Probiotics & Antimicro. Prot. https://doi.org/10.1007/s12602-017-9344-0 Antifungal Activity of Lactobacillus pentosus ŁOCK 0979 in the Presence of Polyols and Galactosyl-Polyols Lidia Lipińska1 & Robert Klewicki2 & Michał Sójka2 & Radosław Bonikowski3 & Dorota Żyżelewicz2 & Krzysztof Kołodziejczyk2 & Elżbieta Klewicka 1 # The Author(s) 2017. This article is an open access publication Abstract The antifungal activity of Lactobacillus pentosus Keywords Antifungal activity . Galactosyl-polyols . ŁOCK 0979 depends both on the culture medium and on the Lactobacillus . Metabolites . Polyols . SEM fungal species. In the control medium, the strain exhibited limited antagonistic activity against indicator food-borne molds and yeasts. However, the supplementation of the bac- Introduction terial culture medium with polyols (erythritol, lactitol, maltitol, mannitol, sorbitol, xylitol) or their galactosyl deriva- Filamentous fungi and yeasts are present in almost all types of tives (gal-erythritol, gal-sorbitol, gal-xylitol) enhanced the an- ecosystems due to their high adaptation ability and low nutri- tifungal properties of Lactobacillus pentosus ŁOCK 0979. Its tional requirements. Filamentous fungi are widespread food metabolites were identified and quantified by enzymatic spoilage microorganisms responsible for significant economic methods, HPLC, UHPLC-MS coupled with QuEChERS, losses in the agri-food industry [6]; they are also a major and GC-MS. The presence of polyols and gal-polyols signif- health concern due to mycotoxin production. The most com- icantly affected the acid metabolite profile of the bacterial mon genera of spoilage fungi include Penicillium, Fusarium, culture supernatant. In addition, lactitol and mannitol were Aspergillus, Cladosporium,andRhizopus [21]. Commercial used by bacteria as alternative carbon sources. A number of foodstuffs are usually protected from such microorganisms by compounds with potential antifungal properties were identi- physical and chemical techniques. However, as chemical pre- fied, such as phenyllactic acid, hydroxyphenyllactic acid, and servatives have become less socially acceptable, natural pres- benzoic acid. Lactobacillus bacteria cultivated with mannitol ervation methods are being sought. Lactic acid fermentation synthesized hydroxy-fatty acids, including 2-hydroxy-4- has been known and used these purposes since antiquity. In methylpentanoic acid, a well-described antifungal agent. recent years, lactic acid bacteria (LAB) have been extensively Scanning electron microscopy (SEM) and light microscopy investigated for their antifungal properties and bioprotective confirmed a strong antifungal effect of L. pentosus ŁOCK cultures have been proposed as a promising biotechnological 0979. approach [22, 24, 25]. Of particular application interest are lactobacilli, which convert carbohydrates into lactic and acetic acids (primary metabolites), as well as a range of secondary * Lidia Lipińska metabolites, such as carbon dioxide, ethanol, hydrogen perox- [email protected] ide, fatty acids, acetoin, diacetyl, cyclic dipeptides, bacterio- cins, and bacteriocin-like inhibitory substances [3]. Since 1 Lodz University of Technology, Institute of Fermentation these metabolites exhibit only weak antifungal properties, Technology and Microbiology, Wolczanska 171/173, many research teams are seeking Lactobacillus strains with a 90-024 Lodz, Poland higher natural ability to inhibit fungal and yeast growth [4, 9, 2 Lodz University of Technology, Institute of Food Technology and 14, 15, 26]. Ryu et al. [26] reported that Lactobacillus Analysis, Stefanowskiego 4/10, 90-024 Lodz, Poland plantarum HD1 synthesizes 5-oxododecanoic acid (MW 3 Lodz University of Technology, Institute of General Food Chemistry, 214), 3-hydroxydecanoic acid (MW 188), and 3-hydroxy-5- Stefanowskiego 4/10, 90-024 Lodz, Poland dodecenoic acid (MW 214), which are considered antifungal. Probiotics & Antimicro. Prot. In turn, Magnusson [14, 16] showed that some LAB can erythritol, gal-xylitol, and gal-sorbitol are modern prebiotics convert glycerol to 1,3-propanediol, which inhibits fungal which confer beneficial effects [8], as related in the blood and growth. While the qualitative and quantitative composition digesta of laboratory rats (Klewicki 2007). of antifungal compounds generated by LAB is species- or even strain-specific, it can be modulated by culture medium Synthesis of Galactosyl Derivatives of Erythritol, Sorbitol, modification. For instance, Lipińska et al. [13] adjusted the and Xylitol antifungal spectrum of lactobacilli by adding polyols and their galactosyl derivatives, proving that the antagonistic activity of Galactosyl derivatives of erythritol, sorbitol, and xylitol were LAB depends on culture medium composition, the LAB obtained by enzymatic transglycosylation using β- species, and the sensitivity of the fungal species. It was found galactosidase EC 3.2.1.23 from Kluyveromyces lactis that in the presence of xylitol and gal-xylitol in the bacterial (Novozymes A/S, Bagsvaerd, Denmark). The procedure for culture medium Lactobacillus pentosus ŁOCK 0979 galactosyl-xylitol synthesis was described by Klewicki [11]. effectively inhibited the growth of A. niger, A. alternata, A. brassicicola, F. lateritium,andM. hiemalis [13]. The Determination of Antifungal Activity of Lactobacillus modulation of LAB metabolism by supplementing the culture pentosus ŁOCK 0979 in the Presence of Polyols medium with various, often atypical, compounds may give and Galactosyl-Polyols rise to new systems inhibiting the growth of spoilage microorganisms. The antagonistic activity of L. pentosus ŁOCK 0979 against The objective of the study was to determine the antifungal the indicator fungi was tested using the double-layer method properties, metabolite profile, and enzymatic activity of the described by Lipińska et al. [13]. First, 10 μL of overnight strain L. pentosus ŁOCK 0979 cultured in the presence of bacterial culture was dropped on MRS agar plates (Merck or polyols, namely, erythritol, xylitol, maltitol, mannitol, sorbi- BTL) supplemented with 1% (m/v) polyols, galactosyl- tol, and lactitol, and their transglycosylation derivatives (gal- polyols, or galactose, separately. The control group consisted erythritol, gal-xylitol, and gal-sorbitol). of MRS agar plates (Merck) with lactobacilli colonies cultured with neither polyols nor gal-polyols. After 18–24 h, the plates were overlaid with Sabouraud 4% dextrose agar (Merck) in- Materials and Methods oculated with an indicator fungal strain (105–106 spores × mL−1). Indicator strain inhibition zones around Microbiological Strains and Polyols Lactobacillus sp. colonies were measured after 24–72 h of cultivation at 30 °C. The results were given as fungal inhibi- The study material consisted of the bacterial strain L. pentosus tion diameters minus the diameter of Lactobacillus sp. ŁOCK 0979 and 10 fungal strains deposited with the Pure colonies. Cultures Collection of Industrial Microorganisms of the Institute of Fermentation Technology and Microbiology, Preparation of Cell-Free Supernatant After Lactic Acid Lodz University of Technology (ŁOCK 105). The indicator Fermentation fungi included the yeasts Candida vini 0008 and 0009 and the molds Mucor hiemalis 0519, Geotrichum candidum 0511, Following lactic acid fermentation by L. pentosus ŁOCK Alternaria alternata 0409, Alternaria brassicicola 0412, 0979 in media with one of polyols or galactosyl-polyols, sam- Aspergillus niger 0433, Fusarium lateritium 0508, ples of cell-free supernatant (CFS) were prepared in order to Aspergillus ochraceus,andPenicillium sp. Two of the tested identify and quantify the antifungal agents produced by the fungi, A. ochraceus and Penicillium sp., were newly isolated bacteria in the modified MRS media. from spoiled food. The media consisted of MRS broth (Merck) containing 1% Fungi were grown in Sabouraud 4% dextrose agar (Merck) (m/v) glucose supplemented with 1% (m/v)ofoneofthe and bacteria in MRS medium (Merck) supplemented with 1% polyols (erythritol, lactitol, maltitol, mannitol, sorbitol, or xy- (m/v) polyols (erythritol, lactitol, maltitol, mannitol, sorbitol, litol) or one of the gal-polyols (gal-erythritol, gal-sorbitol, or xylitol) or galactosyl polyols (gal-erythritol, gal-sorbitol, gal- gal-xylitol). The final pH was 5.7 ± 0.2. In the first step of the xylitol). The microorganisms were cultured at 30 °C under experiment, 200 mL of a medium was inoculated with 3% (v/ aerobic conditions. Fungi were stored at 4 °C on Sabouraud v) of overnight L. pentosus ŁOCK 0979 culture (105– dextrose agar slants (Merck), and bacteria were kept at 106 cfu × mL−1),andincubatedfor48hat30°C. − 20 °C in 20% (v/v) glycerol. Subsequently, the samples were centrifuged (10 min, The polyols used in the study (erythritol, lactitol, maltitol, 12,000×g, 20 °C), and the supernatants were filtered using mannitol, sorbitol, xylitol) occur naturally in some foodstuffs 0.22-μm syringe filters. CFS were stored at − 20 °C for further and may be added to others, e.g., as sweeteners. In turn, gal- study. Probiotics & Antimicro. Prot. Determination of the Content of Polyols and Saccharides Quantification of Antifungal Acids Using UHPLC-MS Using HPLC in Conjunction with QuEChERS Each CFS sample was diluted 10-fold. The obtained solution Antifungal metabolites produced by L. pentosus ŁOCK 0979 was passed through a 5-mL column BAKERBOND® spe in the presence of polyols and their galactosyl derivatives were Octadecyl (18) (J.T. Baker, USA) with