LegalizeCommission Implementing Regulation (EU) 2016/1240 of 18 May 2016 laying down rules for the application of Regulation (EU) No 1308/2013 of the European Parliament and of the Council with regard to public intervention and aid for private storage (Text with EEA relevance)
TITLE I
GENERAL PROVISIONS
CHAPTER I
Introductory provision
Article 1
Scope
This Regulation lays down rules for the application of Regulation (EU) No 1308/2013 and Regulation (EU) No 1370/2013 as regards:
(a) the buying-in and selling from public intervention of products listed in Article 11 of Regulation (EU) No 1308/2013;
(b) the granting of aid for private storage for products listed in Article 17 of Regulation (EU) No 1308/2013.
This Regulation shall apply without prejudice to specific provisions laid down in Implementing Regulations:
(a) opening a tendering procedure for buying-in of products or opening the sale of products from intervention; or
(b) opening a tendering procedure or fixing the amount of aid for private storage in advance.
CHAPTER II
General common rules
Article 2
Submission and admissibility of offers, tenders and applications
An offer, tender or application shall be admissible if it is submitted in the official language or one of the official languages of the Member State in which the offer, tender or application is submitted, and shall include, on a form made available by the paying agency, at least the following information:
(a) the operator's name, address and VAT registration number in the Member State where the operator carries out his main activity;
(b) the product, or type of product, covered with its relevant CN code, if applicable;
(c) the quantity offered, tendered or applied for, subject to the minimum quantities laid down in Article 5, if applicable.
TITLE II
PUBLIC INTERVENTION
CHAPTER I
Specific rules related to public intervention
Article 3
Intervention storage places
Each intervention storage place (‘storage place’) shall have a minimum storage capacity of:
(a) for cereals: 5 000 tonnes, 7 500 tonnes from the public intervention period 2017/18, 10 000 tonnes from the 2018/19 period, 15 000 tonnes from the 2019/20 period;
(b) for rice: 5 000 tonnes, 7 500 tonnes from the public intervention period 2017/18, 10 000 tonnes from the 2018/19 period;
(c) for butter and skimmed milk powder: 400 tonnes, 600 tonnes from the 2017 public intervention period, 800 tonnes from the 2018 period.
Member States with an average annual production of cereals of less than 20 million tonnes may continue to apply from the 2019/20 period a minimum storage capacity of 10 000 tonnes.
Article 4
Establishing the eligibility of products
The eligibility of products for public intervention shall be established in accordance with the methods laid down in the following provisions:
(a) for cereals: in Parts I, II, III and IV of Annex I;
(b) for rice: in Part I of Annex II;
(c) for beef: in Part I of Annex III;
(d) for butter: in Parts I and Ia of Annex IV to this Regulation;
(e) for skimmed milk powder: in Parts I and Ia of Annex V to this Regulation.
CHAPTER II
Buying-in of products into intervention
Section 1
General provisions
Article 5
Minimum quantities of products offered or tendered
The minimum quantity of products offered or tendered for buying-in shall be:
(a) for common wheat, barley and maize: 160 tonnes;
(b) for durum wheat: 20 tonnes;
(c) for rice: 40 tonnes;
(d) for beef: 20 tonnes;
(e) for butter: 30 tonnes;
(f) for skimmed milk powder: 30 tonnes.
Member States with an average annual production of cereals of less than 20 million tonnes may decide to apply a minimum quantity of 120 tonnes for common wheat, barley and maize.
Article 6
Level of the security for buying-in of products
The level of the security required in accordance with Article 4(a) of Delegated Regulation (EU) 2016/1238 when submitting an offer or tender for the buying-in of products into public intervention shall be:
(a) for cereals: EUR 20/tonne;
(b) for rice: EUR 30/tonne;
(c) for beef: EUR 300/tonne;
(d) for butter: EUR 50/tonne;
(e) for skimmed milk powder: EUR 50/tonne.
Article 7
Submission and admissibility of offers and tenders
An offer or tender shall be admissible if it complies with the requirements laid down in Article 2 and, in the case of a tendering procedure, in the Implementing Regulation opening the tendering procedure referred to in Article 12. It shall also meet the following conditions:
(a) it includes at least the following information: (i) for rice, an indication of the type and variety; (ii) except for beef, the place where the product is held at the time of the offer or tender; (iii) for cereals and rice, the storage place for which the offer or tender is made; (iv) for cereals and rice, the year of harvest and the area or areas of production in the Union; (v) for butter and skimmed milk powder, the date of production; (vi) for butter and skimmed milk powder, the name and approval number of the approved undertaking in which it was produced;
(b) the operator has lodged a security in accordance with Article 4(a) of Delegated Regulation (EU) 2016/1238;
(c) for cereals and rice, the operator has declared: (i) that the products are of Union origin; (ii) that the offer or tender refers to a homogeneous lot which, for rice, must comprise paddy rice of the same variety; (iii) whether any post-harvest treatment has been carried out or not, and, where appropriate, the name of the product used, that it has been applied in conformity with the conditions of use, and that the product is authorised under Regulation (EC) No 1107/2009 of the European Parliament and of the Council (1).
Article 8
Verification of offers and tenders by the paying agency
Where the paying agency decides that an offer or tender is inadmissible, it shall inform the operator concerned within three working days of the receipt of the offer or tender. For offers, if the operator does not receive such information, the offer is considered as being admissible.
Article 9
Notifications of offers and tenders to the Commission
Member States shall notify the Commission of all admissible offers and tenders within the following time limits:
(a) in the case of offers, notifications shall be submitted not later than 12.00 (Brussels time) each Tuesday and shall relate to the quantities of the products which, during the preceding week, have been the subject of an admissible offer, and of the related information. When the quantities offered approach the limits fixed in Article 3(1) of Regulation (EU) No 1370/2013, the Commission shall inform Member States as of which date they shall notify the Commission on each working day. As from that date, Member States shall notify the Commission, not later than 14.00 (Brussels time) each working day, of the quantities offered for intervention during the preceding working day.
(b) in the case of tenders, the time limits laid down in the Implementing Regulation opening the tendering procedure shall apply.
Section 2
Buying-in at fixed price
Article 10
Submission of offers for buying-in of common wheat, butter and skimmed milk powder at fixed price
Offers may be submitted to the paying agency from the beginning of the public intervention periods laid down in Article 12 of Regulation (EU) No 1308/2013.
Article 11
Measures for respecting the quantitative limitations
In order to comply with the quantitative limitations fixed in Article 3(1) of Regulation (EU) No 1370/2013, the Commission shall decide as provided for in Article 3(6)(a) of that Regulation:
(a) to close intervention buying-in at fixed price;
(b) where acceptance of the full quantity offered would lead to the maximum quantity being exceeded, to set an allocation coefficient applicable to the total quantity in the offers received and notified to the Commission from each operator on the day of the decision;
(c) where appropriate, to reject pending offers submitted to the paying agencies of the Member States.
The Commission shall decide within two working days of the notification referred to in the first subparagraph of point (a) of Article 9(1), and within five working days of the notification referred to in the third subparagraph of point (a) of Article 9(1).
For the purposes of this Article, when a date of notification is a public holiday for the Commission the counting of the deadline shall start on the first working day after that public holiday. If such public holidays are included in the time period for the Commission's decision, only working days shall be counted.
Section 3
Buying-in via a tendering procedure
Article 12
Tendering procedure
A tendering procedure for the buying-in of products referred to in Article 11 of Regulation (EU) No 1308/2013 shall be opened by way of an Implementing Regulation opening the tendering procedure, which shall contain, in particular, the following information:
(a) the products covered, and: (i) for rice, an indication of the type and variety; (ii) for beef, whether the tender is for the bought-in carcasses to be deboned or for storage without deboning;
(b) the period covered (‘tendering period’) and, if necessary, the different sub-periods during which the tenders can be submitted.
With regard to beef, the following rules shall apply:
(a) the average market price by eligible category in a Member State or in a region thereof shall take account of the prices for qualities U, R and O, expressed in quality R3 using the conversion coefficients set out in Part II of Annex III in the Member State or the intervention region concerned;
(b) the average market prices shall be recorded in accordance with Commission Regulation (EC) No 1249/2008 (2);
(c) the average market price by eligible category in a Member State or a region thereof shall be the average of the market prices for all the qualities referred to in point (b), weighted by the proportion each quality represents in the total number of slaughterings in that Member State or region. The United Kingdom shall consist of two intervention regions as follows: (i) region I: Great Britain; (ii) region II: Northern Ireland.
Article 13
Submission and admissibility of tenders
In the case of cereals and rice, the price tendered per tonne of products shall be a price corresponding to the minimum quality for cereals defined in Part II of Annex I to Delegated Regulation (EU) 2016/1238 or to the standard quality for rice defined in Section A of Annex III to Regulation (EU) No 1308/2013, delivered to the storage place, not unloaded.
In the case of butter and skimmed milk powder, the tendered price shall be the price per 100 kg of products delivered to the loading bay of the storage place.
In the case of beef, tenders shall state the price quoted in accordance with Article 12(5)(a), expressed per tonne of products of quality R3, and if it relates to bone-in meat intended for deboning, or for storage without deboning.
Article 14
Decisions on the buying-in price
On the basis of the tenders notified in accordance with Article 9, the Commission shall decide:
(a) not to fix a maximum buying-in price; or
(b) to fix a maximum buying-in price.
Article 15
Individual decisions on tenders
The paying agency shall only accept tenders that have been notified to the Commission in accordance with Article 9.
The paying agency shall notify the operators of the outcome of their participation in the tendering procedure within three working days of the entry into force of that decision of the Commission.
This notification shall not be necessary when the tender is accepted, if the paying agency issues a delivery order as referred to in Article 17 within five working days of the entry into force of that decision of the Commission. In case of such acceptance, no further extension of the time limit for the issue of the delivery order, as referred to in the second subparagraph of Article 17(1), may be granted.
Article 16
Limitation of buying-in for beef
Where the paying agencies are offered beef in quantities greater than they are able to take over, they may limit the buying-in to the quantities they can take over in their territory or in one of their intervention regions referred to in Article 12(5).
Paying agencies shall ensure equal access for all parties concerned in the event of such limitation.
Section 4
Deliveries and transport costs
Article 17
Delivery order
The paying agency may decide to extend the time limit for the issue of the delivery order when it is necessary due to high quantities of cereals or rice accepted. However, the final date for delivery of the products shall not be later than 65 days after the deadline or entry into force referred to in the first subparagraph. In such cases the paying agency shall inform the affected operators.
The delivery order shall be dated and numbered and shall contain the following information:
(a) the quantity to be delivered;
(b) the final date for delivery of the products;
(c) the storage place to which the products shall be delivered;
(d) the price at which the offer or tender is accepted.
Article 18
Specific provisions for the delivery of cereals and rice
The operator shall bear the costs relating to the following tests carried out for cereals in accordance with the methodology referred to in Part IV of Annex I:
(i) the amylasic activity (Hagberg) test;
(ii) the test for the determination of the protein content of durum wheat and common wheat;
(iii) the Zeleny test;
(iv) the machinability test;
(v) analyses of contaminants.
Article 19
Transport costs for cereals and rice
Beyond that maximum distance, the additional transport costs shall be borne by the paying agency and reimbursed by the Commission at a rate per tonne and per kilometre of EUR 0, 05.
Article 20
Specific provisions for the delivery of beef
Article 21
Specific provisions for the packing, delivery and storage of butter and skimmed milk powder
The butter and skimmed milk powder shall be delivered on pallets of a quality suitable for long-term storage, to be exchanged against equivalent pallets. Alternatively, an equivalent system may be approved by the paying agency.
The costs incurred in unloading the butter or skimmed milk powder at the loading bay of the storage place shall be borne by the paying agency.
Article 22
Delivery
The date of delivery shall be:
(a) for cereals, rice, butter and skimmed milk powder: the date on which it is confirmed that the whole of the quantity referred to in the delivery order entered the designated storage place. This date cannot be earlier than the day following the date of issue of the delivery order;
(b) for each consignment of beef: the date of entrance at the weighing point of the intervention storage place or, where the meat is to be deboned, at the cutting plant.
Section 5
Control and takeover
Article 23
The takeover record
The takeover record shall be issued by the paying agency after the required checks and analyses have established that the eligibility requirements laid down in Article 3 of Delegated Regulation (EU) 2016/1238 are fulfilled. It shall indicate at least:
(a) the quantities delivered and, for rice, the variety;
(b) the characteristics of the products as resulting from the analyses in so far as this is relevant for the calculation of the price;
(c) if applicable, the quantities that have not been taken over. In this case, the operator shall be notified accordingly.
Article 24
Obligations of the operator
The products shall meet the eligibility requirements set out in Article 3 of Delegated Regulation (EU) 2016/1238. Where the required checks show that the products do not fulfil the eligibility requirements, the operator shall:
(a) take back at his expense the products concerned;
(b) pay the associated costs from the date of entry of the products into the storage place until the date of their removal from storage.
The costs to be paid by the operator shall be determined on the basis of standard amounts for entry, removal and storage costs in accordance with Article 3 of Commission Delegated Regulation (EU) No 906/2014 (3).
Article 25
Deboning requirement for beef
Where deboning is required as a condition of the tendering procedure, the paying agency shall have all the beef that is bought-in deboned in accordance with Part III of Annex III.
Section 6
Price adjustments and payments
Article 26
Price adjustments for cereals and rice
The price adjustment as provided for in Articles 2(2) and 3(4) of Regulation (EU) No 1370/2013 shall be made in accordance with:
(a) Parts V and VI of Annex I to this Regulation in the case of cereals;
(b) Part II of Annex II to this Regulation in the case of rice.
Article 27
Payments
CHAPTER III
Sale of products from intervention
Article 28
Opening of the tendering procedure
The first closing date for submission of tenders shall be fixed not earlier than six days after the publication of that Implementing Regulation in the Official Journal of the European Union.
The Implementing Regulation opening the sale shall contain, in particular, the following information:
(a) the products covered, and in particular: (i) for rice, an indication of the type and variety, (ii) for beef, the relevant cuts;
(b) the period covered (‘tendering period’) and the different sub-periods during which the tenders can be submitted;
(c) for beef, butter and skimmed milk powder, the minimum quantity for which a tender may be submitted;
(d) the amount of the security that shall be lodged when submitting a tender. In addition, that Implementing Regulation may contain: (a) the global quantities covered by the tendering procedure; (b) if applicable, provisions concerning transport costs for cereals and rice.
Article 29
Notice of invitation to tender and arrangements related to the invitation to tender
The notice of invitation to tender shall indicate in particular:
(a) the name and address of the paying agency issuing the notice of invitation to tender;
(b) the reference to the Implementing Regulation opening the sale;
(c) the closing dates for the submission of tenders for each partial invitation to tender;
(d) for each storage place, the name and address of the storekeeper, and, as appropriate: (i) for cereals and rice, the quantities available presented in sales lots determined in such a way to ensure equal access to operators, together with a description of the quality of each sales lot; (ii) for beef, the quantities available by cut, in accordance with Part IV of Annex III and the date of delivery; (iii) for butter and skimmed milk powder, the quantities available and the date of delivery;
(e) the delivery stage as referred to in Article 30(1)(d) and, if applicable, the type of packaging;
(f) any facilities at the storage place for loading onto a means of transport;
(g) for butter, where appropriate, the available quantity of sweet cream butter per storage place, as referred to in point 2(d) of Part II of Annex IV.
The paying agency shall make the necessary arrangements to enable interested parties:
(a) to inspect and to take and examine samples of cereals and rice put up for sale at their own expense before submitting a tender;
(b) to consult the results of the analyses referred to in Part IV of Annex I, Part I of Annex II, Part I of Annex IV or Part I of Annex V, as appropriate.
Article 30
Submission and admissibility of tenders
A tender shall be admissible if it complies with the requirements laid down in Article 2 and the Implementing Regulation opening the sale. It shall also meet the following conditions:
(a) it indicates a reference to the Implementing Regulation opening the sale and the expiry date for the sub-period of submission of the tenders;
(b) for beef, it indicates the relevant cuts;
(c) for cereals and rice, it indicates the total quantity of the sales lot;
(d) it indicates the price in euro tendered per unit of measurement, rounded to no more than two decimal places, exclusive of VAT, as follows: (i) in the case of cereals and rice, for the product loaded onto the means of transport; (ii) in the case of butter or skimmed milk powder, for the product supplied on pallets at the loading bay of the storage place or, if necessary, supplied on pallets loaded onto the means of transport where it is a lorry or railway wagon; (iii) in the case of beef, for the product delivered to the loading bay of the storage place.
(e) for beef, butter and skimmed milk powder, that it relates to at least the minimum quantity referred to in the Implementing Regulation opening the sale;
(f) it indicates the storage place where the product is held and, for butter and skimmed milk powder, an alternative storage place may be specified;
(g) the operator has lodged the amount of the security provided for in the Implementing Regulation opening the sale.
For butter, where appropriate, the tender may specify that it is submitted exclusively for sweet cream butter as referred to in Article 29(2)(g).
Article 31
Notification of tenders to the Commission
Article 32
Decisions on the selling price
On the basis of the tenders notified in accordance with Article 31, the Commission shall decide, in accordance with the procedure referred to in Article 229(2) of Regulation (EU) No 1308/2013:
(a) not to fix a minimum selling price; or
(b) to fix a minimum selling price.
For butter and skimmed milk powder the minimum selling price may vary according to the location of the products offered for sale.
Article 33
Individual decisions on tenders
The paying agencies shall only accept tenders that have been notified to the Commission in accordance with Article 31.
The paying agency shall notify the operators of the outcome of their participation in the tendering procedure within three working days of the entry into force of that decision of the Commission.
Article 34
Specific rules for the allocation of beef, butter and skimmed milk powder
Article 35
Payments
Before removing the product and within the period specified in Article 37(2), operators shall pay the paying agency the amount corresponding to their tender for each quantity that they withdraw from the storage place, as notified by the paying agency in accordance with Article 33(3).
Article 36
Sales by Member States
In a Member State where no tendering procedure is open in accordance with Article 28, the paying agency may itself open a tendering procedure for the sale of intervention products when the total quantity remaining in all its storage places is less than:
(a) for each cereal: 10 000 tonnes;
(b) for rice: 2 000 tonnes;
(c) for beef, butter or skimmed milk powder: 200 tonnes.
Article 37
Removal order
On payment of the amount referred to in Article 35, the paying agency shall issue a removal order indicating:
(a) the quantity in respect of which the corresponding amount has been paid;
(b) the storage place in which the product is stored;
(c) the final date for removal of the product.
Article 38
Removal of butter and skimmed milk powder
CHAPTER IV
Disposal of products from intervention for the scheme for food distribution to the most deprived in the Union
Article 38a
Making products from intervention available for the scheme
The Implementing Regulation referred to in paragraph 1 shall contain, in particular, the following information:
(a) the type and quantity of the products that are made available for the scheme;
(b) the location of the products made available for the scheme and the criteria for the distribution of the available lots among the Member States concerned on the basis of their location;
(c) the way in which the products are to be disposed of in accordance with the second subparagraph of Article 23(4) of Regulation (EU) No 223/2014 of the European Parliament and of the Council (4) (direct use, processing or sale) in order to make them available for the scheme in a way that is economically the most favourable option taking into account the nature and characteristics of the products;
(d) where the products are to be sold in accordance with Article 38b of this Regulation, the level of the security to be lodged in accordance with Article 4(a) of Delegated Regulation (EU) 2016/1238 and the price below which the products shall not be sold.
Within 20 working days after the publication of the Implementing Regulation referred to in paragraph 1, the Commission shall adopt an Implementing Regulation without applying the procedure referred to in Article 229(2) or (3) of Regulation (EU) No 1308/2013 by which it:
(a) allocates a quantity to each Member State that submitted a request;
(b) specifies the location of the available lots as distributed to the Member States concerned in accordance with the criteria referred to in point (b) of paragraph 2.
For the purposes of point (a) of the first subparagraph, where the total quantity requested by Member States exceeds the quantity referred to in point (a) of paragraph 2, a quantity shall be allocated to the Member States concerned in proportion to the quantity requested by them.
Article 38b
Sale of intervention products made available for the scheme
Where a Member State has been allocated products held by the paying agency of another Member State, the paying agency holding the products shall provide to the paying agency selling the products the information referred to in Article 29(2)(d) to (g) within 10 working days after the publication of the Implementing Regulation by which the products were allocated.
TITLE III
AID FOR PRIVATE STORAGE
CHAPTER I
Specific rules for aid for private storage
Section I
General provisions
Article 39
Opening of tendering procedures and fixing aid in advance
The Implementing Regulation opening the tendering procedure or fixing the amount of aid in advance may contain the following information:
(a) the products, or types of products, covered with their relevant CN codes, if applicable;
(b) in the case of aid fixed in advance, the amount of aid for storage per unit of measurement for the products covered;
(c) the unit of measurement of the quantities;
(d) if the tender or aid fixed in advance relates to products that have already been placed in storage;
(e) in the case of tenders, the period covered (‘tendering period’) and, if necessary, the different sub-periods during which tenders can be submitted, and in the case of aid fixed in advance the period for submitting an application;
(f) the period of storage;
(g) the global quantity, if applicable;
(h) the minimum quantity per tender or application;
(i) the amount of the security per unit of measurement in the case of tenders and, where applicable, for applications;
(j) the periods of placing into store and of removal from store;
(k) the specifications that have to appear on the packaging, if applicable.
Article 40
Submission and admissibility of tenders and applications for aid for private storage
A tender or application for aid for private storage shall be admissible if it complies with the requirements laid down in Article 2 and the following conditions are met:
(a) it includes at least the following information: (i) a reference to the Implementing Regulation opening the tendering procedure or fixing the amount of aid for private storage in advance; (ii) the storage period where so required in the Implementing Regulation opening the tendering procedure or fixing the amount of aid for private storage in advance; (iii) the quantity of products covered by the tender or application; (iv) where products are already stored, the name and address of each place of private storage, the location of the storage lots/batches/vats/silos with the corresponding quantities and, where appropriate, the number identifying the approved undertaking; (v) in the case of tenders, the expiry date for the sub-period of submission; (vi) in the case of tenders, the amount of aid in euro proposed per unit of measurement rounded to no more than two decimal places, exclusive of VAT;
(b) the operator has lodged the amount of the security referred to in the Implementing Regulation opening the tendering procedure or fixing the amount of aid for private storage in advance.
Article 41
Verification of tenders and applications by the paying agency
Article 42
Notification of tenders and applications to the Commission
Member States shall notify the Commission of all admissible tenders and applications within the following time limits:
(a) in the case of tenders, the time limits laid down in the Implementing Regulation opening the tendering procedure shall apply;
(b) in the case of applications, notifications shall be submitted not later than 12.00 (Brussels time) each Tuesday and shall relate to the quantities of the products which, during the preceding week, have been the subject of an admissible application, and of the related information. The Commission can request that such notifications are made more frequently where such information is necessary for the purposes of managing the scheme.
Section II
Fixing the amount of aid for private storage via a tendering procedure
Article 43
Decisions on the maximum amount of aid for private storage
On the basis of the tenders notified in accordance with Article 42, the Commission shall decide, as provided for in Article 4(2)(a) of Regulation (EU) No 1370/2013:
(a) not to fix a maximum amount of the aid; or
(b) to fix a maximum amount of the aid.
By way of derogation from Article 2(6), an operator to whom an allocation coefficient applies may withdraw his tender within 10 working days of the date of entry into force of the decision fixing the allocation coefficient.
Article 44
Individual decisions on tenders
The paying agency shall only accept tenders that have been notified in accordance with Article 42.
The paying agency shall notify operators of the outcome of their participation in the tendering procedure within three working days of the entry into force of that decision of the Commission.
Section III
Fixing the amount of aid for private storage in advance
Article 45
Decisions on applications for aid for private storage fixed in advance
Where an examination of the situation reveals that excessive use has been made of the aid for private storage scheme, or that there is a risk of excessive use or speculation, the Commission may, without applying the procedure referred to in Article 229(2) or (3) of Regulation (EU) No 1308/2013, decide to:
(a) suspend the application of the scheme for not more than five working days; applications submitted during that period shall not be accepted;
(b) set a single percentage by which the quantities in the applications are reduced, subject to observance of the minimum contractual quantity where appropriate;
(c) reject applications made before the period of suspension the acceptance of which would have been decided during the period of suspension.
By way of derogation from Article 2(6), an operator to whom point (b) of the first subparagraph applies may withdraw his application within 10 working days of the date of entry into force of the decision setting the percentage reduction.
Section IV
Placing of products into private storage
Article 46
Information related to the place of private storage for products not yet in storage
Following receipt of the notification referred to in the second subparagraph of Article 44(3) or the notification of the decision referred to in Article 45(2), the operator shall notify the paying agency of the timeframe for the entry of products into storage, the name and address of each place of private storage and the corresponding quantities. The notification shall be provided to the paying agency at least five working days before the start of the placing lots into storage. The paying agency may decide to accept a shorter period than five working days.
Article 47
Placing into storage of products not yet in storage
For meat, placing in storage shall begin, for each individual lot of the quantity covered by the tender or application, on the day on which it comes under the control of the competent authority. That day shall be the day on which the net weight of the fresh or chilled product is determined:
(a) at the place of private storage, where the product is frozen on the premises;
(b) at the place of freezing, where the product is frozen in suitable facilities outside the place of private storage.
CHAPTER II
Storage contracts
Section I
Conclusion of contracts
Article 48
Contractual storage period
The contractual storage period starts on the day following:
(a) the date of the notification referred to in Article 44(3), or the date of receipt of an admissible application without prejudice to Article 45(1), for products already placed in storage;
(b) the date that placing into storage is considered completed in accordance with Article 47(3) for products which are not yet stored.
By way of derogation from Article 3(4) of Council Regulation (EEC, Euratom) No 1182/71 (5), when the last day of the contractual storage period falls on Saturday, Sunday or a public holiday, the contractual storage period shall end with the expiry of the last hour of that day.
Article 49
Conclusion of contracts
Contracts shall be concluded between the paying agency of the Member State in the territory of which the products are stored or will be stored and operators fulfilling the requirements laid down in Article 2 of Delegated Regulation (EU) 2016/1238 from whom a tender or application has been accepted.
Contracts shall be concluded for the quantity actually placed in storage (the ‘contractual quantity’), which shall not exceed the quantity referred to the second paragraph of Article 44(3) in the case of tenders, or the quantity in the application in respect of products already in store, or the notification of the decision referred to in Article 45(2) in the case of applications for products not yet in store.
Where the quantity actually placed in storage is less than 95 % of the quantity in the tender or application, or the quantity arising from the application of Article 45(3)(b), no contract shall be concluded.
Contracts shall not be concluded where the eligibility of the products is not confirmed.
Article 50
Notification of conclusion of contracts
The paying agency shall notify the successful operator of a contract considered as being concluded within five working days of the date of issue of the control report referred to in Article 61(1), subject to receiving all of the documents necessary for concluding the contract.
The date of conclusion of the contract shall be that on which the paying agency notifies the operator.
Section II
Elements of the contract and obligations of the operator
Article 51
Elements of the contract
The contract shall be construed as including, where appropriate, the elements provided for in Article 52, as well as those provided for in:
(a) the relevant provisions of the Implementing Regulation opening the tendering procedure and in the tender; or
(b) the relevant provisions of the Implementing Regulation fixing the amount of aid for private storage in advance and in the application.
Article 52
Obligations of the operator
Contracts shall cover at least the following obligations for the operator:
(a) to place and to keep the contractual quantity in storage, during the contractual storage period, at their own risk and expense under conditions ensuring the maintenance of the characteristics of the products referred to in Article 3 of Delegated Regulation (EU) 2016/1238, without: (i) substituting the stored products, except in the case of sugar in accordance with paragraph 3; (ii) or transferring them to another place of private storage or, in the case of sugar, to another silo;
(b) to retain the weighing-in documents established at the time of entry into the place of private storage;
(c) to send the documents relating to the operations of placing in storage, including the location of the storage lots/batches/vats/silos with the corresponding quantities, to the paying agency not later than five working days after the date of placing into storage referred to in Article 47(3);
(d) to allow the paying agency to check at any time that all the obligations laid down in the contract are being adhered to;
(e) to make the products stored easily accessible and individually identifiable by storage lot/batch/vat/silo.
By way of derogation from point (a)(ii) of paragraph 1, the paying agency may authorise a relocation of the stored products under the following conditions:
(i) for cheeses benefiting from a protected designation of origin (PDO) or from a protected geographical indication (PGI), where the operator submits a reasoned request;
(ii) for other products, in exceptional cases, where the operator submits a reasoned request.
The operator shall, on request, make available to the paying agency responsible for checking all documentation, for each contract, allowing in particular the following information on the products placed in storage to be verified:
(a) the number identifying the approved undertaking and, if necessary, the Member State of production;
(b) the origin and the date of production of the products, or for sugar the marketing year of production, and for meat the day of slaughtering;
(c) the date of placing into storage;
(d) the weight and, in the case of meat, the number of cuts packaged;
(e) the address of the place of private storage and the means allowing for ready identification of the product within the place of private storage or, for bulk sugar, the identification of the silo designated by the operator;
(f) the end date of the contractual storage period and the actual date of removal from contractual storage.
The operator or, where applicable, the storekeeper shall keep a register at the warehouse covering, by contract number:
(a) the identification of the products placed in storage by lot/batch/vat/silo;
(b) the dates of placing in and removal from storage;
(c) the quantity of products in storage by lot/batch/vat/silo;
(d) the location of the products by storage lot/batch/vat/silo within the store.
CHAPTER III
Removal of products and payment of aid for private storage
Section I
Removal of products from storage
Article 53
Removal from storage
However, in the case of sealed products as referred to in Article 60, only a sealed quantity may be removed from store.
The paying agency may decide to accept a shorter period than five working days.
Section II
Payment
Article 54
Application for payment of aid for private storage
The operator shall lodge an application for payment within three months of the end of the contractual storage period.
Article 55
Payment of aid for private storage
Payment of the aid shall be made not later than 120 days following the date on which an application for payment is lodged provided that the obligations of the contract have been met.
However, if an administrative inquiry is under way, payment shall not be made until entitlement has been recognised.
TITLE IV
CHECKS AND PENALTIES
CHAPTER I
Checks
Article 56
General provisions on checks relating to public intervention and aid for private storage
The measures shall include full administrative checking of offers and tenders for public intervention and tenders and applications for aid for private storage, which shall be supplemented by on-the-spot documentary and physical checks as specified in this Title.
Article 57
Specific provisions on checks relating to public intervention
Article 58
Specific provisions on checks relating to public intervention for cereals and rice
However, if the representative of the paying agency is also the storekeeper, the paying agency shall, within 30 days of the date of delivery, conduct an inspection involving at least a volumetric check. Any difference between the quantity determined by weighing and the quantity estimated in accordance with the volumetric method shall not exceed 5 %.
Where the 5 % tolerance is not exceeded, the storekeeper shall bear all costs relating to any difference observed, at a later weight check, from the weight entered in the accounts on takeover.
Where the 5 % tolerance is exceeded, the cereals or rice shall be weighed forthwith. Where the weight determined is less than that recorded, the costs of weighing shall be borne by the storekeeper. Otherwise, the costs of weighing shall be borne by the paying agency.
However, in the case of ochratoxin A and aflatoxin, if the paying agency concerned is able to prove to the Commission's satisfaction that the standards were met on entry, that normal storage conditions were observed and that the storekeeper's other commitments were respected, the financial liability shall be borne by the Union budget.
Article 59
Specific provisions for the takeover in storage place of the storekeeper for cereals and rice
If the takeover of cereals or rice takes place at the storage place where the products are held at the time the offer or tender is submitted, the quantity taken over shall be established on the basis of the store register, which shall fulfil professional standards which allow to guarantee compliance with Union legislation, in particular with Annex III to Delegated Regulation (EU) No 907/2014, and provided that:
(a) the store register shows: (i) the weight recorded on weighing carried out within a period of no more than 10 months before the takeover; (ii) the physical quality characteristics at the time of weighing and, in particular, the moisture content; (iii) trans-silages, if any, and treatments carried out;
(b) the storekeeper declares that the lot offered corresponds in all respects to the details contained in the store register;
(c) the quality characteristics established at the time of weighing are the same as those of the representative sample made up from the samples taken by the paying agency or its representative at a rate of one for every 60 tonnes.
Within 30 days of takeover the paying agency shall make a volumetric check. Any difference between the quantity determined by weighing and the quantity estimated in accordance with the volumetric method shall not exceed 5 %.
Where the 5 % tolerance is not exceeded, the storekeeper shall bear all costs relating to any difference observed, at a later weight check, from the weight entered in the accounts on takeover.
Where the 5 % tolerance is exceeded, the cereals or rice shall be weighed forthwith. Where the weight determined is less than that recorded, account being taken of the tolerance limits provided for in point 1 of Annex IV to Delegated Regulation (EU) No 907/2014, the costs of weighing shall be borne by the storekeeper. Otherwise, the costs of weighing shall be borne by the European Agricultural Guarantee Fund.
Article 60
Specific provisions on checks relating to aid for private storage
In the case of meat, the checks shall take place at the time of placing into private storage, and for olive oil, prior to the official sealing of the vats.
In duly justified circumstances, the paying agency may extend the period referred to in the first subparagraph by up to 15 days. In such cases, the paying agency shall inform the affected operators.
In the case of cheese, physical checks shall be undertaken on all lots in order to verify the contractual quantity.
The unannounced check referred in the first subparagraph shall not be necessary where the paying agency, with the agreement of the operator, has sealed the products in such a way that the contractual quantities cannot be removed from the place of storage without breaking seal.
In addition to the checks referred to in the first subparagraph, a representative statistical sample of at least 5 % of the lots covering at least 5 % of the total quantities for which contracts have been concluded shall be physically checked to verify the quantity, type, packaging and marking and identity of the products in the place of private storage.
Article 60a
Specific provision on checks relating to public intervention and aid for private storage for milk and milk products
Those methods shall be established by reference to the latest versions of the relevant European or international standards, as the case may be, in force at least 6 months before the first day of the public intervention period as defined in Article 12 of Regulation (EU) No 1308/2013.
Article 61
Reporting of checks
The paying agency shall draw up a control report within five working days of the completion of each on-the-spot check undertaken and, if appropriate, of the checks referred to in Article 56(3). The report shall describe precisely the different items checked and shall set out:
(a) the date and time of commencement of the check;
(b) details of any advance notice given;
(c) the duration of the check;
(d) the responsible persons present;
(e) the nature and extent of the checks carried out, providing, in particular, details of the documents and products examined;
(f) the findings and conclusions;
(g) whether any follow-up is required.
The report shall be signed by the responsible paying agency official and either countersigned by the operator or, where applicable, by the storekeeper, or sent to the operator by registrable means. The report shall be included in the payment file.
CHAPTER II
Penalties and administrative measures
Article 62
Penalties and administrative measures in relation to aid for private storage
TITLE V
NOTIFICATIONS AND FINAL PROVISIONS
CHAPTER I
Notifications
Section I
General provisions on notifications
Article 63
Method of notification
The notifications referred to in this Regulation, as well as in Implementing Regulations referred to in Article 1, shall be made in accordance with Commission Regulation (EC) No 792/2009 (7).
Article 64
Notifications concerning paying agencies
Section II
Notifications as regards public intervention
Article 65
Notifications of information on intervention stocks
Member States whose paying agencies hold intervention stocks shall notify the Commission, not later than the 15th of each month, of the following items:
(a) for cereals and rice: (i) the quantities stored from the beginning of the marketing year; (ii) the cumulative quantities taken over since the beginning of the marketing year; (iii) the cumulative quantities which have left the storage places since the beginning of the marketing year, identified where appropriate by type of use or destination, and the cumulative quantities lost; (iv) the cumulative quantities committed, identified where appropriate by type of use or destination; (v) the quantities under offer at the end of the monthly reporting period;
(b) for butter and skimmed-milk powder: (i) the quantities for each product in storage at the end of the previous month and the quantities entering and leaving the storage places during that month; (ii) a breakdown of the quantities of each product leaving the storage places during the previous month, according to the Regulation opening the tendering procedure for the sale of the products concerned; (iii) a breakdown by age of the quantities in storage at the end of the previous month;
(c) for beef: (i) the quantities for each product in storage at the end of the previous month and the quantities entering and leaving the storage places during that month; (ii) a breakdown of the quantities of each product leaving the storage places during the previous month, according to the Regulation opening the tendering procedure for the sale of the products concerned; (iii) the quantities of each cut covered by contracts of sale concluded in the previous month; (iv) the quantities of each cut covered by removal orders issued in the previous month; (v) the quantities of each cut bought in during the previous month; (vi) the uncommitted stocks and the physical stocks of each cut at the end of the previous month, with details of the length of time the uncommitted stocks have been in storage;
(d) for all products: (i) the opening of a tendering procedure, the quantities awarded and the minimum sale prices fixed in the case of application of Article 36; (ii) information relating to disposals for the scheme for food distribution to the most deprived, including the amount concerned (the difference between the selling price and the accounting value) and the moment at which that amount is transferred to the body to which payments are made by the Commission under Regulation (EU) No 223/2014 in accordance with Article 38b(4) of this Regulation.
For the purposes of point (b) of paragraph 1:
(a) ‘quantities entering’ means quantities physically placed in storage, whether or not taken over by the paying agency;
(b) ‘quantities leaving’ means quantities which have been removed or, if taken over by the purchaser before removal, quantities taken over.
For the purposes of point (c) of paragraph 1:
(a) ‘uncommitted stocks’ means stocks not yet covered by a contract of sale;
(b) ‘physical stocks’ means uncommitted stocks plus stocks covered by a contract of sale but not yet taken over.
Section III
Notifications as regards aid for private storage
Article 66
Notification of information on private storage
Member States in which the aid for private storage scheme is used shall notify the Commission:
(a) at least once a week of the products and quantities for which contracts have been concluded during the preceding week, broken down by storage period;
(b) not later than the 15th of each month for the previous month: (i) of the quantities of products placed into and leaving private storage during the month concerned, where applicable broken down by categories; (ii) of the quantities of products in private storage at the end of the month concerned, where applicable broken down by categories; (iii) of the quantities of products in respect of which the contractual storage period has ended; (iv) if the storage period has been curtailed or extended, as referred to in Article 20(m) of Regulation (EU) No 1308/2013, of the products and quantities in respect of which the storage period has been revised, and of the initial and revised dates for removal from storage;
(c) by 31 March each year for the preceding calendar year, of the results of the on-the-spot checks carried out pursuant to Title IV.
CHAPTER II
Final provisions
Article 67
Entry into force and application
This Regulation shall enter into force on the seventh day following that of its publication in the Official Journal of the European Union.
It shall apply as from 1 October 2016. However, as regards buying-in to public intervention, Tables III and IV of Part V and Point (b) of Part VI of Annex I shall apply as of 1 July 2017.
This Regulation shall be binding in its entirety and directly applicable in all Member States.
ANNEX I
CEREALS
PART I
For durum wheat, common wheat and barley, the definition of ‘broken grains’ is that contained in standard EN 15587.
For maize, the definition of ‘broken grains’ is that contained in standard EN 16378.
For durum wheat, common wheat and barley, the definition of ‘shrivelled grains’ is that contained in standard EN 15587. However, for barley from Estonia, Latvia, Finland and Sweden, ‘shrivelled grains’ means grains with a specific weight of at least 64 kilograms per hectolitre offered or tendered for, or placed in, intervention in those Member States, grains which, after elimination of all other matter referred to in this Annex, pass through sieves with apertures of 2,0 mm.
‘Shrivelled grains’ does not apply to maize.
For durum wheat, common wheat and barley, the definition of ‘other cereals’ is that contained in standard EN 15587.
For maize, the definition of ‘other cereals’ is that contained in standard EN 16378.
For durum wheat, common wheat and barley, the definition of ‘grains damaged by pests’ is that contained in standard EN 15587.
For maize, the definition of ‘grains damaged by pests’ is that contained in standard EN 16378.
For durum wheat and common wheat, the definition is that contained in standard EN 15587.
‘Grains in which the germ is discoloured’ does not apply to barley or maize.
For durum wheat, common wheat and barley, the definition of ‘grains overheated during drying’ is that contained in standard EN 15587.
For maize, the definition of ‘grains overheated during drying’ is that contained in standard EN 16378..
For durum wheat, the definition of ‘mottled grains’ is that contained in standard EN 15587.
‘Mottled grains’ does not apply to common wheat, barley or maize.
For durum wheat, common wheat and barley, the definition of ‘sprouted grains’ is that contained in standard EN 15587.
For maize, the definition of ‘sprouted grains’ is that contained in standard EN 16378.
For durum wheat, common wheat and barley, the definition of ‘extraneous seeds’ is that contained in standard EN 15587.
For maize, the definition of ‘extraneous seeds’ is that contained in standard EN 16378.
‘Noxious seeds’ means seeds which are toxic to humans and animals, seeds hampering or complicating the cleaning and milling of cereals and seeds affecting the quality of products processed from cereals.
For durum wheat, common wheat and barley, the definition of ‘damaged grains’ is that contained in standard EN 15587.
For maize, the definition of ‘damaged grains’ is that contained in standard EN 16378.
In standard EN 15587, for durum wheat, common wheat and barley, the definition of ‘grains affected by fusariosis’ is included in that of ‘damaged grains’.
For durum wheat, common wheat and barley, the definition of ‘extraneous matter’ is that contained in standard EN 15587.
For maize, the definition of ‘extraneous matter’ is that contained in standard EN 16378.
For durum wheat and common wheat, the definition of ‘decayed grains’ is that contained in standard EN 15587.
‘Decayed grains’ does not apply to barley or maize.
Mitadiné grains of durum wheat are grains whose kernels cannot be regarded as entirely vitreous. They are defined in standard EN 15585.
‘Grain impurities’ means shrivelled grains, grains of other cereals, grains damaged by pests, grains in which the germ is discoloured, mottled grains and grains overheated during drying.
‘Miscellaneous impurities’ means extraneous seeds, damaged grains (including grains affected by fusariosis), extraneous matter, husks, ergot, decayed grains and impurities of animal origin.
‘Grain impurities’ means shrivelled grains, grains of other cereals, grains damaged by pests, grains in which the germ is discoloured (only where the content exceeds 8 %) and grains overheated during drying.
‘Miscellaneous impurities’ means extraneous seeds, damaged grains (including grains affected by fusariosis), extraneous matter, husks, ergot, decayed grains and impurities of animal origin.
‘Grain impurities’ means shrivelled grains, grains of other cereals, grains damaged by pests and grains overheated during drying.
‘Miscellaneous impurities’ means extraneous seeds, damaged grains (including grains affected by fusariosis), extraneous matter, husks and impurities of animal origin.
‘Grain impurities’ means grains of other cereals, grains damaged by pests and grains overheated during drying.
‘Miscellaneous impurities’ means extraneous seeds, damaged grains, extraneous matter, cob fragments and impurities of animal origin.
PART II
Pursuant to Article 4, the following methods are to be used to determine the quality of cereals offered or tendered for, or placed in, intervention:
(a) reference method for determining matter other than basic cereals of unimpaired quality: (i) for common wheat, durum wheat and barley: standard EN 15587, (ii) for maize: standard EN 16378;
(b) reference method for determining the moisture content: (i) for maize: standard EN ISO 6540, (ii) for cereals other than maize: standard EN ISO 712, or an infrared technology-based method complying with standard EN 15948. In the event of a dispute, only the results resulting from applying standard EN ISO 6540 for maize and standard EN ISO 712 for cereals other than maize are to be considered valid;
(c) reference method for determining the non-stickiness and machinability of the dough obtained from common wheat: that set out in Part III of this Annex;
(d) reference method for determining the protein content in durum wheat and ground common wheat: that set out in: (i) standard EN ISO 20483, or (ii) standard CEN ISO/TS 16634-2. In the event of a dispute, only the results obtained from applying standard EN ISO 20483 are to be considered valid;
(e) reference method for determining the Zeleny index of ground common wheat: that set out in standard EN ISO 5529;
(f) reference method for determining the Hagberg falling number (amylase activity test): that set out in standard EN ISO 3093;
(g) reference method for determining the rate of loss of the vitreous aspect of durum wheat: that set out in standard EN 15585;
(h) reference method for determining the specific weight: that set out in standard EN ISO 7971/3;
(i) sampling and analysis methods for establishing the rate of mycotoxins: those referred to in the Annex to Commission Regulation (EC) No 1881/2006 (8) and set out in Annexes I and II to Commission Regulation (EC) No 401/2006 (9).
PART III
Method for test baking of wheat flour.
The method is applicable to flour, experimentally milled from wheat for the production of yeast-raised bread.
Dough is made from flour, water, yeast, salt and sucrose, in a specified mixer. After dividing and rounding, the pieces are given 30 minutes' rest; they are moulded, placed on baking sheets and baked after a final proof of fixed duration. Dough-handling properties are noted. The loaves are judged by volume and height.
Active dry yeast of type Saccharomyces cerevisiae DHW-Hamburg-Wansbeck or a product having the same characteristics.
Dissolve 30 ± 0,5 g of sodium chloride (commercial grade), 30 ± 0,5 g of sucrose (commercial grade), and 0,040 ± 0,001 g ascorbic acid in 800 ± 5 g of water. Prepare fresh daily.
Dissolve 5 ± 0,1 g sucrose (commercial grade) in 95 ± 1 g of water. Prepare fresh daily.
Commercial grade.
Controlled to maintain a temperature of 22 to 25 °C.
For maintaining a temperature of 4 ± 2 °C.
Maximum load 2 kg, accuracy 2 g.
Maximum load 0,5 kg, accuracy 0,1 g.
Accuracy 0,1 × 10– 3 g.
Stephan UMTA 10, with mixing arm model ‘Detmold’ (Stephan Soehne GmbH) or similar equipment having the same characteristics.
Controlled to maintain a temperature of 30 ± 1 °C.
Made from polymethylmethacrylate (Plexiglas, Perspex). Inside dimensions: 25 × 25 × 15 cm height, wall thickness 0,5 ± 0,05 cm.
Made from polymethylmethacrylate (Plexiglas, Perspex). At least 30 × 30 cm, thickness 0,5 ± 0,05 cm.
Brabender ball homogeniser (Brabender OHG) or similar equipment having the same characteristics.
According to standard EN ISO 24333.
Determine the water absorption according to ICC Standard No 115/1.
Determine the ‘falling number’ of the flour according to standard EN ISO 3093. If the ‘falling number’ is higher than 250, determine the malt flour addition required to bring it within the range 200 to 250, using a series of mixtures of the flour with increasing quantities of malt flour (point 4.5). If the ‘falling number’ is lower than 250, no malt flour is required.
Adjust the temperature of the sugar solution (point 4.4) to 35 ± 1 °C. Pour one part by weight of the active dry yeast into four parts by weight of this tempered sugar solution. Do not stir. Swirl if necessary.
Allow to stand for 10 ± 1 minute, then stir until a homogeneous suspension is obtained. Use this suspension within 10 minutes.
The temperature of the flour and the water must be adjusted to give a dough temperature of 27 ± 1 °C after mixing.
Weigh, with a precision of 2 g, 10 y/3 g flour on as-is moisture basis (corresponding to 1 kg flour on a 14 % moisture basis), in which ‘y’ is the quantity of flour used in the farinograph test (see ICC Standard No 115/1).
Weigh, with a precision of 0,2 g, the quantity of malt flour necessary to bring the ‘falling number’ within the range 200 to 250 (point 7.2).
Weigh 430 ± 5 g sugar-salt-ascorbic acid solution (point 4.3) and add water to a total mass of (x – 9) 10 y/3 g, (see point 10.2) in which ‘x’ is the quantity of water used in the farinograph test (see ICC Standard No 115/1). This total mass (usually between 450 and 650 g) must be achieved with a precision of 1,5 g.
Weigh 90 ± 1 g yeast suspension (point 7.3).
Note the total mass of the dough (P), which is the sum of the masses of flour, sugar-salt-ascorbic acid solution plus water, yeast suspension and malt flour.
Before starting, bring the mixer to a temperature of 27 ± 1 °C by use of a suitable quantity of tempered water.
Place the liquid dough ingredients in the mixer and place the flour plus malt flour on top.
Start the mixer (speed 1, 1 400 rev/min), and allow to run for 60 seconds. Twenty seconds after the start of mixing, turn the scraper attached to the lid of the mixing bowl two revolutions.
Measure the temperature of the dough. If it is outside the range 26 to 28 °C, discard the dough and mix a new one after adjustment of ingredient temperatures.
Note dough properties using one of the following terms:
— non-sticky and machinable, or
— sticky and non-machinable.
To be considered ‘non-sticky and machinable’ at the end of mixing, the dough should form a coherent mass which hardly adheres to the sides of the bowl and spindle of the mixer. It should be possible to collect the dough by hand and remove it from the mixing bowl in a single motion without noticeable loss.
Weigh, with precision of 2 g, three pieces of dough according to the formula:
Immediately round the pieces for 15 seconds in the moulder (point 5.10) and place them for 30 ± 2 minutes on the square plastic sheets (point 5.9), covered by the inverted plastic boxes (point 5.8) in the proving cabinet (point 5.7).
Do not use dusting flour.
Bring the pieces of dough on the plastic sheets, covered by the inverted boxes, to the moulder (point 5.10), and re-round each piece for 15 seconds. Do not remove cover from a piece of dough until immediately before rounding. Note dough properties again, using one of the following terms:
(a) non-sticky and machinable, or
(b) sticky and non-machinable.
To be considered as ‘non-sticky and machinable’ the dough should adhere hardly, or not at all, to the sides of the chamber so that it can freely rotate around itself and form a regular ball during the operation of the machine. At the end of the operation the dough should not stick to the sides of the dough-moulding chamber when the lid of the chamber is raised.
The test report shall mention:
(a) dough-handling properties at the end of mixing, and at moulding,
(b) the ‘falling number’ of the flour without addition of malt flour,
(c) any anomalies observed.
(d) the method used,
(e) all details required for the identification of the sample.
9.1.The formula for the calculation of the quantity of dough liquid is based on the following considerations:
Addition of x ml water to the equivalent of 300 g flour at 14 % moisture produces the required consistency. As in the baking test 1 kg of flour (14 % moisture basis) is used, whereas x is based on 300 g of flour, for the baking test x divided by three and multiplied by 10 g of water is needed, so 10 x/3 g.
The 430 g sugar-salt-ascorbic acid solution contains 15 g salt and 15 g sugar. This 430 g solution is included in the dough liquid. So to add 10 x/3 g water to the dough, (10 x/3 + 30) g dough liquid composed of the 430 g sugar-salt-ascorbic acid solution and an additional quantity of water must be added.
Although part of the water added with the yeast suspension is absorbed by the yeast, this suspension also contains ‘free’ water. It is arbitrarily supposed that 90 g yeast suspension contains 60 g ‘free’ water. The quantity of the dough liquid must be corrected for this 60 g of ‘free’ water in the yeast suspension, so 10 x/3 plus 30 minus 60 g must finally be added. This can be rearranged as follows: (10 x/3 + 30) – 60 = 10 x/3 – 30 = (x/3 – 3) 10 = (x – 9) 10/3, the formula given in point 7.5. If, for example, a water addition x in the farinograph test was found of 165 ml, this value must be substituted in this formula, so to the 430 g sugar-salt-ascorbic acid solution water must be added to a total mass of:
(165 – 9) 10/3 = 156 × 10/3 = 520 g.
9.2.The method is not directly applicable to wheat. The procedure to be followed for characterising the baking properties of wheat is as follows:
Clean the wheat sample, and determine the moisture content of the cleaned wheat. If the moisture content is within the range 15,0 % to 16,0 %, do not temper the wheat. If the moisture content is outside this range, adjust the moisture content to 15,5 ± 0,5 %, at least three hours prior to milling.
Mill the wheat into flour using a Buehler laboratory mill MLU 202 or a Brabender Quadrumat Senior mill or similar equipment having the same characteristics.
Choose a milling procedure that yields a flour of minimum 72 % extraction, with an ash content of 0,50 to 0,60 % on dry matter basis.
Determine the ash content of the flour according to Annex II to Commission Regulation (EU) No 234/2010 (10) and the moisture content according to this Regulation. Calculate the extraction rate by the equation:
E = (((100 – f) F)/(100 – w) W) × 100 %
where:
Note: Information concerning the ingredients and equipment to be used is published in Document T/77,300 of 31 March 1977 from the Instituut voor Graan, Meel en Brood, TNO — Postbus 15, Wageningen, Netherlands.
PART IV
1.For each lot of cereals, the quality characteristics shall be established on the basis of a representative sample of the lot offered, consisting of samples taken at the rate of once every delivery for at least every 60 tonnes.
2.The reference methods to be used for determining the quality of cereals offered or tendered for, or placed in, intervention are those set out in Parts I, II and III of this Annex.
3.In cases of dispute, the paying agency shall have the necessary tests on the cereals in question carried out again, the cost being met by the losing party.
PART V
| Moisture content (%) | Increases (EUR/tonne) |
|---|---|
| Less than 12.5 to 12 | 0,5 |
| Less than 12 to 11,5 | 1 |
| Less than 11,5 | 1.5 |
| Moisture content (%) | Increases (EUR/tonne) |
| --- | --- |
| Less than 12 to 11,5 | 0,5 |
| Less than 11,5 | 1 |
| Moisture content (%) | Reduction (EUR/tonne) |
| --- | --- |
| More than 13,0 to 13,5 | 0,5 |
| More than 13,5 to 14,0 | 1,0 |
| More than 14,0 to 14,5 | 1,5 |
| Moisture content (%) | Reduction (EUR/tonne) |
| --- | --- |
| More than 12,5 to 13,0 | 0,5 |
| More than 13,0 to 13,5 | 1,0 |
| Protein content (1) (N × 5,7) | Price increase (EUR/tonne) |
| --- | --- |
| More than 12,0 | 2,5 |
| (1) As % of dry matter. | |
| Protein content (1) (N × 5,7) | Price reduction (EUR/tonne) |
| --- | --- |
| Less than 11,5 to 11,0 | 2,5 |
| (1) As % of dry matter. |
PART VI
The price adjustments provided for in Article 26(1) shall be expressed in euro per tonne for offers or tenders for intervention by multiplying the price referred to in that Article by the sum of the established percentage increases or reductions, as follows:
(a) where the moisture content of cereals offered or tendered for intervention is less than 12,0 % for maize and 12,5 % for other cereals, the price increases to be applied shall be those listed in Table I of Part V of this Annex. Where the moisture content of these cereals offered or tendered for intervention is higher than 12,5 % for maize and 13,0 % for other cereals, the price reductions to be applied shall be those listed in Table II of Part V of this Annex;
(b) where the protein content of common wheat is higher than 12,0 %, the increases to be applied shall be those listed in Table III of Part V of this Annex. Where the protein content of common wheat is less than 11,5 %, the reductions to be applied shall be those listed in Table IV of Part V of this Annex.
ANNEX II
RICE
PART I
Methodology of sampling and analyses for paddy rice
1.With a view to verifying the quality requirements as laid down in Part I of Annex II to Delegated Regulation (EU) 2016/1238, samples shall be taken by the paying agency in the presence of the operator or his/her duly authorised agent.
Three representative samples, each weighing a minimum of one kilogram, shall be collected. One each shall go to:
(a) the operator;
(b) the storage place where takeover is to take place;
(c) the paying agency.
To make up the representative samples, the number of individual samples to be taken shall be obtained by dividing the quantity of the lot by 10 tonnes. Each individual sample shall weigh the same. The representative samples shall be made up of the sum of the individual samples, divided by three.
The quality requirements shall be verified using the representative sample intended for the store where takeover is to take place.
2.Representative samples shall be taken of each part-delivery (by lorry, barge, railway wagon) under the conditions laid down in point 1.
Before its entry into the intervention store the examination of each part-delivery can be restricted to a check of the moisture content and impurity level and verification that no live insects are present. However, if it later becomes apparent when the check is finalised that a part-delivery does not satisfy the minimum quality requirements, the concerned quantity shall be refused for takeover. If the paying agency is able to check all the minimum quality requirements for each part-delivery before it enters the store, it shall refuse takeover of any part-delivery that fails to satisfy these requirements.
3.The control of the radioactivity level is performed only if the situation so requires and for a limited period.
4.In cases of dispute, the paying agency shall have the necessary tests on the paddy rice in question carried out again, the cost being met by the losing party.
A new analysis is performed by a laboratory recognised by the paying agency on the basis of a new representative sample made up, in equal parts, by samples preserved by the operator and by the paying agency. In cases where there were part-deliveries of the lot tendered, the result is given by the weighted average of the results of analyses of new representative samples taken for each of the part-deliveries.
PART II
Prices increases and reductions
1.The price adjustments provided for in Article 26(1) shall be expressed in euro per tonne and apply to tenders for intervention by multiplying the price referred to in that Article by the sum of the established percentage increases in Tables I, II and III of this Part.
2.The price increases and decreases shall apply on the basis of the weighted average of the test results on the representative samples as defined in Part I of this Annex.
| Moisture content (%) | Increases (EUR/tonne) |
|---|---|
| Less than 12,5 to 12 | 0,75 |
| Less than 12 to 11,5 | 1,5 |
| Moisture content (%) | Reduction (EUR/tonne) |
| --- | --- |
| More than 13,5 to 14,0 | 0,75 |
| More than 14,0 to 14,5 | 1,5 |
| Yield of whole-grain milled paddy rice | Price increases per yield point (1) |
| --- | --- |
| Above the basic yield | 0,75 % increase |
| Overall yield of milled paddy rice | Price increases per yield point |
| Above the basic yield | 0,60 % increase |
| (1) To be applied where the milling yield of the rice differs from the basic milling yield for the variety concerned as set out in Part II of Annex II to Delegated Regulation (EU) 2016/1238. |
ANNEX III
BEEF
PART I
Conditions and controls for taking over
1.Products delivered shall be taken over subject to verification by the paying agency that they comply with the requirements laid down in Part I of Annex III to Delegated Regulation (EU) 2016/1238. In particular, a systematic check of the presentation, classification, weight and labelling of each carcass, half-carcass and quarter delivered shall be undertaken.
2.The failure to comply with the requirements laid down in Part I of Annex III to Delegated Regulation (EU) 2016/1238 shall result in rejection. Products rejected shall not be presented again for acceptance.
3.The outcome of the checks described in point 1 shall be systematically recorded by the paying agency.
PART II
Conversion coefficients
| Conformation class/fat cover | Coefficient |
|---|---|
| U2 | 1,058 |
| U3 | 1,044 |
| U4 | 1,015 |
| R2 | 1,015 |
| R3 | 1,000 |
| R4 | 0,971 |
| O2 | 0,956 |
| O3 | 0,942 |
| O4 | 0,914 |
PART III
Deboning
1.Deboning may only be carried out in cutting plants approved and operating in accordance with the requirements of Regulation (EC) No 853/2004 of the European Parliament and of the Council (11).
2.For the purposes of this Regulation ‘deboning operations’ means the physical operations for beef/veal as referred to in Annex II to Delegated Regulation (EU) No 906/2014.
3.Boned cuts must meet the requirements laid down in Part IV of this Annex.
1.Deboning shall be carried out under contract on terms laid down by the paying agencies, in accordance with their specifications and in compliance with the requirements of this Regulation.
2.The specifications of the paying agencies shall lay down the requirements to be met by cutting plants, shall specify the plant and equipment required and shall lay down detailed conditions covering deboning operations.
They shall in particular lay down detailed conditions specifying the method of preparation, trimming, packing, freezing and preservation of cuts with a view to their takeover by the paying agency.
The paying agencies shall take all the necessary measures to ensure that deboning operations are carried out in compliance with the requirements of this Regulation and with the contracts and specifications described in Section II of this Part.
In particular, the paying agencies shall put in place a system to ensure the continuous monitoring and verification of all deboning operations. The outcome of such monitoring and verification shall be recorded.
Cuts shall be stored in cold stores located in the territory of the Member State exercising jurisdiction over the paying agency.
Contracts as referred to in Section II of this Part and payments made thereunder shall cover the costs of deboning operations as referred to in point 2 of Section I of this Part.
Deboning, trimming, weighing, packaging and rapid freezing must be completed within 10 calendar days of slaughter. However, the paying agency may set shorter time limits.
1.When as a result of the checks provided for in Section III of this Part, products are found not to be in compliance with the requirements as laid down in this Regulation and with the contracts and specifications described in Section II of this Part they shall be rejected.
2.Without prejudice to the application of penalties, the paying agencies shall recover payments from the responsible parties for an amount equal to the price shown in Part V of this Annex for the cuts that have been rejected.
PART IV
Specifications for intervention deboning
Cutting and deboning: remove by a cut passing through the stifle joint and separating from the topside and the silverside by following the natural seam, leaving the heel muscle attached to the shank. Remove shank bones (tibia and hock).
Trimming: trim sinew tips back to the meat.
Wrapping and packing: these cuts must be individually wrapped before packing in cartons.
Cutting and deboning: separate from the topside by a straight cut down to and along the line of the femur and from the silverside by continuing the cut down in the line of the natural seam; the cap must be left naturally attached.
Trimming: remove the patella, the joint capsule and tendon; the external fat cover must not exceed one centimetre at any point.
Cutting and deboning: separate from the silverside and the shank by a cut following the line of the natural seam and detach from the femur; remove the aitchbone.
Trimming: remove the pizzle butt, the adjacent gristle and the scrotal (superficial inguinal) gland; remove the cartilage and connective tissues associated with the pelvic bone; the external fat cover must not exceed one centimetre at any point.
Cutting and deboning: separate from the topside and the shank by a cut following the line of the natural seam; remove the femur.
Trimming: remove the heavy cartilage adjacent to the bone joint, the popiteal lymph node, attached fat and tendon; the external fat cover must not exceed one centimetre at any point.
Cutting: remove entire length of fillet by freeing the head (butt end) from the hip bone (ilium) and by tracing along the fillet adjacent to the vertebrae, thereby freeing the fillet from the loin.
Trimming: remove gland and de-fat. Leave the silverskin and chain muscle intact and fully attached.
Cutting and deboning: separate from the silverside/thick flank by a straight cut from a point approximately five centimetres from the posterior edge of the fifth sacral vertebra, passing approximately five centimetres from the anterior edge of the aitchbone, taking care not to cut through the thick flank.
Separate from the loin by a cut between the last lumbar and first sacral vertebrae, clearing the anterior edge of the pelvic bone. Remove bones and cartilage.
Trimming: remove the pocket of fat on the internal surface below the eye muscle. The external fat cover must not exceed one centimetre at any point.
Cutting and deboning: separate from the rump by a straight cut between the last lumbar and the first sacral vertebrae. Separate from the fore-rib (five bone) by a straight cut between the eleventh and tenth ribs. Remove the backbones cleanly. Remove the ribs and feather bones by sheeting out.
Trimming: remove any species of cartilage left after deboning. The tendon must be removed. The external fat cover must not exceed one centimetre at any point.
Cutting and deboning: remove the full flank from the eight-rib straight-cut hindquarter by a cut from the point where the flank has been laid back, following the natural seam down around the surface of the hind muscles to a point which is horizontal to the middle of the last lumbar vertebra.
Continue the cut downwards in a straight line parallel to the fillet, through the thirteenth to the sixth rib inclusive along a line running parallel to the dorsal edge of the vertebral column, so that the entire downward cut is no more than five centimetres from the lateral tip of the eye muscle.
Remove all bones and cartilage by sheeting out. The whole flank must remain in one piece.
Trimming: remove the coarse connective tissue sheath covering the goose skirt, leaving the goose skirt intact. Trim fat so that the overall percentage of visible (external and interstitial) fat does not exceed 30 %.
Cutting and deboning: this cut must be separated from the striploin by a straight cut between the eleventh and tenth ribs and must include the sixth to tenth ribs inclusive. Remove the intercostal muscles and pleura in a thin sheet with rib bones. Remove backbone and cartilage, including the tip of the scapula.
Trimming: remove the backstrap (ligamentum nuchae). The external fat cover must not exceed one centimetre at any point. The cap must be left attached.
Cutting and deboning: remove by a cut around the joint separating the shinbone (radius) and clod-bone (humerus). Remove the shinbone (radius).
Trimming: trim sinew tips back to the meat.
Shins must not be packed with shanks.
Cutting and deboning: separate the shoulder from the forequarter by cutting in a line following the natural seam around the edge of the shoulder and the cartilage at the tip of the scapula, continuing around the seam so that the shoulder is lifted from its natural pocket. Remove the scapula. The blade muscle under the scapula must be laid back but left attached so as to allow clean removal of the bone. Remove the clod-bone (humerus).
Trimming: remove cartilage, tendons and joint capsules; trim fat so that the overall percentage of visible (external and interstitial) fat does not exceed 10 %.
Cutting and deboning: separate from the forequarter by cutting in a straight line perpendicular to the middle of the first rib. Remove intercostal muscles and pleura by ‘sheeting out’, with ribs, breastbone and cartilage. Deckle to be left attached to the brisket. Fat underlying the deckle and the sternum must be removed.
Trimming: trim fat so that the overall percentage of visible (external and interstitial) fat does not exceed 30 %.
Cutting and deboning: the cut remaining after removal of the brisket, shoulder and shin is classed as forequarter.
Remove rib bones by sheeting out. Neck bones must be removed cleanly.
The chain muscle must be left attached to this cut.
Trimming: tendons, joint capsules and cartilage to be removed. Trim fat so that the overall percentage of visible (external and interstitial) fat does not exceed 10 %.
PART V
Individual prices of rejected intervention cuts
| (EUR/tonne) | |
|---|---|
| Intervention fillet | 22 000 |
| Intervention striploin | 14 000 |
| Intervention topside, Intervention rump | 10 000 |
| Intervention silverside, Intervention thick flank, Intervention forerib (with five ribs) | 8 000 |
| Intervention shoulder, Intervention forequarter | 6 000 |
| Intervention brisket, Intervention shank, Intervention shin | 5 000 |
| Intervention flank | 4 000 |
ANNEX IV
BUTTER
PART I
Sampling for chemical and microbiological analysis and sensory evaluation
| Quantity of butter (kg) | Minimum number of samples (> 100 g) |
|---|---|
| ≤ 1 000 | 2 |
| > 1 000 ≤ 5 000 | 3 |
| > 5 000 ≤ 10 000 | 4 |
| > 10 000 ≤ 15 000 | 5 |
| > 15 000 ≤ 20 000 | 6 |
| > 20 000 ≤ 25 000 | 7 |
| > 25 000 | 7 + 1 per 25 000 kg or part thereof |
Sampling for microbiological analysis must be carried out aseptically.
Up to five samples of 100 g may be combined into one sample for analysis after thorough mixing.
The samples must be taken randomly from different parts of each lot before or at the time of entry into the cold store designated by the paying agency.
Preparation of composite butter sample (chemical analysis):
(a) using a clean, dry butter trier or similar suitable instrument, extract a core of butter of at least 30 g and place in a sample container. The composite sample must then be sealed and forwarded to the laboratory for analysis;
(b) at the laboratory the composite sample is to be warmed in the original unopened container to 30 °C and shaken frequently until a homogeneous fluid emulsion free of unsoftened pieces is obtained. The container should be one half to two thirds full.
Two samples per year per producer offering butter for intervention must be analysed for non-milk fat.
| Quantity of butter (kg) | Minimum number of samples |
|---|---|
| 1 000 ≤ 5 000 | 2 |
| > 5 000 ≤ 25 000 | 3 |
| > 25 000 | 3 + 1 per 25 000 kg or part thereof |
Following a trial storage period of 30 days, samples are to be taken randomly from different parts of each lot between the 30th and the 45th day following delivery of the butter and graded.
Each sample shall be assessed individually. No resampling or re-evaluation is allowed.
(a)chemical and microbiological analysis:
(i) where individual samples are analysed, one sample showing a single defect out of five to 10 samples or two samples each showing a single defect out of 11 to 15 samples may be allowed. Where a sample shows a defect, two new samples must be taken from either side of the sample showing the defect and checked for the parameter in question. Where neither sample meets the specification, the quantity of butter between the original two samples on either side of the sample showing the defect must be rejected from the quantity offered. Quantity to be rejected where the new sample shows a defect:
(ii) where composite samples are analysed and found to show defects in respect of one parameter, the quantity represented by the composite sample concerned is to be rejected from the quantity offered. The quantity represented by one composite sample may be determined by subdividing the quantity before samples are taken randomly from each part thereof;
(b)sensory evaluation: where a sample fails the sensory evaluation, the quantity of butter between two neighbouring samples on either side of the sample failing is to be rejected from the quantity of the lot,
(c)where samples show a sensory defect and either a chemical or a microbiological defect, the whole quantity is to be rejected.
PART IA
Methods of analysis of unsalted butter for public intervention
| Parameter | Method |
|---|---|
| Fat (1) | ISO 17189 or ISO 3727 part 3 |
| Water | ISO 3727 part 1 |
| Non-fat solids | ISO 3727 part 2 |
| Fat acidity | ISO 1740 |
| Peroxide value | ISO 3976 |
| Non-milk fat | ISO 17678 |
| Sensory characteristics | ISO 22935 parts 2 and 3 and scoring table hereafter. |
| (1) The method to be applied shall be approved by the paying agency. | |
| Appearance | Consistency |
| --- | --- |
| Points | Remarks |
| 5 | Very good Ideal type Highest quality (equal dry) |
| 4 | Good (no evident defects) |
| 1, 2 or 3 | Any defect |
PART II
Delivery and packaging of butter
1.Butter shall be delivered in blocks and packed in new, strong material in such a way as to ensure it is protected throughout transportation, entry into storage, storage and removal from storage.
2.The packing shall show at least the following particulars, where appropriate in code:
(a) the approval number identifying the factory and the Member State of production;
(b) the date of production;
(c) the production batch number and the package number; the package number may be replaced by a pallet number marked on the pallet;
(d) the words ‘sweet cream’ if the butter has a pH of 6,2 or higher.
3.The storekeeper shall keep a register in which the particulars referred to in point 2 are recorded on the date of entry into storage.
ANNEX V
SKIMMED MILK POWDER
PART I
Sampling and analysis of skimmed-milk powder offered for intervention
1.Samples per lot shall be taken in accordance with the procedure laid down in International Standard ISO 707. However, paying agencies may use another method of sampling provided that it complies with the principles of that standard.
2.Number of packages to be selected for taking samples for analysis:
(a) lots containing up to 800 25-kg bags: at least eight;
(b) lots containing more than 800 25-kg bags: at least eight, plus one for each additional 800 bags or fraction thereof.
3.Weight of sample: samples of at least 200 g are to be taken from each package.
4.Grouping of samples: no more than nine samples are to be combined in a global sample.
5.Analysis of samples: each global sample is to undergo an analysis to verify all the quality characteristics laid down in Part II of Annex V to Delegated Regulation (EU) 2016/1238.
6.Where samples show defects:
(a) where a composite sample shows a defect with regard to one parameter, the quantity from which the sample came is rejected;
(b) where a composite sample shows a defect with regard to more than one parameter, the quantity from which the sample came is rejected and samples are taken from the remaining quantities from the same plant; the analysis of those samples shall be decisive. In that case: — the number of samples laid down in point 2 is doubled, — where a composite sample shows a defect with regard to one or more parameters, the quantity from which the sample came is rejected.
PART IA
Methods of analysis of skimmed milk powder for public intervention
| Parameter | Method |
|---|---|
| Protein | ISO 8968 part 1 |
| Fat | ISO 1736 |
| Water | ISO 5537 |
| Acidity | ISO 6091 |
| Lactates | ISO 8069 |
| Phosphatase test | ISO 11816 part 1 |
| Insolubility index | ISO 8156 |
| Scorched particles (1) | ADPI |
| Micro-organisms | ISO 4833-part 1 |
| Buttermilk | Appendix I |
| Rennet whey (2) | Appendix II and III |
| Acid whey (3) | ISO 8069 or On-the-spot inspections |
| Sensory checks (4) | ISO 22935 part 2 and 3 |
| (1) Scorched particles' analyses may be conducted systematically. However, such analyses shall always be conducted if no sensory checks are performed. (2) The method to be applied shall be approved by the paying agency (one or both methods). (3) The method to be applied shall be approved by the paying agency. (4) Sensory checks shall be performed where deemed necessary after risk based analysis approved by the paying agency. |
Appendix I
The method describes a procedure for the quantitative determination of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in skimmed milk powder (SMP) and is suitable for detecting buttermilk solids in SMP.
PS + PE content : the mass fraction of substance determined using the procedure here specified. The result is expressed as milligrams of phosphatidylethanolamine dipalmitoyl (PEDP) per 100 g powder.
Extraction of aminophospholipids by methanol from reconstituted milk powder. Determination of PS and PE as o-phthaldialdehyde (OPA) derivatives by reversed-phase (RP) HPLC and fluorescence detection. Quantification of PS and PE content in the test sample by reference to a standard sample containing a known amount of PEDP.
All reagents shall be of recognised analytical grade. Water shall be distilled or water of at least equivalent purity, unless otherwise specified.
Note: Standard material shall be stored at – 18 °C.
4.2.1. HPLC-grade methanol
4.2.2. HPLC-grade chloroform
4.2.3. Tryptamine-monohydrochloride
4.3.1. Sodium hydroxide, 12 M water solution
4.3.2. Boric acid, 0,4 M water solution adjusted to pH 10,0 with sodium hydroxide (4.3.1)
4.3.3. 2-mercaptoethanol
4.3.4. o-phthaldialdehyde (OPA)
4.4.1. Elution solvents shall be prepared using HPLC-grade reagents.
4.4.2. HPLC-grade water
4.4.3. Methanol of tested fluorimetric purity
4.4.4. Tetrahydrofuran
4.4.5. Sodium dihydrogen phosphate
4.4.6. Sodium acetate
4.4.7. Acetic acid.
5.1. Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg
5.2. Beakers, 25 and 100 ml capacity
5.3. Pipettes, capable of delivering 1 and 10 ml
5.4. Magnetic stirrer
5.5. Graduated pipettes, capable of delivering 0,2, 0,5 and 5 ml
5.6. Volumetric flasks, 10, 50 and 100 ml capacity
5.7. Syringes, 20 and 100 μl capacity
5.8. Ultrasonic bath
5.9. Centrifuge, capable of operating at 27 000 × g
5.10. Glass vials, about 5 ml capacity
5.11. Graduated cylinder, 25 ml capacity
5.12. pH-meter, accurate to 0,1 pH units
Sampling shall be carried out in accordance with ISO Standard 707.
7.1.1. Weigh 30,0 ± 0,1 mg of tryptamine-monohydrochloride (4.2.3) into a 100 ml volumetric flask (5.6) and make up to the mark with methanol (4.2.1)
7.1.2. Pipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6) and make up to the mark with methanol (4.2.1) in order to obtain a 0,15 mM tryptamine concentration
7.2.1. Weigh 1,000 ± 0,001 g of the SMP sample into a 25 ml beaker (5.2). Add 10 ml of distilled water at 40 °C ± 1 °C by a pipette (5.3) and stir with a magnetic stirrer (5.4) for 30 minutes in order to dissolve any lumps
7.2.2. Pipette 0,2 ml (5.5) of the reconstituted milk into a 10 ml volumetric flask (5.6), add 100 μl of the 0,15 mM tryptamine solution (7.1) using a syringe (5.7) and make up to the volume with methanol (4.2.1). Mix carefully by inversion and sonicate (5.8) for 15 min
7.2.3. Centrifuge (5.9) at 27 000 g × g for 10 minutes and collect the supernatant in a glass vial (5.10) Note: Test sample solution should be stored at 4 °C until the HPLC analysis is performed.
7.3.1. Weigh 55,4 mg PEDP (4.1) into a 50 ml volumetric flask (5.6) and add about 25 ml of chloroform (4.2.2) using a graduated cylinder (5.11). Heat the stoppered flask to 50 °C ± 1 °C and mix carefully till the PEDP dissolves. Cool the flask to 20 °C, make up to the volume with methanol (4.2.1) and mix by inversion
7.3.2. Pipette 1 ml (5.3) of this solution into a 100 ml volumetric flask (5.6) and make up to the volume with methanol (4.2.1). Pipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6), add 100 μl (5.7) of 0,15 mM tryptamine solution (7.1) and make up to the volume with methanol (4.2.1). Mix by inversion Note: Reference sample solution should be stored at 4 °C until the HPLC analysis is performed.
Weigh 25,0 ± 0,1 mg of OPA (4.3.4) into a 10 ml volumetric flask (5.6), add 0,5 ml (5.5) of methanol (4.2.1) and mix carefully to dissolve the OPA. Make up to the mark with boric acid solution (4.3.2) and add 20 μl of 2-mercaptoethanol (4.3.3) by syringe (5.7).
Note: The derivatising reagent should be stored at 4 °C in a brown glass vial and is stable for one week.
Solvent A: Solution of 0,3 mM sodium dihydrogen phosphate and 3 mM sodium acetate solution (adjusted to pH 6,5 ± 0,1 with acetic acid): methanol: tetrahydrofuran = 558:440:2 (v/v/v)
Solvent B: methanol
| Time (min) | Solvent A (%) | Solvent B (%) | Flow rate (ml/min) |
|---|---|---|---|
| Initial | 40 | 60 | 0 |
| 0,1 | 40 | 60 | 0,1 |
| 5,0 | 40 | 60 | 0,1 |
| 6,0 | 40 | 60 | 1,0 |
| 6,5 | 40 | 60 | 1,0 |
| 9,0 | 36 | 64 | 1,0 |
| 10,0 | 20 | 80 | 1,0 |
| 11,5 | 16 | 84 | 1,0 |
| 12,0 | 16 | 84 | 1,0 |
| 16,0 | 10 | 90 | 1,0 |
| 19,0 | 0 | 100 | 1,0 |
| 20,0 | 0 | 100 | 1,0 |
| 21,0 | 40 | 60 | 1,0 |
| 29,0 | 40 | 60 | 1,0 |
| 30,0 | 40 | 60 | 0 |
Note: The eluting gradient may require slight modification in order to achieve the resolution shown in figure 1.
Column temperature: 30 °C.
7.5.3. Injection volume: 50 μl derivatising reagent and 50 μl sample solution
7.5.4. Starting up the system on a daily basis, flush the column with 100 % solvent B for 15 minutes, then set at A:B = 40:60 and equilibrate at 1 ml/min for 15 minutes. Perform a blank run by injecting methanol (4.2.1). Note: Before long-term storage flush the column with methanol: chloroform = 80:20 (v/v) for 30 minutes.
7.5.5. Determine the PS + PE content in the test sample
7.5.6. Perform the sequence of the chromatographic analyses keeping constant the run-to-run time in order to obtain constant retention times. Inject the external standard solution (7.3) every 5-10 test sample solutions in order to calculate the response factor Note: The column shall be cleaned by flushing with 100 % solvent B (7.5.1) for at least 30 minutes every 20-25 runs.
PEDP is eluted as a single peak. Determine the peak area by valley-to- valley integration.
Tryptamine is eluted as a single peak (Figure 1). Determine the peak area by valley-to-valley integration.
Under the described conditions (Figure 1), PS elutes as two main partially unresolved peaks preceded by a minor peak. PE elutes as three main partially unresolved peaks. Determine the whole area of each peak cluster setting the baseline as reported in Figure 1.
PS and PE content in the test sample shall be calculated as follows:
C = 55,36 × ((A2)/(A1)) × ((T1)/(T2))
where:
Note: The values for repeatability were calculated according to the IDF International Standard (*).
The relative standard deviation of the repeatability, which expresses the variability of independent analytical results obtained by the same operator using the same apparatus under the same conditions on the same test sample and in a short interval of time, should not exceed 2 % relative. If two determinations are obtained under these conditions, the relative difference between the two results should not be greater than 6 % of the arithmetic mean of the results.
If two determinations are obtained by operators in different laboratories using different apparatus under different conditions for the analysis on the same test sample, the relative difference between the two results should not be greater than 11 % of the arithmetic mean of the results.
10.1. Resmini P., Pellegrino L., Hogenboom J.A., Sadini V., Rampilli M., ‘Detection of buttermilk solids in skimmilk powder by HPLC quantification of aminophospholipids’. Sci. Tecn. Latt.-Cas., 39,395 (1988).
Appendix II
This method allows detection of rennet whey in skimmed milk powder intended for public storage by determination of the caseinomacropeptides.
International Standard ISO 707 - Milk and Milk Products - Guidance on sampling.
The content of rennet whey solids is defined as the percentage by mass as determined by the caseinomacropeptide content by the procedure described.
— Reconstitution of the skimmed milk powder, removal of fat and proteins with trichloroacetic acid, followed by centrifugation or filtration;
— Determination of the quantity of caseinomacropeptides (CMP) in the supernatant by high-performance liquid chromatography (HPLC);
— Evaluation of the result obtained for the samples by reference to standard samples consisting of skimmed milk powder with or without the addition of a known percentage of whey powder.
All reagents shall be of recognised analytical grade. The water used shall be distilled water or water of at least equivalent purity.
Dissolve 240 g of trichloroacetic acid (CCl3COOH) in water and make up to 1 000 ml. The solution should be clear and colourless.
Dissolve 1,74 g of dipotassium hydrogen phosphate (K2HPO4), 12,37 g of potassium dihydrogen phosphate (KH2PO4) and 21,41 g of sodium sulphate (Na2SO4) in about 700 ml of water. Adjust, if necessary, to pH 6,0, using a solution of phosphoric acid or potassium hydroxide.
Make up to 1 000 ml with water and homogenise.
Note: The composition of the eluent can be updated to comply with the certificate of the standards or the recommendations of the manufacturer of the column packing material.
Filter the eluent solution, prior to use, through a membrane filter with a 0,45 μm pore diameter.
Mix one volume acetonitrile (CH3CN) with nine volumes water. Filter the mixture prior to use through a membrane filter with a 0,45 μm pore diameter.
Note: Any other flushing solvent with a bactericidal effect which does not impair the columns' resolution efficiency may be used.
5.4.1. Skimmed milk powder meeting the requirements of this Regulation (i.e. [0])
5.4.2. The same skimmed milk powder adulterated with 5 % (m/m) rennet-type whey powder of standard composition (i.e. [5])
6.1. Analytical balance
6.2. Optional centrifuge capable of attaining a centrifugal force of 2 200 g, fitted with stoppered or capped centrifuge tubes of about 50 ml capacity
6.3. Mechanical shaker
6.4. Magnetic stirrer
6.5. Glass funnels, diameter about 7 cm
6.6. Filter papers, medium filtration, diameter about 12,5 cm
6.7. Glass filtration equipment with 0,45 μm pore diameter membrane filter
6.8. Graduated pipettes allowing delivery of 10 ml (ISO 648, Class A, or ISO/R 835) or a dispensing system capable of delivering 10,0 ml in two minutes
6.9. Dispensing system capable of delivering 20,0 ml water at ca. 50 °C
6.10. Thermostatic water bath, set at 25 ± 0,5 °C
6.11. HPLC equipment, consisting of:
7.1. Samples shall be taken in accordance with the procedure laid down in International Standard ISO 707. However, Member States may use another method of sampling provided that it complies with the principles of the abovementioned standard
7.2. Store the sample in conditions which preclude any deterioration or change in composition
Transfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated inversion of the container.
Weight 2,000 ± 0,001 g of test sample into a centrifuge tube (6.2) or a suitable stoppered flask (50 ml).
8.3.1. Add 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using a mechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C
8.3.2. Add 10,0 ml of the trichloroacetic acid solution (5.1) of ca. 25 °C in two minutes, while stirring vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes
8.3.3. Centrifuge (6.2) for 10 minutes at 2 200 g, or filter through paper (6.6), discarding the first 5 ml of filtrate
8.4.1. Inject 15 to 30 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus (6.11) operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute Note 1. Another flow rate may be used, dependent of the internal diameter of the columns used or the instructions of the manufacturer of the column. Note 2. Rinse the columns with water during each interruption. Never leave the eluent solution in them (5.2). Prior to any interruption of more than 24 hours, rinse the columns with water then wash them with solution (5.3) for at least three hours at a flow rate of 0,2 ml per minute.
| 8.4.2. | The results of chromatographic analysis of the test sample [E] are obtained in the form of chromatogram in which each peak is identified by its retention time RT as follows: Peak II: The second peak of the chromatogram having an RT of about 12,5 minutes. Peak III: The third peak of the chromatogram, corresponding to the CMP, having an RT of 15,5 minutes. The choice of the column(s) can affect the retention times of the individual peaks considerably. The integrator (6.11.6) automatically calculates the area A of each peak: AII: area of peak II, AIII: area of peak III, It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to detect any abnormalities due either to malfunctioning of the apparatus or the columns, or to the origin and nature of the sample analysed. If in doubt, repeat the analysis. |
|---|---|
| Peak II: | The second peak of the chromatogram having an RT of about 12,5 minutes. |
| Peak III: | The third peak of the chromatogram, corresponding to the CMP, having an RT of 15,5 minutes. |
| AII: | area of peak II, |
| AIII: | area of peak III, |
8.5.1. Apply exactly the procedure described from point 8.2 to point 8.4.2 to the standard samples (5.4) Use freshly prepared solutions, because CMP degrade in an 8 % trichloroacetic environment. The loss is estimated at 0,2 % per hour at 30 °C.
8.5.2. Prior to chromatographic determination of the samples, condition the columns by repeatedly injecting the standard sample (5.4.2) in solution (8.5.1) until the area and retention time of the peak corresponding to the CMP are constant
8.5.3. Determine the response factors R by injecting the same volume of filtrates (8.5.1) as used for the samples
| Peak II: | RII = 100/(AII[0]) |
|---|---|
where:
| Peak III: | RIII = W/(AIII[5] – AIII[0]) |
|---|---|
where:
where:
RRTIII[E] = (RTIII[E])/(RTIII[5])
where:
— The RRTIII [E] is < 1,000 when the whey content is > 5 %;
— The RRTIII [E] is ≥ 1,000 when the whey content is ≤ 5 %.
The uncertainty allowed for the values of RRTIII is ± 0,002.
Normally the value of RRTIII [0] deviates little from 1,034. Depending on the condition of the columns, the value may approach 1,000, but it shall always be greater.
W = SIII[E] – [1, 3 + (SIII[0] – 0,9)]
where:
The difference between the results of two determinations carried out simultaneously or in rapid succession by the same analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.
The difference between two single and independent results, obtained in two different laboratories on identical test material shall not exceed 0,4 % m/m.
| 9.4.1. | Assume the absence of whey if the relative area of peak III, SIII [E] expressed in grams of rennet whey per 100 g of the product is ≤ 2,0 + (SIII[0] – 0,9) where 2,0 is the maximum value allowed for the relative area of peak III taking into account the relative average area of peak III, i.e. 1,3, the uncertainty due to variations in the composition of skimmed milk powder and the reproducibility of the method (9.3.2), (SIII [0] – 0,9) is the correction to be made when the area SIII [0] is different from 0,9 (see point 9.2) |
|---|---|
| 2,0 | is the maximum value allowed for the relative area of peak III taking into account the relative average area of peak III, i.e. 1,3, the uncertainty due to variations in the composition of skimmed milk powder and the reproducibility of the method (9.3.2), |
| (SIII [0] – 0,9) | is the correction to be made when the area SIII [0] is different from 0,9 (see point 9.2) |
9.4.2. If the relative area of peak III, SIII [E] is > 2,0 + (SIII[0] – 0,9) and the relative area of peak II, SII [E] ≤ 160, determine the rennet whey content as indicated in point 9.2.
| 9.4.3. | If the relative area of peak III, SIII [E] is > 2,0 + (SIII[0] – 0,9) and the relative area of peak II, SII [E] ≤ 160, determine the total protein content (P %); then examine graphs 1 and 2. 9.4.3.1. The data obtained after analysis of samples of unadulterated skimmed milk powders with a high total protein content have been assembled in graphs 1 and 2. The continuous line represents the linear regression, the coefficients of which are calculated by the least squares method. The dashed straight line fixes the upper limit of the relative area of peak III with a probability of not being exceeded in 90 % of cases. The equations for the dashed straight lines of graphs 1 and 2 are: SIII = 0,376 P % – 10,7 (graph 1), SIII = 0,0123 SII [E] + 0,93 (graph 2), respectively where: SIII is the relative area of peak III calculated either according to total protein content or according to the relative area of peak SII [E], P % is the total protein content expressed as a percentage, by weight, SII [E] is the relative area of sample calculated in point 9.1.2. These equations are equivalent to the figure of 1,3 mentioned in point 9.2. The discrepancy (T1 and T2) between the relative area SIII [E] found and the relative area SIII is given by means of the following: T1 = SIII[E] – [(0,376 P% – 10,7) + (SIII[0] – 0,9)]T2 = SIII[E] – [(0,0123 SII[E] + 0,93) + (SIII[0] – 0,9)] 9.4.3.2. If T1 and/or T2 are zero or less, the presence of rennet whey cannot be determined. If T1 and T2 exceed zero, rennet whey is present. The rennet whey content is calculated according to the following formula: W = T2 + 0,91 where: 0,91 is the distance on the vertical axis between the continuous and dotted straight lines. |
|---|---|
| SIII = 0,376 P % – 10,7 | (graph 1), |
| SIII = 0,0123 SII [E] + 0,93 | (graph 2), |
| SIII | is the relative area of peak III calculated either according to total protein content or according to the relative area of peak SII [E], |
| P % | is the total protein content expressed as a percentage, by weight, |
| SII [E] | is the relative area of sample calculated in point 9.1.2. |
| 9.4.3.2. | If T1 and/or T2 are zero or less, the presence of rennet whey cannot be determined. If T1 and T2 exceed zero, rennet whey is present. The rennet whey content is calculated according to the following formula: W = T2 + 0,91 where: 0,91 is the distance on the vertical axis between the continuous and dotted straight lines. |
| If T1 and/or T2 | are zero or less, the presence of rennet whey cannot be determined. |
| If T1 and T2 | exceed zero, rennet whey is present. |
Appendix III
PURPOSE: DETECTING THE ADDITION OF RENNET WHEY SOLIDS TO SKIMMED MILK POWDER
REFERENCES: INTERNATIONAL STANDARD ISO 707
The content of rennet whey solids is defined as the percentage by mass as determined by caseinomacropeptide content by the procedure described.
Samples are analysed for caseinomacropeptide A by a reversed-phase high-performance liquid chromatography procedure (HPLC procedure). Evaluation of the result is obtained by reference to standard samples consisting of skimmed milk powder with and without a known percentage of whey powder. Results higher than 1 % (m/m) show that rennet whey solids are present.
| 8. | PROCEDURE8.1. Preparation of the test sample Transfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated inversion of the container. 8.2. Test portion Weigh 2,00 ± 0,001 g of test sample into a centrifuge tube (6.2) or suitable stoppered flask (50 ml). Note: In the case of mixtures, weigh such an amount of the test sample that the defatted sample portion corresponds to 2,00 g. 8.3. Removal of fat and proteins 8.3.1. Add 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using a mechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C 8.3.2. Add 10,0 ml of the trichloroacetic acid solution of ca. 25 °C (5.1) constantly over two minutes, while stirring vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes 8.3.3. Centrifuge (6.2) 2 200 g for 10 minutes, or filter through paper (6.6), discarding the first 5 ml of filtrate 8.4. Chromatographic determination 8.4.1. The reversed-phase HPLC method excludes the possibility false-positive results due to the presence of acid buttermilk powder. 8.4.2. Before the reversed phase HPLC-analysis is carried out, the gradient conditions should be optimised. A retention time of 26 ± 2 minutes for CMPA is optimal for gradient systems having a dead volume of about 6 ml (volume from the point where the solvents come together to the volume of the injector loop, inclusive). Gradient systems having a lower dead volume (e.g. 2 ml) should use 22 minutes as an optimal retention time Take solutions of the standard samples (5.4) without and with 50 % rennet whey. Inject 100 μl of supernatant or filtrate (8.3.3) into the HPLC apparatus operating at the scouting gradient conditions given in Table 1. Table 1 Scouting gradient conditions for optimisation of the chromatography Time (min) Flow (ml/min) % A % B Curve Initial 1,0 90 10 * 27 1,0 60 40 linear 32 1,0 10 90 linear 37 1,0 10 90 linear 42 1,0 90 10 linear Comparison of the two chromatograms should reveal the location of the peak of CMPΑ. Using the formula given below, the initial solvent composition to be used for the normal gradient (see 8.4.3) can be calculated % B = 10 – 2,5 + (13,5 + (RTcmpA – 26) / 6) * 30 / 27 % B = 7,5 + (13,5 + (RTcmpA – 26) / 6) * 1,11 Where: RTcmpA : retention time of CMPΑ in the scouting gradient 10 : the initial % B of the scouting gradient 2,5 : % B at midpoint minus % B at initial in the normal gradient 13,5 : midpoint time of the scouting gradient 26 : required retention time of CMPΑ 6 : ratio of slopes of the scouting and normal gradient 30 : % B at initial minus % B at 27 minutes in the scouting gradient 27 : run-time of the scouting gradient. 8.4.3. Take solutions of the test samples Inject 100 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute. The composition of the eluent of the start of the analysis is obtained from 8.4.2. It is normally close to A:B = 76:24 (5.2). Immediately after the injection a linear gradient is started, which results in a 5 % higher percentage of B after 27 minutes. Subsequently a linear gradient is started, which brings the eluent composition to 90 % B in five minutes. This composition is maintained for five minutes, after which the composition is changed, via a linear gradient in five minutes to the initial composition. Depending on the internal volume of the pumping system, the next injection can be made 15 minutes after reaching the initial conditions. Note 1. The retention time of the CMPA should be 26 ± 2 minutes. This can be achieved by varying the initial and end conditions of the first gradient. However, the difference in the % B for the initial and end conditions of the first gradient shall remain 5 % B. Note 2. The eluents should be degassed sufficiently and should also remain degassed. This is essential for proper functioning of the gradient pumping system. The standard deviation for the retention time of the CMPA peak should be smaller than 0,1 minutes (n = 10). Note 3. Every five samples the reference sample [5] should be injected and used to calculate a new response factor R. (9.1.1). 8.4.4. The results of the chromatographic analysis of the test sample (E) are obtained in the form of a chromatogram in which the CMPA peak is identified by its retention time of about 26 minutes The integrator (6.11.6) automatically calculates the peak height H of the CMPA peak. The baseline location should be checked in every chromatogram. The analysis or the integration should be repeated if the baseline was incorrectly located. Note: If the CMPA peak is sufficiently separated from other peaks valley-to-valley baseline allocation should be used, otherwise use dropping perpendiculars to a common baseline, which should have starting point close to the CMPA peak (thus not at t = 0 min!).Use for the standard and the samples the same type integration type and check in case of common baseline its consistency for the samples and the standard. It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to detect any abnormalities due either to malfunctioning of the apparatus or the column, or to the origin and nature of the sample analysed. If in doubt, repeat the analysis. 8.5. Calibration 8.5.1. Apply exactly the procedure described from point 8.2 to point 8.4.4 to the standard samples (5.4.1 to 5.4.2). Use freshly prepared solutions, because CMP degrades in an 8 % trichloroacetic acid environment at room temperature. At 4 °C the solution remains stable for 24 hours. In the case of long series of analyses the use of a cooled sample tray in the automatic injector is desirable Note: 8.4.2. may be omitted if the % B at initial conditions is known from previous analyses. The chromatogram of the reference sample [5] should be analogous to Figure. 1. In this figure the CMPA peak is preceded by two small peaks. It is essential to obtain a similar separation. 8.5.2. Prior to chromatographic determination of the samples inject 100 μl of the standard sample without rennet whey [0] (5.4.1) The chromatogram should not show a peak at the retention time of the CMPA peak. 8.5.3. Determine the response factors R by injecting the same volume of filtrate (8.5.1) as used for the samples. | |||
|---|---|---|---|---|
| 8.3.1. | Add 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using a mechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C | |||
| 8.3.2. | Add 10,0 ml of the trichloroacetic acid solution of ca. 25 °C (5.1) constantly over two minutes, while stirring vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes | |||
| 8.3.3. | Centrifuge (6.2) 2 200 g for 10 minutes, or filter through paper (6.6), discarding the first 5 ml of filtrate | |||
| 8.4.1. | The reversed-phase HPLC method excludes the possibility false-positive results due to the presence of acid buttermilk powder. | |||
| 8.4.2. | Before the reversed phase HPLC-analysis is carried out, the gradient conditions should be optimised. A retention time of 26 ± 2 minutes for CMPA is optimal for gradient systems having a dead volume of about 6 ml (volume from the point where the solvents come together to the volume of the injector loop, inclusive). Gradient systems having a lower dead volume (e.g. 2 ml) should use 22 minutes as an optimal retention time Take solutions of the standard samples (5.4) without and with 50 % rennet whey. Inject 100 μl of supernatant or filtrate (8.3.3) into the HPLC apparatus operating at the scouting gradient conditions given in Table 1. Table 1 Scouting gradient conditions for optimisation of the chromatography Time (min) Flow (ml/min) % A % B Curve Initial 1,0 90 10 * 27 1,0 60 40 linear 32 1,0 10 90 linear 37 1,0 10 90 linear 42 1,0 90 10 linear Comparison of the two chromatograms should reveal the location of the peak of CMPΑ. Using the formula given below, the initial solvent composition to be used for the normal gradient (see 8.4.3) can be calculated % B = 10 – 2,5 + (13,5 + (RTcmpA – 26) / 6) * 30 / 27 % B = 7,5 + (13,5 + (RTcmpA – 26) / 6) * 1,11 Where: RTcmpA : retention time of CMPΑ in the scouting gradient 10 : the initial % B of the scouting gradient 2,5 : % B at midpoint minus % B at initial in the normal gradient 13,5 : midpoint time of the scouting gradient 26 : required retention time of CMPΑ 6 : ratio of slopes of the scouting and normal gradient 30 : % B at initial minus % B at 27 minutes in the scouting gradient 27 : run-time of the scouting gradient. | |||
| Time (min) | Flow (ml/min) | % A | % B | Curve |
| Initial | 1,0 | 90 | 10 | * |
| 27 | 1,0 | 60 | 40 | linear |
| 32 | 1,0 | 10 | 90 | linear |
| 37 | 1,0 | 10 | 90 | linear |
| 42 | 1,0 | 90 | 10 | linear |
| RTcmpA | : | retention time of CMPΑ in the scouting gradient | ||
| 10 | : | the initial % B of the scouting gradient | ||
| 2,5 | : | % B at midpoint minus % B at initial in the normal gradient | ||
| 13,5 | : | midpoint time of the scouting gradient | ||
| 26 | : | required retention time of CMPΑ | ||
| 6 | : | ratio of slopes of the scouting and normal gradient | ||
| 30 | : | % B at initial minus % B at 27 minutes in the scouting gradient | ||
| 27 | : | run-time of the scouting gradient. | ||
| 8.4.3. | Take solutions of the test samples Inject 100 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute. The composition of the eluent of the start of the analysis is obtained from 8.4.2. It is normally close to A:B = 76:24 (5.2). Immediately after the injection a linear gradient is started, which results in a 5 % higher percentage of B after 27 minutes. Subsequently a linear gradient is started, which brings the eluent composition to 90 % B in five minutes. This composition is maintained for five minutes, after which the composition is changed, via a linear gradient in five minutes to the initial composition. Depending on the internal volume of the pumping system, the next injection can be made 15 minutes after reaching the initial conditions. Note 1. The retention time of the CMPA should be 26 ± 2 minutes. This can be achieved by varying the initial and end conditions of the first gradient. However, the difference in the % B for the initial and end conditions of the first gradient shall remain 5 % B. Note 2. The eluents should be degassed sufficiently and should also remain degassed. This is essential for proper functioning of the gradient pumping system. The standard deviation for the retention time of the CMPA peak should be smaller than 0,1 minutes (n = 10). Note 3. Every five samples the reference sample [5] should be injected and used to calculate a new response factor R. (9.1.1). | |||
| 8.4.4. | The results of the chromatographic analysis of the test sample (E) are obtained in the form of a chromatogram in which the CMPA peak is identified by its retention time of about 26 minutes The integrator (6.11.6) automatically calculates the peak height H of the CMPA peak. The baseline location should be checked in every chromatogram. The analysis or the integration should be repeated if the baseline was incorrectly located. Note: If the CMPA peak is sufficiently separated from other peaks valley-to-valley baseline allocation should be used, otherwise use dropping perpendiculars to a common baseline, which should have starting point close to the CMPA peak (thus not at t = 0 min!).Use for the standard and the samples the same type integration type and check in case of common baseline its consistency for the samples and the standard. It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to detect any abnormalities due either to malfunctioning of the apparatus or the column, or to the origin and nature of the sample analysed. If in doubt, repeat the analysis. | |||
| 8.5.1. | Apply exactly the procedure described from point 8.2 to point 8.4.4 to the standard samples (5.4.1 to 5.4.2). Use freshly prepared solutions, because CMP degrades in an 8 % trichloroacetic acid environment at room temperature. At 4 °C the solution remains stable for 24 hours. In the case of long series of analyses the use of a cooled sample tray in the automatic injector is desirable Note: 8.4.2. may be omitted if the % B at initial conditions is known from previous analyses. The chromatogram of the reference sample [5] should be analogous to Figure. 1. In this figure the CMPA peak is preceded by two small peaks. It is essential to obtain a similar separation. | |||
| 8.5.2. | Prior to chromatographic determination of the samples inject 100 μl of the standard sample without rennet whey [0] (5.4.1) The chromatogram should not show a peak at the retention time of the CMPA peak. | |||
| 8.5.3. | Determine the response factors R by injecting the same volume of filtrate (8.5.1) as used for the samples. |
(*)International IDF Standard 135B/1991. Milk and milk products. Precision characteristics of analytical methods. Outline of collaborative study procedure.
PART II
Delivery and packaging of skimmed milk powder
1.Skimmed milk powder shall be packed in new, clean, dry and intact bags meeting the following requirements:
(a) the bags shall have at least three layers, which together correspond to at least 420 J/m2 TEA average;
(b) the second layer shall be covered with a layer of polyethylene of at least 15 g/m2;
(c) inside the paper layers, a polyethylene bag at least 0,08 mm thick shall be fused to the bottom;
(d) bags shall conform to standard EN 770;
(e) when filling, the powder should be well pressed down. Loose powder must on no account be allowed to penetrate between the various layers.
2.The bags shall show the following particulars, where appropriate in code:
(a) the approval number identifying the factory and the Member State of production;
(b) the date or, where appropriate, the week of production;
(c) the number of the production batch;
(d) the description ‘spray skimmed-milk powder’.
3.The storekeeper shall keep a register in which the particulars referred to in point 2 are recorded on the date of entry into storage.
ANNEX VI
Methods of analysis of butter under private storage
| Parameter | Method |
|---|---|
| Fat (1) | ISO 17189 or ISO 3727 part 3 |
| Water | ISO 3727 part 1 |
| Non Fat Solids (excluding salt) | ISO 3727 part 2 |
| Salt | ISO 15648 |
| (1) The method to be applied shall be approved by the paying agency. |
ANNEX VII
Methods of analysis of skimmed milk powder under private storage
| Parameter | Method |
|---|---|
| Fat | ISO 1736 |
| Protein | ISO 8968 part 1 |
| Water | ISO 5537 |
ANNEX VIII
Methods of analysis of cheeses under private storage
1.The method of analysis laid down in the Appendix shall be used to ensure that cheese made exclusively from ewe's milk, goat's milk or buffalo milk or from a mixture of ewe's milk, goat's milk and buffalo milk does not contain cow's milk casein.
Cow's milk casein is considered to be present if the cow's milk casein content of the analysed sample is equal to or higher than the content of the reference sample containing 1 % cow's milk as laid down in the Appendix.
2.Methods for detecting cow's milk casein in cheeses referred to in paragraph 1 may be used provided that:
(a) the detection limit is maximum 0,5 % and
(b) there are no false-positive results and
(c) cow's milk casein is detectable with the required sensitivity even after long ripening periods, as may occur in usual commercial conditions.
If any of the above mentioned requirements is not met, the methods laid down in the Appendix shall be used.
Appendix
METHOD FOR THE DETECTION OF COW'S MILK AND CASEINATE IN CHEESES FROM EWE'S MILK, GOAT'S MILK OR BUFFALO MILK OR MIXTURES OF EWE'S MILK, GOAT'S MILK AND BUFFALO MILK
1. SCOPE
Detection of cow's milk and caseinate in cheeses made from ewe's milk, goat's milk, buffalo milk or mixtures of ewe's, goat's and buffalo milk by isoelectric focusing of γ-caseins after plasminolysis.
2. FIELD OF APPLICATION
The method is suitable for sensitive and specific detection of native and heat-treated cow's milk and caseinate in fresh and ripened cheeses made from ewe's milk, goat's milk, buffalo milk or mixtures of ewe's, goat's and buffalo milk. It is not suitable for the detection of milk and cheese adulteration by heat-treated bovine whey protein concentrates.
3. PRINCIPLE OF THE METHOD
3.1. Isolation of caseins from cheese and the reference standards
3.2. Dissolving of the isolated caseins and submitting to plasmin (EC.3.4.21.7) cleavage
3.3. Isoelectric focusing of plasmin-treated caseins in the presence of urea and staining of proteins
3.4. Evaluation of stained γ3 and γ2 -casein patterns (evidence of cow's milk) by comparison of the pattern obtained from the sample with those obtained in the same gel from the reference standards containing 0 % and 1 % cow's milk.
4. REAGENTS
Unless otherwise indicated, analytical grade chemicals shall be used. Water shall be double-distilled or of equivalent purity.
Note: The following details apply to laboratory prepared polyacrylamide gels containing urea, of dimensions 265 × 125 × 0,25 mm. Where other sizes and types of gel are used, the separation conditions may have to be adjusted.
Dissolve:
in water and make up to 100 ml and store in a brown glass bottle in the refrigerator.
Note: A commercially available pre-blended acrylamide/BIS solution may be used in preference to the quoted fixed weights of the neurotoxic acrylamides. Where such a solution contains 30 % w/v acrylamide and 0,8 % w/v BIS, a volume of 16,2 ml shall be used for the formulation instead of the fixed weights. The shelf life of the stock solution is a maximum of 10 days; if its conductivity is more than 5 μS, de-ionize by stirring with 2 g Amberlite MB-3 for 30 minutes, then filter through a 0,45 μm membrane.
Prepare a gel solution by mixing additives and ampholytes (*) with the stock gel solution (see 4.1.1).
Mix the gel solution and de-gas for two to three minutes in an ultrasonic bath or in vacuum.
Note: Prepare the gel solution immediately prior to pouring it (see 6.2).
4.1.3.1. N, N, N′ N′ — tetramethylethylenediamine (Temed)
4.1.3.2. 40 % w/v ammonium persulphate (PER): Dissolve 800 mg PER in water and make up to 2 ml. Note: Always use freshly prepared PER solution.
Kerosene or liquid paraffin
Dissolve 5,77 g phosphoric acid (85 % w/w) in water and dilute to 100 ml.
Dissolve 2,00 g sodium hydroxide in water and dilute to 100 ml with water.
4.5.1. Dilute acetic acid (25,0 ml of glacial acetic acid made up to 100 ml with water)
4.5.2. Dichloromethane
4.5.3. Acetone
Dissolve
in water and make up to 50 ml
Note: Store in a refrigerator, maximum shelf-life one week.
Titrate a 0,2 mol/l ammonium hydrogencarbonate solution (1,58 g/100 ml water) containing 0,05 mol/l ethylenediaminetetraacetic acid (EDTA, 1,46 g/100 ml with a 0,2 mol/l ammonium carbonate solution (1,92 g/100 ml water) containing 0,05 mol/l EDTA to pH 8.
4.7.2. Bovine plasmin (EC. 3.4.21.7), activity at least 5 U/ml
4.7.3. Dissolve 2,624 g ε-aminocaproic acid (6 amino-n-hexanoic acid) in 100 ml of 40 % (v/v) ethanol.
4.8.1. Certified reference standards of a mixture of renneted ewe's and goat's skimmed milk containing 0 % and 1 % of cow's milk are available from the Commission's Institute for Reference Materials and Measurements, B-2440 Geel, Belgium
4.8.2. Skimmed milk is prepared by centrifuging of either buffalo or bovine raw bulk milk at 37 °C at 2 500 g for 20 minutes. After cooling the tube and contents rapidly to 6 to 8 °C, the upper fat layer is removed completely. For the preparation of the 1 % standard add 5,00 ml of bovine skimmed milk to a 495 ml of buffalo's skimmed milk in a 1 l beaker, adjust the pH to 6,4 by the addition of dilute lactic acid (10 % w/v). Adjust the temperature to 35 °C and add 100 μl of calf rennet (rennet activity 1: 10 000 , c. 3 000 U/ml), stir for 1 minute and then leave the beaker covered with an aluminium foil at 35 °C for one hour to allow formation of the curd. After the curd has formed, the whole renneted milk is freeze-dried without prior homogenization or draining of the whey. After freeze-drying it is finely ground to produce a homogeneous powder. For the preparation of the 0 % standard, carry out the same procedure using genuine buffalo skimmed milk. The standards shall be stored at – 20 °C. Note: It is advisable to check the purity of the buffalo milk by isoelectric focusing of the plasmin-treated caseins before preparation of the standards.
Dissolve 150 g trichloroacetic acid in water and make up to 1 000 ml.
Dilute 500 ml methanol and 200 ml glacial acetic acid to 2 000 ml with distilled water.
Note: Prepare the destaining solution fresh every day; it can be prepared by mixing equal volumes of stock solutions of 50 % (v/v) methanol and 20 % (v/v) glacial acetic acid.
Dissolve 3,0 g Coomassie Brilliant Blue G-250 (C.I. 42655) in 1 000 ml 90 % (v/v) methanol using a magnetic stirrer (approximately 45 minutes), filter through two medium-speed folded filters.
Dissolve 5,0 g copper sulphate pentahydrate in 1 000 ml 20 % (v/v) acetic acid.
Mix together 125 ml of each of the stock solutions (4.11.1, 4.11.2) immediately prior to staining.
Note: The staining solution should be prepared on the day that it is used.
5. EQUIPMENT
5.1. Glass plates (265 × 125 × 4 mm); rubber roller (width 15 cm); levelling table
5.2. Gel carrier sheet (265 × 125 mm)
5.3. Covering sheet (280 × 125 mm). Stick on strip of adhesive tape (280 × 6 × 0,25 mm) to each long edge (see Figure 1)
5.4. Electrofocusing chamber with cooling plate (e.g. 265 × 125 mm) and suitable power supply (≥ 2,5 kV) or automatic electrophoresis device
5.5. Circulation cryostat, thermostatically controlled at 12 ± 0,5 °C
5.6. Centrifuge, adjustable to 3 000 g
5.7. Electrode strips (≥ 265 mm long)
5.8. Plastic dropping bottles for the anode and cathode solutions
5.9. Sample applicators (10 × 5 mm, viscose or low protein-adsorption filter paper)
5.10. Stainless steel or glass staining and destaining dishes (e.g. 280 × 150 mm instrument trays)
5.12. Adjustable rod homogenizer (10 mm shaft diameter), rpm range 8 000 to 20 000
5.13. Magnetic stirrer
5.14. Ultrasonic bath
5.15. Film welder
5.16. 25 μl micropipettes
5.17. Vacuum concentrator or freeze-dryer
5.18. Thermostatically controlled water bath adjustable to 35 and 40 ± 1 °C with shaker
5.19. Densitometer equipment reading at λ = 634 nm
6. PROCEDURE
Weigh the amount equivalent to 5 g dry mass of cheese or the reference standards into a 100 ml centrifuge tube, add 60 ml distilled water and homogenize with a rod homogenizer (8 000 to 10 000 rpm). Adjust to pH 4,6 with dil. acetic acid (4.5.1) and centrifuge (5 minutes, 3 000 g). Decant the fat and whey, homogenize the residue at 20 000 rpm in 40 ml distilled water adjusted to pH 4,5 with dil. acetic acid (4.5.1), add 20 ml dichloromethane (4.5.2), homogenize again and centrifuge (5 minutes, 3 000 g). Remove the casein layer that lies between the aqueous and organic phases (see Figure 2) with a spatula and decant off both phases. Rehomogenise the casein in 40 ml distilled water (see above) and 20 ml dichloromethane (4.5.2) and centrifuge. Repeat this procedure until both extraction phases are colourless (two to three times). Homogenize the protein residue with 50 ml acetone (4.5.3) and filter through a medium-speed folded filter paper. Wash the residue on the filter with two separate 25 ml portions of acetone each time and allow to dry in the air or a stream of nitrogen, then pulverize finely in a mortar.
Note: Dry casein isolates should be kept at –20 °C.
Disperse 25 mg of isolated caseins (6.1.1) in 0,5 ml ammonium carbonate buffer (4.7.1) and homogenize for 20 minutes by e.g. using ultrasonic treatment. Heat to 40 °C and add 10 μl plasmin (4.7.2), mix and incubate for one hour at 40 °C with continuous shaking. To inhibit the enzyme add 20 μl ε-aminoproic acid solution (4.7.3), then add 200 mg of solid urea and 2 mg of dithiothreitol.
Note: To obtain more symmetry in the focused casein bands it is advisable to freeze-dry the solution after adding the ε-aminocaproic acid and then dissolving the residues in 0,5 ml protein dissolving buffer (4.6).
With the aid of a few drops of water roll the gel carrier sheet (5.2) onto a glass plate (5.1), removing any extraneous water with paper towel or tissue. Roll the cover sheet (5.3) with spacers (0,25 mm) onto another glass plate in the same way. Lay the plate horizontally on a levelling table.
Add 10 μl Temed (4.1.3.1) to the prepared and de-aerated gel solution (4.1.2), stir and add 10 μl PER-solution (4.1.3.2), mix thoroughly and immediately pour out evenly onto the centre of the cover sheet. Place one edge of the gel carrier plate (sheet side down) on the cover sheet plate and lower it slowly so that a gel film forms between the sheets and spreads out regularly and free of bubbles (Figure 3). Carefully lower the gel carrier plate completely using a thin spatula and place three more glass plates on top of it to act as weights. After polymerization is complete (about 60 minutes) remove the gel polymerized onto the gel carrier sheet along with the cover sheet by tipping the glass plates. Clean the reverse of the carrier sheet carefully to remove gel residues and urea. Weld the gel sandwich into a film tube and store in a refrigerator (maximum six weeks).
Note: The cover sheet with the spacers can be re-used. The polyacrylamide gel can be cut to smaller sizes, recommended when there are few samples or if an automatic electrophoresis device is used (two gels, size 4,5 × 5 cm).
Set the cooling thermostat to 12 °C. Wipe off the reverse of the gel carrier sheet with kerosene, then drip a few drops of kerosene (4.2) onto the centre of the cooling block. Then roll the gel sandwich, carrier side down, onto it, taking care to avoid bubbles. Wipe off any excess kerosene and remove the cover sheet. Soak the electrode strips with the electrode solutions (4.3, 4.4), cut to gel length and place in the positions provided (distance of electrodes 9,5 cm).
| Step | Time (min.) | Voltage (V) | Current (mA) | Power (W) | Volt-hours (Vh) |
|---|---|---|---|---|---|
| 1. Pre-focusing | 30 | maximum 2 500 | maximum 15 | constant 4 | c. 300 |
| 2. Sample focusing (1) | 60 | maximum 2 500 | maximum 15 | constant 4 | c. 1 000 |
| 3. Final focusing | 60 | maximum 2 500 | maximum 5 | maximum 20 | c. 3 000 |
| 40 | maximum 2 500 | maximum 6 | maximum 20 | c. 3 000 | |
| 30 | maximum 2 500 | maximum 7 | maximum 25 | c. 3 000 | |
| (1) Sample application: After pre-focusing (step 1), pipette 18 μl of the sample and standard solutions onto the sample applicators (10 × 5 mm), place them on the gel at 1 mm intervals from each other and 5 mm longitudinally from the anode and press lightly. Carry out focusing using the above conditions, carefully removing the sample applicators after the 60 minutes of sample focusing. |
Note: If thickness or width of the gels are changed, the values for current and power have to be suitably adjusted (e.g. double the values for electric current and power if a 265 × 125 × 0,5 mm gel is used).
| Step | Voltage | Current | Power | Temp. | Volt-hours |
|---|---|---|---|---|---|
| 1. Pre-focusing | 1 000 V | 10,0 mA | 3,5 W | 8 °C | 85 Vh |
| 2. Sample focusing | 250 V | 5,0 mA | 2,5 W | 8 °C | 30 Vh |
| 3. Focusing | 1 200 V | 10,0 mA | 3,5 W | 8 °C | 80 Vh |
| 4. Focusing | 1 500 V | 5,0 mA | 7,0 W | 8 °C | 570 Vh |
Place sample applicator in step 2 at 0 Vh.
Remove sample applicator in step 2 at 30 Vh.
Remove the electrode strips immediately after turning off the power and put the gel immediately into a staining/destaining dish filled with 200 ml fixative (4.9); leave for 15 minutes, shaking continuously.
Thoroughly drain off the fixative and wash the gel plate twice for 30 seconds each time with 100 ml destaining solution (4.10). Pour off the destaining solution and fill the dish with 250 ml staining solution (4.11.3); allow to stain for 45 minutes with gentle shaking.
Pour off the staining solution, wash the gel plate twice using a 100 ml destaining solution (4.10) each time, then shake with 200 ml destaining solution for 15 minutes and repeat the destaining step at least two or three times until the background is clear and uncoloured. Then rinse the gel plate with distilled water (2 × 2 minutes) and dry in the air (2 to 3 hours) or with a hairdryer (10 to 15 minutes).
Note 1: Carry out fixing, washing, staining and destaining at 20 °C. Do not use elevated temperatures.
Note 2: If more sensitive silver staining (e.g. Silver Staining Kit, Protein, Pharmacia Biotech, Code No 17-1150-01) is preferred, plasmin-treated casein samples have to be diluted to 5 mg/ml.
7. EVALUATION
Evaluation is performed by comparing the protein patterns of the unknown sample with reference standards on the same gel. Detection of cow's milk in cheeses from ewe's milk, goat's milk and buffalo milk and mixtures of ewe's, goat's and buffalo milk is done via the γ3- and γ2-caseins, whose isoelectric points range between pH 6,5 and pH 7,5 (Figures 4 a, b, Figure 5). The detection limit is less than 0,5 %.
For visual evaluation of the amount of bovine milk it is advisable to adjust the concentrations of samples and standards to obtain the same level of intensity of the ovine, caprine and/or buffalo γ2- and γ3-caseins (see ‘γ2 E,G,B’ and ‘γ3 E,G,B’ in Figures 4 a, b and Figure 5). After which the amount of bovine milk (less than, equal to or greater than 1 %) in the unknown sample can be judged directly by comparing the intensity of the bovine γ3- and γ2-caseins (see ‘γ3 C’ and ‘γ2 C’ in Figures 4 a, b and Figure 5) to those of the 0 % and 1 % reference standards (ewe, goat) or, laboratory interim-standards (buffalo).
If available, apply densitometry (5.19) for the determination of the peak area ratio of bovine to ovine, caprine and/or buffalo γ2- and γ3-caseins (see Figure 5). Compare this value to γ2- and γ3-casein peak area ratio of the 1 % reference standard (ewe, goat) or laboratory interim-standard (buffalo) analysed on the same gel.
Note: The method is operating satisfactorily, if there is a clear positive signal for both bovine γ2- and γ3-caseins in the 1 % reference standard but not in the 0 % reference standard. If not, optimize the procedure following the details of the method precisely.
A sample is judged as being positive, if both bovine γ2- and γ3-caseins or the corresponding peak area ratios are equal to or greater than the level of the 1 % reference standard.
8. REFERENCES
Addeo F., Moio L., Chianese L., Stingo C., Resmini P., Berner I, Krause I., Di Luccia A., Bocca A.: Use of plasmin to increase the sensitivity of the detection of bovine milk in ovine and/or caprine cheese by gel isoelectric focusing of γ2-caseins. Milchwissenschaft 45, 708-711 (1990).
Addeo F., Nicolai M.A., Chianese L., Moio L., Spagna Musso S., Bocca A., Del Giovine L.: A control method to detect bovine milk in ewe and water buffalo cheese using immunoblotting. Milchwissenschaft 50, 83-85 (1995).
Krause I., Berner I, Klostermeyer H.: Sensitive detection of cow milk in ewe and goat milk and cheese by carrier ampholyte — and carrier ampholyte/immobilized pH gradient — isoelectric focusing of γ-caseins using plasmin as signal amplifier. in: Electrophoresis-Forum 89 (B. J. Radola, ed.) pp 389-393, Bode-Verlag, München (1989).
Krause Ι., Belitz H.-D., Kaiser K.-P.: Nachweis von Kuhmilch in Schaf and Ziegenmilch bzw. -käse durch isoelektrische Fokussierung in harnstoffhaltigen Polyacrylamidgelen. Z. Lebensm. Unters. Forsch. 174, 195-199 (1982).
Radola B.J.: Ultrathin-layer isoelectric focusing in 50-100 μm polyacrylamide gels on silanised glass plates or polyester films. Electrophoresis 1, 43-56 (1980).
Figure 1
Schematic drawing of the covering sheet
Figure 2
Casein layer floating between aqueous and organic phases after centrifugation
Figure 3
Flapping technique for casting of ultrathin polyacrylamide gels
a = spacer tape (0,25 mm); b = covering sheet (5.3); c, e = glass plates (5.1); d = gel solution (4.1.2); f = gel carrier sheet (5.2)
Figure 4a
Isoelectric focusing of plasmin-treated caseins from ewe's and goat's milk cheese containing different amounts of cow's milk.
% CM = percentage of cow's milk, C = cow, E = ewe, G = goat
Upper half of the IEF gel is shown.
Figure 4b
Isoelectric focusing of plasmin treated caseins from cheese made from mixtures of ewe's, goat's and buffalo milk containing different amounts of cow's milk.
% CM = percentage of cow's milk; 1 + = sample containing 1 % of cow's milk and spiked with pure bovine casein at the middle of the track. C = cow, E = ewe, G = goat, B = buffalo.
Total separation distance of the IEF gel is shown.
Figure 5
Superposition of densitograms of standards (STD) and cheese samples made from a mixture of ewe's and goat's milk after isoelectric focusing.
a,b = standards containing 0 and 1 % of cow's milk; c-g = cheese samples containing 0, 1, 2, 3 and 7 % of cow's milk. C = cow, E = ewe, G = goat.
Upper half of the IEF gel was scanned at λ = 634 nm.
ANNEX IX
Evaluation of the analyses
1. Quality assurance
Analyses shall be performed by laboratories designated in accordance with Article 12 of Regulation (EC) No 882/2004 (**) or designated by the competent authorities of the Member State.
2. Sampling and disputes over the results of analysis
1.Sampling shall be carried out in accordance with the relevant regulation for the product under consideration. If no sampling provisions are expressly provided for, then the provisions laid down in ISO 707, Milk and milk products – Guidance on sampling, shall be used.
2.Laboratory reports of the results of the analysis shall contain sufficient information to allow an evaluation of the results to be carried out in accordance with the Appendix.
3.Duplicate samples shall be taken for analyses required under Union rules.
4.If a dispute arises over the results, the paying agency shall have the necessary analysis on the product in question carried out again, and the cost shall be met by the losing party.
The above mentioned analysis shall be carried out provided that sealed duplicate samples of the product are available and have been stored appropriately with the competent authority. The manufacturer shall send a request to the paying agency to conduct the analysis within 7 working days following the notification of the results of the first analysis. The analysis shall be carried out by the paying agency within 21 working days following receipt of the request.
5.The appeal result shall be the definitive one.
6.If the manufacturer can prove, within five working days of sampling, that the sampling procedure was not carried out correctly, sampling shall be repeated where possible. If sampling cannot be repeated, the consignment shall be accepted.
Appendix
Evaluation of compliance of a consignment with the legal limit
1. Principle
Where public intervention and private storage legislation lay down detailed sampling procedures then those procedures shall be followed. In all other cases a sample of at least 3 sample units taken randomly from the consignment submitted to control shall be used. A composite sample may be prepared. The result obtained shall be compared with the legal limits by calculation of a 95 % confidence interval as 2 x standard deviation, where the relevant standard deviation depends on whether (1) the method is validated through international collaboration with values for σr and σR or (2) in the case of in-house validation, an internal reproducibility has been calculated. This confidence interval will then equate to the measurement uncertainty of the result.
2. The method is validated through international collaboration
In this case, the repeatability standard deviation σr and the reproducibility standard deviation σR have been established and the laboratory can demonstrate compliance with the performance characteristics of the validated method.
Calculate the arithmetic mean
of the n repeated measurements.
Calculate the expanded uncertainty (k = 2) of
as
If the final result x of measurement is calculated using a formula of the form x = y 1 + y 2, x = y 1 – y 2, x = y 1 · y 2 or x = y 1/y 2 the usual procedures for combining standard deviations in such cases shall be followed.
The consignment is judged to be not in compliance with the upper legal limit UL if
otherwise it is judged to be in compliance with UL.
The consignment is judged to be not in compliance with the lower legal limit LL if
otherwise it is judged to be in compliance with LL.
3. In-house validation with calculation of internal reproducibility standard deviation
In cases where methods not specified in this Regulation are used and precision measures have not been established, an in-house validation shall be carried out. Internal repeatability standard deviation σir and the internal reproducibility standard deviation σi R shall be used instead of σr and σR, resp., in the formulae for the computation of the expanded uncertainty U.
The rules to be followed to determine compliance with the legal limit are as set out under point 1. However, if the consignment is judged to be non-compliant with the legal limit, the measurements shall be repeated with the method specified in this Regulation and the result evaluated in accordance to point 1.
(*)The produce Ampholine® pH 3,5-9,5 (Pharmacia) and Resolyte® pH 5-7 and pH 6-8 (BDH, Merck) have proved particularly suitable for obtaining the required separation of γ-caseins.
(**)Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules (OJ L 165, 30.4.2004, p. 1).