Dietary supplementation with lactose or artificial sweetener enhances swine gut Lactobacillus population abundance

Transcription

1 British.Journal of Nutrition (214), 111, S3--S35 The Authors 213 doi:1.117 / Dietary supplementation with latose or artifiial sweetener enhanes swine gut Latobaillus population abundane Kristian Daly\ Alistair C. Darby\ Neil Hall 1, Alexandra Nal?, David Bravo 2 and Soraya P. Shirazi-Beehey 1 * 1 Epithelial Funtion and Development Group, Department of Funtional and Comparative Genomis, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK 2 Panosma SA, Geneva, Switzerland (Submitted 5 Novembe 212- Final revision reeived 73 February 213- Aepted 21 Februmy 213) - ro L :::J Vl. L o Abstrat The ommensal bateria Latobaillus are widely used as probioti organisms onferring a heath benefit on the host. They have been impliated in promoting gut health via the stimulation of host immunity and anti-inflammatory responses, as well as proteting the intestinalmuosa against pathogen invasion. Latobailli grow by fennenting sugars and starhes and produe lati aid as their primary metaboli produt. For effiient utilisation of varied arbohydrates, latobailli have evolved diverse sugar transport and metaboli systems, whih are speifially indued by their own substrates. Many bateria are also apable of sensing and responding to hanges in their environment. These sensory responses are often independent of transport or metabolism and are mediated through membrane-spanning reeptor proteins. We employed DNA-based pyrosequening tehnology to investigate the hanges in the intestinal mirobiota of piglets weaned to a diet supplemented with either a natural sugar, latose or an artifiial sweetener (SUCRAM, onsisting of saharin and neohesperidin dihydrohalone (NHDC); Panosma SA). The addition of either latose or saharin/nhdc to the piglets' feed dramatially inreased the aeal population abundane of Latobaillus, with onomitant inreases in intraluminal lati aid onentrations. This is the first report of the prebioti-like effets of saharin/nhdc, an artifiial sweetener, being able to influene the ommensal gut mirobiota. The identifiation of the underlying mehanism(s) will assist in designing nutritional strategies for enhaning gut immunity and maintaining gut health. Key words: Intestine: Mirobiota: Pig: Artifiial Sweetener: Latobaillus The mirobial olonisation of the gastrointestinal trat at bitth, predominantly by lati aid bateria, inluding Latobaillus, Bifidobateria and Streptoous, is of paramount impottane to health and performane through the stimulation of gut funtion, immunity and maturation - 4 >. After weaning, the gut mirobiota beomes more omplex and is apable of fermenting indigestible dietary omponents to monoarboxylates, mainly aetate, propionate, butyrate and latate. The absorption of aetate, propionate and butyrate by oloni epithelial ells provides a valuable energy soure, with butyrate also regulating homeostasis of the oloni epithelium by ontrolling the expression of genes assoiated with the proliferation, differentiation and apoptosis of oloni epithelial ellss- 9 ) Futthermore, the prodution of lati aid redues oloni ph, thereby inhibiting pathogeni organisms) In today's ommerial pig industty, piglets are weaned between 21 and 35 d of age(jj) Although early weaning inreases the number of piglets born per annum, the sudden and major hange in the diet greatly inreases the risk of enteri disease, diarrhoea and malnutrition(lt) It has been suggested that the high suseptibility of early-weaned piglets to enteri disorders is due to disruption in the establishment of a stable intestinal mirobiota, thereby allowing pathogeni bateria to flourish and ause disease Nutritional strategies, designed to prevent enteri disorders and improve the health and growth of piglets, may have the potential to influene the gastrointestinal mirobiota. These strategies ommonly entail the inlusion of dietary supplements suh as laity produts 5 >, natural sugars< , attifiial sweeteners<js), fermentable arbohydrates< and even Abbreviations: HC, basal hydrolysable arbohydrate diet; HC + L, basal hydrolysable arbohydrate diet supplemented with 5 o/o (w/w) latose; HC + S, basal hydrolysable Gtrbohydrate diet supplemented with 15 % (w/w) suram (saharin/nhdc); NHDC, neohesperidin dihydrohalone: PTS, phosphoenolpyruvate:arbohydrate phosphotransferase system; SGLTl, Na+/gluose o-transporter 1. 'Corresponding author: S. P. Shirazi-Beehey, spsb@liverpool.a.uk

2 Artifiial sweetener enhanes gut Latobaillus S31 ::l z '+- ro :::; probioti miro-organisms21) However, the preise effets of these dietaty supplements on the gastrointestinal mirobiota have not been fully haraterised. We employed 16S ronabased 454 pyrosequening tehnolog/22) to assess the omposition of aeal mirobiota in piglets. The present brief study fouses on the effet of dietaty supplementation of the piglets' feed with either latose or an artifiial sweetener (saharin/nhdc) speifially on the population abundane of gut Latobaillus. Dietaty latose has previously been shown to at as a prebioti, promoting the growth of benefiial ommensal bateria, suh as Bifidobateria and Latobaillus, and improving gut health and growth performane in weaning piglets<15 16) Dietaty supplementation with saharin/nhdc (SUCRAM ), the only artifiial sweetener that has approval for use as a feed additive in the European Union23>, has also been shown to dramatially redue enteri disease and to enhane growth performane in early-weaned piglets 8 ) However, vety few studies24 25) on the prebioti-like effets of sweeteners on the gastrointestinal mirobiota have been published. In the present study, we observed signifiant inreases in the aeal population abundane of Latobaillus in response to the inlusion of attifiial sweetener onsisting of saharine and NHDC in the piglets' feed. The mehanism(s) underlying the inreased abundane of aeal Latobaillus in response to dietaty supplementation with saharin/nhdc are presently unknown. Proesses, suh as the influene of host-derived fators upon the mirobiota26 27) and/or the involvement of a Latobaillus ell membrane-assoiated sensor for reognising attifiial sweetener, are proposed to explain this observation. Methods Animals and olletion of samples Male and female sukling Landrae x Large White piglets aged 28 d were plaed in pairs and housed in standard pens 5 m2, 12 h light-dark yle and 26 7 C). A total of three groups, eah onsisting of eight animals, were weaned to and maintained on the following isoenergeti ( kj/g) diets for 2 weeks: group 1, a ommerial wheat- and soya-based swine basal diet (Target Feeds Limited) ontaining 42% (w/w) hydrolysable arbohydrates (HC); group 2, the same basal diet but ontaining 5% (w/w) latose (in the form of dai1y rest whey) (HC + L); group 3, the same basal diet but supplemented with.15% (w/w) SUCRAM (an artifiial sweetener onsisting of saharin and neohesperidin dihydrohalone) (HC + S). All the animals had free aess to food and water at all times and onsumed the same amount of feed. They all remained healthy throughout the ourse of the feeding trial, and had no signs of enteri disturbanes. After 2 weeks, the piglets were killed with an intravenous injetion of pentobarbitone (2 mg Pentojet/ ml; Animal Care Limited) into the ranial vena ava (aording to UK Home Offie Shedule 1 regulations). National/instinnional guidelines for the are and use of animals were followed, and all experiments were approved by the University of Liverpool Ethis Committee. Immediately post-mottem, aeal and retal ontents were removed, wrapped in aluminium foil and frozen in liquid N2. All samples were subsequently stored at - 8 C until used for mirobial DNA extration or apillary GC analysis. Extration of baterial DNA from aeal ontent samples Nulei aid was extrated from the samples of aeal ontents using the method outlined by Lin & Stahl2 s) and desribed previousl/29) Approximately 1 g aliquots of frozen samples were transferred to srew-ap tubes ontaining SDS, Trisbuffered phenol (ph 8 ) (Sigma-Aldrih Company Limited) and sterile aid-washed glass beads. The samples were immediately homogenised using a mini beadbeater (Biospe Corporation, Strateh Sientifi). The aqueous supernatant was then extrated with phenol-hloroform-isoamylalohol and treated with DNase-free RNase A to remove ontaminating RNA. Total DNA (primarily baterial) was preipitated by the addition of sodium aetate and isopropanol. Purified DNA was resuspended in sterile Tris buffer and stored at - 8 C. DNA integrity was assessed by agarose gel eletrophoresis. We observed that rapid freezing of samples in liquid N2, followed by homogenisation in a buffer ontaining phenol, is an effetive method for inativating nulease ativity. This approah also avoids repeated freeze-thawing of samples that may be deleterious to the effiient isolation of DNA from Gram-negative mirobes. PCR amplifiation of baterial 165 rrna genes (rona) Purified DNA was used as a template for PCR amplifiation of baterial 16S rrna genes using GS FLX + Titanium fusion primers, targeted to flanking regions of the V1-V3 loop of baterial 16S rona, produing amp! ions of approximately 5 bp (sense 5 1 -CAGGCCTAACACATGCAAGTC-3 1 ; antisense 5 1 -ATTACCGCGGCTGCTGG-3 1 ). PCR yling was kept to a maximum of eighteen yles to avoid himera prodution. Amplions from eah group of piglets, labelled by the inlusion of a Multiplex Identifier sequene tag, were pooled in equimolar amounts, and sequened at the Centre for Genomis Researh, University of Liverpool, on a 454 GS FLX + Titanium sequening platform (Rohe). Analysis of 454 GS FLX + sequene reads Post-sequening, raw reads were de-multiplexed using the Multiplex Identifier sequene tags and orreted for PCR and sequening attifats using AmplionNoise3 o) and ChimeraSiayer<31) The reads were aligned using PyNAST and Greengenes32 33) and phylogenies alulated with FAST TREEC34) Operational taxonomi units were defined, using a similarity threshold of 97 %, with UCLUST35) and taxonomy was assigned via RDP Classifier 2.i36) using Qiime version 1.537) to implement analysis workflow.

3 532 K. Daly et at. 3 ** aid onentrations in piglets weaned to the basal HC diet (1 5 (SEM 2)mM) (P< 1; Fig. 2). Effet of saharin/nhdc on the population abundane of swine gut Latobaillus - ""@ ::J.r:. (/) - a HC HC + L HC+ S Fig. 1. Population abundane of aeal total latobailli ( ) and Latobaillus OTU4228 {D) (expressed as a perentage of the total mirobiota) in piglets weaned to a basal hydrolysable arbohydrate (HC) diet and a HC diet supplemented with 5% (w/w) latose (HC + L) or -15 % (w/w) saharin/ NHDC (HC + S). Values are means, with their standard errors represented by vertial bars. Mean values were signifiantly different from those of the HC diet: P< 5, P< 1. Measurement of aeal lati aid onentrations The onentration of lati aid in the aeal ontents of piglets was measured as desribed previousil38) Briefly, thawed aeal ontents were entrifuged to remove partiulates, and lati aid was extrated from supernatants by the addition of onentrated HCI and diethyl ether. The diethyl ether layer extrats were then derivatised and lati aid onentrations determined by apillary GC, quantified in relation to an internal standard. Statistial analysis Data are presented as means with their standard errors. Signifiane of differenes was determined using one-way ANOVA with Bonferroni's multiple omparison test (Graph Pad Prism 5; GraphPad Software, In.). Results were onsidered signifiant if P< 5. Results Effet of latose on the population abundane of swine gut Latobaillus Supplementation w ith the HC + L diet resulted in a signifiant enhanement of the aeal Latobaillus population (expressed as a perentage of the total number of sequenes) from 8 7 (SEM 1 7) to 24 5 (SEM 4 1)%, a 2 8-fold inrease (P< 1; Fig. 1). FUtthermore, this inrease was observed to be almost entirely due to one pattiular phylotype, designated Latobaillus OTU4228, w hih inreased from 7 4 (SEM 1 5) % of the total mirobiota in piglets weaned to the basal HC diet to 21 4 (SEM 4 )% of the total mirobiota in those weaned to the HC + L diet (P< 1; Fig. 1). Moreover, the measurements of lati aid onentrations in the aeal ontents of piglets weaned to the HC + L diet showed lati aid to be present at a onentration of 15 2 (SEM 1 8) mm; a 1-fold inrease ompared with aeal lati A signifiant enhanement of the relative population size of aeal Latobaillus was similarly observed in piglets weaned to the HC + S diet. In these piglets, Latobaillus aounted for 21 6 (SEM 4 9)% of the total mirobiota (expressed as a perentage of the total number of sequenes), ompared with 8 7 (SEM 1 7) % in piglets weaned to the basal HC diet. This represents an inrease of 2 5-fold (P< 5; Fig. 1). Again, this inrease was observed to be almost solely due to an inrease in the population of Latobaillus OTU4228, whih omprised 15 3 (SEM 4 6)% of the total mirobiota in piglets weaned to the HC + S diet (P< 5; Fig. 1). Furthermore, aeal lati aid onentrations in response to the inlusion of saharin/ NHDC were inreased by 2 1-fold over onentrations of aeal lati aid in piglets weaned to the basal HC diet (3 2 (SEM 6) v. 1 5 (SEM 2) 111M, P< 5; Fig. 2). Disussion Latobailli are the predominant lati aid bateria found in the pig intestine and onstitute a major propottion of the entire intestinal mirobiota. As suh, they are of partiular importane to the maintenane of gut health. The presene and ativity of latobailli have a stimulatory effet on both gut immunity and maturation, enhaning immune protetion and reduing gastrointestinal inllammat1y responses<39 4 >. They also display antimirobial ativities that partiipate in host epithelial defene, suh as redution of oloni ph (through the prodution of lati aid), protetion against muosal pathogen invasion and prodution of baterioins< ) We used PCR amplifiation of baterial 16S rrna gene sequenes and subsequent 454 pyrosequening to identify hanges in swine gut mirobiota in response to dietary supplementation. The average number of sequene reads per 2 E *** ;::; Q) u u "lj ::; "' u _J "' HC Fig. 2. Conentration of lati aid in the aeal ontents of piglets fed a basal hydrolysable arbohydrate diet (HC) and a HC diet supplemented with 5 % (w/w) latose (HC + L) or 15 % (w/w) saharin/nhdc (HC + S). Values are means, with their standard errors represented by vertial bars. Mean values were signifiantly different from those of the HC diet: P< 5, P< 1.

4 Artifiial sweetener enhanes gut Latobaillus J.1-J :::::1 z '+- ro ::::: sample was in exess of 15 and using a similarity threshold of 97% allowed lassifiation of over 1 phylotypes. Taxonomi assignment of these phylotypes showed that, in these piglets, the aeal mirobiota is dominated by three major baterial lasses: Bateroidia, Bailli and Clostridia, whih together ontribute over 9% of all sequenes. Major baterial groups represented inlude Porphyromonas and Prevotella (Bateroidia), Latobaillus (Bailli) and Ruminoous, Lahnospira and Veillonella (Clostridia). In the present study, whih fouses exlusively on hanges within the Latobaillus populations, we report that there were signifiant enhanements in the relative population abundane of latobailli in the aeal ontents of piglets in response to dietary supplementation with either a natural sugar, latose or an artifiial sweetener (saharin/ NHDC). The addition of latose (5 %, w/w) to the basal diet resulted in aeal Latobaillus populations inreasing from approximately 9% to over 24% of the total mirobiota. Notably, supplementation of the basal diet with saharin/nhdc ( 15 %, w/ w; a 33-fold lower onentration than latose) also inreased aeal Latobaillus populations to almost the same level (approximately 22% of the total mirobiota) (Fig. 1). In parallel, aeal lati aid onentrations (1 5 mm in piglets weaned to the basal HC diet) inreased 1-fold to over 15 mm in piglets weaned to the same diet ontaining latose and 2-fold to over 3 mm in piglets weaned to the diet ontaining saharin/ NHDC (Fig. 2). Interestingly, the inrease in Latobaillus abundane observed in the aeal ontents of piglets weaned to diets...:ontaining either latose or artifiial sweetener is not a general enhanement in all Latobaillus populations present in the pig aeum. In fat, one partiular phylotype, designated Latobaillus OTU4228, is almost solely responsible for the observed inrease. Latobaillus OTU4228 onstitutes approximately 7 o/o of the total mirobiota in piglets weaned to the basal HC diet (over 85% of the total Latobaillus ommunity). This inreases to over 21 and 15% of the total mirobiota in piglets weaned to the HC +Land HC + S diets, respetively (Fig. 1). Although the response of Latobaillus OTU4228, in terms of inreased population abundane, is similar in piglets weaned to diets supplemented with either latose or saharin/nhdc, the disparity between aeal lati aid onentrations suggests that the underlying mehanisms are quite different. It has been shown that there is a rapid and signifiant derease in pig intestinal latase ativity with both age and weaning< ), indiating that a substantial amount of ingested latose may not be digested by the host< 46 >. Latose is then available as a highly metabolisable substrate readily utilised by bateria (partiularly latobailli), initially in the distal regions of the small intestine as well as in the aeum< 46 ) Inreases in the population abundane of Latobaillus have previously been demonstrated in piglets fed diets supplemented with Jatose 5 16 >, primarily due to the metabolism of latose by latobailli. The highly fermentable nature of latose is refleted in the large inrease in lati aid onentrations seen here in the aeal ontents of piglets weaned to the diet ontaining latose (population abundane of latobailli inreases 2 8-fold; lati aid inreases 1-fold). In ontrast, the inrease in lati aid onentrations measured in the aeal ontents of piglets weaned to the same diet ontaining saharin/ NHDC is in proportion to the inrease in the population abundane of Latobaillus (2 1- and 2 5-fold, respetively). This suggests that, unlike latose whih provides an additional substrate for the growth of latobailli and subsequent lati aid prodution, this anifiial sweetener is not a metabolisable energy soure that an be fermented by Latobaillus populations to produe lati aid. The effets of anifiial sweeteners on gut mirobiota have previously been studied in human subjets. It has been shown that the addition of maltitol, a sugar alohol, to onfetionery signifiantly enhaned the population abundane of both Bifidobateria and Latobaillus< 25 >. However, it is notable that maltitol is a fermentable substrate for these gut mirobes< >. In the mammalian intestine, the sweet taste reeptor, T1R2-T1R3, expressed in enteroendorine ells, an detet the presene of sugars and artifiial sweeteners. This initiates an intraellular signalling pathway leading to the up-regulation of the intestinal gluose transporter, Na+ / gluose o-transponer 1 (SGL Tl), and an inreased apaity of the gut to absorb gluose<4s-so) Likewise, yeasts, suh as Saharomyes erevisiae, possess mutated gluose transponers (Snf3 and Rgt2) that at as transmembrane sweet sensors ontrolling the expression of hexose transporter proteins in the presene of gluose and other sugars< >. Latobailli, and many other enteri bateria, express multiple sugar transport and metaboli systems that allow them to utilise a variety of arbohydrate substrates and adapt quikly to hanges in nutrient availability< 53 ) This versatility is of paltiular importane in an environment suh as the gastrointestinal trat. The predominant sugar transport mehanism in these bateria is the phosphoenolpyruvate: arbohydrate phosphotransferase system (PTS); with over twenty different PTS systems being identified, eah is speifi for only one or a few sugars< 54 >. There are also multiple non-pts sugar transport systems suh as non-pts permeases and ABC transporters for various poly- and oligosaharides< 54 ) The vast majority of these systems are regulated in the presene of a speifi substrate< ) Extraellular sensing is a key method employed by bateria in order to respond to hanges in their environment suh as alterations in ph, hemial omposition or nutrient availability. Many of these sensory responses are independent of transpo!t or metabolism, but involve the binding of hemial ligands to membrane-spanning sensory reeptors in order to initiate intraellular signalling pathways< 5556 ) Notably, reent evidene has shown that transription of genes responsible for utilisation of diverse polysaharides by enteri Bateroides speies an be diretly ativated by the reognition of signature oligosaharide ligands by speifi reeptors >, demonstrating that these systems play a key role in baterial ability to sense and utilise polysaharides in gut eosystems. In the light of results presented here, we propose that latobailli may possess a plasma membraneassoiated sweet sensor apable of sensing anifiial

5 S34 K. Daly et at.. s,_ ;::, z - C\S s,_ ;::, --.. sweeteners and initiating pathways ontrolling arbohydrate transpot1 and metabolism. However, the influene of the host on the omposition and ativity of the gut mirobiota is beoming inreasingly evident and should not be underestimated. Epithelial fators, suh as the seretion of growth-promoting muosal glyans or toxi inhibitory ompounds, have been highly impliated in regulating the omposition of the intestinal mirobiota< 26 >. Funhermore, differenes in mirobial ommunity struture between different host speies have been proposed to arise from distint seletive pressures imposed from within the gut habitat of the respetive host< 59 ) Moreover, host geneti fators have also been shown to ontribute in patt to gut mirobiota omposition< 27 6 ) Whatever the underlying mehanism(s), the data presented here show that dieta1y supplementation with saharin/ NHDC artifiial sweetener an alter the gastrointestinal mirobiota by positively influening the population abundane of latobailli, ommensal bateria that are able to exert a benefiial effet on gut health, immunity and maturation< 39 4 ) The identifiation and haraterisation of the underlying mehanism(s) will assist in the design of nutritional strategies aimed at manipulating the pig ommensal mirobiota, promoting the health of the gut partiularly during the ritial post-weaning period. Aknowledgements The authors gratefully aknowledge the finanial suppon provided by Panosma SA. The authors' ontributions are as follows: K. D. and S. P. S.-B. designed the researh, analysed the data and wrote the paper; K. D. performed the researh; A. D. and N. H. performed the bioinformati analyses of pyrosequening data; A. N. and D. B. provided sientifi and nutritional advie. K. D., A. D., N. H. and S. P. S.-B. delare no onflits of interest. A. N. and D. B. are employees of Panosma SA. Referenes 1. Hooper LV, Wong MH, Thelin A, et a/. (21) Moleular analysis of ommensal host-mirobial relationships in the intestine. Siene 291, Hooper LV, Midtvedt T & Gordon ]I (22) How hostmirobial interations shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 22, Zoetendal EG, Collier CT, Koike S, et a/. (24) Moleular eologial analysis of the gastrointestinal mirobiota: a review. 1 Nutr 134, Bauer E, Williams BA, Smidt H, eta/. (26) Influene of the gastrointestinal mirobiota on development of the immune system in young animals. Curr Issues lntest Mirobial 7, Bergman EN (199) Energy ontributions of volatile fatty aids from the gastrointestinal trat in various speies. Physiol Rev 7, Cummings JH & Mafarlane GT (1997) Coloni miroflora: nutrition and health. Nutrition 13, Cuff MA, Dyer], ]ones M, eta/. (25) The human oloni monoarboxylate transporter Jsoform 1: its potential importane to oloni tissue homeostasis. Gastroenterology 128, Daly K & Shirazi-Beehey SP (26) Miroarray analysis of butyrate regulated genes in oloni epithelial ells. DNA Cell Bioi 25, Putaala H, Barrangou R, Leyer GJ, eta/. (21) Analysis of the human intestinal epithelial ell transriptional response to Latobaillus aidophilus, Latobaillus salivarius, Bifidobaterium /atis and Esherihia oli. Benef Mirobes 1, Liihteinen T, Malinen E, Koort JM, et a/. (21) Probioti properties of Latobaillus isolates originating from porine intestine and fees. Anaerobe 16, Lalles J-P, Bosi P, Smidt H, et at. (27) Nutritional management of gut health in pigs around weaning. Pro Nutr So 66, Hopwood DE & Hampson OJ (23) Interations between the intestinal miroflora, diet and diarrhoea, and their influenes on piglet health in the immediate post-weaning period. In Weaning the Pig: Conepts and Consequenes, pp OR Pluske, J Le Dividih and MWA Verstegen, editors]. Wageningen: Wageningen Aademi Publishers. 13. Konstantinov SR, Favier CF, Zhu WY, eta/. (24) Mirobial diversity studies of the porine gastrointestinal eosystem during weaning transition. Anim Res 53, Konstantinov SR, Awati AA, Williams BA, eta/. (26) Postnatal development of the porine mirobiota omposition and ativities. Environ Mirobial 8, Vente-Spreeuwenberg MAM, Verdonk JMAJ, Bakker GCM, et a/. (24) Effet of dietary protein soure on feed intake and small intestinal morphology in newly weaned piglets. Liuest Prod Si 86, Piere KM, Sweeney T, Brophy PO, eta/. (26) The effet of latose and inulin on intestinal morphology, seleted mirobial populations and volatile fatty aid onentrations in the gastrointestinal trat of the weaned pig. Anim Si 82, Piere KM, Callan]], MCarthy P, eta/. (27) The interation between latose level and rude protein onentration on piglet post-weaning performane, nitrogen metabolism, seleted faeal mirobial populations and faeal volatile fatty aid onentrations. Anim Feed Si Tehno! 132, Sterk A, Shlegel P, Mul AJ, eta/. (28) Effets of sweeteners on individual feed intake harateristis and performane in group-housed weanling pigs. 1 Anim Si 86, Konstantinov SR, Awati A, Smidt H, et at. (24) Speifi response of a novel and abundant Latobaillus amylovoruslike phylotype to dietary prebiotis in the guts of weaning piglets. Appl Environ Mirobio/7, Loh G, Eberhard M, Brunner RM, eta/. (26) Inulin alters the intestinal mirobiota and short-hain fatty aid onentrations in growing pigs regardless of their basal diet. 1 Nutr 136, Huang C, Qiao S, Lifa D, et a/. (24) Effets of latobailli on the performane, diarrhea inidene, VFA onentration and gastrointestinal mirobial flora of weaning pigs. Asian Austral] Anim 17, Meyer M, Stenzel U & Hofreiter M (28) Parallel tagged sequening on the 454 platform. Nat Proto 3, Community Register of Feed Additives, pursuant to Regulation (EC) No. 1831/23, appendies 3 & 4. food/food/animalnutrition/ feedadditives/omm_register_feed_ additives_ pdf

6 Artifiial sweetener enhanes gut Latobaillus S35... ::::i !: Vl :e o 24. Beards E, Tuohy K & Gibson G (21) A human volunteer study to assess the impat of onfetionery sweeteners on the gut mirobiota omposition. BrJ Nutr 14, Thabuis C, Herbomez AC, Desailly F, eta/. (212) Prebiotilike effets of SweetPearl "' maltitol through hanges in aeal and feal parameters. Food Nutr Si (In the Press). 26. Shluter J & Foster KR (212) The evolution of mutualism in gut mirobiota via host epithelial seletion. PLoS Bioi 1, el Benson AK, Kelly SA, Legge R, et a/. (21) Individuality in gut mirobiota omposition is a omplex polygeni trait shaped by multiple environmental and host geneti fators. Pro Nat/ Aad Si US A 17, Lin C & Stahl DA (1995) Taxon-speifi probes for the ellulolyti genus Fibrobater reveal abundant and novel equine-assoiated populations. Appl Environ Mirobial 61, Daly K & Shirazi-Beehey SP (23) Design and evaluation of group-speifi oligonuleotide probes for quantitative analysis of intestinal eosystems: their appliation to assessment of equine oloni miroflora. FEMS Mirobial Eo/44, Quine C, Lanzen A, Curtis TP, et a/. (29) Aurate determination of mirobial diversity from 454 pyrosequening data. Nat Methods 6, Haas BJ, Gevers D, Earl AM, eta/. (211) Chimeri 16S rrna sequene formation and detetion in Sanger and 454- pyrosequened PCR amplions. Genome Res 21, Caporaso JG, Bittinger K, Bushman FD, et a/. (21) PyNAST: a flexible tool for aligning sequenes to a template alignment. Bioinformatis 26, DeSantis TZ, Hugenholtz P, Larsen N, et a/. (26) Greengenes, a himera-heked 16S rrna gene database and workbenh ompatible with ARB. Appl Environ Mirobio/72, Prie MN, Dehal PS & Arkin AP (21) FastTree 2 - approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e Edgar RC (21) Searh and lustering orders of magnitude faster than BLAST. Bioinformatis 26, Wang Q, Garrity GM, Tiedje JM, eta/. (27) Naive Bayesian lassifier for rapid assignment of rrna sequenes into the new baterial taxonomy. Appl Environ Mirobial 73, Caporaso JG, Kuzynski], Stombaugh], eta/. (21) QTIME allows analysis of high-throughput ommunity sequening data. Nat Methods 7, Daly K, Proudman CJ, Dunan SH, eta/. (212) Alterations in mirobiota and fermentation produts in equine large intestine in response to dietary variation and intestinal disease. BrJ Nutr 17, Kimura K, MCartney AL, MConnell MA, eta/. (1997) Analysis of feal populations of bifidobateria and latobailli and investigation of the immunologial responses of their human hosts to the predominant strains. Appl Environ Mirobial 63, Blum S & Shiffrin EJ (23) Intestinal miroflora and homeostasis of the muosal immune response: impliations for probioti bateria? Curr Issues lntest Mirobial 4, Casta D, Requena T & Gomez R (1996) Antimirobial ativity of lati aid bateria isolated from goat's milk and artisanal heeses: harateristis of a baterioin produed by Latobaillus urvatus IFPL Appl Bateriol 81, Varoe ]], Krejarek G, Busta F, et a/. (23) Prophylati feeding of Latobaillus aidophilus NCFM to mie attenuates overt oloni hyperplasia..1 Food Prot 66, Kelly D, Smyth ]A & MCraken KJ (1991) Digestive development of the early-weaned pig. 1. Effet of ontinuous nutrient supply on the development of the digestive trat and on hanges in digestive enzyme ativity during the first week post-weaning. Br.1 Nutr 65, Kelly D, King TP, MFadyen M, et at. 991) Effet of latation on the deline of brush border latase ativity in neonatal pigs. Gut 32, Marion], Petersen YM, Rome V, eta/. (25) Early weaning stimulates intestinal brush border enzyme ativities in piglets, mainly at the posttransriptional level. J Pediatr Gastroenterol Nutr 41, Kim KI, Benevenga NJ & Gntmmer RH (1978) Latase ativity and VFA prodution in the eum and olon of pigs fed a orn-soy or 4o/o whey diet..1 Anim Si 46, Oku T, Akiba M, Lee MH, et a/. (1991) Metaboli fate of ingested [ 14 C]-maltitol in man..1 Nutr Si Vitaminol 37, Dyer], Daly K, Salmon KS, et a/. (27) Intestinal gluose sensing and regulation of intestinal gluose absorption. Biohem So Trans 35, Margolskee RF, Dyer], Kokrashvili Z, eta/. (27) T1R3 and gustduin in gut sense sugars to regulate expression of Na + gluose otransporter 1. Pro Nat/ Aad Si U S A 14, Shirazi-Beehey SP, Moran AW, Bathelor OJ, et a/. (211) Gluose sensing and signalling; regulation of intestinal gluose transport. Pro Nutr So 7, Ozan S &.Johnston M (1999) Funtion and regulation of yeast hexose transporters. Mirobial Mol Bioi Rev 63, Forsberg H & Ljungdahl PO (21) Sensors of extraellular nutrients in Saharomyes erevisiae. Curr Genet 4, Barrangou R, Azarate-Peril MA, Duong T, et a/. (26) Global analysis of arbohydrate utilization by Latobaillus aidophilus using edna miroarrays. Pro Nat/ Aad Si US A 13, Kotrba P, Inui M & Yukawa H (21) Baterial phosphotransferase system (PTS) in arbohydrate uptake and ontrol of arbon metabolism. J Biosi Bioeng 92, Armitage JP & Sakett RE (1987) Sensory transdution in flagellate bateria. Ann NY Aad Si 51, Xu J, Chiang HC, Bjursell MK, eta/. (24) Message from a human gut symbiont: sensitivity is a prerequisite for sharing. Trends Mirobiot12, Sonnenburg ED, Sonnenburg JL, Manhester JK, et at. (26) A hybrid two-omponent system protein of a prominent human gut symbiont ouples glyan sensing in vivo to arbohydrate metabolism. Pro Nat/ A ad Si US A 13, Martens EC, Lowe EC, Chiang H, et a/. (211) Reognition and degradation of plant ell wall polysaharides by two human gut symbionts. PLoS Bio/9, e Rawls JF, Mahowald MA, Ley RE, eta/. (26) Reiproal gut mirobiota transplants from zebrafish and mie to germ-free reipients reveal host habitat seletion. Cell127, Spar A, Koren & Ley R (211) Unravelling the effets of the environment and host genotype on the gut mirobiome. Nat Rev Mirobial 9,