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.

The reformulation of dairy products with plant ingredients poses new food safety challenges, particularly microbiological risks. Due to their proximity to the soil, plant ingredients are susceptible to contamination with spore-forming bacteria such as Bacillus spp., with toxin-producing Bacillus cereus posing a health risk. Here, we examined the contamination of a plant-based raw material used for a cheese alternative with B. cereus at various stages of the production process and how it can be inhibited by fermentation. The raw material consisted of a paste containing 40% shelled sunflower seed oil press cake. The concentration of B. cereus in the untreated paste was below the quantification limit of 50 CFU/g vegetative cells or spores. However, after heat treatment and incubation at room temperature, the vegetative cell counts increased to potentially toxin-forming concentrations of >105 CFU/g within 22 h if no simultaneous fermentation with lactic acid bacteria took place. Simultaneous mesophilic fermentation to a final pH of 4.9 reproducibly inhibited the growth of B. cereus but not at a final pH of 5.7. During the subsequent one- and two-month storage at 4°C, the B. cereus numbers in all fermented masses dropped sharply to <200 CFU/g, despite previous potentially toxigenic B. cereus levels of >105 CFU/g. The decrease in vegetative cells was not associated with a corresponding increase in spores. Based on these findings, we recommend monitoring B. cereus in new dairy alternatives both after heat treatment and immediately following the end of fermentation to support the development of a safe production process.

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.

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/).

Protein is an essential macronutrient in our diet, source of nitrogen and essential amino acids, but the biological utilization of dietary protein depends on its digestibility and the absorption of amino acids and peptides in the gastrointestinal tract. The methods to define the amount and the quality of protein to meet human nutritional needs, such as the Digestible Indispensable Amino Acid Score (DIAAS), require the use of animal models or human studies. These in vivo methods are the reference in protein quality evaluation, but they are expensive and long-lasting procedures with significant ethical restrictions. Therefore, the development of rapid, reproducible and in vitro digestion methods validated with in vivo data is an old demand. This review describes the challenges of the in vitro digestion methods in the evaluation of the protein nutritional quality. In addition to the technical difficulties to simulate the complex and adaptable processes of digestion and absorption, these methods are affected by similar limitations as the in vivo procedures, i.e., analytical techniques to accurately determine bioavailable amino acids and the contribution of the endogenous nitrogen. The in vitro methods used for the evaluation of protein digestibility, with special attention on those showing comparative data, are revised, emphasizing their pros and cons. The internationally harmonized digestion protocol proposed by the INFOGEST network is being adapted to evaluate protein and amino acid digestibility. The inter-laboratory reproducibility of this protocol was demonstrated for dairy products. The in vivo/in vitro comparability results obtained to date with this protocol for several plant and animal sources are promising, but it requires an extensive validation with a wider range of foods and substrates with known in vivo digestibility. These in vitro methods will probably not be applicable to all foods, and therefore, it is important to identify their limitations, not to elude their use, but to apply them within the limits, by using the appropriate standards and references, and always as a complementary tool to in vivo tests to reduce their number.

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.

The effect of the horn status of cows on their milk composition and quality is a controversial research topic. In this study, 128 milk samples from 64 horned and 64 disbudded Brown Swiss and Original Braunvieh cows were collected from alpine farms where both horned and disbudded cows were grazing on mountain pastures. The samples were analyzed for their detailed composition and protein digestion in a simulated in vitro digestion (INFOGEST). To exclude probable influences on digestion, the β-casein genotype with its variants A1 and A2 was also included in the study. The effects of horn status and β-casein genotype were investigated in linear mixed models, which included additional influencing random factors such as Original Braunvieh blood proportion, stage of lactation, and farm. Horn status did not have any effect on milk composition or digestion. In contrast, milk from A1A1 cows showed a different protein digestion than milk of A1A2 and A2A2 cows in the gastric phase, including smaller amounts of β-casomorphin(BCM)21-associated peptides and larger amounts of BCM11-associated peptides. Abundances of BCM7 did not differ between β-casein genotypes. At the end of the intestinal phase, the digested milk of A1A1 and A2A2 b-casein genotypes did not differ.

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).

Healthy, untreated cows of nine dairy herds from the Swiss Canton Tessin were analyzed three times within one year to identify the most abundant species of the intramammary bacteriome. Aseptically collected milk samples were cultured and bacteria identified using MALDI-TOF. Of 256 cows analyzed, 96% were bacteriologically positive and 80% of the 1,024 quarters were positive for at least one bacterial species. 84.5% of the quarters were healthy with somatic cell counts (SCC) < 200,000 cells/mL, whereas 15.5% of the quarters showed a subclinical mastitis (SCC ≥ 200,000 cells/mL). We could assign 1,288 isolates to 104 different bacterial species including 23 predominant species. Non-aureus staphylococci and mammaliicocci (NASM) were most prevalent (14 different species; 73.5% quarters). Staphylococcus xylosus and Mammaliicoccus sciuri accounted for 74.7% of all NASM isolates. To describe the intramammary resistome, 350 isolates of the predominant species were selected and subjected to short-read whole genome sequencing (WGS) and phenotypic antibiotic resistance profiling. While complete genomes of eight type strains were available, the remaining 15 were de novo assembled with long reads as a resource for the community. The 23 complete genomes served for reference-based assembly of the Illumina WGS data. Both chromosomes and mobile genetic elements were examined for antibiotic resistance genes (ARGs) using in-house and online software tools. ARGs were then correlated with phenotypic antibiotic resistance data from minimum inhibitory concentration (MIC). Phenotypic and genomic antimicrobial resistance was isolate-specific. Resistance to clindamycin and oxacillin was most frequently observed (65 and 30%) in Staphylococcus xylosus but could not be linked to chromosomal or plasmid-borne ARGs. However, in several cases, the observed antimicrobial resistance could be explained by the presence of mobile genetic elements like tetK carried on small plasmids. This represents a possible mechanism of transfer between non-pathogenic bacteria and pathogens of the mammary gland within and between herds. The-to our knowledge-most extensive bacteriome reported and the first attempt to link it with the resistome promise to profoundly affect veterinary bacteriology in the future and are highly relevant in a One Health context, in particular for mastitis, the treatment of which still heavily relies on antibiotics.