S.I. No. 200/1984:

EUROPEAN COMMUNITIES (MARKETING OF FEEDINGSTUFFS) REGULATIONS, 1984.

EUROPEAN COMMUNITIES (MARKETING OF FEEDINGSTUFFS) REGULATIONS, 1984.

I, AUSTIN DEASY, Minister for Agriculture, in exercise of the powers conferred on me by section 3 of the European Communities Act, 1972 (No. 27 of 1972), and for the purpose of giving effect to Council Directive No. 77/101/EEC of 23 November, 1976 ¹ as amended in the manner specified in relation thereto in Regulation 2 of the following regulations, and Council Directive No. 79/373/EEC of 2 April, 1979 ² as amended in the manner specified in relation thereto in the said Regulation 2, hereby make the following regulations:

¹ O.J. No. L32/1, 3 February 1977.

² O.J. No. L86/30, 6 April 1979.

1. (1) These Regulations may be cited as the European Communities (Marketing of Feedingstuffs) Regulations, 1984.

(2) Those Regulations, other than Regulation 8 (1) (b) (vi) in so far as it requires to be made declarations of either lysine in compound feedingstuffs for swine or methionine and cystine in compound feedingstuffs for poultry, shall come into effect on the 1st day of August, 1985.

(3) Regulation 8 (1) (b) (vi) of these Regulations shall, in so far as it requires to be made the declarations referred to in paragraph (2) of this Regulation, come into effect on the 1st day of August, 1986.

2. (1) In these Regulations--

"authorised person" means a person appointed in writing by the Minister to be an authorised person for the purposes of these Regulations;

"the Directive of 1976" means Council Directive No. 77/101/EEC of 23 November, 1976 ¹, as amended by Council Directive No. 79/372/EEC of 2 April, 1979³, First Commission Directive No. 79/797/EEC of 10 August, 1979 ⁴, Second Commission Directive No. 80/510/EEC of 2 May, 1980 ⁵, Third Commission Directive No. 82/937/EEC of 21 December, 1982 ⁶, and Commission Directive No. 83/87/EEC of 21 February, 1983 ⁷;

¹ O.J. No. L32/1, 3 February 1977.

³ O.J. No. L86/29, 6 April 1979.

⁴ O.J. No. L239/53, 22 September 1979.

⁵ O.J. No. L126/12, 21 May 1980.

⁶ O.J. No. L383/11, 31 December 1982.

⁷ O.J. No. L56/31, 3 March 1983.

"the Directive of 1979" means Council Directive No. 79/373/EEC of 2 April, 1979 ², as amended by First Commission Directive No. 80/509/EEC of 2 May, 1980 ⁸ Commission Directive No. 80/511/EEC of 2 May, 1980 ⁹, Second Commission Directive No. 80/695/EEC of 27 June, 1980 ¹⁰, and Third Commission Directive No. 82/957/EEC of 22 December, 1982 ¹¹.

² O.J. No. L86/30, 6 April 1979.

⁸ O.J. No. L126/9, 21 May 1980.

⁹ O.J. No. L126/14, 21 May 1980.

¹⁰ O.J. No. L188/23, 22 July 1980.

¹¹ O.J. L386/42, 31 December 1982.

"the Minister" means the Minister for Agriculture;

"straight feedingstuffs" means the various vegetable and animal products in their natural state, fresh or preserved, and their derivatives after industrial processing, as well as the various organic and inorganic substances, intended for oral animal feeding;

"State Chemist" means the head of the State Laboratory or a person authorised by him in writing to perform the functions assigned to the State Chemist by these Regulations.

(2) A word or expression, other than "straight feedingstuff", that is used in these Regulations and is also used in the Directive of 1976 or the Directive of 1979 has the meaning in these Regulations that it has in the Directive of 1976 or the Directive of 1979, as may be appropriate.

(3) The Fertilisers, Feedingstuffs and Mineral Mixtures Regulations, 1957 ( S.I. No. 264 of 1957 ), other than Regulation 13 thereof, shall, in so far as they apply to feedingstuffs, straight feedingstuffs or compound feedingstuffs to which these Regulations apply, cease to have effect.

(4) Subsequent references in these Regulations to feedingstuffs, straight feedingstuffs or compound feedingstuffs shall, unless the context otherwise requires, each be construed as references to feedingstuffs to which these Regulations apply, or, in the case of references to straight feedingstuffs, references to straight feedingstuffs which are feedingstuffs to which these Regulations apply, or, in the case of references to compound feedingstuffs, references to compound feedingstuffs which are feedingstuffs to which these Regulations apply.

3. These Regulations shall apply to any feedingstuff other than--

( a ) a straight feedingstuff which is marked in such a way as to establish that it is intended for export,

( b ) any compound feedingstuff in respect of which--

(i) it is proved at least by some appropriate marking that it is intended for export,

or

(ii) it is proved in the manner specified in Article 14 (c) of the Directive of 1979 that it is intended for animals kept for scientific or experimental purposes,

( c ) wheat, barley, oats, hay, silage, roof crops or any other primary agricultural produce which is sold by the producer for his own benefit and on his own account.

4. Feedingstuffs (whether straight or compound) may be marketed only if they are wholesome, unadulterated and of merchantable quality, and they shall not represent a danger to animal or human health and shall neither be represented nor marketed in a manner liable to mislead.

5. A person shall not market a straight feedingstuff unless--

( a ) the requirements of Regulation 4 of these Regulations are complied with in relation to it,

( b ) it complies with each of the general provisions laid down in Part A of the Annex to the Directive of 1976, other than paragraphs 1.2, 2.5 and 2.6, and paragraph 1.3 in so far as it applies to feedingstuffs listed in items 3.2.8 of Part B of the said Annex,

( c ) where appropriate, the requirements of Article 11 of the Directive of 1976 are complied with in relation to it,

( d ) in case the feedingstuff is not a product or substance referred to in Article 14 (a) of the Directive of 1976, the content of ash insoluble in hydrochloric acid (HCl) does not exceed the maximum specified in column (6) of Part I of the First Schedule to these Regulations opposite the mention of the feedingstuff in column (2) of that Part,

( e ) in case the feedingstuff is a product or substance referred to in the said Article 14 (a) whose content of ash insoluble in hydrochloric acid (HCl) exceeds that specified in the said column (6) opposite the mention in the said column (2) of the feedingstuff and it is marketed to a person who for the time being holds a licence granted under the Fertilisers, Feeding Stuffs and Mineral Mixtures Regulations, 1957 ( S.I. No. 264 of 1957 ), the content of such ash is declared on the package or container or on a label attached thereto or on an accompanying document, and

( f ) in case the feedingstuff is a feedingstuff specified in column (2) of Part I of the First Schedule to these Regulations opposite a reference number in column (1) of that Part--

(i) it is marketed under, and only under, the word or words used as its name in the said column (2), and

(ii) if it has undergone a process which is not indicated by name, the requirements of paragraph 1.1 of Part A of the Annex to the Directive of 1976 are complied with in relation to it, and

(iii) it corresponds to the description set out in column (3) of the said Part I opposite that reference number.

6. (1) A person shall not market a compound feedingstuff unless--

( a ) it is in a sealed package or container, and

( b ) such package or container is sealed in such a way that, when the package or container is opened, the seal is damaged and cannot be reused, and

( c ) each of the requirements specified in paragraph (2) of this Regulation, or where appropriate, each of such requirements as apply to it, is complied with.

(2) The following are the requirements referred to in paragraph (1) of this Regulation--

( a ) the requirements of Regulation 4 of these regulations shall be complied with in relation to the feedingstuff concerned,

( b ) such feedingstuff shall comply with the provisions laid down in points 1 and 2 of the Annex to the Directive of 1979,

( c ) such feedingstuff shall not contain, as an ingredient, any substance specified in Part II of the First Schedule to these Regulations,

( d ) where appropriate, the requirements of Article 11 of the Directive of 1979 are complied with in relation to it,

( e ) the level in such feedingstuff of ash insoluble in hydrochloric acid (HCl) shall not exceed 3.3% of the dry matter in case the feedingstuff is composed mainly of rice by-products, or 2.2% of the dry matter in case the feedingstuff is not so composed:

Provided that the aforesaid levels may be exceeded in, but only in, the following circumstances:

(i) if the relevant feedingstuff is a compound feedingstuff containing mineral binding agents authorised under the European Communities (Feeding Stuffs) (Additives) Regulations, 1974 to 1983, or

(ii) if such feedingstuff is a mineral compound feedingstuff, or

(iii) if such feedingstuff is a compound feedingstuff containing more than 50% of sugar beet chips or pulp, and

(iv) where the level in any such feedingstuff of ash insoluble in hydrochloric acid (HCl) exceeds 3.3% of the dry matter, such level is declared as a percentage of the feedingstuff as such on the relevant package, container or label or on an accompanying document.

7. (1) Notwithstanding Regulation 6 of these Regulations but subject to Regulation 8 (2) (b) of these Regulations, compound feedingstuffs may be marketed in bulk or in unsealed packages or containers in the case of--

( a ) deliveries between producers of compound feedingstuffs,

( b ) deliveries from producers of compound feedingstuffs to packaging firms,

( c ) compound feedingstuffs obtained by mixing grain or whole fruit,

( d ) blocks or licks, or

( e ) quantities of compound feedingstuffs not exceeding 50 kilograms in weight which are intended for the final user and are taken directly from a package or container which before being opened complied with the said Regulation 6.

(2) Notwithstanding Regulation 6 of these Regulations, but subject to Regulation 8 (2) (b) of these Regulations, compound feedingstuffs may be marketed in bulk or in unsealed containers, but not in unsealed packages, in the case of--

( a ) compound feedingstuffs delivered directly from the producer to the final user,

( b ) molassed feedingstuffs consisting of not more than three ingredients, or

( c ) pelleted compound feedingstuffs.

8. (1) Subject to paragraphs (2) and (3) of this Regulation, feedingstuffs shall be marketed only if the following particulars, which shall be clearly visible, legible and indelible, are set out on the package or container or on a label attached thereto:

( a ) in case a feeding stuff is a straight feedingstuff--

(i) unless the feedingstuff is marketed to a person described in Regulation 5 (e) of these Regulations, the words "straight feedingstuff",

(ii) the particulars specified in column (4) of Part I of the First Schedule to these Regulations opposite where the name of the feedingstuff appears in column (2) thereof together with the words or word, as may be appropriate, used as its name in the said column (2),

(iii) the particulars specified in paragraphs (e) and (f) of Article 7 (1) of the Directive of 1976,

(iv) where appropriate, the particulars specified in paragraphs 1.1 and 2 of Part A of the Annex to the Directive of 1976 together with, in case the feedingstuff is a feedingstuff listed in item 2.9.2 of Part B of the said Annex, the particulars specified in paragraph 1.3 of the said Part A,

(v) in case the feedingstuff is a feedingstuff described in paragraph (e) of Regulation 5 of these Regulations, the declaration required by the said Regulation 5.

( b ) in case the feedingstuff is a compound feedingstuff--

(i) where the feedingstuff is a complete feedingstuff, the description "complete feedingstuff",

(ii) where the feedingstuff is a complementary feedingstuff--

(I) if it is a complementary mineral feedingstuff, the description "complementary mineral feedingstuff"

(II) if it is a complementary molassed feedingstuff, the description "complementary molassed feedingstuff"

and

(III) if it is neither a complementary mineral feedingstuff nor a complementary molassed feedingstuff, the description "complementary feedingstuff",

(iii) the particulars listed in paragraphs (b), (c), (d), (f) and (g) of Article 5 (1) of the Directive of 1979,

(iv) except in the case of feedingstuffs which are intended for pet animals, the date of manufacture,

(v) in case the feedingstuff is a milk replacer (suckling feed), the milk powder content,

(vi) the declarations listed in column (2) of part III of the First Schedule to these Regulations opposite the mention in column (1) of that Part of the relevant type of feedingstuff, and

(vii) the declaration required by Regulation 6 (1) of these Regulations and referred to in Regulation 6 (2) (e) (iv) of these Regulations:

Provided that:

(I) in case of small quantities of compound feedingstuffs intended for the final user, in lieu of complying with the requirements of subparagraph (b) of this paragraph, the particulars specified in that subparagraph may be brought to the purchasers' attention by means of an appropriate notice; and

(II) in case the feedingstuff is constituted from no more than three ingredients, the particulars referred to in subparagraph (b) and, where appropriate, either or both of the following, namely, subparagraphs (c) or (d) of the said Article 5 (1) shall not be required if the ingredients used in the feedingstuff appear clearly in a description of the feedingstuff contained in the aforesaid particulars.

(2) ( a ) In relation to straight feedingstuffs marketed in bulk, paragraph (1) of this Regulation shall be construed as requiring the particulars specified in that paragraph to be set out in an accompanying document.

( b ) In relation to compound feedingstuffs duly marketed in tankers or similar vehicles or otherwise in bulk, paragraph (1) of this Regulation shall be construed as requiring the particulars to be set out in an accompanying document.

(3) The declarations referred to in paragraph (1) (b) of this Regulation shall each comply with the requirements of point 1 of the Annex to the Directive of 1979.

9. (1) A person who markets a straight feedingstuff shall not put on the package, container or label thereof or on an accompanying document, in conjunction with any of the particulars set out in compliance with the requirements of Regulation 8 (1) of these Regulations, additional particulars other than the following, namely--

( a ) the particulars listed in Article 7 (5) of the Directive of 1976 (other than paragraph (g)),

and

( b ) the declarations listed in column (5) of Part I of the First Schedule to these Regulations opposite the mention in column (2) thereof of the relevant feedingstuff.

(2) A person who markets a compound feedingstuff shall not put on the package, container, label or accompanying document thereof in conjunction with the particulars set out in compliance with the requirements of Regulation 8 (1) (b) of these Regulations additional particulars other than--

( a ) the particulars listed in Article 5 (5) of the Directive of 1979,

( b ) the trade name of the product,

( c ) each of the ingredients in descending order of its proportion by weight,

( d ) the declarations listed in column (3) of Part III of the First Schedule to these Regulations opposite the mention in column (1) thereof of the relevant type of feedingstuff,

( e ) where it is appropriate, an indication that the compound feedingstuff conforms to a standard or standards regarding analytical characteristics which for the time being stands or stand recommended by the Minister in relation to a particular category or particular categories of compound feedingstuffs.

(3) Paragraph (3) of Regulation 8 of these Regulations shall apply to a declaration, other than a declaration referred to in paragraph (1) (b) of that Regulation, which pursuant to these Regulations is put on a package, container, label or accompanying document as it applies to a declaration so referred to.

10. ( a ) Where information referred to in Article 7 (7) of the Directive of 1976 appears on any packaging, container, label or accompanying document to which the said Article 7 (7) relates, the information shall be given in accordance with the requirements of that Article.

( b ) Where information referred to in Article 5 (8) of the Directive of 1979 appears on any packaging, container, label or accompanying document to which the said Article 5 (8) relates, the information shall be shown in accordance with the requirements of that Article.

11. (1) Where a person has on his premises any feedingstuff (whether straight or compound) which he has purchased and which he proposes to use in the course of his farming operations, he may apply to the Minister to have a sample thereof taken for analysis.

(2) An application under this Regulation shall be--

( a ) made within the period of thirty days beginning on the date on which the feedingstuff to which the application relates was delivered to the applicant, and

( b ) accompanied by a fee which shall be calculated by reference to the rates specified in the Second Schedule to these Regulations.

(3) Where an application is made under this Regulation, an authorised person shall, subject to paragraph (4) of this Regulation--

( a ) take and deal with a sample of the relevant feedingstuff according to the methods described in the Annex to Commission Directive No. 76/371/EEC of 1 March, 1976¹,

¹ O.J. No. L102/1, 15 April 1976.

and

( b ) give or cause to be given, or sent by registered post or by such other method as for the time being stands approved of for the purposes of this paragraph by the Minister, to the State Chemist and to the person whose name or trade name has been given pursuant to paragraph (f) of Article 7 (1) of the Directive of 1976, or, as may be appropriate, paragraph (f) of Article 5 (1) of the Directive of 1979, one of the final samples prepared pursuant to the requirements of subparagraph (a) of this paragraph.

(4) Where an application is made under this Regulation, an authorised person may, if he thinks fit, decline to take a sample if--

( a ) he is not satisfied that the applicant has purchased the feedingstuff to which the application relates,

or

( b ) he is not satisfied that the applicant proposes to use such feedingstuff in the course of his farming operations,

or

( c ) he is not satisfied that such feedingstuff as presented for sampling is fairly representative of the feedingstuff as delivered to the applicant,

or

( d ) the applicant does not furnish such information relating to such feedingstuff as the authorised person may reasonably require.

(5) Where the State Chemist receives a sample taken in pursuance of an application under this Regulation, he shall in making an analysis thereof comply with any of the requirements set out in Part I of the Third Schedule to these Regulations, as apply in the particular case and send to the applicant and to the person (other than the State Chemist) referred to in paragraph (3) (b) of this Regulation a certificate, in the form set out in Part II of the said Third Schedule, of the result of the analysis.

(6) Subject to paragraph (8) of this Regulation, all fees under this Regulation shall be paid into or disposed of for the benefit of the Exchequer in accordance with the directions of the Minister for Finance and, accordingly, the Public Offices Fees Act, 1879, shall not apply in respect thereof.

(7) Nothing in this Regulation shall be construed as requiring the State Chemist to make a test, examination or analysis regarding the presence in or absence from a sample given or sent to him pursuant to these Regulations of any particular substance, product or other thing, if in his opinion there is not in relation to such presence or absence a method of testing, examination or analysis which is sufficiently reliable or if there is not available to the State Chemist the apparatus or other means by which such a test, examination or analysis could be made.

(8) In any case in which he considers it proper so to do (not being a case in which the applicant has received a certificate under this Regulation), the Minister may refund a fee paid in relation to an application under this Regulation.

(9) For the purpose of this Regulation a feedingstuff shall not be regarded as having been delivered to a purchaser until it arrives at the destination to which it is consigned whether the consignment is by direction of the supplier or the purchaser.

12. Every person who carries on, or is employed in connection with, the business of manufacturing, importing or marketing feedingstuffs shall--

( a ) keep records of his transactions in such feedingstuffs;

( b ) produce at the request of an authorised person any books, documents or records relating to such business which are in the possession or under the control of such person;

( c ) permit an authorised person to inspect and take extracts from such books, documents or records and give to the person any information which he may reasonably require in relation to any entries therein;

( d ) afford to an authorised person reasonable facilities for inspecting the stock of any feedingstuff which is for the time being on any premises on which such person carries on such a business;

( e ) give to an authorised person any information he may reasonably require in relation to such transactions including in particular information which he may reasonably require regarding any feedingstuff which is specified by him.

13. Where an authorised person is satisfied that a feedingstuff which is placed on the market or which he believes will be placed on the market, does not comply with any one or more of the requirements of these Regulations, he may require either or both of the following persons, namely, the person who appears to him to have for the time being possession or control of the feedingstuff and the person whose name or trade name has been given pursuant to paragraph (f) of Article 7 (1) of the Directive of 1976, or, as may be appropriate, paragraph (f) of Article 5 (1) of the Directive of 1979, to take such steps as are necessary to ensure that it does not continue to be placed on the market, or, as may be appropriate, is not placed on the market until such authorised person is satisfied that the requirement is complied with.

14. In addition to the powers conferred by Regulation 12 of these Regulations an authorised person may at all reasonable times enter an inspect any premises or any railway wagon, vehicle, ship, vessel or aircraft, or other thing used to transport (e.g. a container), in which he has reasonable grounds for believing that any feedingstuff is being marketed, manufactured for sale, imported, stored or kept for sale, sold or transported and may examine and take samples of any feedingstuff which he finds in the course of his inspection.

15. (1) Where in any proceedings for an offence in which a contravention of these Regulation is alleged the defendant claims that the feedingstuff to which the alleged offence relates was exempted from these Regulations, by reason of paragraph (a) or (b) of Regulation 3 of these Regulations, the onus of proving that such feedingstuff was so exempted shall be on the defendant.

(2) In any proceedings for an offence under these Regulations, evidence of the result of any test, examination or analysis of, or of any report on, a sample taken under these Regulations may be given if, and only if, it is proved that--

( a ) the sample was taken and dealt with in accordance with the methods described in the Annex to Commission Directive No. 76/371/EEC of 1 March, 1976¹,

¹ O.J. No. L102/1, 15 April 1976.

( b ) before the proceedings were instituted one of the final samples prepared pursuant to the requirements of the said Annex was given or caused to be given to the defendant or sent or given to him by registered post or by such other method as stands approved of for the purposes of paragraph (b) of Regulation 11 (3) of these Regulations by the Minister, and

( c ) the test, examination or analysis was carried out in accordance with such of the requirements (if any) specified in Part I of the Third Schedule to these Regulations as applied in the particular case.

(3) In any legal proceedings, other than proceedings to which paragraph (2) of this Regulation applies, evidence, being evidence which relates to an issue regarding the accuracy of a declaration required to be made by these Regulations, of the result of any test, examination or analysis of a sample of a feedingstuff may be given if, and only if, it is proved that--

( a ) the sample was taken and dealt with in accordance with the methods referred to in paragraph (2) (a) of this Regulation,

( b ) before the proceedings were instituted one of the final samples prepared pursuant to the requirements of the Annex to Commission Directive No. 76/371/EEC of 1 March, 1976, was given or caused to be given to the defendant, or sent or given to him by registered post or by any other method which is a method referred to in paragraph (2) (b) of this Regulation, and

( c ) the test, examination or analysis was carried out in accordance with such of the requirements referred to in paragraph (2) (c) of this Regulation as applied in the particular case.

16. The Minister shall furnish an authorised person with a certificate of his appointment and, when exercising any powers conferred by these Regulations, the authorised person shall if requested by any person affected, produce the certificate to that person.

17. In any legal proceedings the production of a certificate in the form specified in Part II of the Third Schedule to these Regulations and purporting to be signed by the State Chemist shall, without proof of any signature on the certificate or that the signatory was the proper person to sign it, be sufficient evidence of the facts stated in the certificate and of the analysis to which it relates having been carried out in accordance with such of the requirements (if any) specified in Part I of the said Third Schedule as applied in the particular case.

18. (1) Any person who--

( a ) contravenes Regulation 5 or 6 of these Regulations,

( b ) in marketing a feedingstuff--

(i) contravenes Regulation 9 of these Regulations,

(ii) fails to comply with a requirement of Regulation 8 or 10 of these Regulations, or of the said Regulation 8, as applied by Regulation 9 (3) of these Regulations, other than a requirement as regards the disclosed composition of a feedingstuff,

( c ) contravenes any of the provisions of Regulation 12 of these Regulations,

( d ) fails to comply with a requirement made of him under Regulation 13 of these Regulations,

( e ) obstructs or interferes with an authorised person in the course of exercising a power conferred on him under Regulation 14 of these Regulations,

shall be guilty of an offence.

(2) Any person who in marketing a feedingstuff fails to comply with a requirement of Regulation 8 or 9 of these Regulations as regards the disclosed composition of a feedingstuff shall, subject to the limits of error specified in the Fourth Schedule to these Regulations, be guilty of an offence.

(3) If any person fraudulently--

( a ) tampers with any thing so as to procure that any sample taken pursuant to Regulation 11 or 14 of these Regulations does not correctly represent the substance sampled, or

( b ) tampers or interferes with any sample taken under these Regulations,

he shall be guilty of an offence.

(4) Any person guilty of an offence under these Regulations shall be liable on summary conviction to a fine not exceeding £500, or, at the discretion of the court, to imprisonment for a term not exceeding six months.

(5) An offence under these Regulations may be prosecuted by the Minister.

FIRST SCHEDULE

PART I

STRAIGHT FEEDINGSTUFFS

PART II

PROHIBITED STRAIGHT FEEDINGSTUFFS

Bagasse including molassed bagasse

Cocoa shells

Corozo nut meal

Dried poultry manure

Grape by-products from wine manufacture (grape pulp, grape follicle meal etc.)

Grass seed cleanings

Hoof or horn meal

Rice hulls

Shea nut meal or karite nut meal

Sal seed meal.

PART III

COMPOUND FEEDINGSTUFFS

(Contents of analytical constituents to be declared as a percentage of the weight of the compound feedingstuff as such)

SECOND SCHEDULE

Fees Referred to in Regulation 11 (2) (b).

THIRD SCHEDULE

PART I

1. GENERAL PROVISIONS ON METHODS OF ANALYSIS FOR FEEDINGSTUFFS.

2. MOISTURE.

2.1 Determination of moisture.

2.2 Determination of moisture in oilseeds and oleaginous fruit.

2.3 Determination of moisture in milk products.

2.4 Determination of moisture in animal and vegetable fats and oils.

3. CARBOHYDRATES.

3.1 Determination of crude fibre

3.2 Determination of starch

3.2.1 Polarimetric method

3.2.2 Pancreatic method.

3.3 Determination of sugar

3.4 Determination of lactose.

4. OILS AND FATS.

4.1 Determination of crude oils and fats.

4.2 Determination of oil content in oleaginous seeds.

4.3 Determination of acid index.

4.3.1 Indicator method

4.3.2 Potentiometric method

4.4 Determination of matter insoluble in light petroleum.

5. PROTEINS.

5.1 Determination of crude protein.

5.2 Determination of crude protein dissolved by pepsin and hydrochloric acid.

5.3 Estimation of pepsin activity.

5.4 Determination of milk powder.

5.5 Determination of volatile nitrogenous bases.

5.5.1 Microdiffusion method.

5.5.2 Distillation method.

6. MINERAL SUBSTANCES.

6.1 Determination of crude ash.

6.2 Determination of ash which is insoluble in hydrochloric acid.

6.3 Determination of water-soluble chlorides.

6.4 Determination of carbonates.

6.5 Determination of calcium.

6.6 Determination of magnesium.

6.6.1 atomic absorption method

6.7 Determination of total phosphorus.

6.7.1 spectrophotometric method.

6.8 Determination of potassium.

6.9 Determination of sodium.

7. MISCELLANEOUS

7.1 Determination of hectolitre weight

7.2 Determination of total solids.

7.3 Determination of the urease activity of products derived from soya beans.

7.4 Determination of vitamin A (Retinol).

1. GENERAL PROVISIONS ON METHODS OF ANALYSIS FOR

FEEDINGSTUFFS.

A. PREPARATION OF SAMPLES FOR ANALYSIS.

1. Purpose

The procedures described below concern the preparation for analysis of final samples, sent to the control laboratories after sampling in accordance with the provisions laid down by First Commission Directive 76/371/EEC of 1 March 1976 establishing Community methods of sampling for the official control of feedingstuffs¹.

¹ OJ No. L 102, 15 April 1976, p.1.

These samples must be prepared in such a way that the amounts weighed out, as provided for in the methods of analysis, are homogeneous and representative of the final samples.

2. Precautions to be taken

All the necessary operations must be performed in such a way as to avoid as far as possible contamination of the sample and changes of its composition. Grinding, mixing and sieving should be carried out as quickly as possible with minimal exposure of the sample to the air and light. Mills and grinders likely to appreciably heat the sample should not be used. Manual grinding is recommended for feedingstuffs which are particularly sensitive to heat. Care should also be taken to ensure that the apparatus itself is not a source of contamination of trace elements.

If the preparation cannot be carried out without significant changes in the moisture content of the sample, determine the moisture content before and after preparation according to the method laid down in method 2.1 of these regulations.

3. Procedure

Mix thoroughly the final sample either mechanically or manually. Divide the sample into two equal portions (the quartering method should be used where applicable). Keep one of the portions in a suitable clean, dry container, fitted with an air-tight stopper, and prepare the other portion or a representative part of it, of at least 100g, as indicated below.

3.1 Feedingstuffs which can be ground as such.

Unless otherwise specified in the methods of analysis, sieve the whole sample through a sieve with a square mesh of 1 mm size (in accordance with recommendation ISO R565) after grinding, if necessary. Avoid any overgrinding.

Mix the sieved sample and collect it in a suitable clean dry container fitted with an air-tight stopper. Mix again, immediately before weighing out the amount for analysis.

Feedingstuffs which can be ground after drying.

Unless otherwise specified in the methods of analysis, dry the sample to bring its moisture content down to a level of 8 to 12% according to the preliminary drying procedure described under point 4.3 of the method of determination of moisture mentioned in section 2 above. Then proceed as indicated in section 3.1.

3.3 Liquid or semi-liquid feedingstuffs.

Collect the sample in a suitable clean, dry container, fitted with an air-tight stopper. Mix thoroughly immediately before weighing out the amount for analysis.

3.4 Other feedingstuffs.

Samples which cannot be prepared according to one of the above procedures should be treated by any other procedure which ensures that the amounts weighed out for the analysis are homogeneous and representative of the final samples.

4. Storage of samples

Samples must be stored at a temperature that will not alter their composition. Samples intended for the analysis of vitamins or substances which are particularly sensitive to light should be stored in brown glass containers.

B. PROVISIONS RELATING TO REAGENTS AND APPARATUS USED IN METHODS OF ANALYSIS.

1. Unless otherwise specified in the methods of analysis, all analytical reagents must be analytically pure (a.p.). When determining trace elements the purity of the reagents must be checked by a blank test. Depending upon the results obtained, further purification of the reagents may be required.

2. Any operation involving preparation of solutions, dilution, rinsing or washing, mentioned in the methods of analysis without indication as to the nature of the solvent or dilutent employed, implies that water must be used. As a general rule, water should be demineralized or distilled. In particular cases, which are indicated in the methods of analysis, it must be submitted to special procedures of purification.

3. In view of the equipment normally found in control laboratories, only those instruments and apparatus which are special or require specific usage are referred to in the methods of analysis. They must be clean, especially when very small amounts of substances have to be determined.

C. APPLICATION OF METHODS OF ANALYSIS AND EXPRESSION OF THE RESULTS.

1. In general a single method of analysis is established for the determination of each substance in feedingstuffs. Where several methods are given, the particular method used by the control laboratory must be indicated on the analysis report.

2. The result given in the analysis report shall be the average value obtained from at least two determinations, carried out on separate portions of the sample, and of satisfactory repeatability.

This result shall be expressed in the manner laid down in the method of analysis to an appropriate number of significant figures and shall be corrected, if necessary, to the moisture content of the final sample prior to preparation.

2. MOISTURE.

2.1 DETERMINATION OF MOISTURE.

1. Purpose and scope

To determine the moisture content of feedingstuffs. It does not cover the analysis of milk products as straight feedingstuffs, the analysis of mineral substances and mixtures composed predominantly of mineral substances, the analysis of animal and vegetable fats and oils or the analysis of the oil seeds and oleaginous fruit defined in Council Regulation No. 136/66/EEC¹ of 22 September 1966 on the establishment of a common organisation of the market in oils and fats.

¹ OJ No. 127. 30 September 1966. P. 3025/66.

2. Principle

The sample is desiccated under specified conditions which vary according to the nature of the feeding stuffs. The loss in mass is determined by weighing. It is necessary to carry out preliminary drying when dealing with solid feeding stuffs which have a high moisture content.

3. Apparatus

3.1 Crusher of non moisture-absorbing material which is easy to clean, allows rapid, even crushing without producing any appreciable heating, prevents contact with the outside air as far as possible and meets the requirements laid down in 4.1.1 and 4.1.2 (e.g. hammer or water-cooled micro-crushers, collapsible cone mills, slow motion or cog-wheeled crushers).

3.2 Analytical balance, accurate to 0.5 mg.

3.3 Dry containers of non-corrodible metal or of glass with lids ensuring airtight closure; working surface allowing the test sample to be spread at about 0.3 g/cm²

3.4 Electrically heated isothermal oven (+1°C) properly ventilated and ensuring rapid temperature regulation.²

3.5 Adjustable electrically heated vacuum oven fitted with an oil pump and either a mechanism for introducing hot, dried air or a drying agent (e.g. calcium oxide).

3.6 Desiccator with a thick perforated metal or porcelain plate, containing an efficient drying agent.

4. Procedure

N.B. The operations described in this section must be carried out immediately after opening the packages of samples. Analysis must be carried out at least in duplicate.

4.1 Preparation

4.1.1 Feedingstuffs other than those coming under 4.1.2 and 4.1.3. Take at least 50g of the sample. If necessary, crush or divide in such a way as to avoid any variation in moisture content (see 6).

4.1.2 Cereals and groats

Take at least 50g of the sample. Grind into particles of which at least 50 per cent will pass through a 0.5 mm mesh sieve and will leave no more than 10 per cent reject on a 1 mm round-meshed sieve.

4.1.3 Feedingstuffs in liquid or paste form, feeding stuffs predominantly composed of oil.

Weigh, to the nearest 10 mg. approximately 25 g of the sample. Add an appropriate quantity of anhydrous sand weighed to the nearest 10 mg and mix until a homogeneous product is obtained.

4.2 Drying

4.2.1 Feedingstuffs other than those coming under 4.2.2 and 4.2.3.

Weigh, to the nearest 0.5 mg, a container (3.3) with its lid. Weigh into the weighed container, to the nearest mg, about 5 g of the sample and spread evenly. Place the container, without its lid, in the oven preheated to 103°C. To prevent the oven temperature from falling unduly introduce the container as rapidly as possible. Leave to dry for 4 hours reckoned from the time when the oven temperature returns to 103°C. Replace the lid on the container, remove the latter from the oven, leave to cool for 30-45 minutes in the desiccator (3.6) and weigh to the nearest mg.

For feeding stuffs composed predominantly of oil, dry in the oven for an additional 30 minutes at 130°C. Cool in a desiccator and weigh. The difference between the two weighings must not exceed 0.1 per cent of moisture.

²For the drying of cereals, flour, groats and meal, the oven must have a thermal capacity such that when pre-set at 131°C it will return to that temperature in less than 45 minutes after the maximum number of lest samples have been placed inside to dry simultaneously. Ventilation must be such that, when as many samples of common wheat as it can contain are dried for 2 hours, the results differ from those obtained after 4 hours of drying by less than 0.15%.

4.2.2 Cereals, flour, groats and meal.

Weigh, to the nearest 0.5 mg, a container (3.3) with its lid. Weigh into the weighed container, to the nearest mg, about 5 g of the crushed sample and spread evenly. Place the container, without its lid in the oven (3.4) preheated to 130°C. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible. Leave to dry for 2 hours reckoned from the time when the oven temperature returns to 130°C. Replace the lid on the container, remove the latter from the oven, leave to cool for 30-45 minutes in the desiccator (3.6) and weigh to the nearest mg.

4.2.3 Compound feedingstuffs containing more than 4 per cent of sucrose or lactose; straight feeding stuffs such as locust beans, hydrolized cereal products, maltculms, dried beet pulp, fish and sugar solubles; compound feeding stuffs containing more than 25 per cent of mineral salts including water of crystallisation.

Weigh, to the nearest 0.5 mg, a container (3.3) with its lid. Weigh into the weighed container, to the nearest mg, about 5 g of the sample and spread evenly. Place the container, without its lid, in the vacuum oven (3.5) preheated to between 80-85°C. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible.

Bring the pressure up to 100 Torr and leave to dry for 4 hours at this pressure, either in a current of hot, dry air or using a drying agent (about 300g for 20 samples). In the latter instance, disconnect the vacuum pump when the prescribed pressure has been reached. Reckon drying time from the moment when the oven temperature returns to 80-85°C. Carefully bring the oven back to atmospheric pressure. Open the oven, place the lid on the container immediately, remove the container from the oven, leave to cool for 30-45 minutes in the desiccator (3.6) and weigh to the nearest mg. Dry for an additional 30 minutes in the vacuum oven at 80-85°C and reweigh. The difference between the two weighings must not exceed 0.1 per cent of moisture.

4.3 Preliminary drying

4.3.1 Feeding stuffs other than those coming under 4.3.2.

Solid feedingstuffs with a high moisture content which makes crushing difficult must be subjected to preliminary drying as follows:

Weigh, to the nearest 10 mg, approximately 50 g of uncrushed sample (compressed or agglomerated feeding stuffs may be roughly divided if necessary) in a suitable container (e.g. a 20x12 cm aluminium plate with a 0.5 cm rim). Leave to dry in an oven from 60-70°C until the moisture content has been reduced to between 8-12 per cent. Remove from the oven, leave to cool uncovered in the laboratory for 1 hour and weigh to the nearest 10 mg. Crush immediately as indicated in 4.1.1 and dry as indicated in 4.2.1 or 4.2.3 according to the nature of the feeding stuff.

4.3.2 Cereals.

Grain with a moisture content of over 17 per cent must be subjected to preliminary drying as follows:

Weigh, to the nearest 10 mg, 50 g of unground grain in a suitable container (e.g. a 20x12 cm aluminium plate with a 0.5 cm rim). Leave to dry for 5-7 minutes in an oven at 130°C. Remove from the oven, leave to cool uncovered in the laboratory for 2 hours and weigh to the nearest 10 mg. Grind immediately as indicated in 4.1.2 and dry as indicated in 4.2.2.

5. Calculation of results

The moisture content, as a percentage of the sample, is calculated by using the following formulae:

5.1 Drying without preliminary drying

where: E=initial mass, in grams, of the test sample;

m=mass, in gramms, of the dry test sample.

5.2 Drying with preliminary drying

6. Repeatability

The difference between the results of two parallel determinations carried out on the same sample should not exceed 0.2% in absolute value.

7. Observation

If crushing proves necessary and if this is seen to alter the moisture content of the product, the results of the analysis of the components of the feeding stuff must be corrected on the basis of the moisture content of the sample in its initial state.

2.2 DETERMINATION OF MOISTURE IN OILSEEDS AND OLEAGINOUS FRUIT.

1. Purpose and Scope

To determine the moisture content and volatile matter in oil seeds and oleaginous fruit.

2. Principle

The sample is dried at 103°C ± 2°C. The loss in mass is determined by weighing.

3. Apparatus

3.1 Analytical balance.

3.2 Flat bottomed containers of non-corrodible metal with lids ensuring airtight closure, and allowing the test sample to be spread to about 0.2g/cm².

3.3 Electrically heated isothermal oven 103°C ± 2°C properly ventilated and ensuring rapid temperature regulation.

4. Procedure

4.1 Preparation

4.1.1 Copra.

Grate the sample wither by hand or mechanically and ensure representativity. The length of the particles may exceed 2 mm but should not be greater than 5 mm. Mix the particles carefully and carry out the determination without delay.

4.1.2 Seeds of medium size (e.g. groundnut, soya etc.) except sunflower seed and cotton seed with adherent linters.

Grind the sample in a mechanical mill fitted with a 2 mm sieve. Mix and carry out the determination without delay.

4.1.3 Small seeds (e.g. linseed, colza, hemp etc.) as well as safflower seed, sunflower seed and cotton seed with adherent linters.

These are analysed without previous grinding.

4.2 Drying.

Weigh to the nearest mg, a container (3.2) previously dried for 30 mins at 103°C ± 2°C and cooled in a desiccator.

Weigh into the container and to the nearest mg, 5 g±0.5 g of products coming under heading 4.1.1 and 4.1.2 and 5 to 10 g for products coming under heading 4.1.3.

Spread the material evenly over the whole base of the vessel and immediately place in the oven (3.3) previously heated to 103°C ± 2°C. Leave to dry for 3 hours (12 to sixteen hours for cotton seed with adherent linters) reckoned from the time the oven temperature returns to 103°C. Replace the lid on the container, remove from the oven, cool in a desiccator and weigh.

Return the uncovered container and lid to the oven for a further hour.

Replace the lid on the container, remove from the oven, cool in a desiccator and weigh.

Repeat the one hour drying operations until the difference between two successive weighings is equal to or less than 5 mg.

5. Calculation of results

The moisture content, as a percentage of the sample, is calculated by using the following formula.

6. Repeatability.

The difference between the results of two parallel determinations carried out on the same sample should not exceed 0.2% in absolute value.

2.3 DETERMINATION OF MOISTURE IN MILK PRODUCTS.

1. Purposes and scope

To determine the moisture content of milk products as straight feeding stuffs.

2. Principle

The sample is dried at 102°C ± 1°C. The loss in mass is determined by weighing.

3. Apparatus

3.1 Analytical balance.

3.2 Flat bottomed dishes of non-corrodible metal or of glass with lids ensuring airtight closure; Suitable dimensions are: diameter 60 to 80 mm and depth about 25 mm.

3.3 Electrically heated isothermal oven (±1°C) properly ventilated and ensuring rapid temperature regulation.

3.4 Desiccator, containing an efficient drying agent.

4. Procedure

Place dishes (3.2) in drying oven at 102°C (3.3) for approximately 60 minutes. Cool in a desiccator (3.4) and weigh accurately. Weigh to the nearest mg, approximately 2 g of sample into the dish. Transfer to oven (102°C) and leave sample uncovered for 2 hours. Replace the lid, transfer the covered dish to the desiccator, allow it to cool to room temperature and accurately weigh to the nearest 0.1 mg as quickly as possible.

Uncover the dish and heat it and its lid for one hour in the oven.

5. Calculation of results

The moisture content, as a percentage of the sample, is calculated by using the following formula:

2.4 DETERMINATION OF MOISTURE IN ANIMAL AND VEGETABLE FATS AND OILS.

1. Purpose and scope

To determine the moisture and volatile substances content of animal and vegetable fats and oils.

2. Principle

The sample is dried to constant weight at 103°C. The loss in mass is determined by weighing.

3. Apparatus

3.1 Flat-bottomed dish, of a corrosion-resistant material, 8-9 cm in diameter and approximately 3 cm high.

3.2 Mercury thermometer with a strengthened bulb and expansion tube at the top end, graduated from approximately 80°C to at least 110°C, and approximately 10 cm in length.

3.3 Sand bath or electric hot-plate.

3.4 Desiccator, containing an efficient drying agent.

3.5 Analytical balance.

4. Procedure

Weigh, to the nearest mg, approximately 20 g of the homogenized sample into the dry, weighed dish (3.1) containing the thermometer (3.2). Heat on the sand bath or hot-plate (3.3), stirring continuously with the thermometer, so that the temperature reaches 90°C in about 7 minutes.

Reduce the heat, watching the frequency with which bubbles rise from the bottom of the dish. The temperature must not exceed 105°C. Continue to stir, scraping the bottom of the dish, until bubbles stop forming.

In order to ensure complete elimination of moisture, reheat several times to 103°C ± 2°C, cooling to 93°C between successive heatings. Then leave to cool to room temperature in the desiccator (3.4) and weigh. Repeat this operation until the loss in mass between two successive weighings no longer exceeds 2 mg.

N.B. An increase in the mass of the sample after repeated heating indicates an oxidation of the fat, in which case calculate the result from the weighing carried out immediately before the mass began to increase.

5. Calculation of results

The moisture content, as a percentage of the sample, is given by the following formula:

6. Repeatability

The difference in moisture between the results of two parallel determinations carried out on the same sample must not exceed 0.05%, in absolute value.

3. CARBOHYDRATES.

3.1 DETERMINATION OF CRUDE FIBRE.

1. Purpose and scope

To determine, in feeding stuffs, the content of fat-free organic substances which are insoluble in acid and alkaline media and are conventionally described as crude fibre.

2. Principle

The sample, defatted where necessary, is treated successively with boiling solutions of sulphuric acid and potassium hydroxide of specified concentrations. The residue is separated by filtration in the presence of asbestos, washed, dried, weighed and ashed at 900°C. The loss of weight resulting from ashing corresponds to the crude fibre present in the test sample.

3. Reagents

3.1 Sulphuric acid, 0.26 N.

3.2 Treated asbestos: add to asbestos of the type used with a Gooch crucible approximately 5 times its weight of dilute hydrochloric acid (1 volume hydrochloric acid, d: 1.19+3 volumes of water). Boil the mixture for approximately 45 minutes, leave to cool and filter through a Buchner funnel. Wash the residue first with water until the washing water is free from acid, and then with acetone (3.6). Dry the asbestos in the drying oven and then ash for 2 hours at 900°C. Leave to cool and keep in a stoppered flask. Asbestos treated in this way may be used several times. It must meet the specifications given in 5 regarding the blank test.

3.3 Antifoam (e.g. silicone).

3.4 Potassium hydroxide solution 0.23 N.

3.5 Hydrochloric acid 0.5 N.

3.6 Acetone.

3.7 Light petroleum, boiling range 40-60°C.

4. Apparatus

4.1 Beakers of at least 600 ml capacity, with measuring marks at the 200 ml level.

4.2 Porcelain discs approximately 80 mm in diameter and approximately 4mm thick, perforated with approximately 32 holes, each approximately 4 mm in diameter.

4.3 Rubber-stoppered vacuum flasks of approximately 2 litre capacity, with measuring marks at the 800 ml level and fitted with glass funnels 120 mm in diameter.

4.4 Filter plates approximately 40 mm in diameter and approximately 4 mm thick, with slanting edges to fit the cone of the funnel (4.3), perforated with approximately 16 holes, each approximately 4 mm in diameter, and covered by a wire mesh, the mesh size being approximately 1 mm. Both plates and wire mesh must be resistant to acids and alkali.

4.5 Platinum or silica ashing crucibles.

4.6 Thermostatically controlled electric muffle-furnace.

4.7 Desiccator.

4.8 Asbestos filter: suspend 2.0 g asbestos (3.2) in 100 ml water. Filter under vacuum over the filter plate covered with a wire mesh (4.4) and placed in the funnel of a vacuum flask (4.3). Collect the filtrate and filter once more through the same filter. Discard the filtrate.

5. Procedure

Weigh, to the nearest mg approximately 3 g of the sample and 2 g treated asbestos (3.2) into a beaker (4.1), add 200 ml sulphuric acid (3.1) and a few drops of antifoam (3.3). Bring rapidly to the boil and leave to boil for exactly 30 minutes. To keep a constant volume, cover the beaker with a cooling device such as a 500 ml round-bottomed flask in which cold water is circulated. Stop boiling by adding approximately 50 ml cold water and filter immediately under vacuum through an asbestos filter previously prepared as shown in 4.8.

Wash the residue with 5 lots of approximately 100 ml of very hot water to obtain a final volume of filtrate of 800 ml. Transfer the residue quantitatively to the beaker (4.1) which has first been fitted with a porcelain disc (4.2) to regulate the boiling. Add 200 ml potassium hydroxide solution (3.4). Bring rapidly to the boil and leave to boil for exactly 30 minutes. Add approximately 50 ml cold water and filter immediately under vacuum through a fresh asbestos filter previously prepared as shown in 4.8. Wash the residue with very hot water until the washing water is neutral (test with litmus paper), then 3 times with acetone (3.6) (approximately 100 ml acetone in all).

Transfer the residue quantitatively to an ashing crucible (4.5), break up if necessary and dry to constant weight in the drying oven at 130°C.

Leave to cool in the desiccator (4.7) and weigh rapidly.

Place the crucible in the muffle-furnace (4.6) and leave to ash for 30 minutes at 900°C. Leave to cool in the desiccator (4.7) and weigh rapidly.

Carry out a blank test applying the same procedure to the treated asbestos (3.2), but without the sample. Loss of weight resulting from ashing of the 6 g asbestos must not exceed 10 mg.

6. Calculation of results

The crude fibre content, as a percentage of the sample, is given by the formula:

where:

a=loss of weight after ashing during the determination;

b=loss of weight after ashing during the blank test.

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed:

0.3%, in absolute value, for crude fibre contents less than 10%;

3%, relative to the higher result, for crude fibre contents equal to or greater than 10%.

8. Observations

8.1 Feeding stuffs containing more than 10% oil must be defatted prior to analysis with light petroleum (3.7). To do this, place the test sample (3 g weighed to the nearest mg) on an asbestos filter (4.8). Cover 3 times with approximately 50 ml light petroleum (3.7) and each time filter carefully under vacuum. Transfer the defatted test sample and the asbestos quantitatively to a beaker (4.1) and continue the analysis as shown in 5.

8.2 Feeding stuffs containing oil which cannot be extracted directly must be defatted as shown in 7.1 and defatted a further time after the acid attack has been washed from the residue.

To do this, wash the residue 3 times with acetone (3.6) (100 ml in all), then 3 times with 50 ml light petroleum (3.7). Then transfer the residue quantitatively to a beaker (4.1) and continue the analysis as shown in the second paragraph of 5 (treatment with potassium hydroxide solution).

8.3 If the feeding stuffs are rich in calcium (more than 2% calcium), place the test sample (3 g, weighed to the nearest mg) in a beaker (4.1) with 100 ml hydrochloric acid 0.5 N (3.5) and leave to stand in a cool temperature for 5 minutes. Filter immediately and wash in cold water. Use as a filtration aid the 2.0 g asbestos specified for boiling with sulphuric acid. If filtration proves difficult, dilute the suspension with acetone (3.6). Then proceed as shown in 5.

3.2 DETERMINATION OF STARCH.

3.2.1 Polarimetric method

1. Purpose and scope

To determine the levels of starch and of high molecular weight starch degradation products in feedingstuffs, with the exception of those feedingstuffs which contain beet chips, beet pulp, dried beet tops or leaves, potato pulp, dehydrated yeast, products rich in inulin (e.g. chips and meal of Jerusalem artichokes) or greaves.

2. 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.

3. Reagents

3.1 Hydrochloric acid, d : 1.126.

3.2 Hydrochloric acid solution, 1.128% (w/v).

The concentration must be checked by titration using a sodium hydroxide solution 0.1 N in the presence of 0.1% (w/v) methyl red in 94% (v/v) ethanol. 10 ml=30.94 ml of NaOH 0.1 N.

3.3 Carrez solution I: dissolve 21.9 g of zinc acetate Zn(CH₃COO)₂.2H₂O 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 K₄[Fe(CN)₆].3H₂O in water. Make up to 100 ml with water.

3.5 Ethanol solution, 40% (v/v).

4. Apparatus

4.1 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 item 7.1).

Weigh, to the nearest mg, 2.5 g of the crushed sample 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 20°C.

Add 5 ml of Carrez solution I (3.3) and shake for 1 minute. Then add 5 ml of Carrez solution II (3.4) and shake again for 1 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 (4.2).

5.3 Determination of the optical rotation (P' or S') of substances soluble in 40% ethanol.

Weigh, to the nearest mg. 5 g of the sample, 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 an 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 20°C.

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 2nd and 3rd paragraphs of 5.2.

6. Calculation of results

The starch content as a percentage of the sample is calculated as follows:

6.1 Measurement by polarimetry

D

where:

+185.9°: rice starch,

+185.7°: potato starch,

+184.6°: maize starch,

+182.7°: wheat starch,

+181.5°: barley starch,

+181.3°: oat starch,

+184.0° : other types of starch and starch mixtures in compound feedingstuffs.

6.2 Measurement by saccharimetry

D D

where:

7 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 starch contents of less than 40%.

-- 1.0% relative to the higher results, for starch contents of 40% or more.

8. Observations

8.1 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.

8.2 In the case of products with a high lactose content, such as powdered whey 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 50°C for 30 minutes. Leave to cool and continue the analysis as indicated in 5.3.

3.2 DETERMINATION OF STARCH.

3.2.2 Pancreatic method

1. Purpose and scope

To determine the content of starch and of starch degradation products of high molecular weight in feedingstuffs containing beet cossettes, beet pulp. dried beet tops or leaves, potato pulp, dried yeasts, products rich in inulin (e.g. cossettes and meal of Jerusalem artichokes) and products containing greaves. The determination should only be carried out when microscopic examination shows that significant quantities of starch are present in the sample.

2. Principle

Sugars present in the sample are extracted with ethanol. The starch in the extracted residue is reduced to sugar with pancreatin. The sugars formed are hydrolysed with hydrochloric acid and the glucose formed is determined by the Luff-Schoorl method. The quantity of glucose thus obtained, multiplied by a constant factor, gives the starch content of the sample.

3. Reagents

3.1 Ethanol solution, 90% (v/v), neutral to phenolphthalein.

3.2 Pentane-1-ol (amyl alcohol).

3.3 Toluene.

3.4 Buffer solution: dissolve in water 9.078 g potassium dihydrogen phosphate KH₂PO₄ and 11.876 g disodium hydrogen phosphate. Na₂HPO₄.2H₂O. Make up to 1 litre with water.

3.5 Sodium chloride solution, 0.2N.

3.6 Carrez I solution : dissolve in water 21.9 g zinc acetate Zn(CH₃COO)₂.2H₂O and 3 g glacial acetic acid. Make up to 100 ml with water.

3.7 Carrez II solution: dissolve in water 10.6 g potassium ferrocyanide K₄[Fe(CN)₆].3H₂O. Make up to 100 ml with water.

3.8 Hydrochloric acid, N.

3.9 Hydrochloric acid approximately 8N, d : 1.125.

3.10 Sodium hydroxide solution approximately 10N, d : 1.33.

3.11 Indicator: 0.1% (w/v) methyl orange solution.

3.12 Pancreatin in powder form, as per the directions in point 8. Keep in stoppered flasks, protected from light and moisture.

3.13 Luff-Schoorl reagent: pour the citric acid solution (3.13.2) into the Sodium carbonate solution (3.13.3) stirring carefully during the addition. Then add the copper sulphate solution (3.13.1) and make up to 1 litre with water. Leave to stand overnight and filter. Check the normality of the reagent thus obtained (Cu, 0.1 N; Na₂CO₃, 2N). The pH of the solution must be approximately 9.4.

3.13.1 Copper sulphate solution: dissolve 25 g copper sulphate CuSo_4.5H₂O in 100 ml water.

3.13.2 Citric acid solution: dissolve 50 g citric acid C₆H₈O₇H₂O in 50 ml water.

3.13.3 Sodium carbonate solution: dissolve 143.8 g anhydrous sodium carbonate in approximately 300 ml hot water. Leave to cool.

3.14 Granules of pumice stone purified by boiling in hydrochloric acid washing in water and drying.

3.15 Potassium iodide solution, 30% (w/v).

3.16 Sulphuric acid, approximately 6N, d : 1.18.

3.17 Sodium thiosulphate solution, 0.1N.

3.18 Starch solution: add a mixture of 5 g soluble starch in 30 ml water to 1 litre boiling water. Boil for 3 minutes, leave to cool. This solution should be freshly prepared.

4. Apparatus

4.1 Extractor (see diagram, fig. 1) consisting of:

4.1.1 A wide-necked 500 ml conical flask;

4.1.2 A reflux condenser fitted with a bung to the conical flask;

4.1.3 A sliding spindle in the centre of the reflux condenser, fitted with a hook at its lower end. A peg to hold the spindle firm;

4.1.4 A metal container for suspending on the spindle hook (4.1.3) and holding the filtration crucible (4.1.5);

4.1.5 A filtration crucible for rapid filtration; maximum size of pores: 90-150 microns (e.g. porosity 1), approximately 30 ml in capacity;

4.1.6 Filter papers, suitable in shape and size for the filtration crucible.

4.2 Incubator, set at 38°C.

4.3 200 ml graduated flasks with a standard ground glass joint and reflux condenser.

4.4 100 ml graduated flasks with a standard ground glass joint and reflux condenser.

5. Procedure

5.1 Preparation of the sample

Crush the sample so that the whole of it will pass through a 0.5 mm sieve. (ISO sieve of R 565 conforms).

5.2 Extraction

Weigh, to the nearest mg approximately 2 g of the sample and place in the filtration crucible (4.1.5), the bottom of which has first been covered with a filter paper (4.1.6) moistened with ethanol (3.1). Place in the conical flask (4.1.1) 55 ml ethanol (3.1) and a few granules of pumice stone (3.14). Place the filtration crucible in the metal container (4.1.4) and suspend the latter on the spindle hook (4.1.3). Place the reflux condenser over the conical flask and lower the spindle so that the bottom of the crucible just touches the surface of the ethanol. Peg the spindle firmly at this level. Bring the ethanol to boiling point and keep boiling for 3 hours. Then leave to cool and raise the spindle (4.1.3) to bring the crucible as high up as possible in the conical flask. Carefully unstopper the conical flask and allow 45 ml water to flow down along the sides of the flask. Replace the reflux condenser over the Erlenmeyer flask and keep the filtration crucible 10 cm above the surface of the liquid. Bring the liquid to boiling point and keep boiling for 3 hours. Then leave to cool, unstopper the conical flask and withdraw the crucible from the container.

5.3 Saccharification and hydrolysis

Place the crucible on a vacuum flask and dry under suction. Transfer the extraction residue to a mortar and grind finely. Using approximately 60 ml water, transfer the powder quantitatively to a 200 ml graduated flask with a standard ground glass joint and add a few drops of amyl alcohol (3.2). Connect a reflux condenser to the flask. Heat to boiling point and keep boiling for 1 hour. Then leave to cool and disconnect the reflux condenser. Add 25 ml buffer solution (3.4), 250 mg pancreatin (3.12), 2.5 ml sodium chloride solution (3.5) and 10 drops toluene (3.3). Shake for 2 minutes, place the flask in the incubator (4.2) and keep there for 21 hours, shaking occasionally. Then leave to cool to room temperature.

Add 5 ml Carrez I solution (3.6) and shake for 1 minute. Then add 5 ml Carrez II solution (3.7) and shake once again for 1 minute. Make up to volume with water, mix and filter. Using a pipette, take 50 ml filtrate and place in a 100 ml graduated flask (one may also work on 100 ml filtrate in a 200 ml graduated flask). Add a few drops of indicator (3.11) and acidify with hydrochloric acid 8 N (3.9) until the indicator turns red. Then add an extra 6.25 ml hydrochloric acid 8 N (3.9) (12.50 ml if working on 100 ml filtrate). Connect the reflux condenser to the flask, bring the solution to boiling point and keep boiling for 1 hour. Leave to cool, neutralise with the sodium hydroxide solution 10 N (3. 10) until the indicator turns yellow. Then acidify slightly by adding a little hydrochloric acid N (3.8), make up to volume with water and mix. Determine the glucose content according to the Luff-Schoorl method as shown in 5.4.

5.4 Titration according to Luff-Schoorl

Using a pipette, take 25 ml Luff-Schoorl reagent (3.13) and place in a conical flask; add 25 ml, accurately measured, of the solution obtained in 5.3; this should not contain more than 60 mg of glucose. Add two granules of pumice stone (3.14), heat, while shaking manually, over a medium flame and bring the liquid to boiling point in approximately 2 minutes. Immediately place the conical flask on a wire gauze fitted with an asbestos screen which has a hole approximately 6 cm in diameter. Under the wire gauze a flame has first been lit, and this is regulated in such a way that only the bottom of the conical flask is heated. Then connect a reflux condenser to the conical flask. As soon as this is done, boil for 10 minutes exactly. Cool immediately in cold water and after approximately 5 minutes, titrate as follows:

Add 10 ml potassium iodide solution (3.15) and, immediately afterwards, but with care, (because of the risk of extensive foaming) 25 ml sulphuric acid 6 N (3.16). Then titrate with the sodium thiosulphate solution 0.1 N (3.17) until a dull yellow colouring appears, add a few drops of starch indicator (3.18) and complete the titration.

Carry out the same titration on an accurately measured mixture of 25 ml Luff-Schoorl reagent (3.13) and 25 ml water, after adding 10 ml Potassium iodide solution (3.15) and 25 ml sulphuric acid 6 N (3.16), without bringing to the boil.

5.5 Blank Test

Carry out a blank test, applying the procedure described in 5.3 and 5.4, but without a sample.

6. Calculation of results

Using table 1 determine the quantity of glucose in mg corresponding to the difference between the results of the two titrations (expressed in ml of sodium thiosulphate 0. 1 N) and relating both to the sample analysis and the blank test.

The content of starch as a percentage of sample is given by the formula:

0.72 (a - b)

where:

a = mg of glucose relating to the sample;

b = mg of glucose relating to the blank test (see item 7.2).

7. Observations

7.1 Where partially or totally dextrinated starch and lactose are simultaneously presented in the sample, the result may be high by 0.53.0%. In such cases, the actual starch content is obtained as follows:

(a) Determine the content of reducing sugars in the ethanolic extract obtained in 5.2, and express the result as a percentage of glucose;

(b) Determine the content of water-soluble reducing sugars in the sample, and express the result as a percentage of glucose;

(c) Deduct the result obtained in (a) from that obtained in (b) and multiply the difference by 0.9;

(d) Deduct the value obtained in (c) from the starch content obtained by applying the method and calculating as shown in 6.

7.2 The quantity of glucose in relation to the blank test is normally 0.25 mg. It may not be greater than 0.50 mg.

8. Directions relating to pancreatin

Physical aspect: yellowish-white, amorphous powder.

Glucose content: the quantity of glucose of the blank test (see 5.5) is normally 0.25 mg. A result greater than 0.50 mg indicates that pancreatin can no longer be used.

Check for iodine consumption: suspend 62.5 mg pancreatin in approximately 50 ml water heated to 2530°C. Add 1 ml iodine solution 0.1 N. Stir for 2 minutes. Titrate with a solution of sodium thiosulphate 0.1 N in the presence of starch indicator. Consumption of iodine solution by pancreatin must not exceed 0.5 ml.

Check for amylolytic activity: mix 100 ml starch solution (3.18), 5 ml buffer solution (3.4), 0.5 ml sodium chloride solution (3.5) and 62.5 mg pancreatin. Heat the mixture to 2530°C, stir for 2 minutes. Add 1 ml iodine solution 0.1 N. The blue colouration must have disappeared within 15 minutes exactly of the addition of the iodine solution.

Fig. 1.

TABLE 1.

Values for 25 ml Luff-Schoorl reagent (ml of Na₂S₂O₃ 0.1 N; 2 minutes heating, 10 minutes boiling)

3.3 DETERMINATION OF SUGAR.

1. Purpose and scope

To determine the amount of reducing sugars and total sugars after inversion expressed as glucose or where appropriate as sucrose, converting by the factor 0.95. It is applicable to compound feedingstuffs. Special procedures are provided for other feedingstuffs. Where necessary, lactose should be measured separately and taken into account when calculating the results.

2. Principle

The sugars are extracted in dilute ethanol; the solution is clarified with Carrez solutions I and II. After eliminating the ethanol, the quantities before and after inversion are determined by the Luff-Schoorl method.

3. Reagents

3.1 Ethanol solution, 40% (v/v), d : 0.948 at 20°C, neutralised to phenolphthalein.

3.2 Carrez solution I: dissolve in water 21.9 g of zinc acetate Zn(CH₃COO)₂.2H₂O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3 Carrez solution II: dissolve in water 10.6 g of potassium ferrocyanide K₄[Fe(CN)₆].3H₂O. Make up to 100 ml with water.

3.4 Methyl orange solution, 0.1% (w/v).

3.5 Hydrochloric acid, 4 N.

3.6 Hydrochloric acid, 0.1 N.

3.7 Sodium hydroxide solution, 0.1 N.

3.8 Luff-Schoorl reagent: stirring carefully, pour the citric acid solution (3.8.2) into the sodium carbonate solution (3.8.3). Add the copper sulphate solution (3.8.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the normality of the reagent thus obtained (Cu, 0.1 N; Na₂CO₃, 2N). The solution's pH should be approximately 9.4.

3.8.1 Copper sulphate solution: dissolve 25 g of copper sulphate, Cu SO₄.5H₂O, free from iron, in 100 ml of water.

3.8.2 Citric acid solution: dissolve 50 g of citric acid, C₆H₈O₇.H₂O in 50 ml of water.

3.8.3 Sodium carbonate solution: dissolve 143.8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

3.9 Sodium thiosulphate solution 0.1 N.

3.10 Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for 3 minutes, leave to cool and if necessary add 10 mg of mercuric iodide as a preservative.

3.11 Sulphuric acid, 6 N.

3.12 Potassium iodide solution, 30% (w/v).

3.13 Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

3.14 3-methylbutane-1-ol.

4. Apparatus

Shaker, approximately 35-40 rpm.

5. Procedure

5.1 Extraction of sample

Weigh, to the nearest mg approximately 2.5 g of the sample and place in a 250 ml volumetric flask. Add 200 ml of ethanol (3.1) and shake in the shaker for 1 hour. Add 5 ml of Carrez solution 1 (3.2) and stir for 1 minute. Add 5 ml of Carrez solution II (3.3) and again stir for 1 minute. Make up to volume with ethanol (3.1), mix and filter. Remove 200 ml of the filtrate and evaporate to approximately half volume in order to eliminate most of the ethanol. Transfer the evaporation residue quantitatively to a 200 ml volumetric flask using warm water, cool, bring up to volume with water, mix and filter if necessary. This solution will be used to determine the amount of reducing sugars, and, after inversion, of total sugars.

5.2 Determination of reducing sugars

Using a pipette, remove not more than 25 ml of the solution containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage content of glucose in the sample.

5.3 Determination of total sugars after inversion

Using a pipette take 50 ml of the solution and transfer to a 100 ml volumetric flask. Add a few drops of methyl orange solution (3.4) then, carefully and stirring continuously, add hydrochloric acid 4 N (3.5) until the liquid turns a definite red. Add 15 ml of hydrochloric acid 0.1 N (3.6), immerse the flask in a fast boiling water bath and keep there for 30 minutes. Cool rapidly to approximately 20°C and add 15 ml of sodium hydroxide solution 0.1 N (3.7). Make up to 100 ml with water and mix. Remove not more than 25 ml containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage of glucose or, where appropriate, sucrose, by multiplying by the factor 0.95.

5.4 Titration by the Luff-Schoorl method

Using a pipette, take 25 ml of Luff-Schoorl reagent (3.8) and transfer to a 300 ml Erlenmeyer flask; add exactly 25 ml of the clarified sugar solution. Add 2 granules of pumice stone (3.13), heat, stirring by hand, over a free flame of medium height and bring the liquid to the boil in approximately 2 minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly 10 minutes. Cool immediately in cold water and after approximately 5 minutes titrate as follows:

Add 10 ml of Potassium iodide solution (3.12) and immediately afterwards (carefully, because of the risk of abundant foaming), add 25 ml of sulphuric acid 6 N (3.11). Titrate with sodium thiosulphate solution 0.1 N (3.9) until a dull yellow colour appears, add the starch indicator (3.10) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (3.8) and 25 ml of water, after adding 10 ml of potassium iodide solution (3.12) and 25 ml of sulphuric acid 6 N (3.11) without boiling.

6. Calculation of results

Using table 1 establish the amount of glucose in mg which corresponds to the difference between the values of two titrations, expressed in mg of sodium thiosulphate 0.1 N.

Express the result as a percentage of the sample.

7. Special procedures

7.1 In the case of feedingstuffs which are rich in molasses and other feedingstuffs which are not particularly homogeneous, weigh out 20 g and place with 500 ml of water in a 1 litre volumetric flask. Mix for 1 hour in the shaker. Clarify using Carrez I (3.2) and II (3.3) reagents as described under 5.1, this time however using four times the quantities of each reagent. Bring up to volume with 80% ethanol (v/v).

Mix and filter. Eliminate the ethanol as described under 5.1. If there is no dextrinised starch, bring up to volume with water.

7.2 In the case of molasses and straight feedingstuffs which are rich in sugar and almost starch-free (carobs, dried beetroot, cossettes, etc.) weigh out 5 g place in a 250 ml volumetric flask, add 200 ml of water and mix in the shaker for 1 hour, or more if necessary. Clarify using Carrez I (3.2) and II (3.3) reagents as described under 5.1. Bring up to volume with cold water, mix and filter. In order to determine the amount of total sugars, continue as described under 5.3.

8. Observations

8.1 In order to prevent foaming it is advisable to add (irrespective of the volume) approximately 1 ml of 3-methylbutane-1-ol (3.14) before boiling with Luff-Schoorl reagent.

8.2 The difference between the content of total sugars after inversion expressed as glucose, and the content of reducing sugars, expressed as glucose, multiplied by 0.95, gives the percentage content of sucrose.

8.3 In order to determine the content of reducing sugars, excluding lactose, two methods may be adopted:

8.3.1 For an approximate calculation, multiply by 0.675 the lactose content established by a different method of analysis and subtract the result obtained from the content of reducing sugars.

8.3.2 For an accurate calculation of reducing sugars, excluding lactose, the same sample must be used for the two final determinations. One of the analyses is carried out on part of the solution obtained under 5.1, the other on part of the solution obtained during the determination of lactose by the method laid down for that purpose (after fermenting the other types of sugar and clarifying).

In both cases the amount of sugar present is determined by the Luff-Schoorl method and calculated in mg of glucose. One of the values is subtracted from the other and the difference is expressed as a percentage of the sample.

TABLE 1.

Values for 25 ml Luff-Schoorl reagent (ml of Na₂S₂O₃ 0.1 N; 2 minutes heating, 10 minutes boiling)

Example

The two volumes taken correspond, for each determination, to a sample of 250 mg.

In the first case 17 ml of sodium thiosulphate solution 0.1 N corresponding to 44.2 mg of glucose is consumed; in the second, 11 ml corresponding to 27.6 mg of glucose.

The difference is 16.6 mg of glucose.

The content of reducing sugars (excluding lactose), calculated as glucose, is therefore:

3.4 DETERMINATION OF LACTOSE.

1. Purpose and scope

To determine the level of lactose in feedingstuffs containing more than 0.5% of lactose.

2. Principle

The sugars are dissolved in water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification and filtration the lactose content of the filtrate is determined by the Luff-Schoorl method.

3. Reagents

3.1 Suspension of Saccharomyces cerevisiae: suspend 25 g of fresh yeast in 100 ml of water. The suspension will keep for a maximum period of 1 week in a refrigerator.

3.2 Carrez solution I: dissolve in water 21.9 g of zinc acetate Zn(CH-₃COO)₂.2H₂O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3 Carrez solution II: dissolve in water 10.6 g of potassium ferrocyanide K4[Fe(CN)6].3H2O. Make up to 100ml with water.

3.4 Luff-Schoorl reagent: stirring carefully, pour the citric acid solution (3.4.2) into the sodium carbonate solution (3.4.3). Add the copper sulphate solution (3.4.1) and make up to 1 litre with water. Leave to settle over night and filter. Check the normality of the reagent thus obtained (Cu 01 N; Na₂CO₃2N). The solution's pH should be approximately 9.4.

3.4.1 Copper sulphate solution: dissolve 25 g of copper sulphate CuSO₄.5H₂O, free from iron, in 100 ml of water.

3.4.2 Citric acid solution: dissolve 50 g of citric acid C₆H₈O₇.H₂O in 50 ml of water.

3.4.3 Sodium carbonate solution: dissolve 143.8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

3.5 Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

3.6 Potassium iodide solution 30% (w/v).

3.7 Sulphuric acid, 6 N.

3.8 Solution of sodium thiosulphate, 0.1 N.

Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for 3 minutes, leave to cool, and if necessary add 10 mg mercuric iodide as a preservative.

4. Apparatus

Water bath with thermostat set at 38-40°C.

5. Procedure

Weigh, to the nearest mg approximately 1 g of the sample and place this portion of the sample in a 100 ml volumetric flask. Add 25-30 ml of water. Place the flask in a boiling water bath for 30 minutes and then cool to approximately 35%C. Add 5 ml of yeast suspension (3.1) and mix. Leave the flask to stand for 2 hours in a water bath, at a temperature of 38-40°C. Cool to approximately 20°C.

Add 2.5 ml of Carrez solution I (3.2) and stir for 30 seconds, then add 2.5 ml of Carrez solution II (3.3) and again stir for 30 seconds. Make up to 100 ml with water, mix and filter. Using a pipette, remove an amount of filtrate which does not exceed 25 ml and which preferably contains from 40-80 mg of lactose and transfer it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.

Carry out a blank test in the same way with 5 ml of yeast suspension (3.1).

Determine the lactose content according to Luff-Schoorl, as follows: add exactly 25 ml of Luff-Schoorl reagent (3.4) and two granules of pumice stone (3.5). Stir by hand while heating over a free flame of medium height and bring the liquid to the boil in approximately 2 minutes. Place the Erlenmeyer flask immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer flask is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly 10 minutes. Cool immediately in cold water and after approximately 5 minutes titrate as follows:

Add 10 ml of potassium iodide solution (3.6) and immediately afterwards (carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid 6 N (3.7). Titrate with sodium thiosulphate solution 0.1 N (3.8) until a dull yellow colour appears, add the starch indicator (3.9) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (3.4) and 25 ml of water, after adding 10 ml. of potassium iodide solution (3.6) and 25 ml of sulphuric acid 6N (3.7) without boiling.

6. Calculation of results

Using the attached table, establish the amount of lactose in mg which corresponds to the difference between the results of the two titrations, expressed in ml of sodium thiosulphate 0.1 N.

Express the result as a percentage of anhydrous lactose in the sample.

TABLE 1.

Values for 25 ml Luff-Schoorl reagent (ml of Na₂S₂O₃ 0.1 N; 2 minutes heating, 10 minutes boiling)

7. Observation

For products containing more than 40% of fermentable sugar, use more than 5 ml of yeast suspension (3.1).

4. OILS AND FATS.

4.1 DETERMINATION OF CRUDE OILS AND FATS.

1. Purpose and Scope

To determine the content of crude oils and fats in feeding stuffs. It does not cover the analysis of the oil seeds and oleaginous fruit defined in Council Regulation 136/66/EEC¹ of 22 September 1966. (See method 4.2)

¹ OJ No. 127, 30 September 1966, P.3025/66.

Depending on the nature of the feedingstuff, either of the two methods described must be used.

1.1 Method A

Applicable to straight feedingstuffs of plant origin, with the exception of those which are known to contain oils and fats which cannot be totally extracted with light petroleum without prior hydrolysis. Among these are glutens, yeasts, soya and potato proteins. This method is also applicable to compound feedingstuffs, with the exception of those which contain milk powder or from which oils and fats cannot be totally extracted with light petroleum without prior hydrolysis.

1.2 Method B

Applicable to straight feedingstuffs of animal origin as well as to feedingstuffs mentioned under point 1.1 as being exceptions for method A.

2. Principle

2.1 Method A

The sample is extracted with light petroleum. The solvent is distilled off and the residue dried and weighed.

2.1 Method B

The sample is treated under heating with hydrochloric acid. The mixture is cooled and filtered. The residue is washed and dried and submitted to the determination according to method A.

3. Reagents

3.1 Light petroleum, boiling range: 40 to 60°C. The bromine value must be less than 1 and the residue of evaporation less than 2 mg/100 ml.

3.2 Sodium sulphate, anhydrous.

3.3 Hydrochloric acid, 3 N.

3.4 Filtration aid, e.g. Kieselgur, Hyflo-supercel.

4. Apparatus

4.1 Extraction apparatus. If fitted with a siphon (Soxhlet apparatus), the reflux rate should be such as to produce about 10 cycles per hour; if of the non-siphoning type, the reflux rate should be about 10 ml per minute.

4.2 Extraction thimbles, free of matter soluble in light petroleum and having a porosity consistent with the requirements of point 4.1.

4.3 Drying oven, either a vacuum oven set at 75±3°C or an air-oven set at 100±3°C.

5. Procedure

5.1 Method A (see point 8.1)

Weigh to the nearest mg, approximately 5 g of the sample and transfer it to an extraction thimble (4.2) and cover with a fat-free wad of cotton wool.

Place the thimble in an extractor (4.1) and extract for six hours with light petroleum (3.1). Collect the light petroleum extract in a dry, weighed flask containing fragments of pumice stone¹.

Distill off the solvent. Dry the evaporation residue maintaining the flask for one and a half hours in the drying oven (4.3). Leave to cool in a dessiccator and weigh. Dry again for 30 minutes to ensure that the weight of the oils and fats remains constant (loss in weight between two successive weighings must be less than 1 mg).

5.2 Method B

Weigh to the nearest mg, approximately 2.5 g of sample (see point 8.2) and place it in a conical flask. Add 100 ml of hydrochloric acid 3 N (3.3), some fragments of pumice stone and fit a reflux condenser. Bring the mixture to a gentle boil over a low flame or a hot-plate and keep it there for one hour. Do not allow the product to stick to the sides of the container.

Cool and add a quantity of filtration aid (3.4) sufficient to prevent any loss of oil and fat during filtration. Filter through a moistened, fat-free, double filter paper. Wash the residue in cold water until a neutral filtrate is obtained. Check that the filtrate does not contain any oil or fats. Their presence indicates that the sample must be extracted with light petroleum, using method A, before hydrolysis.

Place the double filter paper containing the residue on a watch glass and dry for one and a half hours in the oven at 100±3°C.

Place the double filter paper containing the dry residue in an extraction thimble (4.2) and cover with a fat-free wad of cotton wool. Place the thimble in an extractor (4.1) and proceed as indicated in the second and third paragraphs of point 5.1.

6. Expression of result

Express the weight of the residue as a percentage of the sample.

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample by the same analyst should not exceed:

¹Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass bends.

0.2% in absolute value, for contents of crude oils and fats lower than 5%,

4.0% related to the highest result for contents of 5 to 10%,

0.4% in absolute value, for contents above 10%.

8. Observations

8.1 For products with a high content of oils and fats, which are difficult to crush or unsuitable for drawing a homogeneous reduced test sample, proceed as follows.  Weigh 20 g of the sample to the nearest 1 mg and mix with 10 g or more of anhydrous sodium sulphate (3.2). Extract with light petroleum (3.1) as indicated in point 5.1. Make up the extract obtained to 500 ml with light petroleum (3.1) and homogenize. Take 50 ml of the solution and place in a small, dry, weighed flask containing fragments of pumice stone ¹. Distill off the solvent, dry and proceed as indicated in the last paragraph of point 5.1.

Eliminate the solvent from the extraction residue left in the thimble, crush the residue to a fineness of 1 mm, return it to the extraction thimble (do not add sodium sulphate) and proceed as indicated in the second and third paragraphs of point 5.1.

Calculate the content of oils and fats as a percentage of the sample by using the following formula:

(10a + b)x5

where:

a=mass in grams of the residue after the first extraction (aliquot part of the extract),

b=mass in grams of the residue after the second extraction.

8.2 For products low in oils and fats the test sample may be increased to 5 g.

4.2 DETERMINATION OF OIL CONTENT IN OLEAGINOUS SEEDS.

1. Purpose and Scope

To determine the oil content in oleaginous seeds.

2. Principle

The sample is extracted with n-hexane. The solvent is removed by distillation and the residue dried and weighed.

3. Reagents

3.1 n-Hexane.

3.2 Sand, acid washed.

3.3 Hydrochloric acid: d=1.18.

4. Apparatus

4.1 Soxhlet-type extractor or equivalent apparatus.

4.2 Electric oven with temperature regulation.

¹Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass beads.

4.3 Metal vessel, flat-bottomed, diameter about 100 mm, height about 40 mm.

4.4 Porous vessel of ceramic material, cylindrical, internal diameter 68 mm, external diameter 80 mm, height 85 mm, thickness of walls and base 6mm.

4.5 Fumigation oven, with temperature control.

5. Procedure

5.1 Preparation.

5.1.1 Copra.

Grate the product with a hand grater or preferably with the mechanical grater which can deal with the whole sample for analysis. Hand operation does not allow the whole sample for analysis to be grated. Endeavour to obtain a sub-sample which is as representative as possible. The thickness and the colour of the different pieces should be taken into account.

The length of the grated particles may exceed 2 mm, but should not be greater than 5 mm.

Mix the particles carefully and carry out the determination without delay.

5.1.2 Medium-sized seeds (e.g. sunflower, groundnut, soya) with the exception of cottonseed with adherent linters.

Crush the sample for analysis, in the previously well cleaned mechanical mill until particles are obtained with a major dimension not greater than 2 mm. Reject the first grindings (about ½0 of the sample) and collect the rest. Mix carefully and carry out the determination without delay.

5.1.3 Cottonseed with adherent linters weigh into the tared metal vessel (4.3) and to the nearest 10 mg, 60 g of sample. Put the vessel and seed in the oven previously heated to 130°C and leave to dry for two hours at 130E2°C, then remove the vessel from the oven and allow to cool in air for about thirty minutes. Transfer the dried seed to the porous ceramic vessel (4.4), the inside walls and base of which have been previously moistened with 1.5 ml of concentrated hydrochloric acid (3.3) by means of the pipette taking care that the acid is completely absorbed without forming adherent drops. Close the vessel with the watch glass and put in the fumigation oven (4.5). Heat so as to reach 115°C in thirty minutes; do not heat beyond this temperature and maintain it for another thirty minutes.

Take the vessel out of the oven, allow to cool for one hour in air, reweigh the treated seed to the nearest 10 mg, then grind the seed in the mechanical mill and continue as described in clause 5.1.2.

5.1.4 Small seeds (e.g. linseed colza etc.) analyse without grinding.

5.2 Weighing

5.2.1 Seeds coming under, 5.1.1, 5.1.2 and 5.1.3. Weigh to the nearest 10 mg, 10 g of sample, place in an extraction thimble and plug with a wad of cotton wool.

5.2.2 Seeds coming under 5.1.4

Weigh to the nearest 10 mg, 10 g of sample and grind in a microgrinder, taking care not to leave any seeds intact. Transfer, without loss, the ground seeds to the extraction thimble, using a spatula. Wipe the bowl of the micro-grinder and the spatula, with a wad of cotton wool soaked with solvent (3.1) and plug the thimble with this wad.

5.3 Determination.

If the moisture content is greater then 10% place the thimble and sample in an oven set at a temperature not exceeding 80°C and leave there for some time, until the moisture content is reduced to less than 10%.

Place the thimble in the extraction apparatus (4.1) and extract for four hours with n-hexane (3.1). Regulate the heating so that the reflux rate is at least three drops per second. Collect the extract in a dry weighed flask marked A.

After extracting for four hours, allow to cool. Remove the thimble from the extractor and place it in a current of air in order to remove the greater part of the solvent. Empty the thimble into the mortar, add about 10 g of sand (3.2) and triturate as finely as possible. Replace the mixture in the thimble and the latter in the extractor, and continue the extraction for two hours, using the same flask.

Leave to cool, remove the thimble again, eliminate the solvent and repeat the trituration as above (without further addition of sand). Carry out a third extraction for two hours, collecting the product this time in a dried weighed flask marked B.

Remove the greater part of the solvent, by distillation and a boiling water bath, from flask A and B. Remove the last traces of solvent by heating the flasks for twenty minutes at 103±2°C. Assist the elimination either by blowing in air at intervals or by using reduced pressure. Leave the flask to cool in a desiccator, for at least one hour, and weigh to the nearest 1 mg. Heat again for ten minutes under the same conditions, cool and weigh. The difference between these two weighings should not exceed 10 mg. If it does so, heat again for ten minutes, until the difference in mass is not greater than 10 mg. Keep a note of the final mass of flask A.

If the mass of oil in flask B does not exceed 10 mg the operation is complete. If it does so, carry out a fresh extraction for two hours, using flask B, until the mass of oil from the last extraction is not more than 0.010 g.

6. Calculation of results

6.1 For all seeds other than cottonseed with adhering linters.

The oil content, as a percentage of the sample, is calculated by using the following formula.

M1 is the mass in grams of oil in flasks A & B.