Water in freeze-dried lactic acid bacteria: Friend or Foe

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1 EuroFoodWater, March, Reims, France Water in freeze-dried lactic acid bacteria: Friend or Foe Stéphanie Passot, Stéphanie Cenard, Inès Douania, Cristian Tréléa, Fernanda Fonseca Joint Research Unit of Microbiology and food process engineering Thiverval-Grignon, France A G R I C U L T U R E A L I M E N T A T I O N E N V I R O N N E M E N T

2 Outline 1. Context and objective of the work 2. Experimental methods 3. Main results 4. Conclusions

3 Context Concentrates of lactic acid bacteria are used as starters for cheese, fermented milk Freeze-drying is widely used to improved the shelf life of these concentrates. Excipients (sugars, polymers ) Freeze-drying Freezing Active ingredient (Bacteria) Primary Drying Secondary Drying Product Storage Quality criteria The dryer, the better? Water content (<3%) Activity Structure

4 Context Chemical, biological stability Degradation reaction rates Water activity

5 Context State diagram Glass transition temperature Water content Water monolayer parameter Chemical, biological stability Degradation reaction rates Sorption isotherm Water activity Water activity Roos 1993

6 Objective Our objective was to investigate the effect of water activity on: The physical properties of freeze-dried amorphous matrix: Tg, water content The lactic acid bacteria stability

7 Outline 1. Context and objective of the work 2. Experimental methods 3. Main results 4. Conclusions

8 Experimental Lactic acid bacteria : Lactobacillus delbrueckii subsp. bulgaricus CFL1 Fermentation Concentration Protective medium 20% of sucrose Freeze-drying Equilibration under various HR (25 C) Salt HR (%) P 2 O 5 0 LiBr 0.06 LiCl 0.11 CH 3 COOK 0.23 MgCl K 2 CO Mg(NO 3 ) 2.6H 2 O 0.53 NaCl 0.75 KCl 0.84 Storage (25 C)

9 Experimental Lactic acid bacteria : Lactobacillus delbrueckii subsp. bulgaricus CFL1 Fermentation Concentration Protective medium 20% of sucrose Freeze-drying Equilibration under various HR (25 C) Storage (25 C) Physical properties Glass transition temperature: DSC Pyris 1, Perkin Elmer Water content: Karl Fisher titration (Metrohom) Water activity: Aw-meter (Novasina) Biological properties Acidification activity (CINAC system) Viability After equilibration and storage at 25 C

10 Cinac System Acidification activity ph transmeteur T transmeteur Interface ph = f(t) dph/dt = f(t) Sonde ph ph tm Computer temps ph dph dt Heating resistance Controlled T bath Temperature probe Erlenmeyers flasks containing milk + inoc Tmax dph/dt (phu/min) Before FD After FD After storage The higher the Tmax value, The lower the acidification activity time (min) FD: Freeze-drying

11 Outline 1. Context and objective of the work 2. Experimental methods 3. Main results 4. Conclusions

12 Results: Sorption isotherm Determination of the water monolayer (Mo) Water content (%) m = BET equation M CA 0 w ( 1- Aw)( 1- Aw + CAw) Parameter Mo (%) C C200+LAB ,2 0,4 0,6 0,8 1 Water activity Tg = 22 C Aw = BET: Mo: water monolayer parameter

13 Results: Sorption isotherm Determination of the water monolayer (Mo) BET equation Water content (%) C 3.6% ,2 0,4 0, Tg ( C) m = M CA 0 w ( 1- Aw)( 1- Aw + CAw) Parameter Mo (%) C C200+LAB Water activity Tg = 22 C Aw = BET: Mo: water monolayer parameter

14 Results: Acidification activity Aw (Tg=25 C) 0.24 Aw (Mo) Tmax (min) t=0 t=7 days t=10 days t=29 days 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Water activity The higher the Tmax value, The lower the acidification activity

15 Results: Acidification activity Quantification of the rate of loss of acidification activity during storage 1400 Tmax (min) y = 30,623x + 410,8 R 2 = 0,9981 k (min/days) y = 5,6532x + 377,27 R 2 = 0,996 Aw = AW = Time of storage (days)

16 Results: Acidification activity 4 fermentations Various physiological states of bacteria 8 7 F1 F2 F3 F4 Aw (Tg=25 C) 0.24 Aw (Mo) k (min/day) ,05 0,1 0,15 0,2 0,25 0,3 0,35 Water Activity

17 Results: Acidification activity 4 fermentations Various physiological states of bacteria Tg = 51 C F1 F2 F3 F4 Tg 60 k (min/day) Tg = 36 C Tg = 25 C Tg ( C) ,05 0,1 0,15 0,2 0,25 0,3 0,35 Water Activity Aw(Mo) 0-10

18 Results: Acidification activity 4 fermentations Various physiological states of bacteria 10 9 F1 F2 F3 F4 8 7 Mobility transition T < Tg k (min/day) T-Tg ( C)

19 Conclusion The dryer, the better is not always true! Concepts of critical water activity (hydration limit) and critical temperature (mobility transition T < Tg): interesting approach for predicting stability of freeze-dried lactic acid bacteria. Further works are needed to better investigate the water mobility within the glassy matrix: Various storage T and protective excipients. Relating glass transition temperature and bacteria activity to water activity appears as a useful tool to optimize : Freeze-dried lactic acid bacteria stability during processing and storage. Freeze-drying cycle, especially secondary drying

20 Thank you for your attention This work was financially supported by the European Community: CAFÉ Project: Computer-Aided Food processes for control Engineering Joint Research unit Microbiology and food process engineering Grignon, FRANCE

21 titre de partie (Arial taille 28) Titre (Arial taille 44) Titre secondaire (Arial taille 32) > texte avec puce (Arial ou Arial narrow ) Texte courant (Arial ou Arial narrow taille 20) Arial ou Arial narrow taille 18

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