties obtained in this Aim will be fed into the computational simulations in Aim 2.We will characterize the molecular and physicochemical properties of each biopolymer ingredient used to assemble the fish meat analogs. Protein composition will be determined by SDS-PAGE. The molecular weight distribution of the biopolymers will be determined using size exclusion chromatography with multiangle light scattering and refractive index detection. The charge characteristics of the biopolymers will be determined by measuring their zeta-potential versus pH profiles using particle electrophoresis. The water-solubility of the biopolymers will be determined by dispersing them in aqueous solutions with different pH values (pH 3 to 7) and salt concentrations (0, 100, and 200 mM NaCl), centrifuging them, and then measuring the concentration in the supernatant using appropriate spectrophotometric methods. The thermal denaturation and gelation temperatures of the pulse proteins will be determined using differential scanning calorimetry and dynamic shear rheology. Biopolymer solutions ( 15% to 20% protein or 1% polysaccharide) will be heated from 10 to 150 C at 1 C/min and any thermal transitions will be determined. Experiments will be carried out at different pH values (3, 4, 5, 6, and 7) and salt concentrations (0, 100, and 200 mM NaCl) to elucidate the impact of pH and ionic strength on their behavior. The knowledge gained from these experiments will help to select appropriate processing conditions for creating nano- and micro-structures in the plant-based fish meat analogs.Because the bio ink is formed based on controlled phase separation of biopolymer mixtures due to thermodynamic incompatibility, which depends on biopolymer characteristics such as type, concentration, mass ratio, and solution conditions such as pH, ionic strength etc. We will develop a phase diagram for different combinations of biopolymers. In this aim, we will initially evaluate combinations between three proteins (pea protein, soy protein, faba bean protein) against three polysaccharides (pectins, carrageenans, gellan gum, xanthan gum).Aim 2: Identify feasibility regions for coating and laminating protein fibers embedded biopolymer sheets In this aim, high-fidelity simulations will be used to provide insight into physical mechanisms and access to variables that cannot be measured in experiments to guide the design and characterize effective combinations of bio inks and operating conditions for laminating biopolymer sheets. By fine tuning the shearing and stretching effect applied during the sheet forming and lamination process, the fiber interphase can undergo large deformations inside the slit die forming fiber buddles. Wewill also incorporate the presence of a deformable substrate when laminating additional layers. This part of the study will begin by considering the substrate as an impermeable viscoelastic film to study how the flow-induced deformation of the substrate affects the process. This is of critical importance because process parameter space will need a adjusted accordingly in order to maintain the integrity of the structure as additional layers are coated.The fluid dynamic equations governing the problem will be solved simultaneously as an initial-boundary-value problem by a fully implicit, method of lines, arbitrary Lagrangian-Eulerian algorithm using the finite element method with elliptic mesh generation to trace the deforming interfaces and an adaptive time integrator to improve accuracy. Parametric search in the space of dimensionless variables (e.g., Reynolds number, Capillary number, viscosity ratios) with strategies for optimizing the parametric design will help characterize feasibility regions for coating biopolymer sheets with an internal fibrous structure.Aim 3 Fish analog fabrication and texture characterizationWe will customize a bench-top coating system in order to validate and iteratively improve the computer-assisted design of hybrid coating experiments on a limited but useful set of material and operating conditions. In order to maintain the formed internal structure, gelling agent containing calcium ions (such as calcium chloride) will be sprayed after the layer is formed in order to maintain the internarial structure against relaxation. We will characterize internal morphology including fiber orientation and anisotropy using SEM. And we will conduct a texture profile analysis of the developed laminated fish analogs using the a protocal by considering the muscle fiber orientationof . A Texture Analyzer with a flat-ended cylinder probe (25 mm diameter) will be used to characterize the mechanical properties of the fish samples. Double compression will be applied to the constructed samples and the TPA parameters will be obtained from the resulting stress-stain curves according to the method described in previous studies. The texture profile will then be compared against the benchmark salmon texture profile to iteratively optimize the bio ink.

Progress 05/01/22 to 04/30/23

Outputs
Target Audience:The aim of this project is to develop a novel proof of concept additive processing method for creating plant protein-based fish analogs. This endeavor will also contribute to fundamental scientific knowledge about biopolymer physicochemical properties and aid in the development of bio-inks for food additive manufacturing. Additionally, the project will develop computational tools that use numerical simulations and data-driven algorithms to facilitate the development of the new processing technology. The primary audience for this project includes academics, industrial scientists, and engineers interested in food development and processing technologies based on alternative proteins. Furthermore, scientific communities interested in the fundamental physicochemical properties of biopolymers and computational fluid dynamics for free surface flows will also benefit from this research. Findings from this project will be disseminated through peer-reviewed papers and presentations at scientific and professional meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doc (senior scientist) was hired and joined the project in July 2022, the post-doc researcher has worked on the development of computational tools related to this project. In addition, a self-funded master's student participated the project and was involved in evaluating the physicochemical properties of bio-inks early on in this project. How have the results been disseminated to communities of interest?Part of the data-driven approach that evolved from this project was shared at a recent AOCS annual conference in an invited talk. What do you plan to do during the next reporting period to accomplish the goals?In the next phase, the optimized protein-polysaccharide system will be laminated using layer-by-layer printing - the instrument has been assembled in our lab. However, before up-scale production, there are still some key benchmarks to consider during the printing process, such as the time-resolved gelling behavior, internal structure, water and oil holding capacity, and the orientation change of fibrous structure. To this end, the appearance, texture, and cookability should also be considered to increase the printing accuracy and reliability in the following studies. More specifically, we will use post hoc strategies to refine the protein-polysaccharide formula during and after laminating by changing polymer concentration and mass ratio, as well as solution conditions (pH, ionic strength, and temperature). From the modeling side, we will incorporate more features to the current model to better assist the modification and optimization of the processing development. Lastly, and most importantly, we will start creating fish analogs on our newly assembled benchtop testing rig, and characterize the structure-to-texture relationship over a wide range of processing conditions and bio-inks.

Impacts
What was accomplished under these goals? Towards Aim 1 To construct the bio inks using bottom-up strategy, the physicochemical properties of various plant-derived proteins and polysaccharides have been systematically investigated in our study. In particular, the solubility and zeta-potential of faba bean protein (M25010, AGT), pea protein (M22998, AGT), and lentil protein (M17399, AGT), as well as pectin, carrageenan, and xanthan gum were compared at different pH levels. Accordingly, pea protein (10 - 15 wt.%) and citrus pectin (0.1 - 1 wt.%) were chosen to formulate the phase-separated polymer mixture. Transglutaminase (0.1 - 2.5 wt.%) was also used to optimize the texture properties of protein-polysaccharide gels. The result from screening trials shows that the combination of 10 wt.% pea protein, 0.5 wt.% pectin, and 2 wt.% transglutaminase endowed the bio inks with the similar texture attributes of real salmon. Meanwhile, w-3 enriched flaxseed oil (0.5 - 1 wt.%) was successfully encapsulated in the protein-polysaccharide block structure. It should be noted that the hardness, springiness, and chewiness of pea protein-pectin gels can be harnessed by changing the temperature (40 - 55 ?) and ionic strength (e.g., 5 - 50 mM for CaCl2 and 50 - 300 mM for NaCl) of protein-pectin mixture. Towards Aim 2 and 3 We developed and successfully validated a computational model for slot coating, which was tested across a wide range of processing conditions to identify the optimal processing region for various bio-ink material properties. The model also incorporates a complex phase separation process based on the laws of mass conservation and thermodynamics, enabling accurate predictions of texture formation during the laminating process. As a result, this model will offer valuable insights into the interplay between processing conditions and material properties, facilitating the optimization of bio-ink formulations and the development of the proposed processing technology. Lastly, we have built the bench-top coating rig which is ready to test on various bio-inks from Aim 1.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lu, J., Ubal, S., Corvalan, M. C., (2023, Apr. 30 - May 3) Using physics-informed neural networks on inversed problems in food materials. AOCS 2023 Annual Meeting & Expo, Denver, CO, United States