(a)Overlay 1 mL of the digest solution with 0.5 mL of a solution of 1 mL of glacial acetic acid in 10 mL of diluted alcohol: no ring or precipitate forms at the junction of the two liquids, and when shaken no turbidity results (indicating the absence of undigested casein).

(b)Mix 1 mL of the digest solution with 4 mL of a saturated solution of zinc sulfate: a moderate amount of precipitate is formed (indicating the presence of proteoses). Filter, and retain the filtrate.

(c)To 1 mL of the filtrate from the preceding test add 3 mL of water, and follow with 1 drop of bromine TS: a violet-red color is produced, indicating the presence of tryptophane.

(a)2% of digest, in water;

(b)0.1% of digest, in water;

(c)1% of digest, 0.5% of sodium chloride, 0.5% of dextrose, in water;

(d)1% of digest, in water;

(e)2% of digest, 0.5% of sodium chloride, 1.5% of agar, in water.

Adjust all media to a pH of 7.2 to 7.4.

10,000(AU AC)

(a) Overlay 1 mL of the digest solution with 0.5 mL of a solution of 1 mL of glacial acetic acid in 10 mL of diluted alcohol: no ring or precipitate forms at the junction of the two liquids, and on shaking, no turbidity results, indicating the absence of undigested protein.

(b) Mix 1 mL of the digest solution with 4 mL of saturated zinc sulfate: a small amount of precipitate is formed, indicating the presence of proteoses. Filter, and retain the filtrate.

(c) To 1 mL of the filtrate from the preceding test add 1 drop of bromine TS: the light yellow color changes to a red-brown, indicating the presence of tryptophane.

(a) 2% of digest and sufficient phenol red TS to give a perceptible color in water;

(b) 0.1% of digest in water;

(c) 0.1% of digest and 0.5% of dextrose in water;

(d) 1% of digest in water.

Adjust all media to a final pH of 7.2 to 7.4. Place 5 mL of (a) in Durham fermentation tubes, and 5 mL each of (b), (c), and (d) in ordinary test tubes. Autoclave the media at 121 for 15 minutes. After autoclaving, and after standing for 24 hours, all media are clear.

[note—Suitable grades are available as catalog number U 232, from J.T. Baker Chemical Co., www.jtbaker.com.]

For the following tests, prepare a Test solution as follows. Dissolve 6 g in 45 mL of water, add 2 mL of hydrochloric acid, boil gently for 10 minutes, cool, and dilute with water to 60 mL.

Place 0.50-mL portions of the Hyaluronate solution in each of four 16- × 100-mm test tubes, and place 0.50-mL portions of the acetate-buffered Hyaluronate solution in two similar tubes. To two of the four tubes containing Hyaluronate solution add 0.50 mL of Diluent for hyaluronidase solutions, prepared as directed in the Assay under Hyaluronidase for Injection (USP monograph). To the remaining two tubes, on a rigid schedule, at 30-second intervals, add 0.50 mL of Standard solution. Similarly, to the two tubes containing acetate-buffered Hyaluronate solution add at 30-second intervals 0.50-mL portions of acetate-buffered Standard solution. Then proceed as directed in the second paragraph for Procedure, beginning with “Mix the contents,” as far as “Plot the average.” The reduction in absorbance of acetate-buffered Hyaluronate solution is not less than 25% of that observed in the Hyaluronate solution.

Substrate— Dissolve pullulan1 in water to make a 1.25% (w/v) solution. [note—Add pullulan to the water. Clumping will occur if water is added to pullulan.]

Buffer solution A, pH 5.0— Add 0.1 M disodium phosphate (27 g of dibasic sodium phosphate in each L of the solution) to 0.1 M citric acid (21 g of citric acid in each L of the solution) to adjust pH to 5.0.

Buffer solution B, pH 6.0— Add diluted acetic acid to 1 M sodium acetate (136 g of sodium acetate in each L of solution) to adjust the pH to 6.0. Dilute with water to prepare the final buffer solution as 0.01 M acetic acid buffer, pH 6.0.

Somogyi reagent— Add 54 g of disodium phosphate heptahydrate or 28 g of anhydrous disodium hydrogen phosphate and 40 g of potassium sodium tartrate to about 650 mL of water or about 700 mL for anhydrous disodium hydrogen phosphate. Add 100 mL of 1 N sodium hydroxide to this solution and mix. Add 80 mL of 10% cupric sulfate to the solution, and mix. Heat until any solid is completely dissolved. Add 180 g of anhydrous sodium sulfate to the solution and adjust the volume to 1 L. Allow the solution to stand at room temperature for 1 or 2 days to let insoluble matter precipitate. Filter the solution with standard filter paper, and keep the solution in a brown bottle with a ground-glass stopper.

Pullulanase diluent— Dilute pullulanase with Buffer solution B, pH 6.0 to prepare a solution having the enzyme activity of about 0.2 units per mL. [note—The measurement range is between 0.1 and 0.4 units per mL.] Record the dilution factor (FPD). This diluent is used as a diluted enzyme solution.

Procedure— Transfer 4 mL of Substrate to a test tube and add 0.5 mL of Buffer solution A, pH 5.0, mix, and incubate at 30. Add 0.5 mL of Pullulanase diluent, and mix thoroughly. After 30 seconds, transfer 1 mL of this solution to a test tube labeled as “Pullulan test solution 1”, and add 2 mL of Somogyi reagent, and mix. After 30 minutes and 30 seconds, transfer 1 mL of the mixture of Substrate and Pullulanase diluent to a second test tube labeled as “Pullulan test solution 2”, add 2 mL of Somogyi reagent, and mix. In a third test tube labeled as “Standard blank”, mix 2 mL of Somogyi reagent and 1 mL of water. In a fourth test tube labeled as “Glucose standard solution”, mix 2 mL of Somogyi reagent and 1 mL of Glucose standard solution, and add 1 mL of water. Incubate the fourth test tube in a boiling water bath for exactly 10 minutes. Remove the tube and allow it to cool in cold running water. Add 2 mL of Nelson reagent, mix well, and allow the solution to stand for at least 15 minutes. Add 5 mL of water to each of the four test tubes, and mix thoroughly. Determine the absorbance at 520 nm of the Standard blank, Ablank, of the Glucose standard solution, AStd, of the Pullulan test solution 1, A0, and of the Pullulan test solution 2, A30, using water as the blank. One unit is defined as the enzymatic activity that produces 1 µmol of maltotriose (measured as glucose) from pullulan per minute. Calculate the pullulanase activity, PA, in units per mL, using the formula:

Reagent A— Prepare a solution having known concentrations of about 0.1 N sodium hydroxide and about 0.2 M sodium carbonate.

Reagent B— Transfer 0.5 g of cupric sulfate and 1.0 g of sodium citrate dihydrate to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Lowry solution— Mix Reagent A and Reagent B at the proportion of 50:1.

Diluted Folin-Ciocalteu's phenol reagent (for albuminoid quantification)— Prepare a two-fold dilution of 2 N Folin-Ciocalteu's phenol reagent commercially available or prepare a solution by making an appropriate dilution from Folin-Ciocalteu Phenol TS (see Method 2 in Biotechnology-Derived Articles—Total Protein Assay 1057).

Bovine serum albumin standard stock solution— Transfer 0.05 g of bovine serum albumin to a 500-mL volumetric flask, dissolve in and dilute with water to volume, and mix. It contains 100 µg of bovine serum albumin per mL.

Standard solutions— Using appropriate dilutions of Bovine serum albumin standard stock solution in water, prepare five Standard solutions having concentration equally spaced between 5 and 100 µg of bovine serum albumin per mL.

Test solution— Dilute pullulanase with Buffer solution B, pH 6.0 in order to obtain a solution having a concentration between 60 and 70 µg of albuminoid per mL. [note—Water can be used as diluent.] Record the dilution factor, FTS.

Blank solution— Use water.

Procedure— To 0.3 mL in separate tubes of the Standard solutions,the Test solution, and the Blank solution, add 3 mL of Lowry solution, and mix. Allow to incubate at room temperature for 10 minutes. Add 0.3 mL of Diluted Folin-Ciocalteu's phenol reagent to each tube, mix immediately, and allow to stand at room temperature for 60 minutes. Determine the absorbances of the Standard solutions and the Test solution at the wavelength of maximum absorbance at about 750 nm, using the Blank solution as the blank.

Calculation— [note—The relationship of absorbance to protein concentration is nonlinear; however, if the standard curve concentration range is sufficiently small, it will approach linearity.] Using linear regression method, plot the absorbances of the Standard solutions versus the protein (bovine serum albumin) concentrations, in µg per mL, and determine the best fit curve. Using the plot, determine the concentration, Calbuminoid, in µg per mL, of protein (albuminoid amount) in the Test solution. Calculate the albuminoid concentration, in mg per mL, in the pullulanase taken by the formula: Calculate the specific activity, SA, in units per mg, of pullulanase using the formula:

A single spot is exhibited when it is examined by thin-layer chromatography, with the use of plates coated with chromatographic silica gel mixture and a developing system consisting of butyl alcohol, water, and glacial acetic acid (60:25:15).