Screening of a Microbial Culture Collection: Empowering Selection of Starters for Enhanced Sensory Attributes of Pea-Protein-Based Beverages.

Pea-protein-based ingredients are gaining attention in the food industry due to their nutritional benefits and versatility, but their bitter, astringent, green, and beany off-flavors pose challenges. This study applied fermentation using microbial cultures to enhance the sensory qualities of pea-protein-based beverages. Using UHPLC-TOF-MS analyses along with sensory profile comparisons, microbial species such as Limosilactobacillus fermentum, Lactococcus lactis, Lactobacillus johnsonii, Lacticaseibacillus rhamnosus, and Bifidobacterium longum were preselected from an entire culture collection and found to be effective in improving the overall flavor impression by reducing bitter off-notes and enhancing aroma profiles. Notably, L. johnsonii NCC533 and L. fermentum NCC660 exhibited controlled proteolytic activities after 48 h of fermentation, enriching the matrix with taste-active amino acids, nucleotides, and peptides and improving umami and salty flavors while mitigating bitterness. This study has extended traditional volatile analyses, including nonvolatile metabolomic, proteomic, and sensory analyses and offering a detailed view of fermentation-induced biotransformations in pea-protein-based food. The results highlight the importance of combining comprehensive screening approaches and sensoproteomic techniques in developing tastier and more palatable plant-based protein products.

Sensoproteomic Characterization of Lactobacillus Johnsonii-Fermented Pea Protein-Based Beverage: A Promising Strategy for Enhancing Umami and Kokumi Sensations while Mitigating Bitterness.

This study investigated the mechanism underlying the flavor improvement observed during fermentation of a pea protein-based beverage using Lactobacillus johnsonii NCC533. A combination of sensomics and sensoproteomics approach revealed that the fermentation process enriched or generated well-known basic taste ingredients, such as amino acids, nucleotides, organic acids, and dipeptides, besides six new taste-active peptide sequences that enhance kokumi and umami notes. The six new umami and kokumi enhancing peptides, with human recognition thresholds ranging from 0.046 to 0.555 mM, are produced through the degradation of Pisum sativum's storage protein. Our findings suggest that compounds derived from fermentation enhance umami and kokumi sensations and reduce bitterness, thus improving the overall flavor perception of pea proteins. In addition, the analysis of intraspecific variations in the proteolytic activity of L. johnsonii and the genome-peptidome correlation analysis performed in this study point at cell-wall-bound proteinases such as PrtP and PrtM as the key genes necessary to initiate the flavor improving proteolytic cascade. This study provides valuable insights into the molecular mechanisms underlying the flavor improvement of pea protein during fermentation and identifies potential future research directions. The results highlight the importance of combining fermentation and senso(proteo)mics techniques in developing tastier and more palatable plant-based protein products.

In situ interaction of pea peptides and bile salts under in vitro digestion: Potential impact on lipolysis.

The present work evaluated how a native pea protein isolate (PPI) affects the key roles carried out by bile salts (BS) in lipid digestion by means of the in vitro static INFOGEST protocol. Two gastric residence times were evaluated (10 and 60 min), and then the peptides obtained (GPPP) were mixed with BS at physiological concentration in simulated intestinal fluid to understand how they interact with BS both at the bulk and at the interface. Both GPPP give rise to a film with a predominant viscous character that does not constitute a barrier to the penetration of BS, but interact with BS in the bulk duodenal fluid. When the peptides flushing from the stomach after the different gastric residence times undergo duodenal digestion, it was found that for the longer gastric residence time the percentage of soluble fraction in the duodenal phase, that perform synergistically with BS micelles, was twice that of the lower residence time, leading to an increase in the solubilization of oleic acid. These results finally lead to a greater extent of lipolysis of olive oil emulsions. This work demonstrates the usefulness of in vitro models as a starting point to study the influence of gastric residence time of pea protein on its interaction with BS, affecting lipolysis. Pea proteins were shown to be effective emulsifiers that synergistically perform with BS improving the release and bioaccessibility of bioactive lipids as olive oil.

sRAGE-binding and antimicrobial bioactivities of soy and pea protein after heating and in vitro infant digestion.

During infant formula production, proteins are always heated, potentially affecting their digestibility and the bioactivities of resulting peptides. Although plant proteins are a promising dairy alternative for infant formula, they remain understudied, necessitating further investigations. Therefore, this research aimed to fill this gap by assessing the impact of different heating modes on soy protein (SP) and pea protein (PP), focusing on glycation levels, peptide formation during in vitro infant digestion, and immune protection potential (sRAGE-binding and antimicrobial activities) of the resulting peptides. Consequently, dry heating led to increased glycation and glycated peptide production, particularly with higher glycation in PP than SP. Moreover, PP exhibited an overall stronger sRAGE-binding capacity than SP, regardless of heating and digestion conditions. Regarding antimicrobial activity, both SP and PP-derived peptides displayed reduced effectiveness against Enterobacter cloacae after dry heating. Additionally, Staphylococcus epidermidis was differently inhibited, where PP-derived peptides showed inherent inhibition. The primary determinant of sRAGE-binding and antimicrobial potential in digestion-derived peptides was the protein source. Subsequent bioinformatics analysis predicted 519 and 133 potential antimicrobial peptides in SP and PP, respectively. This study emphasises the importance of protein source for infant formula to ensure infant health.

In vitro protein digestibility of RuBisCO-enriched wheat dough: a comparative study with pea and gluten proteins.

Growing demand for sustainable, plant-based protein sources has stimulated interest in new ingredients for food enrichment. This study investigates the nutritional and digestive implications of enriching wheat dough with RuBisCO, in comparison to pea protein-enriched and gluten-enriched doughs. The protein quality and digestibility of these enriched doughs were analysed through dough characterization, in vitro digestion experiments and biochemical analysis of digesta. Our findings indicate that an enrichment at 10% of RuBisCO or pea proteins improves the chemical score and the in vitro PDCAAS (IV-PDCAAS) score of wheat dough as compared to the control dough. Digestibility assays suggest that RuBisCO introduction modifies the protein hydrolysis kinetics: the nitrogen release is lower during gastric digestion but larger during intestinal digestion than other samples. The analysis of the protein composition of the soluble and insoluble parts of digesta, using size-exclusion chromatography, reveals that the protein network in RuBisCO-enriched dough is more resistant to gastric hydrolysis than the ones of other doughs. Indeed, non-covalently bound peptides and disulfide-bound protein aggregates partly composed of RuBisCO subunits remain insoluble at the end of the gastric phase. The digestion of these protein structures is then mostly performed during the intestinal phase. These results are also discussed in relation to the digestive enzymatic cleavage sites, the presence of potential enzyme inhibitors, the protein aggregation state and the secondary structures of the protein network in each dough type.

In vitro gastrointestinal simulated digestion of three plant proteins: determination of digestion rate, free amino acids and peptide contents.

Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

Role of pea protein isolate in modulating pea starch digestibility: insights from physicochemical and microstructural analysis.

BACKGROUND: Understanding the interactions between protein and starch is crucial in revealing the mechanisms by which protein influences starch digestibility. The present study investigated the impact of different contents of pea protein isolate (PPI) on the physicochemical properties and digestibility of pea starch (PS). RESULTS: The results demonstrated that as the content of PPI increased from 0% to 12%, and the digestion of PS decreased by 12.3%. Rheological analysis indicated that PPI primarily interacted with molecular chains of PS through hydrogen bonds. Increasing the content of PPI resulted in a 30.6% decrease in the hardness of the composite gels, accompanied by a 10% reduction in the short-ordered structure of PS. This hindered the formation of molecular aggregation and resulted in a loose and disordered gel network structure. The microstructure confirmed that the attachment of PPI to PS served as a physical barrier, impeding starch digestibility. CONCLUSION: In summary, the primary mechanism by which PPI inhibited PS digestion involved steric hindrance exerted by PPI and its interaction with PS via hydrogen bonds. These findings contribute to a better understanding of the interaction mechanisms between PS and PPI and offer insights for the optimal utilization of pea resources. © 2024 Society of Chemical Industry.

Pea protein extraction method impacts the protein (micro)structural organisation and in vitro digestion kinetics.

There is increasing interest in including pulse proteins into food products due to their nutrient-rich and sustainable character. However, little is known regarding the consequences of different extraction approaches on the pulse protein structure and the subsequent protein (micro)structural organization and protein digestion kinetics. Therefore, three green pea protein extracts were created: (i) cooking followed by cotyledon cell isolation, (ii) alkaline extraction followed by isoelectric precipitation, or (iii) salt extraction, and compared to the original pea flour as well as to sodium caseinate. The results showed that encapsulated, denatured protein inside pea cotyledon cells presented the (s)lowest digestion, while accessible and more native protein (e.g., pea flour, pea protein salt extract) presented much faster and higher digestion. Moreover, the alkali extracted pea protein was denatured to some extent, significantly lowering in vitro digestion kinetics. In the second part, three different in vitro approaches were applied to digest the salt extracted pea protein. Semi-dynamic gastric digestion approaches simulate in vivo conditions more closely which especially impacted the rate of digestion.

Values for the Digestibility of Pea Protein Isolate or Casein Amino Acids Determined using the Dual Isotope Method Are Not Similar to Those Derived with the Standard Ileal Balance Method in Healthy Volunteers.

BACKGROUND: The measurement of ileal amino acid (AA) digestibility is invasive and inappropriate when applied to vulnerable populations. The dual isotope method has been developed over the past 5 y as an alternative method. OBJECTIVE: The aim of this work was to compare the indispensable amino acid (IAA) digestibility values of 2 different proteins obtained using the dual isotope and the standard ileal balance methods in the same subjects. METHODS: Fifteen healthy adults completed the study. Over 4 h, they ingested 9 successive portions of mashed potatoes containing the test protein (pea protein or casein) labeled intrinsically with 15N and 2H, and a 13C-free AA mixture as a reference for the dual isotope method. Plasma was sampled regularly over the 8-h postprandial period, whereas the ileal digesta was collected continuously via a naso-ileal tube. Isotopic enrichments (15N and 13C) were measured in the digesta for the direct determination of ileal IAA digestibility, whereas plasma enrichments (2H and 13C) were measured to determine IAA digestibility using the dual isotope method. RESULTS: The 4-h repeated meal procedure enabled the almost complete digestion of test proteins at 8 h and the attainment of a plasma isotopic plateau between 2.5 and 4 h. These conditions were necessary to perform the ileal balance and dual isotope methods simultaneously. For pea protein, the mean IAA digestibility was similar between the 2 methods, but significant differences (from 10% to 20%) were observed for individual IAA values. For casein, IAA digestibility was significantly lower with the dual isotope method for all the IAA analyzed. CONCLUSIONS: Under our experimental conditions, the degree of agreement between the dual isotope and ileal balance methods varied among AAs and depended on the protein source. Further research is needed to validate the dual isotope method. This study was registered at clinicaltrials.gov as NCT04072770.