The current consumption trends, combined with the expected demographic growth in the coming years, call for a protein transition, i.e., the partial substitution of animal protein-rich foods with foods rich in proteins produced in a more sustainable way. Here, we have discussed some of the most common and promising protein sources alternative to animal proteins, namely: legumes, insects, and microorganisms (including microalgae and fungi). The primary objective was to assess their nutritional quality through the collection of digestible indispensable amino acid score (DIAAS) values available in the scientific literature. Protein digestibility corrected amino acid score (PDCAAS) values have been used where DIAAS values were not available. The ecological impact of each protein source, its nutritional quality and the potential applications in traditional foods or novel food concepts like meat analogues are also discussed. The data collected show that DIAAS values for animal proteins are higher than all the other protein sources. Soybean proteins, mycoproteins and proteins of some insects present relatively high DIAAS (or PDCAAS) values and must be considered proteins of good quality. This review also highlights the lack of DIAAS values for many potentially promising protein sources and the variability induced by the way the proteins are processed.

The escalating global population is anticipated to intensify the demand for high-quality proteins, necessitating the exploration of alternative protein sources. Edible insects are a promising solution, owing to their nutritional richness and sustainability. However, their digestibility and protein quality, particularly after culinary treatment, remains underexplored. In the present study, we investigated the effects of various culinary treatments on the protein digestibility of two insect species, Tenebrio molitor and Gryllus assimilis. Our findings revealed that culinary treatments such as boiling, roasting, drying, and microwave heating significantly influenced the digestibility of both insect species. Notably, drying emerged as the most effective method, leading to a substantial increase in digestibility. Furthermore, we assessed protein quality using the digestible indispensable amino acid score (DIAAS) and found that the choice of the calculation method significantly influenced the evaluation of protein quality. By including the sum of the anhydrous amino acids, we eliminated the potential overestimation of protein content and obtained a more reliable assessment of protein quality. Our results underscore the importance of culinary treatments and calculation methods in determining the suitability of insects as protein sources for human nutrition.

The recommended transition toward more plant-based diets, particularly containing legumes, requires a wider knowledge of plant protein bioavailability. Faba beans are cultivated at different latitudes and are used increasingly in human nutrition.
We aimed to assess the nutritional quality of faba bean protein in healthy volunteers equipped with an intestinal tube to implement the ileal 15N balance method.
Nine volunteers completed the study (7 males, 2 females, aged 33 ± 10 y, BMI: 24.7 ± 2.6 kg/m2). They were equipped with a nasoileal tube. After fasting overnight, they ingested a test meal consisting of cooked mash of dehulled faba bean seeds (20 g protein per serving of approximately 250 g) intrinsically labeled with 15N. Samples of ileal contents, plasma, and urine were collected over an 8-h postprandial period. Undigested nitrogen (N) and amino acids (AAs) were determined using isotopic MS, and subsequently, ileal digestibility and digestible indispensable amino acid score (DIAAS) were calculated. The measurement of postprandial deamination allowed calculation of the net postprandial protein utilization (NPPU).
The ileal N digestibility was 84.1% ± 7.7%. Postprandial deamination represented 19.2% ± 3.6% of ingested N, and the NPPU was 64.7% ± 9.7%. The ileal digestibility of individual AAs varied from 85.1% ± 13.7% for histidine to 94.2% ± 3.6% for glutamine + glutamate. The mean AA digestibility was ∼6 percentage points higher than the digestibility of N, reaching 89.8% ± 5.9%, whereas indispensable AA digestibility was 88.0% ± 7.3%. Histidine and tryptophan were the first limiting AAs [DIAAS = 0.77 (calculated by legume-specific N-to-protein conversion factor 5.4); 0.67 (by default factor 6.25)]. Sulfur AAs were limiting to a lesser extent [DIAA ratio = 0.94 (N × 5.4); 0.81 (N × 6.25)].
Protein ileal digestibility of cooked, dehulled faba beans in humans was moderate (<85%), but that of AAs was close to 90%. Overall protein quality was restricted by the limited histidine and tryptophan content. This trial was registered at clinicaltrials.gov as NCT05047757.

This study aimed to assess the nutritional quality and digestibility of proteins in two red seaweed species, Gelidium corneum and Gracilaropsis longissima, through the application of in vitro gastrointestinal digestions, and evaluate the impact of two consecutive processing steps, extrusion and compression moulding, to produce food snacks. The protein content in both seaweeds was approximately 16 %, being primarily located within the cell walls. Both species exhibited similar amino acid profiles, with aspartic and glutamic acid being most abundant. However, processing impacted their amino acid profiles, leading to a significant decrease in labile amino acids like lysine. Nevertheless, essential amino acids constituted 35-36 % of the total in the native seaweeds and their processed products. Although the protein digestibility in both seaweed species was relatively low (<60 %), processing, particularly extrusion, enhanced it by approximately 10 %. Interestingly, the effect of the different processing steps on the digestibility varied between the two species. This difference was mainly attributed to compositional and structural differences. G. corneum exhibited increased digestibility with each processing step, while G. longissima reached maximum digestibility after extrusion. Notably, changes in the amino acid profiles of the processed products affected adversely the protein nutritional quality, with lysine becoming the limiting amino acid. These findings provide the basis for developing strategies to enhance protein quality in these seaweed species, thereby facilitating high-quality food production with potential applications in the food industry.

The Digestible Indispensable Amino Acid Score (DIAAS) is the new gold standard method for the assessment of protein nutritional quality. The DIAAS is evaluated with in vivo models, that are complex, constraining and costly. There is still no established method to assess it in vitro. In this study, we proposed to add a jejunal-ileal digestion phase to the standardized in vitro gastrointestinal digestion protocol developed by the International Network of Excellence on the Fate of Food in the Gastrointestinal Tract (INFOGEST protocol) to mimic brush border digestion and to enable DIAAS assessment in vitro in a more physiologically relevant manner. This jejunal-ileal digestion phase was performed with a porcine intestinal aminopeptidase as an alternative to brush border membrane extract, which is more difficult to obtain in a standardized way. This modified INFOGEST protocol was applied to various food matrices (faba bean, pea and soy flours, whey protein isolate and caseins) and the results were compared to published in vivo data to assess the model\'s physiological relevance. The addition of the jejunal-ileal digestion phase lead to a significant (p < 0.05) increase of 31 and 29 % in free and total amino acid digestibility, respectively, and of 83 % on average for the in vitro DIAAS values for all food matrices. Although the in vitro DIAAS remained underestimated compared to the in vivo ones, a strong correlation between them was observed (r = 0.879, p = 0.009), stating the relevance of this last digestion phase. This improved digestion protocol is proposed as a suitable alternative to evaluate the DIAAS in vitro when in vivo assays are not applicable.

In wealthy countries, the protein intake of adults is usually considered to be adequate, and considerations of protein quality are often deemed irrelevant. The objective was to examine dietary protein intakes of adults in developed countries in the context of dietary protein quality. An analysis of NHANES population data on actual protein intakes in the United States (a developed country) demonstrated that for a dietary Digestible Indispensable Amino Acid Score (DIAAS) of 100%, 11% of the adult (19-50 y) population had habitual protein intakes below the Estimated Average Requirement (EAR) and 22% below the Recommended Dietary Allowance. The percentage of the population with utilizable protein intakes potentially falling below the EAR increased as the assumed DIAAS declined. Analysis of the NHANES data and several other datasets also indicated that total protein intakes can be limiting or close to limiting for the elderly and some vegetarians and vegans. Here, lower dietary protein quality can potentially lead to inadequate utilizable protein intakes. For many people in specific physiological states (e.g., weight loss, endurance sports, resistance exercise) attempting to meet higher dietary protein targets often with accompanying lowered energy intakes, low dietary protein quality can lead to protein calories expressed as a proportion of total calories, falling outside what may be acceptable limits (maximum of 30% protein calories from total calories). In general, individuals within the adult population may be susceptible to macronutrient imbalance (whenever total protein intakes are high, daily energy intakes low) and for diets with lower protein quality (DIAAS <100%). Our analysis shows that dietary protein quality is relevant in mid- to high-income countries.

During the last years, a strong increase in the sales volume and consumption of plant-based drinks was observed, which were partly used as an alternative to cow\'s milk. As milk is a relevant protein source in many countries, we have investigated the protein bioaccessibility and digestibility of soy, almond, and oat drinks in comparison to milk using the tiny-TIMsg gastrointestinal model. The relative protein digestibility of all products was between 81% (soy drink) and 90% (milk). The digestible indispensable amino acid score (DIAAS) in vitro method was used to estimate the protein nutritional quality. The highest DIAAS values were obtained for milk in tryptophan (117%) and soy drink in sulfur containing amino acids (100%). Oat drink was limited in lysine (73%), almond drink in lysine (34%) and the sulfur containing amino acids (56%). Additionally, the antioxidant activity of the bioaccessible fractions was analyzed using Trolox equivalent antioxidative capacity and oxygen radical absorbance capacity assays, revealing a higher antioxidative potential of milk and soy drink compared to oat and almond drink.

United Nations agencies have a long history of leading work on establishing global human nutrient requirements. Dietary protein contributes to metabolism and homeostasis and plays an essential role in human health for growth, maintenance, reproduction, and immune function (or immunity). Accurately defining the quantity and quality of protein provided by foods and diets required to meet human nutritional needs is essential to achieving global environmental and nutrition goals. There have been many scientific developments related to protein quality over the past decades, with the preferred method being the scoring approach that relates the capacity of protein sources to provide an adequate amount and proportion of nitrogen and indispensable amino acids (IAAs) in a bioavailable form (often referred to as digestibility). Questions surrounding the scoring approach and IAA metabolic availability have been discussed during past and recent expert consultations. Recently, an Food and Agriculture Organization of the United Nations/International Atomic Energy Agency technical meeting, held in Vienna, 10-13 October, 2022, reviewed and updated evidence and related methods on protein requirements and protein quality assessment and designed a framework for the development of a Protein Digestibility Database to aid dialog on the evaluation of protein quality and protein sufficiency in different populations. The database should be a living document and align with national food compositional databases.

There is an urgent need to alleviate protein deficiencies in low-income countries where cereal-based diets dominate. The objective of this study was to use the INFOGEST static digestion method and a recently established analytical workflow to determine the in vitro amino acid digestibility and protein quality of seven maize varieties grown in Malawi. Protein quality was measured using the in vitro digestible indispensable amino acid score (DIAAS). Amino acid digestibility was higher for the dehulled, low fibre, provitamin A maize flour (66%), compared to whole grain maize flours (51-61%), suggesting that the presence of fibre reduced digestibility (p < 0.05). Lysine was the limiting amino acid in all varieties, with the following DIAAS values for each variety; Provitamin A maize - 24, SC 719 - 32, Mtsikinya - 37, SC 167 - 39, Quality protein maize (QPM) - 40, Bantum - 40, SC 403 - 44. In addition to the variety of maize, protein quality was dependent on the level of processing and the agronomic practice applied with higher protein quality for the SC 403 variety in which zinc enriched fertilizer was applied. Comparing protein quality data with published in vivo data showed that DIAAS data were in closer agreement than amino acid digestibility data, which was slightly lower than published values, with mean in vitro amino acid digestibilities of 56-70% compared to a mean in vivo value of 77%. Overall, the in vitro method was able to correctly predict both the direction and magnitude of response. The INFOGEST digestion method coupled with the new analytical workflow will therefore be useful in the screening of high protein cereal crops and subsequent development of cereal-based foods with high protein quality.

Faba bean is an ancient legume that is regaining interest due to its environmental and nutritional benefits. Very little is known on the protein quality of the new faba bean varieties. In this study, the digestibility and the Digestible Indispensable Amino Acid Score (DIAAS) of the protein quality of three Canadian faba bean varieties (Fabelle, Malik and Snowbird) were compared to pea and soy using the harmonized in vitro digestion procedure developed by the International Network of Excellence on the Fate of Food in the Gastrointestinal Tract (INFOGEST). The impact of boiling on the nutritional quality of faba bean flours was also ascertained. Protein content in faba bean (28.7-32.5%) was lower than defatted soy (56.6%) but higher than pea (24.2%). Total phenolics and phytate content were higher (p < 0.05) in faba bean (2.1-2.4 mg/g and 11.5-16.4 mg/g respectively) and soy (2.4 mg/g and 19.8 mg/g respectively) comparatively to pea (1.3 mg/g and 8.9 mg/g). Trypsin inhibitor activity was significantly higher (p < 0.05) in soy (15.4 mg/g) comparatively to pea (0.7 mg/g) and faba bean (0.8-1.1 mg/g). The digestibility of free amino acids of raw faba bean flours ranged from 31 to 39% while the digestibility of total amino acids ranged from 38 to 39%. The in vitro Digestible Indispensable Amino Acid Score (IV-DIAAS) of raw faba bean flours ranged from 13 to 16 (when calculated based on free amino acid digestibility) to 32-38 (when calculated based on total amino acid digestibility) and was in a similar range to pea (13-31) and soy (11-40). Boiling modified the protein electrophoretic profile and decreased trypsin inhibitor activity (30-86% reduction), while total phenolics and phytate content were unaffected. The IV-DIAAS significantly decreased in all boiled legumes, possibly due to an increased protein aggregation leading into a lower protein digestibility (18-32% reduction). After boiling, the nutritional quality of faba bean was significantly lower (p < 0.05) than soy, but higher than pea. Our results demonstrate that faba bean has a comparable protein quality than other legumes and could be used in similar food applications.