No description available.

Many bioactive peptides have been reported from various food proteins through the conventional activity-guided-purification approach; however, the rationale behind the selection of conditions for the production of the bioactive peptides has not been extensively explored. The purposes of the study were to provide the rationale behind the selection of conditions, and to develop an innovative strategy to explore the most potent peptides within egg proteins through an integrated QSAR and bioinformatics approach. Thermolysin-pepsin hydrolysate of ovotransferrin was predicted as the best condition for production of ACE-inhibitory peptides. Three predicted peptides, IRW, LKP and IQW, were successfully released from ovotransferrin. Simulated gastrointestinal incubation showed IQW was stable while IRW and LKP can be degraded into dipeptides (IR and KP respectively). Peptides produced from the study will have the potential to be developed as functional foods and nutraceuticals for the prevention of hypertension, a disease affecting ~ 31% of the adult population. / Food Science and Technology

Il formaggio sta dimostrando possedere, oltre alle caratteristiche nutrizionali classiche, degli aspetti nutrizionali innovati derivanti dalle proprietà di peptidi bioattivi contenuti nella frazione proteica caseinica del formaggio e rilasciati in seguito a proteolisi, ed ancora dalle proprietà di acidi grassi insaturi, quale l’acido linoleico coniugato. L’obiettivo di tale studio di dottorato è stato indagare su questi aspetti nutrizionali innovativi in formaggi tipici italiani quali Grana Padano e TrentinGrana, che pur essendo due formaggi molto simili tra loro, differiscono per l’uso della molecola antibatterica lisozima solo per la produzione del Grana Padano. Simulazioni in vitro della digestione gastrointestinale, nei campioni dei due formaggi a diversi tempi di stagionatura, ha dimostrato che esiste una relazione positiva tra tempo di stagionatura e digeribilità del calcio nel Grana Padano ,quando vengono considerati nella correlazione i risultati dei campioni con stagionatura superiore ai 24 mesi. A tempi di stagionatura inferiori a 24 mesi, i risultati di digeribilità del calcio del Grana Padano sono dispersi come appare nei campioni di TrentinGrana analizzati. Inoltre l’analisi RP-HPLC della distribuzione molecolare degli oligopeptidi dei campioni dei due formaggi, dimostra che la frazione peptidica coinvolta nel legame del calcio e quindi nel suo assorbimento, è quella compresa tra 1000 e 1500 D, e che l’analisi Seldi ha rilevato essere quella dei peptidi attivi fosfocaseinici. Inoltre differenze tra i due formaggi con e senza lisozima, appaiono solo per campioni tra 15 e 20 mesi di stagionatura, in cui campioni di formaggio senza lisozima appaiono più idrolizzati di quelli che lo contengono. Quindi dai risultati ottenuti appare che le differenze del profilo peptidico apportate dal lisozima non modificano le proprietà dei fosfopeptidi di assorbimento del calcio, forse influenzate da altri fattori che intervengono nella produzione del formaggio. L’attività ACE-inibitoria di abbassamento della pressione arteriosa esercitata da peptidi bioattivi è stata testata nei due formaggi, dimostrando che né il tempo di stagionatura e il grado di proteolisi, né il lisozima sono correlati all’attività ACE-inibitoria. Ed infine per l’importante ruolo che ha il minerale calcio nella dieta, indici di digeribilità del minerale in diversi alimenti sono stati considerati, in modo da poter formulare in maniera corretta diete alimentari coprendo il fabbisogno giornaliero con l’adeguato apporto energetico. I formaggi hanno dimostrato possedere indici di digeribilità del calcio superiori ad altri alimenti vegetali o a base di soia. In particolare il valore di digeribilità del Calcio del Grana Padano calcolato in vitro ha confermato il valore ottenuto in vivo , pari a 80%. / This study aimed to investigate about the not common known nutritional aspects of cheeses, which derive from their chemical components. In fact, in addition to the supply of macronutrient, cheeses are gaining interest as a source of bioactive peptides, of conjugated linoleic acid or for the new insight in the metabolic role of calcium. In vitro simulation of human gastrointestinal digestion revealed that cheeses have an higher digestibility of calcium than other foods, because of their casein-derived bioactive phosphopetides (CPPs) content has the ability to carry calcium minerals and avoid calcium precipitation, making it available for intestinal absorption. The in vitro calcium digestibility was calculated for different foods- cheeses, soya based foods and vegetables- to correct cover calcium requirements with an equilibrate energy intake. Calcium digestibility was also assessed in different ripened time Italian locally produced, semi fat, hard cheeses, Grana Padano and TrentinGrana. The main difference between them is the use or not of lysozyme during manufacturing. In Grana Padano samples, produced using lysozyme, there is a positive relationship between aging and dCa (r2 = 0.27; P<0.05) when sample > 24 months aged are considered (Grana Padano dCa results of samples less 24 months aged are quite widespread) , while in TrentinGrana, produced without the use of lysozyme, no significant correlation has been detected. RP-HPLC distribution analysis of oligopeptides molecular weight of these cheese showed that the only difference between them is that cheeses without lyzozyme, aged between 15 and 20 months, are more hydrolyzed than the same ripened time Grana Padano samples. Moreover the fraction of oligopeptides involved in calcium binding ranges between 1000 and 1500 D. SELDI analysis confirmed CPPs presence in this range. Therefore changes in cheese peptidic profiles probably caused by the use of lysozyme do not influence calcium digestibility because according to this study there is not a connection between change in peptidic profile and calcium digestibility results. The difference in calcium digestibility in Grana Padano samples aged over 24 month results should be probably ascribed also to the influence of other factors occurring during cheese manufacturing. Moreover, ACE-inhibitory activity of bioactive peptides was tested on in vitro digested Grana Padano and TrentinGrana samples with different ripening times. Correlation was not found between ACE-inhibitory activity and proteolysis level in different ripened time samples nor the lysozyme influence in releasing ACE-inhibitory bioactive peptides.

<p>  </p> <p>The ever-growing interest in novel food ingredients and their dietary influence on human health and wellbeing has driven the study of bioactive peptides (BAP). BAP are protein-derived fragments composed of (2-20 amino acids) that could positively affect bodily function and chronic diseases. This dissertation explores the health-promoting properties of a novel source of BAPs, namely canary seed, by encompassing three specific aims: 1) evaluate the <em>in vitro </em>potential of hairless canary seed peptides (CSP) as a nutraceutical ingredient, 2) develop an understanding of CSP's bioavailability and molecular interactions with its biological targets, and 3) evaluate CSP's antioxidant and antiobesity activity at the organism level using a nematode (<em>C. elegans</em>) and murine (C57BL/6J mice) model, respectively. First, CSP were generated by implementing mechanical oil extraction followed by commercial enzymatic hydrolysis with Alcalase™. In addition, CSP were also subject to simulated gastrointestinal digestion (SGD) to assess their gastric stability and <em>in vitro</em> bioavailability. The results showed that canary seed proteins were mainly composed of prolamins fractions followed by glutelins, globulins, and albumins. CSP extracts with low molecular weight (< 3 kDa and 3–10 kDa) showed the highest bioactivity. Furthermore, after SGD, CSP inhibitory activity remained stable toward angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPP-IV), and pancreatic lipase but unstable for α-glucosidase. The digested peptides were transported efficiently (>10%) through the Caco-2 monolayer, indicating a potential high absorption capacity through the intestinal epithelium. During kinetic analysis by Lineweaver-Burk plots, it was observed that CSP-SGD interacted by mixed-type inhibition for DPP-IV and α-glucosidase, non-competitive inhibition for ACE, and uncompetitive inhibitor for pancreatic lipase. Furthermore, CSP-SGD were especially potent as antihypertensive (ACE inhibitors) and antiobesity (pancreatic lipase) agents. Consequently, molecular docking and <em>in silico</em> analyses were targeted to understand CSP-SGD interactions with ACE and pancreatic lipase. CSP-SGD with ACE inhibitory activity were found to be rich in proline, glutamine, and cationic residues and could have inhibited ACE by destabilizing the tetrahedral transition state and zinc ions interaction leading to conformational changes in the enzyme structure. For peptides with antiobesity properties, it was also found that arginine, glycine, and hydrophobic amino acids from CSP-SGD hold critical interactions with the lid domain (CYS238-CYS262) of pancreatic lipase, disrupting its proper function and preventing fat hydrolysis.</p> <p>In the second part of this dissertation, the relevant health-promoting properties of CSP were further investigated by testing the effects of peptide supplementation on obesity and oxidative stress animal models. The studies showed that exposure to CSP significantly mitigated the acute and chronic oxidative damage in <em>C. elegans</em>, extending the lifespan of the nematodes by 88 and 61%, respectively. Furthermore, it was established that the CSP prevented oxidative stress by scavenging free radicals and antioxidant gene upregulation. Concerning this, CSP caused a drop in reactive oxygen species (ROS) to safe levels and induce the upregulation of the GST-4 gene encoding antioxidant enzyme Glutathione S-transferase. Concerning antiobesity properties, the daily supplementation with CSP successfully prevented metabolic implications of western diet-induced obesity in C57BL/6J mice, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Likewise, CSP promoted a drop in fatty acid uptake gene, LPL, and fatty acid biosynthesis genes FAS and ACC while unaffecting lipid oxidation genes PPAR-α and ACO in the liver. While both moderate and high CSP supplementation levels exhibit hypolipidemic effects, only moderate levels induce satiety and significantly prevent weight gain. Together, these results suggest that CSP's weight gain prevention depended on a dual mechanism involving lipid metabolism retardation to modulate satiety. Overall, the results presented in this dissertation establish the effectiveness of canary seed peptides as nutraceutical ingredients for antioxidant and antiobesity functional food applications.</p>

<p>Cardiovascular diseases (CVD) and their risk factors remain the leading cause of morbidity and mortality in North America. Food-derived bioactive peptides (BAP) have been shown to play a role in regulating physiological pathways of CVD risk factors including hypertension, diabetes, and chronic inflammation. Common sources of BAP include dairy and plant proteins. In addition to being an alternative protein source, it is now accepted that edible insect proteins can also confer health benefits beyond nutrition. However, as with any novel protein source, allergenicity remains a major concern surrounding edible insect consumption. </p> <p>This dissertation aimed to: 1) Evaluate the bioactive potential of peptides from an edible cricket species and; 2) determine the effects of BAP production methods on immunoreactivity. First, peptide-rich extracts were generated from farmed food-grade crickets via enzymatic hydrolysis techniques with the commercial protease Alcalase™. To measure the <i>in vitro</i> bioavailability, cricket peptides were also subject to simulated gastrointestinal digestion (SGD). Peptides and their digests were tested for relevant bioactivities and active groups were further fractionated by chromatographic methods to identify the major peptides responsible for the bioactivity. When tested for <i>in vitro</i> antihypertensive and anti-glycemic properties, cricket peptides were found to inhibit the activities of angiotensin converting enzyme, dipeptidyl peptidase-4, α-amylase, and α-glucosidase. The anti-inflammatory potential was expounded using RAW-264.7 macrophages and human umbilical vein endothelial cells (HUVEC). Cricket peptides (after SGD) effectively lowered NF-κB, MCP-1, and IL-6 production in cells without affecting their viability. Proteomic analyses identified 18 sequences from the enriched cationic peptide fraction that showed the highest activity. Three novel peptides were identified via molecular docking, as potent ACE-inhibitors and binding was similar to that of the commercial drug captopril. Key binding characteristics included interaction with hydrophobic amino acids (Phe, Pro, Leu) near the C-terminal position and coordination with Zn (II) ions near the ACE active site.</p> <p>Immunological reactivity was measured by IgE-binding from shrimp-allergenic patient sera to antigens present within cricket peptides. Our studies demonstrate that immunoreactivity was impacted by enzymatic hydrolysis, depending on the conditions and heating source used. Tropomyosin (a major shrimp allergen) was extracted from both untreated crickets and protein hydrolysates, and verified as the major reactive protein. Tropomyosin reactivity decreased (under both partial and extensive hydrolysis) or retained (under conditions which prevented epitope cleavage). However, using microwave-assisted enzymatic hydrolysis was effective at decreasing tropomyosin reactivity in all immunoassays tested (IgG and IgE). Proteomic and immunoinformatic analyses revealed prominent allergen binding regions of cricket tropomyosin available for cleavage during enzymatic hydrolysis. Conserved antigen regions showed greater homology with other crustacean species, but not with other well studied allergenic insect proteins (i.e., cockroach). Lastly, LC-MS/MS and FT-Raman spectrometry suggests that reactivity was affected due to distinct epitope cleavage within the protein instead of decreased antigen extractability/solubility. </p> <p>The findings of this dissertation support that edible cricket proteins are a potential source of bioactive peptides for functional food or nutraceutical development. Additionally, using protein extraction methods such as microwave-assisted hydrolysis seems a promising tool for minimizing the immunoreactivity of the allergen present in this edible cricket species.</p>

Herz-Kreislauf-Erkrankungen sind nach wie vor die häufigste Todesursache. Vor allem Bluthochdruck ist in diesem Zusammenhang ein wichtiger Risikofaktor für die Entstehung von koronaren Herzerkrankungen, Myokardinfarkten, Herzinsuffizienz und Schlaganfall. Zur Behandlung der Hypertonie werden unterschiedliche Pharmaka eingesetzt, hauptsächlich Substanzen, die das Renin-Angiotensin-Aldosteron-System (RAAS) hemmen. Dazu gehören synthetische Inhibitoren des angiotensin-converting enzyme (ACE). Für präventive Zwecke können diese ACE-Inhibitoren auf Grund mehrerer Nebenwirkungen nicht eingesetzt werden. Interessant für eine präventive Anwendung sind natürliche ACE-hemmende Peptide, welche in der Sequenz unterschiedlicher Lebensmittelproteine vorliegen und durch enzymatische Hydrolyse freigesetzt werden. Ein besonders potenter ACE-Hemmer ist das Dipeptid Isoleucin-Tryptophan (IW) und damit ein interessanter Kandidat für den Einsatz in einem funktionellen Lebensmittel. Um dies jedoch realisieren zu können, muss IW in einer ausreichenden Menge produziert werden. Durch die enzymatische Hydrolyse ist dies aktuell nicht möglich, da die Peptidsequenz IW sehr selten in Proteinen vorhanden ist. Aus diesem Grund war es Ziel der vorliegenden Arbeit eine innovative biotechnologische Methode zu etablieren, um das ACE-hemmende Dipeptid IW in höheren Mengen und vor allem lebensmittelkonform zu produzieren. Die Produktion des ACE-hemmenden Peptids wurde biotechnologisch mittels rekombinanter DNA-Technologie realisiert. Hierfür wurde eine repetitive IW-Sequenz entworfen (264 bp), welche für ein 10 kDa großes Protein codierte. Dieses IW-Konstrukt enthielt in der Sequenz 16-mal IW. Mit Hilfe von Escherichia coli (E. coli) wurde ein 52 kDa großes Fusionsprotein überexprimiert. Als Fusionstag diente das Maltose Binding Protein (MBP). Dieses rekombinante Fusionsprotein (MBP-IW) lag nach einer Kombination von zwei verschiedenen chromatographischen Verfahren gereinigt vor. Mit dieser Methode war es möglich, 0,52 mg lösliches MBP-IW pro 1 g E. coli Feuchtmasse zu produzieren. MBP-IW wurde enzymatisch mit dem Enzym α-Chymotrypsin hydrolisiert und das Dipeptid IW anschließend chromatographisch isoliert. Nach der Hydrolyse und Isolation lag die Ausbeute des rekombinant produzierten IW (rIW) mit einer Reinheit von ≥ 96 % bei 14 µg. Somit konnten 28 % des möglichen Anteils an rIW vom sauberen MBP-IW gewonnen werden. Die Identifikation von IW erfolgte mit drei unterschiedlichen Methoden, der reversed phase-high performance liquid chromatography-UV-Detektion, der liquid chromatography-electrospray ionisation-tandem mass spectrometry und durch eine N-terminale Derivatisierung des Peptids. Mit diesen Methoden wurde bestätigt, dass es sich bei dem produzierten Peptid um IW handelte. Das rIW wurde im Vergleich zum chemisch produzierten kommerziell erwerblichen L-IW (cIW) und chemisch produzierten kommerziell erwerblichen D-IW (cDIW) auf sein ACE-hemmendes Potential getestet. Um der komplexen und heterogenen Verteilung der ACE-Aktivität im menschlichen Organismus gerecht zu werden, wurde das ACE-hemmende Potential der Dipeptide an verschiedenen ACE-Quellen untersucht. Neben dem nicht-humanen ACE-System (ACE aus der Kaninchenlunge) wurde auch humanes lösliches ACE (aus humanem Plasma) sowie humanes membrangebundenes ACE (aus Human umbilical vein endothelial cells, HUVECs) verwendet. Bei allen getesteten ACE-Systemen zeigte sich kein ACE-hemmendes Potential durch cDIW. Beim Vergleich von rIW mit cIW in Bezug auf deren ACE-hemmendes Potential wurden IC50-Werte von 1,72 ± 0,12 bis 23,30 ± 3,68 µM, abhängig vom getesteten ACE-System, bestimmt. Für alle verwendeten ACE-Quellen konnte gezeigt werden, dass beide unterschiedlich produzierten Dipeptide gleich effektiv waren. Ein weiteres Ziel der Arbeit bestand darin neben einem Peptidmix aus Molkenprotein mit hohem Anteil an IW, noch zwei weitere Peptidmixe pflanzlichen Proteinursprungs hinsichtlich des ACE-hemmenden Potentials zu untersuchen. Auf Grundlage der identifizierten tryptophan- und tyrosinhaltigen Dipeptide in den Hydrolysaten des Molken-, Soja- und Reisproteins wurden drei Peptidmixe hergestellt. Auch hier wurde wieder die Wirkung auf mehrere ACE-Quellen ermittelt. Neben den oben genannten, wurde hier zusätzlich der Einfluss auf membrangebundenes ACE der Rattenaorta untersucht. In allen getesteten ACE-Systemen zeigte der Peptidmix Molke ein signifikant höheres ACE-hemmendes Potential als die Peptidmixe von Soja und Reis. Der Peptidmix Soja war von den getesteten hydrolisierten Pflanzenproteinen der potenteste ACE-Inhibitor. Die IC50-Werte der Peptidmixe lagen, je nach getestetem ACE-System, zwischen 16,60 ± 2,59 und 282,04 ± 18,51 mg/l. Der starke ACE-hemmende Effekt vom Peptidmix Molke wurde mit der hohen Konzentration an IW assoziiert (bis zu 10-fach höher verglichen mit den anderen beiden Peptidmixen). Dies legt nahe, dass das Dipeptid IW hauptverantwortlich für das ACE-hemmende Potential in den getesteten Peptidmixen ist, was nochmals das große Potential des Dipeptids verdeutlicht. Im Rahmen dieser Arbeit konnte gezeigt werden, dass IW aus Molkenprotein im Vergleich mit den bioaktiven Peptidquellen der Proteine aus Soja und Reis, die stärkste ACE-Hemmung aufweist. Des Weiteren ist es erstmals gelungen, das ACE-hemmende Dipeptid IW in hoher Reinheit biotechnologisch mit Hilfe von rekombinanten Proteinen herzustellen. Um den Einsatz als funktionelles Lebensmittel realisieren zu können, müsste im Weiteren die biotechnologische Herstellung von IW optimiert werden, um eine höhere Ausbeute zu generieren. Nach dieser Optimierung könnte in einem Scale-up Verfahren so viel an IW gewonnen werden, dass es industriell einsetzbar wäre. Die ACE-hemmende Wirkung des biotechnologisch hergestellten IWs wurde in dieser Arbeit bestätigt, sodass es in einem innovativen funktionellen Lebensmittel für die tägliche Ernährung eingesetzt werden könnte. Perspektivisch eröffnet sich damit die Möglichkeit IW präventiv zu nutzen, um die Entwicklung von Bluthochdruck und deren Folgeschädigungen zu verzögern oder zu minimieren. / Cardiovascular diseases are still the leading cause of death. Especially hypertension is an important risk factor for the development of coronary heart disease, myocardial infarction, heart failure and stroke. To treat hypertension different drugs are clinically used. This are mainly substances, which inhibit the renin-angiotensin-aldosterone-system (RAAS), such as synthetic inhibitors of angiotensin-converting enzyme (ACE). However these ACE-inhibitors cannot be used for preventive purposes because of several side effects. Therefore natural ACE-inhibitory peptides, which are mostly encrypted in food proteins and released by enzymatic hydrolysis, are of main interest for preventive applications. The dipeptide isoleucine-tryptophan (IW) is a potent ACE-inhibitor and thus an interesting ingredient in functional foods. However, to realize this, IW must be produced in sufficient amounts. This is not possible with the current enzymatic hydrolysis, because the peptide sequence of IW is very rarely present in proteins. For that reason, the aim of the present thesis was to establish an innovative biotechnological method to produce the ACE-inhibitory dipeptide IW in an enlarged amount and especially considering the food-safety. The production of the ACE-inhibitory peptide was realized biotechnologically via recombinant DNA technology. For this, a repetitive IW-sequence (264 bp) was designed, which encoded a 10 kDa protein. In this IW-construct IW was sequenced 16 times. Using Escherichia coli (E. coli) a fusion protein with a size of 52 kDa was overexpressed. The maltose binding protein (MBP) served as fusion tag. This recombinant fusion protein (MBP-IW) was purified by a combination of two different chromatographic methods. It has become possible to produce 0.52 mg of soluble MBP-IW per 1 g wet weight of E. coli. MBP-IW was enzymatically hydrolysed with the enzyme α-chymotrypsin and the dipeptide IW was subsequently isolated by chromatography. After hydrolysis and isolation, the yield of the recombinant produced IW (rIW) with a purity of ≥ 96 % was 14 μg. Thus, 28 % from the possible content of rIW was obtained from the clean MBP-IW. IW was identified by three different methods: reversed phase-high performance liquid chromatography with UV-detection, liquid chromatography-electrospray ionisation-tandem mass spectrometry and N-terminal derivatization of the peptide. These methods confirmed the produced peptide as IW. The ACE-inhibitory potential of rIW was analysed and compared to that of the chemically produced commercially available L-IW (cIW) and of the chemically produced commercially available D-IW (cDIW). To address the complex and heterogeneous distribution of ACE-activity in the human organism, the ACE-inhibitory potential of the dipeptides was investigated in different ACE-sources. Additionally to non-human ACE (from rabbit lung) also human soluble ACE (from human plasma) and human membrane-bound ACE (from human umbilical vein endothelial cells, HUVECs) were used. In all tested ACE-systems cDIW did not show any ACE-inhibitory effect. IC50 values of rIW and cIW ranged from 1.72 ± 0.12 to 23.30 ± 3.68 μM, depending on the investigated ACE-system. In all sources of ACE an equal inhibitory potency of both differently produced dipeptides were determined. The second aim of the thesis was to investigate the ACE-inhibitory effect of two peptide mixes of plant proteins beside the peptide mix of whey protein, containing a high concentration of IW. Based on the identified tryptophan- and tyrosine-containing dipeptides in the hydrolysates of the whey-, soy- and rice-protein, three peptide mixes were prepared. Also here the effect on different ACE-sources was determined. Additionally to the named above, membrane-bound ACE from rat aorta was investigated. In all analysed ACE-systems, the peptide mix of whey showed a significantly higher ACE-inhibitory potential than the peptide mixes of soy and rice. The peptide mix soy was the most potent ACE-inhibitor tested among the hydrolysed plant proteins. The IC50-values of the peptide mixes were between 16.60 ± 2.59 and 282.04 ± 18.51 mg/l, depending on the used ACE-system. The strong ACE-inhibitory effect of the whey peptide mix was associated with the high concentration of IW (10 times higher compared to the other peptide mixes). This indicates that the dipeptide IW is mainly responsible for the ACE-inhibitory potential in the investigated peptide mixes, which demonstrate again the great potential of this dipeptide. It was shown in the present study that IW from whey protein had the strongest ACE-inhibition compared to the bioactive peptides of proteins from soy and rice. Furthermore, for the first time it was possible to produce the ACE-inhibitory dipeptide IW in high purity biotechnologically using recombinant proteins. To use IW as an ingredient in functional foods, the biotechnological production of IW needs to be optimized to receive higher yields. After this optimization, it would be conceivable to increase the production of IW in a scale-up process for industrial application. The ACE-inhibitory effect of the biotechnologically produced IW was confirmed in the present study, thus it could be used in an innovative functional food for daily nutrition. Prospectively, this increases the possibility of using IW preventively in order to delay or minimize the development of hypertension and the consequentially diseases.