Version No :01 Determination of Fat content Standard operating procedure

No.57
Issue date :01/06/2021
Page 1 of 13

) ‫( جدول االعتمادات‬

Preparation Revision Approval

Name Dr . Amr seif Dr. Mohamed Dr . Amr seif
elsenduony
Position Lab technical manager Lab Quality manager Lab manager
Sign
Date

) ‫( جدول التعديالت‬

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫مراجعة‬ ‫إصدار‬

\ ‫إصدار‬
‫مراجعة و اعتماد‬ ‫إعداد‬ ‫مراجعة‬

)‫(جدول التوزيع‬

‫االدارة‬ ‫رقم النسخة‬ ‫االدارة‬ ‫رقم النسخة‬ ‫االدارة‬ ‫رقم النسخة‬

Department Copy No. Department Copy No. Department Copy No.
9 5 1
11 6 2
11 7 3
12 8 4
 Version No :01 Determination of Fat content Standard operating procedure
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1. Purpose:
Determination of Fat content of some Food products and animal feed including :
1- Meat and meat products based on ISO 1444\1996
2- Cheese based on AOAC 933.05:2019
3- Animal feed based on ISO 6492:1999

2. Scope:
This Standard operating procedures is concerned to determination the Fat content on some food items
( meat and meat products , Cheese) and animal feed

3. Responsibility
This procedure shall be applied by all personnel who authorized to carry out this test.
4. Reference

1. ISO 1444\1996
2. AOAC 933.05:2019
3. ISO 6492:1999
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I- Determination of Fat content on Meat and meat products ( SOP 57.1)

1. Purpose.
Determination of fat content in meat and meat products .

2. Scope.
Determination of fat content in meat and meat products according to ISO 1114:1996.

3. Applicability.
This procedure shall be applied by all personnel who authorized to Carry out this test.

4. Environment.
o No air currents
o No dusty atmosphere
o Atmospheric confederation .22±2 and 45-50% humidity

5. Reagents and materials

a. Reagents:
Light petroleum, consisting mainly of hydrocarbons with six carbon atoms, boiling range 400 C to 600 C.
The bromine value shall be less than 1. The evaporation residue shall be less than 20 mg/1.
Alternatively, technical hexane maybe used having an evaporation residue of less than 20 rng/1.
Silicon carbide chips or glass beads.
Acetone.
Hydrochloric acid, c(liCl) = 3 mol/1.
Filtration aid, for example diatomaceous earth (Kieselguhr), balled for 30 min in hydrochloric acid,
c(HCI) = 6 mol/1, washed with water until acid-free, then dried at 1300 C.

b. Apparatus:
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Usual laboratory apparatus and, in particular, the following.

Homogenizing equipment,
Extraction thimbles, free from fats and oils, ether-washed.
Soxhlet-type extractor, with syphoning volume of about 100 ml, or other recirculation extractor.
Heating apparatus, with temperature control, not liable to act as an ignition source.
Drying oven, capable of being maintained at 103 A 2) 0C.
Electrically heated vacuum oven, capable of being maintained at (80 +2) OC and of reducing the
pressure to less than 13,3 kPa, fitted with a device for the introduction of dry air or containing a
desiccant, for example calcium oxide.
Desiccator, containing an efficient desiccant.
- Balance

6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put anything in or near your mouth
 Wash your hands with soap and water.
 Test portion temperature should not exceed 22± 2 oC
 Operation should not be carried out in direct sunlight.
 Mouth pipetting is prohibited

7.Procedure.
Weigh at least 20 g of the prepared test sample to the nearest 1 mg and mix with 10 g of anhydrous sodium
sulfate

-Homogenize the test sample with the appropriate equipment .Take care that the temperature of the sample
material does not rise above 25 °C. If a
mincer is used, pass the sample at least twice through the equipment.
- Fill a suitable air tight container with the prepared sample. Close the container and store in such way that
deterioration and change in composition are prevented.
Analyse the sample as soon as practicable, but always within 24 h of homogenization.

-NOTE 2 If it is required to check whether the repeatability requirement is met, carry

out two single determinations in accordance under repeatability conditions.
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-take a known mass of 5 g to 8 g, weighed to the nearest 0,001 g (mO) of the prepared sample and dry petri
dish and weight 3-4 sample wight sand and dry at 103 °c for 30 min . If desired, the
dried test portion from the determination of moisture content may be used for the determination of free fat.
For reliable measurements, the lowest level of fat
present in the test portion should be 0,05 g.

-Dry the flask of the extraction apparatus containing some boiling-chips for 1 h in the drying oven set at 103
“C.

-Allow the flask to cool to room temperature in the desiccator and weigh to the
nearest 0,001 g (ml).

-Transfer the dried test portion quantitatively from the dish to the extraction thimble . Remove the last traces
of the dried test portion from the dish, using cotton wool moistened with the extraction solvent and also
transfer this cotton wool to the
thimble. Place the thimble in the extraction tube of the
apparatus.
-Pour the extraction solvent into the flask of the extraction apparatus; the amount of solvent shall be at least
one and a half to two times the capacity of
the extraction tube of the apparatus.
- Fit the flask to the extraction apparatus.
When a Soxtec or other similar automatic procedure is
used, the heating period shall be at least 2 h.

-After extraction, take the flask containing the liquid from the extraction apparatus and distil off the solvent
using, for example, the sand bath or water bath. Evaporate
the last traces of solvent using air blowing if desired.

-Dry the flask for 1 h in the oven set at 103 OC and, after allowing it to cool to room temperature in the
desiccator ,weigh to the nearest 0,001 g.

- Repeat the operations of heating, cooling and weighing until the results of two successive weighings,
separated by 1 h of heating, do not differ by more than
0,l % of the mass of the test portion (m*).

-Verify completion of the extraction by taking a second extraction flask and extracting for a further period of
1 h with a fresh portion of the solvent. The increase in
mass shall not exceed 0,l % of the test portion.

Calculation
Calculate the free fat content, Wt I as a percentage by
mass, using the following equation:
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II- Determination of Fat content on animal feed ( SOP 57.2)

1. Purpose.
Determination of fat content in animal feeding stuffs.

2. Scope.
Determination of fat content in animal feeding stuffs according to ISO 6492:1999.

3. Applicability.
This procedure shall be applied by all personnel who authorized to Carry out this test.

4. Environment.
o No air currents
o No dusty atmosphere
o Atmospheric confederation .22±2 and 45-50% humidity

5. Reagents and materials

a. Reagents:
Use only reagents of recognized analytical grade.
Water, complying with at least grade 3 in accordance with ISO 3696.
Sodium sulfate, anhydrous.
Light petroleum, consisting mainly of hydrocarbons with six carbon atoms, boiling range 400
C to 600 C.
The bromine value shall be less than 1. The evaporation residue shall be less than 20 mg/1.
Alternatively, technical hexane maybe used having an evaporation residue of less than 20 rng/1.
Silicon carbide chips or glass beads.
Acetone.
Hydrochloric acid, c(liCl) = 3 mol/1.
Filtration aid, for example diatomaceous earth (Kieselguhr), balled for 30 min in hydrochloric
acid,
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c(HCI) = 6 mol/1, washed with water until acid-free, then dried at 1300 C.

b. Apparatus:

Usual laboratory apparatus and, in particular, the following.

Extraction thimbles, free from fats and oils, ether-washed.
Soxhlet-type extractor, with syphoning volume of about 100 ml, or other recirculation
extractor.
Heating apparatus, with temperature control, not liable to act as an ignition source.
Drying oven, capable of being maintained at 103 A 2) 0C.
Electrically heated vacuum oven, capable of being maintainedat (80 +2) OCand of
reducingthe pressureto less than 13,3 kPa, fittedwith a device for the introductionof dry air
or containinga desiccant, for example calcium oxide.
Desiccator, containingan efficientdesiccant.

6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put anything in or near your mouth
 Wash your hands with soap and water.
 Test portion temperature should not exceed 22± 2 oC
 Operation should not be carried out in direct sunlight.
 Mouth pipetting is prohibited
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7. Procedure.
Weigh at least 20 g of the prepared test sample to the nearest 1 mg and mix with 10 g of
anhydrous sodium sulfate

Hydrolysis:

 Transfer the test portion to a 400 ml beaker or a 300 ml conical flask. Add 100 ml of
hydrochloric acid and silicon carbide chips . Cover the beaker with a watch glass or fit
the conical flask with a reflux condensor. Bring the mixture to a gentle boil over a flame
or a hot plate and maintain it for 1 h. Swirl every 10 min to prevent the product sticking
to the sides of the container.

 Cool to ambient temperature and add a quantity of filtration aid sufficient to prevent any
loss of fat during the filtration. Filter through a moistened, fat-free double filter paper in a
Buchner funnel with suction. Wash the residue with cold water until a neutral filtrate is
obtained.

 CAUTION: If oil or fat appears on the surface of the filtrate, wrong results maybe
obtained. A possible solution is to repeat the procedure applying a smaller test portion or
a higher acid concentration.

 Carefully take out the filter and place the double filter paper containing the residue in an
extraction thimble (6.1) and dry under vacuum for 60 min in the oven (6.5) set at 80 “C.
Remove the thimble from the oven and cover with a fat free wad of cottonwood.

Extraction:

 Transfer some glass beads into dry flask then record its weight.

 Connect the flask with the extractor to collect the light petroleum or Hexane extract.

 Place the thimble in the extractor and extract for 6 h with light petroleum or Hexane.
Regulate the heating apparatus to obtain at least 10 siphoning per hour if a Soxhlet-type
extractor is used, or a reflux rate of at least 5 drops per second (about 10 ml/min) if an
equivalent apparatus is used.

 Distil off the solvent until the flask is nearly free from solvent, Add 2 ml of acetone to
the flask, swirl and gently warm on the heating apparatus to remove the acetone. Blow
off the last traces of acetone.

 Dry the residue for (10) min. in the drying oven set at 103 “C. Cool in the desiccator and
weigh to the nearest 0,1 mg.
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*Calculation:

Calculate the fat content of the test sample, in grams per kilogram, using the equation:
Fat Content = M3-M2
_____ × f
M1
M1…….is the mass of test potion , in grams
M2…….is the mass of flask with glass beads, in grams
M3…….is the mass of the flask with glass beads dried light petroleum or hexane residues
obtained
F………is the units correction factor, in grams per kilogram (f=1000g/kg)
8.Retention And Disposal.

Glass materials, washed by detergent then tap water and distillated water and leave to dry at
room temperature.
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III- Determination of Fat content on Cheese ( SOP 57.3)

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IV- Determination of Fat content on Milk ( SOP 57.4)

AOAC Of fi cial Method 989.05
Fat in Milk (c) Fat extraction.—To test portion in flask add 1.5 mL
NH4OH
and mix thor oughly. NH4OH neu tral izes any acid pres ent
A. Principle and dis solves ca sein. Add 3 drops of phenolphthalein in di ca
Fat is ex tracted with mix ture of ethers from known weight of tor to help sharpen vi sual ap pear ance of in ter face be tween
milk. Ether ex tract is de canted into preweighed dry weigh ng ether and aque ous lay ers dur ing ex trac tion. Add 10 mL
dish, and ether is evaporated. Extracted fat is dried to con stant 95% al co hol, stop per with H2O-soaked cork, and shake
weight. Re sult is ex pressed as % fat by weight. flask 15 s. For first ex trac tion, add 25 mL ethyl ether, stop
B. Apparatus per with cork, and shake flask very vig or ously 1 min,
(a) Flask.—Mojonnier-style ether ex traction flask with volume releasing built-up pressure by loosening stopper as necessary.
of 21–23 mL in lower bulb plus neck at bot tom of flask. Flask Add 25 mL pe tro leum ether, stop per with cork, and re peat
should have smooth, round open ing at top that will seal when vig or ous shak ing for 1 min. Cen tri fuge flasks at ca 600
closed with cork. clean sep a ra tion of aque ous (bright
(b) Weighing dishes.—Metal, 8.5–9.5 cm di am e ter and pink) and ether phases. De cant ether so lu tion into suit able
4.5–5.5 cm tall; or 250 mL glass beak ers. weigh ing dish pre pared as in (b). When ether so lu tion is de
(c) Cal i bra tion weights.—Class S, stan dard cal i bra tion canted into dishes, be care ful not to pour over any
weights to verify balance ac cu racy within weight range to be sus pended sol ids or aque ous phase into weigh ing dish.
used for weighing empty flasks and flask plus test portion and Ether can be evap o rated at
weigh ing empty dishes and dish plus fat. duct ing sec ond extraction.
(d) An a lyt i cal bal ance.—Readability 0.0001 g. Ac cu racy on For sec ond ex trac tion, add 5 mL 95% al co hol, stop per
ver i fi ca tion within 0.0002 g. Check pe ri od i cally and when with cork, and shake vig or ously 15 s. Next, add 15 mL ethyl
ever bal ance is moved or cleaned. Keep re cord of bal ance cal i ether, re place cork, and shake flask vig or ously 1 min. Add
bra tion checks. 15 mL pe tro leum ether, stop per with cork, and re peat vig or
(e) Des ic ca tor.—Room tem per a ture. For cool ing weigh ing ous shak ing for 1 min. Cen tri fuge flasks at ca 600 rpm for
dishes af ter pre lim i nary and fi nal dry ing. Use coarse (bright pink)
desiccant (mesh size 6–16) that con tains min i mum of fine par and ether phases. If in ter face is be low neck of flask,
ti cles and that changes color when mois ture is ab sorbed. add H2O to bring level ca half way up neck. Add H2O slowly
(f) Tongs.—For han dling weigh ing dishes. down in side sur face of flask so that there is min i mum dis
(g) Hot plate.—Steam bath or other heat ing de vice. For tur bance of sep a ra tion. De cant ether so lu tion for sec ond
evap o ra tion of ether at ex trac tion into same weigh ing dish used for first ex trac tion.
hood. For third ex trac tion, omit ad di tion of 95% al co hol and re
(h) Corks.—High qual ity nat u ral cork stop pers (size 5) for peat pro ce dure used for sec ond ex trac tion. Com pletely
flasks. evap o rate sol vents in hood on hot plate at
Soak corks in H2O sev eral hours to im prove seal. spat ter ing). Dry ex tracted fat in weigh ing dish to con stant
(i) Vac uum or forced air oven.—Vac uum oven main tain ing weight in forced air oven at 100+ or -
–75°C at 50.8 cm (20 in.) of vac uum, or –75°C at >50.8 cm (20 in.) of vac uum for
forced air and place in
(j) Wa ter bath for tem per ing test sam ples prior to des ic ca tor to cool to room tem per a ture. Re cord weight
weigh ing.—With ther mom e ter and de vice to main tain milk of each weigh ing dish plus fat.
Run pair of re agent blanks each day tests are con ducted. To
C. Re agents run re agent blank, re place milk test portion with 10 mL H2O
(a) Ethyl ether.—ACS grade, per ox ide free. No res i due on and run test as above. Re cord weight of any dry res i due col
evap o ra tion. lected and use value in cal cu la tion. Re agent blank should be
(b) Pe tro leum ether.— –60°C. <0.0020 g res i due. If re agent blanks for set of tests are neg a
No res i due on evap o ra tion. tive use neg a tive num ber in cal cu la tion. (Note: To sub
(c) Am mo nium hy drox ide.—Con cen trated, ACS grade, spe tract neg a tive num ber [av er age weight blank res i due] in
cific grav ity 0.9. equa tion be low, add it to [(weight dish + fat) -
(d) Ethyl al co hol.—95%. No res i due on evap o ra tion. weight dish].) Neg a tive blank usu ally in di cates that dishes
(e) Dis tilled wa ter.—Free of oil and min eral res i due. were not com pletely dry at start of de ter mi na tion or that bal
(f) Phenolphthalein in di ca tor.—0.5% (w/v) in al co hol. ance cal i bra tion shifted be tween weigh ing of empty pans
Version No :01 Determination of Fat content Standard operating procedure
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D. De ter mi na tion and pans plus fat. Cause of neg a tive blanks should be iden ti
(a) Weighing test portion.—Pre pare by tem per ing milk to fied and cor rected.
38°C as in 925.21 (see 33.2.02). Weigh empty flask with clean,
dry cork stop per. Re move stop per. Pipet ca 10 g milk into
flask.
Place stop per in flask. Weigh to near est 0.1 mg. Check bal
ance zero be tween test portions.
(b) Weighing dishes.—Num ber clean weigh ing dishes and
predry un der same con di tions that will be used for fi nal dry
ing af ter fat ex trac tion. Be sure that all sur faces where weigh
ing dishes will be placed (i.e., hot plate, des ic ca tor, etc.) are
clean and free of particulates. At end of oven dry ing, place
dishes in room tem per a ture des ic ca tor and cool to room tem
per a ture. On same day as fat ex trac tion, (c), weigh dishes to
near est 0.1 mg and re cord weights. Check bal ance zero af ter
weigh ing each dish. Pro tect weighed dishes from con tam i na
tion with ex tra ne ous mat ter.