Purification and characterization of a glycoprotein from Sipunculus nudus and its immune-enhancing activity to RAW 264.7 macrophages.

Sipunculus nudus, an edible marine invertebrate, has long been used as traditional Chinese medicine in folk remedies. In order to assess the immunoregulatory activity of glycoproteins in Sipunculus nudus and conduct a structure-activity relationship, a glycoprotein (SGP1) with molecular mass of 9.26 kDa was purified from Sipunculus nudus, and its chemical structure as well as immune-enhancing activity was investigated in this study. Structure analysis revealed that SGP1, a protein-dominate glycoprotein with O-glycosidic bonds, contained 92.8 % protein and 3.1 % saccharide. GC-MS result indicated that the saccharide moieties of SGP1 basically consisted of lyxose (Lyx), xylose (Xyl) as well as glucose (Glu) at a molar proportion of 0.87:4.16:1.36. The fourier transform infrared specoscopy (FT-IR) result proved that SGP1 have a typical characteristic of glycoprotein. Besides, circular dichroism (CD) result showed that SGP1 contained 4.1 % α-helix, 42.5 % ß-sheet, 21.4 % ß-turn, and 32.0 % random coil, indicating it's mainly a ß-sheet glycoprotein. The amino acid sequence of SGP1 shared a similarity to the Myohemerythrin (sp|Q5K473|HEMTM) with protein sequence coverage of 28.3 %. Moreover, the activity evaluation results showed that SGP1 exhibited significant immune-enhancing activity to the RAW 264.7 macrophages by promoting macrophages proliferation, enhancing phagocytic capacity, and simultaneously stimulating the secretions of nitric oxide (NO), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) via NF-κB pathways. In this study, SGP1 as a novel glycoprotein had an obvious immune-enhancing activity to macrophages, and thus could be applied in the functional foods as a potential immunopotentiator for the hypoimmune population.

The anti-inflammatory activity of GABA-enriched Moringa oleifera leaves produced by fermentation with Lactobacillus plantarum LK-1.

Introduction: Gamma-aminobutyric acid (GABA), one of the main active components in Moringa oleifera leaves, can be widely used to treat multiple diseases including inflammation. Methods: In this study, the anti-inflammatory activity and the underlying anti-inflammatory mechanism of the GABA-enriched Moringa oleifera leaves fermentation broth (MLFB) were investigated on lipopolysaccharide (LPS)-induced RAW 264.7 cells model. The key active components changes like total flavonoids, total polyphenols and organic acid in the fermentation broth after fermentation was also analyzed. Results: ELISA, RT-qPCR and Western blot results indicated that MLFB could dose-dependently inhibit the secretions and intracellular expression levels of pro-inflammatory cytokines like 1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). Furthermore, MLFB also suppressed the expressions of prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS). Moreover, the mRNA expressions of the key molecules like Toll-like receptor 4 (TLR-4) and nuclear factor (NF)-κB in the NF-κB signaling pathway were also restrained by MLFB in a dose-dependent manner. Besides, the key active components analysis result showed that the GABA, total polyphenols, and most organic acids like pyruvic acid, lactic acid as well as acetic acid were increased obviously after fermentation. The total flavonoids content in MLFB was still remained to be 32 mg/L though a downtrend was presented after fermentation. Discussion: Our results indicated that the MLFB could effectively alleviate LPS-induced inflammatory response by inhibiting the secretions of pro-inflammatory cytokines and its underlying mechanism might be associated with the inhibition of TLR-4/NF-κB inflammatory signaling pathway activation. The anti-inflammatory activity of MLFB might related to the relative high contents of GABA as well as other active constituents such as flavonoids, phenolics and organic acids in MLFB. Our study provides the theoretical basis for applying GABA-enriched Moringa oleifera leaves as a functional food ingredient in the precaution and treatment of chronic inflammatory diseases.

Fabrication and characterization of l-ascorbyl palmitate and phospholipid-based hybrid liposomes and their impacts on the stability of loaded hydrophobic polyphenols.

The aim of this study was to evaluate the influence of l-ascorbyl palmitate (LAP) as an additive to liposome formulations by self-assembling with soy lecithin to form hybrid liposomes, in order to enhance the physical stability and bioactivator-loaded retention ratio of the LAP incorporated liposomes (LAP-LP). The addition of LAP significantly increased its surface negative charge and strong hydrophobic interactions occurred between the hydrophobic tails of LAP and phospholipids resulting in more compactly ordered, rigid and hydrophobic phospholipid bilayers as indicated by surface tension, fluorescence probes and DSC. These changes enhanced the stability of hydrophobic polyphenol loaded LAP-LP during storage. Particularly, after four weeks storage at 37 °C for naringenin loaded liposomes, the retention ratio of pure liposome decreased dramatically to 12.5 %, while the LAP-LP remained above 74.5 %. This study opens up the potential for the LAP-LP to be developed as a food-grade multifunctional formulation for encapsulating and delivering bioactivators.

Fabrication and Characterization of W/O/W Emulgels by Sipunculus nudus Salt-Soluble Proteins: Co-Encapsulation of Vitamin C and ß-Carotene.

W/O/W emulsions can be used to encapsulate both hydrophobic and hydrophilic bioactive as nutritional products. However, studies on protein stabilized gel-like W/O/W emulsions have rarely been reported, compared to the liquid state multiple emulsions. The purpose of this study was to investigate the effect of different oil-water ratios on the stability of W/O/W emulgels fabricated with salt-soluble proteins (SSPs) of Sipunculus nudus. The physical stability, structural characteristics, rheological properties, and encapsulation stability of vitamin C and ß-carotene of double emulgels were investigated. The addition of W/O primary emulsion was determined to be 10% after the characterization of the morphology of double emulsion. The results of microstructure and rheological properties showed that the stability of W/O/W emulgels increased with the increasing concentration of SSPs. Additionally, the encapsulation efficiency of vitamin C and ß-carotene were more than 87%, and 99%, respectively, and still could maintain around 50% retention of the antioxidant capacity after storage for 28 days at 4 °C. The aforementioned findings demonstrate that stable W/O/W emulgels are a viable option for active ingredients with an improvement in shelf stability and protection of functional activity.

Intelligent pH-sensing film based on polyvinyl alcohol/cellulose nanocrystal with purple cabbage anthocyanins for visually monitoring shrimp freshness.

We aimed to prepare a new pH-sensing film based on the immobilization of purple cabbage anthocyanins (PCA) into Polyvinyl alcohol (PVA) reinforced by cellulose nanocrystals (CNC). FT-IR, XRD and TGA were used to assess the intermolecular interactions and thermo-stability of films. The addition of CNC and PCA resulted in an enhancement in UV-vis barrier, mechanical properties and moisture resistance. Inclusion of PCA imparted intelligent properties to the films. PCA-loaded films displayed strong visually detectable colorimetric responses to pH (2-13) and volatile ammonia. When applied to monitor shrimp freshness at 4 °C, PVA/CNC films containing 0.6 % PCA exhibited conspicuous color fluctuations from purple to gray blue upon deterioration. As a result, PVA/CNC-PCA colorimetric films were considered as intelligent packaging labels with significant mechanical, water vapor barrier properties and pH-sensing qualities for visual quality evaluation of fresh seafood products.

Formation of Shelf-Stable Pickering High Internal Phase Emulsion Stabilized by Sipunculus nudus Water-Soluble Proteins (WSPs).

To form a stable emulsion system, the water-soluble proteins (WSPs) of Sipunculus nudus were prepared as the sole effective stabilizer for the high internal phase emulsion (HIPEs), of which the influence of the WSPs concentration and environmental stability was investigated. The HIPEs were fabricated using a simple one-pot homogenization process (10,000 rpm/min, 3 min) that involved blending the WSPs (0.1, 1, 2, 3, 4, and 5 wt%) with soybean oil (60, 65, 70, 75, 80, 85, and 90%). The microstructure and properties of stable HIPEs were characterized by particle size, ζ-potential, visual observations, optical microscopy, and dynamic rheology property measurements. As the concentration of WSPs increases, the mean particle diameter of HIPEs decreases, on the contrary, the apparent viscosity and storage modulus gradually increase. At a given emulsifier concentration (3 wt%), the stable and gel-like HIPEs were formed at the oil internal phase (ϕ) values of 70-75%, all the pH range in values from 3 to 9, and the ionic strength from 100 to 500 mM. Furthermore, the HIPEs that were stabilized formed a gel-like state that was relatively stable to heat and storage (30 days). And there was a new phenomenon that the destabilized HIPE of the freeze-thaw treatments could still return to a gel-like state again after homogenizing. The study results suggest that the WSPs of S. nudus as a natural emulsifier could be widely used in the food industry.

Proteomic analysis of body wall and coelomic fluid in Sipunculus nudus.

In order to make clear the protein compositions of Sipunculus nudus and investigate its immune-related proteins, proteomic analysis was performed on body wall and coelomic fluid of Sipunculus nudus. A total of 1659 proteins were identified, and 539 proteins were differentially expressed in the coelomic fluid compared to those in the body wall, of which 415 proteins were up-regulated while 124 proteins were down-regulated. Gene Ontology (GO) analysis showed that the GO terms involved in the two parts of Sipunculus nudus were similar, with metabolic processes, catalytic activity and cell occupying the top categories of biological process, molecular function and cellular component, respectively. KEGG pathway analysis showed that 49 pathways in body wall and 48 in coelomic fluid were mapped respectively, and these pathways were mainly related to cellular processes, environmental information processing, genetic information processing and metabolism. The COG analysis showed that 757 proteins from body wall and 889 from coelomic fluid were classified into 26 COG categories, respectively. Pfam annotation revealed the mainly immune-related proteins contained in Sipunculus nudus, such as insulin-like growth factor binding protein, catalase, basement membrane proteoglycan, titin. Our research provides the first proteomic information of Sipunculus nudus, which contributes to the study of functional proteins in Sipunculus nudus and is of great significance for the application of Sipunculus nudus in functional foods and medicines.

Site specific PEGylation of ß-lactoglobulin at glutamine residues and its influence on conformation and antigenicity.

ß-lactoglobulin (ß-LG) is one of the main allergens in milk. Polyethylene glycol (PEG) modification (PEGylation) was found to have the ability to reduce the antigenicity of proteins. To determine the effect of site specific PEGylation on ß-LG antigenicity and conformation, we applied 5 kDa methoxy polyethylene glycol-amine (mPEG-NH2) to modify ß-LG at glutamine (Gln) residues under the catalysis of transglutaminase. The antigenicity of ß-LG was measured using rabbit IgG antibodies by indirect competitive ELISA. The result indicated that the antigenicity of ß-LG was decreased from 72.2 µg/mL to 22.7 µg/mL after PEGylation. SDS-PAGE and MALDI-TOF-MS showed that the molecular mass of native ß-LG was about 18.3 kDa while the PEGylated ß-LG had a molecular mass of 23.4 kDa, which meant that mono-PEGylated ß-LG was obtained after PEGylation and purification by cation exchange chromatography. Additionally, the circular dichroism spectrum of the PEGylated ß-LG was approximately superimposed on that of ß-LG and the secondary structure content of ß-LG also had no significant changes after PEGylation, which indicated that the secondary structure of ß-LG was preserved. After PEGylation, the intrinsic fluorescence intensity of ß-LG decreased from 6361 to 5159 while the surface hydrophobicity increased, which indicated that the tertiary structure of ß-LG was slightly changed. PEGylation site analysis result showed that Gln 155 or Gln 159 might be the most possible binding site. In conclusion, the decrease of the antigenicity of ß-LG induced by the PEGylation is mainly due to the steric shielding effect of PEG chain rather than conformational changes of ß-LG.