Methods for Microbiome preservation

A. Cell Alive System (CAS) Technique for Intact Microbiome Preservation
The Cell Alive System (CAS), is a novel technique primarily developed to improve the preservation method in food industry, especially for preservation of raw seafood items. In this technique, the spinning motion of water during freezing prevents formation of crystal lattices. The presence of alternating or oscillating magnetic field and mechanical vibrations in this system induces uniform cooling and minimum size ice crystal formation in preserved sample. This process prevents cellular and molecular damage to the preserved sample. CAS freezing technology has been approved as a well-optimized tool for long-term preservation of deep-sea sediment samples for geo-microbiological studies. With suitable improvements to this methodology, researchers might be able to achieve more storage time and viability.

B. Cryopreservation and Lyophilization in Microbiome Preservation
Cryopreservation and lyophilisation are well-known methods for long-term preservation of microbial cultures. Cryoprotectants are a group of chemicals, which prevent the ice crystal formation during freezing process. Also, ultra-low temperature gives a high stability and viability to the microbial cells. Glycerol and DMSO are commonly used cryoprotectants. However, Galacto-oligosaccharides (GOS) another cryoprotectant is getting more attention due to its prebiotic properties. Different microbes behave differently in terms of viability and stability with different cryoprotectants and more study is needed to be done in this area.

C. Gelatine Disk Method: Preservation of Sample During Transportation
This is a simple bacterial preservation technique using gelatine disk. This method gives good result for successful preservation of a number of pathogens but, did not perform well for prolonged preservation of Vibrio and Neisseria species. Gelatine disk method allows survival of fastidious organisms up to one year at – 20 C storage. There is no direct report on the use of the gelatine disk method for the conservation of an intact microbiome, but due to its ability to preserve the viability and functional stability of a variety of bacterial strains, researchers may consider this technique to be a potential alternative to long-term microbiome preservation.

D. Cellular Immobilization or Entrapment
Cellular immobilization or entrapment in the gelling matrix is another alternative for long-term preservation of microbial viability and functionality. Formation of micro-droplets or beads with different gelling agents like alginate and Acacia-gum is well known method of microbial cell entrapment. This method is getting good response in probiotic industry due to better survival and functionality of the entrapped cells compared to other preservation methods and sustained release in gut after intake. Organisms of probiotic importance like Bifidobacterium and Enterococcus have been preserved using this method. Adding cryoprotectants and antioxidants to the gelling matrix ensures long-term survival and stability of entrapped cells. This method is used with pure culture of microbes however like other preservation methods it can be explored for mixed microbiome preservation, and protocols needs to be optimized.

F. Electrospinning and Electrospraying (Microencapsulation) in Microbiome Preservation
Many microbial cells are sensitive to micro-environmental changes and it’s difficult to preserve them using normal preservation techniques. Microencapsulation method works better for such sensitive microbes. Electrospinning and Electrospraying are novel microencapsulation techniques used to maintain the viability and functionality of microorganisms. For Electrospinning, the viscosity of the polymer is high. Polymer solution in Electrospraying is less visocus, offering nano- or microscale fibres of polymer entrapped microbial cells and small droplets (beads) respectively. Unlike other methods, non-involvement of temperature in these processes reduce the protein- denaturation from harsh temperature conditions and consequently increase the cellular viability and functionality during preservation. Since both these methods are used for small groups of microbes, further studies are required to test its preservation efficiency for other group of microbes.

Reference: https://link.springer.com/article/10.1007/s12088-020-00880-9

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