Hi guys,
I have been working to make probiotics using freeze dried L.crispatus. I am using 10% skim milk and 15% trehalose + lactose (1:1) as cryoprotectant. Bacteria seems to survive in skim milk but viable count drops by 100 times to that of before freeze drying and it does not seem to survive at all in trehalose + lactose and I can not wonder as to why it is happening.
Any help would be greatly appreciated. Than k you
Dev - hard to say given the relative lack of information - MANY things could contribute to viable count loss. There is always some loss when freezing, but agree that 100 fold sounds a lot.
15% sugar sounds high, particularly with the milk. Gut feel suggests that the freezing point of such a mixture will be very low, making successful freeze drying a challenge.
Not sure of the stability of your bacteria in a range of pHs, do watch out for pH shift on freezing.
below is a general guide which may help:
https://www.spscientific.com/freeze-drying-lyophilization-basics/
Rob Darrington
Bacteria was cultured in MRS broth anaerobically at 37 degree C for 48 hrs
10% skim milk and 15% sugars are used as two separate cryoprotectants. I had performed viable count before freeze drying when kept in -80 degree C to freeze up and another viable count after freeze drying. Before freeze drying both cryoprotectant showed 10^7 CFU/mL while after freeze drying skim milk was 10^5 CFU/mL and trehalose + lactose was zero viable count. Freeze drying was performed at -46 degree and 0.06mBarr
Bacteria was also resuscitated in MRD broth before performing viable count for freeze dried bacteria
I will check for pH and try to maintain it acidic through out the experiment.
Thank you for your help, much appreciated.
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