Intraspecific Diversity of Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii.

The commensal bacterium Faecalibacterium prausnitzii has unique anti-inflammatory properties, at least some of which have been attributed to its production of MAM, the Microbial Anti-inflammatory Molecule. Previous phylogenetic studies of F. prausnitzii strains have revealed the existence of various phylogroups. In this work, we address the question of whether MAMs from different phylogroups display distinct anti-inflammatory properties. We first performed wide-scale identification, classification, and phylogenetic analysis of MAM-like proteins encoded in different genomes of F. prausnitzii. When combined with a gene context analysis, this approach distinguished at least 10 distinct clusters of MAMs, providing evidence for functional diversity within this protein. We then selected 11 MAMs from various clusters and evaluated their anti-inflammatory capacities in vitro. A wide range of anti-inflammatory activity was detected. MAM from the M21/2 strain had the highest inhibitory effect (96% inhibition), while MAM from reference strain A2-165 demonstrated only 56% inhibition, and MAM from strain CNCM4541 was almost inactive. These results were confirmed in vivo in murine models of acute and chronic colitis. This study provides insights into the family of MAM proteins and generates clues regarding the choice of F. prausnitzii strains as probiotics for use in targeting chronic inflammatory diseases.

Effect of genetic and climatic variability on the metabolic profiles of black gram (Vigna mungo L.) seeds and sprouts.

BACKGROUND: Black gram is becoming increasingly of interest for consumers worldwide. The metabolomics have been conducted to reflect the life history of each individual plant. The metabolic pattern of black gram seeds and sprouts was profiled to investigate genetic and climatic influences on a broad range of chemical constituents. RESULTS: Distinct differences in metabolite profiles among three black gram varieties for both intact seeds and sprouts were observed. The differential impact of climate on metabolite profiles of the variety Chai Nat 80 during both dry and rainy seasons was investigated. Univariate statistical analysis demonstrated that greater maturity due to adequate moisture in the rainy season led to a higher content of nutritionally relevant polar metabolites, whereas the dry season resulted in a high relative amount of storage lipid because of immaturity due to insufficient rain and water supply. CONCLUSION: The investigation confirmed the potential of metabolite profiling to assist in breeding and farming practices.

Gut Microbiota Modulation, Anti-Diabetic and Anti-Inflammatory Properties of Polyphenol Extract from Mung Bean Seed Coat (Vigna radiata L.).

The present study investigated the gut health, anti-diabetic, and anti-inflammatory activities of mung bean seed coat extract (MSE). MSE was obtained by pressurized liquid extraction (PLE) using 50% ethanol as the extracting solvent. After 24 h of in vitro human fecal fermentation, MSE exhibited higher productions of total short-chain fatty acids (SCFA) than those of the control group (CON) and other polyphenol-rich substrates, including gallic acid (GA) and vitexin (VIT) (p > 0.05), but still lower than the fructo-oligosaccharide (FOS). In 16S-rRNA next-generation sequencing, MSE regulated the composition of gut microbiota by stimulating the growth of the beneficial bacteria Enterococcus, Ruminococcus, Blautia, and Bacteroides and decreasing the growth of the potential pathogenic bacteria Escherichia-Shigella. Similarly, qPCR showed increased numbers of Bifidobacterium, Lactobacillus, Faecalibacterium prausnitzii, and Prevotella, compared with those of CON (p < 0.05). MSE also reduced reactive oxygen species and increased glucose uptake in insulin-resistant HepG2 cells dose-dependently. The anti-inflammatory activity of MSE was observed in LPS-stimulated THP-1 monocytes with the reduction of TNFα, IL-1ß, IL-6, and IL-8 genes. The data demonstrated the potential applications of MSE as a dietary supplement with gut health benefits and its ability to mitigate diabetes and inflammatory-related diseases.

Polymeric Packaging Applications for Seafood Products: Packaging-Deterioration Relevance, Technology and Trends.

Seafood is a highly economical product worldwide. Primary modes of deterioration include autolysis, oxidation of protein and lipids, formation of biogenic amines and melanosis, and microbial deterioration. These post-harvest losses can be properly handled if the appropriate packaging technology has been applied. Therefore, it is necessary for packaging deterioration relevance to be clearly understood. This review demonstrates recent polymeric packaging technology for seafood products. Relationship between packaging and quality deterioration, including microbial growth and chemical and biochemical reactions, are discussed. Recent technology and trends in the development of seafood packaging are demonstrated by recent research articles and patents. Development of functional polymers for active packaging is the largest area for seafood applications. Intelligent packaging, modified atmosphere packaging, thermal insulator cartons, as well as the method of removing a fishy aroma have been widely developed and patented to solve the specific and comprehensive quality issues in seafood products. Many active antioxidant and antimicrobial compounds have been found and successfully incorporated with polymers to preserve the quality and monitor the fish freshness. A thermal insulator has also been developed for seafood packaging to preserve its freshness and avoid deterioration by microbial growth and enzymatic activity. Moreover, the enhanced biodegradable tray is also innovative as a single or bulk fish container for marketing and distribution. Accordingly, this review shows emerging polymeric packaging technology for seafood products and the relevance between packaging and seafood qualities.

Polysaccharides from Agaricus bisporus and Agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells.

BACKGROUND: Mushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. Crude mushroom extracts have been tested without detailed chemical analyses of its polysaccharide content. For the present study we decided to chemically determine the carbohydrate composition of semi-purified extracts from 2 closely related and well known basidiomycete species, i.e. Agaricus bisporus and A. brasiliensis and to study their effects on the innate immune system, in particular on the in vitro induction of pro-inflammatory cytokines, using THP-1 cells. METHODS: Mushroom polysaccharide extracts were prepared by hot water extraction and precipitation with ethanol. Their composition was analyzed by GC-MS and NMR spectroscopy. PMA activated THP-1 cells were treated with the extracts under different conditions and the production of pro-inflammatory cytokines was evaluated by qPCR. RESULTS: Semi-purified polysaccharide extracts of A. bisporus and A. brasiliensis (= blazei) were found to contain (1→6),(1→4)-linked α-glucan, (1→6)-linked ß-glucan, and mannogalactan. Their proportions were determined by integration of 1H-NMR signs, and were considerably different for the two species. A. brasiliensis showed a higher content of ß-glucan, while A. bisporus presented mannogalactan as its main polysaccharide. The extracts induced a comparable increase of transcription of the pro-inflammatory cytokine genes IL-1ß and TNF-α as well as of COX-2 in PMA differentiated THP-1 cells. Pro-inflammatory effects of bacterial LPS in this assay could be reduced significantly by the simultaneous addition of A. brasiliensis extract. CONCLUSIONS: The polysaccharide preparations from the closely related species A. bisporus and A. brasiliensis show major differences in composition: A. bisporus shows high mannogalactan content whereas A. brasiliensis has mostly ß-glucan. Semi-purified polysaccharide extracts from both Agaricus species stimulated the production of pro-inflammatory cytokines and enzymes, while the polysaccharide extract of A. brasiliensis reduced synthesis of these cytokines induced by LPS, suggesting programmable immunomodulation.

The Potential of Fractionated Rice Bran Protein Hydrolysates as Antioxidative and Anti-Inflammatory Agents.

Rice bran is generally used as animal feed despite containing numerous nutritional compounds. Small peptides possessing high antioxidant activity can be obtained from rice bran protein via enzymatic hydrolysis. Immune-modulating and antioxidative activity of rice bran protein hydrolysates from crude rice bran protein and its fractions were studied. Albumin, globulin, glutelin, and prolamin proteins were fractionated based on solubility differences and hydrolyzed with two types of enzyme, namely pepsin and protease M. Albumin fraction showed a high degree of hydrolysis in both enzymes. Protease M differently digested rice bran protein fractions, in which it showed low digestion in glutelin and prolamin fractions. After 30 min of hydrolysis time, the reaction slowed down, and antioxidant activity remained constant in pepsin hydrolysis. Due to the high presence of lipopolysaccharide (LPS) in protease M digested fractions (caused by the enzyme), it could not be used to determine immune-modulating activity. THP-1 macrophages were simultaneously stimulated with 100 ng/mL LPS and rice bran protein hydrolysates from 4 h of pepsin digestion. Reduction of pro-inflammatory cytokine IL-1ß and increase of anti-inflammatory cytokine IL-10 were observed from crude rice bran protein and albumin. In conclusion, pepsin-digested rice bran protein could be potentially used as antioxidative and anti-inflammatory agent.

The Effect of Coconut Water on Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells.

Coconut water is reported to have lipid-lowering effects in animal studies. However, there is lack of published reports regarding its effect on adipocytes. This study observed the effect of coconut water on adipocyte differentiation and lipid accumulation in 3T3-L1 cells. The sample used in this study was mature coconut water from tall variety. Based on a preliminary study, the sample was heat-treated and added with certain amino acids as precursors for Maillard reaction to improve its original flavor. As a comparison, aromatic coconut water was used since it is highly preferred as a fresh beverage. Six samples were supplemented to 3T3-L1 cells, which were then analyzed for cell proliferation, lipid accumulation, triglyceride content, and gene expression. Arginine and vitamin C contents of the samples were also determined. The data were analyzed with ANOVA and followed by Tukey's test. Results showed that aromatic coconut water could slightly suppress lipid accumulation, while mature coconut water had a significantly lower percentage of accumulation compared to the control sample (p<0.05). Canned and fresh samples had no significant difference in terms of lipid-lowering activity (p>0.05). Similarly, the addition of lysine and proline in canned samples did not significantly affect the cells' differentiation. There was no significant effect on expressions of C/EBP-α and PPARγ, indicating the possibility of other pathways involved in hypolipidemic effect of coconut water. This study showed that coconut water might have potential to inhibit adipogenesis in 3T3-L1 cells due to its bioactive compounds.

Controlled release and macrophage polarizing activity of cold-pressed rice bran oil in a niosome system.

Phytosterols, α-tocopherol and γ-oryzanol are scientifically recognized as major health promoting compounds found in cold-pressed rice bran oil (CRBO). This study aimed at encapsulating CRBO using a niosome delivery system. Water soluble CRBO niosomes approximately 200 nm in size were generated, remained stable for up to four weeks at 4 °C, and exhibited an encapsulation efficacy >80%. CRBO niosome controlled release was possible until the small intestinal phase in in vitro digestion. To our knowledge, this is the first study indicating a globular morphology of the CRBO niosomes and the location of CRBO. M0, M1, and M2 macrophage phenotypes were stimulated with 25, 50, and 100 µg ml-1 of in vitro digested CRBO niosomes. Gene expression profiles for each macrophage cell were obtained via M1 and M2 marker gene analysis. Changes of M0, M1 and M2 macrophage gene expression profiles occurred after CRBO stimulation and were visualized via principal component analysis (PCA). The results revealed a clear M1 macrophage conversion towards M0 in a digested CRBO niosome dose dependent manner, while this was not the case with M0 and M2 macrophages. Our findings indicated that CRBO niosomes have an ability to reverse M1 pro-inflammatory macrophage transformations back to resting M0 macrophages. Moreover, this study also showed future potential uses of CRBO niosomes, containing rice phytosterols and a co-surfactant, as fabricating materials to deliver and to control the release of oil soluble bioactive compounds for water soluble functional ingredient applications.

Characterization and probiotic properties of Lactobacilli from human breast milk.

Several studies have reported a complex microbial community in human breast milk. This community impacts the shape of the infant gut microbiota and consequently impacts host health. Lactobacillus is an important probiotic and has many applications in the functional food industry. This study isolated and evaluated the potential probiotic bacteria from human milk. Two Lactobacillus species, L. plantarum and L. pentosus, were isolated from the breast milk of Thai women. L. pentosus HM04-22, L. pentosus HM04-3, L. plantarum HM04-80, L. plantarum HM04-88 and L. plantarum HM01-1 showed good adhesion activity (> 55%) and resistance in gastric (pH 2) and bile (pH 8) conditions. Characterization of the probiotic properties indicated that all selected Lactobacillus isolates had anti-adhesion properties against Escherichia coli and Salmonella Typhimurium. Lactobacillus isolates protected Caco-2 cells from pathogen adhesion at 25-40%. In addition, the five selected strains presented anti-inflammatory properties by reducing interleukin (IL)-8 expression at 0.14 ± 0.16 to 0.52 ± 0.117-fold. However, the strains had no effect on the expression of tight junction genes, including zona occludens (ZO)-1, occludin and claudin-1. In conclusion, five selected Lactobacillus isolates from human milk were candidates for use as probiotics to promote health. However, more tests in animal models and clinical trials need to be performed.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1ß, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1ß, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids.

THP-1 cell line: an in vitro cell model for immune modulation approach.

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions.

Characterization of polarized THP-1 macrophages and polarizing ability of LPS and food compounds.

Little is known about the polarizing potential of currently used human macrophage cell lines, while a better understanding phenomena can support the prediction of effects in vivo based on in vitro analysis. To test the polarization capability of PMA differentiated-THP-1 macrophages (M0), cells were stimulated with 20 ng ml(-1) IFNγ + 1 µg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vivo and ex vivo into the M1 and M2 state, respectively. Apart from several well-known M1 and M2 markers, also new possible markers for M1 and M2 polarization were analysed in this study. The expression of M1 marker genes was up-regulated in IFNγ + LPS stimulated-M0 THP-1 macrophages. The IL-4 stimulated-M0 THP-1 macrophages expressed M2 cell membrane receptor genes. However, M2 chemokine and their receptor genes were only slightly up-regulated which might be due to the complexity of the secondary cell-cell interaction of the chemokine system. Lipopolysaccharides from E. coli (LPS) and food compounds [lentinan, vitamin D3 (vD3) and the combination of lentinan + vitamin D3 (Len + vD3)] were investigated for their polarizing ability on M0 THP-1 macrophages towards either the M1 or M2 state. LPS (700 ng ml(-1)) was able to skew M0 THP-1 macrophages towards the M1 direction since all analysed M1 marker genes were strongly expressed. Lentinan, vD3 and Len + vD3 did not induce expression of either M1 or M2 markers, indicating no polarizing ability of these compounds. Based on the expression of M1 and M2 marker genes we concluded that THP-1 macrophages could be successfully polarized into either the M1 or M2 state. Therefore, they can be used as a new macrophage polarizing model to estimate the polarizing/switching ability of test food compounds.

Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs).

In this study, two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIPs (rFIPs) were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to the well-known FIPs, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced rFIPs: rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.

ß-Glucans are involved in immune-modulation of THP-1 macrophages.

SCOPE: We aimed to examine different immunological aspects of ß-glucans derived from different food sources (oat, barley and shiitake) on phorbol myristate acetate (PMA)-differentiated THP-1 macrophages. Commercially purified barley ß-glucan (commercial BG) and lentinan were included to compare ß-glucans from the same origin but different degree of purity and processing. METHODS AND RESULTS: Chemical composition and molecular weight distribution of ß-glucan samples were determined. Inflammation-related gene expression kinetics (IL-1ß, IL-8, nuclear factor kappa B [NF-κB] and IL-10) after 3, 6 and 24 h of stimulation with 100 µg/mL ß-glucan were investigated. All tested ß-glucans mildly upregulated the observed inflammation-related genes with differential gene expression patterns. Similar gene expression kinetics, but different fold induction values, was found for the crude ß-glucan extracts and their corresponding commercial forms. Pre-incubation of THP-1 macrophages with ß-glucans prior to lipopolysaccharide (LPS) exposure decreased the induction of inflammation-related genes compared to LPS treatment. No production of nitric oxide (NO) and hydrogen peroxide (H2O2) was detected in ß-glucan stimulated THP-1 macrophages. Phagocytic activity was not different after stimulation by ß-glucan samples. CONCLUSION: Based on these in vitro analyses, it can be concluded that the analysed ß-glucans have varying levels of immunomodulating properties, which are likely related to structure, molecular weight and compositional characteristic of ß-glucan.

Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds.

An assay was developed to study inflammation-related immune responses of food compounds on monocytes and macrophages derived from THP-1 cell line. First strategy focused on the effects after stimulation with either lipopolysaccharide (LPS) or Concanavalin A (ConA). Gene expression kinetics of inflammation-related cytokines (IL-1ß, IL-6, IL-8, IL-10 and TNF-α), inflammation-related enzymes (iNOS and COX-2), and transcription factors (NF-κB, AP-1 and SP-1) were analyzed using RT-PCR. Time dependent cytokine secretion was investigated to study the inflammation-related responses at protein level. LPS stimulation induced inflammation-related cytokine, COX-2 and NF-κB genes of THP-1 monocytes and THP-1 macrophages with the maximum up-regulation at 3 and 6 h, respectively. These time points, were subsequently selected to investigate inflammation modulating activity of three well known immuno-modulating food-derived compounds; quercetin, citrus pectin and barley glucan. Co-stimulation of LPS with either quercetin, citrus pectin, or barley glucan in THP-1 monocytes and macrophages showed different immuno-modulatory activity of these compounds. Therefore, we propose that simultaneously exposing THP-1 cells to LPS and food compounds, combined with gene expression response analysis are a promising in vitro screening tool to select, in a limited time frame, food compounds for inflammation modulating effects.