**2.6 Centrifugation**

After incubation, the 5 L bacteria culture was aseptically transferred to centrifuge tubes for centrifugation. The centrifuge (CENLEE, Model: CFLR8, made in China) was


#### **Table 1.**

*Composition of the PBS buffer.*

precooled at 4°C and centrifugation was done at 4000 rpm for 5 mins [9]. After centrifugation, the pellet was collected and the supernatant was discarded. Repeated the same procedure twice with PBS buffer. The QC analysis was done by checking CFU of the pellet.

## **2.7 Pre-freezing**

Different ingredients can be used as cryoprotectant and give good survival of bacteria. Here, we used 10% Skim milk and 1% Dimethyl sulfoxide (DMSO) as cryoprotectant [17]. The cell pellets and cryoprotectant ratio was maintained equally and mixed gently. Subsequently, pre-freezing of mixed cell pellets was initiated at 4°C for 2 hrs in the refrigerator, 4 hrs in-20°C and at last it was kept overnight at -86°C.

### **2.8 Lyophilization**

Freeze drying process was completed in a lyophilizer machine [18]. The pre-freeze cell pellet from the −86°C was freeze dried in the lyophilizer (Bioevopeak, model: LYO80V-2S), made in China). Running time can be variable on the basis of sample amount. The freeze drying was done for 50 hrs. After 50 hours, the dry pellets were collected and stored at 4°C for further process. The QC analysis was done by checking CFU of the dried cells.

#### **2.9 Formulation**

Formulations of the lyophilized dried (LP) cells were mixed with different carrier particles such as dextrose, peat soil, and talc and kept it for shelf-life analysis. The CFU of the formulated products was checked initially and frequently with time intervals.

#### **2.10 Dextrose formulation**

The 5% and 10% freeze-dried microbes were mixed with 95% and 90% dextrose, respectively, and kept it for shelf-life analysis at room temperature.

### **2.11 Peat formulation**

We used two different methods, that is, solid and liquid to make peat formulations. For solid formulations, we mixed 5% lyophilized microbes and 95% peat (with less than 5% moisture) and kept it at room temperature for shelf-life analysis. We also *Optimizing Shelf-life of* Pseudomonas fluorescens *after Freeze Drying DOI: http://dx.doi.org/10.5772/intechopen.108034*


**Table 2.** *Pellet solution preparation.*

made liquid formulations by using cell pellets suspended in the following solution (**Table 2**) and mixed with the peat powder. Then 20 ml liquid pellet suspension was mixed with 70 g peat soil fine powder. Initially, the peat was grinded into fine powder, and pH was adjusted to pH 7 with calcium carbonate (CaCO3).

## **2.12 Talc formulation**

The 5% and 10% freeze-dried microbes were mixed with 95% and 90% talc, respectively, and kept it for shelf-life analysis at room temperature.
