Protein Digestibility Assay Kit

Nutritional optimisation of staple food such as pasta with flours from climate-smart crops (i.e. resilient to climatic hazards and requiring minimal inputs) is in line with actual environmental and nutritional challenges. Although the quantity of protein is important, the quality through protein digestibility is essential. The aim of this work was to assess the protein digestibility of four gluten-free climate-smart pasta all based on cowpea flour with or without the addition of teff and/or amaranth leaf flour(s) using two static in vitro methods: static Infogest digestion protocol and Megazyme® enzymatic kit. The effect of both gluten absence and the high fiber content on the in vitro protein digestibility was investigated through the use of three durum wheat semolina pasta controls with increasing fiber content. Statistical analysis were made through ANOVA with p < 0.05. Despite structural differences, induced by protein origin and the disruption of the protein network by fibers, there was no significant difference in in vitro protein digestibility between cowpea-based and traditional durum wheat semolina pasta. Protein quality was then approached with the measurement of the in vitro Protein Digestibility Corrected Amino Acid Score (i-PDCAAS) for the two methodologies. Thanks to their balanced essential amino acid profile, cowpea-based pasta demonstrated i-PDCAAS that was twice as high as that of wheat-based pasta, as determined by both by the Infogest (68-86 versus 35-44) and enzymatic kit (99-104 versus 49-56) methods. In addition, for the first time both methodologies were compared and Infogest protocol appeared more suitable for more detailed studies.

This study addresses the global demand for sustainable protein alternatives by optimizing the texturization of plant-based proteins using saturated steam technology. The optimization of this process was done using a Central Composite Rotatable Design for key process parameters (e.g., dough processing time, thermal exposure time & temperature) that influence the quality of TPPs. Saturated steam-assisted texturization significantly impacted the acceptability attributes such as texture, color, and sensory preferences of TPPs. Thermal exposure time and temperature were critical in optimizing hardness, chewiness, and overall acceptability. Increased exposure (beyond 30 min and 115°C) caused protein denaturation, texture softening, and increased browning. The results indicated that moderate thermal exposure time (30 min) and temperature (118°C) yielded the most desirable texture, color, and sensory acceptance. Finally, TPPs demonstrated superior protein content (39.41%), lower fat (0.50%), and equivalent in-vitro protein quality (0.86) compared to boneless chicken breast, positioning them as a viable, nutritious alternative to traditional meat products.

Multiplex gene editing allows for the simultaneous targeting and mutagenesis of multiple loci in a genome. This tool is particularly valuable for plant genetic improvement, as plant genomes often require mutations at multiple loci to confer useful and/or novel traits. However, the regulation of gene editing can vary depending on the number of loci targeted. In this study, we developed triple-mutant soybean (Glycine max (L.) Merrill) lines using different crop improvement strategies, including conventional backcross breeding of standing variant alleles and clustered regularly interspaced short palindromic repeats-based multiplex editing to introduce new alleles. The mutations were targeted to genes encoding seed antinutritional components, as previously described in a triple null soybean carrying knockout alleles for a Kunitz trypsin inhibitor, a soybean agglutinin, and the allergen P34 protein. The products developed from these respective genetic improvement pipelines were tested for differences between the triple-mutant lines and their parental lines. Analyses included genomics, seed proteomics, trypsin inhibition, seed protein digestibility, and harvestable yield of the different lines. We observed that both multiplex gene editing and conventional breeding approaches produced essentially equivalent products in comparison to their parental lines. We conclude that the multiplex gene editing strategy is not inherently riskier than conventional breeding for developing complex mutant lines of this type.

This study aimed to optimize sourdough preparation from pulse-based flours (faba bean flour, faba bean protein concentrate, and yellow pea flour) using several binary consortia of lactic acid bacteria and yeasts for making new bread formulations. A total of 288 type-II sourdoughs with varying flour substrates and fermentation times were designed. Screening based on optimal sourdough criteria identified seventeen best-performing type-II sourdoughs. The transition to type-IV sourdoughs markedly enhanced their maturity and functionality. Six most promising pulse-based type-IV sourdoughs were selected as tailored inocula for sourdough breadmaking, leading to significant biochemical differences compared to wholewheat bread made with baker's yeast (control). Pulse-based sourdough breads showed higher protein content and superior amino acid profiles. The lowest levels of antinutritional factors were found when Leuconostoc mesenteroides and Saccharomyces cerevisiae were co-cultured in yellow pea. Despite lower in vitro protein digestibility in sourdough breads, the synergistic interaction of Levilactobacillus namurensis and Torulaspora delbrueckii improved protein quality regardless of flour compositions. Additionally, pulse-based sourdoughs enriched the breads with phenolics, like catechin, rutin, epicatechin, sinapic acid, and quercetin, offering substantial functional benefits. Despite the adverse textural characteristics, most sourdough breads exhibited more complex volatile organic compound profiles compared to that of the control.

The combined effects of extrusion and enzymatic hydrolysis with a neutral protease (endoprotease) on quinoa germ flour have been studied in this work. A 2 × 3 factorial design with two temperature levels (60°C-90°C-100°C and 60°C-100°C-120°C) and three protease concentrations (0 %w/v, 0.6%w/v, 1.2 %w/v). The hydrolyzed quinoa germ flours were characterized in terms of their physicochemical and rheological properties, antioxidant capacity by ABTS+, in-vitro digestibility of carbohydrates and protein, infrared spectroscopy, and their rheological behavior in aqueous dispersion at 12% (w/w). The results showed that enzymatic hydrolysis and extrusion temperature influenced hydrolyzed quinoa germ flours properties. The highest protein digestion (65 mg leucine/g protein) was obtained with 0.6% protease and 100°C extrusion. Antioxidant activity increased with protease concentration but decreased with temperature. The solubility of hydrolyzed quinoa germ flours improved after enzymatic hydrolysis, while water absorption decreased. The treatment with 0.6 % protease and 100 °C extrusion produced high amounts of slowly digestible starch and RS, and enhanced protein digestibility compared to the other treatments. Infrared spectroscopy revealed changes in amide functional groups A, B, and I due to hydrolysis. The modification of these flours through enzymatic hydrolysis and extrusion after the tecno-functional properties. This research highlights the importance of understanding the interactions between different sources of proteins and how they contribute to the overall characteristics of the final product.

This study evaluated the nutritional quality of 25 commercial plant-based (PB) yoghurt alternatives (lupin-, soya-, oat-, coconut-based), focusing on protein digestibility and amino acid score and the content of the essential minerals calcium, iron, magnesium, and zinc. The phytates levels were also measured to estimate mineral bioavailability. The content of saponins was examined in soya and oat products. The findings of this study revealed a high variability of Protein Digestibility-Corrected Amino Acid Score (PDCAAS) within product groups, with values ranging from 0.21 to 0.82 for soya-based yoghurt alternatives and from 0.41 to 1.00 for oat-based yoghurt alternatives. Only one oat product exhibited high-quality protein (PDCAAS > 0.9). Most products had low mineral content, which makes them unsuitable as a mineral source. Phytates hindered the low mineral content, with levels ranging from 13 to 193 mg/100 g. However, products enriched with calcium showed satisfactory levels, and phytate content had minimal impact on the estimated calcium bioavailability. Oat- and soya-based products contained 1–8 mg/100 g of avenacosides and 11–18 mg/100 g soyasaponins, respectively. This research underscores the importance of paying more attention to the nutritional value during product development of PB yoghurt alternatives manufactured using various constituents and when incorporating these products into dairy-free diets.

Seaweed biomass is globally underutilized as a source of proteins despite its nutritional potential, with much of its use focused on hydrocolloid extraction. This study evaluated the nutritional quality and digestibility of protein and amino acids from two brown seaweeds (Durvillaea spp. and Macrocystis pyrifera), one green seaweed (Ulva spp.), and a novel mycoprotein derived from Durvillaea spp. through fungal fermentation. Using an in vitro gastrointestinal digestion Megazyme assay kit, protein digestibility-corrected amino acid scores (PDCAASs) and digestible indispensable amino acid scores (DIASSs) were determined. Compared with seaweeds, seaweed-derived mycoprotein presented significantly greater protein contents (~33%) and amino acid profiles (2.2 times greater than those of Durvillaea spp. and M. pyrifera), with greater digestibility (~100%) than seaweeds (<60%). The PDCAAS values were 0.37, 0.41, 0.53, and 0.89 for Ulva spp., Macrocystis pyrifera, Durvillaea spp., and mycoproteins, respectively. The DIASSs highlighted the superior nutritional quality of the mycoprotein, particularly for lysine (0.59) and histidine (0.67). SDS-PAGE revealed soluble peptides (<25 kDa) in Durvillaea spp., Macrocystis pyrifera, and mycoproteins, whereas Ulva spp. proteins exhibited limited solubility due to structural aggregation. These findings highlight the need to characterize the nutritional properties of edible seaweeds in Chile further and emphasize the importance of optimized processing techniques, such as fermentation or bioconversion, to improve the nutritional potential of seaweeds and develop high-quality food ingredients for diverse applications.

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.