Inactivation,freezing

Enzymes not only produce characteristic and desirable flavor (79) but also cause flavor deterioration (80,81) (see Enzyme Applications, Industrial). The latter enzyme types must be inactivated in order to stabilize and preserve a food. Freezing depresses enzymatic action. A more complete elimination of enzymatic action is accompHshed by pasteurization. 

Each interferon preparation was ultracentrifuged at 20,000 revolutions per minute for one hour to remove tissue debris and inactivated virus. The supernatant was dialyzed against distilled water (1 400) for 24 hours at4°C. The material was then freeze-dried. The dried product was reconstituted in one-tenth of the original volume in distilled water and dispensed into ampoules. Reconstituted solutions were assayed for interferon activity, examined for toxicity, and tested for sterility. 

Preparation of Balanoglossus luciferin. The residue of the first pH 6 extraction above was re-extracted with 50 mM potassium phosphate buffer, pH 8. After centrifugation, the supernatant was used as the standard luciferin preparation. Luciferin was highly labile and easily inactivated at an extreme pH, by heat, and also by freezing and thawing. The instability resembled that of certain proteins. 

Although freeze-fracture experiments have demonstrated that monomers are assembled into stable tetramers in the membranes, radiation inactivation studies and, later, expression studies revealed that each monomer is a functional water channel (Fig. lc). 

Thermostable pectinesterases (TSPE), operationally defined as activity that survives 5 min at 70°C, contribute most to cloud loss in juices at low temperatures and juice pH (26). The percentage of total activity that is thermostable is highly variable and differs in kinetic properties, (22, 26), ease of solubilization (28, 29), stability to low pH (25) and stability to freeze-thaw cycles (23). Some of the variability in reported total PE and TSPE may be related to limitations of current methods to quantify activity. Any processing treatment or assay condition that increases cell wall breakdown or release PE from a pectin complex would enhance detection of total and TS-PE activity. Commercially, PE is inactivated by pasteurization in a plate heat exchanger or during concentration in the TASTE evaporator. 

In order to preserve, as much as possible, the phenolic content in fruit and vegetable samples, the literature proposed the application of cold temperatures, even reaching to freezing, when lyophilization is the objective. These procedures also could inactivate the enzymes. The freeze-drying is largely the main preservation technique used in the studies related to the identification and quantification of the phenolic compounds of fruit... 

Freeze-drying is a relatively gentle way of removing water from proteins in solution. However, this process can promote the inactivation of some protein types, and specific excipients (cryopro-tectants) are usually added to the product in order to minimize such inactivation. Commonly used cryoprotectants include carbohydrates (such as glucose and sucrose), proteins (such as HSA), and amino acids (such as lysine, arginine or glutamic acid). Alcohols/polyols have also found some application as cryoprotectants. 

After its purification, sterile filtration and aseptic filling, human urokinase is normally freeze-dried. Because of its heat stability, the final product may also be heated to 60 °C for up to 10 h in an effort to inactivate any undetected viral particles present. The product utilized clinically contains both molecular mass forms, with the higher molecular mass moiety predominating. Urokinase can also be produced by techniques of animal cell culture utilizing human kidney cells or by recombinant DNA technology. 

While Giardia is sensitive to freezing of soil, Cryptosporidium is more resistant. Mahdy et al. (2008) reported Giardia cysts in soil were inactivated after 7 days at -4 °C, but Cryptosporidium could survive for >12 weeks. However, persistence of both protozoa was reduced to 8 weeks at 4 °C and 4 weeks at 25 °C (Mahdy et al., 2008). Cryptosporidium have been shown to be particularly sensitive to drying. Various studies have shown less than 5% viability following 4 h of air drying at room temperature (Anderson, 1986 Nasser et al., 2007 Robertson et al, 1992). 

Sheep serum. Heat-inactivate by incubating in a water bath at 55°C for 30 min. Store in appropriate amount of aliquots at -20°C. Do not freeze-thaw repeatedly. 

Most enzyme powders are prepared by lyophilisation (freeze drying). However, the lyophilization procedure might inactivate the enzyme to some extent. To avoid this and thereby increase the activity of lyophilized enzymes in dry organic solvents, the lyophilization can be carried out in the presence of lyoprotectants such as sorbitol (Dabulis and Klibanov, 1993). The inactivation is believed to be caused at least partly by a reversible conformational change in the enzyme. This process can be reversed and the enzyme reactivated by the addition of small amoimts of water (Dabulis and Klibanov, 1993). 

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