•Three novel ACE inhibitory peptides were isolated from P. esculenta.•Three peptides inhibit ACE in non-competitive manner.•Molecular docking was applied for the illustration of inhibitory mechanism.•Peptides YASGR and GNGSGYVSR significantly decrease the blood pressure of SHRs.Three novel angiotensin-I converting enzyme inhibitory peptides were explored from Sipuncula (Phascolosoma esculenta), a seafood with high protein content. Peptides RYDF, YASGR and GNGSGYVSR were obtained by hydrolysis of the water-soluble protein of Sipuncula with pepsin and trypsin. The peptides were purified through gel filtration and reverse-phase high-performance liquid chromatography, and identified by de novo sequencing method of MALDI-TOF. All three peptides are non-competitive inhibitors of angiotensin-I converting enzyme determined by Lineweaver-Burk plots. Their inhibitory IC50 values were 235, 184 and 29 μM, respectively. The inhibitory mechanism was well illustrated through molecular docking. The docking results showed that the differences of inhibitory activities of the three peptides were due to the degree of non-covalent bond-based interactions between the peptides and angiotensin-I converting enzyme, especially the hydrogen bonds. The antihypertensive effect of peptides was confirmed by their lowering blood pressure in spontaneously hypertensive rats with the oral administration as 5 mg/kg body weight. Peptide GNGSGYVSR decreased systolic blood pressure 31 mmHg at 2 h after oral administration, and maintained the level till 4 h. Therefore, peptides from Sipuncula can be considered as promising candidates for ACE inhibition and hypertension treatment.Download high-res image (206KB)Download full-size image

Chlorella vulgaris: is a nutritional food with high protein content. Thus, 43 protein sequences from C. vulgaris were in silico gastrointestinal digested with the aid of BIOPEP. A peptide library of 468 di- and tri-peptides was built from the produced peptides. Six peptides, AAR, VPA, VPW, IPL, IPR, and PPL, were selected for DPP-IV inhibitory assay based on their sequence feature with Pro or Ala at the second N-terminal site. VPA, VPW, IPL, and IPR had potency of DPP-IV inhibition. VPW and IPR with the same N-terminal and second N-terminal sites as those of diprotin A (IPI) and diprotin B (VPL) achieved relative inhibitory IC50 value of 6.4 and 6.9 versus IPI. These peptides were further demonstrated gastrointestinal stable in vitro and could also inhibit the DPP-IV in mouse serum. Molecular docking illustrated the inhibitory mechanism of VPW and IPR. Both VPW and IPR binding with DPP-IV through hydrogen bonds, van Edward Mars interactions, and hydrophobic interactions. However, VPW formed more stable interaction with DPP-IV. The results suggested that C. vulgaris proteins would be a good source for DPP-IV inhibitory peptides.

An α-l-arabinofuranosidase (Abf) encoding gene was obtained via genomic mining from a Ruminococcus albus strain. The specific activity of this GH 51 Abf was 73.3 U/mg at pH 6.0 and 50 °C. The modification of Abf, aimed at improving thermostability, was performed through different strategies. Structure-based rational design using the PoPMuSiC and the Enzyme Thermal Stability System (ETSS) predicted thermal stability of Abf and enhanced the half-life of thermal inactivation (t1/2) at 50 °C for K208W more than 11.1 times versus the wild-type (WT). Sequence-based rational design was also conducted by substituting histidine with lysine at various sites. Among eight mutants, the t1/2 at 50 °C of H337K was prolonged by 5.0-fold, and the specific activity of this mutant was increased to 121.8 U/mg. In addition, the mutant H337K was utilized with some enzymes to extract pectin from apple pomace. The enzymatically produced pectin got less moisture and ash, milder pH, and higher viscosity than its acid-extracted counterpart, indicating that Abf has an application prospect in pectin production.Keywords: pectin; rational design; site-directed mutagenesis; thermostability; α-l-arabinofuranosidase;

An arabinanase gene was cloned by overlap-PCR from Penicillium sp. Y702 and expressed in Pichia pastoris. The recombinant enzyme was named AbnC702 with 20 U/mg of endo-arabinanase activity toward linear α-1,5-l-arabinan. The optimal pH and temperature of AbnC702 were 5.0 and 50 °C, respectively. The recombinant AbnC702 was highly stable at pH 5.0–7.0 and 50 °C. It could retain about 72.3 % of maximum specific activity at pH 5.0 after incubation for 2.5 h, which indicated AbnC702 was an acid-adapted enzyme. The Km and Vmax values were 24.8 ± 4.7 mg/ml and 88.5 ± 5.6 U/mg, respectively. A three-dimensional structure of AbnC702 was made by homology modeling, and the counting of acidic/basic amino residues within the region of 10 Å around the active site, as well the hydrogen bonds within the area of 5 Å around the active site, might theoretically interpret the acid adaptability of AbnC702. Analysis of hydrolysis products by thin layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) verified that the recombinant AbnC702 was an endo-1,5-α-l-arabinanase, which yielded arabinobiose and arabinotriose as major products. AbnC702 was applied in pectin extraction from apple pomace with synergistic action of α-L-arabinofuranosidase.

In the process of gene mining for novel α-l-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

•Aspergillus oryzae PO showed pectin releasing enzyme activity was isolated.•Protopectinase activity increased 2.38 times after optimization.•Protopectinase from A. oryzae PO contained polygalacturonase and arabinanase.•Expressed in Pichia pastoris GS115, polygalacturonase activity was 1647 U/ml.•Characterization of the purified recombinant polygalacturonase was studied.Protopectinase is an enzyme that solubilizes protopectin forming highly polymerized soluble pectin. Protopectinase activity was detected from Aspergillus oryzae PO isolated from soil of persimmon orchard. Response surface methodology of Box–Behnken Design with three fermentation variables (temperature, NaNO3 and apple pomace concentration) was used to optimize protopectinase production of A. oryzae PO, and protopectinase activity was improved to 270.0 U/ml. Endo-polygalacturonase belonged to A-type PPase from A. oryzae PO was cloned and expressed in Pichia pastoris GS115. The endo-polygalacturonase expression was 0.418 mg/ml and the specific activity of purified recombinant endo-polygalacturonase was 7520 U/mg toward polygalacturonic acid. The optimal temperature and pH of recombinant endo-polygalacturonase were 45 °C and 5.0, respectively. The recombinant endo-polygalacturonase activity was enhanced by the presence of Mg2+, while Ca2+, Ni2+ Mn2+, Cu2+ and SDS strongly inhibited the enzyme activity. The apparent Km value and Vmax value were 5.59 mg/ml and 1.01 μmol/(min ml), respectively.

AbnZ1, with optimal pH of 6.0 and optimal temperature of 40 °C, is a cold-adapted endo-1,5-α-l-arabinanase encoded by the gene abnZ1 from Paenibacillus polymyxa Z6. The specific activity of AbnZ1 remained 54.1% of maximum at 5 °C. To apply AbnZ1 in acidic conditions, three basic hsitidine (His) residues, His48, His218, and His297, around the catalytic domain were selected as mutation sites, which were replaced with Asp, Glu, Arg, and Lys, respectively, to yield 12 mutants, H48D/E/R/K, H218D/E/R/K, and H297D/E/R/K. The optimum pH of mutant H218D shifted toward the acidic direction by 0.5 unit, and the relative activity was enhanced from 20.4 to 55.7% at pH 5.0. Furthermore, the specific activity of H218D in optimal conditions was 82.6 U/mg versus that of wild type, 73.4 U/mg, and the Km decreased from 11.9 to 7.1 mg/mL. This work provided an arabinanase candidate for juice clarification and pectin extraction.

In the present article, a flavonoid was separated and purified from celery leaf through ethanol extraction, column chromatography and crystallization. The product was identified as apiin by LC/ESI-MS, and its antioxidant activities were evaluated in vitro, including by 1,1-diphenl-2-picrylhyrazyl free radical (DPPH˙), superoxide radical (O2−˙) and hydroxyl radical (OH˙) scavenging assays. IC50 values were 68.0 μg ml−1 in the DPPH assay, 0.39 mg ml−1 in the O2−˙ assay and 48.0 μg ml−1 in the OH˙ assay. The antioxidant activities were investigated in vivo with the use of mice models. All data were measured including the contents of maleic dialdehyde (MDA) and lipofuscin (LPF), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (TAOC), in the serum, brain, heart, liver and kidney. Results showed that apiin had a remarkable scavenging activity on MDA and LPF, promoted TAOC and significantly enhanced the activities of SOD, GSH-Px and CAT.

The water-soluble protein from Phascolosoma esculenta was hydrolyzed by pepsin to obtain the hydrolysate with angiotensin I-converting enzyme (ACE) inhibitory activity. The hydrolysate (PEPH) was then further separated by membrane bioreactor system, ion-exchange chromatography, gel filtration, and reversed phase high-performance liquid chromatography (RP-HPLC) and a novel ACE inhibitory peptide named as PeP with the IC50 value of 135 M was isolated. The amino acid sequence, Ala-Trp-Leu-His-Pro-Gly-Ala-Pro-Lys-Val-Phe, was identified by matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF). Inhibitory kinetics study suggested that PeP acted as competitive inhibitor against ACE. Single oral administration of synthesized PeP at 10 mg/kg dose in spontaneously hypertensive rats could reduce the systolic blood pressure around 30 mmHg and the effect could last for more than 8 h. The results suggest that peptide from P. esculenta could be a potent natural ingredient for functional foods or pharmaceuticals against hypertension.Highlights► Pepsin hydrolysate of P. esculenta water-soluble protein had ACE inhibitory activity. ► A novel ACE inhibitory peptide (AWLHPGAPKVF) named as PeP was purified. ► PeP acted as competitive inhibitor against ACE. ► PeP could reduce SBP around 30 mmHg in spontaneously hypertensive rats. ► PeP could maintain the reduced SBP for more than 8 h.

Three sulphated polysaccharides, coded as BEMPA, BEMPB1, BEMPB2, were extracted from the mucilage of mud snail of Bullacta exarata and purified by DEAE-cellulose ion-exchange and size-exclusion chromatography. Structural analysis of purified polysaccharides by chemical and biochemical methods revealed BEMPA was a high (1→3,4)-linked mannose-containing polysaccharide with molecular weight of 22,977 Da. BEMPB1, with molecular weight of 64,117 Da, was a high (1→3)-linked arabinose-containing polysaccharide. BEMPB2 was mainly composed of (1→3,4)-linked mannose with molecular weight of 47,507 Da. The comparison between sulphated polysaccharides and their desulphated products showed that sulphate substitutions of BEMPB1 were deduced to be at the C-3 of (1→4)-linked mannose, while sulphate substitutions of BEMPA and BEMPB2 were at C-4 of (1→3)-linked mannose. Furthermore, BEMPA exhibited highest inhibitory effects on growth of B-16 melanoma cells, and IC50 were 31.1 μg/mL.Highlights► Polysaccharides from the mucilage of mud snail were studied for the first time. ► They were sulphated polysaccharides. ► Chemical composition and structure feature were remarkably distinct. ► BEMPA showed significant inhibitory effects on the growth of cancer cells.

Three sulfated polysaccharides from Bullacta exarata were isolated, purified, and named BEP1, BEP2 and BEP3, respectively. The antitumor and antioxidant activities, in vitro, of three polysaccharides were investigated, including inhibition of cells proliferation, hydroxyl radical scavenging effect, superoxide radical scavenging capacity and reducing power assay. The results suggested that three polysaccharides possessed antioxidant activities in a dose-dependent manner, and BEP3 exhibited stronger antioxidant activities than BEP1 and BEP2. Furthermore, BEP3 showed significant inhibitory effects on growth of Bcap37 breast cancer cells, SW1990 pancreatic cancer cells and HeLa cervical cancer cells, and the IC50 were 135.3, 147.5 and 172.6 μg/ml, respectively. The highest inhibition rates of BEP1 and BEP2 were approximately 10% against three cancer cells. The data obtained in vitro models indicates that polysaccharides from B. exarata could be explored as novel and potential natural antioxidants and cancer prevention agents for use in functional foods.Highlights► Three sulfated polysaccharides from Bullacta exarata have been first fractionated. ► Their molecular weight and monosaccharide compositions were studied. ► The antioxidant and antitumor activities of polysaccharides were evaluated in vitro. ► Three polysaccharides possessed antioxidant activities in a dose-dependent manner. ► One fraction BEP3 showed significant inhibitory effects on growth of cancer cells.

The oil extracted from the viscera of cuttlefish (Sepiella maindroni de Rochebruns) was studied. Fatty acid composition, cholesterol content and volatile compounds composition were analysed. The composition of fatty acids was monounsaturated fatty acids, 50%, followed by polyunsaturated fatty acids, 31%, and finally saturated fatty acids, 19%. The total cholesterol was 1.39 mg/100 g oil. Hexanal, (E,E)-2,4-heptadienal, 2-nonanone, benzothiazole, 2-methyl-4-propylthiazole, 2,3-butanediol, 1-penten-3-ol and ethyl oleate were considered as principal contributors to the distinctive odour of cuttlefish oil.

Carbonyl iron powder (CIP) has been used as a food additive or mineral supplement. However, the effects of CIP on iron deficiency anemia (IDA) and its subchronic toxicity have not been investigated. We found that oral administration of CIP at a dose of 2.96 mg/kg recovered the hemoglobin concentration of erythrocytes of IDA rats to the normal level after 8 days. The no observed adverse effect level of CIP in rats was considered to be > 200 mg/kg. The hematological and serum biochemical parameters of the rats did not differ significantly between the control and treated groups. There were no morphological changes observed in the organs including liver, kidneys, spleen, testes, stomach and intestine. Therefore, CIP might be a safe iron supplement.Download high-res image (212KB)Download full-size image

Highlights•Soy cheese spread was made from soybean without any milk solid added.•Different processing techniques were investigated to produce soy cheese spread.•Rheological, textural, microstructural and sensory properties were evaluated.•Different coagulation processes resulted in significant differences in properties.•Enzymatic hydrolysis process led to effective improvement in cheese properties.In this work, different processes including lactic acid bacteria fermentation, glucono-δ-lactone (GDL) coagulation and enzymatic hydrolysis were applied to produce soy cheese spread without any milk solid and/or alkaline metal caseinates added. All the soy cheese spread samples were studied with respect to the differences in the textural properties – utilizing TPA and rheological behavior, besides their microstructural features and sensory assessment. In addition, urea–SDS-PAGE assay was used to investigate the proteolysis in enzymatic hydrolysis process. As a result, the soy cheese spread sample produced by the combined use of GDL and lactic acid bacteria fermentation together with enzymatic hydrolysis process, was observed with better spread-ability, more acceptable sensory features, and stable homogeneous structure than other samples.