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Lactic Acid Bacteria

Lactic Acid Bacteria. Carbohydrate and Protein Metabolism. Cell Wall. Cell Membrane. Cytoplasm. Milk. Amino Acid Transport System. Amino Acids. Amino Acids. Di- and Tri- Peptidases. ?. Di/Tri Peptide Transport System. Di/Tri peptides. Di/Tri Peptides. ?. Smaller Oligo-

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Lactic Acid Bacteria

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  1. Lactic Acid Bacteria Carbohydrate and Protein Metabolism

  2. Cell Wall Cell Membrane Cytoplasm Milk Amino Acid Transport System Amino Acids Amino Acids Di- and Tri- Peptidases ? Di/Tri Peptide Transport System Di/Tri peptides Di/Tri Peptides ? SmallerOligo- peptides Peptidases ? Oligopeptide Transport System Oligopeptides Large Oligo- peptides Proteinase Casein

  3. LAB proteolytic system • Fastidious, cannot synthesize some amino acids • Utilize exogenous N source • Involves at least a cell wall/membrane associated proteinase • Amino acid, di, tri-peptide and oligopeptide transport systems • Di-, tri-peptidase, oligopeptase

  4. LAB amino acid metabolism • 20 basic amino acids • Amino acids can further be used as building blocks • Or going through metabolism, converting into other intermediates such as pyruvate (C3), oxaloacetic acid (C4), acetyl (C2) • The % of amino acids vary in casein and some insufficient for proper growth, so still rely on biosynthesis

  5. Carbohydrate metabolism Polysaccharide(pectin, cellulose, starch, glycogen, Cp) Enzymes Enzymes PEP-PTS system Disaccharide (C12) Enzymes Monosaccharide (C6) Monosaccharide (C6) PEP-PTS system Enzymes lactate (lactic acid), acetate (acetic acid), formate, CO2, diacetyl,

  6. C6 monosaccharide metabolismGlucose, galactose, fructose, etc.

  7. C6 C5 FDP aldolase PK C2 LDH 2C3 C3

  8. Homolactic fermentation of Glucose(Embden-Meyerhof Pathway, Glycolysis+LDH) Glucose G-P F-6-P F-1,6-P Dihydroxyacetone-P Glyceraldehyde (C3) PEP (2) Pyruvate (2) Lactate FDP aldolase Products: 2ATP 2Lactate Key enzymes FDP aldolase Lactate dehydrogenase (C3) Lactata dehydrogenase (LDH) (C3)

  9. Mixed Acid Fermentation: Alternative end products for pyruvate Glucose FDP aldolase Dihydroxyacetone-phosphate Glyceralderhyde-3-phosphate NAD+ NADH+H+ (2) ADP (2) ATP LDH (2) ADP (2) ATP (2) Lactate (2)Pyruvate (2)CoA (2)NAD+ (2)NADH+(2)H+ PFL (2) Formate (2) Acetyl-CoA NADH+H+ NAD+ Acetaldehyde Products: 3ATP 2 Formate 1 Ethanol 1 Acetate Key enzymes FDP aldolase Pyruvate formate lyase (PFL) Acetyl-phosphate NADH+H+ NAD+ ADP ATP Ethanol Acetate

  10. Heterolactic Fermentation of Glucose (Pentose Phosphate Pathway) Glucose facultative anaerobic Anaerobic, aero-tolerant CO2 Ribulose-5-phosphate Xylulose-5-phosphate PK Glyceralderhyde-3-phosphate Acetyl-phosphate Ethanol Acetaldehyde Acetate Pyruvate Products: 2ATP 2 CO2 1 Lactate 1 Acetate Key enzymes PK LDH (NADH oxidase) LDH Lactate

  11. Group I obligately homofermentative Group II facultatively heterofermentative Group III obligately heterofermentative Character Arrangement of the Genus Lactobacillus - - - + - Lb. acidophilus Lb. delbruckii Lb. helveticus Lb. salivarius + - + + + Lb. casei Lb. curvatus Lb. plantarum Lb. sake + + + - + Lb. brevis Lb. buchneri Lb. fermentum Lb. reuteri Pentose fermentation CO2 from glucose CO2 from gluconate FDP aldolase present Phosphoketolase present

  12. Lactose utilization in LAB • Transport of lactose into cell • Hydrolysis of lactose • Metabolism of the monosaccharides • Efflux of lactic acid and protons from the cell • Unstable

  13. Disaccharide Metabolism(Lactose)

  14. Sugar Sugar Sugar1 H+ Sugar2 H+ F1F0 ATPase Permease Permease EIIS EIIIS HPr H+ Sugar Sugar2 Sugar EI Sugar1 H+ Pyruvate PEP Sugar Transport in LAB Symport Antiport PEP-PTS Out In Energy Source: Proton Motive Force (PMF:∆pH+∆ψ ) Concentration Gradient (∆S1&∆S2 ) PEP

  15. Transport hydrolysis systems • The PEP-PTS system • PEP: phosphoenolpyruvate H2C=COPO32-COO- • PTS: phosphotransferase • Substrate specific system • EII, EIII induced synthesis by the specific sugar • EI, HPr soluble components

  16. Transport hydrolysis systems • The PEP-PTS system • Lactose phosphorylated during transport • Multicomponent group translocation system • Two cytoplasmic proteins: Enz I and HPr • Two lactose-specific components: the membrane-located LacE and the soluble phosphocarrier LacF (or Enz IIlac and Enz IIIlac)

  17. LACTOSE PEP-PTS SYSTEM membrane Medium E-I PEP P-HPr out in pyruvate P-EI HPr LACTOSE P-EIII-lac EII-Lac E-III-lac Lactose-P P-beta-Galactosidase Galactose-6P Glucose

  18. Pathways for Galactose and Lactose Catabolism Galactose Lactose Galactose PEP-PTS Permease PEP-PTS Lactose-P Galactose Galactose-6P P-beta-Gal Gal-1P Glucose Tagatose-6P Glu-1P Glucose-6P Glyceraldehyde-3P +DHAP Tagatose 1,6-diP Glycolysis

  19. Primary and secondary transport systems • Lactose translocated unmodified • Disaccharide hydrolysed by beta–galactosidase (lacz) • Primary-involve a sugar transport ATPase • Agrobacterium radiobacter, Strep. mutans • Secondary-couples with ions or other solutes • L. lactis ATCC 7962 (proton), E.coli (LacY)

  20. Secondary transport systems • Secondary-couples with ions or other solutes • L. lactis ATCC 7962 (proton-coupled), E.coli (LacY) • LacS in Strep. thermophilus • Proton symport or lactose-galactose antiporter

  21. lactose galactose lactose galactose Bata-Gal S. thermophilus Lb. bulgaricus Lb. acidophilus Lb. lactis- don’t have the ability to ferment galactose glucose glycolysis

  22. LACTOSE Beta-Gal LACTOSE Gal Glu Gal-1-P Glu-6-P Glu-1-P Glycolysis Lb. helveticus

  23. Proton pump • Acid tolerant • Inside: pH ~5.3 • Outside: pH~4.2

  24. Summary • Glucose fermentation • Homo- heterolactic fermentation • Lactose utilization trait unstable • Strain dependent diversified pathways • Transport, hydrolysis • Select for proper starters for specific application

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