This protocol describes a standardized in vitro method to determine the digestibility and digestible indispensable amino acid score (DIAAS) of dietary proteins. This ‘INFOGEST Quant’ method is an extension of our previous INFOGEST static digestion model (INFOGEST 2.0) and adds a workflow for the quantification of total protein digestibility, individual amino acid digestibility and DIAAS. The protocol was validated using in vivo data obtained by digesting the same food samples, and the results showed a high degree of agreement, confirming its relevance for nutritional assessments. To establish the DIAAS of a protein source, nonabsorbable peptides and proteins are precipitated after in vitro digestion and the resulting absorbable fraction is analyzed using ultrahigh-performance liquid chromatography with ultraviolet detection (to assess total and individual amino acids). Two alternative quantification strategies are also described: protein titration using the Kjeldahl method (to assess total nitrogen) and spectrophotometric analysis with o-phthalaldehyde (to assess total amino groups). Both alternative methods are valid only for the calculation of total digestibility and the proxy-digestible indispensable amino acid ratio, which gives an approximation of the DIAAS of the tested protein sources. Compared to existing approaches, this protocol is suitable for routine application in nutrition and food science laboratories. The preparatory steps take ~6 d, whereas the full workflow can be completed in triplicate in ~8 d. Analysis of the digesta takes an additional 3–5 d, depending on the method. The procedure requires only standard laboratory equipment and reagents and can be performed by anyone with basic training in biochemistry or a related discipline. This Protocol Extension is an adapted version of the authors’ previous INFOGEST static digestion model (INFOGEST 2.0), with an added workflow for the quantification of total protein digestibility, individual amino acid digestibility and digestible indispensable amino acid score.

Oral iron salts are commonly used as supplements and fortificants to treat iron deficiency and anemia, but they are often poorly absorbed and cause gastrointestinal side effects, highlighting the need for more efficient and better-tolerated alternatives. Bovine lactoferrin (LF), a natural iron-binding glycoprotein, offers a promising option for iron supplementation, as it can deliver iron via receptor-mediated uptake in the small intestine. However, its efficacy may be compromised by proteolytic degradation during gastrointestinal digestion. In this study, we systematically evaluated novel LF-based formulations designed to resist gastric conditions. We focused on complexes with succinylated sodium caseinate, low-methoxyl pectin, and whey protein hydrolysate, each combined with varying iron loadings. Static in vitro digestion was performed using the INFOGEST 2.0 protocol, and LF integrity was assessed by SDS-PAGE and LC-MS/MS. Unprotected LF was extensively hydrolyzed under these conditions. By contrast, ternary complexes of LF with sodium caseinate and pectin at high iron saturation, as well as binary LF-caseinate complexes with high levels of extrinsic iron, showed the highest stability. During intestinal digestion, highly iron-saturated ternary complexes of LF with sodium caseinate released the greatest amount of potential iron-binding peptides. These results indicated that specific LF-protein-polysaccharide-iron combinations are promising next-generation iron supplements and fortificants with improved tolerability and bioavailability.

Water activity (aw) is a critical physico-chemical parameter that influences microbiological and biochemical properties during cheese ripening. This study assessed the use of Raoult's law in estimating aw based on four chemical components (moisture, NaCl, total free amino acids [EFAAs] and lactate) across 14 commercial Swiss hard and semi-hard cheeses. A strong correlation (r = 0.979) was observed between the measured and calculated aw values. Non-protein nitrogen was tested as an alternative to EFAAs but yielded a lower correlation. Additionally, o-phthaldialdehyde (OPA) analysis emerged as a simple, cost-effective alternative to EFAAs for estimating aw values, achieving a comparable correlation coefficient (r = 0.980). The study proved that although moisture and NaCl were primary determinants of aw, EFAAs (or OPA reactive substances) resulting from proteolysis and lactate were also crucial for accurate aw values prediction in ripened hard/semi-hard cheeses. This study provides a robust model for aw prediction in ripened cheeses, particularly when instrumental measurements are unavailable.

This study investigated biogenic amine production in Raclette cheese using Latilactobacillus curvatus strains capable of producing tyramine, beta-phenylethylamine, putrescine, cadaverine, and tryptamine as adjunct cultures. Control cheeses without L. curvatus and with a non-amine-producing strain were included for comparison. During ripening, cheeses with tyramine-producing strains accumulated tyramine up to approximately 300 mg/kg after 120 days, along with strain-dependent levels of beta-phenylethylamine. Strains capable of producing putrescine, cadaverine, and tryptamine also led to the accumulation of these amines. Analyses of free amino acids and carboxylic acids revealed strain-specific effects on amino acid metabolism (tyrosine, ornithine, arginine, aspartic acid, serine) and on D-lactate and formic acid levels. No effect on eye formation was detected by X-ray imaging. These findings demonstrate that L. curvatus is a notable producer of biogenic amines and modulates amino acid and organic acid metabolism in cheese.

Determination of the nutritional quality of dietary proteins requires in vivo or in vitro digestion experiments that evaluate the ileal amino acid digestibility. However, many studies assess protein quality using finely ground flours or isolates rather than the food in its commonly consumed form, which may lead to overestimation of bioaccessibility. This study utilises the standardised INFOGEST Quant protocol to evaluate the impact of sample preparation on protein digestibility and quality of chia and quinoa seeds, popularly known for their high protein quality. Total nitrogen, amino groups and individual amino acids were analysed to calculate digestibility and digestible indispensable amino acid ratios (DIAAR). Results demonstrated that proteins in whole chia seeds, the most prevalent form of consumption, are virtually indigestible (< 4%), rendering their amino acids physiologically unavailable. Only upon grinding did chia’s protein digestibility surge to 63.3%, significantly increasing its DIAAR values and transforming it into a viable amino acid source. In contrast, quinoa exhibited high total protein digestibility (> 90%) upon both traditional processing (boiling) and grinding. These findings highlight that sample preparation is a determinant factor in protein quality assessment. To avoid overestimating the true nutritional value of plant-based “superfoods”, digestibility assays must mimic real-world consumption practices.

BackgroundThe identification of pollen is important in the field of beekeeping for the determination of the botanical origin of bee products and investigations of bee diet. Until now, it has been performed by melissopalynology, the microscopic examination of pollen grains. However, this technique has some limitations, such as the necessity of experienced analysts and identification restricted to the family level for some pollen types. Although many techniques have been proposed as alternatives or complements to melissopalynology and omics techniques have been explored to gather information on the botanical origin of honey, no study has yet been conducted on a large set of pollen types.ResultsThe study dataset consisted of 34 different pollen types of pellets collected by honeybees in Switzerland and analyzed in multiple biological replications, leading to 150 observations. The pollen samples were analyzed after tryptic digestion using a non-targeted mass spectrometry-based method. Liquid chromatography coupled with mass spectrometry (LC-MS) was employed to identify pollen, and melissopalynology was used as a reference method for the identification. We built an OPLS-DA prediction model for the 34 pollen types. The model clearly identified new samples in their membership group (Acer sp., n = 10) and a new pollen type at the species-specific level for Quercus sp. Less predictable results were achieved for Composita H and pollen collected directly from the plant.ConclusionThe use of a non-targeted mass spectrometry-based method and chemometrics resulted in a promising tool for pollen identification as a replacement/supplement method to traditional melissopalynology.

The rise in life expectancies and gap between life span and health span necessitate innovative approaches, interventions, and food solutions to secure healthy aging. This study, conducted within the framework of the EAT4AGE project of the Joint Programming Initiative ‘A Healthy Diet for a Healthy Life’, focuses on the rational design of functional foods for older adults. A co-extruded cereal prototype, fortified with two bioactive moieties, maca root powder and olive leaf extract, aims to address nutritional gaps identified in older adults, with a particular emphasis on high-quality and highly digestible proteins. Analytical determinations reveal the cereals have rich macronutrient profile exceeding 12% (m/m) protein, 20% (m/m) fat, and low sugar (< 5%, m/m), surpassing commercially available products with texture analyses supporting improved hardness, reduced oral friction and oral comfort. An untrained consumer panel (n = 21, age 73 ± 5) confirmed high palatability in various metrics and overall acceptability that were also affirmed through a trained sensory panel. Lastly, the product digestion was explored through an age-tailored in vitro digestion model which consistently demonstrated high protein digestibility, surpassing 80%, across all product formulations. Further, calculation of the in vitro digestible indispensable amino acid score of the product affirms its high nutritional quality. Thus, this study underscores the potential of designing palatable foods that could help promote a balanced and sustainable diet towards healthy aging.

The efficiency and outcomes of mechanically disrupting microalgal cell walls depend on various factors and are case-specific. In general, bead milling is reproducible and scalable, and it can successfully increase the digestibility of microalgal biomass. However, its effects on the aroma of microalgal biomass have not yet been assessed. This study is the first to report the use of the NanoWitt-100 bead mill to break down microalgal biomass. Optimization of processing time and cell concentration revealed that the optimal conditions were a milling time of 15 min, and a cell concentration of 50 g L−1. Under these conditions, we then compared the in vitro protein and amino acid digestibility as well as volatile organic compound (VOC) profiles of undisrupted and disrupted samples. Bead milling substantially increased total protein digestibility from 62.2 ± 2.28 to 99.7 ± 0.06

This study evaluated the impact of x-casein (x-CN) genotypes (AA, AB, BB, BE) and minerals (total, serum, and micellar) on coagulation properties of raw milk in 80 individual Holstein cows. The results showed that the influence of the ratio of x-CN to total casein was significantly different between genotypes (BB > AB > BE > AA), but there was no significant influence on main milk ingredients (proteins, fat or minerals). Of all tested minerals, only total sodium was slightly lower in milk from cows with x-CN genotype B. Rennet induced coagulation properties were statistically evaluated using ANCOVA, with pH as a covariate. The shortest rennet coagulation time (RCT) and the firmest curd were observed with the BE and BB genotypes. The impact of pH on RCT and the x-CN percentages on curd firmness were strong, whereas the minerals showed only a rather small impact on RCT and the curd firmness.

While meat is an established source of high-quality protein, limited data exists on plant-based meat analogues, particularly regarding how specific production steps and extrusion conditions affect their protein quality and ecological footprint. We used the Protéix soybean cultivar to produce dry soy protein intermediate products with varying degrees of refinement and employed them to obtain meat analogues by high-moisture extrusion. As a reference, a commercial soy protein concentrate was used to produce meat analogues by high- and low-moisture extrusion. In vitro amino acid (AA) digestibility and in vitro DIAAS of intermediate products and extrudates were assessed and compared to traditional soy-based foods and chicken breast. The meat analogues had high total protein in vitro digestibility (>95 %) irrespective of extrusion type, energy input, and soybean variety. The extrusion process substantially enhanced protein digestibility of mildly refined soy protein powders which had low protein digestibility (<60 %). Consequently, meat analogues based on these raw materials showed the lowest environmental footprint per kg quality-corrected protein - with a fourfold lower global warming potential than chicken, compared to only a 17 % reduction observed for meat analogues based on soy protein isolate. In vitro DIAAS values for all studied meat analogues ranged from 81 to 102 for children aged 0.5 to 3 years and were only limited in sulfur-containing AA. Soy-based meat analogues were equally digestible as tofu and cooked chicken breast, had similar protein quality as soymilk and tofu, and can be good to excellent protein sources for humans.

Mare milk has a unique protein composition that makes it a preferred option for adult and infant nutrition. Several functional properties have been attributed to this milk but with little evidence yet. In fact, knowledge on mare milk composition is still limited. In particular, studies addressing the performance of mare milk proteins during human gastrointestinal digestion are scarce, which limits the understanding of mare milk nutritional quality and functionality. For this reason, the present study describes the digestibility of mare milk proteins and the release of peptides as affected by management and lactation stage, factors known to affect milk composition. Mare milk samples from 3 different farms, and collected during 6 mo of lactation (n = 54), were subjected to a static in vitro gastrointestinal model to measure peptide release and protein digestibility. In the present study, a detailed description of protein and individual amino acid behavior during the digestion process was given. For the first time, digestion of the 2 equine β-lactoglobulin isoforms (I and II) was described individually. In addition, it was found that lactation stage and management system can significantly affect protein digestibility and peptide release during gastrointestinal digestion of mare milk. Presumably, differences in the composition of mare milk influence the protein structure and enzyme accessibility, which might have an impact on digestion behavior. Despite no specific bioactive peptides were identified, several precursors of previously described bioactive peptides were found. These findings could support the idea of mare milk as a food with added value.

This study evaluated the impact of k -casein (k-CN) protein phenotype (AA (n 1/4 18), AB (n 1/4 18), AE (n 1/4 5), BB (n 1/4 3)) in the raw milk of 44 individual Holstein cows from a single farm on coagulation properties using diffusion wave spectroscopy. k-CN variants were analyzed using a newly developed LC -MS method in individual milk samples, and relative milk protein fractions were quantified by LabChipanalysis. k-CN fractions expressed in the percentage of total milk protein, total casein, and b- and k-CN were then calculated. The gross composition of the individual milk samples and variation between individual cows was analyzed and was rather large, as expected. Traits were analyzed using an ANCOVA model with pH as a covariate. Our results showed that mean percentages of k-CN fractions increased for k-CN phenotypes in the order AA < AE < AB < BB. Rennet coagulation time was slightly affected, but curd firmness was significantly affected by k-CN phenotype (BB > AE > AB > AA) when controlling for pH. (c) 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Efforts towards sustainable food systems have stimulated the development of plant-based alternatives to meat and milk. However, the debate on the nutritional quality of (processed) plant products compared to animal products remains unresolved, and combined assessments are needed to assess the benefits and trade-offs of these products in future diets. Using life cycle assessment (LCA) combined with nutrient density measurements and the Digestible Indispensable Amino Acid Score (DIAAS), this case study evaluates different processing stages of soy-based products, including cooked soybeans, tofu, soy drink, and a processed soy-based meat analogue (SBMA) produced in Switzerland. The nutritional LCA (n-LCA) showed that the environmental impact of all soy-based meat alternatives was 4–20 times lower than that of beef, especially when locally sourced soy was used. The differences were smaller when compared to chicken meat. All soy-based products showed lower DIAAS compared to animal products, but the results from the combined n-LCA were always less favourable for animal products in this case study. Contribution analyses showed that despite the high level of processing, the raw materials contributed significantly to the environmental footprint of SBMA, exceeding 50% in some environmental impact categories. Moreover, comparisons within the soy-based alternatives revealed a lower environmental impact of the minimally processed products. The higher protein quality and quantity of the processed SBMA were not sufficient to offset its higher environmental impact in this case study. SBMA also contained highest level of sodium and saturated fatty acids, highlighting the need for careful food formulation. Overall, this study showed the potential of soy-based alternatives to meat and milk to reduce the environmental impact of food production whilst highlighting the importance of considering their nutritional quality and the role of processing. Overall, this research provides insight into the potential of plant-based alternatives to meat and milk for sustainable diets amid the global challenge of climate change and changing dietary patterns.

To support the transition towards more sustainable and healthy diets, viable alternatives to foods of animal origin need to be identified. Many plant -based protein sources are currently marketed with claims of minimal environmental impact, but very limited consideration has been given to their protein quality and bioavailable mineral content considering the fact that animal -based foods are typically the primary source of both in Western diets. In this study, traditionally consumed soy foods (cooked soybeans, soymilk, tofu) from different Swiss soybean cultivars were nutritionally characterized and the in vitro digestibility of individual amino acids and total protein were assessed using an in vitro model based on the static INFOGEST protocol; the protein quality was evaluated using the in vitro digestible indispensable amino acid score (DIAAS). The results reveal an increase in total protein in vitro digestibility across the traditional soy food production value chain: 52.1-62.7% for cooked soybeans, 84.1-90.6% for soymilk, and 94.9-98.4% for tofu. Protein quality, determined using the recommended amino acid pattern for 0.5-3 years old, was "low" (no claim) for cooked soybeans (DIAAS < 60), while soymilk (DIAAS = 78-88) and tofu products (DIAAS = 79-91) were of similar "good" protein quality, with considerably higher DIAAS values than those of cooked soybeans (P < 0.001). The iron and zinc contents in soy foods were substantial, but high molar ratios of phytic acid (PA) to iron (PA/Fe; >8) and PA to zinc (PA/Zn; >15) indicate a possible strong inhibition of iron and zinc bioavailability. Based on the DIAAS results, soymilk and tofu would be suitable plant -based alternatives to animal -based foods, while future efforts should focus on optimizing soybean preparation to overcome the negative effects of the plant tissue matrix as well as processing steps to reduce mineral absorption inhibiting substances.

Plant-based meat alternatives of high quality and digestibility could be a way to reduce meat consumption and, consequently, the environmental impact. However, little is known about their nutritional characteristics and digestion behaviour. Therefore, in the present study, the protein quality of beef burgers, known as excellent source of protein, was compared with the protein quality of two highly transformed veggie burgers, based on soy or pea-faba proteins, respectively. The different burgers were digested according to the INFOGEST in vitro digestion protocol. After digestion, total protein digestibility was determined, either based on total nitrogen (Kjeldahl) analysis, or after acid hydrolysis based on total amino groups (o-phthalaldehyde method) or total amino acids (TAA; by HPLC). The digestibility of individual amino acids was also determined, and the digestible indispensable amino acid score (DIAAS) was calculated based on in vitro digestibility. The impact of texturising and grilling on in vitro protein digestibility and the digestible indispensable amino acid ratio (DIAAR) was evaluated at the level of the ingredients and the finished products. As expected, the grilled beef burger had the highest in vitro DIAAS values (Leu 124 %), and grilled soy protein-based burger reached in vitro DIAAS values that could be rated as good (soy burger, SAA 94 %) protein source, according to the Food and Agriculture Organization. The texturing process did not significantly affect the total protein digestibility of the ingredients. However, grilling led to a decrease in digestibility and DIAAR of the pea-faba burger (P < 0.05), which was not observed in the soy burger, but led to an increase in DIAAR in the beef burger (P < 0.005).

Identification of food intake biomarkers (FIBs) for fermented foods could help improve their dietary assessment and clarify their associations with cardiometabolic health. We aimed to identify novel FIBs for fermented foods in the plasma and urine metabolomes of 246 free-living Dutch adults using nontargeted LC-MS and GC-MS. Furthermore, associations between identified metabolites and several cardiometabolic risk factors were explored. In total, 37 metabolites were identified corresponding to the intakes of coffee, wine, and beer (none were identified for cocoa, bread, cheese, or yoghurt intake). While some of these metabolites appeared to originate from raw food (e.g., niacin and trigonelline for coffee), others overlapped different fermented foods (e.g., 4-hydroxybenzeneacetic acid for both wine and beer). In addition, several fermentation-dependent metabolites were identified (erythritol and citramalate). Associations between these identified metabolites with cardiometabolic parameters were weak and inconclusive. Further evaluation is warranted to confirm their relationships with cardiometabolic disease risk.

Microalgae are gaining interest as food ingredient. Assessments of functional and nutritional properties are necessary to forward their implementation. In this study, protein content and composition of eight commercially available microalgae biomasses were determined and compared to conventional food proteins. A novel procedure for the determination of the true protein content was proposed: Multiplication of proteinic nitrogen with a sample-specific nitrogen-to-protein conversion factor kA. The proteinic nitrogen was derived from the difference of total nitrogen minus non-protein nitrogen. The average kA for microalgae was 5.3 and considerable variation between different microalgae biomasses were detected. In addition, the content of non-protein nitrogen varied between 3.4% and 15.4%. The amino acid profiles of Chlorella samples were nutritionally superior to the tested plant proteins but indicated lower protein interaction tendency, potentially limiting their structuring functionality. In contrast, Auxenochlorella contained lower amounts of indispensable amino acids while showing comparable interaction potential to plant proteins.

Edible insects, such as mealworms (Tenebrio molitor larvae; TM) and crickets (Acheta domesticus; AD), are a sustainable, protein-dense novel food with a favorable amino acid profile, which might be an alternative to animal proteins. To assess the protein quality of TM and AD, we assessed the digestible indispensable amino acid scores (DIAAS), considering individual amino acids and their ileal amino acid digestibility, using an in vitro model based on the INFOGEST digestion protocol. In addition, we evaluated if various processing and food preparation steps influenced the in vitro digestibility of individual amino acids and the in vitro DIAAS values of TM and AD and compared them to chicken breast as a reference of excellent protein quality. The total protein in vitro digestibility ranged from 91 to 99% for TM and from 79 to 93% for AD and was negatively affected by oven-drying and, to a lesser extent, by chitin-reduction. The in vitro DIAAS values were 113, 89, and 92 for chicken, blanched TM, and blanched AD, respectively, when considering the indispensable amino acid (IAA) requirements of young children between 6 months and 3 years. Across different processing and food preparation methods, the in vitro DIAAS values ranged between 59 and 89 for TM and between 40 and 92 for AD, with the lowest values found in chitin-reduced insects. Due to their similarities to chicken regarding protein composition, total protein in vitro digestibility, and in vitro DIAAS values, TM and AD might be an alternative to traditional animal proteins, provided that suitable processing and food preparation methods are applied. Our in vitro DIAAS results suggest that TM and AD can thus be considered good-quality protein sources for children older than 6 months. The DIAAS calculations are currently based on crude protein (total nitrogen × 6.25), resulting in an overestimation of insect protein content, and leading to an underestimation and potential misclassification of protein quality. The in vitro model applied in this study is a valuable tool for product development to optimize the protein quality of edible insects. Further studies are required to assess the in vivo DIAAS of insects in humans.

The function of the aminotransferase Aat (GenBank Protein WP_159211138) from Pediococcus acidilactici FAM 18098 was studied in vivo. For this purpose, the gene was replaced with an erythromycin resistance gene using the temperature-sensitive Escherichia coli-Pediococcus shuttle plasmid pSET4T_Δaat. The knockout was verified by PCR and genome sequencing. Subsequently, the differences between the metabolism of the knockout and of the wild-type strain were investigated by determining the free amino acids and organic acids in culture supernatants. It was found that the knockout mutant no longer synthesized 3-phenyllactic acid (PLA) and 4-hydroxyphenyllactic acid (HPLA). Additionally, the mutant strain no longer catabolized phenylalanine. Metabolic pathway analysis using the KEGG database indicate that P. acidilactici cannot synthesize α-ketoglutarate that is a predominant amino-group acceptor in many transamination reactions. To study the transfer of the amino group of phenylalanine, the wild-type strain was incubated with [15N] phenylalanine. Mass spectrometry showed that during fermentation, [15N] alanine was formed, indicating that pyruvic acid is an amino group acceptor in P. acidilactici. The present study shows that Aat plays a crucial role in PLA/HPLA biosynthesis and pyruvic acid is an amino acceptor in transamination reactions in P. acidilactici.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.