Glycosylated peptide-calcium chelate: Characterization, calcium absorption promotion and prebiotic effect.

Finding functional preparations that could improve the bioavailability of calcium is one of the keys to solving calcium deficiency. In this study, glycosylated peptides-calcium chelate with calcium absorption promoting activity, named XOS-CSPHs-Ca-MR, was prepared from Crimson Sapper scales protein hydrolysates (CSPHs) and xylooligosaccharides (XOS) via Maillard reaction. Results showed that amino nitrogen, carboxyl oxygen, and carbonyl oxygen atom were the primary calcium chelating sites. Remarkably, XOS-CSPHs-Ca-MR exhibited good calcium phosphate crystallization inhibitory activity, gastrointestinal stability, and could promote calcium transport efficiency in the Caco-2 cell monolayer. In vitro fermentation results showed that XOS-CSPHs-Ca-MR improved the gut microbiota structure of calcium-deficient mice. Its prebiotic effect was achieved by increasing the number of beneficial bacteria, boosting the production of short-chain fatty acids, and improving the colonization ability of microbiota. Therefore, this study could lay a foundation for the study of glycosylated peptide-calcium chelate as a novel calcium supplement with prebiotic effect.

Unveiling metabolic characteristics of an uncultured Gammaproteobacterium responsible for in situ PAH biodegradation in petroleum polluted soil.

Exploring the metabolic characteristics of indigenous PAH degraders is critical to understanding the PAH bioremediation mechanism in the natural environment. While stable-isotopic probing (SIP) is a viable method to identify functional microorganisms in complex environments, the metabolic characteristics of uncultured degraders are still elusive. Here, we investigated the naphthalene (NAP) biodegradation of petroleum polluted soils by combining SIP, amplicon sequencing and metagenome binning. Based on the SIP and amplicon sequencing results, an uncultured Gammaproteobacterium sp. was identified as the key NAP degrader. Additionally, the assembled genome of this uncultured degrader was successfully obtained from the 13 C-DNA metagenomes by matching its 16S rRNA gene with the SIP identified OTU sequence. Meanwhile, a number of NAP degrading genes encoding naphthalene/PAH dioxygenases were identified in this genome, further confirming the direct involvement of this indigenous degrader in the NAP degradation. The degrader contained genes related to the metabolisms of several carbon sources, energy substances and vitamins, illuminating potential reasons for why microorganisms cannot be cultivated and finally realize their cultivation. Our findings provide novel information on the mechanisms of in situ PAH biodegradation and add to our current knowledge on the cultivation of non-culturable microorganisms by combining both SIP and metagenome binning.

The catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation in oil-contaminated soil.

Rhizoremediation is a potential technique for polycyclic aromatic hydrocarbon (PAH) remediation; however, the catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation remain unclear. To address these issues, stable-isotope-probing coupled with metagenomics and molecular ecological network analyses were first used to investigate the phenanthrene rhizoremediation by three different prairie grasses in this study. All rhizospheres exhibited a significant increase in phenanthrene removal and markedly modified the diversity of phenanthrene degraders by increasing their populations and interactions with other microbes. Of all the active phenanthrene degraders, Marinobacter and Enterobacteriaceae dominated in the bare and switchgrass rhizosphere respectively; Achromobacter was markedly enriched in ryegrass and tall fescue rhizospheres. Metagenomes of 13 C-DNA illustrated several complete pathways of phenanthrene degradation for each rhizosphere, which clearly explained their unique rhizoremediation mechanisms. Additionally, propanoate and inositol phosphate of carbohydrates were identified as the dominant factors that drove PAH rhizoremediation by strengthening the ecological networks of soil microbial communities. This was verified by the results of rhizospheric and non-rhizospheric treatments supplemented with these two substances, further confirming their key roles in PAH removal and in situ PAH rhizoremediation. Our study offers novel insights into the mechanisms of in situ rhizoremediation at PAH-contaminated sites.

Radix Pseudostellariae protein-curcumin nanocomplex: Improvement on the stability, cellular uptake and antioxidant activity of curcumin.

Radix Pseudostellariae protein (RPP) with satisfactory antioxidant activity and self-assembled ability was extracted from dried Radix Pseudostellariae. In this study, RPP-curcumin nanocomplex (RPP-Cur) was fabricated, and its improvement on the stability, cellular uptake and antioxidant activity of curcumin was investigated. RPP-Cur with homogeneously spherical structure exhibited good stability, which could maintain the morphology against simulated gastrointestinal digestion and up to 300 mM ionic concentration. After RPP nanoparticles encapsulation, the retention of curcumin increased 1.45 times under UV irradiation for 6 h. Besides, RPP-Cur exhibited additive reducing power of curcumin and RPP. The transport efficiency of hydrophobic curcumin across Caco-2 cells monolayer was greatly improved by RPP nanoparticle by 3.7 folds. RPP-Cur was able to be internalized by Caco-2 cells dose-dependently via macropinocytosis and clathrin-mediated endocytosis. The cellular uptake efficiency of embedded curcumin in RPP nanoparticles by Caco-2 cells was significantly higher than that of free curcumin, which might contribute to the enhanced intracellular antioxidant activity of RPP-Cur. These findings suggest that the proteins from Radix Pseudostellariae have potential to be developed into novel delivery system with intrinsic antioxidant activity for the hydrophobic active molecules in healthy food field.

Protective effects of crimson snapper scales peptides against oxidative stress on Drosophila melanogaster and the action mechanism.

Peptides derived from crimson snapper scales (CSSPs) were reported to possess excellent free radical scavenging activities in vitro. In present study, the anti-aging and anti-oxidative stress effects of CSSPs were evaluated in Drosophila melanogaster models. Results showed that the addition of CSSPs in the diets of normal Drosophila could effectively extend their lifespan and improve the motor ability of aged Drosophila. Moreover, CSSPs could protect Drosophila from oxidative damage induced by H2O2, paraquat and UV irradiation. The extension of lifespan was found to be associated with the effects of CSSPs in improving the antioxidant defense system of Drosophila, manifesting as the reduction of oxidation products MDA and PCO, the elevated activities of T-SOD, CAT and GSH-Px, and the upregulated expression of antioxidant related genes after CSSPs supplemented. Furthermore, CSSPs at 6 mg/mL significantly downregulated mTOR signaling pathway and activated autophagy in aged male Drosophila, and the inhibition on mTOR activation was probably mediated by the antioxidant effects of CSSPs. Our findings suggest that CSSPs have the potential in making dietary supplements against natural aging and oxidative stress in organisms.

pH sensitive doxorubicin-loaded nanoparticle based on Radix pseudostellariae protein-polysaccharide conjugate and its improvement on HepG2 cellular uptake of doxorubicin.

Nanoparticles based on Radix pseudostellariae protein-polysaccharide conjugates were self-assembled via pH adjustment and thermal treatment. The fabricated nanoparticles (CP3) were spherical with narrow size distribution of 125.0 nm in diameter. The doxorubicin (DOX) -loaded CP3 nanoparticles exhibited pH-sensitive release behavior and accelerated the release of DOX under the acidic pH simulating tumor microenvironment and endosomal pH. In HepG2 uptake studies, CP3-DOX nanoparticles notably improved the internalization of DOX, which was 1.56-fold compared with free DOX. CP3-DOX nanoparticles could serve as P-glycoprotein efflux pump inhibitor and be internalized into HepG2 cells via clathrin-dependent endocytosis. Moreover, the cytotoxicity effect of DOX on HepG2 cells was elevated after the encapsulation by CP3, with a lower IC50 value of 0.25 µg/mL. The findings suggested that the pH-sensitive CP3-DOX nanoparticles has a great potential in facilitating the efficacy of DOX in cancer cells, and the obtained CP3 could be a good candidate as nanocarrier for the encapsulation and delivery of functional compounds.

Investigation on activation in RAW264.7 macrophage cells and protection in cyclophosphamide-treated mice of Pseudostellaria heterophylla protein hydrolysate.

Our previous study has demonstrated that Pseudostellaria heterophylla protein hydrolysate (PPH) has immunomodulatory activity on murine spleen lymphocytes. The aim of this study was to investigate the excitation of PPH in RAW264.7 macrophage cells and the protective effect in cyclophosphamide (CTX)-treated mice. The results showed PPH of 50 µg/mL could stimulate macrophages resulting in significant promotions of nitric oxide (NO) production, endocytosis and reactive oxygen species formation. Meanwhile, enzyme-linked immunosorbent assay (ELISA) revealed that the levels of tumor necrosis factor-α and interleukin-10 were significantly upregulated by PPH. Furthermore, 50 mg/kg per day PPH restored the T lymphocyte proliferation and natural killer cell activity, and increased NO production and pinocytosis of peritoneal macrophages in CTX-treated mice. These findings indicate PPH plays a crucial role in RAW264.7 macrophage cells activation and in the protection against immunosuppression in CTX-treated mice and could be used as a potential immunostimulant agent.

Immunomodulatory effects of Pseudostellaria heterophylla peptide on spleen lymphocytes via a Ca2+/CaN/NFATc1/IFN-γ pathway.

Pseudostellaria heterophylla has been becoming a popular research topic because of its functionally active components. The immunomodulatory activity of P. heterophylla peptide (PPH) derived from protein hydrolysate and the molecular mechanism underlying its immunomodulatory effect were investigated in this study. Immunomodulatory PPH achieved the highest stimulation index of 1.53 at a concentration of 100 µg mL-1 for 48 h in spleen lymphocytes and promoted the secretions of tumor necrosis factor-α, interferon-γ, and interleukin-10. Moreover, PPH could elevate the intracellular Ca2+ concentration, calcineurin activity and nuclear factor of activated T cell (NFAT) c1 mRNA expression. Meanwhile these effects could be diminished by the treatment of verapamil and cyclosporin A, suggesting that PPH may activate spleen lymphocytes via the Ca2+/CaN/NFATc1/IFN-γ signaling pathway. These findings demonstrate that the P. heterophylla peptide has the potential to be utilized as a nutraceutical supplement to strengthen the immune system in the human body.

Fabrication of self-assembled Radix Pseudostellariae protein nanoparticles and the entrapment of curcumin.

Simulating the process of traditional Chinese medicine decoction, Radix Pseudostellariae protein (RPP)-based nanoparticles were constructed by combining heat treatment with pH adjustment in succession. The formed nanoparticles were characterized as homogeneously dispersed sphere within 100 nm in diameter. With curcumin as a drug model, the potential application of RPP as a nanocarrier was studied. Curcumin could combine to RPP through hydrophobic interaction and quench the intrinsic fluorescence of RPP. Results of X-ray diffraction revealed that the crystal formation of curcumin was suppressed after the formation of nanocomplexes. In addition, the curcumin-loaded nanocomplexes exhibited good thermal stability and the light stability of curcumin was significantly improved. The curcumin-loaded nanocomplexes had stronger reducing power than free curcumin, which displayed additive effect between curcumin and RPP. In summary, the obtained RPP nanoparticles are potential to become new drug delivery carriers in food field and pharmaceutical applications for the encapsulation of hydrophobic components.

Physico-Chemical and Antifungal Properties of a Trypsin Inhibitor from the Roots of Pseudostellaria heterophylla.

Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the fresh roots of the medicinal herb, Pseudostellaria heterophylla. The purification process involved ammonium sulfate precipitation, gel filtration chromatography on Sephadex G50, and ion-exchange chromatography on DEAE 650M. The PHTI contained 3.7% α-helix, 42.1% ß-sheets, 21.2% ß-turns, and 33% disordered structures, which showed similarity with several Kunitz-type trypsin inhibitors. Inhibition kinetic studies indicated that PHTI was a competitive inhibitor, with a Ki value of 3.01 × 10-9 M, indicating a high affinity to trypsin. The PHTI exhibited considerable stability over a broad range of pH (2⁻10) and temperatures (20⁻70 °C); however, metal ions, including Fe3+, Ba2+, Mn2+, and Al3+, could inactivate PHTI to different degrees. Results of fluorescence spectroscopy and circular dichroism showed that Fe3+ could bind to TI with an association constant of 2.75 × 105 M-1 to form a 1:1 complex, inducing conformation changes and inactivation of PHTI. In addition, PHTI could inhibit the growth of the phytopathogens, Colletotrichum gloeosporioides and Fusarium oxysporum, through disruption of the cell membrane integrity. The present study extended research on Pseudostellaria heterophylla proteins and makes PHTI an exploitable candidate as an antifungal protein for further investigation.

In Vitro Antioxidant Activities of Enzymatic Hydrolysate from Schizochytrium sp. and Its Hepatoprotective Effects on Acute Alcohol-Induced Liver Injury In Vivo.

Schizochytrium protein hydrolysate (SPH) was prepared through stepwise enzymatic hydrolysis by alcalase and flavourzyme sequentially. The proportion of hydrophobic amino acids of SPH was 34.71%. The molecular weight (MW) of SPH was principally concentrated at 180-3000 Da (52.29%). SPH was divided into two fractions by ultrafiltration: SPH-I (MW < 3 kDa) and SPH-II (MW > 3 kDa). Besides showing lipid peroxidation inhibitory activity in vitro, SPH-I exhibited high DPPH and ABTS radicals scavenging activities with IC50 of 350 µg/mL and 17.5 µg/mL, respectively. In addition, the antioxidant activity of SPH-I was estimated in vivo using the model of acute alcohol-induced liver injury in mice. For the hepatoprotective effects, oral administration of SPH-I at different concentrations (100, 300 mg/kg BW) to the mice subjected to alcohol significantly decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and hepatic malondialdehyde (MDA) level compared to the untreated mice. Besides, SPH-I could effectively restore the hepatic superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities and glutathione (GSH) level. Results suggested that SPH was rich in biopeptides that could be exploited as antioxidant molecules against oxidative stress in human body.

A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 µg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells.

Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 µg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives.

Preparation and Evaluation of the Chelating Nanocomposite Fabricated with Marine Algae Schizochytrium sp. Protein Hydrolysate and Calcium.

Marine algae have been becoming a popular research topic because of their biological implication. The algae peptide-based metal-chelating complex was investigated in this study. Schizochytrium sp. protein hydrolysate (SPH) possessing high Ca-binding capacity was prepared through stepwise enzymatic hydrolysis to a degree of hydrolysis of 22.46%. The nanocomposites of SPH chelated with calcium ions were fabricated in aqueous solution at pH 6 and 30 °C for 20 min, with the ratio of SPH to calcium 3:1 (w/w). The size distribution showed that the nanocomposite had compact structure with a radius of 68.16 ± 0.50 nm. SPH was rich in acidic amino acids, accounting for 33.55%, which are liable to bind with calcium ions. The molecular mass distribution demonstrated that the molecular mass of SPH was principally concentrated at 180-2000 Da. UV scanning spectroscopy and Fourier transform infrared spectroscopy suggested that the primary sites of calcium-binding corresponded to the carboxyl groups, carbonyl groups, and amino groups of SPH. The results of fluorescent spectroscopy, size distribution, atomic force microscope, and (1)H nuclear magnetic resonance spectroscopy suggested that calcium ions chelated with SPH would cause intramolecular and intermolecular folding and aggregating. The SPH-calcium chelate exerted remarkable stability and absorbability under either acidic or basic conditions, which was in favor of calcium absorption in the gastrointestinal tracts of humans. The investigation suggests that SPH-calcium chelate has the potential prospect to be utilized as a nutraceutical supplement to improve bone health in the human body.

Isolation of a novel lutein-protein complex from Chlorella vulgaris and its functional properties.

A novel kind of lutein-protein complex (LPC) was extracted from heterotrophic Chlorella vulgaris through aqueous extraction. The purification procedure contained solubilization of thylakoid proteins by a zwitterionic detergent CHAPS, anion exchange chromatography and gel filtration chromatography. Both wavelength scanning and HPLC analysis confirmed that lutein was the major pigment of the protein-based complex, and the mass ratio of lutein and protein was determined to be 9.72 : 100. Besides showing lipid peroxidation inhibition activity in vitro, LPC exerted significant antioxidant effects against ABTS and DPPH radicals with IC50 of 2.90 and 97. 23 µg mL(-1), respectively. Meanwhile, in vivo antioxidant activity of the complex was evaluated using the mice hepatotoxicity model; LPC significantly suppressed the carbon tetrachloride-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and decreased hepatic malondialdehyde (MDA) levels and the hepatosomatic index. Moreover, LPC could effectively restore the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the treated mice livers. Our findings further the progress in the research of natural protein-based lutein complexes, suggesting that LPC has the potential in hepatoprotection against chemical induced toxicity and in increasing the antioxidant capacity of the defense system in the human body.

Purification and characterisation of a glutamic acid-containing peptide with calcium-binding capacity from whey protein hydrolysate.

The bioavailability of dietary ionised calcium is affected by intestinal basic environment. Calcium-binding peptides can form complexes with calcium to improve its absorption and bioavailability. The aim of this study was focused on isolation and characterisation of a calcium-binding peptide from whey protein hydrolysates. Whey protein was hydrolysed using Flavourzyme and Protamex with substrate to enzyme ratio of 25:1 (w/w) at 49 °C for 7 h. The calcium-binding peptide was isolated by DEAE anion-exchange chromatography, Sephadex G-25 gel filtration and reversed phase high-performance liquid chromatography (RP-HPLC). A purified peptide of molecular mass 204 Da with strong calcium binding ability was identified on chromatography/electrospray ionisation (LC/ESI) tandem mass spectrum to be Glu-Gly (EG) after analysis and alignment in database. The calcium binding capacity of EG reached 67·81 µg/mg, and the amount increased by 95% compared with whey protein hydrolysate complex. The UV and infrared spectrometer analysis demonstrated that the principal sites of calcium-binding corresponded to the carboxyl groups and carbonyl groups of glutamic acid. In addition, the amino group and peptide amino are also the related groups in the interaction between EG and calcium ion. Meanwhile, the sequestered calcium percentage experiment has proved that EG-Ca is significantly more stable than CaCl2 in human gastrointestinal tract in vitro. The findings suggest that the purified dipeptide has the potential to be used as ion-binding ingredient in dietary supplements.