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.

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.

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.

Inhibition of DPP-IV Activity and Stimulation of GLP-1 Release by Gastrointestinally Digested Black Soldier Fly Prepupae.

The beneficial effects of an insect-based diet on human health and, in particular, the regulatory ability of digested insects' proteins on the glycaemic response in humans are topics that need to be investigated deeper. In this work, we performed an in vitro study on the modulatory activity of gastrointestinal digested black soldier fly (BSF) prepupae on the enterohormone GLP-1 and its natural inhibitor, DPP-IV. We verified whether actions intended to valorise the starting insect biomass, i.e., insect-optimised growth substrates and prior fermentation, can positively impact human health. Our results highlight that the digested BSF proteins from all the prepupae samples had a high stimulatory and inhibitory ability on the GLP-1 secretion and the DPP-IV enzyme in the human GLUTag cell line. Gastrointestinal digestion significantly improved the DPP-IV inhibitory capacity of the whole insect protein. Moreover, it was seen that optimised diets or fermentation processes preceding the digestion, in any case, did not positively affect the efficacy of the answer. BSF was already considered one of the edible insects more suitable for human consumption for its optimal nutritional profile. The BSF bioactivity here shown, after simulated digestion, on glycaemic control systems makes this species even more promising.

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.

Effectiveness of community hospital post-acute care on mortality, re-admission, institutionalization, and activation of a home care programme in Emilia-Romagna region, Italy.

BACKGROUND: In Italy, there is scant evidence on the impact of Community Hospitals (CHs) on clinical outcomes. AIMS: To assess the effectiveness of CHs versus long-term care hospital or inpatient rehabilitation facilities on mortality, re-admission, institutionalization, and activation of a home care programme in the Emilia-Romagna Region (ERR-Italy) after acute hospitalisation. METHODS: We implemented a cohort study drawing upon the ERR Administrative Healthcare Database System and including hospital episodes of ERR residents subject ≥ 65 years, discharged from a public or private hospital with a medical diagnosis to a CH or to usual care between 2017 and 2019. To control for confounding, we applied a propensity score matching. RESULTS: Patients transferred to CHs had a significantly lower risk of dying but an increased risk of being readmitted to community or acute hospital within 30/90 days from discharge. The hazard of institutionalisation within 30/90 days was significantly lower in the whole population of the CH exposed group but not among patients with cardiac or respiratory chronic diseases or diabetes. The activation of a home care program within 90 days was slightly higher for those who were transferred to a CH. DISCUSSION: The findings of our study show mixed effects on outcomes of patients transferred to CHs compared to those who followed the post-acute usual care and should be taken with cautious as could be affected by the so-called 'confounding by indication'. CONCLUSIONS: The study contributes to the intermediate care available evidence from a region with a well-established care provision through CHs.

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.

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.

Molecular composition of lipid and protein fraction of almond, beef and lesser mealworm after in vitro simulated gastrointestinal digestion and correlation with the hormone-stimulating properties of the digesta.

The current production of meat presents many disadvantages for the environment and much research focuses on alternative protein sources. Insects are novel protein sources highly valued for their nutritional and sustainable potential. However, many aspects concerning biological and nutritional properties of the insects after digestion, in comparison with other protein sources, are still overlooked. In this work, a comparative study on three different protein sources, namely almond, lean beef and insect Alphitobius diaperinus (lesser mealworm), was performed after in vitro simulated gastrointestinal digestion. An in-depth characterization of the chemical composition of the solubilized protein and lipid fractions of the digesta was performed by applying different analytical techniques, including chromatographic methods coupled to mass spectrometry and 1H NMR spectroscopy. Beef and insect were proven to be very similar in amino acid composition and protein solubilization after digestion, when considering the proper corrections for the chitin content. Lipid fraction from insects was solubilized during digestion as the one of almonds, but with a fastest kinetics. Thus, lesser mealworms are a good source of both lipids and highly nutritional proteins. Then, the amino acid composition of raw and digested protein fraction from the three sources was related to the PYY, ghrelin, GLP-1 and CCK release and rats' food intake. The composition of amino acids in insect digesta was found to be related to specific effects on enterohormone release, and the modulation of food intake in rats.

Dynamic changes in molecular composition of black soldier fly prepupae and derived biomasses with microbial fermentation.

Black soldier fly (BSF) is being increasingly used for agro-food by-products valorisation. Adult flies, puparia, and excess of prepupae are the by-products of this process, which could be further valorised. Lactic fermentation of BSF biomasses with two different strains (L. rhamnosus and L. plantarum) has been used for this purpose. Deep changes in the molecular composition were observed, without significant differences related to the different strains used. The lipid and protein fractions were the most impacted. Fermentation enriched the biomass in monounsaturated and polyunsaturated fatty acids and essential amino acids, significantly improving the nutritional properties of the substrates. Although not particularly marked, a proteolytic activity of lactobacilli was observed on the BSF muscular and cuticular proteins, especially in the samples of adult flies and puparia, where fermentation resulted more effective. Conversely, there was no evidence of chitinolytic activity.

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.

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.

General and COVID-19-Related Mortality by Pre-Existing Chronic Conditions and Care Setting during 2020 in Emilia-Romagna Region, Italy.

In 2020, the number of deaths increased in Italy, mainly because of the COVID-19 pandemic; mortality was among the highest in Europe, with a clear heterogeneity among regions and socio-demographic strata. The present work aims to describe trends in mortality and to quantify excess mortality variability over time and in relation to demographics, pre-existent chronic conditions and care setting of the Emilia-Romagna region (Northern Italy). This is a registry-based cross-sectional study comparing the 2020 observed mortality with figures of the previous five years by age, sex, month, place of death, and chronicity. It includes 300,094 deaths in those 18 years of age and above resident in the Emilia-Romagna region. Excess deaths were higher during the first pandemic wave, particularly among men and in March. Age-adjusted risk was similar among both men and women (Mortality Rate Ratio 1.15; IC95% 1.14-1.16). It was higher among females aged 75+ years and varied between sub-periods. Excluding COVID-19 related deaths, differences in the risk of dying estimates tended to disappear. Metabolic and neuropsychiatric diseases were more prevalent among those that deceased in 2020 compared to the deaths that occurred in 2015-2019 and therefore can be confirmed as elements of increased frailty, such as being in long-term care facilities or private homes as the place of death. Understanding the impact of the pandemic on mortality considering frailties is relevant in a changing scenario.

Extraction and Chemical Characterization of Functional Phenols and Proteins from Coffee (Coffea arabica) By-Products.

Not all the coffee produced goes to the roasting stage, because non-compliant green coffee beans are usually discarded by roasters and the silverskin of the coffee is usually removed and discarded. In the present work, non-compliant green coffee beans and coffee silverskins were fully characterized from a chemical point of view. In addition, enzyme-assisted extraction was applied to recover a fraction rich in proteins and polyphenols, tested for antimicrobial, antityrosinase, and antioxidant activities. Non-compliant green coffee beans showed higher amounts of polyphenols, flavanols, flavonoids, and caffeine than coffee silverskins (which were richer in tannins). The enzymatic extraction of non-compliant coffee green beans produced extracts with a good protein content and with a consistent quantity of polyphenols. The extract showed antioxidant, antityrosinase, and antimicrobial activity, thus representing a promising strategy to recover defective green coffee beans. The antioxidant and antimicrobial activity of coffee silver skins is lower than that of non-compliant coffee green beans extracts, while the antityrosinase activity is comparable.

Targeting the Nutritional Value of Proteins From Legumes By-Products Through Mild Extraction Technologies.

Legumes have been known for centuries for their good nutritional properties. Unfortunately, during processing, from 5 to 25% of this production is wasted, generating by-products that can still be a rich source of useful compounds, such as proteins, which can still be used in food and feed formulations. The choice of the extraction technique is important to preserve the nutritional value of proteins since drastic conditions of pH and/or temperature could damage them. In this work, two mild extraction techniques (direct assisted extraction-DAE and enzymatic assisted extraction-EAE) were applied for protein extraction from legume by-products obtained from agro-industrial processes. The quality of proteins was evaluated considering protein integrity [SDS-PAGE, degree of hydrolysis (DH), free amino acid content, racemization degree] and nutritional features [amino acid score (AAS), digestibility]. Direct assisted extraction is the technique that has best preserved protein integrity (1-5% DH and free amino acid content <1%), The digestibility of proteins extracted with EAE is higher (no protein bands detected in SDS-PAGE) than with the one of DAE extracts, making this technique particularly suitable for those food and feed formulation were a high digestibility of proteins is required.

Effect of the Rearing Substrate on Total Protein and Amino Acid Composition in Black Soldier Fly.

Insects are becoming increasingly relevant as protein sources in food and feed. The Black Soldier Fly (BSF) is one of the most utilized, thanks to its ability to live on many leftovers. Vegetable processing industries produce huge amounts of by-products, and it is important to efficiently rear BSF on different substrates to assure an economical advantage in bioconversion and to overcome the seasonality of some leftovers. This work evaluated how different substrates affect the protein and amino acid content of BSF. BSF prepupae reared on different substrates showed total protein content varying between 35% and 49% on dry matter. Significant lower protein contents were detected in BSF grown on fruit by-products, while higher contents were observed when autumnal leftovers were employed. BSF protein content was mainly correlated to fibre and protein content in the diet. Among amino acids, lysine, valine and leucine were most affected by the diet. Essential amino acids satisfied the Food and Agricultural Organization (FAO) requirements for human nutrition, except for lysine in few cases. BSF could be a flexible tool to bio-convert a wide range of vegetable by-products of different seasonality in a high-quality protein-rich biomass, even if significant differences in the protein fraction were observed according to the rearing substrate.

Reduction in the Brining Time in Parmigiano Reggiano Cheese Production Minimally Affects Proteolysis, with No Effect on Sensory Properties.

Brine soaking is one of the most important steps in the production of Parmigiano Reggiano cheese, since it determines the amount of salt in the final product. Reduction in salt in Parmigiano Reggiano cheese might be important for improving its nutritional profile, but it could affect the manufacturing processes by altering proteolysis and consequently the product quality. In this study, for the first time, salt reduction was explored at the industrial level on real cheese samples manufactured in a local dairy. In particular, 20 wheels were produced with conventional (18 days, 10 wheels) and shorter (12 days, 10 wheels) brining steps. In every group, wheels were studied at two different ripening times, 15 and 30 months. A shorter brining time resulted in an average 12% decrease in salt content. A full characterization of free amino acids and peptides was performed by LC-MS on all samples. Free amino acids and peptides, as expected, increased with ripening, due to proteolysis, with samples having low salt content showing a slightly faster increase when compared to standard ones, hinting to a slightly accelerated proteolytic process. Nonetheless, low-salt and conventional cheeses shared similar sensory profiles at both ripening times.