Lyophilization, bacteria

Organisms which have been freeze-dried under vacuum (see above). Some bacteria, for example, can be lyophilized and stored for months at room temperature. They can then be rehydrated on demand and used to conduct bioassays. In the Microtox test, lyophilized Vibrio fischeri are stored in a freezer at -20° C and will be ready for use until the expiration date, which is provided with each batch of Bacterial Reagent. Volume 1(2,3). 

Packaged as a commercial product, NPVs have a remarkable shelf life. The occluded virions, within the protective inclusion-body protein, are among the most stable entomopathogens, compared with other microorganisms and microparasites, such as bacteria, fungi, protozoa, and nematodes. In the laboratory, lyophilized, frozen, or simply refrigerated NPV inclusion bodies, stored in darkness, remain active for many years, even decades... 

Bioluminescence inhibition of bacteria in water samples (method using lyophilized bacteria)... 

These enzymes must have a large activity spectrum, and ideally enantioselectivity for toxic stereoisomers. Their mass production under GMP conditions must be realizable at a reasonable cost. Long-term storage without activity loss (in solution, lyophilized, or adsorbed/bound on a matrix) must be possible under field conditions. Conformational stability can be optimized by chemical modification or addition of stabihzers such as polyols. Thermostable enzymes from thermophilic bacteria (Merone et al., 2005) or mutated/ evolved highly stable enzymes from mesophilic bacteria (Elias et al., 2008) are promising alternatives. 

Pure culture yeasts are available on slants or in lyophilized form. They may be propagated in wineries without complete asepsis (26,27), since the low pH of grape must inhibits many bacteria wine fermentations are mixed cultures naturally. Production of active dry wine yeasts (WADY) began in the 1960s. These are produced by bakers yeast companies and are grown by methods resembling those used for bakers yeast production, described eadier. 

In brief, freeze-drying (lyophilization) consists of preparing the drug solution (with buffers and cryo-protectants), filtering through a bacteria-proof filter, dispensing into containers, removing water in a... 

FIG. 5 Viability of Saccharomyces cerevisiae (o) and Oenococcus oeni strain EQ-54 ( ) inoculated into a Chardonnay juice with the bacteria prepared using a diluted grape juice medium (A) or a lyophilized culture (B). (Adapted from Semon et al, 2001 and with the permission of the Australian Journal of Grape and Wine Research.)... 

In some of our own work, we have developed formulations to lyophilize and preserve bacteria for extended periods of time at relatively high temperatures (Conrad et al., 2000 Ekdawi-Sever and de Pablo, 2003). Some of our data are shovm in Figure 9.2, where it can be seen that freeze-dried samples of Lactobacillus acidophilus in trehalose-phosphate mixtures retain more than 60% of their viability after 3 months of storage at relatively high temperatures. In contrast, commercial processes and formulations lead to complete loss of viability after a few days of storage. 

To estimate water quality, bioluminescent biosensors have been devised and successfully used. They are characterized by rapidity and simplicity of use, high sensitivity, and accuracy. The Collection of Luminous Bacteria IBSO (http //www.bdt.org.brA3dt/msdn/ibso) is being used to develop bioassays for monitoring the environment, using lyophilized luminous bacteria and the luminescent system isolated from them. Bioluminescent assays have an advantage over other biological assays luminescence is easy to measure, the method is rapid, and the measurements can be automated. 

The lyophilized luminous bacteria and lyophilized mixture of luciferase (Lu) from Photobacterium phosphoreum and NADHrFMN-oxidoreductase (R) from P. leiognathi were produced by the Biotechnology sector of the Institute of Biophysics (Krasnoyarsk). One vial of enzymes contained 0.11 mg of Lu and 0.069 units of activity/mL of R. One unit of R activity was defined as 1 mol of NADH degraded per min. All the assays were performed in the 0.1 mol/L phosphate buffer solution at pH 6.8 at room temperature. 

The use of high-speed mixing devices for suspending the bacteria is likely to damage the cell walls. Suspensions of good quality can be obtained by triturating the lyophilized bacteria with a small amount of buffer in an agate mortar, filtration of the diluted suspension over filter paper with coarse pore size, and ultrasonication in cooled water at low frequency (50 kHz). 

Lyophilized products are characterized by prolonged shelf-life, and chemical bacteria or enzymatic changes do not easily occur. The sterility is more guaranteed and solubility assured. In addition, transportation is easier. Finally, certain compounds or radiopharmaceutical kits exist only as lyophilized products. However, freeze-dried products suffer from certain disadvantages. The reentry of moisture may destroy the products. Direct optical control of lyophilized products cannot be performed. Therefore, the risk of particle contamination of the final product is high. Bacterial contamination can only be avoided by using the proper installations (clean rooms) for manufacturing injectable lyophilized products. 

There is some evidence to suggest that soluble compounds produced by lactic acid bacteria may act directly on tumor cells to inhibit their growth. Arimochi et al. (1997) showed (a) an inhibitory effect of L. acidophilus on azoxymethane-induced ACF formation in rat colon and (b) enhanced removal of 0 -methylguanine from the colonic mucosal DNA. This inhibitory effect was found in culture supernatants and not from bacterial cells. Furthermore, other studies have shown that dietary administration of lyophilized cultures of B. longum suppressed azoxymethane-induced colonic tumor development, along with a decrease in colonic mucosal cell proliferation and colonic mucosal and tumor ornithine decarboxylase and ras-p21 activities (Reddy, 1998). 

Preparation of culture supernatants - R haemolytica biotype A1 was grown as previously described (17). Briefly, colonies from a blood agar plate wCTe used to inoculate tryptic soy broth. This culture was incubated at 37 C for 4.5 hours. This broth was used to inoculate fresh tryptic soy broth which was incubated for 1 hour at 370 C with 5% CO2 bubbled throu it. The broth was stirred as rapidly as possible without foaming. After 1 hour of incubation bacteria were removed from the culture by ultrafiltration (Pellicon, Millipore, Inc.). Culture supernatants free of bacteria were lyophilized and stored dessicated at -20 C until loaded into hydrogels. 

---