Enhanced production and immunomodulatory activity of levan from the acetic acid bacterium, Tanticharoenia sakaeratensis Pornpun Aramsangtienchai a,b, ⁎, Titapa Kongmon a , Sirintip Pechroj a , Klaokwan Srisook a,b a Department of Biochemistry and Research Unit of Natural Bioactive Compounds for Healthcare Products Development, Faculty of Science, Burapha University, Chonburi 20131, Thailand b Center of Excellence for Innovation in Chemistry, Burapha University, 20131, Thailand abstract article info Article history: Received 17 May 2020 Received in revised form 30 June 2020 Accepted 1 July 2020 Available online 04 July 2020 Keywords: Levan Exopolysaccharides Acetic acid bacteria Tanticharoenia sakaeratensis Levan is a fructose polymer with β-(2 → 6) glycosidic linkages. It is produced by several microorganisms, and due to its potential biotechnological and industrial applications, various levan-producing bacteria with different levels of production efficiencies have been reported. We investigated the levan-producing ability of the acetic acid bacterium, Tanticharoenia sakaeratensis. The exopolysaccharides produced by the bacterium under a sucrose environment were characterized as levan by FT-IR, and 1 H and 13 C NMR. The molecular weight of levan thus pro- duced range from 1.0 × 10 5 –6.8 × 10 5 Da. The maximum yield of levan from T. sakaeratensis is 24.7 g·L -1 in a liquid medium containing 20% (w/v) sucrose and incubated at 37 °C, 250 RPM for 35 h. The levan produced by T. sakaeratensis can promote nitric oxide production in RAW264.7 macrophage cells in a concentration- dependent manner, suggesting it has immunomodulatory effects. Our study reveals that T. sakaeratensis can be potentially employed as a new source of levan for industrial applications. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Exopolysaccharides (EPS) are a type of polymer synthesized and se- creted by various microorganisms. Well-known examples of bacterial EPS that have been used in the food and drug industries are levan, dex- tran, curdlan, and hyaluronan. Levan is a fructose homopolymer natu- rally produced by certain plants and microorganisms. Microorganisms use levan to enhance survival under stress conditions and it can also be utilized for energy storage under starvation conditions [1]. The de- gree of polymerization of microbial levan varies from 40 to more than 100,000 units, which is higher than that of plant-derived levan [2]. Levan is synthesized by levansucrase (E.C.2.4.1.10) that catalyzes hy- drolytic and transfructosylation activities, using sucrose as its substrate. Thus far, levan displays several interesting biological activities, includ- ing antioxidant, anti-inflammatory, and anti-cholesterol activities. It has also been shown to display biocompatibility, bioadhesivity, low in- trinsic viscosity, and low toxicity [1,3]. In addition, levan-based nano- structured systems for drug delivery, biomedical, and foods industries have gained considerable attention [4]. Levan is, therefore, a polymer with high potential for applications in various industrial sectors. As a consequence, searching for new levan-producing bacterial strains is a constant undertaking. Acetic acid bacteria are gram negative, strictly aerobic, with ellipsoi- dal to rod shapes. They belong to the family Acetobacteraceae and their major characteristic is their ability to oxidize alcohols, sugars, and sugar alcohols to acid products. Several acetic acid bacteria have been recognized for their ability to secrete levan extracellularly in a sucrose-based environment. Examples include Asaia bogorensis [5], Kozakia baliensis DSM14400, Neoasaia chiangmaiensis NBRC101099, Gluconobacter frateurii TMW2.767 [6], Gluconacetobacter xylinus I-2281 [7], and Gluconacetobacter diazotrophicus [8]. Among these strains, K. baliensis produces markedly high molecular weights of levan [6]. In this study, we explored the levan-producing ability of Tanticharoenia sakaeratensis, a bacterium isolated from soil collected in Thailand and recently characterized as a novel, osmotolerant acetic acid bacterium by Yukphan et al. [9]. We characterized the structure and molecular weight of levan from T. sakaeratensis and determined the cultural conditions required to obtain its maximum yield. Fermenta- tion time, agitation rate, and sucrose concentration were optimized by varying one factor at a time. Moreover, the effect of levan from T. sakaeratensis on nitric oxide (NO) production in RAW264.7 macro- phages cells was examined for possible immunomodulatory activity. 2. Materials and methods 2.1. Microorganism, cell culture, and chemical reagents T. sakaeratensis was obtained from the Thailand Bioresource Re- search Center (TBRC; cat#TBRC22). Nutrient broth (NB) and nutrient International Journal of Biological Macromolecules 163 (2020) 574–581 ⁎ Corresponding author at: Department of Biochemistry and Research Unit of Natural Bioactive Compounds for Healthcare Products Development, Faculty of Science, Burapha University, Chonburi 20131, Thailand. E-mail address: pornpun.ar@go.buu.ac.th (P. Aramsangtienchai). https://doi.org/10.1016/j.ijbiomac.2020.07.001 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac