Bioorganic Chemistry 93 (2019) 102787 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Fermentation parameters and conditions affecting levan production and its T potential applications in cosmetics ⁎ Marta Domżał-Kędziaa, Agnieszka Lewińskab, , Anna Jarominc, Marek Weselskib, ⁎ Robert Pluskotad, Marcin Łukaszewicza, a Department of Biotransformation, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland b Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland c Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383 Wroclaw, Poland d InventionBio Sp. z o.o., Wojska Polskiego 65 st., 85-825 Bydgoszcz, Poland ARTICLE INFO ABSTRACT Keywords: Levan is a polysaccharide composed of fructose units with β-2,6-glycoside bonds. Microorganisms synthesize Biocatalysis levan by levansucrase as a mixture of low- and high-molecular-weight fractions. Due to its properties, it has a Bacillus subtilis wide range of applications in cosmetics, pharmaceuticals, food and medicine; it appears that the molecular Levan weight of levan might impact its industrial use. To obtain one fraction of levan after biotransformation, ethanol Isolation precipitation with an increasing volume of alcohol was conducted. This precipitation process was also optimized. Purification Several types of analyses were used. Low-molecular-weight levan was evaluated for toxicity in a normal human Structural characterization Cytotoxicity dermal fibroblast cell line and hemolytic potential on human erythrocytes. Levan was found to be non-cytotoxic Hemolysis and non-hemolytic in concentrations ranging from 0.01 to 1.00 mg/ml. Moreover, levan demonstrated anti- Antioxidant activity oxidant potential expressed as an ability to inhibit of oil/water emulsion oxidation and DPPH radical scavenging. 1. Introduction keratinocyte proliferation [3], it shown no-toxic sensitization for skin or eyes in The Human Repeated Insulin Patch (HRIPT) and Chorioallantoic The current era of human society raised the application of natural Membrane Vascular Assay (CAMVA) tests [4], and it soothes skin irri- sources in personal care especially in cosmetics, which contact with the tations caused by skin irritants such as sodium laureth sulfate (SLS) epidermis, hair system, oral cavity, nails and plays a vital role in en- [3,5,6]. Levan has potential in discolouration products because it can hancing human body appearance, therefore their active ingredients prevent melanin production by reducing tyrosinase activity [7]. The should be biocompatible. Several bacterial polysaccharides possess addition of ascorbic acid to such a levan product results in increased properties that render them suitable for use in cosmetic applications. stability to oxidation [8]. It can also co-create a polymer matrix in The most relevant are xanthan gum and gellan gum that are mostly cosmetics in the form of a solid foil that disappears when applied to the used as base substances in cosmetic vehicles and bacterial cellulose, skin [9]. Levan has high solubility in oil, low viscosity and it is stable in hyaluronic acid, and levan. Biodegradable polymers, especially poly- high temperature [10,11]. Due to the ability of levan to create films and saccharides, attract the attention of scientists looking for new com- its low viscosity, it can be used in hair-fixing products [4,12,13]. The pounds with cosmetic properties, because they can be eliminated by Cosmetic Ingredient Review (CIR) Expert Panels have concluded levan normal metabolic pathways [1,2]. According to the research of many to be safe for use in cosmetic formulations [14]. scientists, levan can be an attractive raw material for cosmetic for- Levan is a polysaccharide consisting of fructose units linked with β- mulations for various purposes. These tests were performed on levan 2,6-glycoside bonds in its main chain and β-2,1 in its branches. It is from other microorganism cultures than B. subtilis, but there is a lack of synthesized from sucrose by a wide range of organisms such as bacteria information about the usefulness of levan in cosmetics of a specific (Bacillus subtilis, Zymomonas mobilis, Erwinia herbicola) and fungi molecular weight [3]. The use of levan in cosmetics, due to its prop- (Aspergillus sydowii, A. versicolor) [15–17]. Levan was described for the erties can be extensive. According to the Transepidermal Water Loss first time in 1881 by Lipmann, who used the term lävulan. The nameof (TEWL) test, the moisturizing effect of levan is comparable to that after the polymer we know today—levan—was proposed in 1901 by bac- using hyaluronic acid and stimulates human fibroblasts and teriologist Greig-Smith [18]. He found a Bacillus strain that grown on ⁎ Corresponding authors. E-mail addresses: [email protected] (A. Lewińska), [email protected] (M. Łukaszewicz). https://doi.org/10.1016/j.bioorg.2019.02.012 Received 14 December 2018; Received in revised form 2 February 2019; Accepted 4 February 2019 Available online 11 February 2019 0045-2068/ © 2019 Published by Elsevier Inc. M. Domżał-Kędzia, et al. Bioorganic Chemistry 93 (2019) 102787 sucrose and produced a polymer. Greig-Smith named the strain levan from BioShop®. Levan standard (E. herbicola), sorbitan sesquioleate, for its levorotation of polarized light and its properties, which are si- fetal bovine serum, 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) and MTT milar to those of dextran [18,19]. Levans of microbial origin are (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were characterized by a high molecular weight exceeding 500 kDa; they can purchased from Sigma-Aldrich (Poznan, Poland). Flaxseed oil was from be linear or branched and have a wide spectrum of applications [16]. Oleofarm (Wroclaw, Poland). L-glutamine, penicillin, streptomycin and Microbial levan is synthesized by levansucrase, an extracellular en- amphotericin B were from Life Technologies (Warsaw, Poland). Normal zyme, which carries out sucrose hydrolysis as well as transfructosyla- human dermal fibroblast cell line and minimum essential medium eagle tion reactions with sucrose molecule or fructooligosaccharide chains as alpha modifications medium were purchased from Lonza (Warsaw, acceptors [20,21]. It was previously reported that the biosynthesis of Poland). All chemicals and reagents not specified in the text were of low- and high-molecular-weight (LMW and HMW, respectively) levan analytical grade. Deionized double distilled water was used for all of co-occurs [22]. The mechanisms by which LMW and HMW levan is the experiments. produced have not yet been determined [23]. High-molecular-weight levan possesses antitumor activity [28,29]. High-molecular-weight 2.1. Bacterial strain and culture conditions particles contribute to lowering blood cholesterol [30]. The use of LMW polymer results in more efficient encapsulation capacity due to the slow B. subtilis natto KB1 strain was isolated from “Soybean Powder” and constant drug release and HMW polymer has a poorer encapsula- Natto Culture Starter produced by Proster®(Korea) [34]. The pre-cul- tion capacity and impairs the release efficiency of the active ingredient ture was incubated for 24 h at 37 °C in 10 ml of LB medium (10 g/L contained in the capsules [14,31]. Levan, a product of levansucrase bacto-tryptone, 5 g/L bacto-yeast extract, 10 g/L NaCl; BioShop® (Ca- biocatalysis, has many applications in biomedicine and health as, for nada); Lab Empire® (Poland) with agitation (180 rpm). For levan pro- example, a drug carrier and a prebiotic [24]. Levan can self-assemble in duction, the KB1 strain was grown in a sucrose-rich medium proposed water into nanoparticles, which makes it an interesting encapsulation by Shih and Yu [35] that consisted of MgSO4·7H2O (0.5 g/L) agent [25,26]. Levan is also used as a color and color enhancer in the NaH2PO4·2H2O (3 g/L), Na2HPO4·12H2O (3 g/L), sucrose (50 g/L) and production of tablets and capsules [27]. The addition of levan to a sodium salt of L-glutamic acid (15 g/L). B. subtilis natto KB1 was culti- matrix supporting capsules enables faster dissolution and a faster vated for 9 days at 37 °C in 50 ml of medium with agitation (180 rpm). therapeutic effect of the active ingredient. These compositions are mainly used in drug formulations that are intended to release the active 2.2. Optimization of the precipitation process ingredient in the oral cavity [28]. Levan production on an industrial scale is associated with many The precipitation process was optimized by setting optimal para- problems such as low process efficiency and pollution of the products meters such as the temperature of the precipitating agent, the rate of its obtained [29]. On the laboratory scale, levan is purified in several ways addition and the speed of the mixing during the precipitation. Three by, for example, re-dissolving the precipitate, removing insoluble par- temperatures of the precipitating agent were tested: 22 °C; +8 ÷ +4°C; ticles from it and re-precipitating, dialyzing the dissolved precipitate −23 °C. The mixing speed of the entire system during the ethanol ad- and ultrafiltration [30–32]. The lack of a suitable way to purify the dition was tested over a range of 200–600 rpm. The speed of ethanol product on a large scale presents a challenge. One must also note a large incorporation was also determined. amount of ethanol left after precipitation from the culture medium. Lyophilization and vacuum drying of obtained levan are commonly 2.3. Fourier-transform infrared analysis employed on a laboratory scale [27,33], but it is not possible to scale up these methods to industrial production. Functional groups and chemical bonds of the obtained levan were This study aimed to obtain levan with a low molecular weight and to determined using Fourier-transform infrared (FT-IR) spectroscopy on determine an easy way to separate LMW and HMW molecules. To separate Spektrometr Bruker Vertex 70 FT-IR.