The Use Of Bacteria In The Chemical Determination Of Total Vitamin c

THE

USE OF BACTERIA IN THE CHEMICAL DETERMINATION OF TOTAL VITAMIN C
BY I. C. GUNSALUS
AND

DAVID

B. HAND College

(From

the Department Agriculture, (Received for

of Dairy

Industry, New York State Cornell University, Ithaca) September 22, 1941)

of

publication,

Based on the discovery of Esselen and Fuller (3) that certain strains of Bacterium coli are able to reducedehydroascorbic acid, a simple rapid method has been worked out for the determination of vitamin C in food products. After 15 minutes incubation with a suspension of resting cells the total vitamin C in the form of ascorbic acid is titrated directly with 2,6-dichlorophenol indophenol dye. The procedure, as applied to milk, is as follows: 10 cc. of milk are measured into a test-tube containing 0.5to 1.Occ. of Bacterium coli suspension. After mixing, the contents of the tube are brought to 40” and held 15 minutes. The tube is then emptied into a beaker and rinsed with 20 cc. of 0.1 N HzS04. The acidified solution is titrated immediately with dye to an end-point remaining definitely pink for 30 seconds (7). Various acids may be us!ed for acidification and the solution may be filtered beforetitration, although this additional step is usually unnecessary. For the determination of total vitamin C in food products other than milk, the extract or juice should be brought to pH 6.2 before reduction. The bacteria are more active if 1.0 cc. of cell suspensionis mixed with 1.0 cc. of 10 per cent glucose 15 minutes before addition of the extract to be analyzed. Pretreatment of the bacterialsuspension with glucose is not necessary when milk is the material for analysis. The Bacterium coli suspension is prepared as follows: An 18 to 24 hour culture of Bacterium coli is added to 5 liters of broth containing 1.0 per cent peptone, 0.1 per cent glucose, and 0.3 per cent secondary potassium phosphate and allowed to grow
853

Downloaded from www.jbc.org by guest, on September 8, 2011854

Determination

of Vitamin

C

14 to 24 hours at 37”. The culture is centrifuged in a continuous centrifuge, the cells washed once with 0.03 M neutral phosphate, then suspended in 50 cc. of the neutral phosphate, and stored in a refrigerator. There is no appreciable deterioration in activity of the cell suspension in 1 month if it is kept cold. Approximately one-half the strains ofBacterium coli isolated from human feces are active in reducing dehydroascorbic acid but the individual strains show considerable differences in activity. A strain designated as “Crookes,” originally presented to the authors through the kindness of Dr. Esselen, has proved consistently active. Besides Bacterium coli certain strains of the closely related Bacterium cloacae were also found to reducedehydroascorbic acid. Negative results were obtained with fifteen strains of streptococci and with bakers’ yeast. Bacterium coli is to a certain extent a specific biological reagent for the determination of oxidized vitamin C in that no oxidation product of ascorbic acid is reduced except dehydroascorbic acid. Oxidation of ascorbic acid with hydrogen peroxide, or treatment of dehydroascorbic acid withalkali or heat yields products which no longer can be reduced with these bacteria. However, the reducing action of Bacterium coli is not entirely specific for oxidized vitamin C, because the oxidation product of d-isoascorbic acid can also be reduced. See Table I. The necessary conditions for the quantitative reduction of dehydroascorbic acid have been elaborated. The sensitivity of the acid toheat and alkali offers the chief difficulty. If conditions of pH and temperature for the most rapid bacterial action are selected, the dehydroascorbic acid disappears so quickly that complete reduction is not obtained. The more alkaline the pH and the higher the temperature the more rapidly the reduction must be carried out to avoid loss. In milk at pH 6.6 and 40” quantitative reduction can be...