Probiotic properties of lactic acid bacteria evaluated in a gastro-intestinal model and in in vivo pig trials

Mare, Louise (2005-03)

Thesis (PhD)--Stellenbosch University, 2005.

Thesis

ENGLISH ABSTRACT: This study describes the use of a gastro-intestinal model to screen lactic acid bacteria isolated from the gastro-intestinal tract of post-weaned piglets (raised on six different diets) for probiotic properties. Intestinal bacteria were isolated from ,the stomach, duodenum, jejunum, caecum, ileum and colon. The highest cell numbers (6 x 107 cfulg) were isolated from the ileum. No significant differences in viable cell counts were recorded for piglets raised on the six diets. Isolates with the best overall probiotic properties were identified as members of Lactobacillus salivarius and Lactobacillus fermentum. The two strains selected for further studies were Lactobacillus plantarum 423 (originally isolated from sorghum beer) and Lactobacillus salivarius 241 (isolated from pig intestine). Enterococcus faecalis FAIR E 92 was originally isolated from pig intestine and was included in this study as a non-pathogenic challenge strain. L. plantarum 423 produces a bacteriocin plantaricin 423, active against E. faecalis FAIRE 92. L. plan/arum 423 and L. salivarius 241 were included in the gastro-intestinal model and their adhesion to the mucus of porcine ileum studied with fluorescent-in-si/u-hybridization (FISH). A decrease in viable cell numbers of L. plan/arum 423 was recorded in the duodenum, jejunum and ileum in the presence of bile and pancreatic juice. However, higher cell numbers were recorded in the caecum and anterior colon, which suggested that strain 423 recovered from these stress factors. Plantaricin 423 was detected for up to 28 hours in the duodenum, jejunum, ileum and middle colon. Lower cell numbers (one log unit) of L. salivarius 241 were recorded in the gastro-intestinal model over seven days, compared to strain 423. Piglets of one, 14 and 28-days-old were dosed with L. plan/arum 423 and L. salivarius 241, separately and in combination (1: 1). In a separate experiment, 14-day-old piglets were challenged twice with E. faecalis FAIRE 92, followed by dosage with strains 423 and 241. New-borne piglets dosed with L. plantarum 423 gained more weight (4 kg over 19 days) compared to piglets dosed with L. salivarius 241 (2.2 kg over 19 days), or a combination of the two strains (2 kg over 19 days). Piglets of 14 and 28-days-old, on the other hand, gained more weight when dosed with a combination of strains 423 and 241. The cell numbers of E. faecalis FAIR E 92 and other enterococci decreased drastically (two log units) when the piglets were dosed with the latter two strains. Overall, piglets of various ages reacted differently when administered L. plantarum 423 and L. salivarius 241, separately or in combination. Fluorescent-in-situ-hybridization (FISH) was used to study the in vivo adhesion of L. plantarum and L. salivarius to mucus in the stomach, duodenum, jejunum, ileum, caecum and colon. The highest number of L. plantarum cells was recorded in the ileum, whereas L. salivarius favoured adhesion to the duodenum. A decrease in cell numbers of E. faecalis in the ileum mucus was recorded when a combination of the probiotic strains 423 and 241 was administered. This study provided a reliable estimation of the presence and/or adhesion of L. plantarum and L. salivarius to various parts of the porcine gastro-intestinal tract, without the use of expensive cultivation techniques. Insight was gained into the co-evolution existing between probiotic bacteria and the porcine gastro-intestinal tract, emphasizing the use of gastro-intestinal models to study the dynamics of the gastro-intestinal tract.

AFRIKAANSE OPSOMMING: Hierdie studie beskryf die gebruik van 'n gastro-intestinale model, om melksuurbakterieë wat geïsoleer is uit die spysverteringskanaal (SVK) van reeds gespeende varkies (gevoed op ses verskillende diëte) vir probiotiese eienskappe te toets. Ingewandsbakterieë is uit die maag, duodenum, jejunum, caecum, ileum en kolon geïsoleer. Die hoogste aantal selle (6 x 107 kve/g) is geïsoleer uit die ileum. Geen betekenisvolle verskille in lewensvatbare seltellings, vir varkies gevoed op ses verskillende voere is aangeteken nie. Isolate met die beste algehele probiotiese eienskappe is as Lactobacillus salivarius en Lactobacillus fermentum geïdentifiseer. Vir verdere studie is twee isolate Lactobacillus plantarum (oorspronklik uit sorghum-bier geïsoleer) en Lactobacillus salivarius (uit die varkdermkanaal geïsoleer) geselekteer. Enterococcus faecalis FAIRE 92, oorspronklik uit die varkdermkanaal geïsoleer, is in hierdie studie as 'n nie-patogeniese indikator gebruik. L. plantarum 423 produseer 'n bakteriosien plantarisien 423 wat aktief is teen E. faecalis FAIR E92. L. plantarum 423 en L. sa/ivarius 241 is ingesluit in die gastro-intestinale model, en vashegting van die bakterieë aan die mukus van vark-ileum is met fluoresensie-in-si/uhibridisasie (FISH) bestudeer. 'n Afname in lewende selgetalle van L. plan/arum 423 in die duodenum, jejunum en ileum is aangetoon in reaksie tot die byvoeging van gal en pankreatiese sappe. Hoër selgetalle is nietemin aangeteken in die caecum en voorste gedeelte van die kolon, wat 'n aanduiding gee dat isolaat 423, ten spyte van hierdie stres-faktore, oorleef. Plantaricin 423 is vir 'n tydperk (28 uur) in die duodenum, jejunum, ileum en sentrale kolon gevind. Laer selgetalle (een logaritmiese eenheid) van L. salivarius 241 is in die gastro-intestinale modeloor 'n tydperk van sewe dae aangetoon, in vergelyking met isolaat 423. Een, 14 en 28 dag oud varkies is met L. plantarum 423 en L. salivarius 241 (afsonderlik en in kombinasie 1:1) twee keer gedaag met E. faecalis FAIR E 92, opgevolg met dosering van 423 en 241. Pasgebore varkies het die hoogste gewigstoename getoon (4 kg oor 19 dae) na dosering met L. plantarum 423 in vergelyking met varkies gedoseer met L. salivarius 241 (2.2 kg oor 19 dae) of 'n kombinasie van die twee isolate (2 kg oor 19 dae). Daarenteen het veertien- en 28 dag oud varkies beter gewigstoename getoon na dosering met 'n kombinasie van isolate 423 en 241. Die selgetalle van E. faecalis FAIRE 92 en ander enterococci het drasties afgeneem (twee logaritmiese eenhede) nadat die varkies met laasgenoemde twee isolate gedoseer is. Varkies van onderskeie ouderdom het verskillend gereageer na dosering met L. plantarum 423 en L. salivarius 241 afsonderlik of in kombinasie. Fluoresensie-in-situ-hibridisasie (FISH) is gebruik om die in vivo vashegting van L plantarum en L. salivarius tot die vark mukus in die maag, duodenum, jejunum, ileum, caecum en kolon te bestudeer. Die hoogste telling van L. plantarum selle is aangeteken in die ileum, terwyl L. salivarius aanhegting tot die duodenum verkies het. 'n Afname in seltellings van E. faecalis in die ileum mukus was aangeteken na toediening met 'n kombinasie van probiotiese isolate 423 en 241. Hierdie studie het 'n betroubare bepaling van die voorkoms en/ofvashegting van L. plantarum en L. sa/ivarius isolate in verskeie gedeeltes van die varkspysverteringskanaal voorsien, sonder die hulp van duur kwekings tegnieke. Probiotiese bakterieë is in 'n gastro-intestinale model, wat die natuurlike omgewing verteenwoordig, bestudeer. Insig oor die ko-evolusie tussen probiotiese bakterieë en die SVK van die vark is verkry. Die gebruik van 'n gastro-intestinale model om die dinamika van die SVK te bestudeer is met hierdie studie beklemtoon.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/50427
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