Commission 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)

• (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

• (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 ([^E0007]).

Article 64

Notifications concerning paying agencies

Section II Notifications as regards public intervention

Article 65

Notifications of information on intervention stocks

• (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.

2 The Commission may request that notifications provided for in paragraph 1 be made with an increased frequency where that is necessary for the purposes of the efficient management of the intervention system.

• (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.

• (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

1. DEFINITION OF MATTER OTHER THAN BASIC CEREALS OF UNIMPAIRED QUALITY

1.1.   Broken grains

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.

1.2.   Grain impurities

(a) Shrivelled grains

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.

(b) Other cereals

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.

(c) Grains damaged by pests

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.

(d) Grains in which the germ is discoloured

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.

(e) Grains overheated during drying

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..

(f) Mottled grains

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.

1.3.   Sprouted grains

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.

1.4.   Miscellaneous impurities

(a) Extraneous seeds

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.

(b) Damaged grains

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’.

(c) Extraneous matter

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.

(d) Husks (cob fragments in the case of maize)

(e) Ergots

(f) Decayed grains

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.

(g) Impurities of animal origin.

1.5.   Live pests

1.6.   Mitadiné grains

Mitadiné grains of durum wheat are grains whose kernels cannot be regarded as entirely vitreous. They are defined in standard EN 15585.

2. SPECIFIC FACTORS TO TAKE INTO CONSIDERATION FOR EACH TYPE OF CEREAL FOR THE DEFINITION OF IMPURITIES

2.1.   Durum wheat

‘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.

2.2.   Common wheat

‘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.

2.3.   Barley

‘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.

2.4.   Maize

‘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

Methods used for determining the quality of cereals offered or tendered for, or placed in, intervention

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 ([^E0008]) and set out in Annexes I and II to Commission Regulation (EC) No 401/2006 ([^E0009]).

PART III

Method for determining the non-stickiness and machinability of the dough obtained from common wheat

1. Title

Method for test baking of wheat flour.

2. Scope

The method is applicable to flour, experimentally milled from wheat for the production of yeast-raised bread.

3. Principle

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.

4. Ingredients

4.1.   Yeast

Active dry yeast of type Saccharomyces cerevisiae DHW-Hamburg-Wansbeck or a product having the same characteristics.

4.2.   Tap water

4.3.   Sugar-salt-ascorbic acid solution

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.

4.4.   Sugar solution

Dissolve 5 ± 0,1 g sucrose (commercial grade) in 95 ± 1 g of water. Prepare fresh daily.

4.5.   Enzyme active malt flour

Commercial grade.

5. Equipment and apparatus

5.1.   Baking room

Controlled to maintain a temperature of 22 to 25 °C.

5.2.   Refrigerator

For maintaining a temperature of 4 ± 2 °C.

5.3.   Balance

Maximum load 2 kg, accuracy 2 g.

5.4.   Balance

Maximum load 0,5 kg, accuracy 0,1 g.

5.5.   Analytical balance

Accuracy 0,1 × 10^{– 3} g.

5.6.   Mixer

Stephan UMTA 10, with mixing arm model ‘Detmold’ (Stephan Soehne GmbH) or similar equipment having the same characteristics.

5.7.   Proving cabinet

Controlled to maintain a temperature of 30 ± 1 °C.

5.8.   Open plastic boxes

Made from polymethylmethacrylate (Plexiglas, Perspex). Inside dimensions: 25 × 25 × 15 cm height, wall thickness 0,5 ± 0,05 cm.

5.9.   Square plastic sheets

Made from polymethylmethacrylate (Plexiglas, Perspex). At least 30 × 30 cm, thickness 0,5 ± 0,05 cm.

5.10.   Moulder

Brabender ball homogeniser (Brabender OHG) or similar equipment having the same characteristics.

6. Sampling

According to standard EN ISO 24333.

7. Procedure

7.1.   Determination of water uptake

Determine the water absorption according to ICC Standard No 115/1.

7.2.   Determination of malt flour addition

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.

7.3.   Reactivation of active dry yeast

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.

7.4.   Temperature adjustment of the flour and the dough liquid

The temperature of the flour and the water must be adjusted to give a dough temperature of 27 ± 1 °C after mixing.

7.5.   Dough composition

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.

7.6.   Mixing

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.

7.7.   Dividing and rounding

Weigh, with precision of 2 g, three pieces of dough according to the formula:

p	=	0,25 P, where:
---	---	---
p	=	mass of scaled dough piece,
---	---	---
P	=	total mass of dough.

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.

7.8.   Moulding

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.

8. Test report

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. General remarks

• 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 ([^E0010]) and the moisture content according to this Regulation. Calculate the extraction rate by the equation:

• E = (((100 – f) F)/(100 – w) W) × 100 %

• where:

E	=	extraction rate,
---	---	---
f	=	moisture of the flour,
---	---	---
w	=	moisture content of the wheat,
---	---	---
F	=	mass of flour produced with moisture content f,
---	---	---
W	=	mass of wheat milled with moisture content w.

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

Methodology of sampling and analyses for cereals

• 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

Price increases and reductions

Table I

Price increases for moisture content for cereals other than maize

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

Price increases for moisture content for maize

Moisture content (%)	Increases (EUR/tonne)
Less than 12 to 11,5	0,5
Less than 11,5	1

Table II

Price reductions for moisture content for cereals other than maize

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

Price reductions for moisture content for maize

Moisture content (%)	Reduction (EUR/tonne)
More than 12,5 to 13,0	0,5
More than 13,0 to 13,5	1,0

Table III

Price increases for protein content of common wheat

Protein content [^E0011] (N × 5,7)	Price increase (EUR/tonne)
More than 12,0	2,5

As % of dry matter.

Table IV

Price reductions for protein content for common wheat

Protein content [^E0012] (N × 5,7)	Price reduction (EUR/tonne)
Less than 11,5 to 11,0	2,5

As % of dry matter.

PART VI

Calculation of prices increases and reductions

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.

Table I

Price increases for moisture content

Moisture content (%)	Increases (EUR/tonne)
Less than 12,5 to 12	0,75
Less than 12 to 11,5	1,5

Table II

Price reductions for moisture content

Moisture content (%)	Reduction (EUR/tonne)
More than 13,5 to 14,0	0,75
More than 14,0 to 14,5	1,5

Table III

Price increases relating to milling yield

Yield of whole-grain milled paddy rice	Price increases per yield point [^E0013]
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

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

I. General conditions governing 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 ([^E0014]).

• 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.

II.   Contracts and specifications

• 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.

III.   Control and monitoring of deboning operations

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.

IV.   Storage of cuts

Cuts shall be stored in cold stores located in the territory of the Member State exercising jurisdiction over the paying agency.

V. Costs of deboning operations

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.

VI.   Time limits for deboning operations

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.

VII.   Checks and rejection of products

• 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

1. HINDQUARTER CUTS

1.1.   Description of cuts

1.1.1.   Intervention shank (code INT 11)

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.

1.1.2.   Intervention thick flank (code INT 12)

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.

1.1.3.   Intervention topside (code INT 13)

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.

1.1.4.   Intervention silverside (code INT 14)

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.

1.1.5.   Intervention fillet (code INT 15)

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.

1.1.6.   Intervention rump (code INT 16)

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.

1.1.7.   Intervention striploin (code INT 17)

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.

1.1.8.   Intervention flank (code INT 18)

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 %.

1.1.9.   Intervention fore-rib (five bone) (code INT 19)

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.

2. FOREQUARTER CUTS

2.1.   Description of cuts

2.1.1.   Intervention shin (code INT 21)

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.

2.1.2.   Intervention shoulder (code INT 22)

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 %.

2.1.3.   Intervention brisket (code INT 23)

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 %.

2.1.4.   Intervention forequarter (code INT 24)

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)	(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

1. Chemical and microbiological analysis

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.

2. Sensory evaluation

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.

3. Guidelines to be followed where samples show defects

• (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: Image omitted; see official source

• (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 [^E0015]	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.

The method to be applied shall be approved by the paying agency.

Scoring table

Appearance	Appearance	Consistency	Consistency	Odour and Flavour	Odour and Flavour
Points	Remarks	Points	Remarks	Points	Remarks
5	Very good Ideal type Highest quality (equal dry)	5	Very good Ideal type Highest quality (equal spreadable)	5	Very good Ideal type Highest quality (absolutely pure finest odour)
4	Good (no evident defects)	4	Good (no evident defects)	4	Good (no evident defects)
1, 2 or 3	Any defect	1, 2 or 3	Any defect	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 [^E0016]	ADPI
Micro-organisms	ISO 4833-part 1
Buttermilk	Appendix I
Rennet whey [^E0017]	Appendix II and III
Acid whey [^E0018]	ISO 8069 or On-the-spot inspections
Sensory checks [^E0019]	ISO 22935 part 2 and 3

Scorched particles' analyses may be conducted systematically. However, such analyses shall always be conducted if no sensory checks are performed.

The method to be applied shall be approved by the paying agency (one or both methods).

The method to be applied shall be approved by the paying agency.

Sensory checks shall be performed where deemed necessary after risk based analysis approved by the paying agency.

Appendix I

SKIMMED MILK POWDER: QUANTITATIVE DETERMINATION OF PHOSPHATIDYLSERINE AND PHOSPHATIDYLETHANOLAMINE

Method: reversed-phase HPLC

1. PURPOSE AND FIELD OF APPLICATION

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.

2. DEFINITION

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.

3. PRINCIPLE OF THE METHOD

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.

4. REAGENTS

All reagents shall be of recognised analytical grade. Water shall be distilled or water of at least equivalent purity, unless otherwise specified.

4.1.   Standard material: PEDP, at least 99 % pure

Note: Standard material shall be stored at – 18 °C.

4.2.   Reagents for standard sample and test sample preparation

• 4.2.1. HPLC-grade methanol

• 4.2.2. HPLC-grade chloroform

• 4.2.3. Tryptamine-monohydrochloride

4.3.   Reagents for o-phthaldialdehyde derivatisation

• 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.   HPLC elution solvents

• 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. APPARATUS

• 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

• 5.13. HPLC equipment

• 5.13.1. Gradient pumping system, capable of operating at 1,0 ml/min at 200 bar

• 5.13.2. Autosampler with derivatisation capability

• 5.13.3. Column heater, capable of maintaining the column at 30 °C ± 1 °C

• 5.13.4. Fluorescence detector, capable of operating at 330 nm excitation wavelength and 440 nm emission wavelength

• 5.13.5. Integrator or data processing software capable of peak area measurement

• 5.13.6. A LiChrospher® — 100 column (250 × 4,6 mm) or an equivalent column packed with octadecylsilane (C 18), 5 μm particle size.

6. SAMPLING

Sampling shall be carried out in accordance with ISO Standard 707.

7. PROCEDURE

7.1.   Preparation of the internal standard solution

• 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.   Preparation of the test sample solution

• 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.   Preparation of the external standard solution

• 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.

7.4.   Preparation of the derivatising reagent

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.

7.5.   Determination by HPLC

7.5.1.   Elution solvents (4.4)

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

7.5.2.   Suggested eluting gradient:

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. Column equilibration

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.

7.6.   Integration mode

7.6.1.   PEDP peak

PEDP is eluted as a single peak. Determine the peak area by valley-to- valley integration.

7.6.2.   Tryptamine peak

Tryptamine is eluted as a single peak (Figure 1). Determine the peak area by valley-to-valley integration.

7.6.3.   PS and PE peaks groups

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.

8. CALCULATION AND EXPRESSION OF RESULTS

PS and PE content in the test sample shall be calculated as follows:

C = 55,36 × ((A2)/(A1)) × ((T1)/(T2))

where:

C	=	PS or PE content (mg/100 g powder) in the test sample
---	---	---
A 1	=	PEDP peak area of the standard sample solution (7.3)
---	---	---
A 2	=	PS or PE peak area of the test sample solution (7.2)
---	---	---
T 1	=	Tryptamine peak area of the standard sample solution (7.3)
---	---	---
T 2	=	Tryptamine peak area of the test sample solution (7.2).

9. ACCURACY OF THE METHOD

Note: The values for repeatability were calculated according to the IDF International Standard (*).

9.1.   Repeatability

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.

9.2.   Reproducibility

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. REFERENCES

• 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).

Figure 1

HPLC pattern of OPA-derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in methanol extract of reconstituted skim-milk powder. Integration mode for the peaks of PS, PE and tryptamine (internal standard) is reported

Image omitted; see official source

Appendix II

DETECTION OF RENNET WHEY IN SKIMMED MILK POWDER FOR PUBLIC STORAGE BY DETERMINATION OF CASEINOMACROPEPTIDES HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

1. SCOPE AND FIELD OF APPLICATION

This method allows detection of rennet whey in skimmed milk powder intended for public storage by determination of the caseinomacropeptides.

2. REFERENCE

International Standard ISO 707 - Milk and Milk Products - Guidance on sampling.

3. DEFINITION

The content of rennet whey solids is defined as the percentage by mass as determined by the caseinomacropeptide content by the procedure described.

4. PRINCIPLE

• — 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.

5. REAGENTS

All reagents shall be of recognised analytical grade. The water used shall be distilled water or water of at least equivalent purity.

5.1.   Trichloroacetic acid solution