INTERNATIONAL PEPPER COMMUNITY

IPC Manual of
Methods of Analysis

2018

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 Table of Content

Page #
Method No. 01.0 Preparation of Sample 1
Method No. 02.0 Moisture (m/m)%, 2–4
Method No. 03.0 Light Berries/Corns(m/m)%, 5–6
Method No. 04.0 Bulk Density (g/l), 7–9
Method No. 05.0 Extraneous Matter (m/m)%, 10 – 11
Method No. 06.0 Whole Insects, dead or alive (by count), 12
Method No. 07.0 Mammalian or/and Other Excreta (by count), 13
Method No. 08.0 Insect Defiled Berries/Corns(% by wt.), 14
Method No. 09.0 Mouldy Berries/Corn(m/m) %, 15
Method No. 10.0 Determination of grey and black berries in white pepper 16
Method No. 11.0 Salmonella Sample Preparation 17 – 18
Method No. 12.0 Salmonella (detection / 25g) 19 – 24
Method No. 13.1 Pinheads % (m/m), 25
Method No. 13.2 Broken berries % (m/m), 26
Method No. 14.0 Total ash, % (m/m), 27 – 28
Method No. 15.0 Acid in Soluble Ash % (m/m), on dry basis 29
Method No. 16.0 Volatile oil % (ml/100 g) on dry basis 30 – 31
Method No. 17.0 Non-volatile ether extract % (m/m), on dry basis 32 – 33
Method No. 18.0 Piperine content, % (m/m), 34 – 35
Method No. 19.0 Crude Fiber insoluble index % (m/m), on dry basis 36 – 37
Method No. 20.0 Sulphur Dioxide % (m/m), or in ppm (mg/Kg), 38 – 40
Method No. 21.0 Aflatoxin Total (B1+B2+G1+G2) (ug/kg) 41 – 43
Method No. 22.0 Escherichia Coli (MPN/g) 44 – 49
 IPC MANUAL OF METHODS OF ANALYSIS

PREPARATION OF SAMPLE
Method No. 1.0

Purpose: To prepare homogeneous sample for the determination of chemical parameters in

black, white and dehydrated green pepper.

A. Apparatus:

1. Sample splitter/dividing apparatus capable of reducing samples into equal portions.

2. Mill capable of grinding sample to particle size U.S. standard No.20/850 micron sieve.
3. U.S. Standard No.20/850 micron sieve

B. Procedure:
1. Mix the samples from a given lot, then split the mixed sample until 100g-200g remains
using the sample splitter/dividing apparatus or by coning and quartering.
2. Grind the sample to pass through the US Standard No.20/850 micron sieve. At least 99% of
sample should pass through the sieve to avoid variation in the composition of the sample.
Grind the sample at a rate that does not excessively heat the sample and cause evaporation of
essential oil.
3. Store the sample in tightly sealed container until the analysis is performed. (Store in
refrigerator if the analysis is not performed immediately).
4. Mix the sample prior to removing aliquot for analysis. (Allow the sample to return to
ambient temperature prior to opening for analysis if stored in refrigerator).

Reference:
1. ASTA Method No. 1.0, 4th edition 1997 (Revised. 2012)

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 IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF MOISTURE
(DISTILLATION METHOD)
Method No. 2.0

Purpose: To determine the moisture content in black, white and dehydrated green pepper (whole
and ground) by co-distillation with toluene.

A. Apparatus:

1. Distillation unit with ground glass joints constructed and assembled as shown in Fig.1 with
a) 500 ml round bottom flask with a T.S. 24/29 joint
b) West condenser with drip tip, 400 mm in length with a T.S. 19/26 joint.
c) Dean and Stark Water estimation trap, TS 24/29 joint, 10 mL capacity graduated in 0.1
mL intervals.

2. Heat source capable of refluxing toluene in the above apparatus. An electric heating mantle
with a variable power control or heating mantle supported by a variable speed stirring plate and
egg shaped teflon covered stir bar can also be used. If not using string plate add boiling chips.
3. Nylon bristle brush, ½ inch in diameter or a wire loop, long enough to extend through the
condenser (approx. 450 mm).
4. Analytical balance of sensitivity 0.01g

B. Reagents:

1. Toluene (Laboratory Reagent Grade)

C. Preparation of Sample:

1. Prepare sample as given in Method No.1.0

D. Procedure:

1. Weigh aliquot of sample sufficient to yield 2-4 mL of water (about 40g) to the nearest 0.01g
2. Transfer sample quantitatively to distillation flask and add sufficient toluene to cover the
sample completely and to middle of distillation flask. Add a stir bar or boiling chips.
3. Assemble the apparatus, and fill the trap with toluene by pouring through the condenser until
it just fills the trap and begins to flow into the flask. Insert a loose non-absorbing cotton plug
into the top of the condenser to prevent condensation of atmospheric moisture into the
condenser.
4. Bring to boil and reflux at about 2 drops per second until most of the water has been collected
in the trap, then increase the reflux rate to about 4 drops per second.
5. Continue refluxing until two consecutive readings 15 min. apart show no change. Turn off the
heat and allow to cool to ambient temperature. Dislodge any water held up in the condenser
with a brush or wire loop. Rinse the condenser carefully with about 5 mL toluene.

6. Read volume of water in the trap.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF MOISTURE
(DISTILLATION METHOD)
Method No. 2.0

E. Calculation:

Vol. of water (mL) 100

1. Moisture, % = X
Correction factor Weight of sample (g)

ML distilled
2. Correction factor =
ML added

F. Reporting:

Report the moisture content to an accuracy of 0.0% (v/wt.)

G. Notes:

1. The apparatus, including the condenser should be cleaned with potassium dichromate sulfuric
acid cleaning solution and rinse with water followed by a rinse with 0.05N KOH solution.
Rinse with alcohol and drain for 10 minutes.

2. A correction blank for toluene must be conducted periodically by adding 1 mL of distilled water
to 150 mL of toluene in the distillation flask and run the method as described above.

H. Reference:

1. ASTA Method No.2.0, Official Analytical Methods of the American Spices Trade
Association, Fourth Edition, 1997 (Revised. 2011).
2. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 986.21 Moisture in
Spices

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IPC MANUAL OF METHODS OF ANALYSIS

Fig.1: Moisture Distillation Apparatus

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IPC MANUAL OF METHODS OF ANALYSIS

LIGHT BERRIES IN BLACK AND WHITE PEPPER

Method No. 3.0

Purpose: To determine the percentage of light berries in black and white pepper.

A. Apparatus:

1. Balance of sensitivity 0.01g

2. Beaker 600 mL. Griffin, Low form approximately 85mm in diameter and 120 mm in height.
3. Blotting paper or other similar absorbent material.

B. Reagents:
1. Alcohol-water solution with a specific gravity of 0.80 to 0.82 at 250C. The alcohol may be
ethanol, denatured ethanol or isopropanol.

C. Procedure:
1. From the composite sample remove two 50g aliquots from opposite quarters after coning
and quartering.
2. Weigh the sample in the 600 mL Griffin, Low form beaker and add 300 mL of alcohol-
water solution.
3. Stir pepper corns with a spoon and allow to settle for 2 minutes. Spoon off the berries that
float.
4. Repeat the stirring, settling and removal of floating berries until two successive additional
stirrings raise no more berries to the surface. (Caution: Some berries may remain suspended
some distance below the surface of the liquid. These are not considered as floaters).

5. Blot the berries, which were removed to free from excessive liquid and spread them out to
dry on a piece of blotting paper or other similar absorbent material.
6. Air dry for one hour and weigh the air dried light berries to the nearest 0.01 g. Calculate
percentage of light berries.

D. Calculation:

% of light berries = Wt. of light berries (g) X 100

Weight of sample (50g)

The range of two determinations shall be averaged and reported as % light berries. If the difference
is greater than 0.8%, determine % light berries in a third aliquot. Average all the three values and
report % light berries.

E. Reporting:

Report the light berries content to an accuracy of 0.0% (by wt.)

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IPC MANUAL OF METHODS OF ANALYSIS

LIGHT BERRIES IN BLACK AND WHITE PEPPER

Method No. 3.0

F. Reference:
1. ASTA Method No. 14.2, Light Berries in Black and White Pepper, Official Analytical
Methods of the American Spice Trade Association, Fourth Edition, 1997 (Revised 2013).
2. ISO 959-1, 1998. Pepper (Piper nigrum L.) Whole or Ground - Specifications – Part I: Black
Pepper and ISO 959-2, 1998. Pepper (Piper nigrum L.) Whole or Ground – Specification –
Part II: White Pepper

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF BULK DENSITY

IN BLACK, WHITE AND DEHYDRATED GREEN PEPPER
Method No. 4.0

Purpose: To determine the bulk density of whole black, white and dehydrated green pepper.

A. Apparatus:

1. Apparatus for measuring bulk density consisting of:

a) Cylinder, of capacity 1L or a cylinder of greater capacity, but equipped with
apparatus allowing levelling of the product to the 1L level.
b) Hopper, of capacity greater than 1L and equipped with a gate;
c) Device, for fixing the hopper above the cylinder at a certain distance, to allow free fall
of the product into the cylinder from a constant height.
Fig-2 shows the example of such an apparatus.

2. Balance – top pan balance of 2 kg. Capacity with a sensitivity of 0.1g.

B. Procedure:

1. Weigh the empty cylinder and tare the balance.

2. Place the cylinder on a horizontal plane and set the hopper on it with the fixing device.
3 Pour the pepper into the hopper until it is filled. Open the gate and allow the pepper berries
to flow freely into the cylinder until the level slightly exceeds the 1L level.
4. Level the pepper using the cut off blade attached to the cylinder.
5. Remove the hopper and its support and weigh the cylinder filled with the pepper.
6. Carry out the above experiment three times.

C. Calculation:

The bulk density of pepper, expressed in grams per litre, is given by the mass of pepper
contained in the cylinder.
The arithmetic mean of three determinations are taken as the result.

D. Reporting:

Report the bulk density as g/L to an accuracy of nearest whole number.

E. Notes:

1. The difference between the results of two determinations carried out in rapid succession by
the same analyst using the same apparatus shall not exceed 5 g per litre.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF BULK DENSITY

IN BLACK, WHITE AND DEHYDRATED GREEN PEPPER
Method No. 4.0

F. Reference:

1. ISO 959-1, 1998. Pepper (Piper nigrum L.) Whole or Ground - Specifications – Part I: Black
Pepper and ISO 959-2, 1998. Pepper (Piper nigrum L.) Whole or Ground – Specification –
Part II: White Pepper

2. ASTA Methods No. 25.0, Bulk Index / Bulk Density, American Spice Trade Association,
4th Edition, revised in 2013

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IPC MANUAL OF METHODS OF ANALYSIS

Fig.2: Apparatus for determination of bulk density

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF EXTRANEOUS MATTER

IN BLACK, WHITE AND DEHYDRATED GREEN PEPPER
Method No. 5.0

Purpose: To determine the amount of extraneous matter in black, white and dehydrated
green pepper.

A. Apparatus:

1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm
in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear
inch or 2 mm square opening).
2. Balance of sensitivity 0.01 g.
3. Tweezers

B. Procedure:

1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time,
with a good light and against a white back ground into a standard pepper sieve. Pick out any
bird, rodent or animal excreta. Do not remove other extraneous/foreign material at this time.

2. Shake the sieve moderately back and forth 10 times. Examine the siftings collected on white
back ground for live and dead insects and for excreta and separate them.
3. Accumulate the siftings. Remove small berries of pepper that pass through the pepper sieve.
Weigh the siftings to the nearest 0.1 g. and calculate the percentage of the
extraneous/foreign matter by sifting.
4. From the composite sample remove two 100g aliquots (A & C) from opposite quarters after
coning and quartering, by designating each quarter as A; B; C; D in a clockwise sequence.
5. Hand pick for any sticks, stones, stems, foreign seeds, other extraneous matter.
6. Weigh the pickings to the nearest 0.1 g and calculate the percentage of extraneous/foreign
matter by picking.

C. Calculation:

% of extraneous/ foreign matter = Wt of siftings (g) X 100

by sifting (Es) Wt of sub-sample (g)

% of extraneous/foreign matter = A (in g) + C (in g)

by hand picking (Ep) 2

% of extraneous/foreign matter = Es + Ep
in the sample

D. Reporting:

Report the average % of extraneous matter into the sample to an accuracy of 0.0% by wt.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF EXTRANEOUS MATTER

IN BLACK, WHITE AND DEHYDRATED GREEN PEPPER
Method No. 5.0

E. Reference:

1 ASTA Method No.14.0; Mold and Extraneous Matter in Black and White Pepper. Official
Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997 (Revised
January, 2013)
2. FDA Technical Bulletin No.5, Macro Analytical Procedures Manual, 1984, electronic
version, 1998, B. Supplemental Method for Black and White Pepper (V-39)
3. ASTA Cleanliness Specifications for Spices, Seeds, and Herbs, 1999

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF WHOLE INSECTS, (DEAD OR LIVE)

IN BLACK, WHITE AND DEHYDRATED GREEN PEPPER
Method No. 6.0

Purpose: To determine the number of whole insects (dead or live) in black, white and
dehydrated green pepper.

A. Apparatus:

1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm
in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear
inch or 2 mm square opening).
2. Tweezers

B. Procedure:

1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time,
with a good light and against a white back ground into a standard pepper sieve. Pick out any
bird, rodent or animal excreta. Do not remove other extraneous/foreign matter at this time.

2. Shake the sieve moderately back and forth. Examine the siftings collected or white back
ground for live and dead insects.
3. Count the number of whole insects live or dead in each sub-sample and record.

C. Reporting:

Report the total number of whole insects live/dead in all the sub-samples.

D. Reference:

1 ASTA Method No.14.0; Mold and Extraneous Matter in Black and White Pepper. Official
Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997 (Revised
January 2013).
2. FDA Technical Bulletin No.5, Macro Analytical Procedures Manual, 1984, electronic
version, 1998, B. Supplemental Method for Black and White Pepper (V-39).
3. ASTA Cleanliness Specifications for Spices, Seeds, and Herbs, 1999.
.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF EXCRETA (MAMMALIAN OR OTHERS)

IN BLACK AND WHITE PEPPER
Method No. 7.0

Purpose: To determine the amount of mammalian or other excreta present in black and
white pepper.

A. Apparatus :

1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm
in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear
inch or 2 mm square opening).
2. Balance of sensitivity 0.01 mg.
3. Tweezers.

B. Procedure:

1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time,
with a good light and against a white back ground into a standard pepper sieve. Pick out any
bird, rodent or animal excreta. Weigh to the nearest 0.1 mg and record. Do not remove other
extraneous/foreign matter at this time.
2. Shake the pepper sieve moderately back and forth 10 times. Examine the siftings collected on
white back ground for excreta. If excreta is present combine the same along with the pickings.

C. Calculation:

Excreta (mammalian Wt. of combined excreta

or others) present in = (mammalian/ others) in mg
the sample across the sub-samples analyzed

D. Reporting:

Report the total amount of excreta (mammalian/others) in mg.

B. Reference:

1 ASTA Method No.14.0; Mold and Extraneous Matter in Black and White Pepper. Official
Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997 (Revised
January 2013).
2. FDA Technical Bulletin No.5, Macro Analytical Procedures Manual, 1984, electronic
version, 1998, B. Supplemental Method for Black and White Pepper (V-39).
3. ASTA Cleanliness Specifications for Spices, Seeds, and Herbs, 1999.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF INSECT DEFILED BERRIES

IN BLACK AND WHITE PEPPER
Method No. 8.0

Purpose: To determine the percentage of insect infested/defiled berries in black and white
pepper.

A. Apparatus:

1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm
in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear
inch or 2 mm square opening).
2. Balance of sensitivity 0.01 g.
3. Tweezers.

B. Procedure:

1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time,
with a good light and against a white back ground into a standard pepper sieve. Pick out any
bird, rodent or animal excreta. Do not remove other extraneous/foreign matter at this time.

2. Shake the sieve moderately back and forth 10 times to remove the siftings.
3. Examine the entire sample for insect defiled/infested berries. Collect all the defiled/infested
berries and record the weight.

C. Calculation:

Percentage of insect Wt. of infested berries

= X 100
defiled/infested Weight of sample

D. Reporting:

Report the average percentage of insect defiled/infested berries to an accuracy of 0.00% by wt.

F. Note:

1. Pepper may be infested by insects in the growing area during production and harvest. Insects
may also attack pepper during storage. Examples of insect infestation and damage range from
webbing and frass, insect tunnels or evidence of surface feeding on the pepper berries.

G. Reference:

1 ASTA Method No.14.0; Mold and Extraneous Matter in Black and White Pepper. Official
Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997 (Revised
January 2013).
2. FDA Technical Bulletin No.5, Macro Analytical Procedures Manual, 1984, electronic
version, 1998, B. Supplemental Method for Black and White Pepper (V-39).
3. ASTA Cleanliness Specifications for Spices, Seeds, and Herbs, 1999.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF MOULD
IN BLACK AND WHITE PEPPER
Method No. 9.0

Purpose: To determine the percentage of mouldy berries in black and white pepper.

A. Apparatus:

1. Standard pepper sieve (U.S. Standard No.8 or B.S.No.8 sieve with 45 cm diameter and 7 cm
in height with round holes of 7/64 inches in diameter with an average of 5 ½ holes per linear
inch or 2 mm square opening).
2. Balance of sensitivity 0.01 g.
3. Tweezers.

B. Procedure:

1. Weigh each sub-sample to the nearest gram. Sprinkle and examine a small portion at a time,
with a good light and against a white back ground into a standard pepper sieve. Pick out any
bird, rodent or animal excreta. Do not remove other extraneous/foreign matter at this time.

2. Shake the sieve moderately back and forth 10 times to remove the siftings.
3. Mix the sub-sample on sieve and weigh 50 g of aliquot into a pan. Hand pick mouldy berries
and record the weight.

C. Calculation:

Percentage of Wt. of mouldy berries

= X 100
mouldy pepper 50 g

D. Reporting:
Report the average percentage of mouldy pepper in all the sub-samples to an accuracy of 0.00%
by wt.

E. Note:
1. Pepper berries bearing mould on more than ¼ ofits surface is considered as mouldy.

F. Reference:

1 ASTA Method No.14.0; Mold and Extraneous Matter in Black and White Pepper. Official
Analytical Methods of the American Spice Trade Association, Fourth Edition, 1997 (Revised
January 2013).
2. FDA Technical Bulletin No.5, Macro Analytical Procedures Manual, 1984, electronic
version, 1998, B. Supplemental Method for Black and White Pepper (V-39).
3. ASTA Cleanliness Specifications for Spices, Seeds, and Herbs, 1999
.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF GREY AND BLACK BERRIES

IN WHITE PEPPER
Method No. 10.0

Purpose: To determine the percentage of grey and black berries in white pepper.

A. Apparatus:

1. Balance of sensitivity 0.01 g.

2. Tweezers

B. Procedure:

1. From the composite sample remove two 50 g. aliquots from opposite quarters after coning
and quartering.
2. Pick-out grey and black berries.
3. Weigh to the nearest 0.01 g.
4. Calculate the percentage of grey and black berries.

C. Calculation:

Percentage of grey & Wt. of grey and black berries (g)

= X 100
black berries Wt. of Sample (50 g.)

D. Reporting:
Report the average of two determinations to accuracy of 0.0% (by wt.)

E. Note:
Black/grey berries are berries with or without pericarp, and are dark in color. Those berries with
or without pericarp, which are light brown or cream in color should not be considered as black.

Reference:
1. ISO 959-2, 1998 Pepper (Piper nigrum L.) , whole or ground – Specification – Part 2: White
Pepper.

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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA SAMPLE PREPARATION

Method No. 11.0

Purpose: To secure a representative sample of a lot in a manner to avoid contamination and

prepare it for Salmonella analysis

A. Apparatus and Reagents :

1. Instruments for opening containers

Sterile scissors, knives, scalpels, can openers, or other hand tools as required.

2. Sample transfer instruments

Sterile spatulas, spoons, forceps, knives, scissors and swabs as required.

3. Sample containers
Pre-sterilized polyethylene bags or other suitable sterile, nontoxic polyethylene bags, that are
clean and dry and do not have a viable bacterial count. Sterile glass containers usually are not
desirable because of possible breakage and consequential glass contamination of the sampling
environment.

4. Microbicide
Medium strength (100 mg/L) hypochlorite solution or other approved disinfectant.

5. Labelling supplies
Pressure-sensitive tapes and labels, tags of adequate size to hold sample information,
indelible marking pens.

6. Sample shipping containers

Containers should be made of sturdy corrugated cardboard or other material capable of
withstanding abusive shipping conditions.

7. Balance
Balance width 2000 g capacity having a sensitivity of 0.1 g with a 200 g load.

B. Procedures:

1. Each sub sample will consist of a minimum of 100 g. Take 100g samples of 30 sub samples
at random to ensure that the total sample is representative of the lot. Collect more than one
sample unit from large institutional or bulk containers when the number of sample units
required exceeds the number of containers in the lot. A sample unit will consist of more than
one container when containers are smaller than 100g (e.g., four 25 g containers could
constitute a sample unit).

2. Make every effort to use aseptic technique when sampling the pepper to minimize its
contamination from hands, nearby materials and air. After sampling, sterilize the sampling
tools and equipment to avoid contaminating subsequent samples. Place the sample in sterile
plastic bags, label clearly and forward to the microbiological laboratory without delay.

3. When a sample is collected by transferring it to sample containers, a sample control must be

submitted which consists of an empty sample container that is exposed to the same conditions
under which the sample is collected.

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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA SAMPLE PREPARATION

Method No. 11.0

4. Take a 25 g analytical unit at random from each 100g sample unit. When a sample unit consists
of more than one container, aseptically mix the contents of each container before taking the
25 g analytical unit. To reduce the analytical workload, the analytical units may be
composited. The maximum size of a composite unit is 375 g or 15 analytical units. The
minimum number of composite units to be tested for is 2 composite units.

C. Reference:

1. Official Microbiological Methods of the American Spice Trade Association (ASTA), First
edition, 1976.
2. Compendium of methods for the microbiological examination of foods of the American
Public Health Association, Fifth edition, 2015.
3. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM), Eighth
edition, 1998.

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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA
Method No. 12.0

Purpose: To detect Salmonella in spices and spice products

A. Apparatus:

1. Conical flasks - 500 mL.

2. Test tubes – 25 X 150 mm and 12 X 100mm.
3. Pipettes (sterile) - 1 mL, 5 mL, 10 mL.
4. Petri dishes (sterile) (at least 15x90mm).
5. Sterile spoons or spatulas.
6. Incubator, 35 + 1°C.
7. Balance, calibrated (with sensitivity of 0.1g).
8. Inoculating loops and needles.
9. Refrigerator, below 8°C.
10. Incubator,42±1°C.

B. Reagents:

1. Pre-enrichment medium -Trypticase Soya Broth.

2. Enrichment media –
(a) Selenite cystine (SC) Broth / Tetra Thionate (TT) Broth.
(b) Rappaport Vassiliadis (RV)Medium.

3. Selective agars -
(a) Bismuth Sulfite(BSA) agar.
(b) Brilliant Green(BGA) agar, Sulphamandelate supplemented.
(c) Xylose lysine desoxycholate(XLD) agar.
(d) Hektoen enteric (HE) agar.

4. Biochemical media -
(a) Triple Sugar Iron (TSI)Agar.
(b) Lysine Iron (LIA)Agar.
(c) IMVIC Media - Peptone water or Tryptone broth, MR-VP medium, Simmons
citrate agar.
(d) Urea agar.
(e) Malonate broth.
(f) Lysine decarboxylase broth.
(g) Phenol red dulcitol broth.
(h) Phenol red sucrose broth.

5 Salmonella polyvalent sera

(a) Salmonella 'O' Antiserum Poly A-I and Vi .
(b) Salmonella 'H'Antiserum Poly A-Z .

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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA
Method No. 12.0

6. IMVIC reagents.
(a) Kovac's Indole reagent.
(b) Methyl Red indicator.
(c) Voges-Proskauer test reagents.

Solution 1 ( -naphthol solution)

- naphthol – 5g
Alcohol (absolute) – 100 mL

Solution 2 (40% KOH)

Potassium hydroxide – 4g
Creatine - 0.3g
Distilled water, sufficient quantity to dilute to 100 mL.

7. Sulpha supplement.

8. Potassium sulphite.

9. Physiological Saline (NaCl), solution,0.85% (Sterile).

Note 1:
All biochemical media prepared are stored in refrigerator for a period of three months.
Pre-prepared culture plates are stored in refrigerator for a period of one month.

Note 2:
For preparation of media and reagents, refer to FDA BAM – 8 th edition.

10. Reagents for Gram – Staining :

(a) Crystal violet
(b) Gram’s iodine
(c) Decolorising agent eg. Alcohol
(d) Safranin

C. Procedure:

1. Pre-enrichment
a) Using aseptic technique homogenize 25 g of food sample with 225 ml of Trypticase
Soya broth. If a larger food sample is required, maintain a sample to broth ratio of 1:9.
b) Before incubating allow it to stand for 60 minutes at room temperature.
c) Loosen cap to allow gas to escape and incubate for 24 h at 35+1°C.

2. Enrichment:
a) Transfer aseptically 1 mL of the pre-enrichment broth into test tube containing 10 mL
of Selenite Cystine broth / Tetra Thionate (TT) Broth and 0.1 mL into 10 mL Rappaport
-vassiliadis medium.
b) Incubate SC broth / Tetra Thionate (TT) Broth at 35±1°C and RV medium at 42±1°C
for 24 hours.

20
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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA
Method No. 12.0

3. Selective Agars:
Streak enrichment media onto three (3) selective agars (HE agar; XLD agar and BS Agar)
and incubate at 35+1°C. Check for typical colonies after 24 and 48 h.

Appearance of colonies:

a) Hektoen enteric (HE) agar: Blue-green to blue colonies with or without black centres.
Many cultures of Salmonella may produce colonies with large, glossy black centres or
may appear as almost completely black colonies.
b) Xylose lysine desoxycholate (XLD) agar: Pink colonies with or without black centres.
Many cultures of Salmonella may produce colonies with large glossy black centres or
may appear as almost completely black colonies.
c) Bismuth sulphite (BS) agar: Brown, gray, or black colonies some times they have a
metallic sheen. Surrounding medium is usually brown at first, but may turn black in
time with increased incubation, producing the so-called halo effect.

4. Gram Stain
Media for gram staining
Brilliant Green agar:
Salmonella - slightly pink - white opaque colonies surrounded by brilliant red medium.
Coliforms:
Yellow-green colony surrounded by yellow green zone.
Pick well isolated suspected colonies from selective agar plates and proceed with the
biochemical tests. Store picked selective agar plates at 5 to 8 °C. (Inoculate the colony into
peptone water, incubate at 35+ 1°C for 5 h. Check whether there is growth and then inoculate
into the media).

Procedure for Gram - Stain as below:

a) In this method of staining, the bacterial films are covered with a solution of the crystal
violet stain and allowed to act for 1 minute.
b) The stain is washed off with water, and a dilute solution of Grams’ iodine is added and
allowed to remain for 1 minute.
c) Next the slide is treated with alcohol or a mixture of alcohol and acetone until almost
all the colour is removed from the film.
d) Stain with Safranin for thirty second and wash off with water.
e) Salmonella is Gram - negative, straight sided, rod-shaped bacteria.

5. Agar Slants

a) Pick well-isolated suspected colonies from selective agar plates and inoculate tubes of
Triple sugar Iron Agar slant (TSI) and Lysine Iron agar (LIA) slants by first stabbing
the butt and then streaking the surface the slants. Suspected colonies that appears to be
mixed culture should be sub cultured on BGA plates.

Urea Slant: Inoculate from selective agar

21
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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA
Method No. 12.0

b) Incubate at 35 + 1 °C, 24 + 2 h.

c) Appearance of slants presumptive positive for Salmonella.

On TSI agar –
red (alkaline) slant and yellow (acid) butt. Note also whether H2S (black) or gas (cracking or
pockets in agar) is formed.

On Lysine Iron agar-

Salmonella - purple (alkaline) slant and butt; may have blackening if H2S is produced.

On Urea Slant-
Formation of red color indicates production of urease (positive test). Salmonella cultures do
not produce urease

6. Biochemical tests for confirmation of Salmonella

a) IMViC tests-inoculate from TSI slants.

(i) Indole test:-

Inoculate peptone / Tryptone water and incubate for 24 h at 35 + 1 °C. After
incubation add 0.5 mL Kovac's Indole reagent, shake well and examine after
1 minute and 5 minutes. A red color in the reagent layer indicates the
presence of Indole. A positive reaction indicates break down the amino - acid
tryptophan with the formation of Indole which forms a highly alcohol
soluble dye complex with Kovac's Indole reagent.

(ii) MR and V.P. tests:-

For both these tests inoculate the above culture into two tubes of MR-
VP medium and incubate them for 48 h + 2 h at 35 + 1 °C.

Methyl red test:

To one of the above incubated tubes add 3-4 drops of 0.04% methyl red
solution. A magenta red color, showing the presence of acid, is regarded as
a positive reaction, a yellow color, a negative and an orange color shows an
equivocal (+) result. Salmonella gives a positive result in this test.

Voges-Proskauer test:
To the second tube of the duplicate culture add 1mL of -Naphthol solution and
0.5 mL 40% KOH. Shake the tube vigorously, keep it in a sloped position and
examine after 1 h and 4 h. The development of an eosin pink color indicates a
positive reaction and a light deep orange brown color indicates an equivocal
(+) result. (To speed up the reaction add 2-3 crystals of Creatine.)

22
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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA
Method No. 12.0

(iii) Citrate Utilization tests:

Inoculate the culture in a Simmon's citrate agar slant and incubate for 48 h at 35
+ 1 °C. Examine the tubes for growth and blue coloration (alkaline reaction).

Reaction Salmonella
indole negative
methyl red positive
Voges-Proskauer negative
Simmons Citrate positive

b) Lysine Decarboxylase broth: Inoculate from TSI. Cotton plug on tightly and
incubate at 35+ 1°C, 2 days. Examine daily. Growth with no change in color is a
positive reaction. Change in color to yellow (acid) is a negative reaction.
Salmonella is positive for lysine decarboxylase.

c) Malonate broth: Inoculate from TSI and incubate at 35+ 1°C, 48 h. A change in color
to blue (alkaline) is a positive test for the utilization of malonate. Salmonella give a
negative test (green or unchanged colour).

d) Phenol red dulcitol broth: Inoculate the broth with small amount of growth from TSI
slant and incubate for 24-48h at 35 + 1°C. Most Salmonella species give a positive test
indicated by gas formation and acidity (yellow) of the medium.

e) Phenol red Sucrose broth: Inoculate the broth with small amount of growth from TSI
slant and incubate for 24-48 h at 35 + 1°C. Salmonella are sucrose-negative.

7. Serological test (Confirmatory test):

Using Salmonella Polyvalent "O" Antisera & Polyvalent "H" Antisera.

a) Prepare a dense suspension of the organism by suspending growth from an 18h TSI
agar slant in 0.5 mL of 0.85% NaCl solution.
b) Using a wax pencil, mark off two circles about 1 cm in diameter on a microscopic
slide.
c) Place a drop of Salmonella Polyvalent "O" Antisera in one of the marked circles and a
drop of 0.85% NaCl solution in the other circle (This will act as a negative control).
d) Using a clean dropper, transfer a drop (0.05 mL) bacterial suspension into each of
the circle and mix thoroughly by gently rocking for 1- 2 min. (Avoid excessive
evaporation).
e) Positive agglutination will be rapid and complete. A delayed or partial agglutination
should be considered negative.

Repeat slide agglutination test with Polyvalent "H" Antisera in the same way.

23
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IPC MANUAL OF METHODS OF ANALYSIS

SALMONELLA
Method No. 12.0

D. Calculation:

Nil.

E. Result & Reporting:

Report results as: “ Salmonella absent/present in 25g of sample"

F. Reference:

1. Official Microbiological Methods of the American Spice Trade Association (ASTA), First
edition, 1976.
2. Compendium of methods for the microbiological examination of foods of the American
Public Health Association, Fifth edition, 2015.
3. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM), Eighth
edition, Online Version 2016.
4. Practical Food Microbiology, Methods for examination of food for micro-organisms of
public health significance, Second edition, 1995.

24
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF PINHEADS IN BLACK PEPPER

Method No. 13.1

Purpose: To determine pinheads in black pepper.

A. Apparatus:

(a) Standard pepper sieve (US No. 8)

(b) Balance - sensitivity 0.1 mg.
(c) Table lamp.
(d) White paper sheet.
(e) Forceps, brushes, Petri dishes.
(f) Binocular - widefield microscope (30 - 60x.)

B. Reagents:

Nil.

C. Procedure:

Determine the weight of sample (50 to 100 grams). Spread the sample on to a white
paper. Pick out the pinheads in black pepper. Collect and weigh.

D. Calculation:

Wt. of pinheads in the sample X 100

% of pinheads= Wt. of sample

E. Result and Reporting:

Report the pinheads content to an accuracy level of 0.0% (by wt).

F. Reference:

1. ISO 959-1: 1998 Pepper (Piper nigrum L) Whole or Ground Specification – Part 1:
Black Pepper

25
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF BROKEN BERRIES

IN BLACK AND WHITE PEPPER 1
Method No. 13.2

Purpose: To determine broken berries in black pepper and white pepper.

A. Apparatus:

(a) Standard pepper sieve (US No. 8)

(b) Balance - sensitivity 0.1 mg.
(c) Table lamp.
(d) White paper sheet.
(e) Forceps, brushes, Petri dishes.
(f) Binocular - widefield microscope (30 - 60x.)

B. Reagents:

Nil.

C. Procedure:

Determine the weight of sample (50 to 100 grams). Spread the sample on to a white paper.
Pick out the broken berries in black pepper and white pepper. Collect and weigh.

D. Calculation:

Wt. of broken berries in the sample

% of broken berries= Wt. of sample X 100

E. Result and Reporting:

Report the broken berries content to an accuracy level of 0.0% (by wt).

F. Reference

1. ISO 959-1, 1998. Pepper (Piper nigrum L.) Whole or Ground - Specifications – Part
I: Black Pepper and ISO 959-2, 1998. Pepper (Piper nigrum L.) Whole or Ground –
Specification – Part II: White Pepper

1
New method added at the 23rd Meeting of Committee on Quality

26
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF TOTAL ASH

(GRAVIMETRIC METHOD)
Method No. 14.0

Purpose: To determine the Total Ash content in black, white and dehydrated green pepper.

A. Apparatus:

i) Flat-bottom dish / crucible, made of silica with a capacity of 50 / 25 mL.

ii) Electric muffle furnace, with indicating pyrometer and automatic thermo regulator or
other control device.
iii) Desiccator containing an efficient desiccant such as anhydrous copper sulphate or silica gel,
preferably of indicating grade.
iv) Analytical balance with accuracy 0.0001g

B. Reagents:

i) Distilled water, Analytical grade HNO3 and HCl.

ii) Ash less filter paper (Whatman No. 42)

C. Procedure:

Prepare aqua regia by mixing Nitric acid and Hydrochloric acid 1:3 ratio and wash the crucibles
with aqua regia.

i) Ignite the flat-bottom dish or crucible to a dull redness, cool to room temperature in a
desiccator and weigh.
ii) Weigh accurately 2 to 10g. of well mixed sample in the tared flat-bottomed dish or
crucible to the nearest 0.001 g.
iii) Place the dish or crucible in the entrance of the open muffle furnace until the sample is well
carbonised. The sample must not catch fire. (Carbonising can be done under an infrared
lamp or over a Bunsen burner, in a hood until the sample is well-charred, taking care that it
does not ignite.)
iv) Place the carbonised sample in the furnace at 600°+ 20°C for about 2 h.
v) Check for the presence of carbon. For this, cool the dish/crucible to room temperature and
wet the ash with several drops of distilled water. If carbon particles are seen, dry the dish
and continue ashing. Repeat wetting and heating until no specks of carbon are visible, and
ignite in the muffle furnace for 1 hour after the disappearance of carbon.
vi) If carbon remains, leach ash with hot water, and ignite in the muffle furnace at 600° +
20°C, until the ash is white.
vii) Cool in a desiccator and weigh.

D. Calculation:

Wt. of ash (g) x 100

Percentage of total ash = Wt. of sample (g)

27
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF TOTAL ASH

(GRAVIMETRIC METHOD)
Method No. 14.0

E. Result and Reporting:

Report the total Ash content to an accuracy of 0.00% (by wt.)

F. Reference:

1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 941.12(b) Ash
of Spices
2. ISO 928:1997. Spices and Condiments – Determination of Total Ash
3. ASTA Method No.4.0, Acid Insoluble Ash; Official Analytical Methods of the
American Spices Trade Association, Fourth Edition, 1997.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF ACID INSOLUBLE ASH

(GRAVIMETRIC METHOD)
Method No. 15.0

Purpose: To determine the Acid Insoluble Ash content in black, white and dehydrated
green pepper.

A. Apparatus:

(1) Funnel 65-75 mm ID.

(2) Filter paper, ashless, 9 to 11 cm in diameter.
(3) Watch glass
(4) Muffle furnace
(5) Silica crucible.
(6) Analytical balance with accuracy 0.0001g

B. Reagents:

1. HCl. solution - 1:2.5 (Dilute100 mL of concentrated HCl with 250 mL of distilled water).
2. Distilled water

C. Procedure:

1. Use total ash obtained in Method No.14.0

2. Add 25 mL of the HCl solution and boil for 5 minutes. Cover the dish with a watch glass to
prevent spattering.
3. Filter through an ashless filter paper quantitatively. Wash with hot water until the washings
are acid free (test with silver nitrate solution).
4. Transfer the filter paper and its contents to the original silica crucible (Method No. 14.0),
dry and ignite in a muffle furnace at 600° + 20°C until the ash is carbon free.
5. When carbon free ash is obtained, transfer the crucible to a desiccator, cool to room
temperature, weigh immediately & record the weight.

D. Calculation:
Wt. of acid insoluble ash (g) X 100
Acid insoluble ash % = Wt. of spice sample (g)

E. Result and Reporting:

Report the result to an accuracy of 0.00% (by wt).

G. Reference:

1. AOAC 20th Edition, 2016, Official Method 941.12 (b) - Ash of Spices
2. ISO 930:1997. Spices and Condiments - Determination of Acid Insoluble Ash
3. ASTA Method No.4.0, Official Analytical Methods of the American Spices
Trade Association, Fourth Edition, 1997. (Revised 2013)

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF VOLATILE OIL

(DISTILLATION METHOD)
Method No. 16.0

Purpose: To determine the Volatile oil content in black and white (whole and ground) pepper
by hydro-distillation.

A. Apparatus:

1. One L round bottom flask with T.S.24/29 ground joint.

2. Suitable electric heating mantle with regulator.
3. Clevenger volatile oil traps [lighter than water] with T.S.24/29 ground joint.
4. West condenser, 400 mm length with drip tip and T.S.19/26 ground joints.

B. Reagents:

1. Deionised / distilled water.

C. Procedure:

a) Clean the trap with chromic acid solution and rinse with deionised water. In extreme cases,
soaking the trap in hot concentrated sodium hydroxide if necessary.

1. Weigh accurately sufficient size sample to yield 1 to 5 mL. of oil.

2. Transfer quantitatively into 1000 mL R.B.flask.
3. Add 500 mL Deionised water.
4. Set the apparatus and place in suitable electric heating mantle and heat the flask
to boiling.
5. Adjust refluxing rate to 1 drop per second by adjusting the regulator of the mantle.
6. Reflux until two consecutive readings taken at one hour interval show no change of oil
volume in the trap.
7. Cool to room temperature by allowing to stand in air.
8. Oil drops sticking to the sides of the condenser can be pulled down to the trap using
a steel rod.
9. Note down the amount of oil collected in the trap in mL.

Note:-
(i) If the separation of the oil is not satisfactory, agitate the liquid in the trap with a
wire inserted through the condenser. If this fails, carefully add a few mL of a
saturated sodium chloride solution to the trap.
(ii) If foaming occurs, add a non-steam distillable anti-foaming agent.

D. Calculation:

volume of oil (mL) X 100

Percentage of volatile oil (v/w) = wt. of sample

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 IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF VOLATILE OIL

(DISTILLATION METHOD)
Method No. 16.0

E. Result and Reporting:

Volatile oil is reported to an accuracy of 0.00% (v/w)

F. Reference
1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 962.17 – Volatile
Oil in Spices
2. ISO 6571:2008. Spices, Condiments and Herbs - Determination of Volatile Oil Content
3. ASTA Method No 5.0, Official Analytical Methods of the American Spices
Trade Association, Fourth Edition, 1997. (Revised 2013)

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF NON VOLATILE ETHER EXTRACT

(SOXHLET EXTRACTION METHOD)
Method No. 17.0

Purpose: To determine the Non-Volatile Ether Extract in black and white pepper (whole
and ground) Soxhlet extraction method

A. Apparatus:

1. Extractor -Soxhlet /Automated soxhlet extractor

2. Desiccator
3. Oven
4. Water bath
5. Balance with accuracy 0.001g

B. Reagents:

Diethyl ether.

C. Procedure:

i) Weigh exactly 2 to 2.5 g of sample into a cellulose extraction thimble / filter paper thimble.
Keep the extraction thimble in soxhlet extractor and extract with diethyl ether for 18 hours.
The ether is removed by evaporating in a water bath after transferring the extract into a pre-
weighed beaker. The beaker with extract is finally dried in airoven at 110°+ 2°C till the loss
in weight between successive weighing is less than 2 mg. The residue is shaken with 2-3 ml
of diethyl ether at room temp. Allow to settle and the ether decanted. The extractions are
repeated until no more residue dissolves. The beaker is dried again until the loss in weight
between two successive weighing is less than 2 mg. The final weight is noted.

ii) USING AUTOMATED SOXHLET EXTRACTOR

i) Prepare the sample as per method no.1.0 when ever applicable
ii) Weigh ca 2.000 g of the sample into a paper extraction thimble
iii) Add approximately 100 to 150 ml of the solvent in the extraction apparatus and set the
equipment with Extraction time: 45 mins, Rinsing time 30 mins, Drying time 20 mins
iv) When last traces of solvent have disappeared (Drying time can be extended based on
the requirement.), place the container in a hot air oven at 110° + 2°C until two
successive weighing taken differ by not more than 1 mg.

D. Calculation:
100 X (W1 – W2)
Percentage of NVEE =
W

W = Weight in g of the material taken for test

W1= Weight in g of the beaker with the non-volatile ether extract
W2= Weight in g of the beaker with ether insoluble residue after decantation.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF NON VOLATILE ETHER EXTRACT

(SOXHLET EXTRACTION METHOD)
Method No. 17.0

E. Result and Reporting:

Report the percentage of NVEE to an accuracy of 0.00%

F. Reference:

1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 940.29 –

Methylene Chloride Extract (Volatile and non-volatile) of spices.
2. ISO 6571:2008. Spices, Condiments and Herbs - Determination of Volatile Oil Content.
3. ASTA Method No 11.0, Non-Volatile Methylene Chloride Extract; Official analytical
Methods of the American Spices Trade Association, Fourth Edition, 1997.

33
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF PIPERINE
(SPECTROPHOTOMETRIC METHOD)
Method No. 18.0

Purpose: To determine the Piperine content in black and white pepper by

Spectrophotometric method.

A. Apparatus:

1. Spectrophotometer - double beam, UV (deuterium) light source, capable of accurately

measuring absorbance at 342-345 nm.
2. Cuvettes, 1 cm. square, silica.
3. Amber Erlenmeyer flask or F. B. flask with 250 mL capacity with T.S.19/26 ground joint.
4. Condenser, West type, with water cooled drip tip, 400 mm., T.S. 19/26 ground joint
5. Amber volumetric flasks, 100 mL & 250 mL with stoppers.
6. Pipettes, transfer type 1,5 and 10 mL.
7. Funnel, 60° angle, 65 to 75 mm. I.D.
8. Filter paper, Whatman No.2 or equivalent.
9. Balance with 0.0001 g accuracy

B. Reagents:

1. SDA no. 3A - Denatured alcohol -Mixture of methyl alcohol and ethyl alcohol (95%) in
the ratio 1:20.
2. Piperine standard (with the purity of minimum 97%)

C. Procedure:

i) Determination of Absorbance Conversion Factor of pure piperine

1. Weigh accurately 0.1000 gram of piperine into a 100 mL. volumetric flask with ca.
70 mL. SDA no. 3A. Shake to dissolve and make up to volume. (Stock standard -
Shelf life 1 year under refrigeration)
2. Pipette 5 mL. aliquot into 50 mL. volumetric flask and make up to volume with SDA no.
3A. Shake well. (Working standard -Shelf life 6 months under refrigeration)
3. Pipette 1,2,3,4,5 and 6 mL. aliquots from solution in 2. and transfer into 100 mL.
volumetric flasks and make up to volume with SDA no. 3A. These solutions represent
1/106, 2/106, 3/106, 4/106 5/106 and 6/106 dilutions.
4. Zero spectrophotomer with SDA no. 3A in both cells and then determine the
absorbance reading of solutions in 3. at absorbance maxima 342 nm.
5. Determine the average absorbance obtained from the readings expressed at
a concentration of 1/106 = 1 ug./mL.
6. The reciprocal of this average absorbance is the number of mg./mL. of piperine required
to obtain an absorbance of 1.000. This reciprocal is used to determine the piperine
content in all future determinations.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF PIPERINE
(SPECTROPHOTOMETRIC METHOD)
Method No. 18.0

ii) Black and White Pepper (whole and ground)

1. Weigh accurately 0.5000 gram of sample prepared under Method 1.0 with
the exception of grinding to pass U.S. No.60 mesh sieve.
2. Place sample in a 250 mL. Erlenmeyer flask / F.B. flask and add ca. 70 mL. SDA
no. 3A & cover with aluminum foil.

3. Reflux one hour, cool to room temperature and filter quantitatively into 250 mL
volumetric flask. Transfer the extracted residue to the filter paper. Wash thoroughly
and dilute to mark with SDA no. 3A.

4. Pipette 1 mL. of this solution into a 25 mL. volumetric flask and make up to volume
with SDA no. 3A. Shake well until dissolved.
5. Determine the absorbance of 4. with SDA no. 3A as blank at 342 nm. within 15
minutes.

D. Calculation:

Percentage of piperine is calculated as follows:

Absorbance X dilution factor

Percentage of = wt. of sample X factor derived from piperine standard
piperine
OR

Percentage of As X F X V
piperine = Ws X 10 6 X 100

Where
As = Absorbance of sample
F = Factor derived from piperine
V = Dilution volume in milli litres
Ws = Sample weight in grams.

E. Result and Reporting:

Report piperine content to an accuracy of 0.00% by wt.

G. Reference:

1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 987.07 – Piperine
in Pepper preparation.
2. ISO 6571:2008. Spices, Condiments and Herbs - Determination of Volatile Oil Content.
3. ASTA Method No 12.1, Piperine content of Black and White Pepper, their oleoresin
and soluble pepper seasoning; Official Analytical Methods of the American Spices
Trade Association, Fourth Edition, 1997.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF CRUDE FIBER

(HYDROLYSIS METHOD)
Method No. 19.0

Purpose: To determine the crude fiber in black and white pepper (whole and ground)
Hydrolysis method

A. Apparatus:

1. Condenser that will maintain a constant volume of solution throughout the digestion period.
An Allihn condenser with 19/26 ground joint is recommended.
2. Digestion flask - one litre RB flask with 19/26 ground joint is recommended.
3. Buchner Funnel. Alternatively a filter cloth, of such character that no appreciable solid matter
can pass through it during rapid filtration, may be used. Retention may be tested by running
filtrate through a Gooch crucible. Butcher's linen, dress linen with ca. 45 threads to an inch,
or No. 40 filter cloth.
4. Electric muffle furnace or a Meeker-type burner.
5. Desiccator containing an efficient desiccant.
6. Gooch crucible-Pour asbestos pulp into Gooch crucible to make a mat of such thickness that
the holes are barely visible (when looking through the crucible at a light source) and ignite.
7. Oven, preferably circulating air type.
8. Balance with 0.001 g accuracy.

B. Reagents:

1. Sulphuric acid solution, 0.255 N, (12.5g. of H2SO4, Sp. gr. 1.84, dilute to 1 liter).
2. Sodium hydroxide solution, 0.312N, (12.5g. carbonate free NaOH per liter). The
concentration of both acid and alkali solutions should be checked by titration. If the
concentration differs by more than +0.01 N from the nominal values adjust to within this
range.
3. Asbestos-Gooch grade, medium fiber, acid washed and ignited asbestos is usually
satisfactory. However, it should be tested before use for chemical stability and filtering speed.
The asbestos may be prepared by digesting it on steam bath or at an equivalent temperature
for at least 8 hours with approximately 5 percent NaOH solution, thoroughly washing it with
hot water, then digest as before for 8 hours with HCl. solution (1+3). Again, wash thoroughly
with hot water, dry and ignite at bright red heat.
4. Ethyl alcohol, 95%.
5. Methylene chloride, anhydrous (dichloromethane).

C. Procedure:

1. Prepare sample as per Method No. 1.0. Extract 2 g. of sample with methylene chloride or use
the fat free residue. Transfer the residue together with ca. 0.5 g. of asbestos to the digestion
flask.

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF CRUDE FIBER

(HYDROLYSIS METHOD)
Method No. 19.0

2. Add 200 mL. of the H2SO4 Solution, connect the digestion flask to the condenser and place
on a preheated hot plate or digestion rack adjusted so that the acid will boil in ca. 5 minutes.
Continue boiling briskly for 28 + 1 minute with frequent rotation of the flask to ensure
thorough wetting and mixing of the sample. Material should not be allowed to remain on the
sides of the flask out of contact with the solution. A blast of air directed into the flask from a
glass tube inserted through the condenser will serve to reduce frothing. Successive sample
digestions should be started at ca. 3 minute intervals to facilitate accurate timing.

3. After boiling 28 minutes, remove the flask and filter immediately through the California
Modified Buchner funnel or through a filter cloth in a fluted funnel using a suction flask to
speed filtration. Wash with boiling water until washings are no longer acid.
4. Transfer the sample and asbestos quantitatively to the digestion flask, washing the filter cloth
or Buchner funnel with 200 mL. of the NaOH solution. A wash bottle marked to deliver 200
mL. is convenient.
5. Connect the flask to the reflux condenser, place on the preheated hot plate or heating mantle
or digestion rack, bring to a boil in ca. 5 minutes, and boil exactly 28 + 1 minutes. Successive
sample digestions should be started at ca. 3 minute intervals to facilitate accurate timing.
6. After 28 minutes, remove the flask and immediately filter through a pre-weighed Gooch
crucible.
7. Wash the residue thoroughly with water and then with ca. 15 ml of ethyl alcohol.
8. Dry the crucible and contents at 110°+ 2°C to a constant weight (ca. one hour). Cool in a
desiccator and weigh.
9. Ignite the crucible and contents in an electric muffle furnace at ca. 600°C + 20°C or over a
Meeker burner at dull red heat for ca. 20 minutes. Cool in a desiccator weigh. Determine the
loss in weight on ignition.

D. Calculation:

Loss in weight on ignition (g)

Crude fibre % = Wt. of original sample (g) X100

E. Result and Reporting:

Report the crude fibre content to an accuracy level of 0.00% (by wt.)

F. Reference:

1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 920.169 –

Fiber (crude) in spices.
2. ISO 6571:2008. Spices, Condiments and Herbs - Determination of Volatile Oil Content.
3. ASTA Method No 7.0, Crude Fiber; Official Analytical Methods of the American
Spices Trade Association, Fourth Edition, 1997.

37
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF SULPHUR DIOXIDE

(Distillation Method)
Method No. 20.0

Purpose: To determine the Sulphur Dioxide content in dehydrated green pepper

Modified Monier William Distillation Method

A. Apparatus:

Three necked 1 L flask with ground joint 24/29.

1. Allihnur condenser with ground joint 24/29 with minimum 30 cm length.

2. Gas inlet tube of minimum 2.5 cm tube with ground joint 24/29.
3. Gas trap of ground joint 29/32.
4. Separatory funnel of minimum capacity of 100 mL with both ends ground joint 24/29.
5. Inlet adapter 24/29 with a hose connector.
6. Gas bubbler with ground joint 19/26.
7. Cylinder column with top ground joint 24/29 and bottom ground joint 19/26.
8. Test tube of minimum 15 mL capacity with ground joint 19/26 and hook.
9. Balance with 0.0001 g accuracy.

B. Reagents & Solutions

1. Nitrogen cylinder with a flow regulator.

2. KOH Solution - Dissolve 65 g of Potassium Hydroxide in 85 mL distilled water.
3. Hydrochloric acid solution - Mix 30 mL con Hydrochloric acid and 60 mL distilled water.
4. 0.02N Sodium hydroxide solution - Dissolve 0.08 g Sodium hydroxide pellets in 100 mL.
distilled water.
5. Methyl red solution - Dissolve 250 mg of methyl red indicator powder in 100 mL alcohol.
6. Hydrogen peroxide Solution - Dilute 30 % solution of Hydrogen peroxide to 3%.
7. Pyrogallo1 - 4.5 g. Prepare pyrogallol trap by adding 4.5 g pyrogallo1 and Potassium
Hydroxide solution to the trap.
8. Barium chloride solution 10% - Dissolve 10g Barium chloride in 100mL distilled water.
9. Sulphuric acid solution - 2N.

C. Procedure:

a. Standardisation of Hydrogen peroxide.

1. Dilute 15 mL of 30% Hydrogen peroxide to 100 mL with distilled water.

2. Pipette 1 mL of this solution into a conical flask. Add 5 mL of dil sulphuric acid and
heat it to bearable warm.
3. Titrate this solution against standard Potassium permanganate solution to a permanent
pink colour. From the titre value calculate the percentage of hydrogen peroxide solution.

38
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF SULPHUR DIOXIDE

(Distillation Method)
Method No. 20.0

b. Standardisation of potassium permanganate.

1. Dissolve about 0.16 g Potassium permanganate in 100 mL distilled water.

2. Prepare a standard solution (0.01) of oxalic acid by accurately weighing 0.063 g of
oxalic acid / equivalent standard oxalic acid solution.
3. Pipette 5 mL of this solution to a conical flask and add 5 mL dil. sulphuric acid and
heated to 60 to 80 °C and titrate against Potassium permanganate solution to a
permanent pink colour. Calculate the Normality of KMnO4 from the litre values.
4. Standardisation of Sodium Hydroxide - Pipette 5 mL 0.01 N Standard oxalic acid in a
250 mL conical flask. Titrate this against Sodium Hydroxide using phenolphthalein as
indicator (end point is just appearance of permanent pink colour). From the titre value
calculate the strength of Sodium hydroxide solution.

c. Determination of Sulphur dioxide.

1 Weigh 25 or 30g powdered sample and transfer to 1 L flask.

2. Add about 400 mL deionised water to it and set the apparatus for the experiment.
3. Take 10 mL of 3% hydrogen peroxide to the test tube. Add one drop of methyl red
indicator and neutralise it with dil. sodium hydroxide and attach it to apparatus.
4. Pass nitrogen gas for about 5 minutes.
5. Then add hydrochloric acid solution from the separator y funnel by applying pressure
and put on the heating mantle.
6. Adjust the temperature such that the reflux rate is about 2 to 3 drops per minute.
7. Pass chilled water through the condenser.
8. Continue the heating for 1 hour and 45 minutes and then put off the water connection
and continue heating till the upper part of the condenser becomes hot. Then put off the
mantle and allow and cool. Close the Nitrogen cylinder.
9. Separate the test tube containing hydrogen peroxide solution from the apparatus and
quantitatively transfer into a conical flask.
10. Titrate this solution against standard sodium hydroxide solution. From the titre Value
calculate the Sulphuric acid formed.

Confirmation by Barium Sulphate

1. Transfer the titrated solution to a 250 mL beaker quantitatively.
2 Add 5 mL dilute Hydrochloric acid (1+2) and heat it on a burner.
3. When it is about to boil add 10% barium chloride solution slowly (about 30 mL).
4. Allow it to stand on burner taking care not to boil it for about 1/2 an hour. Allow this
solution to stand overnight.
5. Filter it through whatman No:41 filter paper. Wash it with hot water till the washings
are free from chlorides (test with Silver Nitrate).
6. When it is free from chlorides place it in a weighed crucible and heat it in a muffle
furnace at 450 degree centigrade.
7. Cool it in a desiccator and weighed. From the wt of Barium sulphate calculate the
concentration of sulphur dioxide in the sample.

39
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF SULPHUR DIOXIDE

(Distillation Method)
Method No. 20.0

D. Calculation:

For sodium hydroxide titration

0.01 N Sodium hydroxide = 0.3203 mg of Sulphur dioxide

0.3203
1 mL 0.01 N NaOH = mg SO2
0.01
0.3203 x X
XN 1 mL NaOH = mg SO2
0.01
0.3203 x X x Y
YmLXN NaOH = mg SO2
0.01
0.3203 x X x Y
1 g sample contains = mg SO2 = Z
Wt. Of sample x 0.01

= Z x 1000 ppm

For BaSO4

233.63 g BaSO4 contains 64 g of SO2

64 g of SO2
1 g BaSO4 contains =
233.63
64 x wt. of BaSO4 (obtained) g of SO2
Total BaSO4 contains =
233.63
64 x wt.of BaSO4 g of SO2
1 g sample contains =
233.63
64 x wt.of BaSO4 g of SO2
SO2 content in the sample = = Z
233.63 wt of sample
SO2 content in the sample in ppm = Z x 10 6 ppm

E. Result and Reporting:

Report the SO2 content in ppm to an accuracy of 0.0

F. Reference:
1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 990.28 – Sulfite
in Foods
2. ISO 5522: 1981 – Fruits, Vegetables and derived products – Determination of total
sulphur dioxide content.

40
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF AFLATOXINS
(HPLC Method)
Method No. 21.0

Purpose: To determine the Aflatoxins contamination in Black, white and dehydrated

green pepper (whole and ground) by HPLC with Fluorescence Detector

A. Apparatus:

1. Blender jars.
2. 250 mL beakers.
3. Glass funnels.
4. 10 mL pipets.
5. 50 mL graduated cylinders
6. 10-100 uL adjustable air displacement pipet.
7. 10 mL volumetric flask.
8. Parafilm.
9. 12 position Pump Stand.
10. HPLC / UPLC systems
11. Scanning Fluorescence Detector
12. Kobra Cell./PHRED
13. Balance with 0.0001 g accuracy

B. Reagents :

1. Aflatoxin Mix Standards

2. HPLC grade Methanol.
3. 80% Methanol/ 20% HPLC grade Water (200 mL of HPLC grade water is added to 800
mL of HPLC grade Methanol and mix thoroughly.
4. 20% Tween-20/ 80% HPLC grade Water (100 mL of Tween-20 is added to a 500
mL volumetric flask and brought to volume with HPLC grade water).
5. 1% Acetic Acid in HPLC water (1mL of HPLC grade Acetic Acid is added to a 100
mL volumetic flask and brought to volume with HPLC grade water).
6. HPLC Water
7. Aflatoxin Immuno Affinity Column [IAC (AFT)].
8. Sodium Chloride Excelar grade.
9. Fluted filter paper.
10. Micro fiber filter paper.
11. Methanol-MS Grade.

C. Procedure

Standard Preparation:
a. Allow Aflatoxin standard to come to room temperature.
b. Transfer one ampule into a 10 mL amber coloured volumetric flask and make up the
volume using HPLC methanol
c. Transfer 2.5 ml of the above standard into a 100 ml amber coloured volumetric flask and
make up the volume using 1:1 (HPLC methanol: 1% acetic acid)

41
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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF AFLATOXINS
(HPLC Method)
Method No. 21.0

Preparation of Sample:
1. In the case of spices, weigh 25 g of sample into a blender jar.
2. Weigh 5 g of salt into blender jar.
3. Add 100 mL of 80% methanol/20% water mix.
4. Cap blender jar and seal with parafilm.
5. Blend at high speed for 1 minute.
6. Filter blender contents through a fluted filter into a 250 mL beaker.
7. Pipet 10 mL of filtrate into 50 mL graduated cylinder.
8. If the sample is black pepper add 40 mL of 20%. Tween-20 solution to the graduated
cylinder. If the sample is not one of the products mentioned, add 40 mL of DI Distilled
water to the cylinder. Mix.
9. Filter the contents of the graduated cylinder through a glass fiber filter into a 250 mL
beaker. This filtrate will be used for the aflatest column.

Immuno-Affinity Column Clean-Up:

a. Attach an IAC [AFT] to the pump stand
b. Pipet 10 mL of filtrate on the column and allow to absorb on column.
c. Once all the sample has passed through the column, rinse the column with 10 mL of
HPLC water. Repeat HPLC water rinse.
d. Place 20 mL stoppered test tube under the tip of the column and add 1 mL of methanol
to the column.
e. Collect all the methanol eluent in the test tube. The sample is now ready for injection into
the HPLC.

Instrument Condition for HPC.

a. Mobile Phase
For Kobra Cell:
63% DI water with 0.1 g/L KBr and 0.02% Nitric
Acid/22% Methanol/ 15% Acetonitrile.

For PHRED Phochemical Reactor

55%HPLC water, 45% HPLC Methanol

b. Flow Rate - 1.2 mL/minute.

c. Column - Symmetry C-18; 4.6x250 mm column (Part No. WAT 054275)
d. Injection Volume - 50 uL
e. Detection - Excitation 365 nm, Emmission 464 nm
f. Kobra Cell at 100 u-amps / PHRED switched on

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IPC MANUAL OF METHODS OF ANALYSIS

DETERMINATION OF AFLATOXINS
(HPLC Method)
Method No. 21.0

Instrument Condition for UPLC

a. Mobile Phase:- 50:50 Water: MeOH

b. Column : UPLC BEH C18 1.7 uM
c. Injection Volume:- 20 uL
d. Detection (Ex- 365 nm, Em-456 nm)

D. Calculation :

Calculation of aflatoxin content in sample.

pG (conc) X 1000 uL X 50 mL X 100 mL

Aflatoxin (ug/kg) = Injection volume (uL) X 10 mL X 10 mL X 25 X10 3 mg

E. Result and Reporting :

Report the value after rounding to the first decimal place

The analytical result is given as

Aflatoxin

B1 (ug/kg) =
B2 (ug/kg) =
G1 (ug/kg) =
G2 (ug/kg) =
Total (ug/kg) =

If the level of aflatoxin is below 0.5 ug/kg, then the result can be given as < 0.5 ug/kg.

F. References:

1. AOAC International, 20th Edition, 2016, Official Methods of Analysis, 991.45– Total
Aflatoxins level in peanut butter.
2. ASTA Method No 24.2, Analysis of Aflatoxins B1, B2, G1, and G2 by HPLC; Official
Analytical Methods of the American Spices Trade Association, Fourth Edition, 1997.

43
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IPC MANUAL OF METHODS OF ANALYSIS

E. Coli [MPN Method]

Method No. 22.0

Purpose: To detect E.coli in black and white pepper by MPN method

To identify and enumerate Escherichia coli in black and white pepper.

A. Apparatus:

1. Balance, calibrated.
2. Incubator 35 + 1°C (forced circulation).
3. Standard (15x100 mm) or atleast (15 x 90 mm) sterile petridishes, glass or
disposable plastic.
4. Sterile spoons or spatulas.
5. Sterile 1mL, 0.2mL, 5mL & 10mL pipettes.
6. Inoculation loop made of Nichrome or Platinum Iridium wire.
7. Tubes, graduated, 18 X 150 mm;
8. 100-1000 variable micro-pipette.
9. Measuring cylinder (10 mL).
10. Covered water bath with circulating system.

B. Reagents:

1. Sterile 225 mL & 9 mL buffered dilution blanks (prepare as per method 30.05
2. Lauryl sulphate Tryptose (LST) broth,
3. Levine's EMB agar
4. Tryptone water
4. MR-VP media
5. Simmon's citrate Agar
6. Reagents for IMVIC test.
a. Kovac's Indole reagent
b. Methyl Red indicator
c. Barrits reagent A & Barrits reagent B
7. Gram Staining Kit
8. Mac Conkey broth
9. EC Broth

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IPC MANUAL OF METHODS OF ANALYSIS

E. Coli [MPN Method]

Method No. 22.0

C. Procedure:

I Sample Preparation, Dilution and Plating:

1. Presumptive Test:
a) Sample and Dilution Preparation
1. Sterile 225 mL & 9 mL Butterfield's Phosphate buffered dilution blank: To
prepare Butter field's Phosphate buffered dilution blank, 1.25 mL of Stock
Butterfeild's Buffer is added to 1L deionized water & (Stock Butterfield
Buffer - 34 g KH2PO4 per litre distilled water) adjusted to pH7.2 with 1N
NaOH. Sterilize 15 min at 121°C.
2. Aseptically weigh 25 g of sample into a 500mL screw capped bottle
containing 225 mL sterile Butterfield's Phosphate Buffer. This dilution
is 10-1. Shake at least 30 sec at 230 rpm in a Stomacher:
3. To prepare 10-2 dilution, transfer 1 mL of 10-1 dilution to 9 mL
dilution blank and shake 25 times in a 1 ft. arc.
4. To prepare 10-3 dilution, pipette 1 mL of 10-2 into 9 mL blank
(use separate sterile pipets for each dilution).
5. Prepare as many decimal dilutions as necessary (10-1 to 10-6) depending
on the expected bacterial load of the material being examined.
6. Shake each dilution before preparing each sub sequential dilution.

b) From dilutions prepared at A above transfer 1 mL portions to 3 LST tubes for

each dilution. Take minimum 3 consecutive dilutions. The dilutions to be
prepared depend on the anticipated coliform density. Shake all suspension 25
times in foot arc. Not more than 15 min should elapse from the time sample is
blended until all dilutions are in appropriate media.
c) Incubate tubes for 24 + 2 hrs at 35 + 0.5°C
d) Examine tubes at 24 + 2 hrs for gas ie displacement of medium in fermentation
vial by gas.
e) Re incubate gas negative LST tubes for an additional 24 hrs and examine and
record the reactions again at 48 + 3 hrs.

2. Confirmation Test for E.coli.

a) From each gassing LST tubes from presumptive test tranfer1 loopful of each
suspension to EC broth and Incubate the tubes at 45.5° C for 24 + 2 hrs in
water bath.
b) After 24 hrs incubation examine for gas production. If negative re incubate
and examine again at 48 + 2 hrs.
c) Count the number of positive tubes in each dilution.

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IPC MANUAL OF METHODS OF ANALYSIS

E. Coli [MPN Method]

Method No. 22.0

3. Complete Test for E.coli.

a) From each gassing EC tubes take loopful of broth and streak on to LEMB
agar plate.
b) Incubate the pates at 35 + 0.5° C for 18-24 hrs
c) After 24 hrs incubation the E.coli colonies are identified bydark
centered colonies with or without metallic sheen.

II Identification of E. coli:-

1. Gram staining
Refer MOA No: 30.08.
Microscopic Examination:
When viewed under the microscope, the cells of E. coli shall be Gram-negative short
rods (i.e. red or pink in color).

2. Biochemical tests

Perform the following bio-chemical IMVIC tests for the identification of E. coli
a. Eijkman test.
b. Indole production test
c. Methyl red test
d. Voges - Proskauer (VP) test.
e. Citrate utilisation test

a. Eijkman test:-
Inoculate Mac Conkey broth, warmed to 37 °C a nd incubate at 45.5 + 0.2 °C
for 48 hours in water bath. Positive result is indicated by the production of both
acid and gas. E. coli gives positive test.
b. Indole test:-
Inoculate in Tryptone water and incubate for 24 + 2hrs at 35 + 0.5 °C. After
incubation add 0.2 to 0.3 mL Kovac's reagent, shake well and examine after 5
minutes. A red color in the reagent layer indicates the presence of Indole. E. coli
produces Indole in this test ie, positive reaction E. coli is able to break down the
amino - acid tryptophan with the formation of Indole which forms a highly
alcohol soluble dye complex with Kovac's reagent.

MR and V.P. tests:-

For both these tests inoculate the above culture into two tubes of MR-
VP medium and incubate them for 48 + 2 hrs at 35 + 0.5 °C.

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IPC MANUAL OF METHODS OF ANALYSIS

E. Coli [MPN Method]

Method No. 22.0

c. Methyl red test:

To one of the above incubated tubes add 5 drops of methyl red solution. A
distinct red color color, showing the presence of acid, is regarded as a positive
reaction, a yellow color, a negative result. E. coli gives a positive result in this
test.

d. Voges-Proskauer test:
To the second tube of the duplicate culture add 0.6 mL of Alpha Naphthol
solution and 0.2 mL 40% KOH. Shake the tube vigorously and examine after 2
hrs. The development of an eosin pink color indicates a positive reaction. (To
speed up the reaction add 2-3 crystals of Creatine.)
e. Citrate Utilization tests:
Inoculate the culture in a Simmon's citrate agar slant and incubate for 48 h
at 35 + 0.5 °C.
Examine the tubes for growth and blue coloration (alkaline reaction).

Reactions *E. coli (Biotype 1) * E. coli (Biotype 2)

Indole + -
MR + +
VP - -
Citrate - -

a) *All cultures that ferment lactose with gas production within 48 hrs at 35°C.
b) *Appear as Gram negative non spore forming rods.
c) *Give IMViC patterns (Biotype 1 or Biotype 2) considered to be E.coli

D. Calculation:

a) Record the number of tubes in each dilution EC tube that were confirmed as positive
for E.coli organisms.
b) Refer to MPN table to find MPN index (organisms per 100

mL); Calculate:

MPN/g = [(MPN/g from Table/100)] X dilution factor of the middle tube.

E. Result and Reporting:

Report counts as MPN per g or mL, as applicable.

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IPC MANUAL OF METHODS OF ANALYSIS

E. Coli [MPN Method]

Method No. 22.0

F. Reference:

FDA, BAM 2013, Chapter 4.

G. Environmental Aspects:

On completion of work, all contaminated liquid and solid wastes should be sterilized,
preferably by autoclave. Lab coats, gloves and mouth covers are must when working with
live microorganisms. It is also important that standard Laboratory Technique be utilized
when working with these hazards.

48
Methods of Analysis and Sampling

10 METHODS OF ANALYSIS AND SAMPLING

1. Methods of Analysis

Provision IPC Methods Principle

Physical
Bulk Density (g/l), Method No. 4.0 Density Determination
Light Berries/Corns(m/m)%, Method No. 3.0 Flotation
Extraneous Matter (m/m)%, Method No. 5.0 Visual Examination
Mouldy Berries/Corn(m/m) %, Method No. 9.0 Visual Examination
Insect Defiled Berries/Corns(% by wt.), Method No. 8.0 Visual Examination
Whole Insects, dead or alive (by count), Method No. 6.0 Visual Examination
Mammalian or/and Other Excreta (by count), Method No. 7.0 Visual Examination
Pinheads % (m/m), Method No. 13.1 Visual Examination
Broken berries % (m/m), Method No. 13.2 Visual Examination
Determination of grey and black berries in white pepper Method No. 10.0 Visual Examination

Chemical
Moisture (m/m)%, Method No. 2.0 Distillation
Total ash, % (m/m), Method No. 14.0 Gravimetric
Acid in Soluble Ash % (m/m), on dry basis Method No. 15.0 Gravimetric
Non-volatile ether extract % (m/m), on dry basis Method No. 17.0 Soxhlet extraction
Volatile oil % (ml/100 g) on dry basis Method No. 16.0 Distillation
Piperine content, % (m/m), Method No. 18.0 Spectrophotometric
Crude Fiber insoluble index % (m/m), on dry basis Method No. 19.0 Gravimetric
Sulphur Dioxide % (m/m), or in ppm (mg/Kg), Method No. 20.0 Titrimetric
Preparation of Sample Method No. 1.0 -

Microbiology
Escherichia coli (MPN/g), Method No. 22.0 Microbiological
Salmonella (detection / 25g) Method No. 12.0 Microbiological
Salmonella Sample Preparation Method No. 11.0 Microbiological

Aflatoxin
Aflatoxin Total (B1+B2+G1+G2) (ug/kg) Method No. 21.0 Chromatography

2. Sampling Methodology
Provision Method Principle
Sampling methodology of Pepper for Chemical analysis Method No. 24.0 -
Sampling methodology of Pepper for Microbiology analysis Method No. 25.0 -

49