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1.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281277

RESUMO

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.


Assuntos
Vitelogeninas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Clonagem Molecular , Gema de Ovo/química , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Interleucina-10/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peso Molecular , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Vitelogeninas/genética , Vitelogeninas/farmacologia
2.
Postepy Hig Med Dosw (Online) ; 71(0): 952-959, 2017 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-29176008

RESUMO

The effect of whey protein hydrolysate (WPH) addition on growth of standard yoghurt cultures and Bifidobacterium adolescentis during co-fermentation and its viability during storage at 4ºC in yoghurts has been evaluated. WPH was obtained with the use of serine protease from Y. lipolytica yeast. Stirred probiotic yoghurts were prepared by using whole milk standardized to 16% of dry matter with the addition of either whey protein concentrate, skim milk powder (SMP), WPH-SMP (ratio 1:1), WPH. The hydrolysate increased the yoghurt culture counts at the initial stage of fermentation and significantly inhibited the decrease in population viability throughout the storage at 4ºC in comparison to the control. The post-fermentation acidification was also retarded by the addition of WPH. The hydrolysate did not increase the Bifidobacterium adolescentis counts at the initial stage. However, the WPH significantly improved its viability. After 21 days of storage, in the yogurts supplemented with WPH, the population of these bacteria oscillated around 3.04 log10 CFU/g, while in samples where SMP or whey protein concentrate was used, the bacteria were no longer detected.


Assuntos
Probióticos , Hidrolisados de Proteína , Soro do Leite , Iogurte , Animais , Bactérias/crescimento & desenvolvimento , Suplementos Nutricionais , Fermentação , Leite , Refrigeração , Proteínas do Soro do Leite
3.
Amino Acids ; 47(11): 2335-43, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26036686

RESUMO

In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 µg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.


Assuntos
Antígenos/química , Caseínas/química , Cucurbita/enzimologia , Proteínas de Plantas/química , Serina Proteases/química , Proteínas do Soro do Leite/química , Animais , Bovinos , Hidrólise , Imunoglobulina E/química , Imunoglobulina G/química
4.
Amino Acids ; 47(2): 369-80, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25408464

RESUMO

An egg yolk protein by-product following ethanol extraction of phospholipids (YP) was hydrolyzed with pepsin to produce and identify novel peptides that revealed antioxidant, ACE inhibitory and antidiabetic (α-glucosidase and DPP-IV inhibitory) activities. The peptic hydrolysate of YP was fractionated by ion-exchange chromatography and reversed-phase high-pressure liquid chromatography. Isolated peptides were identified using mass spectrometry (MALDI-ToF) and the Mascot Search Results database. Four peptides of MW ranging from 1,210.62 to 1,677.88 Da corresponded to the fragments of Apolipoprotein B (YINQMPQKSRE; YINQMPQKSREA), Vitellogenin-2 (VTGRFAGHPAAQ) and Apovitellenin-1 (YIEAVNKVSPRAGQF). These peptides were chemically synthesized and showed antioxidant, ACE inhibitory or/and antidiabetic activities. Peptide YIEAVNKVSPRAGQF exerted the strongest ACE inhibitory activity, with IC50 = 9.4 µg/mL. The peptide YINQMPQKSRE showed the strongest DPPH free radical scavenging and DPP-IV inhibitory activities and its ACE inhibitory activity (IC50) reached 10.1 µg/mL. The peptide VTGRFAGHPAAQ revealed the highest α-glucosidase inhibitory activity (IC50 = 365.4 µg/mL). A novel nutraceutical effect for peptides from an egg yolk hydrolysate was shown.


Assuntos
Proteínas do Ovo/química , Sequestradores de Radicais Livres/química , Inibidores de Glicosídeo Hidrolases/química , Peptídeos/química , Hidrolisados de Proteína/química , Animais , Galinhas , Sequestradores de Radicais Livres/isolamento & purificação , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Peptídeos/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , alfa-Glucosidases/química
5.
Foods ; 10(6)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064052

RESUMO

The aim of the research was the analysis of yoghurts enriched with blue honeysuckle berries dry polyphenolic extract and new preparation of resistant starch. The additives were introduced individually at concentration 0.1% (w/v) and in mixture at final concentration of 0.1 and 0.2% of both components. Yogurt microflora, pH, and its physicochemical and antioxidant properties were examined over 14 days of storage under refrigerated conditions. Studies showed that both substances can be successfully used in yoghurt production. Yoghurt microflora es. S. thermophilus and Lb. delbrueckii subsp. bulgaricus counts appeared to be higher in samples supplemented with these additives comparing to control yoghurt by 3-8%. More stimulating effect on their growth, especially on S. thermophilus, revealed resistant starch. Addition of this polysaccharide improved also the rheological properties of yogurts, which showed higher viscosity than samples produced without it. Addition of honeysuckle berries preparation significantly influenced the yogurts' color, giving them deep purple color, and their antioxidant potential. During storage, contents of anthocyanin and iridoid compounds were decreasing, but antioxidant activity in the products remained stable.

6.
Foods ; 9(9)2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847096

RESUMO

The aim of the study was to evaluate the effect of purified extract from Rosa spinosissima fruits on the quality characteristics and antioxidant properties of yoghurt. The extract, added to yoghurt at a concentration of 0.1% and 0.2%, contained high quantities of phenolic compounds and exhibited high antioxidant activity due to the presence of anthocyanins flavan-3-ols, flavonols and ellagitannins. Yoghurt physicochemical properties, microbiology and antioxidant properties were evaluated after 1, 7 and 14 days of storage at a temperature of 4 °C. The data revealed a positive influence of rose preparation on yoghurt's microflora and on its other properties. The highest count of traditional yoghurt microflora was observed in samples with 0.2% of extract. Its addition had a positive effect on the yoghurts' color, giving them a characteristic pink color of an intensity dependent on additive concentration. It also significantly affected the yoghurts' antioxidant properties, which were stable during storage, as well as the content of the introduced phenolic compounds.

7.
Food Chem ; 302: 125350, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415999

RESUMO

The aim of the study was to investigate the use of serine protease from Yarrowia lipolytica yeast for reduction of milk proteins allergenicity. Whey protein concentrate (WPC-80), αs-casein and their hydrolysates were analyzed for the capacity to bind IgE and IgG antibodies present in sera from patients with cow milk protein allergy using a competitive ELISA. The hydrolysis of αs-casein and whey protein concentrate contributed to a significant reduction of their immunoreactive epitopes. In case of IgE antibodies, the lowest binding capacity was detected in the 24 h hydrolysates of both proteins in which the inhibition of the reaction was ≤20 and ≤68% for αs-casein and whey protein concentrate respectively. One hour hydrolysis of WPC-80 reduced the protein antigenicity, while the longer time (5 h) might lead to the exposure of new IgE - reactive epitopes.


Assuntos
Hipersensibilidade a Leite/imunologia , Proteínas do Leite/imunologia , Hidrolisados de Proteína/imunologia , Serina Proteases/metabolismo , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Caseínas/imunologia , Caseínas/metabolismo , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Cabras/imunologia , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Proteínas do Leite/metabolismo , Hidrolisados de Proteína/metabolismo , Proteínas do Soro do Leite/imunologia , Proteínas do Soro do Leite/metabolismo , Yarrowia/enzimologia
8.
Artigo em Inglês | MEDLINE | ID: mdl-18598777

RESUMO

Four forms of chymotrypsin (Chtr1, Chtr2, Chtr3, Chtr4), one form of trypsin and one form of elastase were purified from a slightly alkaline extract of ostrich (Struthio camelus) pancreas. The zymogens in the crude extract were activated with immobilized trypsin and then separated by affinity chromatography using immobilized inhibitors and ion exchange chromatography. One of the purified forms of chymotrypsin (Chtr1) exhibited an unusual interaction with the highly selective protein trypsin inhibitor from Cucurbita maxima (CMTI). Interactions with other protein trypsin inhibitors such as basic pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (STI), trypsin inhibitors from Cyclanthera pedata (CyPTI), Cucurbita pepo (CPTI), Cucurbita pepo var. giramontia (CPGTI) and Linum usitatissimum (LUTI) were also investigated. This study demonstrated the affinity of Chtr1 to inhibitors containing Arg at P1 position. Studies of substrate specificity of Chtr1 using oxidized B-chain of insulin revealed four susceptible bonds: Tyr15-Leu16, Phe24-Phe25, Phe25-Tyr26 and, surprisingly, Arg22-Gly23. The amino acid composition, as well as the first 13 residues of the N-terminal amino acid sequence, was determined. Studies of ostrich elastase showed that it can interact with immobilized CMTI in the presence of 5 M NaCl. This unusual characteristic is reported for the first time and suggests that elastase specificity depends on ionic strength. The kinetic constants K(M), k(cat) and k(cat)/K(M) for purified ostrich trypsin, chymotrypsin 4 and elastase were also determined.


Assuntos
Quimotripsina/isolamento & purificação , Quimotripsina/metabolismo , Pâncreas/enzimologia , Struthioniformes/metabolismo , Inibidores da Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Quimotripsina/análise , Quimotripsina/química , Eletroforese , Cinética , Dados de Sequência Molecular , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/isolamento & purificação , Elastase Pancreática/metabolismo , Ligação Proteica , Especificidade por Substrato , Tripsina/isolamento & purificação , Tripsina/metabolismo
9.
Acta Biochim Pol ; 64(2): 245-253, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28388696

RESUMO

Deriving non-conventional enzymes from cheaper sources than those used for commercially available enzymes may result in the production of hydrolysates with beneficial features, while drastically reducing the cost of hydrolysis. This is especially significant for enzymatic hydrolysis as a method of protein waste utilization. We have previously described the ability of non-commercial serine protease from Yarrowia lipolytica yeast to produce/release bioactive peptides from egg white protein by-products (EP). The enzymatic hydrolysis of EP was carried out for 24 h using the serine protease at an enzyme: substrate ratio of 1:30 (w/w). The obtained hydrolysate was characterized by protein degradation of 38% and also exhibited an antioxidant and cytokine-inducing activity. The isolation procedure (ultrafiltration and RP-HPLC) of bioactive peptides from the EP hydrolysate provided peptide fractions with significant antioxidant and ACE inhibitory activities. Three homogeneous and three heterogeneous peptide fractions were identified using MALDI-TOF/MS and the Mascot Search Results database. The peptides, mainly derived from ovalbumin, were composed of 2-19 amino-acid residues. We have thus demonstrated a novel ability of serine protease from Y. lipolytica to release biopeptides from an EP by-product.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Antioxidantes/química , Proteínas do Ovo/química , Peptídeos/química , Serina Proteases/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Proteínas do Ovo/efeitos dos fármacos , Hidrólise , Proteólise/efeitos dos fármacos , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologia , Yarrowia/enzimologia
10.
Int J Food Microbiol ; 108(1): 36-41, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16380185

RESUMO

Extensive analysis of the Staphylococcus aureus genome has allowed the identification of new genes encoding enterotoxin-like superantigens (SEls). Some of these are thought to be involved in staphylococcal food poisoning, while others do not elicit any emetic effect. The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity. In this paper the distribution of the genes coding for enterotoxin-like superantigens in S. aureus isolated from food was studied. Fifty isolates of S. aureus were examined and 27 were shown to be enterotoxigenic. Only 9 of the 27 strains carried genes encoding enterotoxins SEA-SEE. In 18 SEA-SEE-negative strains the presence of newly described enterotoxin genes was detected. All SEA-SEE-positive strains simultaneously carried genes of new SEls. We show that the gene encoding SElH (staphylococcal enterotoxin-like enterotoxin H) was the most frequently detected (n=14), while genes encoding SElI together with SElG accompanied by the other genes of the egc locus were detected in three strains. We also detected the presence of three less investigated genes: sep, sel, and sek. These genes were present in eight, two, and one isolate, respectively. In one strain, sep was accompanied by genes of other SEls, while in the remaining seven it was the only enterotoxin-like gene detected. The high prevalence of newly discovered enterotoxin genes, including the genes encoding emetic toxins, was demonstrated in food-derived strains. This supports the need for additional work on its role in food poisoning and, alternatively, to monitor its presence in S. aureus isolated from food. Our results suggest that yet unknown genetic elements encoding enterotoxin genes may exist.


Assuntos
DNA Bacteriano/análise , Enterotoxinas/genética , Microbiologia de Alimentos , Intoxicação Alimentar Estafilocócica/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Sequência de Bases , Qualidade de Produtos para o Consumidor , Primers do DNA , Contaminação de Alimentos/análise , Humanos , Reação em Cadeia da Polimerase
11.
J Proteomics ; 110: 107-16, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25138009

RESUMO

In the present study angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from egg-yolk protein preparation (YP). Enzymatic hydrolysis conducted using unconventional enzyme from Cucurbita ficifolia (dose: 1000 U/mg of hydrolyzed YP (E/S (w/w)=1:7.52)) was employed to obtain protein hydrolysates. The 4-h hydrolysate exhibited a significant (IC50=482.5 µg/mL) ACE inhibitory activity. Moreover, hydrolysate showed no cytotoxic activity on human and animal cell lines which makes it a very useful multifunctional method for peptide preparation. The compiled isolation procedure (ultrafiltration, size-exclusion chromatography and RP-HPLC) of bioactive peptides from YP hydrolysate resulted in obtaining peptides with the strong ACE inhibitory activity. One homogeneous and three heterogeneous peptide fractions were identified. The peptides were composed of 9-18 amino-acid residues, including mainly arginine and leucine at the N-terminal positions. To confirm the selected bioactive peptide sequences their analogs were chemically synthesized and tested. Peptide LAPSLPGKPKPD showed the strongest ACE inhibitory activity, with IC50 value of 1.97 µmol/L. BIOLOGICAL SIGNIFICANCE: Peptides with specific biological activity can be used in pharmaceutical, cosmetic or food industries. Because of their potential role as physiological modulators, as well as theirhigh safety profile, they can be used as natural pharmacological compounds or functional food ingredients. The development of biotechnological solutions to obtain peptides with desired biological activity is already in progress. Studies in this area are focused on using unconventional highly specific enzymes and more efficient methods developed to conduct food process technologies. Natural peptides have many advantages. They are mainly toxicologically safe, have wide spectra of therapeutic actions, exhibit less side effects compared to synthetic drugs and are more efficiently absorbed in the intestinal tract. The complexity of operation of large scale technologies and high cost of purification techniques are limiting factors to the commercialization of food-derived bioactive peptides. Research on the isolation of bioactive peptides in order to reduce the processing time and costs is continuously developing. Bioactive peptides can also be released from protein by-products of the food industry, which reduce the substrate expense and production cost as well as provide the added advantage of an efficient waste disposal. Moreover, proteins as precursors of food-derived peptides are well-tolerated by the human body and therefore their application in drug development may reduce costs and duration of toxicological studies during research, development and clinical trials.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Cucurbita/enzimologia , Proteínas do Ovo/química , Peptídeo Hidrolases/química , Peptídeos/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Cucurbita/classificação , Proteínas do Ovo/isolamento & purificação , Ativação Enzimática , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Especificidade da Espécie
12.
Acta Biochim Pol ; 60(1): 117-22, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23520577

RESUMO

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (µmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.


Assuntos
Caseínas/metabolismo , Cucurbita/química , Cucurbita/enzimologia , Serina Proteases/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Hidrólise , Peptídeos/metabolismo
13.
Food Sci Nutr ; 1(2): 184-95, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24804027

RESUMO

Enzymatic hydrolysis led to improve functional properties and biological activity of protein by-products, which can be further used as protein ingredients for food and feed applications. The effects of proteolytic enzyme modification of egg-yolk protein preparation (YP) and white protein preparation (WP), obtained as the by-products left during the course of lecithin, lysozyme, and cystatin isolation on their biological and functional properties, were evaluated by treating a commercial Neutrase. The antihypertensive and antioxidative properties of YP and WP hydrolysates were evaluated based on their angiotensin-converting enzyme (ACE)-inhibitory activity and radical scavenging (DPPH) capacity, ferric reducing power, and chelating of iron activity. The functionality of obtained hydrolysates was also determined. Neutrase caused a degree of hydrolysis (DH) of YP and WP by-products: 27.6% and 20.9%, respectively. In each of them, mixture of peptides with different molecular masses were also observed. YP hydrolysate showed high levels of antioxidant activity. The scavenging capacity, ferric reducing power, and chelating capacity were observed at the level: 0.44 µmol/L Trolox mg(-1), 177.35 µg Fe(2+) mg(-1), and 549.87 µg Fe(2+) mg(-1), respectively. YP hydrolysate also exhibited significant ACE-inhibitory activity, in which the level was 59.2 µg. Protein solubility was significantly improved as the DH increased. WP hydrolysate showed high water-holding capacity of 43.2. This study indicated that YP and WP hydrolysates could be used in foods as natural antioxidants and functionality enhancers.

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