Development)of)a)synergistic)synbioticfor Lactobacillus+ plantarum...
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Stephens K | Kolida S | Hernandez O | OHara S | Gibson GR | Rastall RA Department of Food and Nutritional Sciences Introduction and Aims Lactobacillus plantarum LPLDL is a probiotic selected from 4000 bacterial strains for its high bile salt hydrolase (BSH) and in vitro cholesterol reduction activities. Its efficacy in lowering cholesterol and systemic blood pressure, both contributing factors for the development of coronary heart disease, has been shown in vivo in mildly hypercholesterolaemic adults. The aim of this work was to use reverse enzyme technology to synthesize galactooligosaccharides (GOS) that will selectively enhance the growth and cholesterol lowering activity of Lactobacillus plantarum LPLDL. Methods A systematic approach was used to identify bacterial strains with high in vitro BSH activity and cholesterol reducing activity. L. plantarum LPLDL was further tested for its galactosidase hydrolytic activity using the ONPG test. Crude cell extracts of L. plantarum LPLDL were used to synthesize GOS (LPGOS) from lactose. The effect of purified LPGOS alone and in combination with L. plantarum LPLDL (LP) on the human faecal microbiome was evaluated in pHcontrolled, micro scale batch cultures in the presence of cholesterol. Purified, commercially available bifidobacterial GOS (BGOS) was used as control. Micro scale faecal batch cultures (10ml) testing in parallel: Cultures (24h) were run in triplicate, using faeces from each of three healthy adults. Samples were obtained for bacteriology (FISH), metabolite, BSH activity and cholesterol reduction determination. Development of a synergistic synbiotic for Lactobacillus plantarum LPLDL targeting cholesterol reduction Results LPGOS significantly and selectively increased Lactobacillus populations, BSH and cholesterol reducing activities. The effect was further enhanced by combining LPGOS with L. plantarum LPLDL. • Faeces • Faceses + LP • Faeces + LPGOS • Faeces + LPGOS + LP • Faeces + BGOS • Faeces + BGOS + LP 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 Faeces Faeces+LP Faeces +PGOS Faeces + PGOS+LP Faeces+BGOS Faeces+BGOS+LP Concentration (Log10 cells/ml culture) 0h 8h 24h 0 5 10 15 20 25 30 35 Faeces Faeces+LP Faeces Faeces+LP Faeces+PGOS Faeces+PGOS+LP Faeces+BGOS Faeces+BGOS+LP Faeces Faeces + LP PGOS PGOS+LP BGOS BGOS+LP3LDL 0h 8h 24h % cholesterol removal * * * * Contact information OptiBiotix Health plc | Science and Technology Centre, Whiteknights Campus, Reading (UK) | Email: [email protected] | +44(0) 7585001450| www.optibiotix.com The effect on metabolic activity correlated with Lactobacillus concentrations indicating a true synergistic effect that was not observed when LPLDL was used in combination with BGOS. BGOS did not have an impact on cholesterol reduction. Average Lactobacillus concentrations (±SD) in fecal batch culture (n=3)enumerated using the Lab158 FISH probe Average cholesterol reduction (±SD) in faecal batch culture (n=3). LPLDL significantly reduces cholesterol even in the absence of carbohydrate source. Effect is significantly enhanced in the presence of PGOS. * * Conclusions We have used a systematic approach to select a probiotic, L. plantarum LPLDL, targeting cholesterol reduction. Using galactosidases expressed by L. plantarum LPLDL we achieved the synthesis of GOS that works in true synergy with the parent strain, not only increasing its population but also impacting on the biological activity the probiotic was selected for. This is the first time true synergy is demonstrated for a synbiotic in faecal culture. Study Highlights Selecting a probiotic to target a specific health biomarker and using its enzymatic artillery to generate GOS, resulted in the formulation of a true synergistic synbiotic enhancing: • BSH activity • Cholesterol reduction potential
Transcript of Development)of)a)synergistic)synbioticfor Lactobacillus+ plantarum...
Stephens K | Kolida S | Hernandez O | OHara S | Gibson GR | Rastall RA
Department of Food and Nutritional Sciences
Introduction and AimsLactobacillus plantarum LP-‐LDL is a probiotic selected from4000 bacterial strains for its high bile salt hydrolase (BSH) and invitro cholesterol reduction activities. Its efficacy in loweringcholesterol and systemic blood pressure, both contributingfactors for the development of coronary heart disease, has beenshown in vivo in mildly hypercholesterolaemic adults.
The aim of this work was to use reverse enzyme technology tosynthesize galactooligosaccharides (GOS) that will selectivelyenhance the growth and cholesterol lowering activity ofLactobacillus plantarum LP-‐LDL.
MethodsA systematic approach was used to identify bacterial strainswith high in vitro BSH activity and cholesterol reducing activity.L. plantarum LP-‐LDL was further tested for its b-‐galactosidasehydrolytic activity using the ONPG test.
Crude cell extracts of L. plantarum LP-‐LDL were used tosynthesize GOS (LPGOS) from lactose.
The effect of purified LPGOS alone and in combination with L.plantarum LP-‐LDL (LP) on the human faecal microbiome wasevaluated in pH-‐controlled, micro scale batch cultures in thepresence of cholesterol. Purified, commercially availablebifidobacterial GOS (BGOS) was used as control.
Micro scale faecal batch cultures (10ml) testing in parallel:
Cultures (24h) were run in triplicate, using faeces from each ofthree healthy adults. Samples were obtained for bacteriology(FISH), metabolite, BSH activity and cholesterol reductiondetermination.
Development of a synergistic synbiotic for Lactobacillus plantarum LP-‐LDL targeting cholesterol reduction
ResultsLPGOS significantly and selectively increased Lactobacilluspopulations, BSH and cholesterol reducing activities. The effectwas further enhanced by combining LPGOS with L. plantarumLP-‐LDL.
• Faeces• Faceses + LP• Faeces + LPGOS
• Faeces + LPGOS + LP• Faeces + BGOS• Faeces + BGOS + LP
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7.2
7.4
7.6
7.8
8
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8.4
8.6
8.8
Faeces, Faeces+LP Faeces,+PGOS Faeces,+,PGOS+LP Faeces+BGOS Faeces+BGOS+LP
Concentra
tion,(Log10,ce
lls/m
l,culture)
0h 8h 24h
0
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Faece
s
Faece
s+LP
Faece
s
Faece
s+LP
Faece
s+PGO
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Faece
s+PGO
S+LP
Faece
s+BGO
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Faece
s+BGO
S+LP
Faece
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Faece
s2+2LP
PGOS
PGOS
+LP BGOS
BGOS
+LP3LD
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0h 8h 24h
%2choles
terol2
removal
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Contact informationOptiBiotix Health plc | Science and Technology Centre, Whiteknights Campus, Reading (UK) | Email: [email protected] | +44(0) 7585001450| www.optibiotix.com
The effect on metabolic activity correlated with Lactobacillusconcentrations indicating a true synergistic effect that was notobserved when LP-‐LDL was used in combination with BGOS.BGOS did not have an impact on cholesterol reduction.
Average Lactobacillus concentrations (±SD) in fecal batch culture(n=3)enumerated using the Lab158 FISH probe
Average cholesterol reduction (±SD) in faecal batch culture (n=3). LP-‐LDLsignificantly reduces cholesterol even in the absence of carbohydratesource. Effect is significantly enhanced in the presence of PGOS.
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ConclusionsWe have used a systematic approach to select a probiotic, L.plantarum LP-‐LDL, targeting cholesterol reduction. Using b-‐galactosidases expressed by L. plantarum LP-‐LDL we achievedthe synthesis of GOS that works in true synergy with the parentstrain, not only increasing its population but also impacting onthe biological activity the probiotic was selected for. This is thefirst time true synergy is demonstrated for a synbiotic in faecalculture.
Study HighlightsSelecting a probiotic to target a specific health biomarker andusing its enzymatic artillery to generate GOS, resulted in theformulation of a true synergistic synbiotic enhancing:• BSH activity• Cholesterol reduction potential
