standard testing procedure serratiopeptidase

Storage Requirements:
Store protected from light and moisture at temperature not exceeding 30º.
Sampling:
Sample equal quantity from each container / bag. Collect a minimum of 5g from each container/bag sample into individual, self –sealing clear polythene bag kept in another clear self sealing polythene bag bearing ‘Sample for Analysis,lable.After completion of sampling return rest sample on same container. Collect control sample in Pet Bottle/Glass Bottle.
Quantity of Composite Sample:
30 g
Quantity of individual Identification
0.0100 g from each and every container /bag.
Quantity of Control Sample:
2 X 30 g

Description: A grayish white to pale brown pow4er with characteristic odour.

Solubility: Soluble in water, practically insoluble in alcohol and in solvent ether.

Identification: The activities of Solution A and Bare almost same and the activity of C is not more than 5 % of that
of activity of A.

Arsenic: NMT 5 ppm

Heavy Metals: NMT 20 ppm

Sulphated Ash: NMT 7.0%w/w

Loss on Drying: NMT 7.0%w/w

Microbial contamination:
Total Viable Count: Not more than 1000 cfu/g
Total Fungal Count: Not more than 1000 cfu/g
Escherichia coli: Absent/g
Salmonella: Absent/10g
Shigella: Absent/10g

Assay: Not less than 2000 IU and Not more than 2600 IU.

Description: A grayish white to pale brown powered with characteristic odour.

Examine the individual samples by visually.

Reporting: Report as Complies/Does not comply.

Solubility: Soluble in water, practically insoluble in alcohol and in solvent ether.

Reporting: Report as Complies/Does not comply.

Identification:
Dissolve 0.4 g of serratiopeptidase in 100 ml of acetic acid sodium acetate buffer solution pH5.0, transfer exactly 1 ml of
this solution into 3 test tubes, refer them as A, Band C. Add 1 ml of water in tube A. In tube Band C add 1 ml of0.04 ml of
disodium edetate solution. Mix gently and allow them to stand at 4±10, for 1 hour. Add 2 ml of water in tube A and C. Add 2 ml of 0.04 M zinc chloride solution in tube B. Mix gently and allow them to stand at 4±10, for 1 hour. Pipette 1ml of A and B and make up the volume to 200 ml with borate buffer pH 9.0. Pipette 1ml of C and make up the volume to 50 ml with borate buffer pH9.0. Proceed with these solutions as sample solutions as directed in the assay. The activities of solution A and B are almost same and the activity of C is not more than 5 per cent of that of activity of A.

Reporting: Report as Complies/Does not comply.

Arsenic:
Dissolve 0.4 g in 50 ml of water, and add 10 ml of Stannated hydrochloric acid. The resulting solution complies with the limit test for arsenic (5 ppm).

Reporting: Report as value.

Heavy metals: 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).

Reporting: Report as value.

Sulphated Ash: Determine on 1.0 gm sample.

Reporting: Report as value.

Loss on drying: Determined on 1 g by drying in an Oven at 105° for 4 hours.

Reporting: Report as value.

Microbial contamination: Total viable count, not more than 1000 micro-organisms per g and total fungal cont is not more than 100 micro-organisms per g, determined by plate count. 1 ml is free from Escherichia coli and 10 g is free from Salmonella and Shigella.

Reporting: Report as value.

Assay:
Sodium borate hydrochloric acid buffer pH 9.0. : Dissolve 19 g sodium borate in 900.ml of water. Adjust the pH 9.0 with 1M hydrochloric acid and dilute with water to 1000 ml.

Substrate solution: Dissolve 1.2 g dried casein in 100 ml of sodium borate hydrochloric acid buffer pH 9.0. Keep it on boiling water-bath for 1-2 minutes to get clear solution. Cool and filter through cotton and dilute with sodium borate hydrochloric acid buffer pH9. 0 to 200 ml.

Protein precipitating solution: To 18 g of trichloroacetic acid add 30 g of sodium acetate and 20 ml of glacial acetic acid, and dilute with water to 100 ml.

Dilute folin’s reagent: 1 ml of folin’s reagent, add 2 ml of water.

Reference tyrosine solution: Dissolve 10 mg of tyrosine in 1 ml of1M hydrochloric acid and dilute to 100 ml with sodium borate hydrochloric acid buffer pH 9.0.

Reference tyrosine curve: To 1, 2, 3, 4 and 5 ml of reference tyrosine solution, add 5, 4, 3, 2 and 1 ml of sodiul1~ borate
hydrochloric acid buffer pH 9.0 respectively. Add 5 ml of protein precipitating reagent in each tube. To 2 ml of these
solutions, add 5 ml ofsodium carbonate solution. Add 1 ml of diluted folin’s reagent mix and allow to stand at 37° for 30
minutes, measure the absorbance at 660 nm. Plot a graph of µm of tyrosine per system against the absorbance.

Stock test solution: Weigh about 0.1 g of the substance under examination, dissolve in 100 ml of sodium borate hydrochloric acid buffer pH 9.0 (solution A). Mix and keep it for 5 minutes. Take 1ml of solution A and dilute to 200 ml with sodium borate hydrochloric acid buffer pH9.0 (solution B).

Test solution: Pipet 1.0 ml of stock test solution, allow to stand in a water-bath at 37° for 5 minutes. Add 5 ml of substrate solution shake immediately and allow to stand in water-bath at 37° for 20 minutes. Add 5 ml of protein precipitating solution. Mix and allow tostand in water-bath at 37° for 30 minutes and filter. Take 2 ml of the filtrate, add 5 ml of0.6 per cent w/v sodium carbonate solution. Add 1 ml of diluted folin’s reagent mix and allow to stand at 37° for 30 minutes. Measure the absorbance at 660 nm.

Blank solution: Pipet 1.0 ml of stock test solution, allow to stand in a water-bath at37º for 5 minutes, add 5ml of protein
precipitating solution, shake immediately and allow to stand in water-bath at 37° for 20 minutes. Add 5 ml of substrate
solution. Mix and allow standing in water-bath at 37° for 30 minutes and filtering. Take 2 ml of the filtrate; add 50 ml of 0.6 per cent w/v sodium carbonate solution. Add 1 ml of diluted folin’s reagent mix and allow stand at 37° for 30 minutes.
Measure the absorbance at 660 nm.

Calculate the serratiopeptidase IU/mg by using concentration of tyrosine from graph considering the reaction time and
dilution.

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