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.

Hot Air Drying of Sipunculus nudus: Effect of Microwave-Assisted Drying on Quality and Aroma.

The present study aimed to investigate the effect of different microwave pre-drying times under hot-air-drying processes on the quality properties and sensory evaluation of Sipunculus nudus (S. nudus). The colour, proximate analysis, amino acid content, fat oxidation, and volatile components of dried S. nudus were determined. Microwave pre-drying could significantly (p < 0.05) increase the drying rate and shorten the drying time. The results of colour, proximate analysis, and amino acid content indicated that microwave pre-drying could improve the quality of the product, resulting in dried S. nudus with less of a loss in nutrients. The samples that underwent microwave pre-drying had a high degree of fatty acid oxidation and low monounsaturated fatty acid content, which facilitated the formation of volatile components. Additionally, the MAD-2 and MAD-3 groups had high relative contents of aldehydes and hydrocarbons, and the FD group had the highest relative content of esters found in the samples. The relative content of ketones and alcohols did not differ significantly between the different drying groups. The finding of this study has important potential for enhancing the quality and aroma of dry S. nudus products with microwave pre-drying during the drying process.

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.

Polypeptides Isolated from Lactococcus lactis Alleviates Lipopolysaccharide (LPS)-Induced Inflammation in Ctenopharyngodon idella.

The main purpose of the present study was to evaluate the anti-inflammatory activity of Lactococcus lactis BL52 and isolate active substances responsible for anti-inflammatory activity. Head-kidney (HK) macrophages were used for in vitro bioassay-guided isolation, and the structure of the two peptides was identified by mass spectrometry analysis. Lipopolysaccharide (LPS)-induced inflammatory responses in Ctenopharyngodon idella were also examined to evaluate the in vivo anti-inflammatory activity of active substances. Two active peptides were isolated by HPLC from L. lactis BL52, and an in vitro anti-inflammatory assay demonstrated that peptide ALBL1 and ALBL2 dose-dependently inhibited LPS-induced inflammatory cytokines TNF-α, IL-6, and IL-1ß and inflammatory factors NO and PGE 2 production in macrophages (p < 0.05). After being treated with 20 mg/Kg peptide ALBL1 and ALBL2, the expression levels of TNF-α, IL-6, IL-1ß, NO, and PGE 2 were significantly inhibited (p < 0.05). Results from the in vivo test showed that when the concentration of peptide ALBL1 and ALBL2 reached 30 mg/Kg, the LPS-induced upregulations of TNF-α, IL-6, IL-1ß, NO, and PGE 2 were prevented. In addition, peptide ALBL1 and ALBL2 blocked the expression of Toll-like receptor 2 (TLR2) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of IκBα. Moreover, C. idella treated with peptide ALBL1 and ALBL2 can relieve pathological inflammatory responses caused by LPS. These results suggest that the anti-inflammatory properties of peptide ALBL1 and ALBL2 might be a result from the inhibition of IL-6, IL-1ß, and TNF-α expressions through the downregulation of Toll2/NF-κB signaling pathways.

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.

Comparison of hydrability, antioxidants, microstructure, and sensory quality of barley grass powder using ultra-micro-crushing combined with hot air and freeze drying.

To explore the physicochemical characters of barley grass, ultra-micro-crushing (UMC) technology combined with air drying or freeze drying was carried out. After barley grass was air-dried at 70°C or freeze-dried at 15°C, it was grinded for 30, 60, 90, and 120 min using UMC, respectively. After combined processing, moisture content, particle size, odor, color, microstructure, water and oil-holding capacity, the content of flavonoid and chlorophyll, water activity, and sensory qualities were determined. The particle size of barley grass powder decreased, and lightness value was increased; water and oil-holding capacity decreased significantly (p ≤ .05), whereas swelling and dissolving capacity increased in the processed grass powder. On the other hand, the total flavonoid content increased significantly (p ≤ .05). Barley grass odor features sulfide aroma, and its microstructure demonstrates lamellar morphology with some fewer fragmented pieces. The results suggested combined UMC at 90-120 min will be suitable for processing barley grass powder.

Encapsulation of Hydrophobic and Low-Soluble Polyphenols into Nanoliposomes by pH-Driven Method: Naringenin and Naringin as Model Compounds.

Naringenin and naringin are a class of hydrophobic polyphenol compounds and both have several biological activities containing antioxidant, anti-inflammatory and anti-tumor properties. Nevertheless, they have low water solubility and bioavailability, which limits their biological activity. In this study, an easy pH-driven method was applied to load naringenin or naringin into nanoliposomes based on the gradual reduction in their water solubility after the pH changed to acidity. Thus, the naringenin or naringin can be embedded into the hydrophobic region within nanoliposomes from the aqueous phase. A series of naringenin/naringin-loaded nanoliposomes with different pH values, lecithin contents and feeding naringenin/naringin concentrations were prepared by microfluidization and a pH-driven method. The naringin-loaded nanoliposome contained some free naringin due to its higher water solubility at lower pH values and had a relatively low encapsulation efficiency. However, the naringenin-loaded nanoliposomes were predominantly nanometric (44.95-104.4 nm), negatively charged (-14.1 to -19.3 mV) and exhibited relatively high encapsulation efficiency (EE = 95.34% for 0.75 mg/mL naringenin within 1% w/v lecithin). Additionally, the naringenin-loaded nanoliposomes still maintained good stability during 31 days of storage at 4 °C. This study may help to develop novel food-grade colloidal delivery systems and apply them to introducing naringenin or other lipophilic polyphenols into foods, supplements or drugs.

The Formation of Chitosan-Coated Rhamnolipid Liposomes Containing Curcumin: Stability and In Vitro Digestion.

There is growing interest in developing biomaterial-coated liposome delivery systems to improve the stability and bioavailability of curcumin, which is a hydrophobic nutraceutical claimed to have several health benefits. The curcumin-loaded rhamnolipid liposomes (Cur-RL-Lips) were fabricated from rhamnolipid and phospholipids, and then chitosan (CS) covered the surface of Cur-RL-Lips by electrostatic interaction to form CS-coated Cur-RL-Lips. The influence of CS concentration on the physical stability and digestion of the liposomes was investigated. The CS-coated Cur-RL-Lips with RL:CS = 1:1 have a relatively small size (412.9 nm) and positive charge (19.7 mV). The CS-coated Cur-RL-Lips remained stable from pH 2 to 5 at room temperature and can effectively slow the degradation of curcumin at 80 °C; however, they were highly unstable to salt addition. In addition, compared with Cur-RL-Lips, the bioavailability of curcumin in CS-coated Cur-RL-Lips was relatively high due to its high transformation in gastrointestinal tract. These results may facilitate the design of a more efficacious liposomal delivery system that enhances the stability and bioavailability of curcumin in nutraceutical-loaded functional foods and beverages.

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.