How D-amino acids embedded in the protein sequence modify its digestibility: Behaviour of digestive enzymes tested on a model peptide used as target.

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to ß-lactoglobulin, position 48-60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.

Formulation of new sourdough bread prototypes fortified with non-compliant chickpea and pea residues.

Purpose: Nowadays, the promotion of a circular economy is fundamental to reduce food losses and waste. In this context, the possibility of using food supply chains non-compliant residues emerges. Much interest has been directed toward legume residues, in general and, in particular, to the possibility of combining different plant-matrices to improve nutritional profile, providing high-quality products. Methods: Five different formulations of breads, with a combination of seeds and cereals, were fortified with chickpea and pea protein concentrates. Samples were analyzed and compared with their relative control recipe to determine differences in composition, actual protein quality and integrity, and protein digestibility (performed with the INFOGEST method). Results: Samples showed a clear improvement in the nutritional profile with higher values of proteins, from averagely 12.9 (control breads) to 29.6% (fortified breads) (17.7-24.7 g/100 g of dry matter respectively), and an improvement in amino acidic profile, with a better balancing of essential amino acids (lysine and sulfur amino acid contents), without affecting protein integrity. Regarding in vitro gastro-intestinal digestibility, sample C (19% chickpea proteins) showed the best results, having a comparable protein digestibility to its control bread-48.8 ± 1.1% versus 51.7 ± 2.3%, respectively. Conclusion: The results showed how the fortification with chickpea and/or pea protein concentrate improved the nutritional profile of bread. These prototypes seem to be a valid strategy to also increase the introduction of high biological value proteins. Furthermore, the not-expected lower digestibility suggested the possible presence of residual anti-nutritional factors in the protein concentrates interfering with protein digestibility. Therefore, it seems of fundamental importance to further investigate these aspects.

Exploitation of bones-rich poultry by-products to produce protein hydrolysates: optimization of hydrolysis parameters and chemical characterization.

A significant quantity of bone-rich poultry by-products must be disposed of by poultry processors. These products still contain a significant amount of nutritionally valuable animal proteins. In the present work, a hydrolysis protocol was optimized to recover the protein fraction of bone-rich poultry by-products while simultaneously minimizing the amount of water required for hydrolysis (thus reducing drying costs) and recycling the hydrolytic broth up to 3 times, to reduce the cost of the proteolytic enzyme. The final hydrolysis conditions involved the use of (protease from B. licheniformis, ≥2.4 U/g; 0.5 V/w of raw material) and a hydrolysis time of 2 h at 65°C. The protein hydrolysate obtained has a high protein content (79-86%), a good amino acid profile (chemical amino acid score equal to 0.7-0.8) and good gastric digestibility (about 30% of peptide bonds are already hydrolyzed before digestion). This supports its use as an ingredient in food, pet food or animal feed formulations.

Assessment of Protein Quality and Digestibility in Plant-Based Meat Analogues.

In this first work, commercial steak-like (n = 3) and cured meat (n = 3) analogues with different legume and cereal formulations were studied and compared to their animal-based (n = 3) counterparts. Plant-based products showed lower protein content than meat controls but a good amino acidic profile even though the sum of essential amino acids of plant-cured meats does not fulfill the requirements set by the Food and Agriculture Organization for children. A comparable release of soluble proteins and peptides in the digestates after in vitro digestion was observed in meat analogues as meat products, whereas the digestibility of proteins was lower in plant-based steaks and higher in plant-based cured meats than their counterparts. The overall protein quality and digestibility of products are related to both the use of good blending of protein sources and processes applied to produce them. An adequate substitution of meat with its analogues depends mostly on the quality of raw materials used, which should be communicated to consumers.

Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

SCOPE: Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. METHODS AND RESULTS: This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. CONCLUSION: The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Molecular characterization and allergenicity assessment of different samples of Mung Bean.

Legumes represent a promising nutritional alternative source of proteins to meat and dairy products. Additionally, Novel Foods (Regulation EU 2015/2283) can help meet the rising protein demand. However, despite their benefits, emerging allergenicity risks must be considered. The aim of this work is the molecular characterization of the Novel Food Mung bean protein isolate for allergenicity prediction with High Resolution Mass Spectrometry analysis. The assessment of the allergenicity was evaluated in silico by comparing protein sequences of the Novel Food with other known legume allergens, using bioinformatic databases. The results highlighted similarity higher than 60 % of the protein structure of Mung bean with two known allergens of soybean and pea. Furthermore, enzymatic hydrolysis effects on allergenic potential was evaluated by immunoblotting analysis using sera of patients allergic to legumes. The protein hydrolysates obtained showed a high nutritional quality and a reduced allergenic potential, making them suitable for hypoallergenic food formulations.

Comparison of protein quality and digestibility between plant-based and meat-based burgers.

Nowadays, consumers are increasingly inclined toward plant-based meat analogues for sake of food security, safety, and sustainability. This growing interest, not only from consumers but also from food companies, brought the offer on the market to be wide and vast. From our previous study it emerged that the market supply, especially the Italian one, is diversified both in terms of protein sources and nutrient content. Although these products are increasingly consumed, for most of the meat analogues today on the market, little is still known about their actual protein quality and digestibility. To fill this gap, in this study different commercial plant-based burgers (2 soy-based and 2 pea-based) were selected and compared to two beef burgers, as controls, in terms of protein quality and digestibility. The findings of this study demonstrated the essential amino acidic profile lacks lysine for almost all burgers (including the meat-based ones) compared to the amino acid scoring pattern set by FAO/WHO (for older children and adults), even if the sum of essential amino acids was within the range of sufficiency. All samples showed good initial protein integrity with low hydrolysis (above 6%) and percentage of D-enantiomers (above 15%). The study of the digestibility, performed by the validated INFOGEST in vitro model, showed better protein solubilisation in the case of meat burgers (63 ± 3% and 61 ± 8%), but a good digestibility also in the case of plant-based ones (from 55% to 40%). The degree of hydrolysis of the solubilised proteins was very high in all samples (from 65% to 40%) indicating a very good protein accessibility to digestive enzymes. The analysis of the peptide fraction of digestates indicated a high prevalence of collagen proteins in beef burgers and of reserve proteins in plant-based burgers. This study showed that the differences between these products are mostly dependent on the quality of the raw materials used, rather than on the vegetal or animal protein source. Therefore, to have a product with a good protein quality and digestibility, independently from the protein origin, the consumer needs to make an accurate choice, carefully reading the ingredient list.

Protein Quality and Protein Digestibility of Vegetable Creams Reformulated with Microalgae Inclusion.

Microalgae are considered a valuable source of proteins that are used to enhance the nutritional value of foods. In this study, a standard vegetable cream recipe was reformulated through the addition of single-cell ingredients from Arthrospira platensis (spirulina), Chlorella vulgaris, Tetraselmis chui, or Nannochloropsis oceanica at two levels of addition (1.5% and 3.0%). The impact of microalgae species and an addition level on the amino acid profile and protein in vitro digestibility of the vegetable creams was investigated. The addition of microalgae to vegetable creams improved the protein content and the amino acid nutritional profile of vegetable creams, whereas no significant differences were observed in protein digestibility, regardless of the species and level of addition, indicating a similar degree of protein digestibility in microalgae species despite differences in their protein content and amino acid profile. This study indicates that the incorporation of microalgae is a feasible strategy to increase the protein content and nutritional quality of foods.

Label-Free Quantification by Liquid Chromatography-Tandem Mass Spectrometry of the Kunitz Inhibitor of Trypsin KTI3 in Soy Products.

The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources. Among these, of relevance both for human food use and for animal feed, is soy. However, its high protein content is unfortunately accompanied by the presence of antinutritional factors, including Kunitz's trypsin inhibitor (KTI). Now there are few analytical methods available for its direct quantification, as the inhibitory activity against trypsin is generically measured, which however can be given by many other molecules and undergo numerous interferences. Therefore, in this work, a direct label-free liquid chromatography-mass spectrometry (LC-MS) method for the identification and quantification of trypsin Kunitz inhibitor KTI3 in soybean and derivative products has been developed. The method is based on the identification and quantification of a marker peptide, specific for the protein of interest. Quantification is achieved with an external calibration curve in the matrix, and the limit of detection and the limit of quantification of the method are 0.75 and 2.51 µg/g, respectively. The results of the LC-MS method were also compared with trypsin inhibition measured spectrophotometrically, highlighting the complementarity of these two different pieces of information.

Evaluation of in vitro whey protein digestibility in a protein-catechins model system mimicking milk chocolate: Interaction with flavonoids does not hinder protein bioaccessibility.

Flavonoids are largely present in plant food such as cocoa and derived products. These compounds can interact with proteins inherently contained in the food matrix and/or the proteolytic enzymes involved in gastrointestinal digestion. The flavonoid/protein interaction might hamper protein bioaccessibility and digestibility, affecting the nutritional quality. However, information on the digestion fate of proteins in food matrices containing both proteins and flavonoids is limited. The aim of this work was to evaluate the interaction between proteins and flavonoids and verify the potential effects of this interaction on protein digestibility. Taking milk chocolate as model, first a simple whey proteins/catechins mixed system was evaluated, and then the effects on digestibility were also verified in a real sample of commercial milk chocolate. The effects of the catechins/whey proteins interaction in the model system were evaluated by optical and chiro-optical spectroscopy, outlining a slight protein structure modification upon interaction with catechins. The digestibility of the protein fraction both in the model system, with and without catechins, and also in milk chocolate, was then determined by the application of INFOGEST in vitro digestion method: the bioaccessibility was evaluated in terms of protein hydrolysis and protein solubilisation, and major peptides generated by the digestion were also determined by LC/HR-MS. Despite the slight interaction with proteins, flavonoids were found to not hinder nor modify protein solubilization, protein hydrolysis and peptide profile by digestive enzymes. Also protein digestibility in milk chocolate, evaluated by SDS-PAGE, was found to be complete. The present data clearly indicate that the interaction of the proteins with the flavonoids present in the cocoa matrix does not to affect protein bioaccessibility during digestion.

Allergenicity of tropomyosin variants identified in the edible insect Hermetia illucens (black soldier fly).

Insect consumption could address the increasing protein demand in compliance with environmental sustainability. Hermetia illucens (black soldier fly, BSF) is a promising insect for human diet and it is essential to assess the related allergenic risk, meant as primary sensitization or cross-reactivity with known allergens. In this work, we investigate the allergenicity of two tropomyosin variants identified in the BSF genome and produced as recombinant proteins. Immunoblot experiments showed that both proteins were recognized by sera of patients allergic to shrimp or mites highlighting the cross-reactivity risk. CD spectroscopy, cross-linking assays and size-exclusion chromatography showed a structure composed of alpha-helices oligomers for both variants. These proteins were quite stable to pH but sensitive to increasing temperatures. In vitro simulated digestion associated to mass-spectrometry allowed the identification of peptides resistant to gastrointestinal conditions which were compared with epitopes of Arthropoda and Mollusca allergens to predict the persistence of allergenicity upon digestion.

Effectiveness of enzymatic hydrolysis for reducing the allergenic potential of legume by-products.

The interest in agri-food residues and their valorization has grown considerably, and many of them are today considered to be valuable, under-exploited sources of different compounds and notably proteins. Despite the beneficial properties of legumes by-products, there are also some emerging risks to consider, including their potential allergenicity. In this work the immunoreactivity of chickpea, pea, and white bean by-products was assessed, and whether the production of enzymatic hydrolysates can be an effective strategy to reduce this allergenic potential. The results presented clearly indicate that the efficiency of this strategy is strongly related to the enzyme used and the food matrix. All legume by-products showed immunoreactivity towards serum of legume-allergic patients. Hydrolysates from alcalase did not show residual immunoreactivity for chickpea and green pea, whereas hydrolysates from papain still presented some immunoreactivity. However, for white beans, the presence of antinutritional factors prevented a complete hydrolysis, yielding a residual immunoreactivity even after enzymatic hydrolysis with alcalase.

Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami.

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing "anti-nitrite feeling" has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative "clean label" salamis without negatively affecting their nutritional value.

Assessing food authenticity through protein and metabolic markers.

This chapter aims to address an issue of ancient origins, but more and more topical in a globalized world in which consumers and stakeholders are increasingly aware: the authenticity of food. Foods are systems that can also be very complex, and verifying the correspondence between what is declared and the actual characteristics of the product is often a challenging issue. The complexity of the question we want to answer (is the food authentic?) means that the answer is equally articulated and makes use of many different analytical techniques. This chapter will consider the chemical analyses of foods aimed at guaranteeing their authenticity and will focus on frontier methods that have been developed in recent years to address the need to respond to ever-increasing guarantees of authenticity. Targeted and non-targeted approaches will be considered for verifying the authenticity of foods, through the study of different classes of constituents (proteins, metabolites, lipids, flavors). The numerous approaches available (proteomics, metabolomics, lipidomics) and the related analytical techniques (LC-MS, GC-MS, NMR) are first described from a more general point of view, after which their specific application for the purposes of authentication of food is addressed.

Peptide profile of Parmigiano Reggiano cheese after simulated gastrointestinal digestion: From quality drivers to functional compounds.

Time of ripening has a strong impact on shaping the valuable and recognizable characteristics of long-ripened types of cheese such as Parmigiano Reggiano (PR) due to the interrelationship between microbiota and proteolysis that occurs during ripening. The derived peptide profile is linked to cheese quality and represents the canvas for enzymes upon digestion, which could be responsible for the release of potentially bioactive peptides (BPs). In this study, we aimed at investigating the presence of BP in 72 PR cheese samples of different ripening times, from curd to 24 months of ripening, produced in six different dairies, and following their fate after simulated gastrointestinal digestion. A small number of peptide sequences sharing 100% similarity with known antimicrobial, antioxidant, and ACE-inhibitor sequences were found in PR cheeses, while a higher number of potential BPs were found after their simulated gastrointestinal digestion, in different amounts according to ripening time. Taking advantage of the complex organization of the sampling plan, we were able to follow the fate of peptides considered quality drivers during cheese ripening to their release as functional compounds upon digestion.

Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment.

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

Nutritional Quality of Meat Analogues: Results From the Food Labelling of Italian Products (FLIP) Project.

Nowadays, the interest in meat substitutes is increasing, and consumers perceive their nutritional quality better than that of the animal products they intend to resemble. Therefore, this work aimed to investigate the overall nutritional quality of these new products. Regulated information [Regulation (EU) 1169/2011], the presence/absence of nutrition or health claim and organic declarations, the gluten-free indication, and the number of ingredients were collected from the food labels of 269 commercial meat analogues currently sold on the Italian market. Nutritional information of reference animal meat products was used to compare the nutrition profile. As an indicator of the nutritional quality, the Nutri-Score of meat analogues and counterparts was also determined. Plant-based steaks showed significantly higher protein, lower energy, fats and salt contents, and better Nutri-Scores than the other analogues. All the meat analogues showed a higher fibre content than meat products, while plant-based burgers and meatballs had lower protein contents than meat counterparts. Ready-sliced meat analogues showed a lower salt content than cured meats. Overall, all these plant-based products showed a longer list of ingredients than animal meat products. Results from this survey highlighted that plant-based steaks, cutlets, and cured meats have some favourable nutritional aspects compared to animal-based products. However, they cannot be considered a "tout-court" alternative to meat products from a nutritional point of view.

Structural and chemical changes induced by temperature and pH hinder the digestibility of whey proteins.

In the food and feed industry, protein extraction is commonly performed under acid or basic conditions, combined with heat, in order to increase the extraction yield. Under severe processing conditions, proteins may undergo molecular modifications. Here, the effects of heating (30, 60, 90 °C) at different pH values (2, 7, 9, 11, 13) were evaluated on commercial whey proteins, used as a simplified protein model. The main structure and chemical modifications concerning protein aggregation, hydrolysis, insolubilization, amino acid degradation and racemization were investigated in detail. Using in vitro static models, the degree of protein hydrolysis and the released peptides were determined after the digestive process. Accumulation of molecular modifications was mostly observed after basic pH and high temperatures treatments, together with a marked decrease and modification of the digestibility profile. Instead, protein digestibility increased in neutral and acidic conditions in a temperature-dependent manner, even if some modification in the structure occurs.

Impact of a Shorter Brine Soaking Time on Nutrient Bioaccessibility and Peptide Formation in 30-Months-Ripened Parmigiano Reggiano Cheese.

Reducing the salt content in food is an important nutritional strategy for decreasing the risk of diet-related diseases. This strategy is particularly effective when applied to highly appreciated food having good nutritional characteristics, if it does not impact either upon sensory or nutritional properties of the final product. This work aimed at evaluating if the reduction of salt content by decreasing the brine soaking time modifies fatty acid and protein bioaccessibility and bioactive peptide formation in a 30-month-ripened Parmigiano Reggiano cheese (PRC). Hence, conventional and hyposodic PRC underwent in vitro static gastrointestinal digestion, and fatty acid and protein bioaccessibility were assessed. The release of peptide sequences during digestion was followed by LC-HRMS, and bioactive peptides were identified using a bioinformatic approach. At the end of digestion, fatty acid and protein bioaccessibility were similar in conventional and hyposodic PRC, but most of the bioactive peptides, mainly the ACE-inhibitors, were present in higher concentrations in the low-salt cheese. Considering that the sensory profiles were already evaluated as remarkably similar in conventional and hyposodic PRC, our results confirmed that shortening brine soaking time represents a promising strategy to reduce salt content in PRC.

In vitro simulated semi-dynamic gastrointestinal digestion: evaluation of the effects of processing on whey proteins digestibility and allergenicity.

The effect of thermal processing on digestibility of milk proteins should be better understood as this can greatly affect their immunoreactivity. The aim of this study was to evaluate the effects of thermal processing and lactosylation on digestibility and allergenicity, by comparing non heat-treated with industrially processed whey proteins. A semi-dynamic model was used to mimic the kinetics of digestion, and ELISA inhibition tests against human specific serum IgE were performed on the mass-spectrometry characterized products. A quicker gastric digestion of the industrially treated sample produced a lower immunogenic response in comparison with the raw sample, where intact conformational epitopes remained. In later digests, greater IgE reactivity was shown in the heat treated product, probably due to the release of linear epitopes, while at intestinal level the immunogenic response was negligible. Moreover, transepithelial transport of a reported ß-lactoglobulin-derived allergen, KIDALNENVLVL, produced during digestion was assayed. It was found that the epitope-belonging peptide could be transported through the cell monolayer, both in the native and mono-lactosylated forms, with a favored passage of the native peptide.