Protein Digestibility Assay Kit

Societal Impact Statement: Pulse crops, including dry pea, lentil, and chickpea, are rich sources of protein, low digestible carbohydrates, and micronutrients. With the increasing demand for plant-based protein with gluten-free and allergen-free foods, pulse crops have become of global importance for meeting the nutritional demand of growing populations. Breeding for nutritional quality is becoming a bottleneck for most breeding programs globally due to the cost of these available tools. Therefore, low-cost, high-throughput phenotyping tools will be a focus of interest for the selection of elite germplasm for cultivar development and gene identification for pulse cultivar development. This publication explains the emerging and future trends of phenotyping tools that are feasible for pulse breeding and improving nutritional quality. Summary: Precision agriculture tools based on spectroscopic and imaging techniques now contribute to high-throughput phenotyping (HTP) pipelines for nutritional and agronomic traits to speed breeding and selection for cultivar development. Fourier transform mid-infrared (FT-MIR) spectroscopy has been a reliable HTP tool for macro nutritional traits in pulse crops. Hyperspectral, multispectral, and RGB (red-green-blue) imaging with unmanned aerial systems (UAVs) have been developed to measure agronomic traits for cereals, but these techniques have yet to be developed and validated for pulse crops. This review summarizes different phenotyping techniques applied to nutritional and agronomic traits for crop breeding and reviews applications of machine learning tools for optimizing HTP.

The standard method of estimating in vitro protein digestibility, the protein digestibility corrected amino acid score (PDCAAS) assay, does not support the expected workflow of a pulse breeding program. This is mainly due to its low-throughput design and long processing time (~16-24 h) per sample. Fourier-transform mid-infrared (FT-MIR) spectroscopy has been developed as a high-throughput phenotyping tool to estimate protein digestibility in pulses. The mid-infrared region representing the amide I band (1756.81-1586.27 cm⁻¹) was utilized to perform chemometric modeling with partial least squares regression (PLSR) to estimate in vitro protein digestibility in dry pea (Pisum sativum L.), lentil (Lens culinaris Medik.), and chickpea (Cicer arietinum L.) flours. The root mean square error of predictions of the developed PLSR models for dry pea, lentil, and chickpea were 0.00039, 0.00024, and 0.00017, respectively. Phenotyping with the FT-MIR approach is more rapid than with the PDCAAS assay and estimates protein digestibility from the flour of a single seed in a shorter time (~1-2 min). The FT-MIR approach is robust as the spectroscopic data are consistent and chemically fingerprint this nutritional trait. Accordingly, FT-MIR can resolve the phenotypic bottleneck of pulse breeding related to in vitro protein digestibility measurements by enabling a high-throughput phenotyping workflow.

To meet global protein demand sustainably in the future, we have to move to alternative, non-animal sources. A problem of many plant-derived foods is their low protein quality compared to animal-based proteins, in particular their deficiency in lysine. To improve the protein quality, plant proteins could be converted to fungal proteins using solid-state fungal fermentation (SSFF). In this project Rhizopus microsporus var. oligosporus and Aspergillus oryzae were used to ferment barley and rice, producing tempeh and koji, respectively. SSFF yielded products with 6–13% (dry weight basis) fungal biomass. Protein quality was defined by the parameters indispensable amino acid index (IAAI) and protein digestibility corrected amino acid score (PDCAAS). SSFF improved both parameters in all samples. Lysine was the limiting amino acid in unfermented barley and rice, with PDCAAS of 0.54 and 0.57, respectively. SSFF increased the PDCAAS of lysine by approximately 30% in barley koji, barley tempeh and rice koji and 10% in rice tempeh. These results demonstrate the use of SSFF to improve the protein quality of staple foods. Thereby, this fermentation method can aid in meeting protein demands sustainably in the future through plant-based diets.

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. This research provided, for the first time, a comprehensive evaluation of different protein extraction protocols to produce a functional PcPI that can compete with soy protein for various food applications, such as acidic beverages, meat and dairy products, and emulsified systems.

House crickets (Acheta domesticus) were processed into a seasoning paste, the so-called fermented cricket paste (FCP), by mimicking the production process of Thai fermented shrimp paste (Kapi). Whole house crickets were ground with solar salt (crickets to salt ratio = 10:1 w/w), sun-dried to ~50% moisture content and fermented at 30°C for 4 weeks using Kapi (5% w/w) as a starter culture. Results showed that salting and drying steps might contribute greatly to eradicating undesirable microorganisms by lowering the A_w of cricket from 0.9 to 0.7. The changes in physicochemical properties during FCP preparation could be attributed to the activity of lactic acid bacteria, which were predominant microorganisms in the FCP (5.66 log CFU/g DW). Overall, the FCP had similar characteristics to commercial Kapi products and should be well-preserved without refrigeration. Despite the lower overall nutritive value, the FCP possessed an improved amino acid profile and protein digestibility compared with the raw material (93.80% vs 81.91%), indicating that the FCP preparation process dealing with fermentation could enhance the protein quality of house crickets. The FCP could be used as a protein-rich seasoning and an alternative for fermented shrimp paste, thus helping to promote the consumption of insect-based foods.

In Canada and the United States, front-of-package protein content claims require data to support the quality of the protein. In general, protein quality reflects the product of the amino acid composition of the food protein relative to human amino acid requirements and a measure of digestibility. The currently accepted method in both jurisdictions is the protein digestibility-corrected amino acid score (PDCAAS) that requires the measurement of true fecal protein (nitrogen) digestibility. The latter must be measured in vivo using a rat model. This requirement for animal testing is inconsistent with international efforts to reduce the usage of animals in testing for regulatory purposes. The current commentary positions four options to remove the need to use animal testing for determining protein quality, when considering protein content claim substantiation. These options include (i) a focus on protein quantity alone; (ii) the use of the amino acid score alone, with no correction for digestibility; (iii) the use of a fixed digestibility coefficient to estimate protein quality; and (iv) the use of in vitro methods to measure protein and/or amino acid digestibility. The relative merits and deficiencies of the options are positioned with the goal of encouraging dialogue within the regulatory agencies to move towards alternative approaches for substantiating protein content claims on foods, including those derived from plant-based sources.

The world is increasingly looking to plant-based sources to meet its protein needs. Multiple factors are driving this progression, ranging from nutritional and ethical considerations to climate change and population growth. As a pulse crop, lentil is ideal to help meet this change in demand. However, plant-based proteins have limiting amino acids and lower protein digestibility compared to animal-based proteins. This research identifies genetic markers that can be used to accelerate breeding of protein quality traits in lentil to ultimately help meet the rising demand in high-quality plant-based protein and bolster global food and nutritional security.

Black soldier fly (Hermetia illucens L., BSF) larvae are a promising alternative for future sustainable nutrient sources both as feed and food. However, the scientific investigation of BSF larvae is still limited, especially on the flavor chemistry aspects of understanding consumer acceptance. This research examined the odor-active compounds and protein content of BSF larvae. Whole and partially defatted BSF larvae meals were compared to an anchovy fish meal. Gas chromatography/mass spectrometry and gas chromatography/olfactometry were employed to determine and identify the odor-active compounds of BSF larvae and observe the differences in volatile composition between fat-reduced samples. 95 volatile and more than 40 odor-active compounds were detected in the BSF larvae and anchovy fish meal. Overall BSF larvae meal odor profile was characterized by having more fishy, earthy, cheesy, and roasted notes. Trimethylamine, acetic acid, 3-methylbutanoic acid were considered by assessors as key off-flavor compounds contributing undesirable smell in BSF larvae meal. The defatting process slightly affected the overall odor profile; however, it did not change the volatile composition.

Dry pea (Pisum sativum L.) is a cool-season food legume rich in protein (20-25%). With increasing health and ecosystem awareness, organic plant-based protein demand has increased; however, the protein quality of organic dry pea has not been well studied. This study determined the genetic variation of individual amino acids (AAs), total AAs (liberated), total protein, and in vitro protein digestibility of commercial dry pea cultivars grown in organic on-farm fields to inform the development of protein-biofortified cultivars. Twenty-five dry pea cultivars were grown in two USDA-certified organic on-farm locations in South Carolina (SC), USA, for two years (two locations in 2019 and one in 2020). The concentrations of most individual AAs (15 of 17) and the total AA concentration significantly varied with dry pea cultivar. In vitro protein digestibility was not affected by the cultivar. Seed total AA and protein for dry pea ranged from 11.8 to 22.2 and 12.6 to 27.6 g/100 g, respectively, with heritability estimates of 0.19 to 0.25. In vitro protein digestibility and protein digestibility corrected AA score (PDCAAS) ranged from 83 to 95% and 0.18 to 0.64, respectively. Heritability estimates for individual AAs ranged from 0.08 to 0.42; principal component (PCA) analysis showed five significant AA clusters. Cultivar Fiddle had significantly higher total AA (19.6 g/100 g) and digestibility (88.5%) than all other cultivars. CDC Amarillo and Jetset were significantly higher in cystine (Cys), and CDC Inca and CDC Striker were significantly higher in methionine (Met) than other cultivars; CDC Spectrum was the best option in terms of high levels of both Cys and Met. Lysine (Lys) concentration did not vary with cultivar. A 100 g serving of organic dry pea provides a significant portion of the recommended daily allowance of six essential AAs (14-189%) and daily protein (22-48%) for an average adult weighing 72 kg. Overall, this study shows organic dry pea has excellent protein quality, significant amounts of sulfur-containing AAs and Lys, and good protein digestibility, and thus has good potential for future plant-based food production. Further genetic studies are warranted with genetically diverse panels to identify candidate genes and target parents to develop nutritionally superior cultivars for organic protein production.

Introduction: One key approach to achieve zero hunger in Sub-Saharan Africa (SSA) is to develop sustainable, affordable, and green technologies to process nutritious food products from locally available sources. Soybeans are an inexpensive source of high-quality protein that may help reduce undernutrition, but it is underutilized for human consumption. This research evaluated the feasibility of a low-cost method developed initially at the United States Department of Agriculture to produce soy protein concentrate (SPC) from mechanically pressed soy cake and thus create a more valuable ingredient to improve protein intake in SSA. Methods: The method was initially tested in the bench scale to assess process parameters. Raw ingredients comprised defatted soy flour (DSF), defatted toasted soy flour (DTSF), low-fat soy flour 1 (LFSF1; 8% oil), and LFSF2 (13% oil). Flours were mixed with water (1:10 w/v) at two temperatures (22 or 60°C) for two durations (30 or 60 min). After centrifugation, supernatants were decanted, and pellets were dried at 60°C for 2.5 h. Larger batches (350 g) of LFSF1 were used to examine the scalability of this method. At this level, protein, oil, crude fiber, ash, and phytic acid contents were measured. Thiobarbituric acid reactive substances (TBARS), hexanal concentration and peroxide value were measured in SPC and oil to evaluate oxidative status. Amino acid profiles, in vitro protein digestibility, and protein digestibility corrected amino acid score (PDCAAS) were determined to assess protein quality. Results: Bench scale results showed accumulation of protein (1.5-fold higher) and reduction of oxidative markers and phytic acid to almost half their initial values. Similarly, the large-scale production trials showed high batch-to-batch replicability and 1.3-fold protein increase from initial material (48%). The SPC also showed reductions in peroxide value (53%), TBARS (75%), and hexanal (32%) from the starting material. SPC's in vitro protein digestibility was higher than the starting material. Conclusion: The proposed low-resource method results in an SPC with improved nutritional quality, higher oxidative stability, and lower antinutrient content, which enhances its use in food-to-food fortification for human consumption and is thus amenable to address protein quantity and quality gaps among vulnerable populations in SSA.

Introduction: One key approach to achieve zero hunger in Sub-Saharan Africa (SSA) is to develop sustainable, affordable, and green technologies to process nutritious food products from locally available sources. Soybeans are an inexpensive source of high-quality protein that may help reduce undernutrition, but it is underutilized for human consumption. This research evaluated the feasibility of a low-cost method developed initially at the United States Department of Agriculture to produce soy protein concentrate (SPC) from mechanically pressed soy cake and thus create a more valuable ingredient to improve protein intake in SSA. Methods: The method was initially tested in the bench scale to assess process parameters. Raw ingredients comprised defatted soy flour (DSF), defatted toasted soy flour (DTSF), low-fat soy flour 1 (LFSF1; 8% oil), and LFSF2 (13% oil). Flours were mixed with water (1:10 w/v) at two temperatures (22 or 60°C) for two durations (30 or 60 min). After centrifugation, supernatants were decanted, and pellets were dried at 60°C for 2.5 h. Larger batches (350 g) of LFSF1 were used to examine the scalability of this method. At this level, protein, oil, crude fiber, ash, and phytic acid contents were measured. Thiobarbituric acid reactive substances (TBARS), hexanal concentration and peroxide value were measured in SPC and oil to evaluate oxidative status. Amino acid profiles, in vitro protein digestibility, and protein digestibility corrected amino acid score (PDCAAS) were determined to assess protein quality. Results: Bench scale results showed accumulation of protein (1.5-fold higher) and reduction of oxidative markers and phytic acid to almost half their initial values. Similarly, the large-scale production trials showed high batch-to-batch replicability and 1.3-fold protein increase from initial material (48%). The SPC also showed reductions in peroxide value (53%), TBARS (75%), and hexanal (32%) from the starting material. SPC's in vitro protein digestibility was higher than the starting material. Conclusion: The proposed low-resource method results in an SPC with improved nutritional quality, higher oxidative stability, and lower antinutrient content, which enhances its use in food-to-food fortification for human consumption and is thus amenable to address protein quantity and quality gaps among vulnerable populations in SSA.

The impact of whey protein (WP) denaturation on the in vitro digestibility and biological activity of milk protein concentrate-85 (MPC85) was investigated. MPC85S1 and MPC85S2 having undenatured WP levels equal to 16.6 and 6.0 g/100 g overall protein, respectively, had similar in vitro protein digestibility corrected amino acid scores equal to 1.14. The samples were subjected to in vitro simulated gastrointestinal digestion while sampling was performed every 30 min during gastric (GD) followed by intestinal (GID) digestion. Liquid chromatography–mass spectroscopy showed that MPC85S1-GD, MPC85S2-GD, MPC85S1-GID and MPC85S2-GID had 50, 38, 47 and 66 unique peptides, respectively. The degree of hydrolysis, molecular mass distribution, dipeptidyl peptidase-IV inhibition, oxygen radical absorbance capacity and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity of the digests were compared. Overall, the results showed higher digestibility and bioactivities for low-denatured MPC85 compared to high-denatured MPC85 upon GD, however, following GID, both samples were digested to a similar extent.

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 PG-PP 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.

Insects, especially crickets, have been proposed as a novel source of nutrients in human nutrition since they possess bioactive molecules, including high protein content, lipids, chitin, vitamins and minerals. In this work, the nutritional and functional properties of a novel Italian spray-dried (SD) cricket powder were evaluated. The powder was characterized by physico-chemical properties (morphology, size distribution, solid state, thermal profiles, and surface zeta potential), and antioxidant properties. Moreover, preclinical properties (cytocompatibility and pro-inflammatory immune response) were assessed. The powder was characterized by microparticle structure with bulges and rough surfaces, showing distinctive antioxidant properties. The preclinical results suggested that the SD crickets were biocompatible towards Caco-2 and macrophages without immune response, representing an interesting material for the food industry that could provide health benefits in addition to the basic nutritional value of traditional foods.

A deficit in protein intake can severely halt children growth, and is associated with both structural and functional pathologies of the brain, as well as increase the risk of contracting infectious diseases. Protein-enriched defatted meals from sacha inchi (Plukenetia volubilis) (sdSI) and chocho (Lupinus mutabilis) (sdCH) were obtained by a solvent defatting procedure. The concentration of protein was higher in sdSI (64%) than in sdCH (60%). Lysine and tryptophan were the limiting amino acid (AA) in each case. However, the AA profile of the protein meals met daily intake FAO requirements for adults, while their blends met the requirements for infants. The sdSI had an in vitro digestibility of 100. Regarding functional properties, sdSI presented higher values for water absorption index (3.5), emulsifying activity (56%), and foaming capacity (167%), while the sdCH had a slightly better oil absorption capacity (2.8). However, when mixing sdSI with sdCH some improvements in functional properties were observed. The nutritional and functional properties of the mixtures of meals can be used to develop protein-enriched foods and beverages.