METHODS FOR ANALYSIS IN THE OFFICIAL CONTROL OF FEEDINGSTUFFS RULES *

METHODS FOR ANALYSIS

IN THE OFFICIAL CONTROL OF FEEDINGSTUFFS

SUBSIDIARY LEGISLATION 437.63

METHODS FOR ANALYSIS IN THE OFFICIAL

CONTROL OF FEEDINGSTUFFS RULES *

5th April, 2005

LEGAL NOTICE 93 of 2005.

(1) The title of these rules is the Methods for Analysis in

the Official Control of Feedingstuffs Rules.

(2) The scope for these rules is to implement the rules found

under EU Commission Directive 72/199/EEC establishing methods

of analysis for the official control of feedingstuffs.

(3) Malta shall require that analyses for official controls of

feedingstuffs, as regards their level of starch, crude protein, crude

protein which can be dissolved by Pepsin and hydrochloric acid, of

free and total gossypol and as regards pepsin activity be carried out

using the methods described in Schedule I.

(4) Malta shall require that analyses for official controls of

feedingstuffs for the purpose for determining the levels of tylosin,

and virginiamycin in feedingstuffs, shall be carried out using the

methods described in Schedule II.

*These rules have been repealed by Legal Notice 37 of 2025.

1. DETERMINATION OF STARCH

POLARIMETRIC METHOD

1. Purpose and scope

This method makes it possible to determine the levels of starch and of high

molecular weight starch degradation products in feedingstuffs for the purpose of

checking compliance with EU Commission Directive 86/174/EEC and EU Council

Directive 96/25/EC.

Principle

The method comprises two determinations. In the first, the sample is treated when

hot with dilute hydrochloric acid. After clarification and filtration the optical

rotation of the solution is measured by polarimetry.

In the second, the sample is extracted with 40% ethanol. After acidifying the

filtrate with hydrochloric acid, clarifying and filtering, the optical rotation is

measured as in the first determination.

The difference between the two measurements, multiplied by a known factor,

gives the starch content of the sample.

4. DETERMINATION OF TYLOSIN

-

by diffusion on agar -

1. Purpose and scope

This method makes it possible to determine the tylosin content of feedingstuffs,

concentrates and premixes where more than 2 ppm are present.

2. Principle

The sample is treated with a pH 8 phosphate buffer solution, previously heated to

80ºC, and then extracted with methanol. After centrifuging, the extract is diluted and

its antibiotic activity determined by measuring the diffusion of the tylosin on an agar

medium seeded with Sarcina lutea. The diffusion is made evident by the formation

of inhibition zones in the presence of the micro-organism. The diameter of these

zones is directly proportional to the logarithm of the antibiotic concentration.

3. Micro-organism: Sarcina lutea ATCC No. 9341

3.1

Maintenance of the parent strain

Inoculate with Sarcina lutea a tube of sloped agar taken from the culture medium

(4.1), adjust to pH 7ꞏ0. Incubate overnight at approximately 35ºC. Keep the culture

in a refrigerator and re-inoculate sloped agar with it every month.

3.2

Preparation of the bacteria suspension

Collect the bacteria from a recently prepared tube of sloped agar (3.1) using 2 to 3

ml of physiological saline (4.4). With this suspension seed a Roux flask containing

250 ml of the culture medium (4.1), adjusted to pH 7ꞏ0. Incubate for 24 hours at

35ºC, then collect the bacteria in 25 ml of physiological saline (4.4). Homogenize,

and dilute this suspension to obtain approximately 75% light transmission at 650 nm.

If kept in a refrigerator this suspension may be used for one week.

By preliminary tests on plates with the basic medium for the determination (4.1),

establish the quantity of inoculum which, for the different concentrations of tylosin

used, will give the largest possible inhibition zones that are still clear. The culture

medium is inoculated at 48 to 50ºC.

4. Culture media and reagents

Basic medium for the determination) *

4.1

Glucose

1 g

Tryptic peptone

10 g

Meat extract

1ꞏ5 g

Yeast extract

3 g

Agar, according to quality

10 to 20 g

Distilled water to

1000 ml

Adjust immediately before use to pH 7.0 for maintenance of the parent strain and

preparation of the bacteria suspension, and to pH 8ꞏ0 for the determination.

4.2

Phosphate buffer solution, pH 8

Potassium dihydrogen phosphate KH 2 PO 4 A.R.

0ꞏ523 g

diPotassium hydrogen phosphate K 2 HPO 4 A.R.

16ꞏ730 g

Distilled water to

1000 ml

4.3

Phosphate buffer solution, pH 7

Potassium dihydrogen phosphate KH 2 PO 4 A.R.

5ꞏ5 g

di Potassium hydrogen phosphate K 2 HPO 4 A.R.

13ꞏ6 g

Distilled water to

1000 ml

4.4

Sterile physiological saline.

4.5

Pure methanol.

4.6

40% (v/v) methanol.

4.7

Mixture of phosphate buffer solution (4.2)/pure methanol: 60/40 by

volume.

4.8

Standard substance: tylosin of known activity.

5. Standard solutions

Dry the standard substance (4.8) for 3 hours at 60ºC in a vacuum oven (5 mm of

mercury). Weigh 10 to 50 mg into a graduated flask, dissolve in 5 ml of methanol

(4.5) and dilute the solution with the phosphate buffer solution, pH 7 (4.3), to obtain

a tylosin-base concentration of 1000 µg per ml.

Prepare a standard working solution S 8 containing 2 µg per ml of tylosin base

from this stock solution by diluting with the mixture (4.7).

Then prepare by successive dilutions (1 + 1), using the mixture (4.7), the

following concentrations

1 µg/ml

0ꞏ5 µg/ml

0ꞏ25 µg/ml

6. Extraction

*Any commercial culture medium of similar composition and giving the same results may be used

For concentrates, take a 10 g test sample[ for premixes and feedingstuffs, a 20 g

test sample. Add 60 ml of phosphate buffer solution, pH 8 (4.2), previously heated to

80ºC, and homogenize for 2 minutes (domestic mixer, Ultra-turrax, etc.).

Leave to stand for 10 minutes, add 40 ml of methanol (4.5) and homogenize for 5

minutes. Centrifuge the extract and dilute an aliquot part with the mixture (4.7) to

obtain a presumed tylosin concentration of 2 µg per ml (= U 8 ). Then prepare the

concentrations U 4 , U 2 and U 1 by successive dilutions (1 + 1) using the mixture (4.7).

For contents of less than 10 ppm, evaporate the extract until dry in a rotary

evaporator at 35ºC and dissolve the residue in 40 % methanol (4.6).

7. Determination method

7.1

Inoculation of the culture medium

Inoculate at 48 to 50ºC the basic medium for the determination (4.1), adjust to pH

8ꞏ0, with the bacteria suspension (3.2).

7.2

Preparation of the trays

Diffusion on agar is carried out in trays using 4 concentrations of the standard

solution (S 8 , S 4 , S 2 , S 1 ) and 4 concentrations of the extract (U 8 , U 4 , U 2 , U 1 ). The 4

concentrations of standard solution and of extract must be placed in each tray.

Choose trays, therefore, which are large enough to allow at least 8 holes 10 to 13

mm in diameter to be made in the agar medium. Calculate the quantity of inoculated

culture medium (7.1) needed to provide a uniform covering approximately 2 mm

thick. The test should preferably be carried out on flat trays consisting of glass plates

filled with a perfectly level aluminium or plastic ring, 200 mm in diameter and 20

mm high.

Pipette into the holes accurately measured quantities of between 0ꞏ10 and 0ꞏ15 ml

of antibiotic solution, depending on the diameter of the holes.

For each sample repeat the diffusion at least 4 times with each concentration so

that each determination comprises an evaluation of 32 inhibition zones.

7.3

Incubation

Incubate the trays overnight at 35 to 37ºC.

8. Evaluation

Measure the diameter of the inhibition zones, preferably by projection.

Record the measurements on semi-logarithmic paper, plotting the logarithm of the

concentrations against the diameter of the inhibition zones. Trace the lines of the

standard solution and of the extract. Provided there is no interference the two lines

will be parallel.

The logarithm of the relative activity is calculated by using the following formula

Real activity = presumed activity × relative activity.

9. Repeatability

The difference between the results of two parallel determinations carried out on

the same sample must not exceed 10% in relative value.

5. DETERMINATION OF VIRGINIAMYCIN

- by diffusion in an agar medium -

1. Purpose and scope

The method is for the determination of virginiamycin in feedingstuffs and

premixes. The lower limit of determination is 2 mg/kg (2 ppm). *

2. Principle

The sample is extracted with a methanolic solution of Tween 80. The extract is

decanted or centrifuged and diluted. Its antibiotic activity is determined by

measuring the diffusion of virginiamycin in an agar medium inoculated with

Micrococcus luteus. Diffusion is shown by the formation of zones of inhibition of

the micro-organism. The diameter of these zones is taken to be in direct proportion

to the logarithm of the antibiotic concentration over the range of antibiotic

concentrations employed.

3. Micro-organism: Micrococcus luteus ATCC 9341 (NCTC 8340, NCIB

8553)

3.1.

Maintenance of stock culture

Inoculate tubes containing slopes of culture medium (4.1) with Micrococcus

luteus and incubate for 24 hours at 30ºC. Store the culture in a refrigerator at about

4ºC. Reinoculate every two weeks.

Preparation of the bacterial suspension †

3.2.

Harvest the growth from a recently prepared agar slope (3.1) with 2 to 3 ml of

sodium cloride solution (4.3). Use this suspension to inoculate 250 ml of culture

medium (4.1) contained in a Roux flask and incubate for 18 to 20 hours at 30ºC.

Harvest the growth in 25 ml of sodium chloride solution (4.3) and mix. Dilute the

suspension to 1/10 with sodium chloride solution (4.3). The light transmission of the

suspension must be about 75 %, measured at 650 nm in a 1 cm cell against sodium

chloride solution (4.3). This suspension may be kept for one week at about 4ºC.

4. Culture media and reagents

Culture and assay medium ‡

4.1.

Meat peptone

6,0 g

Tryptone

4,0 g

Yeast extract

3,0 g

Meat extract

1,5 g

Glucose

1,0 g

Agar

10,0 to 20,0 g

Water

1 000ml

pH 6.5 (after sterilization)

4.2.

Phosphate buffer, pH 6

Potassium hydrogen phosphate, K 2 HPO 4

2,0 g

Potassium dihydrogen phosphate, KH 2 PO 4

8,0 g

*1mg virginiamycin is equivalent to 1000 UK units

†Other methods may be used provided that it has been established that they give similar bacterial

suspensions.

‡Any commercial culture medium of similar composition and giving the same results may be used.

Water to

1 000 ml

4.3.

Sodium chloride solution 0,8% (w/v): dissolve 8 g sodium chloride in

water and dilute to 1 000 ml; sterilize.

4.4.

Methanol.

4.5.

Mixture of phosphate buffer (4.2)/methanol (4.4): 80/20 (v/v).

4.6.

Tween 80 methanolic solution 0,5% (w/v): dissolve 5 g Tween 80 in

methanol (4.4) and dilute with methanol to 1 000 ml.

4.7.

Standard substance: virginiamycin of known activity.

5. Standard solutions

Dissolve an accurately weighed quantity of the standard substance (4.7) in

methanol (4.4) and dilute with methanol (4.4) to give a stock solution containing

1000 µg virginiamycin per ml.

Stored in a stoppered flask at 4ºC this solution is stable for up to five days.

From this stock solution prepare by successive dilution with the mixture (4.5) the

following solutions

µg/ml

0,5

µg/ml

0,25

µg/ml

0,125 µg/ml

6. Preparation of the extract and assay solutions

6.1.

Extraction

6.1.1. Products with a virginiamycin content up to 100 mg/kg

Weigh out a quantity of sample of 50 g, add 200 ml of solution (4.6) and shake for

30 minutes. Leave to settle or centrifuge, take 20 ml of the supernatant solution and

evaporate to about 5 ml in a rotary evaporator at a temperature not exceeding 40ºC.

Dilute the residue with the mixture (4.5) to obtain an expected virginiamycin content

of 1 µg/ml (= u 8 ).

6.1.2. Products with a virginiamycin content greater than 100 mg/kg

Weigh out a quantity of sample not exceeding 10,0 g and containing between 1 and

50 mg virginiamycin, add 100 ml of solution (4.6) and shake for 30 minutes. Leave

to settle or centrifuge, then dilute the supernatant solution with the mixture (4.5) to

obtain an expected virginiamycin content of 1 µg/ml (= u 8 ).

6.2.

Assay solutions

From solution u 8 prepare solutions u 4 (expected content: 0,5 µg/ml), u 2 (expected

content: 0,25 µg/ml) and u 1 (expected content: 0,125 µg/ml) by means of successive

dilution (1 + 1) with the mixture (4.5).

7. Assay procedure

7.1.

Inoculation of the assay medium

Inoculate the assay medium (4.1) with the bacterial suspension (3.2) at about

50ºC. By preliminary trials on plates with the medium (4.1) determine the quantity

of bacterial suspension required to give the largest and clearest zones of inhibition

with the various concentrations of virginiamycin.

7.2.

Preparation of the plates

Diffusion through agar is carried out in plates with the four concentrations of the

standard solution (s 8 , s 4 , s 2 and s 1 ) and the four concentrations of the assay solution

(u 8 , u 4 , u 2 and u 1 ). These four concentrations of extract and standard must

necessarily be placed in each plate. To this effect, select plates big enough to allow

at least eight holes with a diameter of 10 to 13 mm and not less than 30 mm between

centres to be made in the agar medium. The test may be carried out on plates

consisting of a sheet of glass with a faced aluminium or plastic ring placed on top,

200 mm in diameter and 20 mm high.

Pour into the plates a quantity of the medium (4.1) inoculated as in point 7.1, to

give a layer about 2 mm thick (60 ml for a plate of 200 mm diameter). Allow to set in

a level position, bore the holes and place in them exactly measured volumes of assay

and standard solutions (between 0,10 and 0,15 ml per hole, according to the

diameter). Apply each concentration at least four times so that each determination is

subject to an evaluation of 32 zones of inhibition.

7.3.

Incubation

Incubate the plates for 16 to 18 hours at 30 ± 2ºC.

8. Evaluation

Measure the diameter of the zones of inhibition to the nearest 0,1 mm. Record the

mean measurements for each concentration on semi-logarithmic graph paper

showing the logarithm of the concentrations in relation to the diameters of the zones

of inhibition. Plot the best fit lines of both the standard solution and the extract, for

example as below

Determine the ‘best fit’ point for the standard lowest level (SL) using the formula

7 S1 + 4 S2 + S4 - 2 S8

(a) SL =

Determine the ‘best fit’ point for the standard highest level (SH) using the

formula

7 S8 + 4 S4 + S2 - 2 S1

(b) SH =

Similarly calculate the ‘best fit’ points for the extract lowest level (UL) and the

extract highest level (UH) by substituting u 1 , u 2 , u 4 and u 8 for s 1 , s 2 , s 4 and s 8 in the

above formulae.

Record the calculated SL and SH values on the same graph paper and join them to

give the ‘best fit’ line for the standard solution. Similarly record UL and UH and join

them to give the ‘best fit’ line for the extract.

In the absence of any interference the lines should be parallel. For practical

purposes the lines can be considered parallel if the values (SH-SL) and (UH-UL) do

not differ by more than 10% from their mean value.

If the lines are found to be non-parallel either u1 and s1 or u8 and s8 may be

discarded and SL, SH, UL and UH calculated, using the alternative formulae, to give

alternative ‘best fit’ lines

5 S1 + 2 S2 - S4

5 + 2 S4 - S8

OR S2

(a') SL =

5 S4 + 2 S2 - S1

5 + 2 S4 - S2

OR S8

(b') SH =

and similarly for UL and UH. The same criteria of parallelism should be satisfied.

The fact that the result has been calculated from three levels should be noted on the

final report.

When the lines are considered as being parallel, calculate the logarithm of the

relative activity (log A) by means of one of the following formulae, depending upon

whether three or four levels have been used for the assessment of parallelism.

For four levels

(c) Log A =

For three levels

(d) Log A =

or

(d') Log A =

Activity of sample extract = activity of relevant standard × A

(U 8 = s 8 × A)

If the relative activity is found to be outside the range of 0,5 to 2,0, then repeat the

assay making appropriate adjustments to the extract concentrations or, if this is not

possible, to the standard solutions. When the relative activity cannot be brought into

the required range, any result obtained must be considered as approximate and this

should be noted on the final report.

When the lines are considered as not being parallel, repeat the determination. If

parall el ism i s sti ll no t achieved, the d etermi nation must be considered as

unsatisfactory.

Express the result in milligrams of virginiamycin per kilogram of feedingstuff.

9. Repeatability

The difference between the results of two parallel determinations carried out on

the same sample by the same analyst should not exceed

-

2 mg/kg, in absolute value, for contents of virginiamycin up to 10 mg/kg,

-

20% related to the highest value for contents of 10 to 25 mg/kg,

-

5 mg/kg, in absolute value, for contents of 25 to 50 mg/kg,

-

10% related to the highest value for contents above 50 mg/kg.

Reagents

3.1.

25% (w/w) hydrochloric acid, d: 1,126 g/ml.

3.2 1. 28% (w/v) hydrochloric acid.

The concentration must be checked by titration using a sodium hydroxide solution

0,1 mol/litre in the presence of 0,1% (w/v) methyl red in 94% (v/v) ethanol. 10 ml =

Apparat

4.1

erlenmeyer flask ta’ 250ml b’standard ground glass joint u reflux

condenser

4.2

polarimeter jew saccharimeter

Pro`edura

5.1

Preparazzjoni tal-Kampjun

Farrak il-kampjun sakemm ikun biżżejjed fin biex jgħaddi kollu minn għarbiel

b’xibka b’toqob tondi ta’ 0.5mm.

5.2

Determinazzjoni tat-total optical rotation (P jew S) ara osservazzjoni

(7.1)

Iżen 2.5 grammi tal-kampjun midħun sa l-eqreb milligram u poggih ġo graduated

flask tal-100ml. Żid 25ml hydrochloric acid (3.2) u ħawwad biżżejjjed biex il-

kampjun jitħallat sew u erġa’ żid 25ml hydrochloric acid . Għaddas il-flask f’water

bath jagħli waqt li tħawwad bil-qawwa għall-ewwel tliet minuti biex tiġi evitata l-

formazzjoni ta’ l-agglomerati. Irid ikun hemm biżżejjed ilma fil-water bath biex

jibqa’ jagħli meta l-flask jitpoġġa fih. Il-flask m’għandux jitneħħa mil-banju waqt li

tkun qed tħawwad. Wara 15-il minuta bl-eżatt neħħi l-flask minn ġol-banju u żid

30ml ilma kiesaħ u kessaħ mill-ewwel sa 20ºC.

Żid 5ml Carrez Solution I (3.3) u ħawwad għal minuta. Wara żid 5ml Carrez

Solution II (3.4) u erġa’ ħawwad għal minuta. Żid l-ilma sal-marka ta’ 100ml,

ixxejkja u ffiltra. Jekk il-filtrat ikun għadu mdardar (li hu rari) irrepeti d-

determinazzjoni billi tuża kwantità akbar ta’ Carrez Solution I u II, per eżempju

10ml.

Kejjel l-optical rotation tas-solution f ’tubu ta’ 200mm bil-polarimeter jew

saccharimeter.

5.3

Determinazzjoni ta’ l-Optical Rotation (P jew S) ta’ Sustanzi li jinħallu

f ’40% Ethanol

Iżen 5 grammi mill-kampjun sa l-eqreb milligram u poggih f’graduated flask tal-

100 ml u żid xi 80ml ethanol (3.5) (ara osservazzjoni 7.2). Ħalli l-flask joqgħod għal

xi siegha f’temperatura ambjentali filwaqt li tħawwad bis-saħħa għal xi sitt darbiet

biex il-kampjun ittestjat jiġi mħallat sewwa ma’ l-ethanol. Żid sal-marka b’40%

ethanol, ħallat u iffiltrah. Iġbed b’pipetta 50ml mil-filtrat (=2.5g mill-kampjun) u

poggih f ’Erlenmeyer flask ta’ 250ml, żid 2.1ml hydrochloric acid u ħawwad bis-

saħħa.

Qabbad reflux condenser ma’ l-erlenmeyer flask u għaddas il-flask ġo banju

misħun jagħli. Wara eżattament 15-il minuta, neħħi l-erlenmeyer flask mill-banju,

laħalħu ġo ftit ilma kiesaħ, u kesħu sa 20ºC. Saffi permezz ta’ Carrez Solution I (3.3)

u Carrez Solution II (3.4) u żid l-ilma sal-volum meħtieġ, ħawwad, iffiltra u kejjel l-

optical rotation kif indikat fit-tieni u t-tielet paragrafu ta’ 5.2.

Kalkolu tar-Riżultati

Il-kontenut ta’ lamtu (%) huwa kalkulat kif imniżżel hawn taħt

Miżura bi polarimeter

2000(P - P')

Kontenut ta’ lamtu (%) =

ġ α ħ 20°

D

Fejn

P = Optical rotation totali fi angle degrees

P' = Optical rotation f’angle degrees tas-sustanza solubbli fi 40% (V/V) ethanol

ġ α ħ 20°

= optical rotation specifika tal-pure starch. Il-valuri numeri`i

konvenzjonalment a``ettati għall-fattur huma dawn li ġejjin

D

+ 185.9º: lamtu tar-ross

+ 185.4º: lamtu tal-patata

+ 184.6º: lamtu tal-qamħirrum

+ 182.7º: lamtu tal-qamħ

+ 181.5º: lamtu tax-xgħir

+ 181.3º: lamtu tal-ħafur

+ 184.0º: tipi oħra ta’ lamtu u taħlitiet ta’ lamtu f’għalf kompost.

6.2.

Miżuri bis-saccharimeter

(2N × 0,665) × (S - S')

6N × (S - S')

×

-

ġ α 20°

Kontenut ta’ lamtu (%) =

20°

ġ α ħ

ħ D

D

Fejn

S = optical rotation totali f’saccharimeter degrees

S' = optical rotation fi saccharimeter degrees tas-sustanzi solubbli fi 40% (V/V)

ethanol

N = piż (g) ta’ saccharose fi 100ml ta’ ilma u li jagħti optical rotation ta’ 100

saccharimeter degrees meta mkejjel permezz ta’ 200mm tube

16,29 g għal saccharimeters

26,00 g għal saccharimeters Ġermaniżi

20,00 g għal saccharimeters mħallta.

ġ α ħ 20°

= specific optical rotation ta’ pure starch (ara 6.1)

D

6.3

Kif jiġi ripetut

Id-differenza bejn ir-riżultati ta’ żewġ determinazzjonijiet paralleli meħudin mill-

istess kampjun ma jridux je``edu 0.4 tal-valur assolut għall-kontenut tal-lamtu inqas

minn 40% u 1.1% relattivament għall-kontenut ta’ lamtu ta’ 40% jew aktar.

7. Osservazzjonijiet

7.1

Jekk il-kampjun ikun fih aktar minn 6% carbonates, ikkalkulati fis-sens

ta’ calcium carbonate dawn iridu jiġu distrutti b’trattament b’kwantità eżatta u

xierqa ta’ dilute sulphuric acid qabel ma tiġi mkejla t-total optical rotation.

7.2

Fil-każ ta’ prodotti li għandhom kontenut għoli ta’ lactose bħal trab tas-

serum tal-ħalib jew trab tal-ħalib xkumat imxi hekk

Wara li żżid 80ml ta’ ethanol (3.5) qabbad reflux condenser mal-flask u għadsu ġo

water bath f ’50ºC għal nofs siegħa. Ħallih jiksaħ u kompli l-analiżi skond il-

paragrafu 5.3.

7.3

F’każ fejn il-materjali tal-għalf segwenti jkunu preżenti f’ammonti kbar

f’għalf imhallat, jistgħu jkunu ta’ tfixkil fid-determinazzjoni tal-kontenut ta’ lamtu

bil-metodu polarimetriku u allura joħorġu riżultati inkorretti

-

prodotti ta’ sugar beet bħal (sugar) beet pulp, (sugar) beet molasses,

(sugar) beet pulp-molassed, (sugar) beet vinasse, (beet) zokkor,

-

polpa ta`-`itru,

-

kittien, expeller tal-kittien, extraxtor tal-kittien,

-

rape seed; rape seed expeller; rape seed extracted; rape seed hulls,

-

żerriegħa tal-ġirasol; żerriegħa tal-ġirasol estratti; żerriegħa tal-girasol,

parzjalment dekortikati u estratti,

-

copra expeller; copra extracted,

-

polpa tal-patata,

-

ħmira mnixxfa,

-

prodotti mimlijin fl-inulin (eż. chips u meal tal-qaqo`` ta’ Ġerusalemm);

-

greaves.

2. DETERMINAZZJONI TAL- PROTEINA KRUDA

1. Għan u Skop

Dan il-metodu jiddetermina l-kontenut tal-proteina kruda tal-għalf fuq il-bażi tal-

kontentut nitroġenu determinat skond il-metodu Kjeldahl.

2. Prinċipju

Il-kampjun jiġi iddiġerit mis-sulphuric acid fil-preżenza ta’ katalist. Is-soluzzjoni

a`iduża ssir alkalina b’soluzzjoni ta’ sodium hydroxide. L-ammonia tiġi distillata u

miġbura f ’kwantità mkejla ta’ sulphuric acid, iż-żejjed jgħaddi minn titration

f’soluzzjoni standard ta’ sodium hydroxide.

3. Reaġenti

3.1

Potassium sulfate.

3.2

Katalista: copper (II) oxide CuO jew copper (II) sulfate pentahydrate,

CuSO 4 5H 2 O

3.3

Żingu granulat.

Sulfuric acid, ρ 20 = 1,84 g/ml.

3.4.

3.5

Sulfuric acid c(½H 2 SO 4 ) = 0,5 mol/l.

3.6

Sulfuric acid c(½H 2 SO 4 ) = 0,1 mol/l.

Methyl red indicator: ħoll 300 mg methyl red ġo 100 ml ethanol, σ 95-96%

3.7

(v/v)

Soluzzjoni ta’ sodium hydroxide (Technical grade tista’ tintuża) ß = 40 g/

3.8

100 ml (m/v: 40%).

3.9

Soluzzjoni ta’ sodium hydroxide c = 0,25 ml/l.

3.10

Soluzzjoni ta’ sodium hydroxide c = 0,1 mol/+l.

3.11

Ħaffiefa granulata, maħsula fil- hydrochloric acid u maħruqa f’nar qawwi.

3.12

Acetanilide (m.p. = 114ºC, N = 10,36%)

3.13

Sucrose (bla nitroġenu).

4. Apparat

Dan l-apparat huwa adattat biex jitwettqu d-diġestjoni, id-distillazzjoni u t-

titration skond il-pro`edura Kjeldahl.

5. Proċedura

5.1

Diġestjoni

Iżen 1g tal-kampjun sa l-eqreb 0.001 g u poġġi l-kampjun ġol-flask ta’ l-apparat

tad-diġestjoni. Żid 15g potassium sulfate (3.1), kwantità xierqa ta’ katalizzatur (3.2)

(0.3 sa 0.4 gr copper II oxide jew 0.9g sa 1.2 g copper II sulfate pentahydrate, 25ml

sulphuric acid (3.4), u ftit granuli ta’ ħaffiefa (3.11) u ħallat kollox. Għall-ewwel

saħħan il-flask moderament, ħawwad minn ħin għall-ieħor sakemm it-taħlita

tikkarbonizza u r-ragħwa tkun għebet; wara saħħan fuq nar iktar qawwi sakemm

jibda jagħli sewwa. Ikun issaħħan biżżejjed jekk l-a`idu jagħli jibda jikkondensa

mal-ġnub tal-flask. Tħallix li l-ġnub jisħnu ż-żejjed u li l-parti`elli organi`i jeħlu

magħhom. Meta s-soluzzjoni ti``ara u ssir tixgħel ħadra, kompli għalli għal sagħtejn

u wara ħalliha tiksaħ.

5.2

Distillazzjoni

Żid attentament biżżejjed ilma biex tassigura li s-sulphates idubu kompletament.

Ħalli t-taħlita tiksaħ u żid ftit granuli taż-żingu (3.3).

Poġġi kwantità pre`iża sulphuric acid (3.5) jew (3.6) fil-collecting flask ta’ l-

apparat tad-distillazzjoni skond il-kontenut preżunt ta’ nitroġenu li hemm fih. Żid

ftit qtar ta’ methyl red indicator (3.7).

Għaqqad id-digestion flask mal-kondensatur ta’ l-apparat tad-distillazzjoni u

għarraq it-tarf tal-kondensatur fil-likwidu li hemm fil-collecting flask għal fond ta’

mill-inqas 1cm (ara osservazzjoni 8.3) Ferra bil-mod 100ml soluzzjoni ta’ sodium

hydroxide (3.8) fid-digestion flask mingħajr telf ta’ ammonia.

Saħħan il-flask sakemm l-ammonia kollha tiddistilla ruħha.

5.3

Titration

Għamel titration b’soluzzjoni ta’ sodium hydroxide (3.9) jew (3.10) tas-sulphuric

acid żejjed li hemm fil-collecting flask, skond il-kon`entrazzjoni ta’ sulphuric acid

użata, sakemm jidher il-qiegħ.

5.4

Blank test

Biex tikkonferma li r-reaġenti huma ħielsa min-nitroġenu għamel blank test

(diġestjoni, distillazzjoni u titration) billi tuża 1gr sucrose (3.13) minflok il-

kampjun.

6. Kalkolu tar-Riżultati

Il-kontenut tal-proteina kruda huwa ikkalkolat skond il-formula segwenti

(V 0 - V 1 ) × c × 0.014 × 100 × 6.25

m

Fejn

V 0 = volum (ml) ta’ NaOH (3.9 jew 3.10) li ġie użat fil-Blank test

V 1 = volum (ml) ta’ NaOH (3.9 jew 3.10) li ġie użat fit-titration tal-kampjun

c = Kon`entrazzjoni (mol/l) tas- sodium hydroxide (3.9 jew 3.10)

m = massa (gr) tal-kampjun

7. Verifikazzjoni tal-Metodu

7.1

Kif jiġi ripetut

Id-differenza bejn iż-żewġ riżultati meħuda fl-istess ħin ma tistax taqbeż

0.2% tal-valur assolut, fil-każ ta’ kontenut ta’ proteina kruda ta’ inqas

minn 20%;

1% relattiv għall-ogħla valur, fil-każ ta’ kontenut ta’ proteina kruda

minn 20% sa 40%;

0.4% tal-valur assolut, fil-każ ta’ kontenut ta’ proteina kruda ta’ iktar

minn 40%.

7.2

Eżattezza

Għamel l-analiżi (diġestjoni, distillazzjoni u titration) fuq 1.5gr sa 2gr acetanilide

(3.12) fil-preżenti ta’ 1gr sucrose (3.13); 1gr acetanilide jikkonsma 14.80ml

sulphuric acid (3.5). L-irkupru għandu jkun ta’ mill-inqas 99%.

8. Osservazzjonijiet

8.1

L-apparat jista’ jkun ta’ tip manwali, semi-awtomatiku jew awtomatiku.

Jekk l-apparat jeħtieg transference bejn id-diġestjoni u d-distillazzjoni, it-

trasferiment irid isir bla telf. Jekk il-flask ta’ l-apparat tad-distillazzjoni mhux

mgħammar b’dropping funnel, mill-ewwel żid is-sodium hydroxide qabel ma

tqabbad il-flask mal-condenser waqt li tferra l-likwidu minn mal-ġenb.

8.2

Jekk id-digest jagħqad, erġa’ ibda d-determinazzjoni billi tuża ammont

akbar ta’ sulphuric acid (3.4) milli kien spe`ifikat l-ewwel darba.

8.3

Fil-każ ta’ prodotti li għandhom kontenut ta’ nitroġenu baxx il-valur ta’

sulphuric acid (3.6) li jrid jitpoġġa fil-collecting flask jista’ jekk ikun hemm bżonn

jiġi mnaqqas għal 10ml sa 15ml u miżjud sa 25ml bl-ilma.

3. DETERMINAZZJONI TAL-PROTEINA KRUDA MAĦLULA BIL-

PEPSIN U L-HYDROCHLORIC ACID

1. Għan u Skop

Dan il-metodu jiddetermina l-frazzjoni ta’ proteina kruda maħlula bil-pepsin u

hydrochloric acid taħt kondizzjonijiet iddeterminati. Japplika għal materjali tal-

għalf kollha.

2. Prinċipju

Il-kampjun irid jissaħħan għal 48 siegħa f ’soluzzjoni ta’ Pepsin hydrochloride

f’40ºC. Is-sospensjoni trid tiġi ffiltrata u l-kontenut ta’ nitroġenu tal-filtrat irid jiġi

ddeterminat skond il-metodu għad-determinazzjoni tal-proteina kruda.

3. Reaġenti

3.1

Hydrochloric acid, d: 1:25

3.2

hydrochloric acid 0.075 N

3.3

2.0 U/mg Pepsin: l-attività tal-Pepsin hi definita fil-metodu deskritt fil-

Parti 4 ta’ din l-Iskeda

3.4

Xi 2% w/v soluzzjoni ppreparata friska ta’ Pepsin fil-hydrochloric acid

(3.2); attività 400U/l.

3.5

anti-foaming emulsion

3.6

Ir-reaġenti kollha fil-lista taħt 3 fil-metodu tad-determinazzjoni tal-

proteina kruda.

4. Apparat

4.1

Water bath jew inkubatur, issettjat fuq 40ºC ± 1ºC,

4.2

apparat tal-Kjeldahl digestion u distillation.

5. Proċedura

5.1

Preparazzjoni tas-soluzzjoni (ara osservazzjoni 7.2)

Iżen 2gr tal-kampjun sa l-eqreb mg u poġġih ġo graduated flask tal-500ml. Żid

450ml ta’ soluzzjoni ta’ Pepsin hydrochloride (3.4) li qabel tkun issaħnet sa 40ºC u

ixxejkja biex ma jkunx hemm mil-formazzjoni ta’ agglomerate. Ara li l-pH tas-

sospensjoni tkun inqas minn 1.7. Poġġi l-flask ġo water bath jew inkubatur u ħallih

hemm għal 48 siegħa. Ixxejkja wara 8, 24 u 32 siegħa. Wara 48 siegħa, żid 15ml

hydrochloric acid (3.1), mkessaħ sa 20ºC , żid sal-marka bl-ilma u iffiltrah.

5.2

Diġestjoni

Ħu 250ml tal-filtrat u poġġih fil-flask ta’ l-apparat tad-distillazzjoni (4.2). Żid ir-

reaġenti meħtieġa għad-diġestjoni indikati fit-tieni sentenza ta’ 5.1 tal-metodu għad-

determinazzjoni tal-proteina kruda. Omoġenizzah u għallih. Jekk tifforma xi ragħwa,

żid xi qtar ta’ anti-foaming emulsion (3.5). Kompli għallih bil-qawwa sakemm kważi

l-ilma kollu jkun evapora. Naqqas in-nar u elimina b’attenzjoni l-aħħar tra``i ta’

ilma.

Meta s-soluzzjoni ti``ara u tkun bla kulur (jew aħdar `ar jekk intuża katalista

bbażat fuq i`-`omb), kompli għallih għal siegħa oħra. Ħalliha tiksaħ.

5.3

Distillazzjoni u titration

Ippro`edi kif indikat f’5.2 u 5.3 tal-metodu tal-proteina kruda.

5.4

Blank test

Għamel Blank test bl-istess pro`edura iżda mingħajr il-kampjun għall-analiżi.

5. Kalkolu tar-Riżultati

Naqqas il-volum ta’ sulphuric acid ikkunsmat fil-Blank test minn dak ikkunsmat

mill-kampjun tat-test. 1ml sulphuric acid 0.1N jikkorrispondu għal 1.4mg nitroġenu.

Immoltiplika l-kwantità ta’ nitroġenu bil-fattur 6.25. Esprimi r-riżultat bħala fattur

ta’ l-eżempju.

6. Kif jiġi ripetut

Id-differenza bejn ir-riżultati taż-żewġ determinazzjoniiet paralleli magħmula fuq

l-istess kampjun ma jistgħux je``edu

-

0.4%, valur assolut, fil-każ ta’ kontenut inqas minn 20%;

-

2.0%, valur relattiv, fil-każ ta’ kontenut ta’ mhux inqas minn 20% u

mhux inqas minn 40%;

-

0.8, valur assolut, fil-każ ta’ kontenut ta’ iktar minn 40%

7. Osservazzjonijiet

7.1

Il-valuri miksubin b’dan il-metodu m’għandhom ebda konnessjoni diretta

mad-digestibilità in vivo.

7.2

Prodotti b’kontenut ta’ żejt jew xaħam ogħla minn 10% iridu l-ewwel

jgħaddu minn pro`ess ta’ defatting permezz ta’ estrazzjoni bil-petroleum ether. (B.P.

40 sa 60ºC )

4. ESTIMAZZJONI TA’ PEPSIN ACTIVITY

1. Għan u Skop

Dan il-metodu jistabbilixxi l-attività tal-Pepsin użata fid-determinazzjoni tal-

proteina kruda maħlula bil-Pepsin u l-hydrochloric acid.

2. Prinċipju

Il-haemoglobin tiġi ttrattata bil-Pepsin ġo hydrochloric acid medium taħt

kondizzjonijiet definiti. Il-non-hydrolyzed fraction tal-Pepsin tiġi pre`ipitata ġo

trichloroacetic acid. Reaġenti ta’ Sodium hydroxide u Folin-Ciocalteu huma miżjuda

mal-filtrat. L-optical density tas-soluzzjoni hija mkejla f ’750nm u l-kwantità

korrispondenti ta’ tyrosine tinqara minn calibration curve.

Definizzjoni: L-unità tal-Pepsin hija definita bħala l-kwantità ta’ dak l-enżima li,

taħt il-kondizzjonijiet ta’ dan il-metodu, tillibera kull minuta, numru ta’ hydroxyl

groups li, meta jittebbgħu bil-Folin-Ciocaltue reagent, għandhom optical density li

tikkorrispondi ma’ umole tyrosine imtebba’ fl-istess mod.

3. Reaġenti

3.1

Hydrochloric acid 0.2 N

3.2

Hydrochloric acid 0.06 N

3.3

Hydrochloric acid 0.025 N

3.4

5% soluzzjoni (w/v) trichloroacetic acid

3.5

soluzzjoni ta’ sodium hydroxide 0.5N

3.6

Folin-Ciocalteu reagent. Poġġi 100gr sodium tunsgate (Na 2 WO 4 . 2H 2 O),

25gr sodium molybdate (Na 2 MoO 4 . 2H 2 O) u 700 ml ilma ġo flask bil-qiegħ tond ta’

2 litri ffittjat b’standard ground glass joint. Żid 50ml phosphoric acid (d:1.71) u

100ml concentrated hydrochloric acid (d:1.19), qabbad reflux condenser mal-flask,

għallih u żomm is-soluzzjoni tagħli bil-mod għal 10 sigħat. Ħalliha tiksaħ, aqla’ r-

reflux condenser, żid 175gr lithium sulphate (Li 2 SO 4 . 2H 2 O), 50ml ilma u 1ml

bromine. Għallih għal 15-il minuta biex telimina bromine żejjed.

3.7

Soluzzjoni tal-haemoglobin: Iżen kwantità ta’ haemoglobin (xi 2gr

substratum tal-proteina ddeterminat skond Anson) li jikkorrispondu għal 354mg

nitroġenu * u poġġih ġo flask tal-200ml iffittjat b’standard ground glass joint. Żid ftit

ml hydrochloric acid (3.2), qabbad il-flask mal-vacuum pump u ixxejkja sakemm il-

haemoglobin tinħall kompletament. Itlaq il-vacuum u waqt li tkun qed tixxejkja, żid

hydrochloric acid (3.2) sa 100ml. Ippreparah eżatt qabel ma tuzah.

3.8

Standard tyrosine solution: Ħoll 181.2 mg tyrosine fil-hydrochloric acid

(3.1) u żid bl-istess a`idu sakemm ikollok 1l (stick solution). Ħu 20.0ml u ħallat sa

100 ml bil-hydrochloric acid (3.1). 1ml ta’ din is-soluzzjoni fih 0.2 µmole tyrosine.

4. Apparat

4.1

Water bath issetjat fuq 25ºC ± 0ꞏ1ºC fuq l-ultrathermostat

4.2

spectrophotometer

4.3

Kronometru, eżattezza: sekonda

4.4

pH-meter

5. Proċedura

5.1

Preparazzjoni tas-Soluzzjoni (ara osservazzjoni 12)

Ħoll 150mg pepsin f’100 ml hydrochloric acid (3.2). Permezz ta’ pipetta itfa’ 2ml

mis-soluzzjoni ġo graduated flask tal-50ml u imla sal-marka bil-hydrochloric acid

(3.3). Il-pH, meta tiġi eżaminata bil-pH meter trid tkun 1ꞏ6 ± 0ꞏ1. Għaddas il-flask

ġol-water bath.

5.2

Hydrolysis

Itfa b’pipetta 5ml soluzzjoni ta’haemoglobin (3.7) ġo test tube, saħħan sa 25ºC ġo

water bath (4.1), żid 1ml soluzzjoni tal-Pepsin meħuda minn 5.1 u ħawwad b’glass

rod għandha tarf wieħed eħxen mill-ieħor, b’qisu 10 daqqiet minn naħa għall-oħra.

Ħalli t-test tube ġol-water bath f ’25ºC għal għaxar minuti eżatti, ikkalkulati miż-

żieda tas-soluzzjoni tal-pepsin (durazzjoni u temperatura jridu jkunu osservati

attentament). Żid 10ml soluzzjoni ta’ trichloroacetic acid (3.4) li tkun qabel issaħnet

sa 25ºC, omoġenizza u għaddih minn filter niexef.

5.3

Żvilupp tal-Kulur u Tkejjil ta’ l-Optical density

Itfa b’pipetta 5.0 ml mill-filtrat ġo Erlenmeyer Flask tal-50ml, żid 10ml soluzzjoni

tas-sodium hydroxide (3.5), u waqt li tixxejkja mingħajr ma tieqaf, 3.0ml Folin-

Ciocalteu reagent imħallat (3.6). Wara 5 sa 10 minuti, determina l-optical density

*Iddetermina il-kontenut ta’ nitroġenu permezz tal-metodu semi-micro ta’ Kjeldahl (kontenut

teoretiku: 17.7% nitroġenu).

tas-soluzzjoni b’spectrophometer fuq 750nm f’`ellulli ħoxnin 1cm kontra l-ilma.

5.4

Blank test

Għal kull determinazzjoni, għamel Blank test f’dan il-mod

Itfa’ b’pipetta 5ml soluzzjoni tal-haemoglobin (3.7) ġo test tube, saħħan sa 25ºC

ġo water bath (4.1), żid 10ml soluzzjoni tat-trichloroacetic acid (3.4) li qabel tkun

issaħnet sa 25ºC, omoġenizza u żid 1.0ml soluzzjoni tal-Pepsin meħuda minn 5.1.

Ħawwad b’glass rod u ħalli t-test tube ġol-water bath (4.1) f ’25ºC għal għaxar

minuti eżatti. Omoġenizza u għaddi minn filter niexef. Imxi mal-pro`edura f’5.3

5.5

Calibration curve

Itfa’ 1.0, 2.0, 3.0, 4.0, 5.0 ml aliquots soluzzjoni tat-tyrosine standard (3.8) li

tikkorrispondi għal 0.2, 0.4, 0.6, 0.8 u 1.0 µmoles tat-tyrosine repettivament ġo

Erlenmeyer flasks tal-50ml. Ikkompleta s-serje b’reference solution mingħajr

tyrosine. Tella l-volumi sa 5.0ml bil-hydrochloric acid (3.1). Żid 10.0ml soluzzjoni

tas-sodium hydroxide (3.5) u filwaqt li tixxejkja mingħajr waqfien, 3.0ml Folin-

Ciocalteu reagent imħallat (3.6). Kejjel l-optical density kif indikat fl-aħħar

sentenza ta’ 5.3. Sib il-calibration curve billi tipplottja l-optical densities kontra l-

kwantitajiet tat-tyrosine.

6. Kalkolazzjoni tar-Riżultati

Mill-Calibration curve aqra l-kwantità tat-tyrosine, f ’µmoles, li tikkorrispondi

ma’ l-optical density tas-soluzzjoni ikkulurita, korretta fuq il-bażi tal-blank value.

L-attività tal-Pepsin, f ’µmoles ta’ tyrosine f ’25ºC, f ’mg u kull minuta, tiġi

kkalkulata permezz tal-formula

0ꞏ32a

Units għal kull mg (U/mg) =

P

Fejn

a = kwantità ta’ tyrosine, f’moles, moqrija mil-Calibration curve;

p = massa f’mg tal-kwantità ta’ Pepsin miżjuda f’5.2

7. Osservazzjonijiet

7.1

Il-kwantità ta’ Pepsin li trid tinħall trid tkun tali li, fit-tkejjil fotometriku

ta’ l-aħħar, optical density ta’ 0.35 +/- 0.035 tiġi miksuba.

7.2

Żewġ units għal kull mg meħuda b’dan il-metodu jikkorispondu għal

3.64 Anson milliunits/mg (µmoles tyrosine/mg . min f’35.5ºC) jew

36 400 commercial units/g (µmoles tyrosine/g f’10 minuti f’35.5ºC).

DETERMINAZZJONI TAL-FREE AND TOTAL GOSSYPOL

1. Għan u Skop

Dan il-metodu jiddetermina l-livelli ta’ free gossypol, total gossypol u sustanzi

kimi`i oħra li huma relatati ma’ dawn fil-cottonseed, cottonseed meal u l-cottonseed

cakes fl-għalf kompost li fihom dawn is-sustanzi fejn iktar minn 20ppm huma

preżenti.

2. Prinċipju

Il-gossypol jiġi estratt fil-preżenti ta’ 3-aminopropane-1-ol, jew b’taħlita ta’

p ro p a n - 2 - o l u h e x a n e , g ħ a d - d e t e r m i n a z z j o n i t a l - f re e g o s s y p o l , j e w b i d -

dimethylformamide, għad-determinazzjoni tat-total gossypol. Il-gossypol jiġi

konvertit bl-anilina f’gossypol dianiline, li l-optical density tagħha hija mkejla bħala

440 nm.

3. Reaġenti

3.1

Taħlita ta’ propan-2-ol u hexane, ħallat 60 parti volum ta’ propan-2-ol

A.R. ma’ 40 parti volum ta’ n-hexane.

3.2

Solvent A: Poġġi qisu 500ml taħlita ta’ propan- 2-ol-hexane ġo graduated

flask tal-1l (3.1), 2ml 3-aminopropan-1-ol, 8ml glacial acetic acid u 50ml ilma. Żid

sal-marka bis-soluzzjoni tal-propan-2-ol-hexane (3.1) Dan ir-reaġent huwa stabbli

għal ġimgħa.

3.3

Solvent B: Itfa’ b’pipetta 3ml ta’ 3-aminopropan-1-ol u 10ml glacial

acetic acid fi graduated flask tal-100ml. Kessaħ sat-temperatura ambjentali u imla

sal-marka b’N, N-dimethylformamide. Dan ir-reaġent huwa stabbli għal gimgħa.

3.4

Anilina A.R.: Jekk l-optical density fil-blank test te``edi 0.022, iddistilla l-

anilina bit-trab taż-żingu, u armi l-ewwel u l-aħħar 10% tad-distillat. Jekk tinżamm

fil-friġġ ġo stoppered flask tal-ħġieġ kannella, dan ir-reaġent għandu jżomm għal

xhur.

3.5

Soluzzjoni Standard tal-gossypol A: Poġġi 27.9 mg gossypolacetate

f’graduated flask tal-250ml. Ħoll u imla sal-marka bis-solvent (3.2). Itfa’ b’pipetta

50ml minn din is-soluzzjoni ġo graduated flask tal-250ml u imla sal-marka b’solvent

A. Il-kon`entrazzjoni ta’ gossypol ta’ din is-soluzzjoni hi 0.02 mg għal kull ml.

Ħalliha toqgħod għal siegħa f’temperatura ambjentali qabel ma tużaha.

3.6

Soluzzjoni Standard tal-gossypol B: Poġġi 27.9 mg gossypol acetate fi

graduated flask tal-50 ml u imla sal-marka b’solvent A. Il-kon`entrazzjoni ta’

gossypol f’din is-soluzzjoni hija 0.5mg għal kull ml.

Is-soluzzjonijiet standard tal-gossypol A u B jibqgħu stabbli għal 24 siegħa jekk

dawn ma jarawx dawl.

4. Apparat

4.1

Mixer (tumbler): madwar 35 rpm.

4.2

Spectrophotometer.

5. Proċedura

5.1

Kampjun tat-test

L-ammont ta’ kampjun għat-test li jiġi użat jiddependi fuq il-kontenut tal-gossypol

preżunt fil-kampjun. Preferibilment għandu jintuża kampjun żgħir u parti aliquot

mil-filtrat relattivament żgħira, biex jiġi miksub biżżejjed gossypol biex ikun

possibbli tkejjil fotometriku pre`iż. Għad-determinazzjoni tal-free gossypol, fil-

cottonseed meal u l-cottonseed cake, il-kampjun għat-test ma għandux je``edi 1 gr;

fil-każ ta’ għalf kompost, jista’ jkun daqs 5 gr. Parti aliquot mill-filtrat ta’ 10 ml hija

adegwata f ’ħafna mill-każijiet; għandu jkun fiha 50 sa 100 µg gossypol. Għad-

determinazzjoni tat-total gossypol, il-kampjun għat-test għandu jkun bejn 0.5 u 5 gr.

Parti aliquot mill-filtrat fiha 40 to 200 µg gossypol.

L-analiżi għandha ssir f’temperatura ambjentali ta’ madwar 20ºC.

5.2

Determinazzjoni tal-free gossypol

Poġġi l-kampjun tat-test ġo ground-necked flask tal-250ml, bil-qiegħ tal-flask

miksi bil-ħġieġ imkisser. Permezz ta’ pipetta żid 50ml solvent A (3.2), poġġi stopper

fuq il-flask u ħallat għal siegħa bil-mixer. Iffiltrah b’filter niexef u iġbor il-filtrat ġo

round-necked flask żgħir. Waqt il-filtrazzjoni, għatti l-funnel b’watch glass. Itfa’

b’pipetta partijiet ta’ l-aliquot identi`i tal-filtrat li fihom 50 sa 100 µg gossypol

f’żewġ graduated flasks tal-25ml (A u B). Jekk ne`essarju, imla sal-marka ta’ 10ml

b’solvent A (3.2). Mbagħad imla l-kontenut ta’ flask A (3.2) sal-marka bit-taħlita tal-

propan-2-ol-hexane (3.1). Din is-soluzzjoni tiġi użata bħala soluzzjoni ta’ referenza

li magħha titkejjel is-soluzzjoni tal-kampjun.

Itfa’ b’pipetta 10ml solvent A (3.2) ġo żewġ graduated flasks oħra tal-25 ml (C u

D). Imla l-kontenut ta’ flask C sal-marka bit-taħlita tal-propanol-2-ol-hexane (3.1).

Din is-soluzzjoni tiġi użata bħala soluzzjoni ta’ referenza ma xiex titkejjel is-

soluzzjoni tal-blank test.

Żid 2ml anilina (3.4) ma’ żewġ flasks (D u B). Saħħan għal 30 minuta fuq water

bath jagħli biex jiżviluppa l-kulur. Kessaħ sat-temperatura ambjentali, żid sal-marka

bit-taħlita tal-propan-2-ol-hexane (3.1), omoġenizzaha u ħalliha toqgħod għal

siegħa.

Iddetermina l-optical density tas-soluzzjoni tal-blank test (D) billi tqabbilha mas-

soluzzjoni ta’ referenza (C) u l-optical density tas-soluzzjoni tal-kampjun (B) li

titqabbel mas-soluzzjoni ta’ referenza (A) , fil-spectrophotometer f’440nm b’`elluli

tal-ħġieġ ta’ 1cm.

Naqqas l-optical density tas-soluzzjoni tal-blank test minn dik tas-soluzzjoni tal-

kampjun (= corrected optical density). Minn dan il-valur ikkalkula l-kontenut tal-

free gossypol kif indikat f’6.

5.3

Determinazzjoni tat-total gossypol

Poġġi l-kampjun tat-test li fih 1 sa 5 mg gossypol ġo graduated flask tal-5-ml u żid

10ml solvent B (3.3). Fl-istess ħin, ipprepara blank test, u itfa’ 10 ml solvent B (3.3)

ġo graduated flask ieħor tal-50ml. Saħħan iż-żewġ flasks fuq water bath jagħli għal

30 minuta. Kessaħ sat-temperatura ambjentali u żid il-kontenut taż-żewġ flasks sal-

marka bit-taħlita tal-propan-2-ol-hexane (3.1). Omoġenizzah u ħallih joqgħod bejn

10 u 15-il minuta, iffiltra u iġbor il-filtrati fi ground-necked flasks.

Itfa’ b’pipetta 2ml tal-filtrat tal-kampjun ġo żewġ graduated flasks tal-25ml, u

2ml filtrate tal-blank test ġo żewġ graduated flasks oħra tal-25ml. Imla l-kontenut

ta’ flask minn kull serje sal-marka ta’ 25ml bit-taħlita tal-propan-2-ol-hexane (3.1).

Dawn is-soluzzjonijiet jintużaw bħala soluzzjonijiet ta’ referenza.

Żid 2ml anilina (3.4) f ’żewġ flasks oħra. Saħħanhom għal 30 minuta fuq waer

bath jagħli biex jiżviluppa l-kulur. Kessaħ sat-temperatura ambjentali, żid sal-marka

ta’ 25ml tat-taħlita tal-propan-2-ol-hexane, omoġenizzhom u ħallihom joqogħdu

għal siegħa.

Iddetermina l-optical density kif indikat f ’5.2 għal free gossypol. Minn dan il-

valur ikkalkula l-kontenut tat-total gossypol kif indikat f’6.

6. Kalkolu tar-Riżultati

Ir-riżultati jistgħu ikunu kkalkulati jew mil-optical density (6.1), jew b’referenza

għal Calibration Curve (6.2).

6.1

Mis-specific optical density

Is-specific optical densities, taħt il-kondizzjonijiet deskritti, ikunu dawn li ġejjin

1%

free gossypol: E

= 625

1cm

1%

total gossypol: E

= 600

1cm

Eꞏ1250

% gossypol =

1%

ꞏ p ꞏ

E

1cm a

fejn

E = corrected optical density, skond kif imniżżel fi 5.2;

p = test sample fi g;

a = aliquot part tal-filtrate f’ml.

6.2

Minn calibration curve

6.2.1

Free gossypol

Ipprepara żewġ serje ta’ ħames graduated flasks tal-25ml. Itfa’ b’pipetta aliquots

ta’ 2.0, 4.0, 6.0, 8.0 u 10.0 soluzzjoni tas-standard gossypol A (3.5) ġo kull serje ta’

flasks. Żid il-volum sal-marka ta’ 10ml b’solvent A (3.2). Ikkompleta kull serje bi

graduated flask b’10ml solvent A (3.2) (blank test).

Żid il-volum tal-flasks fl-ewwel serje (inkluż il-flask tal-blank test) sal-marka ta’

25ml bit-taħlita tal-propan-2-ol-hexane (3.1) (serje ta’ referenza). Żid 2ml anilina

(3.4) f ’żewġ flasks oħra. Saħħanhom għal 30 minuta fuq water bath jagħli biex

jiżviluppa l-kulur. Kessaħ sat-temperatura ambjentali, żid sal-marka ta’ 25ml tat-

taħlita tal-propan-2-ol-hexane, omoġenizzhom u ħallihom joqogħdu għal siegħa.

(serje standard).

Iddetermina kif indikat f’5.2 l-optical density tas-soluzzjonijiet fis-serje standard

mqabbla mas-soluzzjonijiet korrispondenti tas-serje ta’ referenza. Sib il-calibration

curve billi tipplottja l-optical densities kontra l-kwantitajiet ta’ gossypol. (f’µg)

6.2.2

Gossypol totali

Ipprepara 6 graduated flasks tal-50ml. Fl-ewwel flask poġġi 10ml solvent B (3.3),

u fl-oħrajn 2.0, 4.0, 6.0, 8.0, 10.0 ml soluzzjoni tas-standard gossypol (3.6)

respettivament. Żid il-kontenut ta’ kull flask sal-marka ta’ 10ml b’solvent B (3.3).

Saħħan għal 30 minuta fuq water bath jagħli. Kessaħ sat-temperatura ambjentali, żid

il-kontenut taż-żewġ flasks sal-marka bit-taħlita tal-propan-2-olhexane (3.1) u

omoġenizzah.

Itfa’ 2ml ta’ dawn is-soluzzjonijiet f’żewġ serje ta’ sitt graduated flasks tal-25ml.

Imla l-kontenut ta’ kull flask ta’ l-ewwel serje sal-marka bit-taħlita tal-propan-2-ol-

hexane (3.1). (soluzzjonijiet ta’ referenza.)

Żid 2ml anilina (3.4) f ’kull flask fit-tieni serje. Saħħanhom għal 30 minuta fuq

water bath jagħli. Kessaħ sat-temperatura ambjentali, żid sal-marka bit-taħlita tal-

propan-2-ol-hexane, omoġenizzhom u ħallihom joqogħdu għal siegħa (serje

standard)

Iddetermina kif indikat f’5.2 l-optical density tas-soluzzjonijiet fis-serje standard

kif imqabbla mas-soluzzjonijiet korrispondenti fis-serije ta’ referenza.

Sib il-calibration curve billi tipplottja l-optical densities kontra l-kwantitajiet ta’

gossypol. (f’µg).

6.3

Kif jiġi ripetut

Id-differenza bejn ir-riżultati ta’ żewġ determinazzjonijiet paralleli meħuda mill-

istess kampjun ma jistgħux je``edu

-

15%, valur relattiv, fil-każ ta’ kontenut ta’ gossypol ta’ inqas minn

500ppm;

-

75 ppm, valur relattiv, fil-każ ta’ kontenut ta’ mhux inqas minn 500ppm

u mhux iktar minn 750ppm;

-

10%, valur relattiv, fil-każ ta’ kontenut ta’ iktar minn 750ppm.

94 ml of NaOH 0.1 mol/lite.

3.3

Carrez solution I: dissolve 21,9 g of zinc acetate Zn (CH 3 COO) 2 2H 2 O

and 3 g of glacial acetic acid in water. Make up to 100 ml with water.

3.4

Carrez solution II: dissolve 10,6 g of potassium ferrocyanide [K 4

(Fe(CN) 6 ].3H 2 O in water. Make up to 100 ml with water.

3.5

40% (v/v) ethanol, d: 0,948 g/ml at 20ºC.

4. Apparatus

4.1.

250 ml erlenmeyer flask with standard ground-glass joint and with reflux

condenser.

4.2.

Polarimeter or saccharimeter.

5. Procedure

5.1.

Preparation of the sample

Crush the sample until it is fine enough for all of it to pass through a 0,5 mm

round-meshed sieve.

5.2.

Determination of the total optical rotation (P or S) (see observation 7.1)

Weigh 2,5 g of the crushed sample to the nearest mg and place in a 100 ml

graduated flask. Add 25 ml of hydrochloric acid (3.2), shake to obtain even

distribution of the test sample and add a further 25 ml of hydrochloric acid (3.2).

Immerse the flask in a boiling water bath shaking vigorously and steadily for the

first three minutes to prevent the formation of agglomerates. The quantity of water in

the water bath must be sufficient for the bath to remain at boiling point when the

flask is introduced into it. The flask must not be taken out of the bath whilst being

shaken. After exactly 15 minutes, remove from the bath, add 30 ml of cold water and

cool immediately to 20ºC.

Add 5 ml of Carrez solution I (3.3) and shake for one minute. Then add 5 ml of

Carrez solution II (3.4) and shake again for one minute. Make up to volume with

water, mix and filter. If the filtrate is not perfectly clear (which is rare), repeat the

determination using a larger quantity of Carrez solutions I and II, for example 10 ml.

Measure the optical rotation of the solution in a 200 mm tube with the polarimeter

or saccharimeter.

5.3.

Determination of the optical rotation (P' or S') of substances soluble in

40% ethanol

Weigh 5 g of the sample to the nearest mg, place in a 100 ml graduated flask and

add about 80 ml of ethanol (3.5) (see observation 7.2). Leave the flask to stand for 1

hour at room temperature; during this time, shake vigorously on six occasions so that

the test sample is thoroughly mixed with the ethanol. Make up to volume with

ethanol (3.5), mix and filter. Pipette 50 ml of the filtrate (= 2,5 g of the sample ) into

a 250 ml erlenmeyer flask, add 2,1 ml of hydrochloric acid (3.1) and shake

vigorously. Fit a reflux condenser to the erlenmeyer flask and immerse the latter in a

boiling water bath. After exactly 15 minutes, remove the erlenmeyer flask from the

bath, transfer the contents to a 100 ml graduated flask, rinsing with a little cold

water, and cool to 20ºC. Clarify using Carrez solutions I (3.3) and II (3.4), make up

to volume with water, mix, filter and measure the optical rotation as indicated in the

second and third paragraphs of 5.2.

6. Calculation of results

The starch content (%) is calculated as following

6.1.

Measurement by polarimeter

2000(P - P')

Starch content (%) =

[ α ] 20°

D

Where

P = total optical rotation in angle degrees

P' = optical rotation in angle degrees of the substances soluble in 40% (V/V)

ethanol

[ α ] 20°

= specific optical rotation of pure starch. The numerical values

conventionally accepted for factor are the following

D

+ 185.9º: rice starch

+ 185.4º: potato starch

+ 184.6º: maize starch

+ 182.7º: wheat starch

+ 181.5º: barley starch

+ 181.3º: oat starch

+ 184.0º: other types of starch and starch mixtures in compound

feedingstuffs

6.2.

Measurement by saccharimeter

(2N × 0,665) × (S - S')

26.6N × (S - S')

×

-

Starch content (%) =

20°

20°

[ α ]

[ α ] D

D

S = total optical rotation in saccharimeter degrees

S' = optical rotation in saccharimeter degrees of the substances soluble in 40% (V/

V) ethanol

N = weight (g) of saccharose in 100 ml of water yielding an optical rotation of 100

saccharimeter degrees when measured using a 200 mm tube

16,29 g for the French saccharimeters

26,00 g for the German saccharimeters

20,00 g for mixed saccharimeters.

[ α ] 20°

= specific optical rotation of pure starch (see 6.1)

D

6.3.

Repeatability

The difference between the results of two parallel determinations carried out on

the same sample must not exceed 0,4 in absolute value for a starch content lower

than 40% and 1,1% relative for starch contents equal or higher than 40%

7. Observations

7.1.

If the sample contains more than 6% of carbonates, calculated in terms of

calcium carbonate, they must be destroyed by treatment with an exactly appropriate

quantity of dilute sulphuric acid before determination of the total optical rotation.

7.2.

In the case of products with high lactose content, such as powdered milk

serum or skimmed milk powder, proceed as follows after adding 80 ml of ethanol

(3.5). Fit a reflux condenser to the flask and immerse the latter in a water bath at

50ºC for 30 minutes. Leave to cool and continue the analysis as indicated in 5.3.

7.3.

Following feed materials, in case they are present in significant amount in

feedingstuffs, are known to give rise to interferences when determining the starch

content by polarimetric method and whereby incorrect results could be yielded

-

(sugar) beet products such as (sugar) beet pulp, (sugar) beet molasses,

(sugar) beet pulp-molassed, (sugar) beet vinasse, (beet) sugar, citrus

pulp,

-

linseed; linseed expeller; linseed extracted,

-

rape seed; rape seed expeller; rape seed extracted; rape seed hulls,

sunflower seed; sunflower seed extracted; sunflower seed, partially

decorticated, extracted,

-

copra expeller; copra extracted,

-

potato pulp,

-

dehydrated yeast,

-

products rich in inulin (e.g. chips and meal of Jerusalem artichokes);

-

greaves.

2. DETERMINATION OF CRUDE PROTEIN

1. Purpose and scope

This method makes it possible to determine the crude protein content of

feedingstuffs on the basis of the nitrogen content, determined according to the

Kjeldahl method.

2. Principle

The sample is digested by sulfuric acid in the presence of a catalyst. The acid

solution is made alkaline with sodium hydroxide solution. The ammonia is distilled

and collected in a measured quantity of sulfuric acid, the excess of which is titrated

with a standard solution of sodium hydroxide.

3. Reagents

3.1

Potassium sulfate.

3.2

Catalyst: copper (II) oxide CuO or copper (II) sulfate pentahydrate, CuSO 4

ꞏ 5H 2 O

3.3

Granulated zinc.

Sulfuric acid, ρ 20 = 1,84 g/ml.

3.4.

3.5

Sulfuric acid c(½H 2 SO 4 ) = 0,5 mol/l.

3.6

Sulfuric acid c(½H 2 SO 4 ) = 0,1 mol/l.

3.7

Methyl red indicator; dissolve 300 mg of methyl red in 100 ml of ethanol,

σ 95-96% (v/v)

Sodium hydroxide solution (Technical grade may be used) ß = 40 g/100 ml

3.8

(m/v: 40%).

3.9

Sodium hydroxide solution c = 0,25 ml/l

3.10

Sodium hydroxide solution c = 0,1 mol/l.

3.11

Granulated pumice stone, washed in hydrochloric acid and ignited.

3.12

Acetanilide (m.p. = 114ºC, N = 10,36%)

3.13

Sucrose (nitrogen free).

4. Apparatus

Apparatus suitable for performing digestion, distillation and titration according to

the Kjeldahl procedure.

5. Procedure

5.1

Digestion

Weigh 1 g of the sample to the nearest 0,001 g and transfer the sample to the flask

of the digestion apparatus. Add 15 g of potassium sulfate (3.1.), an appropriate

quantity of catalyst (3.2) (0,3 to 0,4 g of copper (II) oxide or 0,9 to 1,2 g of copper

(II) sulfate pentahydrate), 25 ml of sulfuric acid (3.4) and a few granules of pumice

stone (3.11) and mix. Heat the flask moderately at first, swirling from time to time if

necessary until the mass has carbonized and the foam has disappeared[ then heat

more intensively until the liquid is boiling steadily. Heating is adequate if the boiling

acid condenses on the wall of the flask. Prevent the sides from becoming overheated

and organic particles from sticking to them. When the solution becomes clear and

light green continue to boil for another two hours, then leave to cool.

5.2

Distillation

Add carefully enough water to ensure complete dissolution of the sulfates. Allow

to cool and then add a few granules of zinc (3.3).

Place in the collecting flask of the distillation apparatus an exactly measures

quantity of 25 ml of sulfuric acid (3.5) or (3.6) depending on the presumed nitrogen

content. Add a few drops of methyl red indicator (3.7).

Connect the digestion flask to the condenser of the distillation apparatus and

immerse the end of the condenser in the liquid contained in the collecting flask to a

depth of at least 1 cm (see observation 8.3). Slowly pour 100 ml of sodium

hydroxide solution (3.8) into the digestion flask without loss of ammonia (see

observation 8.1).

Heat the flask until the ammonia has distilled over.

5.3

Titration

Titrate the excess sulfuric acid in the collecting flask with sodium hydroxide

solution (3.9) or (3.10) depending on the concentration of the sulfuric acid used,

until the end point is reached.

5.4

Blank test

To confirm that the reagents are free from nitrogen, carry out a blank test

(digestion, distillation and titration) using 1 g of sucrose (3.13) in place of the

sample.

6. Calculation of results

The content of crude protein is calculated according to the following formula

(V 0 - V 1 ) × c × 0.014 × 100 × 6.25

m

Where

V 0 = Volume (ml) of NaOH (3.9 or 3.10) used in the blank test.

V 1 = Volume (ml) of NaOH (3.9 or 3.10) used in the sample titration.

c = Concentration (mol/l) of sodium hydroxide (3.9 or 3.10).

m = Mass (g) of sample.

7. Verification of the method

7.1

Repeatability

The difference between the results of two parallel determinations carried out on

the same sample must not exceed

0,2% in absolute value, for crude protein contents of less than 20%;

1,0% relative to the higher value, for crude protein contents from 20%

to 40%;

0,4% in absolute value, for crude protein contents of more than 40%.

7.2

Accuracy

Carry out the analysis (digestion, distillation and titration) on 1,5 to 2,0 g of

acetanilide (3.12) in the presence of 1 g of sucrose (3.13); 1 g acetanilide consumes

14,80 ml of sulfuric acid (3.5). Recovery must be at least 99%.

8. Observations

8.1

Apparatus may be of the manual, semi-automatic or automatic type. If the

apparatus requires transference between the digestion and distillation steps, this

transfer must be carried out without loss. If the flask of the distillation apparatus is

not fitted with a dropping funnel, add the sodium hydroxide immediately before

connecting the flask to the condenser, pouring the liquid slowly down the side.

8.2

If the digest solidifies, recommence the determination using a larger

amount of sulfuric acid (3.4) than that specified above.

8.3

For products with a law nitrogen content, the volume of sulfuric acid (3.6)

to be placed in the collecting flask may be reduced, if necessary, to 10 or 15 ml and

make up to 25 ml with water.

3. DETERMINATION OF CRUDE PROTEIN DISSOLVED BY PEPSIN

AND HYDROCHLORIC ACID

1. Purpose and scope

This method makes it possible to determine the fraction of crude protein dissolved

by pepsin and hydrochloric acid under defined conditions. It is applicable to all

feedingstuffs.

2. Principle

The sample is heated for 48 hours at 40ºC in a solution of pepsin hydrochloride.

The suspension is filtered and the nitrogen content of the filtrate determined

according to the method for the determination of crude protein.

3. Reagents

3.1

Hydrochloric acid, d: 1ꞏ125.

3.2

Hydrochloric acid 0ꞏ075 N.

3.3

2ꞏ0 U/mg pepsin; pepsin activity is defined in the method described in Part

4 of this Annex and must be established according to that method.

3.4

About 0ꞏ2% (w/v) freshly prepared solution of pepsin in hydrochloric acid

(3.2); activity: 400 U/l.

3.5

Anti-foaming emulsion (eg. silicone).

3.6

All the reagents listed under 3 in the method for the determination of crude

protein.

4. Apparatus

4.1

Water bath or incubator, set at 40ºC ± 1ºC.

4.2

Kjeldahl digestion and distillation apparatus.

5. Procedure

5.1

Preparation of solution (see observation 7.2)

Weigh 2 g of the sample to the mearest mg and place in a 500 ml graduated flask.

Add 450 ml of pepsin hydrochloride solution (3.4) previously heated to 40ºC and

shake to prevent the formation of agglomerates. Check that the pH of the suspension

is less than 1ꞏ7. Place the flask in the water bath or incubator (4.1) and leave there

for 48 hours. Shake after 8, 24 and 32 hours. After 48 hours, add 15 ml of

hydrochloric acid (3.1), cool to 20ºC, make up to volume with water and filter.

5.2

Digestion

Take 250 ml of the filtrate and place in the flask of the distillation apparatus (4.2).

Add the reagents necessary for digestion indicated in the second sentence of 5.1 of

the method for the determination of crude protein. Homogenize and bring to the boil.

If any foam should form, add a few drops of anti-foaming emulsion (3.5). Continue

to boil vigorously until the water has been almost completely evaporated. Reduce the

heat and carefully eliminate the last traces of water.

When the solution becomes clear and colourless (or light green if a copper-based

catalyst is used), continue to boil for another hour. Leave to cool.

5.3

Distillation and titration

Proceed as indicated in 5.2 and 5.3 of the method for the determination of crude

protein.

5.4

Blank test

Carry out a blank test applying the same procedure but omitting the sample to be

analyzed.

6. Calculation of results

Subtract the volume of sulphuric acid consumed in the blank test from that

consumed by the test sample. 1 ml of sulphuric acid 0ꞏ1 N corresponds to 1ꞏ4 mg of

nitrogen.

Multiply the quantity of nitrogen by the factor 6ꞏ25. Express the result as a

percentage of the sample.

Repeatability

The difference between the results of two parallel determinations carried out on

the same sample must not exceed

-

0ꞏ4, in absolute value, for contents of less than 20%;

-

2ꞏ0%, in relative value, for contents of not less than 20% and not more

than 40%;

-

0ꞏ8, in absolute value, for contents of more than 40%.

7. Observations

7.1

The values obtained by this method have no direct connection with

digestibility in vivo.

7.2

Products with an oil or fat content exceeding 10% must first be defatted by

extraction with petroleum ether (B.P. 40 to 60ºC).

4. ESTIMATION OF PEPSIN ACTIVITY

1. Purpose and scope

This method makes it possible to established the activity of the pepsin used in the

determination of crude protein dissolved by pepsin and hydrochloric acid.

2. Principle

Haemoglobin is treated with pepsin in a hydrochloric acid medium under defined

c o n d i t i o n s . T h e n o n - h y d r o l y z e d f r a c t i o n o f t h e p r o t e i n i s p r e c i p i t at ed i n

trichloroacetic acid. Sodium hydroxide and Folin-Ciocalteu reagent are added to the

filtrate. The optical density of this solution is measured at 750 nm and the

corresponding quantity of tyrosine is read from a calibration curve.

Definition: The unit of pepsin is defined as being the quantity of that enzyme

which, under the conditions of the method, liberates per minute, a quantity of

hydroxyaryl groups which, when stained with the Folin-Ciocalteu reagent, has an

optical density corresponding to that of one µmole of tyrosine stained in the same

manner.

3. Reagents

3.1

Hydrochloric acid 0ꞏ2 N.

3.2

Hydrochloric acid 0ꞏ06 N.

3.3

Hydrochloric acid 0ꞏ025 N.

3.4

5% solution (w/v) of trichloroacetic acid.

3.5

Sodium hydroxide solution 0ꞏ5 N.

3.6

Folin-Ciocalteu reagent. Place 100 g of sodium tungstate (Na 2 WO 4 .

2H 2 O), 25 g of sodium molybdate (Na 2 MoO 4 . 2H 2 O) and 700 ml of water in a 2

litre round-bottomed flask fitted with a standard ground-glass joint. Add 50 ml of

phosphoric acid (d: 1ꞏ71) and 100 ml of concentrated hydrochloric acid (d: 1ꞏ19),

connect a reflux condenser to the flask, bring to the boil and keep the solution gently

boiling for 10 hours. Leave to cool, detach the reflux condenser, add 175 g of lithium

sulphate (Li 2 SO 4 . 2H 2 O), 50 ml of water and 1 ml of bromine. Boil for 15 minutes

to eliminate excess bromine.

Leave to cool, transfer the solution to a 1 litre graduated flask, make up to volume

with water, homogenize and filter. No greenish coloration must remain. Before use,

dilute 1 volume of the reagent with 2 volumes of water.

3.7

Haemoglobin solution: Weigh a quantity of haemoglobin (approx. 2 g of

protein substratum determined according to Anson) corresponding to 354 mg of

nitrogen * and place in a 200 ml flask fitted with a standard ground-glass joint. Add a

few ml of hydrochloric acid (3.2), connect the flask to the vacuum pump and shake

until the haemoglobin has completely dissolved. Release the vacuum and, while

shaking, add hydrochloric acid (3.2) to make up to 100 ml. Prepare immediately

before use.

3.8

Standard tyrosine solution: Dissolve 181ꞏ2 mg of tyrosine in the

hydrochloric acid (3.1) and make up to 1 litre with the same acid (stick solution).

Take 20ꞏ0 ml and dilute to 100 ml with hydrochloric acid (3.1). 1 ml of this solution

contain 0ꞏ2 µmole of tyrosine.

4. Apparatus

4.1

Water bath set at 25ºC ± 0ꞏ1ºC by ultrathermostat.

4.2

Spectrophotometer.

4.3

Chronometer, accuracy: 1 second.

4.4

pH-meter.

5. Procedure

5.1

Preparation of the solution (see observation 7.1)

Dissolve 150 mg of pepsin in 100 ml of hydrochloric acid (3.2). Pipette 2 ml of the

solution into a 50 ml graduated flask and make up to volume with hydrochloric acid

(3.3). The pH, checked with the pH-meter, must be 1ꞏ6 ± 0ꞏ1. Immerse the flask in

the water bath (4.1).

5.2

Hydrolysis

Pipette 5ꞏ0 ml of haemoglobin solution (3.7) into a test tube, heat to 25ºC in the

water bath (4.1), add 1ꞏ0 ml of the pepsin solution obtained in 5ꞏ1 and mix with a

glass rod thickened at one end, with about 10 back-and-forth movements. Leave the

*Determine the nitrogen content by the semi-micro Kjehdahl method (theoretical content:17.7 % of

nitrogen).

test tube in the water bath at 25ºC for exactly 10 minutes, timed from the addition of

the pepsin solution (duration and temperature must be strictly observed). Then add

10ꞏ0 ml of trichloroacetic acid solution (3.4) previously heated to 25ºC, homogenize

and filter through a dry filter.

5.3

Development of coloration and measurement of optical density

Pipette 5ꞏ0 ml of the filtrate into a 50 ml Erlenmeyer flask, add 10ꞏ0 ml of sodium

hydroxide solution (3.5) and, shaking constantly, 3ꞏ0 ml of dilute Folin-Ciocalteu

reagent (3.6). After 5 to 10 minutes, determine the optical density of the solution

with the spectrophotometer at 750 nm in cells 1 cm thick against water.

5.4

Blank test

For each determination, carry out a blank test as follows

Pipette 5ꞏ0 ml of haemoglobin solution (3.7) into a test tube, heat to 25ºC in the

water bath (4.1), add 10ꞏ0 ml of trichloroacetic acid solution (3.4) previously heated

to 25ºC, homogenize, then add 1.0 ml of the pepsin solution obtained in 5.1. Mix

with a glass rod and leave the test tube in the water bath (4.1) at 25ºC for exactly 10

minutes. Homogenize and filter through a dry filter. Follow the procedure indicated

in 5.3.

5.5

Calibration curve

Place 1ꞏ0, 2ꞏ0, 3ꞏ0, 4ꞏ0 and 5ꞏ0 ml aliquots of standard tyrosine solution (3.8),

corresponding to 0ꞏ2, 0ꞏ4, 0ꞏ6, 0ꞏ8 and 1ꞏ0 µmoles of tyrosine respectively in 50 ml

Erlenmeyer flasks. Complete the series with a reference solution free from tyrosine.

Make up the volumes to 5ꞏ0 ml with hydrochloric acid (3.1). Add 10ꞏ0 ml of sodium

hydroxide solution (3.5) and, shaking constantly, 3ꞏ0 ml of dilute Folin-Ciocalteu

reagent (3.6). Measure the optical density as indicated in the last sentence of 5.3.

Trace the calibration curve by plotting the optical densities against the quantities of

tyrosine.

6. Calculation of results

From the calibration curve read the quantity of tyrosine, in µmoles, corresponding

to the optical density of the coloured solution, corrected on the basis of the blank

value.

The pepsin activity, in µmoles, of tyrosine at 25ºC, per mg and per minute, is

calculated by using the formula

0ꞏ32a

Units per mg (U/mg) =

P

Where

a = quantity of tyrosine, in µmoles, read from the calibration curve;

p = weight in mg of the quantity of pepsin added in 5.2.

7. Observations

7.1

The quantity of pepsin to be dissolved must be such that, on final

photometric measurement, an optical density of 0ꞏ35 ± 0ꞏ035 is obtained.

7.2

Two units per mg obtained by this method correspond to

3ꞏ64 Anson milliunits/mg (µmoles of tyrosine/mg ꞏ min at 35ꞏ5ºC) or

36 400 commercial units/g (µmoles of tyrosine/g in 10 min at 35ꞏ5ºC).

5. DETERMINATION OF FREE AND TOTAL GOSSYPOL

1. Purpose and scope

This method makes it possible to determine the levels of free gossypol, total

gossypol and chemically related substances in cottonseed, cottonseed meal and

cottonseed cake and in compound feedingstuffs containing these substances where

more than 20 ppm are present.

2. Principle

The gossypol is extracted in the presence of 3-aminopropan-1-ol, either with a

mixture of propan-2-ol and hexane, for the determination of free gossypol, or with

dimethylformamide, for the determination of total gossypol. The gossypol is

converted by aniline into gossypoldianiline, the optical density of which is measured

at 440 nm.

3. Reagents

3.1

Propan-2-ol-hexane mixture: mix 60 parts by volume of propan-2-ol A.R.

with 40 parts by volume of n-hexane.

3.2

Solvent A: Place in a 1 litre graduated flask approximately 500 ml of

propan-2-ol-hexane mixture (3.1), 2 ml of 3-aminopropan-1-ol, 8 ml of glacial acetic

acid and 50 ml of water. Make up to volume with the propan-2-ol-hexane mixture

(3.1). This reagent is stable for one week.

3.3

Solvent B: Pipette 2 ml of 3-aminopropan-1-ol and 10 ml of glacial acetic

acid into a 100 ml graduated flask. Cool to room temperature and make up to volume

with N, Ndimethylformamide. This reagent is stable for one week.

3.4

Aniline A.R.: If the optical density in the blank test exceeds 0ꞏ022, distil

the aniline over zinc dust, discarding the first and last 10 % fractions of the distillate.

Refrigerated and stored in a brown, stoppered glass flask, this reagent will keep for

several months.

3.5

Standard gossypol solution A: Place 27ꞏ9 mg of gossypol acetate in a 250

ml graduated flask. Dissolve and make up to volume with solvent A (3.2). Pipette 50

ml of this solution into a 250 ml graduated flask and make up to volume with solvent

A. The gossypol concentration of this solution is 0ꞏ02 mg per ml. Leave to stand for

one hour at room temperature before use.

3.6

Standard gossypol solution B: Place 27ꞏ9 mg of gossypol acetate in a 50

ml graduated flask, Dissolve and make up to volume with solvent B (3.3). The

gossypol concentration of this solution is 0ꞏ5 mg per ml. Standard gossypol solutions

A and B will remain stable for 24 hours if protected from the light.

4. Apparatus

4.1

Mixer (tumbler): approximately 35 rpm.

4.2

Spectrophotometer.

5. Procedure

5.1

Test sample

The amount of test sample used depends on the presumed gossypol content of the

sample. It is preferable to work with a small test sample and a relatively large aliquot

part of the filtrate, so as to obtain sufficient gossypol for precise photometric

measurement to be possible. For the determination of free gossypol in cottonseed,

cottonseed meal and cottonseed cake, the test sample should not exceed 1 g; for

compound feedingstuffs, it may be as much as 5 g. A 10 ml aliquot part of filtrate is

suitable in most cases; it should contain 50 to 100 µg of gossypol. For the

determination of total gossypol, the test sample should be between 0ꞏ5 and 5 g, that a

2 ml aliquot part of filtrate will contain 40 to 200 µg of gossypol.

The analysis should be carried out at a room temperature of about 20ºC.

5.2

Determination of free gossypol

Place the test sample in a ground-necked 250 ml flask, the bottom of the flask

having been covered with crushed glass. Using a pipette, add 50 ml of solvent A

(3.2), stopper the flask and mix for one hour in the mixer. Filter through a dry filter

and collect the filtrate in a small ground-necked flask. During filtration, cover the

funnel with a watch glass.

Pipette identical aliquot parts of filtrate containing 50 to 100 µg of gossypol into

each of two 25 ml graduated flasks (A and B). If necessary, make up the volume to

10 ml with solvent A (3.2). Then make the contents of flask (A) up to volume with

the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference

solution against which to measure the sample solution.

Pipette 10 ml of solvent A (3.2) into each of two other 25 ml graduated flasks (C

and D). Make the contents of flask (C) up to volume with the propan-2-ol-hexane

mixture (3.1). This solution will be used as a reference solution against which to

measure the blank test solution.

Add 2 ml of aniline (3.4) to each of flasks (D) and (B). Heat for 30 minutes over a

boiling water bath to develop the colour. Cool to room temperature, make up to

volume with the propan-2-ol-hexane mixture (3.1), homogenize and leave to stand

for one hour.

Determine the optical density of the blank test solution (D) by comparison with

the reference solution (C), and the optical density of the sample solution (B) by

comparison with the reference solution (A), in the spectrophotometer at 440 nm

using 1 cm glass cells.

Subtract the optical density of the blank test solution from that of the sample

solution (= corrected optical density). From this value calculate the free gossypol

content as indicated in 6.

5.3

Determination of total gossypol

Place a test sample containing 1 to 5 mg of gossypol in a 50 ml graduated flask

and add 10 ml of solvent B (3.3). At the same time, prepare a blank test, placing 10

ml of solvent B (3.3) in another 50 ml graduated flask. Heat the two flasks for 30

minutes over a boiling water bath. Cool to room temperature and make the contents

of each flask up to volume with the propan-2-ol-hexane mixture (3.1). Homogenize

and leave to settle for 10 to 15 minutes, then filter and collect the filtrates in ground-

necked flasks.

Pipette 2 ml of the sample filtrate into each of two 25 ml graduated flasks, and 2

ml of the blank test filtrate into each of two other 25 ml flasks. Make the contents of

one flask from each series up to 25 ml with the propan-2-ol-hexane mixture (3.1).

These solutions will be used as reference solutions.

Add 2 ml of aniline (3.4) to each of the other two flasks. Heat for 30 minutes over

a boiling water bath to develop the colour. Cool to room temperature, make up to 25

ml with the propan-2-ol-hexane mixture (3.1), homogenize and leave to stand for

one hour.

Determine the optical density as indicated in 5.2 for free gossypol. From this value

calculate the total gossypol content as indicated in 6.

6. Calculation of results

Results may be calculated either from the specific optical density (6.1), or by

reference to a calibration curve (6.2).

6.1

From the specific optical density

The specific optical densities, under the conditions described, will be the

following

1%

free gossypol: E

= 625

1cm

1%

total gossypol: E

= 600

1cm

Eꞏ1250

% gossypol =

1%

ꞏ p ꞏ

E

1cm a

where

E = corrected optical density, determined as indicated in 5.2;

p= test sample in g;

a = aliquot part of the filtrate in ml.

6.2

From a calibration curve

6.2.1

Free gossypol

Prepare 2 series of five 25 ml graduated flasks. Pipette aliquots of 2ꞏ0, 4ꞏ0, 6ꞏ0,

8ꞏ0 and 10ꞏ0 ml of standard gossypol solution A (3.5) into each series of flasks.

Make up the volumes to 10 ml with solvent A (3.2). Complete each series with a 25

ml graduated flask containing only 10 ml of solvent A (3.2) (blank test). Make the

volume of the flasks in the first series (including the flask for the blank test) up to 25

ml with the propan-2-ol-hexane mixture (3.1) (reference series).

Add 2 ml of aniline (3.4) to each flask in the second series (including the flask for

the blank test). Heat for 30 minutes over a boiling water bath to develop the colour.

Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture

(3.1), homogenize and leave to stand for one hour (standard series).

Determine as indicated in 5.2 the optical density of the solutions in the standard

series by comparison with the corresponding solutions in the reference series. Trace

the calibration curve by plotting the optical densities against the quantities of

gossypol (in µg).

6.2.2

Total gossypol

Prepare six 50 ml graduated flasks. In the first flask place 10 ml of solvent B (3.3),

and in the others 2ꞏ0, 4ꞏ0, 6ꞏ0, 8ꞏ0 and 10ꞏ0 ml of standard gossypol solution B (3.6)

respectively. Make the contents of each flask up to 10 ml with solvent B (3.3). Heat

for 30 minutes over a boiling water bath. Cool to room temperature, make up to

volume with the propan-2-ol-hexane mixture (3.1) and homogenize. Place 2.0 ml of

these solutions in each of two series of six 25 ml graduated flasks. Make the contents

of the flasks in the first series up to 25 ml with the propan-2-ol-hexane mixture (3.1)

(reference series).

Add 2 ml of aniline (3.4) to each flask in the second series. Heat for 30 minutes

over a boiling water bath. Cool to room temperature, make up to volume with the

propan-2-ol-hexane mixture (3.1), homogenize and leave to stand for one hour

(standard series).

Determine as indicated in 5.2 the optical density of the solutions in the standard

series by comparison with the corresponding solutions in the reference series. Trace

the calibration curve by plotting the optical densities against the quantities of

gossypol (in µg).

6.3

Repeatability

The difference between the results of two parallel determinations carried out on

the same sample must not exceed

-

15%, in relative value, for gossypol contents of less than 500 ppm;

-

75 ppm, in absolute value, for contents of not less than 500 ppm and not

more than 750 ppm;

-

10%, in relative value, for contents of more than 750 ppm.