Peptídeos com ação inibitória sobre a enzima conversora de angiotensina I (ECA) e com o potencial de reduzir a pressão arterial têm sido obtidos a partir de diferentes tipos de alimentos ou matérias-primas, sendo grande o interesse em aproveitar resíduos da indústria alimentícia como fontes desses peptídeos. Neste aspecto, as amêndoas de cupuaçu (Theobroma grandiflorum S.), que são em sua maioria descartadas pela indústria, apresentam um teor considerável de proteínas e poderiam ser aproveitadas como fontes de peptídeos inibidores da ECA. Assim, o objetivo deste trabalho foi verificar se o concentrado proteico obtido a partir de amêndoas fermentadas de cupuaçu após ser submetido à hidrólise com a enzima pepsina poderia gerar peptídeos com ação inibitória sobre a ECA in vitro. Foi observado que após a hidrólise do concentrado proteico com pepsina por 1h foi obtido um efeito de 50 % de inibição da ECA, em ensaio realizado com o substrato Abz-FRK(Dnp)-P-OH. Posteriormente, esse hidrolisado foi submetido ao fracionamento por cromatografia em fase reversa (RP-HPLC) e resultou em cinco frações (F1-F5), das quais a terceira teve uma subfração (F3.1) com quatro novos peptídeos identificados por LC-MS/ MS com potencial em inibir a ECA. Esses quatro peptídeos (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) foram sintetizados e submetidos aos ensaios para a determinação do IC50 e Ki. Os peptídeos YRLAF e LGYFK, que apresentaram mecanismo de inibição do tipo competitivo e acompetitivo, respectivamente, tiveram valores de IC50 de 4.73 e 11.11µM, e de Ki de 9.14 e 8.15 µM. Dentre os peptídeos identificados merece destaque VTTVVTGLTF que demonstrou ser um inibidor do tipo acompetitivo e apresentou as menores IC50 (0.70 µM) e Ki (2.79 µM). Em contraste, FWVAM atuou como substrato da ECA e não peptídeo inibidor. A partir dos resultados obtidos neste estudo fica demonstrado que as amêndoas fermentadas de cupuaçu podem ser fonte de peptídeos com ação inibitória da ECA, com potencial efeito anti-hipertensivo a ser, futuramente, investigado a partir de estudos in vivo. / Angiotensin converting enzyme (ACE) inhibitory peptides with the potential to reduce blood pressure have been obtained from different types of food or raw materials, and there is a great interest in utilize residues from the food industry as sources of peptides. In this regard, cupuassu almonds (Theobroma grandiflorum S.), which are mostly discarded by the industry, has a considerable protein content and could be used as source of ACE-inhibiting peptides. Thus, the objective of this work was to verify if the protein concentrate obtained from fermented almonds of cupuassu after being submitted to the hydrolysis with the enzyme pepsin could generate peptides with inhibitory activity on ACE in vitro. In this study it was observed that after hydrolysis of the protein concentrate with pepsin for 1 h, a 50% effect of ACE inhibition was obtained in an assay performed with the Abz-FRK (Dnp) -P-OH substrate. Posteriorly, the hydrolyzate was subjected to fractionation by reverse phase chromatography (RP-HPLC) and resulted in five fractions (F1-F5), of which the third had a subfraction (F3.1) with four new peptides identified by LC-MS / MS with the potential to inhibit ACE. These four peptides (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) were synthesized and assayed for IC50 and Ki. The YRLAF and LGYFK peptides, which showed a competitive and uncompetitive type inhibition mechanism respectively, presented IC50 values of 4.73 and 11.11µM, and the values for Ki were 9.14 and 8.15 µM. Among the peptides identified, it is possible to highlight VTTVVTGLTF, which was shown to be an inhibitor of the uncompetitive type and presented the lowest value for IC50 (0.70 µM) and Ki (2.79 µM). While FWVAM acted as a substrate of the ACE and not as an inhibitory peptide. From the results obtained in this study it is demonstrated that cupuassu fermented almonds can be a source of peptides with ACE inhibitory activity with potential antihypertensive effect to be further investigated from in vivo studies.

Peptídeos com ação inibitória sobre a enzima conversora de angiotensina I (ECA) e com o potencial de reduzir a pressão arterial têm sido obtidos a partir de diferentes tipos de alimentos ou matérias-primas, sendo grande o interesse em aproveitar resíduos da indústria alimentícia como fontes desses peptídeos. Neste aspecto, as amêndoas de cupuaçu (Theobroma grandiflorum S.), que são em sua maioria descartadas pela indústria, apresentam um teor considerável de proteínas e poderiam ser aproveitadas como fontes de peptídeos inibidores da ECA. Assim, o objetivo deste trabalho foi verificar se o concentrado proteico obtido a partir de amêndoas fermentadas de cupuaçu após ser submetido à hidrólise com a enzima pepsina poderia gerar peptídeos com ação inibitória sobre a ECA in vitro. Foi observado que após a hidrólise do concentrado proteico com pepsina por 1h foi obtido um efeito de 50 % de inibição da ECA, em ensaio realizado com o substrato Abz-FRK(Dnp)-P-OH. Posteriormente, esse hidrolisado foi submetido ao fracionamento por cromatografia em fase reversa (RP-HPLC) e resultou em cinco frações (F1-F5), das quais a terceira teve uma subfração (F3.1) com quatro novos peptídeos identificados por LC-MS/ MS com potencial em inibir a ECA. Esses quatro peptídeos (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) foram sintetizados e submetidos aos ensaios para a determinação do IC50 e Ki. Os peptídeos YRLAF e LGYFK, que apresentaram mecanismo de inibição do tipo competitivo e acompetitivo, respectivamente, tiveram valores de IC50 de 4.73 e 11.11µM, e de Ki de 9.14 e 8.15 µM. Dentre os peptídeos identificados merece destaque VTTVVTGLTF que demonstrou ser um inibidor do tipo acompetitivo e apresentou as menores IC50 (0.70 µM) e Ki (2.79 µM). Em contraste, FWVAM atuou como substrato da ECA e não peptídeo inibidor. A partir dos resultados obtidos neste estudo fica demonstrado que as amêndoas fermentadas de cupuaçu podem ser fonte de peptídeos com ação inibitória da ECA, com potencial efeito anti-hipertensivo a ser, futuramente, investigado a partir de estudos in vivo. / Angiotensin converting enzyme (ACE) inhibitory peptides with the potential to reduce blood pressure have been obtained from different types of food or raw materials, and there is a great interest in utilize residues from the food industry as sources of peptides. In this regard, cupuassu almonds (Theobroma grandiflorum S.), which are mostly discarded by the industry, has a considerable protein content and could be used as source of ACE-inhibiting peptides. Thus, the objective of this work was to verify if the protein concentrate obtained from fermented almonds of cupuassu after being submitted to the hydrolysis with the enzyme pepsin could generate peptides with inhibitory activity on ACE in vitro. In this study it was observed that after hydrolysis of the protein concentrate with pepsin for 1 h, a 50% effect of ACE inhibition was obtained in an assay performed with the Abz-FRK (Dnp) -P-OH substrate. Posteriorly, the hydrolyzate was subjected to fractionation by reverse phase chromatography (RP-HPLC) and resulted in five fractions (F1-F5), of which the third had a subfraction (F3.1) with four new peptides identified by LC-MS / MS with the potential to inhibit ACE. These four peptides (FWVAM, YRLAF, LGYFK, VTTVVTGLTF) were synthesized and assayed for IC50 and Ki. The YRLAF and LGYFK peptides, which showed a competitive and uncompetitive type inhibition mechanism respectively, presented IC50 values of 4.73 and 11.11µM, and the values for Ki were 9.14 and 8.15 µM. Among the peptides identified, it is possible to highlight VTTVVTGLTF, which was shown to be an inhibitor of the uncompetitive type and presented the lowest value for IC50 (0.70 µM) and Ki (2.79 µM). While FWVAM acted as a substrate of the ACE and not as an inhibitory peptide. From the results obtained in this study it is demonstrated that cupuassu fermented almonds can be a source of peptides with ACE inhibitory activity with potential antihypertensive effect to be further investigated from in vivo studies.

<p>Proizvodnja fermentisanih mliječnih napitaka unapreijeđenih funkcionalnih karakteristika postala je jedan od glavnih fokusa u industriji prerade mlijeka. Cilj doktorske disertacije je ispitivanje biolo&scaron;ke aktivnosti fermentisanih mliječnih napitaka dobijenih primjenom nekonvencionalne starter kulture, kombuhe (kultivisane na crnom čaju zaslađenim saharozom u koncentraciji od 10%) i poređenje sa karakteristikma proizvoda dobijenih primenom konvencionalnih starter kultura, jogurtne odnosno probiotske, tokom skladi&scaron;tenja. Za fermentaciju je kori&scaron;ćeno mlijeko sa 2,8% mliječne masti na temperatura 42&deg;C.<br />Promjene tokom fermentacije mlijeka primjenom kombuhe i konvencionalnih starter kultura praćene su određivanjem stepena proteolize, sadržaja laktoze, D&ndash; galaktoze, D&ndash;glukoze i D&ndash;fruktoze i masnih kiselina pri sledećim pH vrijednostima: 6,4; 6,0; 5,5; 5,0 i 4,6. Promjene antihipertenzivne aktivnosti (AKE inhibitorna aktivnost), antioksidativne aktivnosti (ABTS i DPPH metod) kao i promjene stepena proteolize, reolo&scaron;kih i senzornih karakteristika sve tri vrste fermentisanih mliječnih napitaka praćene su tokom 21-og dana skladi&scaron;tenja. Osim toga praćene su i promjene sadržaja &scaron;ećera, masnih kiselina, minerala (kalcijuma, natrijuma i kalijuma), vitamina C i biogenih amina.<br />Tokom procesa fermentacije mlijeka primjenom različitih starter kultura može se zaključiti da postoji razlika u promjenama udijela pojedinačnih proteinskih frakcija analiziranih metodom kapilarne elektroforeze.<br />Različite starter kulture utiču na različitu AKE inhibitornu aktivnost tokom skladi&scaron;tenja, &scaron;to ukazuje na različitu proteolitičku aktivnost kori&scaron;ćenih starter kultura. Utvrđeno je da AKE inhibitorna aktivnost raste tokom skladi&scaron;tenja, pri čemu uzorci proizvedeni primjenom kombuhe imaju najveću AKE inhibitornu aktivnost na kraju 14 dana skladi&scaron;tenja i ona iznosi 79,4%, dok su u jogurtu i probiotskom jogurtu te vrijednsoti 63,4 i 64,6% redom. Takođe, tokom skladi&scaron;tenja stepen proteolize raste u svim uzorcima sa značajnim međusobnim varijacijama. Antiksidativna aktivnost svih uzoraka opada tokom skladi&scaron;tenja ali je u svim uzorcima zabilježena veća aktivnost na ABTS nego na DPPH slobodne radikale. Nakon 21-og dana skladi&scaron;tenja najveći antioksidativni potencijal određen metodom stabilizacije ABTS.+ katjona imali su uzorci sa jogurtnom starter kulturom (TEAC vrijednost 8,922 mmolmg-1). U pogledu sastava masnih kiselina, tokom 14 dana skladi&scaron;tenja u kombuha fermentisanim mliječnim napicima<br />kao i napicima dobijenim sa jogurtnom i probiotskom starter kulturom dolazi do porasta udjela zasićenih (SFA) i opadanje mononezasićenih (MUFA) i polinezasićenih masnih kiselina (PUFA). Nakon 21-og dana skladi&scaron;tenja sadržaj SFA; MUFA i PUFA u kombuha fermentisanom mliječnom napitku iznosio je 65,94; 30,73 i 3,33% redom, dok su te vrijednosti kod jogurta iznosile 66,02; 30,77 i 3,21% i probiotskog jogurta 66,04; 30,66 i 3,30 % redom. Najveći sadržaj vitamina C nakon proizvodnje i 14 dana skladi&scaron;tenja imali su uzorci sa kombuha starter kulturom (0,5457 &plusmn; 0,017 mg100g-1). Uzorci dobijeni upotrebom konvencionalnih startera pokazali su bolje reolo&scaron;ke osobine pri ispitivanim uslovima tokom 21 dana skladi&scaron;tenja. Kombuha fermentisani mlečni proizvod imao je karakterističan, blago kiseli, osvežavajući ukus i nagla&scaron;enu aromu.<br />Na osnovu dobijenih rezultata biolo&scaron;ke aktivnosti i promjena kvaliteta kombuha fermentisanog mliječnog napitka tokom skladi&scaron;tenja, u odnosu na karakteristike proizvoda dobijenih upotrebom konvencionalnih starter kultura može se objasniti opravdanost upotrebe kombuha starter kulture u fermentaciji mlijeka sa ciljem dobijanja novog funkcionalnog fermentisanog mliječnog proizvoda.</p> / <p><span style="font-size:11px;">Nowadays, production of fermented dairy products with elevated benefits on human health has become one of the major focuse in dairy industry. The aim of the PhD thesis is to examine the biological activity of fermented milk products obtained using non-conventional starter culture kombucha (cultivated on black tea with 10% of sucrose) and comparision with products obtained by conventional starter cultures, probiotic/yoghurt during storage. Milk with 2.8% of milk fat was used for the samples production at temperature of 42 &deg;C.<br />The changes of components content during the milk fermentation by kombucha and conventional starter cultures were monitored at the following pH values: 6.4; 6.0; 5.5; 5.0 and 4.6., by determining the degree of proteolysis, lactose, D-galactose, D-glucose and D-fructose, fatty acids. The antihypertensive activity (ACE inhibitory activity), antioxidant activity (ABTS and DPPH tests) and the degree of proteolysis, sensory and rheological characteristics of all three types of fermented milk products were observed during 21 days of storage. Moreover, the chemical qualities of samples were monitored analyzing the contents of sugars, fatty acids, minerals (calcium, sodium and potassium), vitamin C and biogenic amines.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; There were differences in protein fractions (analyzed by capillary electrophoresis) of products obtained by using different starter cultures during the milk fermentation. Different starter cultures affect different ACE inhibitory activity during the storage, which implies different proteolytic activity of used starter cultures. It has been found that the ACE inhibitory activity was increased during the storage; wherein the samples obtained using kombucha starter culture have the highest ACE inhibitory activity at the 14th day of storage, 79,4%, while in yogurt and probiotic yoghurt it was 63.4 and 64.6% respectively. Also, the degree of proteolysis during the storage was increased in all samples with significant mutual variations. In all products, higher ABTS than 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined, while both activities slightly decreased during the storage. The antioxidant activity of all samples decreases during storage. After 21 days of storage, the highest antioxidant potential, determined by the ABTS. + method had a yoghurt samples (TEAC value of 8.922 mmolmg-1). In terms of the fatty acids composition during 14 days of storage in all type of fermented dairy products relative content of SFA (saturated fatty acids - SFA) increased, while relative contents of MUFA (monounsaturated fatty acids) and PUFA (polyunsaturated fatty acids) decreased during that period of storage. After 21 days of storage the content of SFA; MUFA and PUFA in kombucha fermented milk product was 65.94; 30.73 and 3.33% respectively. In yogurt sample their content was 66.02, 30.77 and 3.21%, while in probiotic 66.04; 30.66 and 3.30% respectively. In all fermented milk products, long chain fatty acids were dominant with a total share of about 45% in all varieties of fermented dairy products. The highest content of vitamin C after production and 14 days of storage was in samples obtained by kombucha starter culture (0.5457 &plusmn; 0.017 mg100g-1). Samples obtained by conventional starter showed better overall rheological properties at the tested conditions for 21 days of storage. Kombucha fermented milk product had a characteristic, distinctive mild sour, refreshing taste and conspicuous aroma.<br />The obtained results of biological activity and the quality of kombucha fermented milk products during storage in comparison to the same characteristics of the products obtained using conventional starter culture, could explain that kombucha is convenient starter for milk fermentation with the aim of obtaining new functional fermented milk products with pronounced bioactive characteristics and distinctive sensory and rheology properties.</span></p>