Real-time PCR-based quantitative microbiome profiling elucidates the microbial dynamic succession in backslopping fermentation of Taiwanese pickled cabbage.

BACKGROUND: Microbiota succession determines the flavor and quality of fermented foods. Quantitative PCR-based quantitative microbiome profiling (QMP) has been applied broadly for microbial analysis from absolute abundance perspectives, transforming microbiota ratios into counts by normalizing 16S ribosomal RNA (16S rRNA) gene sequencing data with gene copies quantified by quantitative PCR. However, the application of QMP in fermented foods is still limited. RESULTS: QMP elucidated microbial succession of Taiwanese pickled cabbage. In the spontaneous first-round fermentation (FR), the 16S rRNA gene copies of total bacteria increased from 6.1 to 10 log copies mL-1. The dominant lactic acid bacteria genera were successively Lactococcus, Leuconostoc and Lactiplantibacillus. Despite the decrease in the proportion of Lactococcus during the succession, the absolute abundance of Lactococcus still increased. In the backslopping second-round fermentation (SR), the total bacteria 16S rRNA gene copies increased from 7.6 to 9.9 log copies mL-1. The addition of backslopping starter and vinegar rapidly led to a homogenous microbial community dominated by Lactiplantibacillus. The proportion of Lactiplantibacillus remained consistently around 90% during SR, whereas its absolute abundance exhibited a continuous increase. In SR without vinegar, Leuconostoc consistently dominated the fermentation. CONCLUSION: The present study highlights that compositional analysis would misinterpret microbial dynamics, whereas QMP reflected the real succession profiles and unveiled the essential role of vinegar in promoting Lactiplantibacillus dominance in backslopping fermentation of Taiwanese pickled cabbage. Quantitative microbiome profiling (QMP) was found to be a more promising approach for the detailed observation of microbiome succession in food fermentation compared to compositional analysis. © 2024 Society of Chemical Industry.

Feasibility of Utilizing Stable-Isotope Dimethyl Labeling in Liquid Chromatography⁻Tandem Mass Spectrometry-Based Determination for Food Allergens-Case of Kiwifruit.

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography- tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5-109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86-9.92%. The limit of quantification (LOQ) was set at 8 pg mL-1 for SPA1-H and at 8 ng mL-1 for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175-0.0515 mg g-1 of Act d 1 and 0.0252-0.0556 mg g-1 of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.

The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Interleukin-21 suppresses the differentiation and functions of T helper 2 cells.

T helper type 2 (Th2) cells, which produce interleukin-4 (IL-4), IL-5 and IL-13, control immunity to all forms of allergic inflammatory responses. Interleukin-21 (IL-21) reduces allergic symptoms in murine models and inhibits IL-4-induced IgE secretion by B cells. However, whether or not IL-21 directly affects Th2 cells, which leads to reduced allergic symptoms, is unclear. In this study, we investigated the effects of IL-21 on the differentiation and effector functions of Th2 cells. We found that IL-21 reduced the number of differentiated Th2 cells and these Th2 cells showed a diminished Th2 cytokine production. Interleukin-21 suppressed Th2 cytokine production of already polarized Th2 cells by down-regulation of transcription factor GATA-3. It also induced apoptosis of Th2 cells with decreased anti-apoptotic factor Bcl-2. Intranasal administration of IL-21 at the beginning of ovalbumin (OVA) sensitization or before OVA challenge decreased Th2 cytokines in the bronchoalveolar lavage fluid of OVA/alum-immunized allergic mice. In addition, the inhibitory effects of IL-21 on Th2 effector functions can also be found in allergic patients. Our results demonstrate that IL-21 suppresses the development of Th2 cells and functions of polarized Th2 cells. Hence, the administration of IL-21 may be considered for use as a preventive and therapeutic approach when dealing with Th2-mediated allergic diseases.

A novel exopolysaccharide from the biofilm of Thermus aquaticus YT-1 induces the immune response through Toll-like receptor 2.

Bacterial polysaccharides are known to induce the immune response in macrophages. Here we isolated a novel extracellular polysaccharide from the biofilm of Thermus aquaticus YT-1 and evaluated its structure and immunomodulatory effects. The size of this polysaccharide, TA-1, was deduced by size-exclusion chromatography as 500 kDa. GC-MS, high performance anion-exchange chromatography with pulsed amperometric detection, electrospray ionization-MS/MS, and NMR revealed the novel structure of TA-1. The polysaccharide is composed of tetrasaccharide-repeating units of galactofuranose, galactopyranose, and N-acetylgalactosamine (1:1:2) and lacked acidic sugars. TA-1 stimulated macrophage cells to produce the cytokines TNF-α and IL-6. Screening of Toll-like receptors and antibody-blocking experiments indicated that the natural receptor of TA-1 in its immunoactivity is TLR2. Recognition of TA-1 by TLR2 was confirmed by TA-1 induction of IL-6 production in peritoneal macrophages from wild-type mice but not from TLR2(-/-) mice. TA-1, as a TLR2 agonist, could possibly be used as an adjuvant and could enhance cytokine release, which increases the immune response. Furthermore, TA-1 induced cytokine release is dependent on MyD88/TIRAP.

Structure and immunological characterization of the capsular polysaccharide of a pyrogenic liver abscess caused by Klebsiella pneumoniae: activation of macrophages through Toll-like receptor 4.

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-ß-D-Glc-(1→4)-[2,3-(S)-pyruvate]-ß-D-GlcA-(1→4)-α-L-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C(2)-OH or C(3)-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.

Administration of recombinant Reishi immunomodulatory protein (rLZ-8) diet enhances innate immune responses and elicits protection against nervous necrosis virus in grouper Epinephelus coioides.

Nervous necrosis virus (NNV) infection during larvae and juvenile stage in grouper (Epinephelus coioides) has caused severe economic losses in the aquaculture industry in Asia. The aims of this study were to evaluate the influence of recombinant Reishi protein, rLZ-8, on the innate immune responses and the viral resisting ability in fish. Groupers were fed with rLZ-8 supplemented diet (1.25-37.5 mg (rLZ-8)/kg(diet)), and the cytokine gene expression, innate immune responses, and survival rate after NNV challenge were examined. The fish fed with rLZ-8 diet showed 6- to 11-fold upregulated TNF-α and IL-1ß gene expression, along with significant increased respiratory burst and phagocytic activity. Moreover, feeding the fish with 37.5 mg/kg rLZ-8 diet elicited significant improvement in post viral challenge survival rate (85.7%). These discoveries indicated that rLZ-8 could be utilized as an ant-pathogen immunostimulant, and provided a new candidate to fight against NNV infection in fish.

Determination of the soybean allergen Gly m 6 and its stability in food processing using liquid chromatography-tandem mass spectrometry coupled with stable-isotope dimethyl labelling.

A cost-effective method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable-isotope dimethyl labelling was used for the determination of Gly m 6. The validation results revealed that the recoveries and precisions obtained from five spiked levels were in the ranges of 88.8-113.0% and 8.3-22.0%, respectively. The content and stability of the major soybean allergen Gly m 6 in various food processing procedures were evaluated by the quantification results of its surrogate signature peptide. The Gly m 6 content in soybean decreased by 42% after natto fermentation, and by 31% and 35% in pasteurised soymilk and sterilised soymilk, respectively, relative to the raw soymilk. Only 19% of Gly m 6 in raw soymilk was retained in the soymilk film. This study extended the feasibility of dimethyl labelling to soy-based food samples and examined the proteolysis of Gly m 6 in natto fermentation and its thermal instability.

Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway.

Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.

Rapid screening and determination of pesticides on lemon surfaces using the paper-spray mass spectrometry integrated via thermal desorption probe.

This paper presents a novel thermal desorption probe integrated with the corona-discharged assisted paper-spray mass spectrometry (PS-MS) for rapid detecting the residual pesticides on fruit surfaces. Pesticide detection can be simply achieved by scratching the fruit surface and then placed in front of the inlet of the MS for target pesticides screening. A novel ionization method comprising the electrospray ionization and the corona discharged is generated on the paper tip to simultaneously ionize the pesticide of high and low polarities for MS detection. Six pesticides composed of polar acetamiprid, azoxystobin, pyridaben and low polar chlorfenapyr, pyriproxyden, λ-cyhalothrin are successfully detected in seconds. The results are also validated with the LC-MS/MS and GC-MS/MS spectra performed via the standard protocols by a certificated laboratory of Eurofins Taiwan. The developed method provides a rapid, simple yet efficient way for screening residual pesticides on fruits.

Microfluidic colorimetric analysis system for sodium benzoate detection in foods.

Sodium benzoate (SBA) is a widely-used additive for preventing food spoilage and deterioration and extending the shelf life. However, the concentration of SBA must be controlled under safe regulations to avoid damaging human health. Accordingly, this study proposes a microfluidic colorimetric analysis (MCA) system composing of a wax-printed paper-microchip and a self-made smart analysis equipment for the concentration detection of SBA in common foods and beverages. In the presented method, the distilled SBA sample is mixed with NaOH to obtain a nitro compound and the compound is then dripped onto the reaction area of the paper-microchip, which is embedded with two layers of reagents (namely acetophenone and acetone). The paper-microchip is heated at 120 °C for 20 min to cause a colorimetric reaction and the reaction image is then obtained through a CMOS (complementary metal oxide semiconductor) device and transmitted to a cell-phone over a WiFi connection. Finally, use the self-developed RGB analysis software installed on the cell-phone to obtain the SBA concentration. A calibration curve is constructed using SBA samples with known concentrations ranging from 50 ppm (0.35 mM) to 5000 ppm (35 mM). It is shown that the R + G + B value (Y) of the reaction image and SBA concentration (X) are related via Y = -0.034 X +737.40, with a determination coefficient of R2 = 0.9970. By measuring the SBA concentration of 15 commercially available food and beverage products, the actual feasibility of the current MCA system can be demonstrated. The results show that the difference from the measurement results obtained using the macroscale HPLC method does not exceed 6.0%. Overall, the current system provides a reliable and low-cost technique for quantifying the SBA concentration in food and drink products.

4-Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

Effects of water-soluble carbohydrate concentrate from Chinese jujube on different intestinal and fecal indices.

This study was to investigate the effects of water-soluble carbohydrate concentrate (WSCC) prepared from Chinese jujube ( Zizyphus jujuba) on different intestinal and fecal indices in a hamster model. WSCC contained carbohydrates (771 g kg(-1) of WSCC) including glucose, fructose, pectin polysaccharide, and hemicellulose. The administration of WSCC (5.0 and 15 g kg(-1) of diet) effectively ( P<0.05) shortened gastrointestinal transit time (by 34.2-57.3%), reduced cecal ammonia (by 58.1-60.3%), elevated total short-chain fatty acid concentrations in cecum (3-4-fold), increased fecal moisture (147-170%), reduced daily fecal ammonia output (by 31.9-75.8%), and decreased the activities of beta-D-glucuronidase (by 73.0-73.8%), beta-D-glucosidase (by 58.2-85.7%), mucinase (by 46.2-72.6%), and urease (by 31.9-48.7%) in feces. This study suggested that adequate consumption of jujube WSCC (at least 5.0 g kg(-1) of diet or 40 mg day(-1)) might exert favorable effects on improving the gastrointestinal milieu and reducing the exposure of intestinal mucosa to toxic ammonia and other detrimental compounds.

High processing tolerances of immunomodulatory proteins in Enoki and Reishi mushrooms.

This study investigated the processing tolerances of two mushroom proteins with immunomodulatory activities, including FVE from Enoki ( Flammulia velutipes ) and LZ8 from Reishi ( Ganoderma lucidum ) mushrooms, under food processing treatments such as heating, sterilization, frozen storage, extraction in acid/alkaline conditions, and dehydration. Results showed that the capability of these two proteins to induce IFN-gamma secretion by murine splenocytes remained after 100 degrees C heating for 30 min, 121 degrees C autoclaving for 15 min, and -80 degrees C freezing. The retained activities of both proteins on cell proliferation and IFN-gamma production did not decrease at 0.6 M hydrochloric acid (at pH 2) but strikingly dropped at 5 M sodium hydrate (at pH 13). After vacuum dehydration, FVE and LZ8 retained most of their activities on cell proliferation; nevertheless, the IFN-gamma secretion decreased to about half of the initial values. These findings suggest that these two mushroom proteins have a good thermal/freezing resistance, acid tolerance, and dehydration stability and are candidates for processing in food and nutraceutical utilization.

A novel protein from edible fungi Cordyceps militaris that induces apoptosis.

Cordyceps militaris is a dietary therapeutic fungus that is an important model species in Cordyceps research. In this study, we purified a novel protein from the fruit bodies of C. militaris and designated it as Cordyceps militaris protein (CMP). CMP has a molecular mass of 18.0 kDa and is not glycosylated. Interestingly, CMP inhibited cell viability in murine primary cells and other cell lines in a time- and dose-dependent manner. Using trypan blue staining and a lactate dehydrogenase release assay, we showed that CMP caused cell death in the murine hepatoma cell line BNL 1MEA.7R.1. Furthermore, the frequency of BNL 1MEA.7R.1 cells at the sub-G1 stage was increased by CMP. Apoptosis, as determined by Annexin V and propidium iodide analysis, indicated that CMP could mediate BNL 1MEA.7R.1 apoptosis, but not necrosis. After coincubation with CMP, a decrease in mitochondria potential was detected using 3,3'-dihexyloxacarbocyanine iodide. These results suggest that CMP is a harmful protein that induces apoptosis through a mitochondrion-dependent pathway. Stability experiments demonstrated that heat treatment and alkalization degraded CMP and further destroyed its cell-death-inducing ability, implying that cooking is necessary for food containing C. militaris.

Application of thermal stability difference to remove flammutoxin in fungal immunomodulatory protein, FIP-fve, extract from Flammulina velutipes.

Fungal immunomodulatory protein (FIP-fve) is a potential functional food ingredient. However, undesirable component flammutoxin (FTX) would occur in the extracted fraction of FIP-fve. In this paper, an application of heating processing instead of the intensive separation process was employed in fractionation of FIP-fve, meanwhile, exclusion of FTX was reached. Contents of FIP-fve and FTX were monitored by HPLC-UV-ESI-MS. Both FIP-fve and FTX had higher thermal stability in a lower concentration solution. Cold water could effectively extract FIP-fve and FTX from fresh mushroom without acetic acid and disulfide-bond breaking agent ß-mercaptoethanol commonly used in biochemical studies. Heating cold water extract contained 580 µg/mL FIP-fve and 452 µg/mL FTX at 60 °C for 5 min could effectively exclude FTX and remain 75% of FIP-fve. Adding 0.1 M trehalose or 20% ethanol did not significantly alter the stability of both proteins. The method developed is an applicable procedure for preparing FIP-fve solution free of FTX.

Combination of on-line desalting and HPLC-UV-ESI-MS for simultaneous detection and identification of FIP-fve and flammutoxin in Flammulina velutipes.

A rapid analytical approach, on-line desalting HPLC-UV-ESI-MS method, for the analysis of FIP-fve and flammutoxin (FTX), two important bioactive proteins in the fruiting bodies of Flammulina velutipes, was developed. In this study, a highly efficient desalting method is provided using molecular weight cut-off centrifugal filtration and on-line desalting. Sample preparation followed by an on-line desalting HPLC-UV-ESI-MS system was employed for simultaneous desalting and detection and identification of FIP-fve and FTX. Results indicated that using trifluoroacetic acid as a modifier on a C18 reversed-phase column renders effective separation. ESI-MS revealed that the apparent molecular masses of FIP-fve and FTX were 12,749.1 Da and 21,912.5 Da, respectively. Eleven milligrams of FIP-fve was obtained from 100 g of fresh fruiting bodies, and UV detection was performed at 280 nm using bovine serum albumin as the standard protein. The calibration curve was linear in the concentration range of 0.29-4.69 mg/mL (r2 = 0.9999). FTX and a series of degradation products were isolated from F. velutipes using 35% saturated ammonium sulfate on a DEAE cellulose column. The complete identification of FTX and a series of degradation products were carried out by precipitation of various ammonium sulfate concentrations (0-45%, 45-65% and 65-90%), in-gel trypsin digestion, and MS analysis with combined database search. The molecular weights of FTX and a series of degradation products were 29,957.2 Da, 27,480.2 Da, 26,512.5 Da, and 21,912.5 Da.

Dose effects of the food spice cardamom on aspects of hamster gut physiology.

The dose effects of pectic polysaccharide-rich extract from the food spice cardamom (Amomum villosum Lour.) on intestinal environment were investigated. The results showed that pectic polysaccharides and hemicellulose were the major polysaccharides in the cardamom extract. The administration of cardamom extract (0.5 and 1.5 g/100 g diet) effectively (p < 0.05) shortened hamster gastrointestinal transit time by approximately 58%, increased fecal moisture contents (148-174%), increased SCFA concentrations in hindgut (4.0- to 7.8-fold), decreased the activities of beta-D-glucuronidase (by 71.4-85.7%), beta-D-glucosidase (by 24.3-51.5%), mucinase (by 63.6-72.7%), and urease (by 88.8-90.4%) in feces, and reduced the production of toxic ammonia (by 16.1-64.5%). These findings suggested that the consumption of cardamom extract (at least 0.5 g/100 g diet or 40 mg/day) might exert a favorable effect on improving the gastrointestinal milieu, and also provide a clue to substantiate its traditional therapeutic uses and dosage for intestinal health improvement.

Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a Polyketide Pathway Using Orsellinic Acid as a Ring Precursor in Antrodia cinnamomea.

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from the polyketide pathway, we cultivated this fungus in a culture medium containing [U-13C]oleic acid, and then we analyzed the crude extracts of the mycelium using UHPLC-MS. We found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2] fragments on the ring backbone were detected in the mass spectrum for [13C2]AQ, [13C2]4-AAQB, and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A. cinnamomea.

Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis.

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1ß, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.