Preservation of bacteria and micro-organisms by freeze-drying

The unstoppable advance of biotechnology makes new working methods necessary to preserve the properties of cultures. Although we may not be aware of it, microorganisms are essential in day-to-day laboratory work, whether for obtaining medicines, for the production of foodstuffs such as bread, cheese or liquors, or for multiple research projects and for the manufacture of solvents and reagents.

In this sense, the preservation of microorganisms is a fundamental task to ensure the stability and purity of microbial cultures for as long as possible. Various preservation techniques are available, with freeze-drying being a method that fulfils all the necessary requirements.

What can freeze-drying contribute to these processes? Well, the truth is that one of the basic principles of freeze-drying is precisely the interruption of microbial growth, a factor that ensures the genetic stability of the culture and ensures its viability for a prolonged period of time.

As you may already know, freeze-drying is the removal of the water present in a substance by a process of freezing and subsequent sublimation (the water changes from a solid to a gaseous state directly). The freeze-drying process of cultures involves the following steps:

1. FORMULATION OF THE SAMPLE: a correct formulation of the sample to be freeze-dried is very important to ensure the survival of the cells during the process. In short, it consists of adding the micro-organisms to a solution composed of water and a lyoprotective substance. What is the function of these lyoprotective substances? To protect the cell from harmful effects during subsequent freezing and drying. For example, when water freezes, it forms ice crystals that can puncture cells and damage them both inside and out, so lyoprotectants are placed around them to prevent these effects. Examples of lyoprotectants are skimmed milk, horse whey, some polyols or various disaccharides such as trehalose, lactose, maltose or sucrose. Skimmed milk is considered an ideal lyoprotectant because of its composition, low cost and easy availability.
2. FREEZING: of course, this stage is essential to obtain a quality product that is completely dehydrated. Thus, the sample, already formulated and placed in glass vials, is placed in the freezer at -20ºC for a few hours.
3. DRYING: in order to be able to carry out the drying process, special conditions of pressure and temperature must be met. Once the samples have been transferred to the freeze-dryer, the vacuum system is turned on until the necessary pressure is reached for sublimation to occur (the ice turns into vapour) and the solvent evaporates.
4. SEALING THE Vials: it is also important that the vials are properly sealed under vacuum conditions, once dehydration of the sample becomes evident.

Freeze-drying is the most widely used method for the preservation of microorganisms as it paralyses cell metabolism, which is very important when working with bacteria, for example, which are very sensitive to heat and environmental changes.

This method therefore allows optimal survival of micro-organisms over long periods of time without altering their morphological and biological characteristics.

The advantages of freeze-drying are obvious:

• It allows the STANDARDISATION of the process and the replication of the freeze-dried strains.
• It minimises the risk of genetic change in the cells by limiting their metabolism due to lack of water and keeps them VIABLE FOR A LONG TIME, up to ten years.
• The genetic characteristics of the formulated samples are NOT ALTERED.
• The decrease in moisture content results in a compact material that can be EASILY DISSOLVED AGAIN.
• It is sometimes difficult to preserve certain micro-organisms using liquid substrates. In these cases, it is recommended to use SOLID SUBSTRATES such as sterilised and chemically treated seeds to preserve the strain.

Once the freeze-drying process is finished, in order to keep the glass vials with the strains of microorganisms for a long time, it is advisable to store them in drawers or drawers made of shock- and fire-resistant material, especially if we are talking about large productions. There are different types of furniture similar to metal safety cabinets, which will allow you to restrict access to their contents, as well as to control that the freeze-dried materials are not damaged.

Although it is true that freeze-drying allows long-term preservation without cold chain with less than 15% humidity and high microbiological stability, if the ambient temperature is high, it is best to store the vials in a refrigerator.

Preserving microorganisms over time is essential for research work and in industries such as pharmaceuticals, agriculture and food. Before the application of more advanced preservation methods such as freeze-drying, culture collections suffered from various complications such as adaptation to the in vitro environment, genetic mutations, accidental loss of cultures or contamination of cultures.

Thanks to freeze-drying, long-term storage with suspended metabolic processes was achieved, causing minimal damage to the cells and ensuring maximum viability and genetic stability. In the biological products industry, it is used to preserve blood plasma, serum, hormone solutions, drugs, surgical transplants of arteries, skin or bones, as well as bacteria, viruses and yeasts. In the food industry, it is used, for example, to preserve probiotics and prebiotics.

Probiotics are live micro-organisms (bacteria and yeasts) that are beneficial to health once ingested. They act primarily in the digestive tract, protecting it from harmful micro-organisms and improving digestion and intestinal function.

However, not all dietary supplements and not all strains of micro-organisms can be considered probiotics, even if they are labelled as such. There are 4 conditions that must be met:

1. They must be correctly identified in terms of genus, species and strain.
2. They must be safe for their intended use.
3. Supported by at least one human clinical trial demonstrating efficacy.
4. Be alive in the product, in sufficient quantities to be effective, throughout the shelf life.

It is in this aspect, that of conservation over time, that freeze-drying is considered fundamental for the preservation of these probiotic micro-organisms. Through freeze-drying, by eliminating most of the water from the cells, the micro-organisms remain dormant or inactive, which ensures their shelf life.

In this way, probiotics found in food supplements (usually in the form of white powder) or added to certain foods (yoghurts, cheeses, fermented milks, etc.) ARE ALIVE in their own way. Their metabolic activity is suspended: by freeze-drying in the case of food supplements and by a combination of low temperature and acidity in the case of dairy foods.

Here are some examples of foods containing natural probiotics: sauerkraut, natural yoghurt, miso or fermented soya, kimchi, kefir, pickles, buttermilk, etc.

CURIOSITIES BARNALAB freeze-dried products:

Speaking of yeasts, you can get real sourdough bread from certain sachets of freeze-dried micro-organisms in fine powder form (available on the market) containing millions of yeasts and lactic acid bacteria that reactivate on contact with water. Freeze-drying makes life easier!