100A-16 |
Preparation of cryoprotected Escherichia coli culture for a lysine bioavailability assay |
X. LI and S. C. Ricke. Poultry Science Department, Texas A&M University, Kleberg Building, Room 101, College Station, TX 77843-2472 Lysine is one of the essential amino acids and can be used as an indicator of biological value of food protein. Food processing and storage can cause loss of lysine bioavailability. Among the existing assays to determine amino acid content, microbiological assays have shown to be more rapid, simple, inexpensive and relatively as accurate as animal assays to quantify availability of amino acids in protein sources or food samples. The objective of the study was to compare various cryoprotective agents to preserve the Escherichia coli lysine auxotroph culture for an E. coli lysine microtiter assay. The overnight culture of the E. col lysine auxotroph were harvested by centrifugation. The bacterial pellet was washed twice using M9 medium and finally resuspended in M9 media plus different cryoprotective agents (0.3 M trehalose, 0.3 M glycerol, 0.3 M lactose or 0.3 M sucrose). In order to examine the optimal effect of induced cryotolerance of the cryoprotective agents, the bacterial suspensions were preincubated at room temperature following by freezing immediately at –20 0C for 7 days before estimation of the survivability by enumeration on LB plates. Cryoprotective agents improved the resistance of the culture to freezing and thawing. Glycerol had the greatest protective action yielding 98% survivors followed by sucrose 35%, trehalose 34% and lactose 10%. The E. coli lysine assay using the cryoprotected lysine auxotroph culture was compared with serial sub-cultured inocula. The standard curve for lysine estimation generated by using sucrose cryoprotective culture was not significantly (p>0.05) different to the standard curve from using serial sub-cultured inocula. Using the cryoprotected culture was less labor intensive than using the conventional serial culture for the lysine microtiter assay because the bacterial culture could be grown in large quantities and frozen for future use.
Session 100A, Food Microbiology: General II
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