An exopolysaccharide (EPS) synthesizing potentially probiotic Gram-positive bacterial strain was isolated from fish (Tor putitora) gut, and its EPS was structurally characterized. The isolate, designated as FW2, was identified as Lactobacillus reuteri through 16S rRNA gene sequencing and phylogenetic analysis. This isolate produces fructan-type EPS using sucrose as a substrate. Based on 13C-NMR spectroscopy, methylation analysis and monosaccharide composition, the EPS was identified as a linear levan polymer with fructose as main constituent linked via β(2 → 6) linkages. Based on molecular weight (MW) distribution, two groups of levan were found to be produced by the isolate FW2: one with high MW (4.6 × 106 Da) and the other having much lower MW (1.2 × 104 Da). The isolate yielded about 14 g/L levan under optimized culturing parameters including aeration conditions, pH, temperature and substrate concentration. The obtained bimodal molecular weight linear levan is the first of its type to be synthesized by a L. reuteri isolate from fish gut. Bimodal molecular weight prebiotic levan together with the probiotic potential of the producing strain would provide a new promising synbiotic combination for use in aqua culture.

Department of Microbiology Abbottabad University of Science and Technology Havelian Abbottabad Pakistan

Industrial Biotechnology Division National Institute for Biotechnology and Genetic Engineering College Pakistan Institute of Engineering and Applied Sciences Faisalabad 38000 Punjab Pakistan

School of Chemical and Biomedical Engineering Nanyang Technological University Block N1 2 62 Nanyang Drive Western Part of Singapore 637459 Singapore

Zobrazit více v PubMed

Abdel-Fattah AM, Gamal-Eldeen AM, Helmy WA, Esawy MA (2012) Antitumor and antioxidant activities of levan and its derivative from the isolate Bacillus subtilis NRC1aza. Carbohydr Polym 89:314–322. https://doi.org/10.1016/j.carbpol.2012.02.041 PubMed DOI

Abid Y, Azabou S, Casillo A, Gharsallah H, Jemil N, Lanzetta R, Attia H, Corsaro MM (2019) Isolation and structural characterization of levan produced by probiotic Bacillus tequilensis-GM from Tunisian fermented goat milk. Int J Biol Macromol 133:786–794. https://doi.org/10.1016/j.ijbiomac.2019.04.130 PubMed DOI

Ammar YB, Matsubara T, Ito K, Iizuka M, Limpaseni T, Pongsawasdi P, Minamiura N (2002) Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature. J Biotechnol 99:111–119. https://doi.org/10.1016/S0168-1656(02)00160-8 PubMed DOI

Anadón A (2006) WS14 The EU ban of antibiotics as feed additives (2006): alternatives and consumer safety. J Vet Pharmacol Ther 29:41–44. https://doi.org/10.1111/j.1365-2885.2006.00775_2.x DOI

Anwar MA, Kralj S, Pique AV, Leemhuis H, van der Maarel MJ, Dijkhuizen L (2010) Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products. Microbiol 156:1264–1274. https://doi.org/10.1099/mic.0.036616-0 DOI

Anwar MA, Kralj S, van der Maarel MJ, Dijkhuizen L (2008) The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme. Appl Environ Microbiol 74:3426–3433. https://doi.org/10.1128/AEM.00377-08 PubMed DOI PMC

Ashfaq I, Amjad H, Ahmad W et al (2020) Growth inhibition of common enteric pathogens in the intestine of broilers by microbially produced dextran and levan exopolysaccharides. Curr Microbiol 77:2128–2136. https://doi.org/10.1007/s00284-020-02091-3 PubMed DOI

Badel S, Bernardi T, Michaud P (2011) New perspectives for Lactobacilli exopolysaccharides. Biotechnol Adv 29:54–66. https://doi.org/10.1016/j.biotechadv.2010.08.011 PubMed DOI

Byun BY, Lee SJ, Mah JH (2014) Antipathogenic activity and preservative effect of levan β→ (2, 6 fructan), a multifunctional polysaccharide. Int J Food Sci Tech 49:238–245. https://doi.org/10.1111/ijfs.12304 DOI

Calazans GM, Lima RC, de França FP, Lopes CE (2000) Molecular weight and antitumour activity of Zymomonas mobilis levans. Int J Biol Macromol 27:245–247. https://doi.org/10.1016/S0141-8130(00)00125-2 PubMed DOI

Carpita NC, Shea EM (1989) Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. Analysis of Carbohydrates by GLC and MS 9:157–216

Chau K, Lau E, Greenberg S, Jacobson S, Yazdani-Brojeni P, Verma N, Koren G (2015) Probiotics for infantile colic: a randomized, double-blind, placebo-controlled trial investigating Lactobacillus reuteri DSM 17938. J Pediatr 166:74-78 e71. https://doi.org/10.1016/j.jpeds.2014.09.020 PubMed DOI

Chen M-H, Kaur P, Dien B, Below F, Vincent ML, Singh V (2013) Use of tropical maize for bioethanol production. World J Microbiol Biotechnol 29:1509–1515. https://doi.org/10.1007/s11274-013-1317-1 PubMed DOI

Combie J (2006) Properties of levan and potential medical uses. In: Marchessault RH, Ravenelle F, Zhu XX (eds) Polysaccharides for drug delivery and pharmaceutical applications. ACS Publications, Washington, p 263–269. https://doi.org/10.1021/bk-2006-0934

Djurić A, Gojgić-Cvijović G, Jakovljević D, Kekez B, Kojić JS, Mattinen M-L, Harju IE, Vrvić MM, Beškoski VP (2017) Brachybacterium sp. CH-KOV3 isolated from an oil-polluted environment–a new producer of levan. Int J Biol Macromol 104:311–321. https://doi.org/10.1016/j.ijbiomac.2017.06.034 PubMed DOI

Dong CX, Zhang LJ, Xu R, Zhang G, Zhou YB, Han XQ, Zhang Y, Sun YX (2015) Structural characterization and immunostimulating activity of a levan-type fructan from Curcuma kwangsiensis. Int J Biol Macromol 77:99–104. https://doi.org/10.1016/j.ijbiomac.2015.03.009 PubMed DOI

Dong C, Wang J (2013) Immunostimulatory effects of dietary fructooligosaccharides on red swamp crayfish, Procambarus clarkii (Girard). Aquac Res 44:1416–1424. https://doi.org/10.1111/j.1365-2109.2012.03146.x DOI

Faggio C, Fazio F, Marafioti S, Arfuso F, Piccione G (2015) Oral administration of Gum Arabic: effects on haematological parameters and oxidative stress markers in Mugil cephalus. Iran J Fish Sci 14:60–72. https://doi.org/10.22092/IJFS.2018.114423

French AD (1989) Chemical and physical properties of fructans. J Plant Physiol 134:125–136. https://doi.org/10.1016/S0176-1617(89)80044-6 DOI

Gatlin III D (2002) Nutrition and fish health. In: Halver J, Hardy RW (eds) Fish Nutrition. Academic Press, San Diego, California

Gibson GR, Hutkins R, Ellen M et al (2017) Expert consensus document: the International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol 14:491–502. https://doi.org/10.1038/nrgastro.2017.75 PubMed DOI

Gojgic-Cvijovic G, Jakovljevic D, Loncarevic B, Todorovic N, Pergal MV, Ciric J, Loos K, Beskoski V, Vrvic M (2019) Production of levan by Bacillus licheniformis NS032 in sugar beet molasses-based medium. Int J Biol Macromol 121:142–151. https://doi.org/10.1016/j.ijbiomac.2018.10.019 PubMed DOI

Goncalves BCM, Baldo C, Celligoi M (2015) Levan and levansucrase–a mini review. Int J Sci Technol Res 4:100–104

Han J, Feng H, Wang X, Liu Z, Wu Z (2021) Levan from Leuconostoc citreum BD1707: production optimization and changes in molecular weight distribution during cultivation. BMC Biotechnol 21:1–14. https://doi.org/10.1186/s12896-021-00673-y DOI

Han X, Yang Z, Jing X, Yu P, Zhang Y, Yi H, Zhang L (2016) Improvement of the texture of yogurt by use of exopolysaccharide producing lactic acid bacteria. Biomed Res Int. https://doi.org/10.1155/2016/7945675 PubMed DOI PMC

Hou Y, Huang F, Yang H et al (2021) Factors affecting the production and molecular weight of levan in enzymatic synthesis by recombinant Bacillus subtilis levansucrase SacB-T305A. Polym Int 70:185–192. https://doi.org/10.1002/pi.6112 DOI

Kırtel O, Lescrinier E, Van den Ende W, Öner ET (2019) Discovery of fructans in Archaea. Carbohydr Polym 220:149–156. https://doi.org/10.1016/j.carbpol.2019.05.064 PubMed DOI

Kralj S, van Geel-Schutten G, Dondorff M, Kirsanovs S, Van Der Maarel M, Dijkhuizen L (2004) Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains. Microbiol 150:3681–3690. https://doi.org/10.1099/mic.0.27321-0 DOI

Louis P, Flint H, Michel C (2016) Microbiota of the human body. Springer Basel, Switzerland

Martín R, Langella P (2019) Emerging health concepts in the probiotics field: streamlining the definitions. Front Microbiol 10:1047. https://doi.org/10.3389/fmicb.2019.01047 PubMed DOI PMC

McGinnis GD, Lave ML, Biermann CJ (1988) High performance liquid chromatography (HPLC) of carbohydrates. Analysis of Carbohydrates by GLC and MS. CRC Press Boca Raton, Florida, pp 87–117

Morales-Arrieta S, Rodríguez ME, Segovia L, López-Munguía A, Olvera-Carranza C (2006) Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F. Gene 376:59–67. https://doi.org/10.1016/j.gene.2006.02.007 PubMed DOI

Mozzi F, Savoy de Giori G, Font de Valdez G (2003) UDP-galactose 4-epimerase: a key enzyme in exopolysaccharide formation by Lactobacillus casei CRL 87 in controlled pH batch cultures. J Appl Microbiol 94:175–183. https://doi.org/10.1046/j.1365-2672.2003.01821.x PubMed DOI

Nasir A, Sattar F, Ashfaq I et al (2020) Production and characterization of a high molecular weight levan and fructooligosaccharides from a rhizospheric isolate of Bacillus aryabhattai. LWT 123:109093. https://doi.org/10.1016/j.lwt.2020.109093 DOI

Ozimek LK, Kralj S, Van der Maarel MJ, Dijkhuizen L (2006) The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions. Microbiol 152:1187–1196. https://doi.org/10.1099/mic.0.28484-0 DOI

Peran L, Sierra S, Comalada M et al (2007) A comparative study of the preventative effects exerted by two probiotics, Lactobacillus reuteri and Lactobacillus fermentum, in the trinitrobenzenesulfonic acid model of rat colitis. Br J Nutr 97:96–103. https://doi.org/10.1017/S0007114507257770 PubMed DOI

Phengnoi P, Charoenwongpaiboon T, Wangpaiboon K et al (2020) Levansucrase from Bacillus amyloliquefaciens KK9 and its Y237S variant producing the high bioactive levan-type fructooligosaccharides. Biomolecules 10:692 DOI

Porras-Domínguez JR, Ávila-Fernández Á, Miranda-Molina A, Rodríguez-Alegría ME, Munguía AL (2015) Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight. Carbohydr Polym 132:338–344. https://doi.org/10.1016/j.carbpol.2015.06.056 PubMed DOI

Ritsema T, Smeekens S (2003a) Engineering fructan metabolism in plants. J Plant Physiol 160:811–820. https://doi.org/10.1078/0176-1617-01029 PubMed DOI

Ritsema T, Smeekens S (2003b) Fructans: beneficial for plants and human. Curr Opin Plant Biol 6:223–230. https://doi.org/10.1016/S1369-5266(03)00034-7 PubMed DOI

Runyon JR, Nilsson L, Ulmius M, Castro A, Ionescu R, Andersson C, Schmidt C (2014) Characterizing changes in levan physicochemical properties in different pH environments using asymmetric flow field-flow fractionation. Anal Bioanal Chem 406:1597–1605. https://doi.org/10.1007/s00216-013-7388-x PubMed DOI

Seesuriyachan P, Kuntiya A, Techapun C (2011) Exopolysaccharide production by Lactobacillus confusus TISTR 1498 using coconut water as an alternative carbon source: the effect of peptone, yeast extract and beef extract. Songklanakarin J Sci Technol 33:379

Shih L, Chen LD, Wang TC, Wu JY, Liaw KS (2010) Tandem production of 826 levan and ethanol by microbial fermentation. Green Chem 12:1242–1247. https://doi.org/10.1039/b924765c DOI

Shih L, Wang TC, Chou SZ, Lee GD (2011) Sequential production of two biopolymers-levan and poly-ε-lysine by microbial fermentation. Bioresour Technol 102:3966–3969. https://doi.org/10.1016/j.biortech.2010.11.066 PubMed DOI

Silva LA, Neto JHPL, Cardarelli HR (2019) Exopolysaccharides produced by Lactobacillus plantarum: technological properties, biological activity, and potential application in the food industry. Ann Microbiol 69:321–328. https://doi.org/10.1007/s13213-019-01456-9 DOI

Silva P, Garcia S, Baldo C, Celligoi M (2017) Prebiotic activity of fructooligosaccharides produced by Bacillus subtilis natto CCT 7712. Acta Aliment 46:145–151. https://doi.org/10.1556/066.2016.0004 DOI

Sims IM, Frese SA, Walter J et al (2011) Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100–23. ISME J 5:1115–1124. https://doi.org/10.1038/ismej.2010.201 PubMed DOI PMC

SN W (2008) Sugarcane bagasse: how easy is it to measure its constituents? In Proceedings of the South African Sugar Technologists Association 81:266–273

Soleimani N, Hoseinifar SH, Merrifield DL, Barati M, Abadi ZH (2012) Dietary supplementation of fructooligosaccharide (FOS) improves the innate immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach (Rutilus rutilus) fry. Fish Shellfish Immunol 32:316–321. https://doi.org/10.1016/j.fsi.2011.11.023 PubMed DOI

Tanaka T, Oi S, Yamamoto T (1980) The molecular structure of low and high molecular weight levans synthesized by levansucrase. J Biochem 87:297–303. https://doi.org/10.1093/oxfordjournals.jbchem.a132737 PubMed DOI

Van Hijum S, Szalowska E, Van Der Maarel M, Dijkhuizen L (2004) Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri. Microbiol 150:621–630. https://doi.org/10.1099/mic.0.26671-0 DOI

Van Hijum SA, Bonting K, van der Maarel MJ, Dijkhuizen L (2001) Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced. FEMS Microbiol Lett 205:323–328. https://doi.org/10.1111/j.1574-6968.2001.tb10967.x PubMed DOI

Vieira AT, Teixeira MM, Martins FdS (2013) The role of probiotics and prebiotics in inducing gut immunity. Front Immunol 4:445. https://doi.org/10.3389/fimmu.2013.00445 PubMed DOI PMC

Vijn I, Smeekens S (1999) Fructan: more than a reserve carbohydrate? Plant Physiol 120(2):351–360. https://doi.org/10.1104/pp.120.2.351 PubMed DOI PMC

Wang T, Zheng N, Luo Q, Jiang L, He B, Yuan X, Shen L (2019) Probiotics Lactobacillus reuteri abrogates immune checkpoint blockade-associated colitis by inhibiting group 3 innate lymphoid cells. Front Immunol 10:1235. https://doi.org/10.3389/fimmu.2019.01235 PubMed DOI PMC

Wu FC, Chou SZ, Shih L (2013) Factors affecting the production and molecular weight of levan of Bacillus subtilis natto in batch and fed-batch culture in fermenter. J Taiwan Inst Chem Eng 44:846–853. https://doi.org/10.1016/j.jtice.2013.03.009 DOI

Xu J, Chen D, Liu C, Wu XZ, Dong CX, Zhou J (2016) Structural characterization and anti-tumor effects of an inulin-type fructan from Atractylodes chinensis. Int J Biol Macromol 82:765–771. https://doi.org/10.1016/j.ijbiomac.2015.10.082

Yamamoto Y, Takahashi Y, Kawano M, Iizuka M, Matsumoto T, Saeki S, Yamaguchi H (1999) In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats. J Nutr Biochem 10:13–18. https://doi.org/10.1016/S0955-2863(98)00077-1 PubMed DOI

Yao T, Chen MH, Lindemann SR (2020) Carbohydrate complexity structures stable diversity in gut-derived microbial consortia under high dilution pressure. bioRxiv. https://doi.org/10.1101/2020.01.31.929760

Yoo SH, Yoon EJ, Cha J, Lee HG (2004) Antitumor activity of levan polysaccharides from selected microorganisms. Int J Biol Macromol 34:37–41. https://doi.org/10.1016/j.ijbiomac.2004.01.002 PubMed DOI

Zannini E, Waters DM, Coffey A, Arendt EK (2016) Production, properties, and industrial food application of lactic acid bacteria-derived exopolysaccharides. Appl Microbiol Biotechnol 100:1121–1135. https://doi.org/10.1007/s00253-015-7172-2 PubMed DOI

Zhang T, Li R, Qian H, Mu W, Miao M, Jiang B (2014) Biosynthesis of levan by levansucrase from Bacillus methylotrophicus SK 21.002. Carbohydr Polym 101:975–981. https://doi.org/10.1016/j.carbpol.2013.10.045 PubMed DOI