Research Projects

Plant Protein Blending: Inducing Molecular Interactions to Enhance Texturization

Update:
A mild alkaline extraction coupled with isoelectric precipitation (AE-IEP) method was successful in producing both benchtop and scaled up (SU) oat, pea, and chickpea protein isolates (OPI, PPI, and ChPI) with acceptable purity, yield, and structure and functional characteristics. The OPI had good gelling properties at pH 7 and good solubility at acidic pH, while ChPI had a relatively high solubility at neutral pH and good emulsification capacity, and PPI had good solubility at both neutral and acidic pH. These findings support the success and scalability of the mild AE-IEP method, as well as the potential of OPI and ChPI as functional protein ingredients. (July 2024)

AI-Driven Prediction of Plant Protein Flavor Biotransformation during Fermentation and Experimental Validation

Principal Investigator: Yonghui Li
Co-PIs: Bo Hu, Fernanda Dias, B. Pam Ismail

Pea protein consumption has increased in recent years as consumers are looking for plant-based products for environmental and health benefits. However, the presence of off-flavor compounds, primarily beany and green notes, are limiting its widespread adoption. Fermentation of plant proteins has the potential to reduce beany flavors, and generate desirable flavor compounds, contributing to improved consumer acceptability. However, traditional fermentation experiments and fermentation product identification methods are excessively time-consuming. The recent breakthroughs in AI and increasing availability of fermentation-related data such as microbial strains, enzymes, and metabolite profiles, indicate that AI can be a promising tool for predicting molecular biotransformation during fermentation. Our overall goal is to develop an AI prediction model for the utilization of fermentation to mitigate off flavors in pea protein ingredients. An integrated platform will be built based on large datasets and machine learning algorithms for predicting microbial enzymes-substrate interaction potential and metabolite profiles during fermentation. Pea flour will be used as a model system to conduct fermentation experiments and validate the developed AI model. Changes in pea protein (off)flavor compounds and techno-functional properties during fermentation will be monitored. The proposed platform will offer a robust tool for predicting fermentation outcomes and metabolites-related product quality biotransformation. (July 2024)

Next-generation food processing: Developing environmentally friendly strategies to produce chickpea protein isolates

Principal Investigator: Fernanda Dias
Co-PIs: B. Pam Ismail, Gary Reineccius

Consumers' growing preference for plant-based diets has led to a surge in pulse crop production, driven by the development of new plant-based products by the food industry. Among pulses, chickpeas have gained prominence as a nutritious and sustainable protein source suitable for a variety of food applications. However, the utilization of chickpea protein faces several challenges that hinder its widespread adoption. Firstly, the use of flammable solvents in the defatting process of chickpea flour raises significant safety and environmental concerns. Additionally, the intrinsic off-flavor and reduced functionality of chickpea protein pose a hurdle that restricts its potential for food applications. To address these issues, it is crucial to explore alternative extraction methods that eliminate the need for flammable solvents, enhance the flavor profile, and improve the functionality of chickpea protein. Natural deep eutectic solvents (NADES) have emerged as promising eco-friendly alternatives. These solvents are not only inexpensive and easy to prepare but also biocompatible and biodegradable, aligning with sustainability principles. The main objective of this proposal is to investigate the efficacy of flammable-solvent-free extraction approaches for maximizing the extractability of protein from chickpeas while simultaneously improving its flavor profile and functionality, creating new opportunities for its application as food ingredients. (July 2024)

Impact of Pea Storage Protein Fractions and Their Ratio on Functionality and Nutritional Quality

Principal investigator: B. Pam Ismail
Co-PIs: Daniel Gallaher, Robert Stupar
Researcher: Holly Husband

The overall goal of this project is to determine the impact of varying the proportion of pea storage proteins on functional and nutritional properties for enhanced breeding efforts. Storage pea protein components, namely legumin, vicilin, and convicilin, as well as the albumin fraction, will be separated and purified. The isolated fractions will be reconstituted into protein isolates in different ratios (from 0 to 1 for each fraction). Separated fractions as well as reconstituted protein isolates will be subjected to structural characterization, including protein denaturation, protein profiling, surface charge, surface hydrophobicity, and protein secondary structure. Additionally, the functional properties (solubility, emulsification, foaming, and gelation) of the fractions and isolates will be determined. Commercial PPI, lab produced PPI, and commercial SPI will be used as references. The in vitro PDCAAS will be determined for each of the fractions and isolates, using a newly available commercial kit. Data generated will aid in determining the association between different pea protein components and functional properties, as well as identifying the optimal protein profile. Understanding this association is critical when screening for pea protein cultivars that could potentially have superior functionality and nutritional quality, which could be used as parental cultivars in breeding programs aimed at improving the functional aspects of pea protein and not just yield.

Update:
Since beginning this project, the conditions of alkaline protein extraction have been optimized to produce pea protein isolate from pea flour with ideal purity and yield. This optimization is necessary to supply the pea protein isolate needed as a reference sample when analyzing the molecular and structural properties, functionality, and nutritional quality of fractionated pea protein isolate. The next phase of the project is in progress and is focused on the selection of methodology for pea protein fractionation. Various methods of fractionation including alkaline separation, size-exclusion chromatography, and membrane filtration will be evaluated to select the most efficient fractionation method. Work is currently being done on alkaline separation with reference to a published method for soy protein fractionation. If similar fractionation results are observed using pea, this method may be chosen as the final fractionation method for the project. (03/2021)

Executive Summary of Findings:
The relatively low abundance of 11S legumin in pea protein, coupled with the wide diversity in 7S vicilin to 11S legumin ratio among pea protein ingredients, are assumed contributors to pea protein’s inferior and inconsistent functionality and nutritional quality relative to soy protein. To improve the performance of pea protein ingredients in food and beverage applications, an optimum protein profile must be identified. Therefore, this work followed a holistic approach to determine the impact of 7S/11S ratio on pea protein structure, functionality, and nutritional quality. Vicilin- and legumin-rich fractions were isolated and combined in different proportions to produce pea protein samples of varying 7S/11S ratios. For the first time, pea protein isolate was also enriched with 11S legumin to evaluate the impact of 11S abundance on functionality within an unfractionated protein matrix. Results revealed the isolated 7S vicilin had 6-fold higher gel strength and 5-fold higher emulsification capacity, but significantly lower nutritional quality, than the isolated 11S legumin. Despite having significantly higher sulfur-containing amino acids, high protein polymerization in the isolated 11S contributed to the relatively low functionality. Further, results indicated that fractionation induced unique changes to amino acid composition, which decreased the amino acid score of isolated 7S and 11S relative to pea protein isolate. Accordingly, legumin enrichment of pea protein isolate did not improve functionality or nutritional quality. Nevertheless, this work contributed foundational knowledge that will provide direction for future studies aiming at devising strategies to improve the quality and consistency of pea protein ingredients. (April 9, 2023)

(RFP 2)Flavor Reactions with Plant Proteins

Principal investigator: Gary Reineccius
Co-PIs: B. Pam Ismail, Daniel Gallaher
Researcher: Igor Shepelevs

The overall goal of this work is to develop analytical methodology to measure the covalent bonding that occurs between flavor compounds and the very complex and diverse plant proteins (this project will focus on pea protein). There are two competing approaches to achieve our goals – radioisotope labelling and mass spectrometry (MS). The MS approach begins with an enzyme digestion and then uses tandem mass spectrometry to identify the sites of post translational modifications that take place on the various proteins after reacting with a flavor molecule. A targeted m/z approach on a triple quad LC/MS will be used for quantitation. The alternative and potentially complimentary approach is to use radioactively labeled flavor compounds to measure covalent bond formation. A radio labelled flavor compound will be reacted with pea protein isolate (PPI), the unreacted isotopes separated from the protein, and the radioactivity will be measured with a scintillation counter. The MS method has the advantage of providing an understanding of where flavor compounds react on each protein even in a mixture of proteins. This knowledge can provide information that will help us design ways to mitigate these reactions. The radioisotope approach has the advantage of being a rapid, highly quantitative measure of the degree of bonding with whatever proteins are present in the sample.  

Update:
The objective of this project is to develop a method to accurately measure the covalent reactions that occur between flavor molecules and plant proteins. These reactions result minimally in undesirable changes in flavor character and in many cases, in flavor loss that can bring about end of shelf-life. We have a LC-MS method that is well suited to measure covalent reactions with small proteins but not large proteins common to plants. Thus, we have been working on a radioisotope method whereby we can get good measurements of flavor – protein reactions with larger (plant) proteins. Our method of choice is to use C14 labelled flavor molecules to monitor this reaction.

In principle, one would simply add a C14 labelled flavor molecule to a protein, allow a reaction period, and then extract the unreacted flavor from the protein reaction system (commonly an aqueous system) using a non-polar solvent. Unfortunately, proteins bind flavors by two mechanisms: hydrophobic bonding and covalent bonding. The hydrophobic bonding can be sufficiently strong to make extraction uncertain. Also, one cannot solvent extract most protein-containing systems since they typically serve as good emulsifiers which creates a stable solvent: water emulsion that cannot be separated easily. After much effort, we have been able to work with low protein concentrations, salt addition and pH adjustments to keep the proteins from forming stable emulsions.  We have just received two radiolabeled flavor molecules: one to serve as a control that will bind via hydrogen bonding and a second that bonds primarily by covalent reaction.  We will be testing the extraction method the week of April 5. (03/2021)

Executive Summary of Findings:
 A major challenge in obtaining data on how flavorings covalently react with plant proteins has been the lack of methodology to make this measurement. It is relatively simple to measure total protein bonded flavor molecules, but historically we cannot distinguish between type of bond and this discrimination is critical. In this work we have developed a 14C labeled method to selectively measure covalently bonded flavor molecules. That said, there are limitations in using radioisotopes in research. Radioisotopes can only be used in laboratory spaces designed for working with radioisotopes. In addition, many commonly used lab techniques (e.g. centrifugation and freeze drying) cannot be used due to the potential for radioisotope contamination. Using the methodology developed in our laboratory, we have been able to gather substantial information on covalent bond formation between several classes of flavor compounds and plant/dairy proteins (a benchmark). The plant proteins studied include pea, partially hydrolyzed pea, soy, camelina and pennycress. Protein/model flavor compound reaction was measured during a storage study (25, 45, and 65ºC) and a single time/temp heat retreatment adequately severe to detect the potential for reaction (aqueous solution heated at 65°C for 2 hrs). The flavor compounds chosen for study were based on other work we did on beta lactoglobulin. They included 2-undecanone (a ketone), decyl aldehyde (an aldehyde), and an isothiocyanate (2-phenylethyl isothiocyanate). This work showed that isothiocyanates are extremely reactive with all proteins. The decyl aldehyde was moderately reactive and the 2-undecanone was just measurably reactive. There is no question that plant proteins will react with flavor components during processing and storage. Related work carried out on beta lactoglobulin (previously published) has provided additional insights on reactions that will occur with plant proteins and provided some information on reaction rates. (November 3, 2023)

*Shepelev, I.; Reineccius, G. A. 14C-Isotope Use to Quantify Covalent Reactions between Flavor Compounds and β-Lactoglobulin. J. Agric. Food Chem. 2024, 72 (18), 10579–10583. https://doi.org/10.1021/acs.jafc.4c00134.

(RFP 1)Impact of Cold Plasma Treatment on Pea Protein Structural and Functional Characteristics

Principal investigator: B. Pam Ismail
Co-PIs: Peter Bruggeman, George Annor, Chi Chen
Researcher: Fan Bu

While pea protein is gaining traction, functionality limitations are hindering its market growth. Improving pea protein functionality will enable its successful utilization in various food products. There are several reports on pea protein functionality and applications, but much is still not known about the effect of different processing and modifications on the structural and associated functional changes in pea protein. Cold plasma, a non-thermal processing technique, is being explored as a novel means for protein functionalization. Cold plasma technology involves the exposure of plasma, a partially ionized gas, to proteins. The reactive species generated by cold plasma can induce several chemical reactions including oxidation, bond cleavage, and/or polymerization. The overall goal of this project is to evaluate the effect of various cold plasma treatments on pea protein structural and functional properties. Reactive species and changes due to potential chemical reactions will be monitored. Specifically, changes in the protein tertiary, secondary, and primary structure will be evaluated, and chemical reactions will be elucidated. The impact of structural change on protein functionality will be determined. This research will provide for the first time a controlled evaluation of the impact of cold plasma on pea protein structural and functional characteristics. Cold plasma treatment may lead to the production of a viable pea protein ingredient with functional properties that are comparable or better than those of traditional protein ingredients. The information gathered from this short-term project, as well as the joined interdisciplinary effort will definitely lead to designing future long-term studies with a targeted outcome.

Update:
Pea (Pisum sativum) protein is gaining more interest as a potential replacement of high-allergenicity soy, whey, and egg protein in food applications. However, limitations in functionality limit the use of pea protein isolate in food products. Cold atmospheric plasma (CAP), a non-thermal processing and anti-microbial technology is a novel method that can be used to change the primary, secondary, and tertiary structure of protein. OH radical, H₂O₂, O₃, and reactive nitrogen species (RNS) are major components in plasma. Plasma generated with different gas mixtures and by different configurations results in different profile of reactive species, leading to various changes in protein structure and consequently protein functionality. Therefore, the objectives of this project were to 1) investigate the impact of OH radical, H₂O₂, O₃, and RNS on the structure and functionality of pea protein isolate, and 2) investigate the impact of atmospheric pressure plasma jet (APPJ), dielectric barrier discharge (DBD), and nanosecond pulsed discharge (ns-pulsed) on the structure and functionality of pea protein isolate.  Pronounced structural and functional effects were observed when PPI was treated with plasma reactive species at pH 2 compared to pH 7. All plasma species induced the formation of disulfide-linked soluble aggregates with molecular weights around 1200 kDa, as demonstrated by SE-HPLC and SDS-PAGE. Protein denaturation was observed after treatment with all plasma species. However, a significant increase in β-sheet content and surface hydrophobicity was only induced by treatment with O3 and OH, which resulted in the greatest enhancement in gelation and emulsification properties of PPI. Additionally, we found that the gel strength and emulsification capacity of PPI were significantly improved after all plasma treatments whereas the solubility of PPI was decreased significantly. Denaturation enthalpy of all APPJ-, DBD-, and ns-pulsed- treated PPI were significantly lower than that of non-treated PPI, indicating that plasma triggered the unfolding of protein. The significant incremental increase in soluble aggregates content after plasma treatment was revealed by SE-HPLC. Overall, DBD- 30min was the optimal plasma modification condition for improving gel strength and emulsification capacity of pea protein isolates. Soluble pea protein aggregates obtained as a result of plasma treatment can potentially result in enhanced texturization potential for the formation of fibrous meat-like structures. Future work will focus on correlating findings to texturizing properties. (03/2021)

Final Report:
The impact of plasma-produced reactive oxygen and nitrogen species and in particular O₃, N_xO_y, H₂O₂ and OH on the structure and functionality of pea protein isolate (PPI) was evaluated. The species were produced through a combination of controlled measurements and plasma treatments. Pronounced structural and functional changes were observed upon treatment with reactive species at pH 2. All reactive species induced protein denaturation and the formation of disulfide-linked soluble aggregates. A significant increase in β-sheet content and surface hydrophobicity was only induced by treatment with O₃ and OH, which resulted in the greatest enhancement in gelation and emulsification. While H₂O₂ enhanced PPI color by increasing whiteness, it had the least impact on protein structure and functionality. Results of this work can be used to optimize cold atmospheric plasma treatment of PPI to induce specific structural changes and a directed enhancement in functionality. In a subsequent study, the impact of three cold atmospheric pressure (CAP) sources, atmospheric pressure plasma jet (APPJ), two- dimension dielectric barrier discharge (2D-DBD), and nanosecond pulsed discharge (ns-pulsed), on the color, protein structure and functionality, as well as amino acid composition of PPI was evaluated. Different plasma sources and associated reactive species resulted in protein denaturation, increased surface hydrophobicity, formation of soluble aggregates mostly by disulfide linkages, and changes in the protein secondary structure. Enhancement in surface properties, presence of soluble aggregates, and increase in β-sheet resulted in significant enhancement in gelation and emulsification properties. Differences among CAP treated samples were attributed to composition and intensity of plasma species. Treatment with 2D-DBD (Ar + O₂) for 30 min could be a comparatively appreciable functionalization approach due to its modest and desirable structural changes and insignificant effect on amino acid composition. (3/31/2021)

Investigation of novel cold atmospheric plasma sources and their impact on.pdf (umn.edu)

Impact of plasma reactive species on the structure and functionality of pea protein isolate (umn.edu)

(RFP 1)An Interdisciplinary Strategy for Improving Hemp Protein as a Food Ingredient through Plant Breeding and Processing

Principal investigator: Tom Michaels
Co-PIs: B. Pam Ismail, James House
Researcher: Laura Echkardt

Hemp seed protein benefits human diets through its high digestibility, potential free radical-scavenging activity, and hypoallergenicity. However, it is considered an incomplete protein due to limiting amino acids lysine, leucine, and tryptophan. In food preparations, hemp protein exhibits poor solubility, relative to soybean protein. Genetic variance among current industrial hemp genotypes for protein nutritional quality and structural/functional properties for food applications is unknown. Therefore, the potential for breeding new industrial hemp varieties with improved protein characteristics is unexplored. The objectives of this project are to develop hemp seed protein extraction protocols, determine protein quantity, nutritional quality and structural/functional properties for food applications, and identify differences among industrial hemp genotypes and breeding lines for these characteristics. Once these protocols have been optimized and genetic differences characterized, our team will develop a pragmatic, interdisciplinary strategy for improving hemp protein as a food ingredient that integrates plant breeding and optimization of processing protocols. Our team will then pursue this strategy in subsequent research projects.

Update:
The conditions for optimized protein extraction using alkaline solubilization/isoelectric precipitation and salt solubilization coupled with membrane filtration have been determined. Extraction at pH 11 resulted in higher protein purity (88% protein) and yield (87% yield) compared to extraction at pH 10.  The protein purity and yield are quite high, which indicates industrial feasibility. Salt extraction using coupled with ultrafiltration similarly resulted in high and appreciable protein purity and yield.  Both isolates have a bland off white color, which is also desirable. These protein extraction methods have been used to produce two larger batches of hemp protein isolate (HPI) for characterizing structural and functional attributes. Soy protein isolate (SPI) and pea protein isolate (PPI) produced by the Ismail lab has been procured along with commercially produced SPI and PPI as reference samples for the structure/function work. Structure/function work is currently underway. (03/2021)

Executive Summary of Findings:
The objectives of this project were 1) optimize conditions for protein extraction from hemp seeds and characterize the resulting isolates and 2) compare hemp protein isolates produced from four different cultivars. Whole hemp seeds were dehulled, separated, defatted, and milled, which resulted in a defatted hemp meal (DHM) high in protein (60% protein). Optimized alkaline solubilization with isoelectric precipitation (pH-HPI) and salt solubilization coupled with membrane filtration (salt-HPI) resulted in hemp protein isolates (HPI) with high purities and yields (>80%) and acceptable color. While both isolates demonstrated poor solubility at neutral pH compared with commercially produced soy and pea protein isolates, HPI demonstrated similar solubility to that of soy protein under acidic conditions. Solubilization in 0.5 M NaCl improved HPI solubility and other functional performance. While emulsification and foaming properties were improved with solubilization in salt, overall, pH-HPI was more functional than salt-HPI regardless of solubilization medium. Accordingly, alkaline solubilization with isoelectric precipitation was used to produce HPI from four hemp cultivars (CFX-2, Grandi, Joey, Picolo). While minimal differences among cultivars were observed, HPI demonstrated promising gelation and water holding properties at neutral pH, and again, promising solubility at acidic pH. Regardless of variety, protein digestibility was ~90%, and the first limiting amino acid was lysine, bringing PDCAAS score to 0.62-0.64. This work demonstrated that 1) it is feasible to produce HPI with acceptable color, purity, and yield, 2) hemp protein has some promising functional attributes for food use, and 3) there is minimal differences among the four hemp cultivars studied, suggesting the need to explore other hemp cultivars.  Grain-type breeding lines were successfully selected from naturalized hemp populations collected in Minnesota, demonstrating the value of naturalized hemp in breeding.  Protein characterization of these lines will be undertaken in subsequent projects. (October 30, 2021)

Read the full publication on our Outputs Page

(RFP 2)Enhancing Pennycress Oilseeds as a New Protein Source by Improving Flavor and Protein Extractability

Principal investigator: M. David Marks
Co-PIs: B. Pam Ismail, Gary Reineccius, Adrian Hegeman, Ratan Chopra, Krishan Mohan Rai
Researcher: Ratan Chopra & Devon McDonald

Pennycress is being domesticated as a new eco-friendly winter/spring oilseed. Ongoing work has led to the development of pennycress varieties that produce canola-like oil. This oil can be used for human nutrition or can be used for the creation of biofuels and products. Additional work has identified varieties that have reduced glucosinolates and reduced condensed tannins. We propose to make further enhancements in pennycress that will allow it to become a new source of proteins for human use. Current varieties of pennycress contain chemicals such as phytic acid, sinapine, and dimethyl methoxypyrazine that adversely affect the flavor and function of plant proteins. The separation of these compounds from plant proteins requires extra extraction procedures that can alter protein functionality and increase processing costs. We have created a large pool of mutant lines that have been characterized by whole genome sequencing, and within this pool we have identified lines that are predicted to have reductions in the undesirable compounds. The proposed changes to pennycress seed chemistry will greatly enhance the value and utility of pennycress seed proteins. We will present these new pennycress varieties to the food science members of our team for testing. 

Update:
The winter annual plant species pennycress is being domesticated as a new cash/cover crop that can protect farmlands between the harvest and establishment of traditional summer crops and provide farmers with a new source of income. To enhance the value of pennycress seed meal efforts are being made to improve pennycress meal flavor. Candidate pennycress varieties have been identified that may reduce the quantities of known compounds that adversely affect flavor. Currently, several of these varieties are being grown to produce seeds for flavor analyses. Sufficient seeds should be available this coming summer to begin testing these candidates.

The aroma profiles of pennycress and its extracts are being collected using the purge and trap method that offers solvent-free, highly reproducible results. The volatile compounds are collected from the seeds, defatted meal, and multiple protein extracts to show what the original profile is and how volatile flavor compounds change with different extraction techniques. The current line of pennycress still has considerable amounts of off-flavors, such as isothiocyanates, sulfides, and cyanides, that give it an undesirable mustard aroma. Current extraction protocols lower the influence of some of the undesirable compounds, but could possibly impart new or concentrated off-flavors that were not originally in the seed. Identifying all of these compounds that can be minimized to improve the flavor of pennycress products will be helpful for extraction and processing work in the future to turn pennycress into a desirable plant protein source.  (03/2021)

Scalability, Sustainability and Economic Feasibility of a Green Process for Co-Extraction of Protein and Oil from Sustainable Sources

Funded by UMN Institute on the Environment Impact Goal Grant
Co-PIs: Fernanda Dias and Pam Ismail as Co-PIs

Abstract: The goal of the proposed work is to assess the scalability, sustainability, and economic feasibility of a novel green, flammable solvent-free process for co-extracting protein and oil from an oilseed, namely hemp, that has a potential impact on both the agricultural system and the food industry in Minnesota. Scaling up of the proposed green process from a bench scale to a pilot scale production will be evaluated while collecting data for life cycle assessment (LCA) and techno-economic analysis (TEA) in comparison to a conventional process. We believe that the evaluation of the proposed process will demonstrate its environmental and economic impact within and outside Minnesota while incentivizing the scaling of hemp production on the land. We will develop strategies for disseminating the knowledge and provide the basis for commercializing this technology with the support of our community partner, the Agricultural Utilization Research Institute (AURI), ultimately providing stakeholders with sustainable solutions for food ingredient production.

Developing high-quality protein ingredients from dry beans for food applications

Funded by MN Department of Agriculture - Specialty Crop Block Grant
Principal Investigator: B. Pam Ismail
Co-PIs: Gary Reineccius and Fernanda Dias 

Abstract: The overall goal of this project is to increase the value of two specialty crops, kidney and pinto beans by expanding their use as food sources, banking on their high-value protein component. Quality driven process to produce functional, nutritional, and acceptable protein ingredients from these beans will be developed through a series of holistic evaluations. Potential differences in quality traits among select varieties grown in two locations will be evaluated. 

Enhancing Pea Protein Functionality through Glycation Following a Novel and Efficient Upcycling Approach

Funded by USDA ARS
Principal Investigator: B. Pam Ismail
Co-PIs: Mirko Bunzel, Gary Reineccius
Researcher: Andressa Maria Suzin

Update:
The proposal addresses the first and the sixth research priorities listed in the RFP. The overall goal is to develop a sustainable and scalable approach to produce a pea protein isolate with improved functionality and nutrition through the employment of a natural reaction and valorization of by-products. Pea protein isolate (PPI) will be produced on a pilot scale. Pea hulls and pea inner fiber will be hydrolyzed separately using a mechanical and enzymatic treatment to produce fractions with reducing power. The fractions will be individually incubated with PPI, and the extent of glycation will be monitored. Conditions that induce controlled glycation will be selected for scaled-up production and purification of glycated protein. The efficiency of the purification will be monitored by mass balance, with a target of at least 80% protein purity. The glycated protein will be subjected to structural, functional, and nutritional characterization. Additionally, the impact of glycation on flavor development will be evaluated. This research will provide for the first time a controlled glycation process with targeted isolation, while upcycling pea by-products, to impart improved functionality, nutritional quality, and flavor. Accordingly, the proposed work will enhance the prospects of pea protein as a lead ingredient in the market. (July 2024)

Legumes of the Future: Developing Methodologies and Germplasm to Enhance the Functionality and Nutritional Quality of Pea Protein

Funded by Foundation of Food Agricultural Research
Principal Investigators: Robert Stubar & B. Pam Ismail
Researcher: Sungil Ferreira, Matheus Gouveia

Plants are an important source of protein in the human diet, but most plant-derived proteins are not as nutritionally balanced and are not as readily digested as animal proteins. Dried yellow peas are a good source of protein with applications in many food products. Pea protein has lower allergenicity and fewer potential side effects than soy protein, the most common plant protein ingredient, but lags behind soy protein in terms of protein functionality and nutritional quality. This project focuses on the development of peas with improved protein profiles and higher productivity in diverse environments. The project will survey the genetic variation of public yellow pea accessions and develop improved and high-throughput methods for predicting pea seed protein functionality. We will also assess yellow pea accessions for protein digestibility and amino acid profile. Furthermore, we will identify regions of the pea genome that control storage protein content and distribution ratios and initiate breeding activities to select for improved pea protein functionality and digestibility. Products from this work will include new tools and knowledge to support the breeding of pea cultivars with these traits. These products will find immediate applications in a rapidly expanding plant-based food market. (03/2021)

Update:
Yellow peas are a valuable source of protein with applications in many food products. Pea protein has lower allergenicity and fewer potential side effects than soy protein, the most common plant protein ingredient, but it lags behind soy protein in terms of functionality and nutritional quality. This project focuses on developing peas with improved protein profiles and higher productivity in diverse environments. Efforts in breeding to improve the protein profile of yellow peas require a fast and high-throughput method to determine protein profiles and storage protein ratios. Near-infrared spectroscopy (NIR) is a powerful tool for determining protein profiles. However, it is a secondary method, so it requires standards and calibration against biochemical assays. A salt extraction process combined with precipitation of fractions at low temperature has been found successful in producing vicilin- and legumin-rich fractions from pea flour, as validated by SDS-PAGE. In addition to selecting and optimizing a fractionation method, anion exchange chromatography (AXC) and size exclusion chromatography (SEC) were performed to determine the purity of each fraction. Results showed that the combination of AXC and SEC is a successful method of purification for both 7S and 11S fractions. We are now working on the purification of the fractions using the developed 2D-chromatography method. Once the proteomics on the 7S and 11S fractions confirm their purity, this method will be an appropriate approach to produce purified 7S and 11S fractions to be used as standards for ratio determination in different pea cultivars using NIR and SDS-PAGE. (July 2024)

Evaluating navy beans as a source of high quality/value protein ingredients

Funded by Northarvest Beans Growers Association
Principal investigator: B. Pam Ismail
Co-PI: Gary Reineccius
Researcher: Matheus Gouveia

Update:
An interest in plant proteins as a replacement for animal proteins is currently growing due to the increasing consumer demand attributable to various factors (health problems, vegetarianism, religious restrictions). Among these protein sources, legumes, navy beans are excellent sources of protein, are easy to grow, have environmental benefits, and can be planted in a crop rotation. However, the use of these and other beans has been generally limited to canned beans and dry beans. Therefore, producing protein ingredients from beans, such as navy beans, will increase their utilization, thus increasing their value. The aim of this project is to expand the use of this underutilized crop and explore its potential as a source of functional and nutritional protein ingredients to be incorporated in various food products. For this purpose, two varieties of navy beans, each grown in two different locations, were selected. Cleaned, dehulled and milled beans will be subjected to two protein extraction procedures, alkaline and salt extraction, varying pH, salt concentration, solubilization time, and membrane filtration conditions. While there has been some work done on the production of protein ingredients from navy beans, the research has been limited in scope and applicability. Identifying feasible means of efficiently extracting the protein from the bean’s matrix, while maintaining protein functionality and flavor, is essential. (July 2024)

Protein profiling and characterization of diverse oat lines

Funded by PepsiCo
Principal Investigator: B. Pam Ismail
Researchers: Sungil Ferreira & Nigel Kang

Oat (Avena sativa) is the seventh most abundant cereal produced globally; however, it accounts for only under 1% of total cereal production. In recent years, the demand for sustainable, protein-rich, and nutrient-dense foods has increased due to shifts in consumer interests. Oats are notable among cereal grains for their superior nutritional value and well-balanced amino acid composition. Oats separate themselves from other cereals by having lower allergenicity and a higher content of protein (15-20%), unsaturated fatty acids, soluble fiber (beta-glucans), and antioxidants. For these reasons, there are opportunities for oat research and production to grow, especially for human food applications. Therefore, the objective of this study is to evaluate a set of genetically diverse oat samples and correlate protein profiles with environment and genetic variation parameters. About 200 oat lines will be screened to characterize structural differences by proximate analysis, Osborne fractionation, SDS-PAGE, size-exclusion HPLC, and LC-MS/MS. In particular, the percent distribution of protein fractions in oats—namely, albumin, globulin, prolamin, and glutelin—will be contrasted and studied closely. Following the screening, a subset of oat lines will be further characterized for protein quality by in vitro assays and functionality by evaluating solubility, gelation, emulsification, and foaming properties. Variability within lines will be assessed between environment and growing location by genetic marker-based clusters. This research will be used to ultimately cross-breed oat varieties with enhanced protein nutritional quality and functionality. (03/2021)

Update:
Oats are widely accepted globally as a grain with numerous health benefits. While this recognition is primarily attributed to β-glucan, consumer demands for good-tasting, high-quality plant protein with low allergenicity and diverse food applications are driving attention towards oat protein. However, to establish a prosperous oat protein market, oat proteins must be extracted profitably at a large scale, fulfill techno-functional needs of the final food product, and meet nutritional requirements. We hypothesize that the protein profile (i.e., relative distribution of albumin, globulin, prolamin, and glutelin) of oat groats has a profound impact on the production and performance of oat protein ingredients, including protein yield, purity, techno-functionality, and nutritional quality. It is also hypothesized that oat genetic variance and growing environment affect its protein profile. Thus, the overall goal is to investigate how oat protein profile can be altered for food applications through breeding and growing. Genetically diverse oat lines grown in two locations will be evaluated, and the protein profiles will be correlated with genotype and growing location. A subset of samples with the greatest variation in protein profile will be selected for protein isolate production and the protein yield, purity, profile, structural, functional, and nutritional properties will be compared. This research will provide a framework for understanding the complexity and significance of oat protein profile as affected by breeding, growing environment, and processing. Research findings will inform the development of relevant oat breeding programs to cultivate oats with ideal protein profiles that impart superior functional and nutritional properties for food applications. (June 2024)

Functionalization of Plant Proteins

Funded by Several External Sources
Principal Investigator: B. Pam Ismail

Plant protein (other than soy) functionality may not outperform commonly used protein ingredients. Accordingly, we are investigating ways to enhance functionality. Functionalization approaches of various plant proteins currently investigated include targeted and limited enzymatic modification utilizing various common and unique enzymes, Maillard-induced glycation, and chromatographic fractionation. We will also be investigating physical means such as the use of cold plasma. Functionalization is assessed via various analytical tools including spectroscopy, microscopy, proteomics, and chemical assays to evaluate the impact of functionalization on molecular changes. Structural/functional relationships are assessed to achieve desired modification for targeted and unique applications.

Maximizing the Value of Alfalfa Protein by Controlling Endogenous Protease Activity and Evaluating the Quality of all Streams from Protein Extraction

Funded by Forever Green Initiative
Principal Investigator: B. Pam Ismail
Researcher: Shreevats Mor

The global population is expected to reach ~ 9.7 billion by the year 2050. Consumers are increasingly considering plant sources to satisfy their protein requirements. Alfalfa, the fourth most harvested crop in the United States, is poised to fill that protein niche and be a leading climate smart crop. However, there are key challenges for alfalfa in joining the plant protein market which include protein characterization, stability, and scalability. The overall goal of this proposal is to address two of those challenges by characterizing alfalfa proteins and evaluating the potential value of the by-products from the protein extraction process. Specifically, we proposed to: 1) Evaluate post-harvest treatments to limit protease activity and breakdown of RuBisCO; 2) Isolate alfalfa proteins using various extraction methods and determine the impact on structural properties; 3) Isolate alfalfa proteins from 5 unique lines and characterize the protein functionality, nutrition, flavor and lipid profile; 4) Quantify waste products of the protein extraction process for animal and human nutritional and pharmaceutical applications. Our team is composed of agronomists, and food chemists who have experience working with alfalfa and the challenges that alfalfa protein extraction poses. Working as a team, this proposal aims to advance the integration of alfalfa protein into the plant-based food market.

Hemp grain characterization for food applications

Funded by MN Dept of Agriculture
Principal Investigator: B. Pam Ismail
Researcher: Yi Chen

With the rise in popularity of plant-based proteins, the appeal of hemp proteins is increasing across commercial and scientific domains, because of their high digestibility and notable amino acid compositions. However, given the poor solubility of hemp protein, high alkalinity is required to ensure a high protein extraction yield. Such high alkalinity is detrimental to the protein quality and functionality. Also, the use of flammable solvents in the defatting process causes the significant health, safety, and environmental concerns. This study aims to optimize both of the enzyme-assisted alkaline extraction and two-phase natural deep eutectic solvent (NADES)-assisted extraction of hemp protein and impacts on the structral, functional and nutritional properities. 

Update:
Hemp protein concentrate was treated with and without a specific enzyme (non-hydrolytic enzyme) in triplicate, followed by alkaline solubilization with isoelectric precipitation at pH 7 - 11 and precipitation at pH 5. Solubility of HPI was measured at pH 7 and pH 3.4 under heated and non-heated conditions. The success of the enzyme treatment was assessed based on the protein’s yield, purity, and solubility. Control and reference hemp protein isolates (HPIs) were also produced for comparison with the enzyme treated HPIs; Control samples were produced using heat treatment along with the alkaline extraction and references were extracted by alkaline extraction. There was no significant difference at solubilization pH 11 for all the samples. Enzyme-treated HPIs had significantly higher yields than the control and reference HPI’s. At pH 9 and 10, protein yield for enzyme treated HPIs was higher than 80%. Even at pH 7, enzyme-treated HPI had a significantly higher yield than that of the reference sample, at 43% and 3.9% respectively. Overall, high purities above 87% were observed for all pH conditions for enzyme-treated HPIs. The solubility for enzyme-treated HPIs were markedly higher than the references under both heated and non-heated conditions at pH 11, 10, and 9. The highest solubility for enzyme-treated HPIs was at pH 8, which was significantly higher on both heated and non-heated condition than the other tested pHs. We can conclude that enzyme treatment significantly increased the solubilities at both non-heated and heated conditions at pH 7, with the highest solubility observed for enzyme-treated HPI at a solubilization pH of 8. 

NADES-extracted HPIs were assessed in comparison to a control protein isolate prepared using hexane-defatted hemp flour and following alkaline (pH 8) extraction and isoelectric precipitation. The NADES extraction significantly enhanced protein extraction yield (83% vs. 8%) and purity (94% vs. 78%) in comparision to the control. It also reduced residual lipid content of the NADES-extracted HPI (1.2% vs. 5.3%) and had significantly higher protein solubility at acidic pH in comparision to the control (92.4% vs. 52%). This increased solubility was attributed to the relatively high surface charge and slightly lower hydrophobicity of the NADES sample. Protein profiling revealed that NADES-extracted isolate lacked vicilin-like protein, compared to the control. 

Protein Blending: Impact on Functionality Nutrition and Flavor Attributes

Funded by Forever Green Initiative
Principal Investigators: 
Researchers: Jeanne Goh & Saajewa Dasent

Given the increase in global demand for protein, novel plant protein sources are being explored. In this project, winter crops, specifically winter peas, camelina, and pennycress are being investigated. Winter peas, camelina, and pennycress can be used as short-season cover crops, providing many environmental benefits. These crops also contain a high amount of protein, presenting attractive alternative sources of protein. For the first time, this research will provide a study on the protein characterization of winter peas.  In addition to their roles as cover crops, we are exploring their potential as plant protein sources for human consumption. However, plant protein often has functional and nutritional limitations due to its inherent properties. One of the approaches to overcome some of its limitations is to blend the protein strategically. Therefore, the objectives of this project are: 1) produce winter peas and evaluate their protein characteristics in comparison to spring peas; 2) optimize the blending ratio of camelina, pennycress, and winter pea protein for optimal nutrition and functionality; 3) evaluate the texturization potential of the optimal protein blends (using a twin-screw extruder), and the resultant nutritional quality and sensory perception of the texturized product.

Winter pea, camelina, and pennycress protein isolates will be produced in-house using optimized bench-top extraction methods. Commercial pea and soy protein isolates will be compared as a reference. These protein isolates will be subjected to structural, functional, and nutritional characterization. The results of this study will provide us with some foundational knowledge of how blending can impact its structural properties while improving protein functionality and nutritional quality. (August 2024)

Characterizing and Texturizing Proteins from Pulses to Form Fibers with Textures that Mimic Chicken

Funded by The Good Food Institute
Principal Investigators: Zata Vickers, Job Ubbink, & B. Pam Ismail
Researchers: Brigitta Yaputri & Kenzie Mcclintic

Pea protein has gained traction in the alternative meat sector in addition to soy and gluten. Chickpea is also gaining interest due to its low cost, high protein content (~20%), and good nutritional value. Though the application of pea protein in alternative meat products is increasing, pulse protein, in general, don’t have good texturization properties. Thus, there is a need to develop functionalization techniques. In addition, protein isolation method plays a fundamental role in dictating the structural and functionality properties of the protein. Production of pea protein isolates (PPI) and chickpea protein isolates (ChPI) are commonly done via pH-based extraction, but no data have been collected from salt extraction process coupled with ultrafiltration/ diafiltration.

Therefore, the first objective of this research is to characterize and functionalize pulse proteins isolates produced by salt extraction coupled with ultrafiltration/diafiltration and relate functional properties to texturization potential. Two functionalization techniques – cold plasma and transglutaminase – will be conducted to induce polymerization that could potentially enhance texturization. The second objective is to evaluate the impact of different extrusion parameters of twin-screw extruder on the physicochemical and mechanical properties of the texturized proteins. The last objective is to determine the sensory quality of the texturized proteins. This research is the first study to investigate the impact of mild isolation process and polymerization treatments on the texturization potential of pulse proteins.

Currently, structural and functional characterization are being conducted on all in-house and commercial protein ingredients. Results so far indicated that ChPI have higher level of legumin and potentially superior functional properties compared to PPI. (03/2021)

The demand for pulse proteins as alternatives to soy protein has been steeply increasing over the past decade.  Pulse proteins, such as pea and chickpea proteins, have inferior functionality, specifically gelation, compared to soy protein, hindering their applications in different food products, such as meat analogs. The overall goal of this project was to link pulse protein characteristics with their suitability for extrusion into fibrous structures with meat-like texture. Additionally, the focus of the texturization work was to optimize extrusion and microcompounding methods and a working protocol for the use of the microcompounder with high moisture samples was established.

Harsh extraction and processing conditions adversely impact the functional performance of pea and chickpea protein. Therefore, a mild protein extraction method involving salt extraction coupled with ultrafiltration (SE-UF) was evaluated for the production of chickpea protein isolate (ChPI). Produced ChPI was compared to pea protein isolate (PPI) produced following the same extraction method in terms of functionality and feasibility of scaling. Scaled-up (SU) ChPI and PPI were produced under industrially relevant settings and evaluated in comparison to commercial pea, soy, and chickpea protein ingredients. Controlled scaled-up production of the isolates resulted in mild changes in protein structural characteristics and comparable or improved functional properties. Partial denaturation, modest polymerization, and increased surface hydrophobicity were observed in SU ChPI and PPI compared to the benchtop counterparts. The unique structural characteristics of SU ChPI, including its ratio of surface hydrophobicity and charge, contributed to superior solubility at both a neutral and acidic pH compared to both commercial soy protein and pea protein isolates (cSPI and cPPI) and significantly outperformed cPPI in terms of gel strength. These findings demonstrated both the promising scalability of SE-UF and the potential of ChPI as a functional plant protein ingredient.

To close the functionality gap, protein polymerization via targeted modification can be pursued. Two modification techniques (cold-atmospheric plasma and transglutaminase treatments) were conducted on the scaled-up pea and chickpea protein isolates. Transglutaminase (TGase)-modified PPI and ChPI were evaluated in comparison to unmodified counterparts and to commercial protein ingredients. Protein denaturation and polymerization were observed in the TG PPI and TG ChPI. In addition, the TGase modification led to the formation of intermolecular β-sheet and β-turn structures that contributed to an increase in high-molecular-weight polymers, which, in turn, significantly improved the gel strength. The TG ChPI had a significantly higher gel strength but a lower emulsification capacity than the TG PPI. These results demonstrated the impact of the inherent differences in the protein fractions on the functional behavior among species.

Publications:

Transglutaminase-Induced Polymerization of Pea and Chickpea Protein to Enhance Functionality (umn.edu)

Salt Solubilization Coupled with Membrane Filtration-Impact on the Structure/Function of Chickpea Compared to Pea Protein (umn.edu)

Optimization of Extraction of Camelina (Camelina sativa) Protein and Screening for Varietal Differences

Funded by Forever Green Initiative
Principal Investigator: B. Pam Ismail
Researcher: Vaidehi Narkar

This research focuses on developing and optimizing industry-feasible extraction methodology to produce camelina protein isolates with optimal yield and purity as well as structural integrity. The extraction methods have been optimized to produce camelina protein isolates from Joelle line with a purity of 83-84% and a protein yield of 55-63%. The conditions for pH extraction were optimized to improve the color of the protein isolate without any protein polymerization. Currently, there are concerns about the functional properties of plant-based proteins. Addressing these concerns is important for the application of plant-based protein ingredients in food systems. Thus, this research project assesses the functional properties including solubility, water holding, gelation, and emulsification, as impacted by the structural characteristics of camelina protein. The camelina protein isolates from Joelle line demonstrated superior or comparable emulsification properties to commercial soy and pea protein isolates. Gelation properties of alkaline extracted camelina protein was acceptable and better than that of pea protein. 

Diverse camelina lines are also being evaluated for protein functionality. Traditional plant breeding efforts at the University of Minnesota have identified camelina lines with potential differences in protein composition and profile. The protein profiles of 40 camelina lines, representing different growing conditions and origins, were screened using SDS-PAGE. Protein isolates produced from selected camelina lines will be used for further investigation of effect of breeding line on protein functionality. Structural and functional characterization of camelina lines will feed into the breeding program that aim at developing camelina protein for different food applications.

The development of quality-driven protein isolation protocols and optimal camelina lines for food use will advance its commercialization as an alternative and functional plant-based protein source. The knowledge and methodologies acquired through this research could also be applied to other protein sources and systems. (04/2021)

Current consumer trends such as vegan and flexitarian diets and interest in sustainably sourced ingredients are driving the demand for plant proteins. Producers are seeking alternative plant proteins that are non-allergenic and non-GMO. Camelina sativa, a novel oilseed crop with several environmental benefits, is high in protein content (20-30%). The objectives of this research were: 1) Optimize protein extraction methodology to produce camelina protein isolates (CPI) with optimal yield, purity, structural characteristics, and functional properties; 2) Screen camelina lines for protein profile, structure, functionality, and nutritional quality to develop optimal lines for food use. This study showed that CPI with optimal purity, yield, color, and functionality can be produced using industry feasible extraction methods, thus advancing its commercialization as an alternative plant protein. The structural and functional characterization of the different camelina lines provided useful information to the camelina breeding programs that aim at developing camelina protein for food use.

Impact of Selective Breeding, Extraction Conditions, and Modification Technologies on Pennycress Protein Structural, Functional, and Nutritional Characteristics

Funded by Forever Green Initiative
Principal Investigator: B. Pam Ismail
Researcher: Rachel Mitacek

Pennycress (PC), a sustainable, short-season cover crop with vast environmental benefits, is a novel protein source. To develop a commercially-viable protein ingredient, effective protein extraction methods that preserve structural, functional, and nutritional attributes, as well as possible applications, need to be investigated. The objectives of this research were to 1) optimize protein extraction methods that maximize purity and yield, and characterize the influence of extraction method and scale-up on structural, functional, and nutritional attributes 2) evaluate the effect of traditional breeding on PC protein structure & functionality 3) investigate the enzymatic and atmospheric cold plasma (APC) effects on PC protein potential for texturization. Alkaline extracted protein was more denatured and polymerized, with higher surface hydrophobicity and aggregation, resulting in lower overall functionality compared to salt extracted protein. Scaled up, salt extracted protein had comparable and sometimes better functionality compared to commercial soy protein isolate. Enhancing texturization potential is currently underway. (03/2021)

As the consumer demand for plant proteins continues to grow, the food industry is seeking novel and sustainable protein sources to incorporate in various food products. Pennycress (Thlaspi arvense), a sustainable cover crop, produces oilseeds high in protein, warranting investigation. Accordingly, protein extraction from pennycress was evaluated under various extraction conditions, using alkaline extraction and salt solubilization coupled with ultrafiltration. Given the superior color and functionality of the salt extracted pennycress protein isolate (PcPI), its production was scaled-up about two hundred folds in a pilot plant. Furthermore, a new pennycress accession bred to have zero erucic acid (0EA) was evaluated to determine the impact of seed variety on protein characteristics. Structural and functional characterization was performed on PcPI and compared to native (nSPI) and commercial (cSPI) soy protein isolates. Salt extracted PcPI had comparable gel strength to cSPI, three times higher solubility under acidic conditions, and 1.5 times better emulsification capacity. PcPI extracted from 0EA was mildly different in structure and functionality from that extracted from wildtype pennycress, with the slight variation attributed to genetic variance. Finally, the protein digestibility-corrected amino acid score (PDCAAS) of the salt extracted PcPI, calculated in vivo (0.72) and in vitro (0.87), was superior or comparable to other plant protein sources.

Pennycress protein isolate (PcPI) has inferior gelation and water holding properties compared to market leader soy protein, preventing its use in food applications such as meat analogues. Therefore, this work aimed to induce polymerization of PcPI by either cold atmospheric plasma (CAP) or transglutaminase (TG) to improve functionality and texturization potential. Micro-compounding was utilized to determine bench-scale texturization potential of unmodified, CAP, and TG modified PcPI hydrated at 50% moisture. CAP treatment following dielectric barrier discharge (DBD) induced polymerization primarily through disulfde linkages. Whereas, TG resulted in a relatively higher extent of polymerization induced by disulfde linkages and other covalent interactions involving mostly cruciferin acidic subunits. Compared to unmodified PcPI, the gel strength doubled and tripled post CAP and TG treatments, respectively. TG treatment caused a significant increase in water holding capacity by 20%. Unmodified PcPI did not form fibrous structures upon micro-compounding, instead it formed a soft mass with low resilience and cohesiveness. CAP modified PcPI (PcPI-CP) had the lowest water holding properties, which resulted in a relatively hard (significantly highest mechanical energy), dense fibrous structures. Due to high gelation strength and water holding capacity, TG modified PcPI (PcPI-TG) resulted in less dense fibrous structures with more air incorporation (significantly higher void %) relative to PcPI control and PcPI-CP. PcPI-TG texturized mass had significantly higher chewiness that is desirable for meat analogues.

improving_functionality_and_texturization_potential_of_novel_pennycress_protein_by_inducing_polymerization_-mitacek_2.pdf (umn.edu)

Impact of extraction conditions and seed variety on the characteristics of pennycress (Thlaspi arvense) protein: a structure and function approach (umn.edu)

Extraction and Modification of Pea Protein for Improved Functionality, Flavor, and Nutrition

Funded by USDA ARS
Principal Investigators: B. Pam Ismail, Gary Reineccius, & Daniel Gallaner
Researchers: Lucy Hansen, Yara Benavides-Paz, & Joseph Eggers

Extraction conditions including solubilization pH, isoelectric precipitation pH, solubilization duration, number of solubilizations, and use of dialysis were optimized for pea protein isolate (PPI) production by alkaline solubilization coupled with isoelectric precipitation (pH-extraction), based on protein purity and yield as well as industrial feasibility. Similarly, purification conditions including use of ultrafiltration (UF) and dialysis, individually or combined, were optimized for PPI production by salt solubilization coupled with membrane filtration (salt-extraction). Optimized benchtop methods were then scaled-up to pilot plant production. The structural characteristics and functional properties of the scaled up PPIs were compared to commercial PPI. Results demonstrated successful optimization and scalability of two pea protein extraction methods: alkaline solubilization with isoelectric precipitation and salt solubilization coupled with membrane filtration. Both methods achieved high protein purity and yield, and superior functionality to commercial isolates. Nutritional evaluation of the isolates is underway. Samples during pilot plant production were collected for flavor analysis as described below.

Three different methods to isolate aroma compounds from pea flour were tested: SBSE (Stir Bar Sorptive Extraction), SAFE (Solvent Assisted Flavor Evaporation) and SPME (Solid Phase Microextraction). SAFE was found to provide a more complete aroma profile of pea flour compared to SBSE and SPME. In order to monitor the aroma profile during the production of pea protein isolates by salt and pH extraction, samples were collected at several steps throughout the extraction process. The aroma compounds present in each sample were then isolated by using SAFE and analyzed by GC-MS-O (Gas Chromatography – Olfactometry). Three panelists were recruited and instructed to record the retention time, sensory descriptors and to rate the odor intensity of each odorant eluting from the sniffing port.  The aroma compounds detected by at least two panelists with an odor intensity ≥ 3 (using a general magnitude scale where 0 corresponds to “no sensation” and 6 corresponds to “strongest imaginable sensation”) in at least one of the steps were then identified using retention indices, mass spectral data. The tables below show the aroma compounds that were detected and rated with a high odor intensity by the panelists (≥ 3) during the production of pea protein by salt and pH extraction.

We are currently working on analyzing the effect of processing conditions on the relative amount of the aroma compounds present in the samples. Preliminary results showed that there are not compounds that suddenly appear or disappear along the process and therefore, the processing steps do not completely remove or promote the formation of aroma compounds. (03/2021)

Update:
The aroma profile was monitored during an optimized pH-extraction method (alkaline solubilization coupled with isoelectric precipitation) to produce pea protein isolates (PPIs). Samples were taken at different steps throughout the protein extraction. The aroma compounds were isolated from these samples using solvent-assistant flavor evaporation (SAFE) and were identified by gas chromatography-mass spectrometry-olfactometry (GC-MS-O) and gas chromatography-time-of-flight-mass spectrometry (GC-TOF-MS). A sensory evaluation of pea flour (PF) and PPI aqueous solutions was also conducted. From the instrumental analysis, 13 compounds were found to be likely the main contributors to the aroma profile of the samples examined. This hypothesis was also supported by the sensory data, which showed that the PF and PPI aqueous solutions were described with some of the odor descriptors used during the instrumental analysis. No new aroma compounds appear to be produced via the optimized pH-extraction and no existing compounds were completely removed from making a sensory contribution as determined by the olfactory analysis.

The volatile profile was monitored during an optimized salt extraction process (salt solubilization coupled with membrane filtration) to produce a pea protein isolate (PPI). Aroma compounds from samples collected at different steps of the manufacturing process were isolated using solvent-assisted flavor evaporation (SAFE) and analyzed by gas chromatography−mass spectrometry−olfactometry (GC−MS−O) and GC−time-of-flight mass spectrometry (GC−TOF-MS). A sensory evaluation of pea flour (PF) and PPI aqueous solutions was also conducted. Twelve aroma compounds were perceived with a “moderate” odor intensity by panelists from the sniffing port of GC−MS−O. From the sensory evaluation, the aroma descriptors used to describe the PF and PPI testing solutions were also used to describe individual compounds eluting from the sniffing port. This observation supports the hypothesis that the 12 compounds identified in this study by GC−MS−O are likely to be the main contributors to the aroma profile of the samples analyzed. (3/2023)

Monitoring the Aroma Profile During the Production of a Pea Protein Isolate by Alkaline Solubilization Coupled with Isoelectric Precipitation (umn.edu)

Monitoring the Aroma Profile during the Production of a Pea Protein Isolate by Salt Solubilization Coupled with Membrane Filtration (umn.edu)

Protein-Flavor Interactions in Food Matrices

Funded by Several External Sources
Principal Investigator: Gary Reineccius
Researchers: Vaidhy Anantharamkrishnan & Igor Shepelevs

Adding flavorings to high protein foods and beverages leads to interactions, generally reducing overall product acceptance and shelf life. Therefore, it is pertinent to understand protein-flavor interactions to formulate consumer acceptable products. There has been a lot of research studying these multifaceted interactions, particularly on the “temporary” protein-flavor interactions (hydrophobic, hydrophilic, and ionic), but very little research has been done on a more permanent interaction: covalent bonding. Studying the covalent linkages is important because they are irreversible, and therefore will alter the flavor profile in an irreversible manner. This research aims to determine the covalent bonds that are formed between the side chains and terminal amino acids of plant proteins (soy, pea, canola) and aroma compounds. The extent and rate of these chemical reactions are monitored by MALDI-TOF MS and UPLC/ESI/TOF-MS. This study will provide understanding of the broad range of interactions of different flavor molecules with proteins under different conditions, as the type and rate of interaction are expected to vary with functional groups, flavor compound usage levels, protein structure, amino acid composition, pH, water activity, storage temperature, and food composition

Sweetening the Crop: Perennial Flax for Ecosystem Benefits

Funded by Environment and Natural Trust Fund
Principal Investigators: B. Pam Ismail, Neil Anderson
Researcher: Anastasia Natania

Perennial flax has numerous environmental benefits including soil stabilization and requiring less input of energy and pesticides. Domestication of perennial flax varieties has the potential to fulfill the demand for alternative and sustainable plant-based protein sources. Farmers will thus be incentivized to grow the perennial crop with consumer interest and high commercial value.

Flax protein has the potential to be a good source of plant-based proteins as it contains a complete amino acid profile. However, the presence of mucilage -- water soluble polysaccharides that swell during protein extraction -- poses a challenge in obtaining good protein purity and yield. Therefore, enzymatic deamidation using protein-glutaminase (PG) = prior to alkaline protein extraction will be investigated. The aim of this research is to optimize the alkaline protein extraction method of annual and perennial flax varieties using enzymatic deamidation.

Update: 
Optimization of alkaline protein extraction with glutaminase enzyme is currently in progress. Deamidation using PG may allow for double alkaline solubilization to be done at a lower pH, minimizing protein denaturation and improving extraction purity and yield. Future research will compare the protein purity and quality of FPC extracted at lower alkaline pH values. The structural, functional and nutritional characteristics of FPC from perennial and annual varieties will also be determined and compared. The optimized methodology and resulting data will be used for perennial flax screening to improve flax characteristics for human consumption. (8/29/2024)

Enzymatic modification of pea protein isolate for improved functionality

Funded by USDA ARS
Principal Investigator: B. Pam Ismail
Researcher: Maneka Malalgoda

In recent years, the demand for plant protein ingredients has increased rapidly. Although ingredients such as soy protein isolate are widely used as a food ingredient, novel ingredients such as pea protein isolate are gaining interest. However, in comparison to soy protein isolate, pea protein isolate is not as functional. In this study, seven enzymes were investigated under optimized conditions for hydrolysis, to produce hydrolysates with degree of hydrolysis between 5-7%, and of these, two enzymes were used in a sequential hydrolysis. In general, upon hydrolysis smaller proteins and peptides were produced, which displayed lower charge and higher surface hydrophobicity. In terms of functional properties, hydrolysis did not improve gelation, and solubility was generally lower in the hydrolysates, with the exception of Trypsin-flavorzyme hydrolysate at pH 3.4 under non-heated conditions. However, emulsification properties were improved as a result of hydrolysis. This study shows the versatility of enzymes in the context of improving functionality of pea protein isolate. Additionally, the results show that targeted improvement of functionality can be achieved through the use of specific enzymes under optimum conditions for partial hydrolysis. For example, the Trypsin-flavorzyme showed improved EC, EAI and ES as well as FC and FC. Overall, this work emphasizes the need to investigate novel enzymes as well as hydrolysis with multiple enzymes as a potential way of improving pea protein functionality in food systems. (03/2021)

Improving the Functionality of Low Protein Streams for High Value Applications

Funded by Midwest Dairy Association
Principal Investigator: B. Pam Ismail
Researcher: Chelsey Hinnenkamp

The industry solution to the increased consumer appetite for protein and protein enriched products has been to utilize protein concentrates (> 80% protein) and isolates (>90% protein) in their formulations to enhance nutrition, functionality, and overall protein content. However, the production of these high protein ingredients generates coproducts, such as Procream, a phospholipid-rich whey coproduct, that are underutilized due to lower protein content and functionality.  A potential valorizing opportunity for Procream is to blend it with functionally enhanced WPC for high value applications like microencapsulation of oils rich in omega 3 fatty acids, such as fish oil. By leveraging inherent characteristics, blending Procream with hydrolyzed whey protein has the potential to improve interfacial activity and antioxidant activity of whey protein, which have an augmentative effect on microcapsule stability and overall success. There exists a need to not only understand the performance of these blended systems in microcapsule systems but to understand the interactions between Procream and intact and hydrolyzed WPC driving that performance. This project employed a combination of proteomics and bioinformatics to better characterize protein hydrolysis and related protein structural characteristics with inherent properties of the blends to understand the impact of blending on emulsion and encapsulation systems.

Results demonstrated for the first time that blending of Procream with hydrolyzed WPC had a synergistic effect on emulsion stability, while Procream demonstrated potential as an acceptable replacement for more expensive whey protein ingredients in microcapsule systems. The “molecular to applied” approach employed allowed for better optimization of whey protein hydrolysis for emulsification and encapsulation application. This approach also provided an understanding on how interactions between Procream and intact or hydrolyzed WPC contributed to emulsion stability and microcapsule performance. The “molecular to applied” approach of this project can be expanded to valorize plant protein coproducts and to help create markets for new plant protein streams, while providing clean-label, protein-based solutions to food industry challenges. (03/2021)

Extraction, Modification, and Structural/Functional Characterization of Camelina Protein

Funded by GMI
Principal Investigator: B. Pam Ismail
Researcher: Claire Boyle

Camelina sativa, a sustainable, short-season cover crop high in protein (30%), is gaining interest due to the increasing global demand for protein-rich products and for novel plant proteins. In order to create a functional, market-viable ingredient, an efficient means of protein extraction needs to be established for camelina. The aim of this project was to evaluate two protein extraction approaches, alkaline and salt extraction, and determine their impact on the structural and functional properties of camelina protein. Protein yield, content, structural characteristics, and functional properties of the produced camelina protein concentrates (CPC, 70-80% protein) and hydrolysates (CPH) were assessed and compared to soy protein isolate (SPI). Compared to alkaline pH extraction, data showed that salt extraction produces less denatured and more functional CPC, composed mainly of cruciferin and napin proteins. The functionality of the salt extracted CPC was comparable and sometimes better than that of SPI. Overall, results revealed the potential of camelina as a novel source of functional plant protein that might gain a position in the protein market, and possibly compete with soy protein for several applications targeting the use of plant proteins. Further functionalization of camelina protein for various applications is underway.

The Effect of Limited Enzymatic Hydrolysis and Maillard-Induced Glycation on Soy Protein Immunoreactivity and Functionality

Funded by Several External Sources
Principal Investigator: B. Pam Ismail
Researcher: Chelsey Hinnekamp

Soy protein is a functional protein of high nutritional quality, yet it is one of the “Big 8” food allergens. Accordingly, food manufacturers are seeking alternatives or solutions to the allergenicity of soy protein. To explore hypoallerginization of soy protein while maintaining functionality, this study evaluated the potential of combining two controlled protein modification approaches, limited/targeted enzymatic hydrolysis and partial Maillard-induced glycation. The impact of molecular/structural changes on immunoreactivity and functionality was assessed. Changes in immunoreactivity were assessed with immunoassays utilizing human sera. Limited enzymatic hydrolysis significantly reduced immunoreactivity, with reductions ranging from 20 – 75%, depending on the enzyme used and sera variability, while Maillard-induced glycation had a variable, at times negative, effect on immunoreactivity. To determine the effect of molecular changes upon modification on immunoreactivity, a proteomics approach was employed: two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were coupled with two-dimensional immunoblotting. The modification did not cleave/mask all of the allergenic epitopes. The residual immunoreactivity was due to intact epitopes found on peptides cleaved from both β-conglycinin and glycinin subunits. Moreover, individual variability among sera was observed due to differential reactivity to various subunits. On the other hand, the targeted, dual modification had minimal effect on functionality of the protein. This study highlighted the importance of targeting individual variability in immunoreactivity and understanding the structural impact of modification when optimizing conditions to develop a functional, hypoallergenic soy protein ingredient. Additional work to achieve maximal reduction in immunoreactivity while maintaining functionality is under way. 

Proteomics Approach to Characterize Intermediate Wheatgrass Proteins

Funded by The Forever Green Initiative
Principal Investigator: B. Pam Ismail
Researcher: Chathurada Gajadeera

Thinopyrum intermedium, commonly known as intermediate wheatgrass (IWG), is a perennial crop with favorable agronomic characteristics and nutritional benefits. IWG is relatively high is protein content compared to wheat (17-21% compared 10-15% on dry basis), yet it is deficient in high molecular weight glutenins (HMWG), responsible for dough strength. The aim of this study is to characterize IWG proteins using a proteomics approach to elucidate impact on functionality and identify potential applications. Molecular weight distribution of IWG proteins are analyzed using size exclusion high performance chromatography. The figure below shows SDS-PAGE visualization of gluten proteins selected for chymotrypsin in-gel digestion for subsequent molecular identification by LC-MS. Isolated protein fractions are analyzed and identified by gel electrophoresis and LC-MS/MS respectively. Preliminary results showed that, in contrast to wheat, IWG had lower percentage of SDS-unextractable high molecular weight polymeric proteins (uHMWPP). Relative quantity of soluble albumins and globulins were higher in IWG samples than that of wheat. HMWG, low molecular weight glutenin (LMWG), α, β, γ and ω–gliadins, and albumins and globulins were identified by LC/MS-MS as major proteins in IWG. These proteins were considerably different in their molecular weights in contrast to those of wheat controls. Similarly, the protein distribution and molecular weights of HMWG subunits in the IWG bulk sample were considerably different from wheat. HMWG present in IWG were of lower molecular weight, approximately 60 kDa compared to 80-120 kDa in wheat. The lower uHMWPP content in IWG,  suggest that it might not be suitable for applications that require dough rising properties. The detailed protein characterization of IWG, will be used to identify other unique applications to expand its market potential.

Identification of key volatile compounds contributing to pennycress aroma

Funded by Several Funding Sources 
Principal Investigator: Gary Reineccius
Researcher: Peishan Luo

As a member of the mustard family (Brassicaceae), pennycress has an unpleasant “mustard-like” aroma, which is hard to be accepted by consumers, largely limiting its application in food industry. However, no published literatures has reported the volatiles analysis of pennycress. This research aims to identify key volatile compounds that contribute to pennycress aroma, thus making it more possible to design breeding programs or processes to minimize the undesirable aroma. To extract volatile compounds present in wild type pennycress seeds, solvent extraction and solvent assisted flavor evaporation (SAFE) are used, and subsequently, volatiles are detected and analyzed by gas chromatography-olfactometry (GC-O), coupled with aroma extraction dilution analysis (AEDA). Consequently, key volatile compounds are identified using retention indices, aroma descriptors, and standard chemicals verification. Identification of these volatiles may lead to determining the metabolic pathways of their production in the seed and changes in flavor development during protein isolation.

Enhancement of pea protein solubility and thermal stability for acidic beverage applications via endogenous Maillard-induced glycation and chromatography purification

Funded by Schwan’s Corporate Giving Foundation, PPIC
Principal Investigator: B. Pam Ismail
Researchers: Alissa A. Schneider

Update: 
A clean-label process to endogenously glycate and purify pea protein was investigated. The production of maltodextrin from pea starch with a specific dextrose equivalent (DE) was optimized. The produced maltodextrin (14.6 DE) was used to initiate a limited and controlled Maillard-induced glycation of pea protein. The partially glycated pea protein (PG-PP) was subjected to hydrophobic interaction chromatography to remove unreacted carbohydrate, followed by characterization of the purified product. The extent of Maillard-induced glycation was monitored by assessing changes in color, free amino groups, and protein/glycoprotein profiles. The purified PGPP was evaluated for thermal denaturation, surface properties, protein secondary structure, protein solubility, thermal stability, and digestibility. Maillard-induced glycation was limited to initial stages and resulted in a moderate blockage of amine groups (~30%). The purified PG-PP had a relatively low surface hydrophobicity, a markedly enhanced protein solubility (~90%) at pH 3.4, and a nonimpacted protein in vitro digestibility (~100%). This work provided the impetus needed for future scale-up and process optimization for the production of value-added pea protein ingredient intended for high protein beverage applications. (March 2023)

PPIC Patents

Method for Producing Functional Pea Protein. Alissa Schneider, B. Pam Ismail, 2020. Provisional Patent Filed Oct 30^th 2020

Scaling of Endogenous Maillard-Induced Glycation to Produce Pea Protein Isolate with Enhanced Solubility and Thermal Stability

Funded by Schwan’s Corporate Giving Foundation, PPIC
Principal Investigator: B. Pam Ismail
Researchers: Allison Boerboom

The primary aim of this study is to verify the enhanced solubility and thermal stability of pea protein through partial glycation using pea protein and pea maltodextrin produced on a pilot plant scale. This research intends to establish a viable and scalable method for producing purified, partially glycated pea protein (PG-PP). This involves utilizing Maillard-induced glycation of pea protein isolate with pea maltodextrin derived from the inherent starch-rich byproduct of a pH-based pea protein extraction process. The glycated pea proteins will undergo purification using hydrophobic interaction chromatography (HIC) and alkaline extraction combined with isoelectric precipitation (AE-IEP). Structural characterization of the pea protein isolates will encompass protein denaturation, protein profiling, surface charge, surface hydrophobicity, and protein secondary structure analysis. Furthermore, the solubility of the isolated pea protein from AE-IEP will be assessed at pH 7.0 and 3.4. Commercial pea protein isolate (PPI) will serve as a comparative benchmark. Additionally, the study aims to determine the in vitro Protein Digestibility-Corrected Amino Acid Score (PDCAAS) of the isolated protein utilizing a newly available commercial kit. The data generated from this project will lay the essential groundwork necessary for scaling up toward commercial integration. 

Update:
Since the beginning of this project, we have fine-tuned the HIC and AE-IEP processes to obtain purified, partially glycated pea protein with optimal purity and yield. Notably, protein isolated via AE-IEP exhibits markedly enhanced solubility at pH 7.0 compared to commercial pea protein isolate (cPPI) and our pilot plant's scale-up pea protein isolate (SUPPI). Our current efforts are directed towards evaluating protein digestibility. (July 2024)