The inulin has been regarded as a good stabiliser for proteins during freeze-drying.
e.g. these two papers used 10% w/v, i.e. 100 mg/ml
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The inulin product (I2255) from Sigma-Aldrich has a solubility of 100 mg in 1 ml pure water. So the final maximum concentration is between 90-100 mg/ml, depending on the solution density.
I have a big problem with dissolving inulin (I2255) around 90 mg/ml. I asked many questions to Sigma-Aldrich. They told and suggested me:
) reported solubility in water is 100 mg/ml, this means you can dissolve up to 100 mg of Inulin in 1 ml of ultra pure water
) the average molecular weight of this product is approximately 5000 Da.
) Inulin is only slightly soluble in cold water but freely soluble in hot water. The solid matter should be completely redissolved by heating not more than 15 minutes, then cooling before use. The solution should not be reheated.
) Solubility specification for this material is 100 mg/mL. This solution is heated at 105°C for ~ 5 minutes.
I tried using 42°C water bath or 105°C oven to heat the inulin solution. The white solution slightly become clearer after heating, but turned back to white colour after several hours. I can also see some sediments at the bottom of the tube. When it was filtered through 0.2 μm filter, the filter easily blocked, so the inulin is not actually in monomeric forms of 5000 Da. Because the filters are blocked, so large quantities would be retained in the filter.
I need to use inulin for freeze-drying formulation. So I need to make sure the inulin solution is stable at room temperature. It should also be fully dissolved, so the desired concentration can be achieved. The papers above did not mention how to dissolve inulin (e.g. if heating is used). So can I ask how to actually handle this?
You can check this link
https://www.medicalnewstoday.com/articles/318593
Also check the preparation of inulin as therapeutic agent in pharmacy
https://www.tandfonline.com/doi/pdf/10.5661/bger-28-33
Inulin as filler-binder for tablets prepared by direct compaction
https://www.researchgate.net/publication/11556198_Inulin_as_filler-binder_for_tablets_prepared_by_direct_compaction
Stjepan Krešimir Kračun
Thank you very much for your information. At least this is a common issue. But I am curious this heating procedure is "never" mentioned in papers, and many papers still use 10% w/v, which exceeds the theoretical solubility.
"When cooled down, most of them will crash out and precipitate"
) So it is difficult to maintain a desired concentration in a homogeneous solution.
"not to heat up the sugars above 80 degrees centrigrade - because you risk caramelization"
) I see your point. No worries, the solution temperature is not that high.
"getting dissolved" and "staying dissolved"
) This is a good differentiation.
MW distribution of inulin
) I can understand the MW distribution. However, its average MW is 5000 Da, even it is 100 kDa, it should still pass through 0.2 μm filters?
"1. Dialyze your sample with a 3000 kDa dialysis membrane against water"
) This would be a complicated procedure, and I could not know the exact quantity that diffuse out of the membrane.
"2. If you need it to be stable only until it's frozen - why is it a problem that it crashes out after a few hours when you're going to freeze it and freeze-dry it? Just freeze it before it crashes out."
) Inulin is only one of the excipients I am using. I also use sucrose, trehalose, Tween, etc. So I make stock solutions for them and mix them together, which takes hours to do. If the inulin is such unstable, I could not control the time very well. I need to have the data reproducible between different batches.
The last 4 steps of heating-cooling-centrifuge-FD
) This seems to be the only way to quantify the "staying dissolved". But I feel the "crashes out" is an equilibrium. As the sediment is removed, more inulin would crash out.
Thank you Chinaza Godswill Awuchi . But I did not see any guidance about dissolving inulin in solution.
Dissolve in demineralised water (neutral pH) and heat until inulin completely dissolves. Upon coolong and prolonged storage at room temperature or in the fridge you might observe precipitation (depending on the concentration, since the strongly temperature dependent solubility of inulin might cause it to reprecipitate.
Roland Beck thank you! I can get this phenomenon. But what I am wondering now is, how the people actually do in their papers, especially for 10% w/v? Do they cool down the stock solution, only take the supernatant? If the supernatant is taken, there are two variables, 1) even the supernatant has minor particles, so pipetting would give batch-to-batch difference; 2) the supernatant concentration is lower than the initial concentration.
10% w/v solution of inulin with a molecular weight of 5000 Dalton is certainly not stable for a long time before reprecipitation.
You may want to try fructo-oligosaccharides which have a much better solubility and solution stability, since they are much smaller in molecular weight.
You can use the shorter chain of inulin instead of the longer one of Sigma inulin or you partially hydrolyze it so as to easily dissolve it and avoid precipitation.
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