5th Asian Conference on Lactic Acid Bacteria

1,002 views

Published on

The Conference was held at National University of Singapore, Singapore during 1-3 July 2009

Published in: Education, Technology, Business
0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
1,002
On SlideShare
0
From Embeds
0
Number of Embeds
9
Actions
Shares
0
Downloads
0
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide

5th Asian Conference on Lactic Acid Bacteria

  1. 1. Possible Synergistic Effect between High Lactate and Depletion of Essential Peptides Caused Biomass Reduction during High-Cell Starter Culture Production Mallika BOONMEE Khon Kaen University, Thailand
  2. 2. Backgrounds • Some starters subjected to end-product inhibition on growth – Limit to low initial sugar  [low biomass] per batch • Production of high cell starter culture – Batch cultivation with high initial sugar – In situ removal of lactate ions using anion exchange resin  reduce end-product inhibition on growth Asian Conference on Lactic Acid Bacteria 2009 2 NUS, Singapore
  3. 3. Unexpected profiles Batch 80 g/l Biomass unchanged until lactose depleted IXF 180 g/l Same lactate & lactose Biomass dropped Asian Conference on Lactic Acid Bacteria 2009 3 NUS, Singapore
  4. 4. Questions for 180 g/l IXF What causes the drop in biomass concentration while there was still Shouldn’t the biomass lactose available? remain unchanged like at 80 g/l when lactate reached the growth inhibition level? Asian Conference on Lactic Acid Bacteria 2009 4 NUS, Singapore
  5. 5. What have been investigated? • Comparing C:N ratio between batch and ion exchange fermentation (IXF) – Sugar : protein ratio as C:N ratio – Batch 80 g/l lactose – Ion exchange fermentation at 80, 120 & 180 g/l lactose • Protein (polypeptides) profile of cell- free medium Asian Conference on Lactic Acid Bacteria 2009 5 NUS, Singapore
  6. 6. Experiment in brief • Bacteria: Lactococcus lactis NZ133 – End-product inhibition on growth at about 50 g/l lactate • Cultivation medium: modified M17 – Based formulation @ 40 g/l lactose – Organic nitrogen increased proportionally to the increase in lactose. Asian Conference on Lactic Acid Bacteria 2009 6 NUS, Singapore
  7. 7. Experimental works … continued • Cultivation system – Glass jar fermenter with pH control unit attached. • 1-litre fermenter with 500-ml working volume – Temperature control using water bath – Stirring by magnetic stirrer • pH controls – Automatic addition of NaOH – Manual addition of IRA-67, anion exchange resin • Double action as the removal of lactate ions. Asian Conference on Lactic Acid Bacteria 2009 7 NUS, Singapore
  8. 8. Glass jar fermenter in water bath Asian Conference on Lactic Acid Bacteria 2009 8 NUS, Singapore
  9. 9. Setup for fermentation system Asian Conference on Lactic Acid Bacteria 2009 9 NUS, Singapore
  10. 10. Experimental works … continued • Analysis – Protein in supernatant  Lowry’s method – Protein (polypeptide) profiles  SDS-PAGE • 12% and 16% gel • Markers: 10-200 kDa & 1,423-26,625 Da – Lactose concentration  Reducing sugar by DNS – Lactate concentration  HPLC Asian Conference on Lactic Acid Bacteria 2009 10 NUS, Singapore
  11. 11. Experimental plans • Cultivate L. lactis NZ133 (IXF) – 80, 120 and 180 g/l lactose – IRA-67 to remove lactate & control pH – Partial use of NaOH for pH control • Batch cultivation at 80 g/l lactose as control • Analysis of supernatants Asian Conference on Lactic Acid Bacteria 2009 11 NUS, Singapore
  12. 12. Effect of Carbon:Nitrogen ratio Does particular value of C:N ratio cause the biomass drop? If such, carbon to nitrogen balance could be the key to high cell production. RESULTS Asian Conference on Lactic Acid Bacteria 2009 12 NUS, Singapore
  13. 13. 80 g/l 80 g/l iXC C:N 120 g/l iXC 180 g/l iXC Asian Conference on Lactic Acid Bacteria 2009 13 NUS, Singapore
  14. 14. 80 g/l 80 g/l IXF Protein 120 g/l IXF 180 g/l IXF Asian Conference on Lactic Acid Bacteria 2009 14 NUS, Singapore
  15. 15. • C:N ratio does not give indication to the drop in ratio biomass while there was C:N still residual sugar. • Drop in biomass seemed to be related to protein utilisation. Asian Conference on Lactic Acid Bacteria 2009 15 NUS, Singapore
  16. 16. Protein (polypeptides) profiles Look at how the polypeptide profile changes during fermentation period. What may cause the biomass drop? RESULTS Asian Conference on Lactic Acid Bacteria 2009 16 NUS, Singapore
  17. 17. Protein profiles (12% gel) Batch 80 g/l Batch 80 g/l + ion exchange Fermentation time Fermentation time kDa 50 40 30 25 20 15 Asian Conference on Lactic Acid Bacteria 2009 17 NUS, Singapore
  18. 18. Protein profiles (12% gel) Batch 120 g/l + ion Batch 180 g/l + ion exchange exchange Fermentation time Fermentation time kDa 50 40 30 25 20 15 Asian Conference on Lactic Acid Bacteria 2009 18 NUS, Singapore
  19. 19. Protein in cell (12% gel) Cell kDa 80 g/l IXF kDa 50 50 40 40 30 30 25 25 20 20 15 15 Possible secretion by cell Asian Conference on Lactic Acid Bacteria 2009 19 NUS, Singapore
  20. 20. Batch 80 g/l Asian Conference on Lactic Acid Bacteria 2009 20 NUS, Singapore
  21. 21. + ion exchange Batch 180 g/l Asian Conference on Lactic Acid Bacteria 2009 21 NUS, Singapore
  22. 22. Protein profiles (16% gel) Batch 80 g/l Batch 80 g/l + ion exchange Fermentation time Fermentation time Da 14,437 6,512 3,496 1,423 Asian Conference on Lactic Acid Bacteria 2009 22 NUS, Singapore
  23. 23. Protein profiles (16% gel) Batch 120 g/l + ion Batch 180 g/l + ion exchange exchange Fermentation time Fermentation time Da 14,437 6,512 3,496 1,423 Asian Conference on Lactic Acid Bacteria 2009 23 NUS, Singapore
  24. 24. L. lactis autolysis Pillidge et al. International Dairy Journal. 12 (2002) 133-140 • Studies related to cheese ripening properties • Complex multifactorial phenomenon • N-acetyl muramidase (AcmA) as major autolysin – degrade peptidoglycan for cell separation during growth – 46,564 Da (40,264 Da mature form after cleavage of an N- terminal leader sequence) • Other autolysin with the reported molecular mass – 30-40 kDa – ≈ 30 kDa Asian Conference on Lactic Acid Bacteria 2009 24 NUS, Singapore
  25. 25. • Ion exchange resin adsorbs lactate ion as well as essential proteins/polypeptides • Lack of nitrogen source seems to affect the cell more than carbon source (sugar) Protein profile does. • High lactate may enhance the cell drop during lack of essential N-source. Asian Conference on Lactic Acid Bacteria 2009 25 NUS, Singapore
  26. 26. Future points worth investigating • Batch at 180 g/l lactose with ion exchange, spike concentrate sugar-free medium before cell drops.  supply extra essential peptides • Batch at 80 g/l lactose or higher with no additional N-source (use normal based medium).  induce N limitation • Batch at 180 g/l lactose with ion exchange but more resin to keep low lactate.  control lactate at low level Asian Conference on Lactic Acid Bacteria 2009 26 NUS, Singapore
  27. 27. National Science and Technology Development Agency (NSTDA) Ministry of Science, Thailand. Asian Conference on Lactic Acid Bacteria 2009 27 NUS, Singapore

×