Abstract
We have previously shown that galacto-rhamnogalacturonan fibers can be enzymatically extracted from potato pulp and that these fibers have potential for exerting a prebiotic effect in piglets. The spore-forming Bacillus species are widely used as probiotics in feed supplements for pigs. In this study, we evaluated the option for further functionalizing Bacillus feed supplements by selecting strains possessing the enzymes required for extraction of the potentially prebiotic fibers. We established that it would require production and secretion of pectin lyase and/or polygalacturonase but no or limited secretion of galactanase and β-galactosidase. By screening a library of 158 Bacillus species isolated from feces and soil, we demonstrated that especially strains of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus mojavensis have the necessary enzyme profile and thus the capability to degrade polygalacturonan. Using an in vitro porcine gastrointestinal model system, we revealed that specifically strains of B. mojavensis were able to efficiently release galacto-rhamnogalacturonan from potato pulp under simulated gastrointestinal conditions. The work thus demonstrated the feasibility of producing prebiotic fibers via a feed containing Bacillus spores and potato pulp and identified candidates for future in vivo evaluation in piglets.
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Alexopoulos C, Georgoulakis IE, Tzivara A, Kyriakis CS, Govaris A, Kyriakis SC (2004) Field evaluation of the effect of a probiotic-containing Bacillus licheniformis and Bacillus subtilis spores on the health status, performance, and carcass quality of grower and finisher pigs. J Vet Med A Physiol Pathol Clin Med 51:306–312
Altmeyer S, Kröger S, Vahjen W, Zentek J, Scharek-Tedin L (2014) Impact of a probiotic Bacillus cereus strain on the jejunal epithelial barrier and on the NKG2D expressing immune cells during the weaning phase of piglets. Vet Immunol Immunopathol 161:57–65
Bano S, Qader SA, Aman A, Syed MN, Durrani K (2013) High production of cellulose degrading endo-1,4-β-D-glucanase using bagasse as a substrate from Bacillus subtilis KIBGE HAS. Carbohydr Polym 91:300–304
Barbosa TM, Serra CR, La Ragione RM, Woodward MJ, Henriques AO (2005) Screening for Bacillus isolates in the broiler gastrointestinal tract. Appl Environ Microbiol 71:968–978
Bhandari SK, Xu B, Nyachoti CM, Giesting DW, Krause DO (2008) Evaluation of alternatives to antibiotics using an Escherichia coli K88+ model of piglet diarrhea: effects on gut microbial ecology. J Anim Sci 86:836–847
Cutting SM (2011) Bacillus probiotics. Food Microbiol 28:214–220
de Lima EA, Machado CB, Zanphorlin LM, Ward RJ, Sato HH, Ruller R (2016) GH53 endo-β-1,4-galactanase from a newly isolated Bacillus licheniformis CBMAI 1609 as an enzymatic cocktail supplement for biomass saccharification. Appl Biochem Biotechnol 179:415–426
Fogel GB, Collins CR, Li J, Brunk CF (1999) Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microb Ecol 38:93–113
Ghazala I, Sayari N, Romdhane MB, Ellouz-Chaabouni S, Haddar A (2015) Assessment of pectinase production by Bacillus mojavensis I4 using an economical substrate and its potential application in oil sesame extraction. J Food Sci Technol 52:7710–7722
Hamza A, Fdhila K, Zouiten D, Masmoudi AS (2016) Virgibacillus proomii and Bacillus mojavensis as probiotics in sea bass (Dicentrarchus labrax) larvae: effects on growth performance and digestive enzyme activities. Fish Physiol Biochem 42:495–507
Huang JM, La Ragione RM, Nunez A, Cutting SM (2008) Immunostimulatory activity of Bacillus spores. FEMS Immunol Med Microbiol 53:195–203
Jers C, Kobir A, Søndergaard EO, Jensen PR, Mijakovic I (2011) Bacillus subtilis two-component system sensory kinase DegS is regulated by serine phosphorylation in its input domain. PLoS One 6:e14653
Kang TJ, Basu S, Zhang L, Thomas KE, Vogel SN, Baillie L, Cross AS (2008) Bacillus anthracis spores and lethal toxin induce IL-1β via functionally distinct signaling pathways. Eur J Immunol 38:1574–1584
Kashyap DR, Vohra PK, Chopra S, Tewari R (2001) Applications of pectinases in the commercial sector: a review. Bioresour Technol 77:215–227
Larsen N, Thorsen L, Kpikpi EN, Stuer-Lauridsen B, Cantor MD, Nielsen B, Brockmann E, Derkx PM, Jespersen L (2014) Characterization of Bacillus spp. strains for use as probiotic additives in pig feed. Appl Microbiol Biotechnol 98:1105–1118
Latorre JD, Hernandez-Velasco X, Kallapura G, Menconi A, Pumford NR, Morgan MJ, Layton SL, Bielke LR, Hargis BM, Téllez G (2014) Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens. Poult Sci 93:1793–1800
Leser TD, Knarreborg A, Worm J (2008) Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs. J Appl Microbiol 104:1025–1033
Majeed M, Nagabhushanam K, Natarajan S, Sivakumar A, Eshuis-de Ruiter T, Booij-Veurink J, de Vries YP, Ali F (2016) Evaluation of genetic and phenotypic consistency of Bacillus coagulans MTCC 5856: a commercial probiotic strain. World J Microbiol Biotechnol 32:60
Nakano MM, Dailly YP, Zuber P, Clark DP (1997) Characterization of anaerobic fermentative growth. J Bacteriol 179:6749–6755
Ochiai A, Itoh T, Kawamata A, Hashimoto W, Murata K (2007) Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization. Appl Environ Microbiol 73:3803–3813
Okamoto K, Fujiya M, Nata T, Ueno N, Inaba Y, Ishikawa C, Ito T, Moriichi K, Tanabe H, Mizukami Y, Chang EB, Kohgo Y (2012) Competence and sporulation factor derived from Bacillus subtilis improves epithelial cell injury in intestinal inflammation via immunomodulation and cytoprotection. Int J Color Dis 27:1039–1046
Prieto ML, O'Sullivan L, Tan SP, McLoughlin P, Hughes H, Gutierrez M, Lane JA, Hickey RM, Lawlor PG, Gardiner GE (2014) In vitro assessment of marine Bacillus for use as livestock probiotics. Mar Drugs 12:2422–2445
R Core Team (2012) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna ISBN 3-900051-07-0, URL http://www.R-project.org/
Rasmussen LE, Meyer AS (2010) Size exclusion chromatography for the quantitative profiling of the enzyme-catalyzed hydrolysis of xylo-oligosaccharides. J Agric Food Chem 58:762–769
Schaeffer P, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54:704–711
Scharek-Tedin L, Pieper R, Vahjen W, Tedin K, Neumann K, Zentek J (2013) Bacillus cereus var. toyoi modulates the immune reaction and reduces the occurrence of diarrhea in piglets challenged with Salmonella Typhimurium DT104. J Anim Sci 91:5696–5704
Strube ML, Meyer AS, Boye M (2013) Mini review: basic physiology and factors influencing exogenous enzymes activity in the porcine gastrointestinal tract. Anim Nutr Feed Techn 13:441–459
Strube ML, Ravn HC, Ingerslev HC, Meyer AS, Boye M (2015a) In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galacto-rhamnogalacturonan. Appl Environ Microbiol 81:1668–1678
Strube ML, Jensen TK, Meyer AS, Boye M (2015b) In situ prebiotics: enzymatic release of galacto-rhamnogalacturonan from potato pulp in vivo in the gastrointestinal tract of the weaning piglet. AMB Express 5:66
Tam NK, Uyen NQ, Hong HA, Duc le H, Hoa TT, Serra CR, Henriques AO, Cutting SM (2006) The intestinal life cycle of Bacillus subtilis and close relatives. J Bacteriol 188:2692–2700
Thite VS, Nerurkar AS (2015) Xylanases of Bacillus spp. isolated from ruminant dung as potential accessory enzymes for agro-waste saccharification. Lett Appl Microbiol 60:456–466
Thomassen LV, Vigsnæs LK, Licht TR, Mikkelsen JD, Meyer AS (2011) Maximal release of highly bifidogenic soluble dietary fibers from industrial potato pulp by minimal enzymatic treatment. Appl Microbiol Biotechnol 90:873–884
Torres S, Sayago JE, Ordoñez RM, Isla MI (2011) A colorimetric method to quantify endo-polygalacturonase activity. Enzym Microb Technol 48:123–128
Trejo M, Douarche C, Bailleux V, Poulard C, Mariot S, Regeard C, Raspaud E (2013) Elasticity and wrinkled morphology of Bacillus subtilis pellicles. Proc Natl Acad Sci U S A 110:2011–2016
Tsukahara T, Tsuruta T, Nakanishi N, Hikita C, Mochizuki M, Nakayama K (2013) The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets. Anim Sci J 84:316–321
van den Broek LAM, den Aantrekker ED, Voragen AGJ, Beldman G, Vincken JP (1997) Pectin lyase is a key enzyme in the maceration of potato tuber. J Sci Food Agric 75:167–172
Yadav S, Yadav PK, Ydav D, Ydav KDS (2009) Pectin lyase: a review. Process Biochem 44:1–10
Acknowledgements
We would like to express our gratitude to Annette Eva Jensen for expert technical assistance pertaining to the HPSEC analyses. APHA Scientific (UK) is acknowledged for the provision of a strain (B. mojavensis 10894) used in this study. This work was supported by a grant from the Green Development and Demonstration Programme (GUDP), Ministry of Environment and Food of Denmark (grant no. 34009-13-0700).
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This work was supported by a grant from the Green Development and Demonstration Programme (GUDP), Ministry of Environment and Food of Denmark (grant no. 34009-13-0700). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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M.D.C. and B.K.K.N. are employed at Chr. Hansen A/S that sells Bacillus probiotics and O.B.S. is employed at KMC amba, a producer of FiberBind. The authors declare that they have no conflict of interest.
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Jers, C., Strube, M.L., Cantor, M.D. et al. Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets. Appl Microbiol Biotechnol 101, 3605–3615 (2017). https://doi.org/10.1007/s00253-017-8176-x
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DOI: https://doi.org/10.1007/s00253-017-8176-x