Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Food Science
Non Technical Summary
?The US consumer demand for plant-based meat analogs is increasing rapidly. Even though some select plant proteins have shown promise in imitating the textures of select meat analog products, most current plant-based meat analogs are still far away from imitating textures of various animal-based meats. This is primarily due to the gaps in understanding the texturizing process in extrusion and the impacts of the protein chemistry and quality on texturization. It is critical to address this gap to meet the needs of US consumers. This project aims to evaluate and identify the chemical and physicochemical characteristics of select proteins that play a significant role in the development of the desired texture during extrusion processing. The fundamental understanding generated from this project can be applied to a broad range of plant protein sources. This information can assist the broader food industry tailor the products to have specific textures the consumers desire in the plant-based meat analog, along with the protein nutrition.
Animal Health Component
20%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
Our long-term goal is to develop a fundamental understanding of the quality of plant proteins that affect their extrusion processing performance (Fig. 1). This, we believe, will assist the food industry in developing quality and nutritious foods that consumers desire, but this will also provide information to the plant breeding community to develop varieties that are nutritious and perform well in processing. Our goal of this proposal is to determine the relationships between physicochemical and functional characteristics of pulse proteins and their texturizing abilities in extrusion processing.Objective 1: Evaluate the influence of the extrusion processing parameters on the texturizing abilities of the select plant protein isolates (pea, wheat,and soy) in making high moisture meat analogs.Hypothesis: The combination of the different extrusion processing variables can induce different levels of mechanical and thermal energies into the proteins resulting in different levels of protein texturization. `Methodology: A state-of-the-art twin-screw extruder will be utilized for this work. Extrusion processing parameters that affect mechanical and thermal energy input to the protein material (e.g., material feed rate, feed moisture, screw speed, screw configuration, and die dimensions) will be varied. The extent of texturization and the potential protein modifications in the samples produced under different mechanical energies will be analyzed using physicochemical analyses, microscopic techniques (confocal and scanning electron), and rheological techniques.Objective 2: Explore the mechanisms of texturization as influenced by the protein-protein interactions during extrusion processing. Hypothesis: The extent of thermo-mechanical processing through the extruder will modify the proteins to enable their texturization. Methodology: The select proteins will be extruded under pre-determined conditions from Objective 1 that impart calculated energy inputs to the material yielding in desired texturized proteins. While running under stable conditions, the extruder will be subjected to an emergency stop, and samples will be extracted along the length of the screws and the die-set-up. The samples will be analyzed for the changes in the protein characteristics and correlated to the final texturized product characteristics.Objective 3: Develop structure-processing-property relationships between molecular protein structure, extrusion processing conditions, and ability to texturize.Hypothesis: Texturization occurs when protein disulfides reduce, leading to macromolecule unraveling and the subsequent reassembly via hydrogen bond and disulfide formation between thiols into lamellar sheets; modification of the protein structure at these sites would impact the texture of the final product.Methodology: Analytical techniques to identify and quantify the molecular structure and modifications to the protein structure will be utilized to determine the characteristics of the proteins (e.g., number of disulfides or thiol groups) to identify critical structures that lead to texturization. Protection of disulfides and thiols via an alkylation route (with 2-iodoacetamide) would reduce the extent of texturization by preventing reassembly, confirming the proposed mechanism. The texturized protein samples will be analyzed using various analytical techniques (physicochemical, spectroscopy, and microscopy) to determine the degree of texturization and protein modifications.
Project Methods
Following is the list of the methods to be used in this research project:Extrusion trials: A laboratory-scale co-rotating twin-screw extruder (TSE 20/40, 7.5 HP, CW Brabender, S. Hackensack, NJ, USA) will be used for all the extrusion studies. An aspect ratio of the screw (L/D) of 40.5 will be used, and five different barrel zones will be utilized to control the temperatures.Protein solubility: The solubility will be determined at seven different pH values. 250 g of the sample will be suspended in 50 mL of 50 mM sodium citrate buffers (pH 3, 4, 5, and 6) or 50 mM sodium phosphate buffers (pH 7, 8, and 9). The slurries will be stirred and centrifuged; then, the protein in the supernatant will be measured according to the biuret method with a standard of BSA stock solution.Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE): SDS-PAGE will give insight into which subclasses of proteins are present and at what percentage (relative to total protein). The molecular weights of proteins for all samples will be determined by SDS-PAGE gel electrophoresis. Proteins will be extracted with a 1% SDS solution (Fisher Biotech, USA) containing 2% β-mercaptoethanol (BME). The mixture will be vortexed for 30 minutes, followed by centrifugation at 15,000 g. 4 µL of the supernatant will be transferred to an Agilent protein 230 chips (microfluidic gel electrophoresis chips), and the molecular weights will be determined according to the manufacturing company's assay guide. The 2100 Expert software, provided by a manufacturing company, will be used to analyze, integrate, and collect the data.Texture analysis: Texture profiles analysis (including hardness, springiness, gumminess, and chewiness) and cutting strengths (transverse and longitudinal directions) will be determined using a texture analyzer.Proximate analysis: Protein isolates will be subjected to proximate analysis to understand and correlate the differences in composition to their texturization ability. Raw materials will be analyzed for their crude content of ash (AACCI 2000 08?01.01a), moisture (AACC 44?15.02), protein (AACC 46?30.01), fat (AACC 30?25.01), and crude fiber (Ankom 2000 fiber analyzer, Ankom Technology, NY, USA; AOCS 2017, Ba 6a?05). The total carbohydrate will be calculated as 100% - the sum of the other crude values.Physicochemical properties: Analyses of physicochemical properties of the raw protein isolates will allow us to understand the differences in the samples better. Physicochemical properties that will be measured include oil holding capacity (OHC), as well as foaming capacity and stability, water holding capacity (AACC 56-30.01), emulsion stability, and index, surface hydrophobicity as well as pasting properties (Brabender Micro Visco?Amylo?Graph; AACC 76?21.01). Correlations between the physicochemical characteristics and their ability to texturize will be made.Amino acid composition: High-performance liquid chromatography (HPLC) will be used to measure amino acid composition. Hydrolysis and amino acid analysis will follow methods described by Yufei (2017), which updated protocols first described in the Hewlett Packard Amino Quant Operator's Handbook (1990). Cysteine will be assessed by creating cysteine- 3-mercaptopropionic acid (Cys-MPA) complexes as described by the Amino Quant Operator's Handbook (1990). This will limit cysteine degradation during hydrolysis, which has an issue in the preliminary optimization of this procedure. Correlations between amino acid composition and the ability to texturize will be made.Sulfhydryl (SH) and disulfide (SS) groups: Quantitative analysis of thiol and disulfide groups in the raw protein isolates and the samples collected after the immediate stop will be conducted spectrophotometrically using Ellman's reagent. Correlations between the content of such groups and the ability to texturize will be made. Changes along the barrel will give insights into the importance of these groups during the mechanism of texturization.Texture analysis: Extrudate samples will be measured for texture using TA.XT2 Texture analyzer (Stable Micro Systems, UK) with three different probes, one for compression, one for cutting strength, and one for extensibility. Extrudates will be cut into squares (23 x 23 mm) and compressed to 50% of their original thickness to determine the hardness, springiness, gumminess, and chewiness. For cutting strength, square samples (23 x 23 mm) will be cut 80% of the way through perpendicular to and parallel to the extrudate fibers. For extensibility, extrudate samples will be placed in metal grips attached to the texture analyzer and pulled apart until completely separated. Each independent replicate will have 20 repeated measures. All textural characteristics of extrudates will be measured within 48 hours of extrusion processing. Current high moisture meat analogs available at the market will also be tested as reference points for quality.Protection of thiol and disulfide bonds: Sulfur-containing amino acids in the protein isolates will be alkylated before extrusion processing. Alkylation prevents thiol groups from forming new disulfide bonds after the unfolded proteins have aligned towards the end of the extruder. Alkylation will be performed with and without the prior reduction of existing disulfides to evaluate the essential sulfur-containing bonds. Existing disulfide bonds in soy, wheat, and pea protein isolates will be reduced with BME. After reduction, protein isolates will be subjected to alkylation reactions. Our strategy is to protect the thiol groups via an SN2 alkylation reaction with 2-iodoacetamide. However, we will also explore other alkylation reagents (including 2-chloroacetamide, iodoacetate, and maleimide) to find the most efficient balance between costs, alkylation efficiency, and human resources.Micrographic properties and visual assessment: Samples with significantly different texturization abilities will be subjected to microscopic testing to access the physical structures of the samples. A scanning electron microscope, confocal laser microscope, and optical microscope will be used for testing. As reported in the literature, standard procedures will be used 41,42. Correlations between the texturization ability obtained from microscopic images and the importance of sulfur-containing groups will be established. Additionally, samples will be visually assessed by a trained panel.FTIR analysis: The raw material and the milled samples will be analyzed by Fourier-Transform Infrared spectroscopy (FTIR) to determine any conformational changes induced by extrusion processing. FTIR provides insights into the secondary structure of proteins 43. Stretching vibrations of the amide C=O bond and bending vibrations of the amide N-H bond result in characteristic protein bands in infrared spectra. Both bonds (the C=O and the N-H bond) are essential constituents of the secondary structure as they are involved in hydrogen bonding responsible for diverse structural elements. The wavelengths at which the two bands, Amide I and Amide II, arise are sensitive to the secondary structure of the protein. Potassium bromide pellets will be analyzed using an infrared spectrophotometer. Because the amide bands are relatively broad and secondary structure-related shifts are comparatively small, Fourier self-deconvolution will be applied to the spectra. Extrudates will be analyzed in triplicates to determine the effect of the raw material, specifically the importance of thiol and disulfide groups, by comparing spectra of the unprotected and the protected samples.Data analysis:Analyses will be conducted in triplicates, except for the texture analysis, which will be conducted 20 times for each sample for any of the three probes. Results will be analyzed using analysis of variance (ANOVA); differences between samples will be determined using Tukey's HSD at a significance level of p<0.05.