Recent advances in polysaccharide biomodification by microbial fermentation: production, properties, bioactivities, and mechanisms.

Polysaccharides are natural chemical compounds that are extensively employed in the food and pharmaceutical industries. They exhibit a wide range of physical and biological properties. These properties are commonly improved by using chemical and physical methods. However, with the advancement of biotechnology and increased demand for green, clean, and safe products, polysaccharide modification via microbial fermentation has gained importance in improving their physicochemical and biological activities. The physicochemical and structural characteristics, biological activity, and modification mechanisms of microbially fermented polysaccharides were reviewed and summarized in this study. Polysaccharide modifications were categorized and discussed in terms of strains and fermentation techniques. The effects of microbial fermentation on the physicochemical characteristics of polysaccharides were highlighted. The impact of modification of polysaccharides on their antioxidant, immune, hypoglycemic, and other activities, as well as probiotic digestive enhancement, were also discussed. Finally, we investigated a potential enzyme-based process for polysaccharide modification via microbial fermentation. Modification of polysaccharides via microbial fermentation has significant value and application potential.

Adding genistein or luteolin decreased the yield of citrinin and without reducing pigments in yam solid-fermentation by Monascus.

BACKGROUND: Chinese yam fermented by Monascus, namely red mold dioscorea (RMD), has the potential of treating diseases. However, the production of citrinin limits the application of RMD. In the present study, the fermentation process of Monascus was optimized by adding genistein or luteolin to reduce citrinin yield. RESULTS: The results showed that citrinin in 25 g of Huai Shan yam was reduced by 48% and 72% without affecting the pigment yield by adding 0.2 g of luteolin or genistein, respectively, to a 250-mL conical flask after fermentation for 18 days at 28 °C, whereas the addition of luteolin increased the content of yellow pigment by 1.3-fold. Under optimal conditions, citrinin in 20 g of iron bar yam decreased by 55% and 74% after adding 0.2 g of luteolin or genistein. Luteolin also increased yellow pigment content by 1.2-fold. Ultra HPLC coupled to quadrupole time-of-flight mass spectrometry was used for the preliminary analysis of Monascus fermentation products. It was found that the amino acid types in RMD are similar to those in yams, but there are fewer polysaccharides and fatty acids. CONCLUSION: The results obtained in the present study showed that the addition of genistein or luteolin could reduce citrinin on the premise of increasing pigment yield, which laid a foundation for the better use of yams in Monascus fermentation. © 2023 Society of Chemical Industry.

Alleviating effects of essential oil from Artemisia vulgaris on enteritis in zebrafish via modulating oxidative stress and inflammatory response.

Artemisia vulgaris (A. vulgaris) is a traditional Chinese medicine widely distributed in China and contains many bioactive compounds with pharmacological effects. However, the anti-inflammatory effects and mechanism of essential oil from A. vulgaris on enteritis in fish are still unclear. In this study, in order to elucidate the underlying mechanism of essential oil from A. vulgaris on zebrafish enteritis, zebrafish were used for establishing animal models to observe the histopathological changes of intestines, determine the activities of immune-related enzymes and oxidative stress indicators, and the mRNA expression of genes in MyD88/TRAF6/NF-KB signaling pathways. The results showed that different doses of A. vulgaris essential oil could effectively alleviate zebrafish enteritis in a dose- and time-dependent manner by improving the intestinal histopathological damage, decreasing the intestinal oxidative stress, repairing the intestinal immune ability, changing the expression levels of IL-1ß, IL-10 and genes in MyD88/TRAF6/NF-κB pathway. In addition, co-treatment with oxazolone and MyD88 inhibitor could alleviate the morphological damage, the induction of oxidative stress, and the levels of immune-related enzymes and the mRNA expression of genes in MyD88/TRAF6/NF-κB signaling pathway. Moreover, essential oil from A. vulgaris had more significantly therapeutic effects on enteritis of male zebrafish than that of female zebrafish. This result will clarify the therapeutic effect and anti-inflammatory mechanism of essential oil from A. vulgaris on zebrafish enteritis, and provide a theoretical basis for further research on the rationality of A. vulgaris to replace feed antibiotics.

Development of an immunochromatographic test strip for the rapid detection of aristolochic acid A in herbal medicinal materials.

INTRODUCTION: Plants containing aristolochic acid and its derivatives are nephrotoxic, mutagenic, and carcinogenic to humans; chronic diet poisoning caused by the aristolochic acid is the cause of endemic (Balkan) nephropathy and related cancers. OBJECTIVE: To develop a colloidal gold immunochromatographic test strip (ICS) based on the competitive format for the rapid detection of aristolochic acid A (AA-A) in herbal medicinal materials. MATERIALS AND METHODS: For the ICS based on gold nanoparticles (AuNPs), the antigen [AA-A-bovine serum albumin (BSA)], and goat anti-mouse IgG were drawn on the nitrocellulose membrane as the test line (T line) and the control line (C line), respectively. Monoclonal antibody (MAb)-AuNP conjugates were sprayed onto the conjugate pad. The sensitivity of the ICS was 6 ng/mL, and the test was completed in 10 min. The analysis of AA-A in traditional Chinese medicine samples showed that the ICS results were in good agreement with those obtained by high-performance liquid chromatography methods. CONCLUSION: These results demonstrated that the ICS test could be used as a reliable, rapid, cost-effective, and convenient qualitative tool for on-site screening techniques to detect AA-A in herbal medicinal materials without any special instrumentation.

Rapid simultaneous detection of fumonisin B1 and deoxynivalenol in grain by immunochromatographic test strip.

Mycotoxins, such as fumonisin B1 (FB1) and deoxynivalenol (DON), are toxic secondary metabolites produced by fungi. Herein, the simultaneous detection of FB1 and DON (both of which are water-soluble) in grain samples by immunochromatographic methods was used to probe whether the levels of these mycotoxins exceeded the limit of 1 mg/kg. Two types of immunochromatographic test strips (ICSs) were developed for this purpose, namely, those based on Au nanospheres (prepared via reduction with trisodium citrate) and Au nanoflowers (prepared by Au seed-mediated growth) as markers, with the respective sensitivities to both FB1 and DON equalling 20 and 5.0 ng/mL. The former ICS was used to detect FB1 and DON in grain (51 wheat samples and 18 maize samples), providing results consistent with those of high-performance liquid chromatography and enzyme-linked immunosorbent assays. The developed technique was suitable for the on-site screening of large-scale samples for FB1 and DON.

Soybean isoflavones reduce citrinin production by Monascus aurantiacus Li AS3.4384 in liquid state fermentation using different media.

BACKGROUND: Monascus, a filamentous fungus, produces many bioactive substances. However, in the process of fermentation, Monascus also produces the mycotoxin citrinin. Owing to the presence of citrinin, the safety of Monascus products has been questioned and their wide application limited. Using soybean isoflavones (SI) as exogenous additives, alterations in citrinin production by Monascus aurantiacus Li AS3.4384 (MALA) in different media used for liquid state fermentation were investigated. RESULTS: Results showed that the citrinin concentration was 95.98% lower than that of the control group after 16-days fermentation when 20.0 g L-1 SI were added to rice powder and inorganic salt medium. Citrinin production was reduced by 97.24% after 12-days fermentation with 10.0 g L-1 SI in starch inorganic salt medium; 82.52% after 20-days fermentation with 20.0 g L-1 SI in starch peptone medium with high starch content; 45.07% after 14-days fermentation with 5.0 g L-1 SI in starch peptone medium with low starch content; and 82.21% after 14-days fermentation with 20.0 g L-1 SI in yeast extract sucrose medium. CONCLUSION: The developed method of removing citrinin is simple, safe, and effective, and it can be applied to reduce the citrinin content of Monascus products. © 2019 Society of Chemical Industry.

Determination of two potential toxicity metabolites derived from the disruption of the pksCT gene in Monascus aurantiacus Li As3.4384.

BACKGROUND: We previously demonstrated that disruption of the pksCT gene of Monascus led to a greater than 98% decrease in its citrinin production capacity in Monascus (PHDS26). Two potentially toxic compounds, monascopyridine A (MPA) and monascopyridine B (MPB), were found in the fermentation products of the pksCT gene-disrupted Monascus. Moreover, a rapid and reliable high-performance liquid chromatography method was developed for the simultaneous determination of MPA and MPB. We studied the effects of various extraction parameters and designed an orthogonal experiment to investigate the importance of each factor. RESULTS: The optimal extraction conditions were: methanol concentration, 90%; extraction temperature, 40 °C; extraction time, 10 min; two extraction cycles; and a solid-liquid ratio of 1:25. Under the optimal chromatographic conditions, good linearity was reached over the concentration ranges 0.5-200 µg mL-1 and 0.5-300 µg mL-1 for MPA and MPB, respectively, and the corresponding determination coefficients were 0.9999 and 0.9997. The percentage relative standard deviation values of within-day and between-day precision for MPA were 2.0% and 2.1%, respectively; the corresponding values for MPB were 4.8% and 4.6%. The average recovery for MPA and MPB was 99.9% and 94%, respectively. CONCLUSION: Maximum MPA and MPB yields (2073.7 and 1961.7 µg g-1 , respectively) were observed after 16 days of cultivation. © 2017 Society of Chemical Industry.

Comprehensive Quantitative Analysis of SQ Injection Using Multiple Chromatographic Technologies.

Quality control of Chinese medicine injections remains a challenge due to our poor knowledge of their complex chemical profile. This study aims to investigate the chemical composition of one of the best-selling injections, Shenqi Fuzheng (SQ) injection (SQI), via a full component quantitative analysis. A total of 15 representative small molecular components of SQI were simultaneously determined using ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS); saccharide composition of SQI was also quantitatively determined by high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) on an amino column before and after acid hydrolysis. The existence of polysaccharides was also examined on a gel permeation chromatography column. The method was well validated in terms of linearity, sensitivity, precision, accuracy and stability, and was successfully applied to analyze 13 SQI samples. The results demonstrate that up to 94.69% (w/w) of this injection product are quantitatively determined, in which small molecules and monosaccharide/sucrose account for 0.18%-0.21%, and 53.49%-58.2%, respectively. The quantitative information contributes to accumulating scientific evidence to better understand the therapy efficacy and safety of complex Chinese medicine injections.

Urchin-like gold nanoparticle-based immunochromatographic strip test for rapid detection of fumonisin B1 in grains.

An immunochromatographic strip (ICS) using urchin-like gold nanoparticles (UGNs) for sensitive detection of fumonisin B1 (FB1) was developed to meet the requirement for rapidly monitoring FB1 in grain samples. The sensitivity of the ICS was 5.0 ng/mL, which represents a fourfold increase in sensitivity over conventional strip preparation using colloidal gold as the antibody-labeled probe. Analysis of FB1 in grain samples showed that data obtained from the strip tests were in a good agreement with those obtained from HPLC and enzyme-linked immunosorbent assays (ELISAs). This qualitative test did not require any specialized equipment, and the detection time was less than 5 min, which is suitable for on-site testing of FB1 in grain samples. Overall, to our knowledge, this is the first report of using a UGN as the antibody-labeled probe for sensitive detection of FB1 in grains using an ICS. Graphical Abstract Preparation of ICS using conventional colloidal gold and urchin-like gold nanoparticle, respectively.

Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1ß, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1ß, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

Effects of synergistic modification using alkalis and guar gum on the pasting, rheological, and microstructural properties of germinated highland barley starch gels.

Germination treatment of highland barley enhances its nutritional value while weakening the starch gel properties. This study aims to enhance the characteristics of germinated highland barley starch (GBS) by exploring the synergistic effects of two alkalis (Na2CO3 and NaHCO3) and guar gum (GG) on GBS gel properties. The combined action of alkalis and GG significantly improved the peak viscosity, setback viscosity, and hardness compared with GG alone. The highest G' and G" reached 998 and 204 Pa at 0.4% Na2CO3 addition, which were increased by nearly 44% and 50%, respectively. Fourier-transform infrared spectral analysis revealed that the alkalis strengthened interaction forces, particularly with intensified absorption peaks at 3200-3700 cm-1 and 1550-1750 cm-1. The Na2CO3 and NaHCO3 reduced the spin-spin relaxation time (T2), resulting in a dense starch gel network. This study contributes to enhancing the market application of GBS and offers innovative insights for modifying other starches.

An Exopolysaccharide from Genistein-Stimulated Monascus Purpureus: Structural Characterization and Protective Effects against DSS-Induced Intestinal Barrier Injury Associated with the Gut Microbiota-Modulated Short-Chain Fatty Acid-TLR4/MAPK/NF-κB Cascade Response.

Inflammatory bowel disease is a major health problem that can lead to prolonged damage to the digestive system. This study investigated the effects of an exopolysaccharide from genistein-stimulated Monascus purpureus (G-EMP) in a mouse model of colitis to clarify its molecular mechanisms and identified its structures. G-EMP (Mw = 56.4 kDa) was primarily consisted of → 4)-α-D-Galp-(1 →, → 2,6)-α-D-Glcp-(1→ and →2)-ß-D-Manp-(1 → , with one of the branches being α-D-Manp-(1 →. G-EMP intervention reduced the loss of body weight, degree of colonic damage and shortening, disease activity index scores, and histopathology scores, while restoring goblet cell production and oxidative homeostasis, repairing colonic functions, and regulating inflammatory cytokines. RNA sequencing and Western blot analysis indicated that G-EMP exerts anti-inflammatory properties by suppressing the TLR4/MAPK/NF-κB inflammatory signaling pathway. G-EMP modulated the gut microbiota by improving its diversities, elevating the relative abundances of beneficial bacteria, declining the Firmicutes/Bacteroidota value, and regulating the level of short-chain fatty acids (SCFAs). Correlation analysis demonstrated strong links between SCFAs, gut microbiota, and the inflammatory response, indicating the potential of G-EMP to prevent colitis.

Metabolomics analysis based on UHPLC-QTOF-MS/MS to explore the synthesis mechanism and culture conditions optimization of Penicillium EF-2 exopolysaccharide.

Penicillium exopolysaccharide (EPS) inhibits galactose lectins and enhances immunity. However, EPS production is low and its synthesis mechanism remains unclear. Penicillium EF-2 strains with high EPS production were selected for this study, and Penicillium fermentation conditions were subsequently improved. The optimal culture conditions were 30 g/L lactose, 6 g/L yeast extract powder, 4 d seed age, 10 % inoculation amount, 3 d of secondary fermentation time, and the final EPS yield was 3.97 g/L. UHPLC-Q-TOF-MS/MS was used to explore the mechanism of EPS synthesis at the metabolic level. Optimal carbon source: lactose and optimal nitrogen source: yeast extract can provide precursors for EPS synthesis through related metabolic pathways. Moreover, regulating the energy, vitamin, and lipid metabolic pathways created favourable conditions for EPS synthesis and secretion. These findings explain the mechanism of EPS synthesis at the metabolic level and provide a theoretical basis for optimising and industrialising EPS production.

Rapid and Simultaneous Detection of Aflatoxin B1, Zearalenone, and T-2 Toxin in Medicinal and Edible Food Using Gold Immunochromatographic Test Strip.

(1) Background: Medicinal and edible food and traditional Chinese medicine have been used to treat various diseases. However, their safety has not been thoroughly assessed. (2) Methods: An immunochromatographic test strip (ICS) was used for the first time to screen some mycotoxins, including aflatoxin B1 (AFB1), zearalenone (ZEN), and T-2 toxin, in medicinal and edible food and traditional Chinese medicine. Antibody/nano-gold particle coupling was used with the prepared ICS, and the pH, monoclonal antibody concentration, and antigen amount were optimized. The extraction sample solution was diluted 10 times with phosphate-buffered saline containing 0.5% Tween-20 and 0.05% sodium dodecyl sulfate to remove the complex matrix in medicinal and edible food. (3) Results: Under optimal conditions, the sensitivities of the developed ICS for AFB1, ZEN, and T-2 were 0.5, 5.0, and 5.0 ng/mL, respectively. Among the 30 medicinal and edible food samples tested, two samples (both of sand jujube kernels) were positive, and the results were verified by high-performance liquid chromatography and enzyme-linked immunosorbent assay and were consistent with the ICS test results. (4) Conclusions: The ICS could be used for rapid screening and simultaneous detection of mycotoxins at medicinal and edible food storage facilities.

Contribution of Quercetin to the Composition and Antioxidant Properties of Monascus Exopolysaccharides.

Exopolysaccharides are important metabolites of Monascus with healthy activities. However, the low production level limits their applications. Hence, the aim of this work was to increase the yield of exopolysaccharides (EPS) and optimize liquid fermentation by adding flavonoids. The EPS yield was optimized via both medium composition and culture conditions. The optional fermentation conditions achieved for EPS production of 7.018 g/L were 50 g/L sucrose, 3.5 g/L yeast extract, 1.0 g/L MgSO4·7H2O, 0.9 g/L KH2PO4, 1.8 g/L K2HPO4·3H2O, 1 g/L quercetin, and 2 mL/L Tween-80, with pH 5.5, inoculum size 9%, seed age 52 h, shaking speed 180 rpm, and fermentation culture 100 h, respectively. Furthermore, the addition of quercetin increased EPS production by 11.66%. The results also showed little citrinin residue in the EPS. The exopolysaccharides' composition and antioxidant capacity of quercetin-modified exopolysaccharides were then preliminarily investigated. The addition of quercetin changed the composition of the exopolysaccharides and the molecular weight (Mw). In addition, the antioxidant activity of Monascus exopolysaccharides was monitored using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+), and -OH. Monascus exopolysaccharides have good scavenging ability of DPPH and -OH. Furthermore, quercetin increased the scavenging ABTS+ ability. Overall, these findings provide a potential rationale for the application of quercetin in improving the EPS yield.

Development of two immunochromatographic test strips based on signal amplification and selenium nanoparticles for the rapid detection of T-2 mycotoxin.

Conventional immunochromatographic test strips (ICSs) based on gold nanoparticle (AuNP) probes offer limited sensitivity. Here, AuNPs were separately labeled with monoclonal or secondary antibodies (MAb or SAb). In addition, spherical, homogeneously dispersed, and stable selenium nanoparticles (SeNPs) were also synthesized. By optimizing the preparation parameters, two ICSs based on the dual AuNP signal amplification (Duo-ICS) or SeNPs (Se-ICS) were developed for the rapid detection of T-2 mycotoxin. The detection sensitivities of the Duo-ICS and Se-ICS assays for T-2 were 1 ng/mL and 0.25 ng/mL, respectively, which were 3-fold and 15-fold more sensitive, respectively, than a conventional ICS. Furthermore, the ICSs were applied in the detection of T-2 in cereals, which requires higher sensitivity. Our findings indicate that both ICS systems can be used for rapid, sensitive, and specific detection of T-2 toxin in cereals and potentially other sample types.

Monacolin K Induces Apoptosis of Human Glioma U251 Cells by Triggering ROS-Mediated Oxidative Damage and Regulating MAPKs and NF-κB Pathways.

Monacolin K (MK), a polyketo secondary metabolic compound of the mold genus Monascus, can promote the apoptosis of malignant cancer cells, possessing potential antitumor properties. However, its mechanism of action on gliomas remains unclear. Here, we explored and investigated the potential of the monacolin K's antitumor effect on human glioma U251 cells and its possible molecular mechanism. Results showed that the application of 10 µM monacolin K inhibited the proliferation of U251 cells, with an inhibitory rate of up to 53.4%. Additionally, monacolin K induced the generation of reactive oxygen species and activated mitochondria-mediated pathways, including decreased MMP, activation of caspase3/caspase9, decreased Na+/K+-ATPase and Ca2+-ATPase activities, and disruption of the antioxidant system, resulting in the disruption of intracellular reduction-oxidation homeostasis. Monacolin K also activated MAPK and NF-κB pathways, upregulating P38 activity and downregulating JNK/ERK/P65/IκBα expression, ultimately leading to apoptosis of U251 cells. Importantly, monacolin K was not cytotoxic to normal human cells, hUC-MSCs. We concluded that monacolin K can induce apoptosis in U251 cells by triggering ROS-mediated oxidative damage and regulating MAPKs and NF-κB pathways.

Exopolysaccharides from Genistein-Stimulated Monascus purpureus Ameliorate Cyclophosphamide-Induced Intestinal Injury via PI3K/AKT-MAPKs/NF-κB Pathways and Regulation of Gut Microbiota.

Exopolysaccharides from genistein-stimulated Monascus purpureus (G-EMP) exhibited immunomodulatory potential in vitro, but whether it had immune-enhancing effects in vivo and its potential mechanism are not yet known. Here, the immunomodulatory effects of G-EMP were investigated by establishing an immunosuppressed mouse model treated with cyclophosphamide (Cy). The results suggested that G-EMP effectively alleviated the signs of weight reduction and diet reduction caused by Cy, increased fecal water content and splenic index, and decreased the oxidative stress of the liver. Simultaneously, G-EMP improved Cy-induced intestinal injury by restoring villus length, increasing the number of cupped cells, upregulating the expression of mucin and tight junction proteins, and downregulating the ratio of apoptotic proteins (Bax/Bcl-2). It also boosted the levels of mouse colonic cytokines, CD4+ and CD8+ T cells. Additionally, G-EMP markedly enhanced immunomodulation via the activation of PI3K/AKT-MAPKs/NF-κB signal pathways. Furthermore, G-EMP intervention displayed a positive association with most immunological indexes by elevating the levels of short-chain fatty acids, varying gut microbiota composition, and enhancing beneficial bacteria (Lactobacillaceae, Prevotellaceae, and S24-7). These findings demonstrated that G-EMP can strengthen immunity, repair intestinal mucosal damage, regulate gut microbiota, and be a potential source of prebiotics.

Isolation and characterization of the citrinin biosynthetic gene cluster from Monascus aurantiacus.

Monascus aurantiacus produces high amounts of citrinin which is a mycotoxin with nephrotoxic activity. Six putative citrinin biosynthesis genes have been discovered in M. purpureus and at least 10 genes are responsible for its biosynthesis. However, the sequence of citrinin pathway gene cluster in M. aurantiacus has not been reported. Here, the putative sequence of citrinin biosynthetic gene cluster was obtained by a PCR-based strategy for screening a genome fosmid library of M. aurantiacus. A sequence of 43 kb revealed 16 ORFs including the six putative biosynthetic genes reported previous. The putative gene cluster consists of a polytekide synthetase encoding one PKS module, an oxidoreductase gene, three dehydrogenase genes, an acyl-coenzyme A synthetase gene, a membrane transport protein gene, a transcriptional activator gene as well as genes encoding proteins of undefined function.

Immunomodulation effect of polysaccharides from liquid fermentation of Monascus purpureus 40269 via membrane TLR-4 to activate the MAPK and NF-κB signaling pathways.

A polysaccharide (EMP) was prepared from Monascus purpureus 40,269 through liquid fermentation, and its immunoregulatory effect was investigated to find clues regarding its potential applicability. Structural characterization demonstrated that EMP, with a molecular weight of 83.1 kDa, consists of Xyl, Man, Gal, Ara, Rha, Glc, GalA, and GlcA at a molar ratio of 2.6: 22: 35.1: 7: 1: 29.2: 2.7: 0.6. Immunomodulatory assays involving RAW264.7 cells indicated that EMP exhibits significantly enhanced pinocytic and phagocytic capacities and promotes the secretion of reactive oxygen species, nitric oxide, and cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) by activating RAW264.7 cells. Moreover, EMP is recognized by Toll-like receptor 4 (TLR-4) and has an immunomodulatory effect by activating the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways in RAW264.7 cells. Collectively, these results demonstrate that EMP could be used as a functional food for immunological reagents.