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

Highlights

• An exopolysaccharide (EMP) is prepared from Monascus spp. by liquid fermentation.

• Its physicochemical features and structural characterization are investigated.

• EMP has immunoregulation effects on RAW 264.7 cells.

• EMP exerts these effects via TLR-4-MAPK/NF-κB signaling pathway.

Abstract

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-1β, 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.

Introduction

Polysaccharides, which are a class of important biomacromolecules found widely in plants, animals, and microorganisms, have drawn worldwide attention for their relatively low toxicity and various bioactivities, including antioxidant, hepatoprotective, preventing lipid absorption, and immunomodulatory activities [1], [2]. Several studies have proven the potential application of polysaccharides as immune system adjuvants [3], [4], [5]. For example, the polysaccharides from Laminaria ochroleuca and Porphyraum bilicalis exhibited immunomodulation effects in a dose-dependent manner [6]. Ganoderma lucidum polysaccharides could attenuate microglia-mediated neuroinflammation, regulate cell phagocytosis, and induce behavioral responses [1]. However, owing to overdevelopment, habitat destruction, long growth cycles, high demand, and other reasons, the wildlife sources of polysaccharide resources are slowly declining. Therefore, it is necessary to find methods other than the scaling up of artificial breeding for obtaining the same or similar bioactive polysaccharides that are found in nature. Microbial fermentation is an alternative method for producing large amounts of active substances, which has several notable advantages, including fast growth rate, easy cultivation, cost-effectiveness, and environmental friendliness in comparison with plant extraction [7]. As a result, an increasing number of studies have focused on the production of polysaccharides by either liquid or solid microbial fermentation.

Monascus purpureus is a medicinal and edible fungus with a long history of use, whose fermentation metabolites are widely applied in the food and pharmaceutical industries [8]. M. purpureus secretes a series of beneficial secondary metabolites, including dimerumic acid, γ-aminobutyric acid, Monacolin K, and Monascus pigments, which possess many biological effects, including anti-inflammatory, antifungal, hypoglycemic, anti-oxidant, and anti-tumor properties [9]. M. purpureus polysaccharides, as another important class of metabolites, have also been attracting significant research attention recently. Wang et al. [10] optimized the extraction conditions of M. purpureus polysaccharides, and reported that mildly alkaline extraction conditions [extraction time of 2.3 h, material: solvent ratio of 1:23 (g/mL), 7% NaOH, and 49 °C] significantly increased the yield (10.1%) of Monascus polysaccharides compared with that obtained using hot-water extraction (4.8%). Jia et al. [11] successfully extracted different exopolysaccharides from Monascus, and compared their structures and antioxidant effects. As the important bioactive components, there is accumulating evidence that M. purpureus polysaccharides can exert the immunoregulatory activities. For example, mycelium polysaccharide from Monascus purpureus TY02 could significantly promote cytokines secretion including IL-6, TNF-α, and IL-10 [12]. Polysaccharides from Monascus purpureus was able to activate blood mononuclear cells to inhibit the growth of human leukemic U937 cells [13]. Another study has demonstrated that the polysaccharide obtained from NTU 568 fermentation can elevate reactive oxygen species production and immune activity, including phagocytosis, cytokines productions in leukemic THP-1 cells [14]. In addition, an exopolysaccharide from M. purpureus (Hong Qu) also showed immunoregulatory effects on RAW264.7 cells via promoting cytokines secretion [9]. However, the underlying signaling pathways of the immunological functions of Monascus polysaccharides remain unclear.

Compared with the solid fermentation method, liquid fermentation methods have several advantages, including shorter fermentation times and higher growth rates, along with less labor-intensive processes and easier manipulation. Moreover, it is relatively simple to change the metabolic pathway to increase the production of a metabolite during liquid fermentation [15]. Therefore, in this study, we used liquid fermentation to rapidly produce polysaccharides from M. purpureus in large amounts. We obtained an exopolysaccharide (EMP), and determined its chemical composition, molecular weight (Mw), and monosaccharide composition. Moreover, the immunomodulatory effects of EMP on RAW264.7 cells were examined along with the underlying mechanism. These findings can enhance the value of M. purpureus in future applications.