1 -
Reach:
1.1.
Objective:
To establish an identity
and the minimum quality requests to be attended as a Propolis
extract.
1.2.
Ambit
of Application: The present
Regulation refers to Propolis Extract destined to national and
international commerce’s.
2.Description:
2.1.
Definition: Propolis Extract
understands as a product that comes from soluble components of
propolis
extraction in neutral alcohol (alimentary grade) by ad equated
technological process.
2.2.
Designation (Selling
Denomination): Propolis Extract.
3.
References:
-
ABNT
(Brazilian Technical Norms
Association).
-
AOAC.
Official Methods of Analysis of
the Association of Analytical Chemists, Arlington, 1992.
-
AOAC.
Official Methods of Analysis of
the Association of Analytical Chemists, 16th Edition,
Chapter
4.1.03, 1995.
-
ASIS,
M. Propoleo – El Oro Purpura
de las Abejas. Cuba, 1989.
-
BRAZIL.
Ministerio da Agricultura e
do Abastecimento. Portaria nº 368, dated 04.09.97 – Technical
Regulation about Sanitarian and Hygienic Conditions.
-
BRASIL.
RIISPOA – Regulation of
Industrial Inspection and Sanitarian of Products from Animal
Origin.
Decree nº 30.691 dated March 29th, 1952.
-
BRAZIL
– Ministerio da Agricultura
e do Abastecimento. Portaria SIPA nº 06/84. Hygienic-Sanitarian
and
Technological Norms for Honey, Bee wax, and Derivatives.
-
FAO/OMS. Organización de
las Naciones Unidas para la Agricultura y la Alimentación.
Codees
Alimentarius, CAC/vol. A, 1985.
-
ICMSF
– Microorganisms in Foods. 2.
Sampling for microbiological analysis: Principles and specific
applications. University of Toronto. Press, 1974.
-
ICMSF.
Compendium of Methods for
Microbiological Examination of Foods, 1992.
4.
Composition and Requisites:
4.1 Composition:
- Propolis Extract is composed by soluble elements from propolis
in
hydro alcoholic solution, alcohol and water.
4.2 Requisites:
4.2.1. Sensorial Characteristics:
4.2.1.1. Aroma: characteristic, depending on botanic origin
(balsamic
and resinous);
4.2.1.2. Color: it varies, depending on origin and concentration
(amber,
reedy and greeny tones);
4.2.1.3. Flavor: characteristic, from suave to strong, bitter
and whet
(piquant);
4.2.1.4. Aspect: limpid and homogeneous liquid.
4.2.2. Physical and Chemical Requisites;
4.2.2.1. Dry Extract: minimum of 11% (m/v);
4.2.2.2. Wax: maximum 1% from dry extract (m/m);
4.2.2.3. Flavonoids composition: minimum 0,25% (m/m);
4.2.2.4. Phenolic Composition: minimum 050% (m/m);
4.2.2.5. Activity of Oxidation: maximum 22 seg.;
4.2.2.6. Alcoholic contents; maximum 70º GL (v/v);
4.2.2.7. Methanol: maximum 0,40mg/l;
4.2.2.8. Spectrum of Absorption of Radiations UV visible; the
propolis
extract must be present characteristic peaks of principal
flavonoids
classes between 200nm and 400nm;
4.2.2.9. Lead Acetate: positive;
4.2.2.10. Sodium Hydroxide: positive;
4.2.3. Conditioning:
- Must be package in material bromatologicly adequate and it
confers to
the product an adequate protection.
5.
Addictives:
It doesn’t admit.
6.
Contaminators:
The organic and inorganic contaminators don’t have to be present
in
superior quantities to the to established limits by effective
Regulation.
6.1. Other Contaminators:
Research of spurs (esporos) of Paenibacillus larvae, in 25ml of
propolis
extract (using the methodology described in the Portaria 248,
from
12.30.1998). Accepted Result: absence of spurs (esporos) in
25ml.
7. Hygiene:
7.1. General Considerations:
- The hygienic practices to elaborate the product have to be in
accordance with the Technical Regulation on the
Hygienic-Sanitarians
Conditions and the Good Practices of Manufacturing for
Elaborating/Industrialized Establishments of Foods.
7.2. Macroscopic Criterions:
- The product mustn’t contain strange matters, of any nature.
7.3. Microscopic Criterions:
- The product mustn’t contain strange matters, of any nature.
8. Weights
and Measures:
It must be applied a specific Regulation.
9. Labeled:
It must be applied the specific Regulation with the following
additional
information – Dry Extract: minimum of ….%.
10. Analysis
Methods:
- Official Analytical Methods for Quality Control;
- Validated Methodologies by The Ministry of Agriculture;
-
Microbiologic Analysis Methods for Foods. Research of
Paenibacillus
larvae in honey and bee products. Portaria 248 date 12.30.1998.
Ministério da
Agricultura e do Abastecimento.
11.
Sampling:
Following the procedures recommended by effective
norm
(ABNT).
Published
in Official Diary of 01.23.2001, pages 18-20.
Ministério
da Agricultura e do Abastecimento.
Required Analyses:
A)
Dry Extract: minimum of 11% (m/v)
Weight
5,0g of propolis in a
capsule of porcelain previous heating in stove at 105º C for
three
hours and “tarada”. Put the sample of propolis in the stove at
105º
during three hours. Take it off from the stove and put it to
desiccate
for 1 hour, weight it and repeat the operation until the
constant
weight.
%
resin = 100 x (A1 – A2)
Weight
of sample
A1 – Weight of capsule + sample (before heating)
A2 – Weight of capsule + sample (after heating in stove)
P – Weight of propolis sample.
B) Wax (maximum of 1% of dry
extract)
Weight 1 kg of propolis and put it in a “becker” (recipient) of
250ml. Add 100ml of distillated water and put it in water-bath
(banho-maria) at 40º for 30 minutes. Filter it in a previous dry
and
hard paper filter, dry the filter paper (more than 80º C) until
the
constant weight.
Total
insoluble solids % =
100 x (A2 – A1)
Weight
A1- Paper filter weight.
A2 - Paper filter weight + sample after filtered and dried in
stove.
P – Weight of propolis sample.
% wax = % total insoluble solids.
C)
Flavonoids
composts
Reagents:
Quercetin PA
Ethanol PA
Aluminum Nitrate Solution 10%. Weight 17,6g of aluminum nitrate monohydrated
– AL
(NO3) 3 and 9H2O and dissolve it in distillated water, and
complete the
volume for 100ml.
Sample
Preparing
Get 0,5ml of the sample and dilute it for 50ml with ethanol 80º C
GL.
Preparing
the standard curve:
Weight
50mg of quercetin,
converting the weight on quercetin anhydrous. Dissolving in 100
ml of
ethanol to 95% (stock solution). Prepare three samples from the
stock
solution that should contain quercetin anhydrous on proportions
of 0,01
and 0,05 and 0,1mg/ml respectively. Measure the absorbance of
each
aliquot following the same procedure described for sample and
makes the
standard curve (Graphic of absorbance in relation to quercetin
concentration).
Procedure:
Get 0,5ml of sample and add 4,3ml of ethanol at 80% 0,21ml of
aluminum
nitrate solution 10% and 0,1ml of acetate potassium solution 1M;
let it
resting at environment temperature during 40 minutes, measure
the
absorbance in 415nm, determine the content of quercetin in the
sample,
using a standard curve and considering the dilutions done in the
sample.
D) Phenolic Composition
(minimum 0,50% (m/m)
Reagents
Reagent of Folin-Cicalteau
Saturated solution of sodium carbonate
Standard of phenol. Solution.
Dissolve 0,5000g of dry gallic acid in 100ml of water, inside a
volumetric balloon of 100ml.
Procedure
C – Standard curve
Pipetting
aliquot of 0 to 10ml of
solution of tannic acid standard in volumetric balloon of 100ml
and
complete the volume with distilled water.
Pipetting 1 ml of each dissolution and transfer it for a balloon
of
100ml.
Add 60ml of distilled water and agitate it.
Add 5ml of Folin Reagent and agitate again.
After 30 seconds and before 8 minutes add 10ml of sodium
carbonate
solution and complete the volume with distilled water and
agitate it
well.
Let it resting for 2 hours and do a reading at 765nm.
D- Sample
Dilute
the sample in proportion of
1:25.
Transfer it for a balloon of 100ml, 1ml of the sample and add
60ml of
distilled water.
Add 5ml of Folin Reagent and agitate it.
After 30 seconds and before 8 minutes add 10ml of sodium
carbonate
solution.
Complete the volume with distilled water and agitate it well.
Let it resting for 2 hours and the do a reading at 765nm.
E) Oxidation Activity:
Get
5ml of propolis extract and add
100ml of distilled water. After constant agitation, it has to be
filtered in filter paper. Get 1ml of a filtered sample and put
it in
proof tube of 5ml. Add to this 1ml of distilled H20; after
agitate it
again, mix to this 1ml of sulfuric acid at 20%. This mix has to
be
agitated for 1 minute more, in which should be add 1 drop of
permanganate potassium 0.1 N. With the help of a chronometer
instrument,
should be measured the time, in seconds, that corresponds to the
time
expensed for the coloring changing from yellow to pink. It is
what
determines oxidation indices. The determinations are done in
duplicate
(ASIS, 1989).
Spectrum of
Absortions’Radiations UV visible.
(It
presents characteristic peaks
of the principal flavonoids classes between 200 and 400 nm).
An
aliquot of 25ul of propolis
extract is diluted in 30ml of ethanol at 80%. So, is analyzed
the
spectrum of absorption in the band of length wave of 200 to
400nm;
(analyzed in spectrophotometer HP 8451A (PARK et al, 1995,
1997).
F) Plumb Acetate and Sodium
Hydroxide
Solutions:
Plumb Acetate at 10%
Putting
in a volumetric balloon of
50ml, approximately 30ml of distilled water and 10g of plumb
acetate.
Agitate and complete the volume until reach 100ml of distilled
water.
The solution of plumb acetate at 10% remains stable during six
months.
Sodium
Hydroxide at 20%
Get
a “becker” (recipient) and
put inside 20g of sodium hydroxide and dissolve it with 50ml of
distilled water. After that, transfer it to a balloon 100ml and
complete
the volume with distilled water, (with the solution in
environment
temperature).
Putting in a test glass 5ml of propolis extract, and take it to
water-bath during 3 minutes. Filter it through paper filter with
the
solution in the state of environment temperature. Retire with a
pipette,
1ml of filtered material and put it in “Erlenmeyer” of 50ml. Add
to
this 10ml of ethylic alcohol and agitate it carefully. Put 1ml
of this
solution in two glass tubes:
Tube 1-
Add 0,5ml of sodium hydroxide solution at 20%;
Tube
2
– Add 0,5ml of Plumb Acetate solution at 10%.
Positive Reaction: The solution of the first glass tube should get color so fast with a coloring yellow/orange/ and / or darkness. The second glass tube solution will get a yellow greeny and precipitated, or, should present disturbance; (if it becomes disturbance, the precipitation will appear after 30 to 60 minutes.
Translation,
Tarcísio Martins / February 8th