Dec 03, 2025
Freeze Drying Plant and Food Samples
- Alexander Hernandez1,
- Michael Dzakovich1
- 1USDA-ARS Children's Nutrition Research Center
Protocol Citation: Alexander Hernandez, Michael Dzakovich 2025. Freeze Drying Plant and Food Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.261gek12jg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 31, 2025
Last Modified: December 03, 2025
Protocol Integer ID: 231278
Keywords: freeze dry, food, plants, plant foods, dry matter content, freeze-drying, moisture, lyophilization, freeze drying, drying plant, plant biochemistry, controlled primary drying, food sample, food science, based food sample, standardized method for freeze, food samples this protocol, retention of delicate nutrient, lyophilized material, nutritional content, secondary drying, primary drying under vacuum, bound moisture, nutrition, freeze, delicate nutrient, phytochemical, downstream biochemical analysis, large ice crystal
Funders Acknowledgements:
USDA-ARS CRIS Funds
Grant ID: 3092-10700-066-001S
Abstract
This protocol describes a standardized method for freeze-drying plant-based food samples and is optimized to preserve structural integrity, nutritional content, and bioactive compounds. Samples are first gradually frozen to form large ice crystals, then subjected to controlled primary drying under vacuum, followed by secondary drying to remove bound moisture. By carefully managing sublimation and temperature profiles, this method minimizes collapse and volume loss, while maximizing retention of delicate nutrients and phytochemicals. The resulting lyophilized materials are ideal for long-term storage, downstream biochemical analyses, or other studies. This approach balances process efficiency with sample quality, making it broadly applicable for research in food science, nutrition, and plant biochemistry.
Materials
Harvest Right Freeze-Dryer Mk 10.967a
Troubleshooting
Problem
Dark spots or signs of residual moisture
Solution
If dark spots or other signs of residual moisture are present, the system may have been overloaded. Re-freeze samples immediately and start the process over the following day. Depending on the amount of remaining moisture, the samples may only need to be freeze dried for an additional 48-72 hours
Preparation
14h 10m
Prior to freeze drying, create and save the freeze drying profile (details are found in "run profile" section)
10m
Ensure samples are frozen (ex: leave samples in a -20 ℃ freezer for at least one night)
10h
Freezing samples more slowly will result in relatively larger ice crystals making lyophilization more efficient
Power on freeze dryer (Harvest Right Freeze Dryer Mk 10.967a in this protocol, but these parameters will likely work on other systems) and set it to chill to -30 °F
3h
The freeze dryer will take ~3 hours to reach temperature
gTo chill more efficiently, install the insulating cover and gasket, close the door, and close the drain hose
Insulating cover and gasket
Closed freeze dryer with insulating cover and gasket installed
Drainage hose in the closed position
Move your samples to a walk-in refrigerator. There, uncap all your samples and cover each sample tube opening with a piece of cheese cloth
Example of tubes covered with cheese cloth
20m
Secure the cheese cloth in place with a rubber band
5m
Move the samples back to a freezer. Allow the samples enough time to become frozen again (typically at least 20 minutes in a -80 ℃ freezer)
20m
Once the freeze dryer reaches a temperature of -30 °F, close the vacuum pump line and start the vacuum pump.
Vacuum pump in the closed position
15m
Allow the vacuum pump to warm up for 15 minutes before loading your samples.
Loading samples
9m
Remove your samples from your freezer and then place them inside the freeze dryer on the shelf. Replace the insulating cover and latch the freeze dryer door closed
3m
Make sure you place the sample tube caps on the lower freeze dryer shelf. They may have a bit of sample material or residual moisture on them that also need to be freeze dried. Not freeze-drying the caps will alter total weight and moisture content estimates.
Set the freeze dryer to have a maximum pressure of 700 mTorr and a minimum pressure of 300 mTorr
Freeze dryer screen. The pressure range is set to 300 (Low) and 700 (High)
1m
Ensure the drainage hose is closed. Then, open the vacuum pump line slowly to allow the pressure to decrease (~1 minute)
Vacuum pump in the partially open
1m
Wait until the pressure reaches your pressure range (300-700 mTorr) then proceed to next section
4m
Run Profile
5d 0h 2m
Start the profile to begin freeze drying. The profile needs to have a ramp rate of 5 °F per minute and must consist of the following steps and settings:
Freeze dry protocol steps
"Vacuum" and "Chiller" should be activated for all steps
Pressure should range from 300 to 700 and the ramp rate (℉/min) should be 5
2m
step 0: temperature set to -20 °F for 2 hours
step 1: temperature set to 0 °F for 2 hours
step 2: temperature set to 20 °F for 2 hours
step 3: temperature set to 40 °F for 2 hours
step 4: temperature set to 60 °F for 99 hours
step 5: temperature set to 60 °F for 99 hours
Depending on how much total water content is in your samples, leave the samples in the freeze dryer anywhere from one day to five days
5d
The more total water content your samples are, the longer you should leave the samples in the freeze dryer. Five days is recommended for most batches of ~200-250 g of total plant material
Unloading freeze dryer
10m
Close the vacuum pump line, then end the freeze dryer profile
2m
Make sure you close the vacuum pump first to avoid rapidly pressurizing the samples
Ensure that vacuum pump is powered off once isolation valve is in the closed position
Slowly open the drainage hose (~1 minute) to allow the pressure in the freeze dryer to equalize
1m
Remove samples from the freeze dryer shelf. Immediately remove the cheese cloth and reinstall the sample caps
3m
Turn off the freeze dryer completely. Leave the door open and keep the insulating cover removed to allow the the inside to defrost and dry out
Freeze dryer post shutdown
2m
Weigh capped samples if necessary and move samples to their storage destination. For some applications, room temperature can be suitable, but do sufficient testing to ensure any compounds of interest are stable over time.
2m