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1.
J Sci Food Agric ; 99(15): 6731-6740, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31350863

RESUMO

BACKGROUND: Common oil-in-water plant-based emulsions are allergenic and unstable to environmental stress, leading to increased consumer concerns about the food industry. To solve the problem of safety and instability, we investigated the influence of environmental stress on the stability of emulsions containing various rice protein hydrolysates, and compared the performance to whey protein, a common food emulsifier. RESULTS: Rice protein hydrolysates were obtained by enzymatic hydrolysis with different proteases (neutrase, trypsin and alcalase). We evaluated the stability of emulsions produced with different hydrolysates according to storage, pH, ionic strength and thermal processing. Trypsin hydrolysates formed emulsion as stable as emulsion containing whey protein against a range of environmental stress containing pH (pH 6 to 7), salt (< 150 mmol L-1 NaCl) and temperature (30-90 °C). Moreover, a higher partition coefficient of protein in emulsion showed that the trypsin hydrolysates were easy to adsorb at the oil-water droplet interface, indicating its higher stability. CONCLUSION: The results obtained in the present study suggest that trypsin hydrolysates could be utilized as natural emulsifiers to stabilize emulsion instead of traditional animal-based emulsifiers, opening many opportunities with respect to hypoallergenic emulsion systems in the food industry. © 2019 Society of Chemical Industry.


Assuntos
Metaloendopeptidases/química , Oryza/química , Hidrolisados de Proteína/química , Subtilisinas/química , Tripsina/química , Biocatálise , Emulsões/química , Concentração de Íons de Hidrogênio , Concentração Osmolar
2.
Appl Biochem Biotechnol ; 189(2): 680-689, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31102127

RESUMO

Ovomucoid (OVM) is a protein found in chicken egg white. When it is hydrolyzed by a protease, subtilisin A from Bacillus licheniformis, it possesses Cu2+-chelating activity. In the present work, we demonstrate that the resulting OVM hydrolysates bind to reverse-phase chromatography media in pipette tips and can be applied to remove Cu2+ within microdroplets. 1.4 nmol of purified OVM was digested in the presence of 17 pmol of subtilisin A at 55 °C for 3 h. The OVM hydrolysates efficiently removed 2.1 and 2.4 nmol of Cu2+ in the droplets by binding to the C4 and C18 chromatography media, respectively. Conversely, 0.6 and 1.0 nmol of Cu2+ were removed by the non-digested OVM bound to the C4 and C18 media, respectively. The removal ratio of Cu2+ increased as more OVM was digested by subtilisin A. The digested OVM polypeptides were stained with Cu2+ after they were separated by non-denaturing electrophoresis. These results indicate that OVM hydrolysates bound to chromatography media in a pipette tip can be applied to remove Cu2+ within microdroplets of biological samples.


Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Cromatografia de Fase Reversa , Cobre/química , Ovomucina/química , Subtilisinas/química
3.
J Agric Food Chem ; 67(23): 6625-6632, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31117491

RESUMO

Fava bean protein isolate (FBPI) was hydrolyzed by Alcalase with different degrees of hydrolysis (DHs), and the role of hydrolysates in oil-in-water (O/W) emulsion stability was investigated. Four emulsions, DH0, DH4, DH9, and DH15, were prepared by 1% (w/v) FBPI hydrolysates with different DHs (0% as the control and 4, 9, and 15%) and 5% (w/v) purified rapeseed oil. The emulsions were monitored for physical and oxidative stability at 37 °C for 7 days. DH4 and DH0 exhibited better physical stability than DH9 and DH15, indicated by droplet size, morphology, and Turbiscan stability index. More importantly, FBPI hydrolysates with DH of 4% most effectively inhibited lipid oxidation (i.e., formation of conjugated dienes and hexanal) while maintaining protein oxidative stability compared to the native and extensively hydrolyzed FBPI. Higher DHs (9 and 15%) induced unduly decreased surface hydrophobicity and increased surface load, which might negatively affect the emulsifying activity. FBPI hydrolysates with DH of 4% had suitable molecular weight for better interfacial layer stability, increased surface net charge for more repulsive electrostatic force, and increased hydrophobicity for better adsorption at the interface and, therefore, may serve as potential natural emulsifiers to maintain both physical and oxidative stability of O/W emulsions.


Assuntos
Proteínas de Plantas/química , Óleo de Brassica napus/química , Sementes/química , Vicia faba/química , Biocatálise , Emulsões/química , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Proteínas de Plantas/isolamento & purificação , Estabilidade Proteica , Subtilisinas/química
4.
J Agric Food Chem ; 67(22): 6313-6323, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31070910

RESUMO

Gliadins are major allergens responsible for wheat allergies. Food processing is an effective strategy to reduce the allergenicity of gluten. In the present study, we determined the secondary and tertiary structures of gluten and gliadins treated by chemical, physical, and enzymatic means through FTIR, surface hydrophobicity, intrinsic fluorescence spectra, and UV absorption spectra. The results showed that the three treatments of phosphorylation and alcalase and papain hydrolyses significantly changed the conformational structures of gliadins, especially the secondary structure. Then, the potential allergenicity of the phosphorylated and alcalase and papain hydrolyzed gliadins were further characterized, and we observed a significant decrease in the allergenicity through the results of the index of spleen, serum total IgE, gliadin-specific IgE, histamine, and serum cytokine concentrations. An elevation of Th17 cells, the absence of Treg cells, and an imbalance in Treg/Th17 are associated with allergy. On the basis of the expression levels of related cytokines and key transcription factors, we also confirmed that phosphorylation and alcalase and papain hydrolysis could effectively reduce the allergenicity of gliadins by improving the imbalance of both Th1/Th2 and Treg/Th17 in the spleens of sensitized mice. This study suggested that the changes in conformational structure contribute to gliadin hyposensitization and that phosphorylation and alcalase and papain hydrolysis may be promising strategies for the production of wheat products with low allergenicity.


Assuntos
Gliadina/química , Gliadina/imunologia , Papaína/química , Subtilisinas/química , Hipersensibilidade a Trigo/imunologia , Alérgenos/química , Alérgenos/imunologia , Animais , Biocatálise , Histamina/imunologia , Humanos , Hidrólise , Imunoglobulina E/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Baço/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Triticum/química , Triticum/imunologia
5.
J Agric Food Chem ; 67(20): 5772-5781, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31046268

RESUMO

This study aimed to purify and identify antioxidant peptides from the low-molecular-weight fraction (SPH-I, MW < 3 kDa) of Alcalase-hydrolyzed soybean ( Glycine max L.) hydrolysate and further evaluate the cytoprotective effects of synthesized peptides against oxidative stress in human intestinal Caco-2 cells. After purification by gel filtration chromatography and reversed-phase HPLC, four major peptides were sequenced by nano-LC-ESI-MS/MS as VVFVDRL (847 Da, SPH-IA), VIYVVDLR (976 Da, SPH-IB), IYVVDLR (877 Da, SPH-IC), and IYVFVR (795 Da, SPH-ID). The antioxidant peptides were synthesized and displayed desirable DPPH radical-scavenging activity (from 16.5 ± 0.5 to 20.3 ± 1.0 µM Trolox equivalent (TE)/µM), ABTS•+ radical-scavenging activity (from 3.42 ± 0.2 to 4.24 ± 0.4 mM TE/µM), ORAC (from 143 ± 2.1 to 171 ± 4.8 µM TE/µM), and FRAP (from 54.7 ± 1.2 to 79.0 ± 0.6 mM Fe2+/µM). Moreover, the synthesized peptides protected Caco-2 cells against H2O2-induced oxidative damage via significantly downregulating intracellular ROS generation and lipid peroxidation ( p < 0.05). Additionally, SPH-IC and SPH-ID statistically upregulated total reduced glutathione synthesis, enhanced activities of catalase and glutathione reductase, and suppressed ROS-mediated inflammatory responses via inhibiting interleukin-8 secretion ( p < 0.05).


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Soja/química , Antioxidantes/isolamento & purificação , Biocatálise , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Hidrólise , Intestinos/citologia , Intestinos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/isolamento & purificação , Substâncias Protetoras/isolamento & purificação , Subtilisinas/química
6.
Food Chem ; 289: 568-574, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955650

RESUMO

In this work, a facile approach was developed to synthesized alcalase-inorganic hybrid nanocomposite (alcalase@CaHPO4) by immobilizing alcalase with calcium hydrogen phosphate (CaHPO4). The nanocomposite possessed flower-like morphological features with excellent hydrolysis activity on soybean protein isolates (SPI) with 1.57 fold higher compared to free alcalase. The experiment was evident of alcalase@CaHPO4 hybrid nanoflowers with 90% sustainability after the seven cycles of reusability and 80-100% relative activity at 50-70 °C and with 65% at pH 4 in acidic condition. Soybean protein hydrolysates (SPHs) produced by immobilized alcalase possessed 70% radical-scavenging capacity at 0.8 mg/mL concentration and 20% calcium-binding capacity at pH 6. The solubility of SPHs produced by alcalase@CaHPO4 hybrid nanoflowers was also improved by 15% compared to free alcalase. The high radical scavenging capability, good calcium binding capacity and improved solubility of SPHs prepared through alcalase@CaHPO4 hybrid nanoflowers would be highly promising in food industries.


Assuntos
Nanoestruturas/química , Hidrolisados de Proteína/química , Proteínas de Soja/isolamento & purificação , Subtilisinas/química , Cálcio/metabolismo , Fosfatos de Cálcio/química , Enzimas Imobilizadas/química , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Hidrolisados de Proteína/farmacologia , Solubilidade , Proteínas de Soja/química , Soja/química
7.
J Med Food ; 22(3): 286-293, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30835154

RESUMO

Mojarra of Nile tilapia (Oreochromis niloticus) skeleton was used as protein source for the preparation of protein hydrolysates and peptide fractions with angiotensin-converting enzyme (ACE) inhibitory activity. The flour presented a content of 34.92% protein and a brightness (luminosity, L*) of 82.29. Protein hydrolysates were obtained from the protein-rich flour with the enzymes Flavourzyme® and Alcalase® reaching degree of hydrolysis (%DH) of 52% and 67% at 100 min of reaction, respectively. Both hydrolysates showed low-molecular-weight (MW) peptides estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hydrolysates obtained with Flavourzyme at 60 min and at 80 min with Alcalase showed greater ACE inhibitory activity with IC50 values of 0.238 and 0.344 mg/mL, respectively. The peptide fraction A (MW >10 kDa) with Flavourzyme and fraction B (MW = 10-5 kDa) with Alcalase obtained by ultrafiltration of hydrolysates with higher DH presented IC50 of 0.728 and 0.354 mg/mL, respectively, whereas peptide fraction C (MW = 5-3 kDa) with both enzymes hydrolysates with greater ACE inhibitory activity showed IC50 values of 0.470 and 0.634 mg/mL. The components obtained in this study could be used as functional ingredients in the design and development of functional foods.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Ciclídeos , Proteínas de Peixes/química , Peptídeos/química , Animais , Biocatálise , Hidrólise , Cinética , Peptidil Dipeptidase A/química , Hidrolisados de Proteína/química , Subtilisinas/química
8.
Se Pu ; 37(3): 274-278, 2019 Mar 08.
Artigo em Chinês | MEDLINE | ID: mdl-30900855

RESUMO

In this paper, the separation and purification of nattokinase have been reviewed by primarily focusing on solvent precipitation, adsorption column chromatography, magnetic microspheres, expanded bed, reverse micelle, and three-phase separation methods. The different methods for determination of the enzyme activity have been discussed and compared. Finally, the feasibility that the nucleic acid-based identification techniques can be used for nattokinase purification and enzyme activity assay has been proposed.


Assuntos
Subtilisinas/química , Subtilisinas/isolamento & purificação , Cromatografia , Magnetismo , Micelas , Solventes
9.
Food Chem ; 285: 266-274, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797344

RESUMO

The present study demonstrated the hydrolysis of peony seed protein isolate (PSPI) by using alcalase and resulted in the generation of an anti-oxidative peptide. In brief, a model was used to illustrate the enzymolysis of PSPI with the determination of kinetic factors as per investigation information. The model proved suitable to explain the PSPI hydrolysis by alcalase. A novel anti-oxidative peptide was obtained successfully by ultrafiltration and a series of chromatography techniques. Subsequently, a purified fragment was identified with the amino acid sequence of SMRKPPG followed by its synthesis and evaluation of its anti-oxidative activities. After hydrolysis, the peony seed protein hydrolysate (PSPH) with the degree of hydrolysis of 18% displayed the most significant antioxidant action which was further used to isolate the anti-oxidative peptide.


Assuntos
Antioxidantes/farmacologia , Paeonia/química , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/farmacologia , Sementes/química , Sequência de Aminoácidos , Antioxidantes/química , Antioxidantes/isolamento & purificação , Hidrólise , Cinética , Modelos Teóricos , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Hidrolisados de Proteína/análise , Subtilisinas/química , Subtilisinas/metabolismo , Ultrafiltração
10.
J Biol Chem ; 294(13): 4806-4814, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30692196

RESUMO

Mycobacteria use type VII secretion systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to ESX-5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, mycosin protease (MycP), is not part of the core complex but is essential for secretion, as it stabilizes this membrane complex. Here we investigated which MycP domains are required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane domain are required for the ESX system-specific function of mycosins. In addition, we observed that the transmembrane domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in interaction with and stabilization of their respective ESX complexes.


Assuntos
Proteínas de Bactérias , Mycobacterium marinum , Mycobacterium tuberculosis , Subtilisinas , Sistemas de Secreção Tipo IV , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycobacterium marinum/enzimologia , Mycobacterium marinum/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
11.
Colloids Surf B Biointerfaces ; 175: 586-595, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30580149

RESUMO

Two anticoagulant proteins, nattokinase and lumbrokinase, were successfully immobilized onto the dopamine-coated polysulfone (PSf) membrane surface by covalent bonding. X-ray photoelectron spectroscopy (XPS) and Zeta potential measurement confirmed the immobilization of these anticoagulant proteins. The immobilization yield of nattokinase and lumbrokinase reached to 35.2 and 33.4 µg/cm2, respectively. The water contact angle measurement revealed that the membrane surface hydrophilicity was improved after immobilizing the anticoagulant proteins. Meanwhile, the immobilized proteins retained their biological activity. Then blood compatible tests demonstrated that the modified membranes had lower static protein adsorption, platelet/erythrocyte adhesion, hemolysis, as well as longer blood clotting time, compared to virgin PSf membrane. In addition, the nattokinase-immobilized membrane showed a higher blood compatibility than BSA and lumbrokinase immobilized memrbanes, due to its' high bioactivity. Our research demonstrates that the dopamine-assisted immobilization of nattokinase and lumbrokinase can endow the membranes with improved blood compatibility as well as high biological activity, which may be expected to apply to blood-contacting materials.


Assuntos
Anticoagulantes/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Endopeptidases/farmacologia , Enzimas Imobilizadas/farmacologia , Polímeros/farmacologia , Subtilisinas/farmacologia , Sulfonas/farmacologia , Animais , Anticoagulantes/química , Coagulação Sanguínea , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Adesão Celular , Materiais Revestidos Biocompatíveis/química , Dopamina/química , Endopeptidases/química , Enzimas Imobilizadas/química , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Membranas Artificiais , Polímeros/química , Ratos , Subtilisinas/química , Sulfonas/química
12.
Food Chem ; 274: 261-267, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30372937

RESUMO

Antioxidant casein hydrolysates were produced from buffalo casein (CB) and bovine casein (CN) using alcalase, trypsin, pepsin, or papain. The highest degree of hydrolysis (molecular weights <3.5 kDa) were observed in CN (87.28%) and CB (83.90%) using alcalase, and the next highest was using trypsin (65.84% and 63.42%, respectively). In comparison with bovine casein hydrolysates (CNH), buffalo casein hydrolysates (CBH) contained more hydrophobic amino acids when pepsin was used, followed by trypsin, alcalase and papain. Hydroxyl radical scavenging capacity, superoxide scavenging activity, oxygen radical absorbance capacity and Fe3+ reducing power for alcalase-CBH and trypsin-CNH were 81.16% and 84.55%, 66.84% and 70.30%, 2.45 and 2.23 mM BHA, and 140.73 and 136.59 µM Fe2+/mg protein, respectively. With the exception of papain hydrolysates, all hydrolysates (1 mg protein) exhibited protection against plasmid DNA nicking. Thus, alcalase-CBH and trypsin-CNH could offer beneficial antioxidant properties in functional foods and pharmaceuticals.


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Caseínas/química , Peptídeos/química , Animais , Búfalos , Bovinos , Hidrólise , Peso Molecular , Papaína/química , Papaína/metabolismo , Pepsina A/química , Pepsina A/metabolismo , Peptídeos/farmacologia , Subtilisinas/química , Subtilisinas/metabolismo , Tripsina/química , Tripsina/metabolismo
13.
Food Chem ; 270: 25-31, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30174042

RESUMO

This work was carried out to identify accurate methods that could be recommended for the quantification of proteins in food protein hydrolysates. Following hydrolysis with 4% alcalase, the amount of protein in various hydrolysate samples was measured using seven different analytical methods. Although the data obtained using different methods varied, HPLC amino acid analysis with a Pico-Tag column indicated that the highest concentration of amino acids in the protein hydrolysates was present in the casein sample while the lowest amount of protein was found in the sample of hempseed studied. However, the amino acid analysis data was mostly positively correlated with the Dumas and Lowry methods. We conclude that where available, amino acid analysis provides the best estimate of protein content of hydrolysates but the Dumas and Lowry methods can also be recommended as alternatives.


Assuntos
Análise de Alimentos/métodos , Hidrolisados de Proteína/análise , Aminoácidos , Hidrólise , Peptídeos , Subtilisinas/química
14.
Food Chem ; 277: 655-663, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30502200

RESUMO

The low solubility of wheat gluten (WG) considerably limits its application. Owing to its high hydrolytic efficiency, alcalase was the protease selected for the enzymatic hydrolysis of WG. The functional properties of WG hydrolysate prepared by alcalase (AHWG) with a hydrolysis degree (DH) of 10% were better than those with DH 5% and DH 15%. The application of AHWG was hindered by its bitterness. To mask the bitterness of AHWG, WG was respectively deamidated with acetic acid, tartaric acid, and citric acid, followed by being hydrolyzed by alcalase to DH 10%. The citric acid deamidation-alcalase hydrolysis WG hydrolysate (CDAH) exhibited the best functional properties. Partial least squares regression analysis results indicated that CDAH exhibited an enhanced bitter-masking property attributable to a high content of umami taste amino acids (glutamic acid and aspartic acid). Thus, CDAH showed the greatest potential as a modified WG product to expand the application of WG.


Assuntos
Ácido Acético/química , Proteínas de Bactérias/metabolismo , Glutens/metabolismo , Subtilisinas/metabolismo , Paladar , Triticum/metabolismo , Ácido Aspártico/química , Proteínas de Bactérias/química , Ácido Glutâmico/química , Glutens/química , Hidrólise , Análise dos Mínimos Quadrados , Solubilidade , Subtilisinas/química , Tartaratos/química
15.
J Agric Food Chem ; 66(37): 9738-9749, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30142267

RESUMO

Hydrolyzed protein-rich byproducts from food production may find a variety of applications, for example, as rich ingredients of fermentation media. We have conducted a study of the enzymatic hydrolysis of three byproducts from Norwegian food industries: chicken byproducts, mixed pork and beef byproducts, and salmon viscera. The efficiency and optimization of the enzymatic hydrolysis were evaluated using endogenous enzymes alone and in combination with commercial proteases. Hydrolysis reactions were conducted with freshly thawed raw materials using short incubation times and including an initial temperature gradient from 4 to 60 °C to both harness the power of endogenous enzymes and minimize microbial contamination. Subsequently, hydrolysates were characterized by analyzing the total recovery of protein, the peptide molecular-weight distribution, and the composition of total and free amino acids. The action of endogenous enzymes played an important role in raw-material hydrolysis, particularly when hydrolyzing salmon viscera but less so when hydrolyzing chicken byproducts. For pork-beef and chicken byproducts, the addition of Alcalase or Papain improved protein recovery, reaching levels up to 90%. Next to showing efficient hydrolysis protocols, the present data also provide a comparison of the amino acid compositions of hydrolysates derived from these three different protein-rich byproducts. Growth studies showed that the obtained protein-rich hydrolysates from meat and fish industries are a promising alternative for expensive nitrogen sources that are commonly used for fermenting yeasts.


Assuntos
Resíduos Industriais/análise , Papaína/química , Peptídeos/química , Hidrolisados de Proteína/química , Subtilisinas/química , Resíduos/análise , Animais , Biocatálise , Biotecnologia , Bovinos , Galinhas , Hidrólise , Noruega , Salmão , Suínos
16.
World J Microbiol Biotechnol ; 34(9): 135, 2018 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-30128628

RESUMO

Heterologous expression is an efficient strategy for target protein production. Dlt operon plays the important role in the D-alanylation of lipoteichoic acid, which might affect the net negative charge of cell wall for protein secretion. In this study, dlt operon was deleted to improve the target protein production, and nattokinase, α-amylase and ß-mannanase with different isoelectric points (PIs) were served as the target proteins. Firstly, our results implied that deletions of dltA, dltB, dltC and dltD improved the net negative charge of cell wall for extracellular protein secretion respectively, and among which, the dltB deficient strain DW2ΔdltB showed the best performance, its nattokinase (PI: 8.60) activity was increased by 27.50% compared with that of DW2/pP43SacCNK. Then, the dltABCD mutant strain was constructed, and the net negative charge and nattokinase activity were increased by 55.57% and 37.13% respectively, due to the deficiency of dltABCD. Moreover, it was confirmed that the activities of α-amylase (PI: 6.26) and ß-mannanase (PI: 5.75) were enhanced by 44.53% and 53.06% in the dltABCD deficient strains, respectively. Collectively, this study provided a strategy that deletion of dlt operon improves the protein secretion in B. licheniformis, and which strategy was more conducive to the target protein with lower PI.


Assuntos
Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Proteínas de Bactérias/biossíntese , Lipopolissacarídeos/metabolismo , Ácidos Teicoicos/metabolismo , Bacillus licheniformis/química , Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte , Parede Celular/metabolismo , Fermentação , Deleção de Genes , Técnicas de Inativação de Genes , Ponto Isoelétrico , Óperon/genética , Eletricidade Estática , Subtilisinas/química , Subtilisinas/genética , Tioléster Hidrolases , alfa-Amilases/química , alfa-Amilases/genética , beta-Manosidase/química , beta-Manosidase/genética
17.
J Agric Food Chem ; 66(19): 4902-4912, 2018 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-29706068

RESUMO

The aim of this work was to determine the T2R4 bitter taste receptor-blocking ability of enzymatic beef protein hydrolysates and identified peptide sequences. Beef protein was hydrolyzed with each of six commercial enzymes (alcalase, chymotrypsin, trypsin, pepsin, flavourzyme, and thermoase). Electronic tongue measurements showed that the hydrolysates had significantly ( p < 0.05) lower bitter scores than quinine. Addition of the hydrolysates to quinine led to reduced bitterness intensity of quinine with trypsin and pepsin hydrolysates being the most effective. Addition of the hydrolysates to HEK293T cells that heterologously express one of the bitter taste receptors (T2R4) showed alcalase, thermoase, pepsin, and trypsin hydrolysates as the most effective in reducing calcium mobilization. Eight peptides that were identified from the alcalase and chymotrypsin hydrolysates also suppressed quinine-dependent calcium release from T2R4 with AGDDAPRAVF and ETSARHL being the most effective. We conclude that short peptide lengths or the presence of multiple serine residues may not be desirable structural requirements for blocking quinine-dependent T2R4 activation.


Assuntos
Aromatizantes/química , Peptídeos/química , Proteínas/química , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Animais , Biocatálise , Cálcio/metabolismo , Bovinos , Quimotripsina/química , Nariz Eletrônico , Endopeptidases/química , Aromatizantes/metabolismo , Células HEK293 , Humanos , Peptídeos/metabolismo , Quinina/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Carne Vermelha/análise , Subtilisinas/química , Tripsina/química
18.
Food Chem ; 261: 301-310, 2018 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-29739598

RESUMO

In this study, the hydrolysis of a pecan protein isolate (PPI) with Alcalase was carried out to generate antioxidant peptides. We proposed a kinetic model to illustrate the enzymolysis process of PPI, which was found suitable for depiction of the kinetic behavior for PPI hydrolysis by Alcalase. The PPI hydrolysis products were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 10, 5 and 3 kDa and their antioxidant activities were evaluated in vitro. Further, the strongest antioxidant fraction (<3 kDa) and novel antioxidative peptide were successfully purified. The amino acid sequence of the purified peptide was identified as LAYLQYTDFETR. The purified fraction exhibited appreciable scavenging activities on ABTS radical (67.67%), DPPH radical (56.25%) and hydroxyl radical (47.42%) at 0.1 mg/mL. The results suggested that this novel peptide may serve as a potential antioxidant and it should be evaluated for development of functional foods and pharmaceuticals products.


Assuntos
Antioxidantes/química , Carya/química , Peptídeos/química , Subtilisinas/química , Sequência de Aminoácidos , Antioxidantes/isolamento & purificação , Biocatálise , Hidrólise , Cinética , Peso Molecular , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/química , Hidrolisados de Proteína/isolamento & purificação , Ultrafiltração
19.
Food Chem ; 262: 39-47, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29751919

RESUMO

Food-derived bioactive peptides have gained attention for their role in preventing chronic diseases. Edible insects are viable sources of bioactive peptides owing to their high protein content and sustainable production. In this study, whole crickets (Gryllodes sigillatus) were alcalase-hydrolyzed to a degree of hydrolysis (DH) ranging from 15 to 85%. Antioxidant activity, angiotensin converting enzyme (ACE), and dipeptidyl peptidase-4 (DPP-IV)- inhibition of the cricket protein hydrolysates (CPH) were evaluated before and after simulated gastrointestinal digestion (SGD). Antioxidant activity was similar among CPH, whereas ACE and DPP-IV inhibition was greater (p < 0.05) in CPH with 60-85% DH. Bioactivity improved after SGD. CPH allergenicity was evaluated using human shrimp-allergic sera. All sera positively reacted to tropomyosin in the unhydrolyzed cricket and CPH with 15-50% DH, whereas 60-85% DH showed no reactivity. In conclusion, CPH (60-85% DH) had the greatest bioactive potential and lowest reactivity to tropomyosin, compared with other CPH and the unhydrolyzed control.


Assuntos
Inibidores da Dipeptidil Peptidase IV/farmacologia , Hipersensibilidade Alimentar/imunologia , Gryllidae/química , Proteínas de Insetos/imunologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Digestão , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Humanos , Hidrólise , Soros Imunes , Proteínas de Insetos/farmacologia , Hidrolisados de Proteína/química , Hidrolisados de Proteína/imunologia , Hidrolisados de Proteína/farmacologia , Subtilisinas/química , Tropomiosina/imunologia
20.
J Sci Food Agric ; 98(14): 5225-5234, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29635691

RESUMO

BACKGROUND: Lupin seeds are rich in proteins, which are utilized in the food industry. There is an increased interest in lupin research due to its association with health-related benefits, such as reduction of hypertension and hyperglycemia. However, studies on the peptides derived from lupin proteins are rare. RESULTS: Lupin protein hydrolysates (LPHs) were prepared by proteolysis using alcalase, trypsin and pepsin, respectively. All the hydrolysates demonstrated higher antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities compared to lupin proteins. The hydrolysates were fractionated into three fractions based on molecular weight (MW), and the peptides with MW < 3 kDa (LPH3) had the highest antioxidant and ACE inhibitory activities compared to other fractions. Cell model study revealed that LPH3 fraction had the highest protection against the generation of reactive oxygen species in HepG2 cells, which was associated with increased activities of superoxide dismutase and glutathione peroxidase through upregulation of SOD1, GPX1, GCLM, SLC7A11 and SRXN1 expression. CONCLUSIONS: The analysis of amino acid composition indicated that the peptides were characterized with high content of hydrophobic amino acids, which may be responsible for the greatest antioxidant activity. This study highlights the promising potential of lupin peptides as a functional ingredient in healthy foods. © 2018 Society of Chemical Industry.


Assuntos
Lupinus/química , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas de Armazenamento de Sementes/química , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Células Hep G2 , Humanos , Hidrólise , Peso Molecular , Pepsina A/química , Peptídeos/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Proteínas de Armazenamento de Sementes/farmacologia , Sementes/química , Subtilisinas/química
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