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1.
Food Chem ; 334: 127475, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32688176

RESUMO

Although numerous types of organisms have been used to enrich selenium, a low-cost and efficient organism is yet to be identified. This study aimed to develop a new means of selenium enrichment using Tenebrio molitor larvae. Our results indicated that the total selenium content in larvae was increased 83-fold to 54.21 ± 1.25 µg/g, and of this content, organic selenium accounted for over 97% after feeding the larvae with 20 µg/g of sodium selenite. Selenium was distributed unequally in the protein fraction with following order: alkali-soluble protein-bound selenium (36.32%) > salt-soluble protein-bound selenium (19.41%) > water-soluble protein-bound selenium (17.03%) > alcohol-soluble protein-bound selenium (3.21%). Additionally, 81% of the selenium within the soluble proteins was distributed in subunits possessing molecular weights of <40 kDa. After hydrolysis by alcalase, the protein hydrolysate of selenium-enriched larvae possessing 75% selenium recovery exhibited stronger antioxidant and immunoregulatory activities than those of regular larvae.


Assuntos
Antioxidantes/farmacologia , Fatores Imunológicos/farmacologia , Proteínas de Insetos/metabolismo , Hidrolisados de Proteína/farmacologia , Selênio/farmacocinética , Tenebrio/metabolismo , Adulto , Aminoácidos/análise , Aminoácidos/metabolismo , Animais , Antioxidantes/metabolismo , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Hidrólise , Fatores Imunológicos/metabolismo , Proteínas de Insetos/farmacologia , Larva/efeitos dos fármacos , Larva/metabolismo , Camundongos , Hidrolisados de Proteína/metabolismo , Células RAW 264.7 , Selênio/análise , Subtilisinas/química , Subtilisinas/metabolismo , Tenebrio/efeitos dos fármacos
2.
J Food Sci ; 85(6): 1772-1780, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32484970

RESUMO

The combined application of CaCl2 and Alcalase 2.4 L to the aqueous extraction process of peanuts was evaluated as a method to destabilize the oil body (OB) emulsion and improve the oil yield. After adding 5 mM CaCl2 , the oil yield was reached to 92.0% which was similar with that obtained using Alcalase 2.4 L alone, and the required enzyme loading was decreased by approximately 60 times. In addition, the demulsification mechanism during aqueous extraction process was also investigated. Particle size and zeta-potential measurements indicated that the stability of the peanut OB emulsion dramatically decreased when CaCl2 was added. Under these conditions, the demulsification of Alcalase 2.4 L performed was more efficiently. SDS-PAGE results showed that adding CaCl2 changed the subunit structure of the peanut OB interface proteins and promoted the cross-linking among the arachin Ara h3 isoforms, resulting in unstable emulsions.


Assuntos
Arachis/química , Óleo de Amendoim/análise , Óleo de Amendoim/isolamento & purificação , Subtilisinas/química , Biocatálise , Cloreto de Cálcio/química , Emulsões/química , Gotículas Lipídicas/química , Tamanho da Partícula
3.
Food Chem ; 321: 126686, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32247182

RESUMO

Impacts of 2-butanol and ß-cyclodextrin (ß-CD) at various ratios and treatment times on bitterness, physicochemical and functional properties of Alcalase salmon frame protein hydrolysate (ASF) were investigated. ASF treated with 2-butanol at a ratio of 1:4 (w/v) for 20 min (ASFB) or with ß-CD at a ratio of 1:1 (w/w) for 30 min (ASF-C-1) had lower bitterness score than ASF (p < 0.05). Bitterness score of ASF (8.45) was reduced to the lowest score (1.32) when ASFB was subsequently treated with ß-CD at a 1:1 ratio (w/w) for 30 min (ASFB-C-1). Surface hydrophobicity of all debittered samples was lower than that of ASF sample (p < 0.05). The level of aromatic amino acids-containing peptides was reduced in ASFB-C-1 as shown by gel permeation chromatography. ASFB-C-1 sample had higher overall-likeness score but lower antioxidant properties than ASF (p < 0.05). The desired antioxidant activity could be achieved via increasing the amount of protein hydrolysate without imparting undesirable taste.


Assuntos
Antioxidantes/química , Proteínas de Peixes da Dieta/química , Salmo salar , Subtilisinas/química , beta-Ciclodextrinas/química , Animais , Butanóis/química , Proteínas de Peixes da Dieta/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Salmo salar/metabolismo , Alimentos Marinhos , Subtilisinas/metabolismo , Paladar
4.
Food Chem ; 320: 126654, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32222661

RESUMO

Plastein is defined as a protease-induced peptide aggregate and has been explored for over a century. This study investigated the effects of Alcalase and papain on plastein formation in protein hydrolysates of porcine hemoglobin and meat by measuring turbidity, particle size distribution, free amino groups and chemical interactions, as well as identifying the soluble peptides remaining in solution by LC-MS/MS. The results showed that Alcalase induced more peptide aggregation than papain in terms of increases in turbidity and particle size. Porcine hemoglobin was better than meat in inducing plastein formation in a short reaction time. Besides, covalent bonds involving peptide bonds and disulfide bonds were not crucial in the plastein reaction, instead a high proportion of hydrophobic interactions dominated the plastein. Not all peptides of both hydrolysates took part in plastein formation, and the regions of sequence that were prone to aggregation were visualized by Peptigram.


Assuntos
Hemoglobinas/metabolismo , Papaína/metabolismo , Hidrolisados de Proteína/metabolismo , Subtilisinas/metabolismo , Animais , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Carne , Papaína/química , Tamanho da Partícula , Peptídeos/química , Peptídeos/metabolismo , Hidrolisados de Proteína/química , Subtilisinas/química , Suínos , Espectrometria de Massas em Tandem
5.
J Food Sci ; 85(4): 1045-1059, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32112435

RESUMO

Highland barley brewer's spent grain (BSG), being China's brewing industry's major by-product is the focus of current research. The aim of the present study was to scrutinize the effects of ultrasound and heat pretreatments on enzymatic hydrolysis of highland barley BSG protein hydrolysates (HBSGPH) and evaluate the effect of enzymatic hydrolysis time on the antioxidant activity of hydrolysates by Alcalase. Different ultrasonic waves (40 and 50 kHz) and heat pretreatment temperatures (50 and 100 °C) were chosen and the pretreatment time was 15, 30, and 60 min. The obtained results revealed that the ultrasound pretreatment of highland barley BSG protein at 40 and 50 kHz has significantly (P < 0.05) enhanced about 57 and 67% of oxygen radical absorption capacity of obtained hydrolysate over the untreated substrate. The 1,1-diphenyl-2-picrylhdrazl (DPPH) radical scavenging activity (DRSA) 28%, metal chelating activity (MCA) 54%, superoxide radical scavenging activity (SRSA) 18%, and hydroxyl radical scavenging activity (HRSA) 25% of HBSGPH at 50 kHz were also improved (P < 0.05) significantly. HBSGPH from heat treatment at 100 °C showed no SRSA and HRSA scavenging activities but improved significantly (P < 0.05) about 27% ferric reducing antioxidant power (FRAP) assay values. In the present work, the resultant HBSGPH had stronger antioxidant properties with ultrasound pretreatment at 50 kHz and the enzymatic hydrolysis after 4 hr was facilitating the enzymatic release of antioxidant peptides from HBSGPH. PRACTICAL APPLICATION: Highland barley BSG is attracting toward natural food products due to its potent natural antioxidants to overcome the risk of diseases and are beneficial for human health.


Assuntos
Antioxidantes/análise , Hordeum/química , Extratos Vegetais/análise , China , Manipulação de Alimentos , Hidrólise , Hidrolisados de Proteína/química , Subtilisinas/química , Ultrassom , Resíduos/análise
6.
J Biol Chem ; 295(7): 2068-2083, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31915245

RESUMO

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the trans-Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, EXEXXXL725, critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the EXEXXXL725 motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1.


Assuntos
Antígenos CD/genética , Citosol/metabolismo , Transporte Proteico/genética , Receptores da Transferrina/genética , Subtilisinas/genética , Fator de Transcrição AP-2/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Antígenos CD/química , Membrana Celular/genética , Movimento Celular/genética , Citosol/química , Endossomos/genética , Células HEK293 , Humanos , Receptores da Transferrina/química , Subtilisinas/química , Rede trans-Golgi/genética
7.
J Sci Food Agric ; 100(3): 1320-1327, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31742702

RESUMO

BACKGROUND: Potato protein hydrolysates (PPHs) were preparedwith Alcalase on intact potato protein isolates (PPI), with differenthydrolysis times (0.5-4 h), and functional and conformational properties of resultant hydrolysates were investigated. RESULTS: The degree of hydrolysis changed during incubation. Peptide bond cleavage increased and hydrolysis progressed rapidly. Gel electrophoresis showed that, by increasing the hydrolysis time, peptides with an apparent molecular weight below 20 kDa increased. It also revealed that, among potato protein components, patatin was more sensitive to Alcalase® hydrolysis than protease inhibitors. Enzymatic hydrolysis significantly enhanced the solubility and foam capacity of PPHs, but impaired foam stability (P < 0.05). Limited enzymatic hydrolysates (0.5PPH) at the interface improved the emulsion activity and stability index. These emulsions also had the smallest z-average and polydispersity index and showed the highest zeta potential. Fourier-transform infrared spectrometry (FTIR) analysis indicated extensive disruption of hydrogen bonds in PPHs, besides augmentation of α-helices and ß-turns, and a decline in the ß-sheets in the secondary structure of the PPHs was shown. CONCLUSION: Potato protein isolate, especially 0.5PPH, has good functional and conformational properties. Overall, our results provide new insights into the use of potato protein hydrolysates as a functional food component in the food industry. © 2019 Society of Chemical Industry.


Assuntos
Proteínas de Plantas/química , Solanum tuberosum/química , Subtilisinas/química , Biocatálise , Manipulação de Alimentos , Hidrólise , Peso Molecular , Peptídeos/química , Hidrolisados de Proteína/química , Solubilidade
8.
J Sci Food Agric ; 100(1): 50-58, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31435937

RESUMO

BACKGROUND: Protein hydrolysate powder was prepared from non-penaeid shrimp (Acetes indicus) by enzymatic hydrolysis using Alcalase enzyme. Extraction conditions such as pH (6.5, 7.5 and 8.5), enzyme to substrate ratio (1.0, 1.5 and 2.0) and temperature (40, 50 and 60 °C) were optimized against the degree of hydrolysis using response surface methodology. RESULTS: Protein hydrolysate comprised of 740 g kg-1 protein, 150 g kg-1 ash and 90 g kg-1 fat contents. The amino acid score showed superior attributes with 56% essential amino acids. Furthermore, the functional properties of spray-dried protein hydrolysates were evaluated. Protein solubility was found to be the 90.20% at pH 2 and 96.92% at pH 12. Emulsifying properties were found to vary with the concentration of protein hydrolysates and the highest emulsifying capacity (26.67%) and emulsion stability (23.33%) were found at a concentration of 20 mg mL-1 . The highest and the lowest foaming capacity were observed at pH 6 and pH 10 with a concentration of 20 mg mL-1 . The water holding capacity of protein hydrolysate was found to increase with concentration, with a value of 5.4 mL g-1 at a concentration of 20 mg mL-1 . CONCLUSION: The results of the present study indicate that the use of A. indicus for the production of protein hydrolysate has good functional properties and nutritional value, rendering it suitable for broad industrial food applications. © 2019 Society of Chemical Industry.


Assuntos
Crustáceos/química , Proteínas de Frutos do Mar/química , Aminoácidos/análise , Animais , Biocatálise , Emulsões/química , Manipulação de Alimentos , Hidrólise , Valor Nutritivo , Hidrolisados de Proteína/química , Solubilidade , Subtilisinas/química
9.
Mar Drugs ; 17(12)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801228

RESUMO

In the present manuscript, various by-products (heads, trimmings, and frames) generated from salmonids (rainbow trout and salmon) processing were evaluated as substrates for the production of fish protein hydrolysates (FPHs), potentially adequate as protein ingredients of aquaculture feeds. Initially, enzymatic conditions of hydrolysis were optimized using second order rotatable designs and multivariable statistical analysis. The optimal conditions for the Alcalase hydrolysis of heads were 0.1% (v/w) of enzyme concentration, pH 8.27, 56.2°C, ratio (Solid:Liquid = 1:1), 3 h of hydrolysis, and agitation of 200 rpm for rainbow trout and 0.2% (v/w) of enzyme, pH 8.98, 64.2 °C, 200 rpm, 3 h of hydrolysis, and S:L = 1:1 for salmon. These conditions obtained at 100 mL-reactor scale were then validated at 5L-reactor scale. The hydrolytic capacity of Alcalase and the protein quality of FPHs were excellent in terms of digestion of wastes (Vdig > 84%), high degrees of hydrolysis (Hm > 30%), high concentration of soluble protein (Prs > 48 g/L), good balance of amino acids, and almost full in vitro digestibility (Dig > 93%). Fish oils were recovered from wastes jointly with FPHs and bioactive properties of hydrolysates (antioxidant and antihypertensive) were also determined. The salmon FPHs from trimmings + frames (TF) showed the higher protein content in comparison to the rest of FPHs from salmonids. Average molecular weights of salmonid-FPHs ranged from 1.4 to 2.0 kDa and the peptide sizes distribution indicated that hydrolysates of rainbow trout heads and salmon TF led to the highest percentages of small peptides (0-500 Da).


Assuntos
Produtos Pesqueiros/análise , Oncorhynchus mykiss , Hidrolisados de Proteína/química , Salmão , Animais , Anti-Hipertensivos/isolamento & purificação , Anti-Hipertensivos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Aquicultura , Óleos de Peixe/isolamento & purificação , Hidrólise , Subtilisinas/química
10.
ACS Appl Mater Interfaces ; 11(47): 43902-43919, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31718141

RESUMO

Biofilms are prevalent in chronic wounds and once formed are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Here, we propose a novel nanotechnology to do this, based on protease-functionalized nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase 2.4 L FG, "digest" their way through the biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls, which overwhelms their defenses. We demonstrated their effectiveness against six wound biofilm-forming bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis. We confirmed a 6-fold decrease in the biofilm mass and a substantial reduction in bacterial cell density using fluorescence, atomic force, and scanning electron microscopy. Additionally, we showed that co-treatments of ciprofloxacin and Alcalase-coated Carbopol nanogels led to a 3-log reduction in viable biofilm-forming cells when compared to ciprofloxacin treatments alone. Encapsulating an equivalent concentration of ciprofloxacin into the Alcalase-coated nanogel particles boosted their antibacterial effect much further, reducing the bacterial cell viability to below detectable amounts after 6 h of treatment. The Alcalase-coated nanogel particles were noncytotoxic to human adult keratinocyte cells (HaCaT), inducing a very low apoptotic response in these cells. Overall, we demonstrated that the Alcalase-coated nanogels loaded with a cationic antibiotic elicit very strong biofilm-clearing effects against wound-associated biofilm-forming pathogenic bacteria. This nanotechnology approach has the potential to become a very powerful treatment of chronically infected wounds with biofilm-forming bacteria.


Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Biofilmes/efeitos dos fármacos , Subtilisinas/química , Infecção dos Ferimentos/tratamento farmacológico , Antibacterianos/química , Infecções Bacterianas/microbiologia , Biocatálise , Ciprofloxacino/química , Ciprofloxacino/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/fisiologia , Testes de Sensibilidade Microbiana , Nanogéis/química , Polietilenoglicóis/química , Polietilenoimina/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Infecção dos Ferimentos/microbiologia
11.
Int J Mol Sci ; 20(20)2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31635129

RESUMO

Velvet antler has a long history in traditional medicine. It is also an important healthy ingredient in food as it is rich in protein. However, there has been no report about antioxidant peptides extracted from velvet antler by enzymatic hydrolysis. Thus, the objective of this study was to hydrolyze velvet antler using different commercial proteases (Acalase, Neutrase, trypsin, pepsin, and α-chymotrypsin). Antioxidant activities of different hydrolysates were investigated using peroxyl radical scavenging assay by electron spin resonance spectrometry. Among all enzymatic hydrolysates, Alcalase hydrolysate exhibited the highest peroxyl radical scavenging activity. Alcalase hydrolysate was then purified using ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. The purified peptide was identified to be Trp-Asp-Val-Lys (tetrapeptide) with molecular weight of 547.29 Da by Q-TOF ESI mass spectroscopy. This purified peptide exhibited strong scavenging activity against peroxyl radical (IC50 value, 0.028 mg/mL). In addition, this tetrapeptide showed significant protection ability against AAPH-induced oxidative stress by inhibiting of reactive oxygen species (ROS) generation in Chang liver cells in vitro and in a zebrafish model in vivo. This research suggests that the tetrapeptide derived from Alcalase-proteolytic hydrolysate of velvet antler are excellent antioxidants and could be effectively applied as functional food ingredients and pharmaceuticals.


Assuntos
Antioxidantes/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Subtilisinas/química , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Chifres de Veado/química , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/farmacologia , Humanos , Hidrólise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
12.
J Dairy Sci ; 102(12): 10748-10759, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31548068

RESUMO

Novel bioactive peptides from camel milk protein hydrolysates (CMPH) were identified and tested for inhibition of cholesterol esterase (CEase), and their possible binding mechanisms were elucidated by molecular docking. Papain-generated CMPH showed the highest degree of hydrolysis. All CMPH produced upon enzymatic degradation demonstrated a dramatic enhancement of CEase inhibition compared with intact camel milk proteins, with papain-generated hydrolysate P9 displaying the highest inhibition. Peptide identification and their modeling through PepSite 2 revealed that among 20 potential bioactive peptides in alcalase-generated hydrolysate A9, only 3 peptides, with sequences KFQWGY, SQDWSFY, and YWYPPQ, showed the highest binding toward CEase catalytic sites. Among 43 peptides in 9-h papain-generated hydrolysate P9, 4 peptides were found to be potent CEase inhibitors. Molecular docking revealed that WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR from P9 hydrolysates were able to bind to the active site of CEase with good docking scores and molecular mechanics-generalized born surface area binding energies. Overall, this is the first study reporting CEase inhibitory potential of peptides generated from milk proteins.


Assuntos
Camelus , Inibidores Enzimáticos/isolamento & purificação , Proteínas do Leite/química , Peptídeos/química , Esterol Esterase/antagonistas & inibidores , Animais , Camelus/metabolismo , Inibidores Enzimáticos/química , Feminino , Leite/química , Simulação de Acoplamento Molecular , Papaína/química , Peptídeos/isolamento & purificação , Hidrolisados de Proteína/química , Subtilisinas/química
13.
J Oleo Sci ; 68(9): 881-891, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31413240

RESUMO

Enzyme-assisted aqueous extraction of rice germ oil (RGO) was performed in this study. The physicochemical properties, fatty acid composition, bioactive substances and antioxidant activity of RGO were analyzed. An enzyme composed of alcalase and cellulase (1:1, w/w) was found to be the most effective in the extraction yield of oil. The optimal oil yield of 22.27% was achieved under the conditions of an enzyme concentration of 2% (w/w), incubation time of 5 h, incubation temperature of 50°C, water to seed ratio of 5:1, and pH 6.0. The predominant fatty acids of RGO were oleic acid (39.60%), linoleic acid (34.20%) and palmitic acid (20.10%). The total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) composition of RGO were 22.50%, 39.60% and 36.00%, respectively. RGO yielded a high content of γ-oryzanol (530 mg/100 g oil), tocotrienol (62.96 mg/100 g oil), tocopherol (23.24 mg/100 g oil) and a significant amount of phytosterol (372.14 mg/100 g oil). It exhibited notable antioxidant activities with IC50 values of 32.37 and 41.13 mg/mL, according to the DPPH radical scavenging assay and ß-carotene/linoleic acid bleaching test, respectively.


Assuntos
Depuradores de Radicais Livres/química , Oryza/química , Óleos Vegetais/química , Sementes/química , Celulase/química , Ácidos Graxos/análise , Depuradores de Radicais Livres/análise , Depuradores de Radicais Livres/isolamento & purificação , Fitosteróis/análise , Óleos Vegetais/análise , Óleos Vegetais/isolamento & purificação , Extração em Fase Sólida/métodos , Subtilisinas/química , Tocoferóis/análise
14.
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
15.
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
16.
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
17.
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
18.
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
19.
J Biol Chem ; 294(25): 9888-9900, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31076508

RESUMO

Vibrio cholerae, the causative agent of the human diarrheal disease cholera, exports numerous enzymes that facilitate its adaptation to both intestinal and aquatic niches. These secreted enzymes can mediate nutrient acquisition, biofilm assembly, and V. cholerae interactions with its host. We recently identified a V. cholerae-secreted serine protease, IvaP, that is active in V. cholerae-infected rabbits and human choleric stool. IvaP alters the activity of several host and pathogen enzymes in the gut and, along with other secreted V. cholerae proteases, decreases binding of intelectin, an intestinal carbohydrate-binding protein, to V. cholerae in vivo IvaP bears homology to subtilisin-like enzymes, a large family of serine proteases primarily comprised of secreted endopeptidases. Following secretion, IvaP is cleaved at least three times to yield a truncated enzyme with serine hydrolase activity, yet little is known about the mechanism of extracellular maturation. Here, we show that IvaP maturation requires a series of sequential N- and C-terminal cleavage events congruent with the enzyme's mosaic protein domain structure. Using a catalytically inactive reporter protein, we determined that IvaP can be partially processed in trans, but intramolecular proteolysis is most likely required to generate the mature enzyme. Unlike many other subtilisin-like enzymes, the IvaP cleavage pattern is consistent with stepwise processing of the N-terminal propeptide, which could temporarily inhibit, and be cleaved by, the purified enzyme. Furthermore, IvaP was able to cleave purified intelectin, which inhibited intelectin binding to V. cholerae These results suggest that IvaP plays a role in modulating intelectin-V. cholerae interactions.


Assuntos
Cólera/metabolismo , Intestinos/enzimologia , Serina Endopeptidases/metabolismo , Subtilisinas/química , Vibrio cholerae/enzimologia , Animais , Cólera/microbiologia , Humanos , Intestinos/microbiologia , Coelhos , Serina Endopeptidases/genética
20.
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
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