Extraction microbiological assay

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

The use of mutant 34486 of Neurospora crassa for the microbiological assay of ch oline has been described (8). A physiological method has also been used in which the ch oline is extracted after hydrolysis from a sample of biological material and acetylated. The acetylcholine is then assayed by a kymographic procedure, in which its effect in causing contraction of a piece of isolated rabbit intestine is measured (33). 

Goyer, A., Navarre, D. A. (2007). Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay. J. Agric. Food Chem., 55, 3523-3528. 

The amount of total residues is generally determined by study with radiolabeled drugs and is expressed as the parent drug equivalent in milligrams per kilogram of the food. Bound metabolites can be measured as the difference between the total and extractable residue. Microbiological assays measure the parent molecule and its bioactive metabolites immunochemical assays measure the parent molecule and closely chemically related metabolites. 

Later studies concerned the detection of both sulfonamides and sulfonamide potentiators in fish tissues. Mixtures containing ethyl acetate, sodium sulfate, and sodium hydroxide (80), or ethyl acetate and sodium hydroxide (115), have been all used for extracting residues of sulfadiazine and trimethoprim from trout and salmon tissues. In the former approach, para-aminobenzoic acid was employed to neutralize sulfadiazine prior to the microbiological assay of trimethoprim, whereas in the latter approach Bacillus subtilis (ATCC) was directly used for sulfadiazine detection. The limit of detection for sulfadiazine using Bacillus subtilis was found to be 0.04 ppm. The test indicator organism used in both approaches to detect trimethoprim down to 0.1 ppm was Bacillus pumilus (CN60 Welcome Research Laboratories, London) (115,116). 

A titre of ca. 20 pg/ml, determined by microbiological assay on agar plates seeded with Candida albicans, was reached in 5 days. The fermentation was stopped. The fermentation mixture was extracted twice with 1 v/v of n-butanol. The combined butanol extracts were washed with 1 v/v of water, dried with anhydrous sodium sulfate and evaporated to dryness under reduced pressure to yield a residue. The oily residue was extracted 3 times with 2 L of methanol. The combined methanol extracts were passed through diatomaceous earth (Celite) and evaporated to dryness to yield an oily residue containing crude Rapamycin. 

Tamura, T., and Hyun, T.H., 2005. Trienzyme extraction in combination with microbiologic assay in food folate analysis An updated review. Experimental... 

Fazili, Z., PfeifTer, C.M., Zhang, M., and Jain, R., 2005. Erythrocyte folate extraction and quantitative determination by Kquid chromatography-tandem mass spectrometry Comparison of results with microbiologic assay. Clinical Chemistry. 51 2318-2325. 

S DeSouza, R Eitenmiller. Effects of different enzyme treatments on extraction of total folate from various foods prior to microbiological assay and radioassay. J Micronutr Anal 7 37-57, 1990. 

JI Rader, CM Weaver, G Angyal. Use of microbiological assay with tri-enzyme extraction for measurement of pre-fortification levels of folates in enriched cereal-grain products. Food Chem 62 451 65, 1998. 

The sample (10-50 fig.) in 1-2 ml. of solution is added to a large test tube containing 150 mg. of ascorbic acid freshly dissolved in 2 ml. of 1 ilf acetate buffer pH 5.0. The tube is placed in a water bath at 70°C. After 30 minutes the solution is diluted for microbiological assay. The method may be used for detecting the proportions of these vitamins (Bn and Bnb) in concentrates from fermentation sources. With liver extracts it has not been found to give effective separation. 

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