There are almost all the companies that sold the bromelain enzyme powder is mixed with papain.It is difficult to purify the bromelain to 5000GDU/g. but it is very easy to purify the papain to 1000,000GDU/g. even to 2000,000GDU/g. But the functions of bromelain is different from papain. the color, ordour, water solubility, and taste contrast with the fake and true products. They are different the right bromelain enzyme powder is gray, odorless, tasteless and water insoluble.
But the false bromelain has the following features:
● It is light-yellow, aromatic and mild sweet, and water soluble. It is the characteristic of papain.
● It is used for reducing inflammation and relieve pain. but there are almost no these functions in papain.
● It is used as dietary supplement only, not food additive, but indeed can be pharma grade. But papain is only used as meat tenderizer powder and cosmetics.
We tested the bromelain according to SDS-PAGE gel and GELATIN DIGESTION UNIT ANALYTICAL and HPLC methods, there is less target molecules in SDS-PAGE gel method for fake product.
Following are the SDS Page electrophoresis results
The speckle in the above show that only the right product has the same speckle on the right place in the picture, but the fake product has little speckles on it, that means the molecular weight is barely visible.
The molecular weight of Bromelain is about 33000, but the molecular weight of papain is 21000. And the HPLC Chromatogram:
You will see that only the right extract contains the right molecular weights of bromelain and is detectable by HPLC.
But if we only tested by Gelatin Digestion Unit Analytical Method (GDU) see Annex II. Both the right and the false product have the same results as below:
Pineapple stem is very cheap, and the rate from stem to bromelain is very low, there are some pollution in the manufacturing process.
Almost all the bromelain enzyme powder factory are near field. They use fresh stem. It is impossible to ship the stem in long distance. The raw material should be handled as soon as possible when it is collected form the field or it should be stored in a low tempreture environment(4-8°C) to avoid protein degradation. In real life, the manufacturer actually can't meet any of the two above conditions due to too many of stems pineapples. The reasonable method is a balance between handling time and protein activity loss. Usually, the manufacturing the crude raw materials in the Pineapple harvest season, and store the crude raw materials in a low temperature environment(4-8°C).
We are doing some test about the SDS Page electrophoresis, In theory we can raise it to 10,000 or 20,000.but it will be very expensive. 5000GDU/g and make it stable maybe on a malto support / microencapsulated one or something + organic grade extraction/purification.
Annex I
● Method 5 SDS polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE is a denatured polyacrylamide gel electrophoresis method. The principle of protein separation in this method is based on the fact that most proteins can combine with the anionic surfactant sodium dodecyl sulfate (SDS) in a weight ratio to form a complex
The negative charge carried by protein molecules far exceeds the net charge of natural protein molecules, eliminating the charge effect of different protein molecules and separating proteins according to their molecular size.
This method is used for qualitative identification, purity and impurity control, and quantitative determination of proteins.
1 Instrument device
Constant voltage or constant current power supply, vertical plate electrophoresis tank, and adhesive making mold.
2. Reagents
(1) Water.
(2) Separation gel buffer (4x, solution A) 1.5mol/L trihydroxymethyl aminomethane hydrochloric acid buffer. Weigh 18.15g of trihydroxymethyl aminomethane and dissolve it with an appropriate amount of water. Adjust the pH value to 88 with hydrochloric acid and dilute to 100ml with water.
(3) Weigh 58.0g of acrylamide and 20g of N, N '- methylene bisacrylamide in 30% acrylamide solution (B solution), dissolve in warm water and dilute to 200ml, filter with filter paper (stored in dark).
(4) Weigh 10g of sodium dodecyl sulfate in 10% SDS solution (C solution), dissolve in water, and dilute to 100ml
(5) Commercialization reagent of tetramethylethylenediamine solution (TEMED, D solution).
10% ammonium persulfate solution (E solution) Weigh 10g of ammonium persulfate, dissolve in water, and dilute to 100ml. It is recommended to prepare it before use or store it separately at -20 ℃ for 2 weeks.
(7) Concentrated rubber buffer (4 ×, F solution) 0.5mol/L Trihydroxymethylaminomethane Hydrochloric acid buffer, weigh 6.05g of Trihydroxymethylaminomethane, add an appropriate amount of water to dissolve, adjust the pH value to 6.8 with hydrochloric acid, and dilute with water to 100ml.
(8) Electrode buffer (10 ×) Weigh 30g of trimethylaminomethane, 144g of glycine, and 10g of sodium dodecyl sulfate, dissolve them in water and dilute to about 800ml. Adjust the pH value to 8.1-8.8 with hydrochloric acid, and dilute to 1000ml with water.
(9) Non reduced sample buffer (4 ×) Weigh 303g of trimethylaminomethane, 20mg of bromophenol blue, and 8.0g of sodium dodecyl sulfate, measure 40ml of glycerol, dissolve in water and dilute to about 80ml. Adjust the pH to 68 with hydrochloric acid, and dilute to 100ml with water.
(8) Electrode buffer (10 ×) Weigh 30g of trimethylaminomethane, 144g of glycine, and 10g of sodium dodecyl sulfate, dissolve in water and dilute to about 800ml. Adjust the DH value to 81-88 with hydrochloric acid, and dilute to 1000ml with water
(9) Non reduced sample buffer (4 ×) Weigh 3.03g of trimethylaminomethane, 20mg of bromophenol blue, and 80g of sodium dodecyl sulfate, measure 40ml of glycerol, dissolve in water, and dilute to about 80ml. Adjust the pH to 6.8 with hydrochloric acid, and dilute to 100ml with water.
(10) Reduced test substance buffer (4 ×) Weigh 3.03g of trimethylaminomethane, 20mg of bromophenol blue, and 80g of sodium dodecyl sulfate, measure 40ml of glycerol, dissolve and dilute with water to about 80ml, and add β- 20ml mercaptoethanol, adjust pH to 6.8 with hydrochloric acid, dilute with water to 100ml (or 3.03g of trimethylaminomethane, 20mg of bromophenol blue, 8.0g of sodium dodecyl sulfate, measure 40ml of glycerol, dissolve in water and dilute to 80ml, adjust pH to 6.8 with hydrochloric acid, dilute with water to 100ml, and before use, add dithiothreitol to 100mmol/L).
● Annex II
Gelatin Digestion unit Analytical Method (GDU)
A. Purpose: This procedure is used to determine the proteolytic activity of Bromelain Enzyme Powder.
B. Equipment:
1. pH Meter
2. Constant Temperature Water Bath at 45.0° ± 0.1°C
3. Analytical Balance
4. Volumetric flasks
5. Volumetric pipettes
6. Timer
7. Digital Burette (Accuracy of 0.1 ml)
8. Fume hood
9. Automatic Pipetters
C. Safety Precautions:
This test must be performed in the fume hood.
1. Utilize standard laboratory safety practices.
2. Formaldehyde: Prepare and keep in a fume hood at all times. Known carcinogen and teratogen.
3. Peroxide: Strong oxidizer
D. Reagents and Reagent preparations:
1. Distilled water: approximately 500 ml: adjust to a pH of 4.5 with 0.1 N HCI.
2. Gelatin substrate:
a. Dissolve 25 grams of gelatin (Mikrobiologie. 1.04070) in 375 ml hot water, bring to a boil. Cool to 45°C.
b. Adjust the pH to 4.5 with 0.1 N HCI and dilute to 500 ml pH 4.5 distilled water.
c. Keep the gelatin substrate at 45°C.
3. Buffer Solution:
a. Add 15 gm NaCl slowly to 40-50 ml water in a 150ml beaker and stir to dissolve.
b. Add 0.570 ml acetic acid.
c. Adjust the pH to 4.5 with 0.2N HCL (volume will be greater than 100ml.)
4. 3 % Hydrogen peroxide:
a. Pipette 2.5 ml of 30% Hydrogen peroxide (stock solution) into a 25 ml volumetric flask and dilute to volume with pH 4.5 distilled water
5. 37 % Formaldehyde pH 9.0: a. Adjust a sufficient volume (100 ml) of formaldehyde to pH 9.0 with 0.1 N NaOH (approximately 20 ml of formaldehyde per sample prior to pH adjustment.)
Gelatin Digestion Unit Analytical Method (GDU) - cont.
6. 0.100 N NaOH: (Purchased standardized) a. Stock solution: standardized at 0.100 N
E. Procedure:
1. Enzyme Preparation:
a. Calculating Enzyme Preparation
Weight = 100/Target activity (approximately 0.05 g or 50 mg)
A. Weigh the enzyme precisely into 50 ml volumetric flask
B. Add 8.3 ml of buffer solution.
b. Let stand for 30 minutes at room temperature.
C. Dilute to 50 ml using pH 4.5 distilled water, add a small stir bar and stir for an additional 10 to 15 minutes
2. Enzyme Procedure:
a. Pipette 25 ml of Gelatin substrate into each of two 100 ml beakers containing stir bars and place them in a water bath at 45°C for 5 minutes, one for the Test Solution and the other for the Blank Solution.
b. Test Solution:
1) Add 1.0 ml of bromelain enzyme powder solution into the beaker designated for the test solution, start timing and swirl.
2) After exactly 20 minutes of incubation at 45°C, add 0.1 ml of 3 % hydrogen peroxide and swirl.
3) Incubate for an additional 5 minutes.
4) Remove the beaker from the water bath and with constant stirring insert the pH probe.
5) Record the pH after 10 seconds (Initial pH)
6) Adjust to pH 6.0 with 0.1 N NaOH. (Approximately 2-4 ml)
*Note: When adjusting the pH to 6.0 be cautious at pH 5.8; the pH increases slowly and minute additions of NaOH at this point will significantly increase the pH.
7) Continuing constant stirring, add 10 ml of 37 % formaldehyde pH 9.0.
8) Record the pH after 10 seconds and 1 minute.
9) Titrate to pH 9.0 with 0.1 N NaOH.
10) Record the titration volume.
This is the test titer, T. c.
Blank Solution: The Blank Solution should be run concurrently with the Test solution. This is accomplished by starting the Blank Solution determination 12 minutes after the Test Solution is started. That gives you time to complete the assay on the Test Solution before having to proceed with the Blank Solution. 1) Add 0.1 ml of 3 % hydrogen peroxide to the beaker designated for the blank solution and swirl.
2) After exactly 20 minutes of incubation at 45°C add 1.0 ml of bromelain solution and swirl.
3) Incubate for an additional 5 minutes.
GELATIN DIGESTION UNIT ANALYTICAL METHOD (GDU) - cont.
4) Remove the beaker from the water bath and with constant stirring insert the pH probe.
5) Record the pH after 10 seconds (Initial pH)
6) Adjust to pH 6.0 with 0.1 N NaOH. (Approximately 2-4 ml)*See Note above.
7) Continuing constant stirring, add 10 ml of 37 % formaldehyde pH 9.0.
8) Record the pH after 10 seconds and 1 minute.
9) Titrate to pH 9.0 with 0.1 N NaOH.
10) Record the titration volume. This is the blank titer, B.
F. Calculation:
1. Definition: One Gelatin Digestion Unit is that amount of enzyme which will liberate, after 20 minutes digestion at 45°C, 1 mg of amino nitrogen from a standard gelatin solution at pH 4.5 or pH 5.5(GDU pH 4.5 or pH 5.5).
GDU/g = (T-B) x 14 x N x 50 Wt (g) Where: T = Test titer (ml 0.1 N NaOH) B = Blank titer (ml 0.1 N NaOH) N = Normality of standardized NaOH (i.e. 0.100) Wt (g) = Initial weight of enzyme
G. Testing Parameters:
1. Enzyme preparations are diluted to a concentration of approximately 0.001 g/ml (1 mg/ml) or for bromelain enzyme powder concentrate approximately 1-2 GDU/ml. A Reference Material is assayed with each run to ensure method accuracy.
Guanjie provides bulk bromelain powder, using the GDU test method.Our production process operates under CGMP standard workshops, utilizing three production lines across two factories, and two independent laboratories. For any inquiries regarding our products, please contact us at info@gybiotech.com.
