Elsevier

Process Biochemistry

Volume 72, September 2018, Pages 162-169
Process Biochemistry

Lasiosan, a new exopolysaccharide from Lasiodiplodia sp. strain B2 (MTCC 6000): Structural characterization and biological evaluation

https://doi.org/10.1016/j.procbio.2018.06.014Get rights and content

Highlights

Abstract

Ascomycete fungi are promising biofactories producing bioactive compounds exhibiting diverse biological activities. In an ongoing search for bioactive potential of microorganisms from different ecological niches, a promising Lasiodiplodia sp. strain B2 (MTCC 6000) was identified as a producer of exopolysaccharide (designated as Lasiosan). The exopolysaccharide was purified and structurally characterized as glucomannan having glucose and mannose residues (ratio 1:1) with average molecular mass of 29.3 kDa. Lasiosan demonstrated promising antimicrobial and anti-biofilm activities by inhibiting the growth of different Gram-negative, Gram-positive, drug-resistant bacteria and different Candida strains. The purified exopolysaccharide exhibited potential antioxidant activity in terms of good reducing power and scavenging of DPPH free radicals, superoxide anions and lipid peroxyl radicals. Further, the in vitro immune responses of Lasiosan were demonstrated in mouse RAW 264.7 macrophages. Lasiosan inhibited the LPS-stimulated reactive oxygen species (ROS) and nitric oxide (NO) generation in RAW 264.7 macrophages without affecting cell proliferation. Furthermore, Lasiosan significantly down-regulated the production of LPS stimulated proinflammatory mediators such as TNF-α and IL-6 from RAW 264.7 macrophages. This is the first report on Lasiosan exhibiting broad spectrum antimicrobial, anti-biofilm, antioxidant and immunomodulatory activities, which could be explored as a promising candidate for application in biotechnological and biomedical fields.

Introduction

Exopolysaccharides (EPS) produced by some fungal species have recently garnered renewed interest since they exhibited biological response modifying (BRM) activities and possibly function as leads for application in medical therapy [1]. There is growing evidence that bioactivities exhibited by certain EPS are dependent on the chemical structural features of the carbohydrate biopolymer, including monosaccharide composition, molecular weight, the type and configuration of the glycosidic linkages, the degree and size of the branch points, the conformation of the EPS itself, solubility and charge on the polymer [2]. Furthermore, the physiological roles of EPS are mainly related to pathogenicity, quorum sensing, biofilm formation, etc. [3]. The knowledge on biological properties, the structures and fermentation process of different microbial-derived EPS is very important for understanding their physiological activities and industrial applicability. These macromolecular EPS are mainly composed of sugars such as glucose, mannose and uronic acids [4]. EPS acts as a physical barrier to many pathogenic bacteria [5], apart from their anti-tumor [6] and immunostimulatory activities [7]. The fungal diversity is estimated to have 1.5 million species [8] and among them a few fungal genera produce EPS exhibiting diverse biological activities and chemical properties can be explored from an application perspective. Basidiomycete fungi including Ganoderma lucidum, Agaricus blazei, Lentinus edodes and Grifola frondosa were reported to produce a repertoire of diverse bioactive polysaccharides having anti-tumor and immunomodulating properties [9].

In continuation to our earlier efforts on microbial EPS from a biotechnological perspective [[10], [11], [12]], different fungal strains isolated from diverse biosphere zones of India and maintained as in-house culture collection were screened for EPS production. Among them, Lasiodiplodia sp. strain B2 (GenBank accession number of 18S rDNA sequence: KX878985) identified as a EPS producer was isolated earlier from a spoiled banana sample and maintained in our laboratory and also deposited in the Microbial Type Culture Collection, CSIR-Institute of Microbial Technology, Chandigarh, India, bearing accession number MTCC 6000. Lasiodiplodia sp. strain B2, a ascomyceteous fungus belongs to the family Botryosphaeriaceae. The culture on potato dextrose agar (PDA) medium grows rapidly at 28–30 °C, with a light to dark gray aerial mycelium and later produces abundant black pigments, which can be easily noticed from the reverse side of the PDA culture. Based on lactophenol cotton blue staining, the conidia were ellipsoidal, one-celled, hyaline when immature, becoming dark brown and formed septa with age, whilst enclosed within the pycnidia. Normally, the pigmentation and septation happen after they get discharged. Furthermore, the conidia invariably took on a striate appearance. Externally the walls were smooth, but melanin deposited on the inner surface of the walls often gave the conidia a verruculose appearance. The main characteristics were the presence of aseptate paraphyses within the pycnidial conidiomata, but these paraphyses developed septa as they matured. In the present study, the EPS produced by Lasiodiplodia sp. strain B2 was purified, structurally characterized and further evaluated for various biological activities.

Section snippets

Fungal species and fermentation conditions

EPS production by Lasiodiplodia sp. strain B2 was carried out in a minimal salts medium (pH 8) containing (per litre): 20 g maltodextrin, 2 g NaNO3, 5 g MgSO4 and 1 g KH2PO4 and incubated at 30 °C with agitation at 150 rev min−1 in an Innova 43R (New Brunswick Scientific Co. Inc., Edison, New Jersey, USA) rotary shaker for 120 h.

Purification and structural characterization of EPS

After fermentation, the medium was filtered through a muslin cheese cloth to remove the fungal biomass and then the infiltrate was centrifuged at 2000 ×g to obtain a

Purification and structural elucidation of EPS

Lasiodiplodia theobromae is an endophyte to many different fruits and vegetables, while its teleomorphic form, Botryosphaeria rhodina is a plant pathogen causing spoilage of fruits and vegetables worldwide. In the present study, the purified EPS from Lasiodiplodia sp. strain B2 was a white fibrous solid. The final pure EPS of 1.405 g L−1 was obtained from 3 g L−1 of the original isopropanol precipitated EPS. The average molecular weight (Mw) recorded was 29.3 kDa based on HPLC analysis using

Conclusions

Lasiodiplodia sp. strain B2 isolated from spoiled banana sample produced a bioactive exopolysaccharide. After isolation, purification and structural elucidation based on biochemical and spectral analysis, the EPS was identified as a glucomannan and it was named as Lasiosan. Results demonstrated that the exploration for novel fungal polysaccharides seems to be very interesting from a medicinal application perspective, since Lasiosan purified from Lasiodiplodia sp. strain B2 exhibited

Conflicts of interest

The authors declare that there is no conflict of interest with any researcher or funding agency.

Acknowledgement

The authors acknowledge the financial assistance provided in the form of a Senior Research Fellowships by the Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India to Ms. Poornima Mongolla and Ms. Sujitha Pombala.

References (52)

  • F. Liu et al.

    Free radical scavenging activities of mushroom polysaccharide extracts

    Life Sci.

    (1997)
  • H. Wang et al.

    Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader

    Free Radic. Biol. Med.

    (1999)
  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival; application to proliferation and cytotoxicity assays

    J. Immunol. Methods

    (1983)
  • Y. Zhang et al.

    Schizophyllan: a review of its structure, properties, bioactivity and recent developments

    Bioact. Carbohydr. Diet. Fibre

    (2013)
  • J.I. Farina et al.

    Isolation and physicochemical characterization of soluble scleroglucan from Sclerotium rolfsii. Rheological properties, molecular weight and conformational characteristics

    Carbohydr. Polym.

    (2001)
  • Z.M. Wang et al.

    Structural characterization and immunomodulatory property of an acidic polysaccharide from mycelial culture of Cordyceps sinensis fungus Cs-HK1

    Food Chem.

    (2011)
  • E.C. Giese et al.

    Free-radical scavenging properties and antioxidant activities of botryosphaeran and some other β-D-glucans

    Int. J. Biol. Macromol.

    (2015)
  • S. Guo et al.

    Preparation, structural characterization and antioxidant activity of an extracellular polysaccharide produced by the fungus Oidiodendron truncatum GW

    Process Biochem.

    (2013)
  • J.Q. Zheng et al.

    Characterization and antioxidant activity of exopolysaccharide from submerged culture of Boletus aureus

    Process Biochem.

    (2014)
  • K.-Y. Lee et al.

    Macrophage activation by polysaccharide isolated from Astragalus membranaceus

    Int. Immunopharmacol.

    (2005)
  • J. Luo et al.

    Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immunostimulating activity of exopolysaccharides

    Carbohydr. Polym.

    (2009)
  • M.M. Cordova et al.

    Polysaccharide glucomannan isolated from Heterodermia obscurata attenuates acute and chronic pain in mice

    Carbohydr. Polym.

    (2013)
  • B.B.C. Weng et al.

    Toxicological and immunomodulatory assessments of botryosphaeran (β-glucan) produced by Botryosphaeria rhodina RCYU 30101

    Food Chem. Toxicol.

    (2011)
  • K.S.M. Oliveira et al.

    (1→6)- and (1→3) (1→6)-β-glucans from Lasiodiplodia theobromae MMBJ: structural characterization and pro-inflammatory activity

    Carbohydr. Polym.

    (2015)
  • B. Vu et al.

    Bacterial extracellular polysaccharides involved in biofilm formation

    Molecules

    (2009)
  • M.J. Franklin et al.

    Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa which is required for alginate acetylation

    J. Bacteriol.

    (1993)
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