ANNEX I

(Article 1)

LIST OF REFERENCE METHODS

Index Min. = minimum, Max. = maximum, Annex = Annex to quoted Regulation, SNF = solids non fat, PV = peroxide value, A = appearance, F = flavour, C = consistency, TBC = total bacterial count, Therm = thermophilic bacterial count, MS = Member State, IDF = International Dairy Federation, ISO = International Standards Organisation, IUPAC = International Union of Pure and Applied Chemistry, ADPI = American Dairy Products Institute, SCM = sweetened condensed milk, EMC = evaporated milk or cream.

PART A

Commission Regulation	Product	Parameter	Limit (1)	Reference method	Remark
Regulation (EC) No 2771/1999 — Public storage	Unsalted butter	Fat	Min. 82 % m/m	ISO 17189:2003|IDF 194:2003
		Water	Up to 16 % m/m	ISO 3727-1:2001|IDF 80-1:2001
		SNF	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
		Fat acidity	1,2 mmole/100 g of fat	ISO 1740:2004|IDF 6:2004
		PV (max.)	0,3 meq. oxygen/1 000  g fat	ISO 3976:2006|IDF 74:2006	Note 1
		Coliforms	Not detectable in 1 g	Annex X	Note 3
		Non-milk fat	Not detectable by triglyceride analysis	Annex XX
		Sterol tracers	Not detectable, β-sitosterol ≤ 40 mg/kg	Annex VIII
		Other tracers
		— vanillin	Not detectable	Annex VI
		— ethyl ester of carotenic acid	≤ 6 mg/kg	Annex VII
		— triglycerides of enanthic acid	Not detectable	Annex V
		Sensory characteristics	At least 4 out of 5 points for A, F and C	Annex IV
		Water dispersion	At least 4 points	ISO 7586:1985 — IDF 112A:1989
Regulation (EC) No 2771/1999 — Private storage	Unsalted butter	Fat	Min. 82 % m/m	ISO 17189:2003|IDF 194:2003
		Water	Up to 16 % m/m	ISO 3727-1:2001|IDF 80-1:2001
		SNF	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
Regulation (EC) No 2771/1999 — Private storage	Salted butter	Fat	Min. 80 % m/m	ISO 17189:2003|IDF 194:2003
		Water	Up to 16 % m/m	ISO 3727-1:2001|IDF 80-1:2001
		SNF (excluding salt)	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
		Salt	Up to 2 % m/m	ISO 15648:2004|IDF 179:2004
Regulation (EC) No 1898/2005 chapter II	Unsalted butter	Fat	Min. 82 % m/m	ISO 17189:2003|IDF 194:2003
		Non-milk fat		Annex XX
		Water	Up to 16 % m/m	ISO 3727-1 2001|IDF 80-1:2001
		SNF	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
		Tracers:
		— sterols	See Annex VIII	Annex VIII
		— vanillin	See Annex VI	Annex VI
		— ethyl ester of carotenic acid	See Annex VII	Annex VII
		— triglycerides of enanthic acid	See Annex V	Annex V
Regulation (EC) No 1898/2005 chapter II	Salted butter	Fat	Min. 80 % m/m	ISO 17189:2003|IDF 194:2003
		Non-milk fat		Annex XX
		Water	Up to 16 % m/m	ISO 3727-1:2001|IDF 80-1:2001
		SNF (excluding salt)	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
		Salt	Up to 2 % m/m	ISO 15648:2004|IDF 179:2004
		Tracers:
		— sterols	See Annex VIII	Annex VIII
		— vanillin	See Annex VI	Annex VI
		— ethyl ester of carotenic acid	See Annex VII	Annex VII
		— triglycerides of enanthic acid	See Annex V	Annex V
Regulation (EC) No 1898/2005 chapter II	Concentrated butter	Fat	Min. 99,8 % m/m	IDF 24:1964
		Water and SNF	Up to 0,2 % m/m	ISO 5536:2002|IDF 23:2002 (moisture) IDF 24:1964 (SNF)
		Fat acidity	1,2 mmole/100 g of fat	ISO 1740:2004|IDF 6:2004
		PV (max.)	0,5 meq. oxygen/1 000  g fat	ISO 3976:2006|IDF 74:2006	Note 1
		Non-milk fat	Absence	Annex XX
		Flavour	Clean
		Smell	Absence of foreign odours
		Other	Absence of neutralising agents, anti-oxidants and preservatives
		Tracers:
		— sterols	See Annex VIII	Annex VIII
		— vanillin	See Annex VI	Annex VI
		— ethyl ester of carotenic acid	See Annex VII	Annex VII
		— triglycerides of enanthic acid	See Annex V	Annex V
Regulation (EC) No 1898/2005 chapter II	Cream	Fat	Minimum 35 % m/m	ISO 2450:1999|IDF 16 C:1987
		Non-milk fat		Annex XX
		Tracers:
		— sterols	See Annex VIII		Note 2
		— vanillin	See Annex VI	Annex VI
		— ethyl ester of carotenic acid	See Annex VII		Note 2
		— triglycerides of enanthic acid	See Annex V	Annex V
Regulation (EC) No 1898/2005 chapter III	Concentrated butter	Fat	Min. 96 % m/m		Note 2
		Non-milk fat		Annex XX
		SNF	Up to 2 % m/m		Note 2
		Tracers:
		— stigmasterol (95 % m/m)	15 g/100 kg of concentrated butter	Annex VIII
		— stigmasterol (85 % m/m)	17 g/100 kg of concentrated butter	Annex VIII
		— triglycerides of enanthic acid	10,34 kg/t of concentrated butter	Annex V
		— ethyl ester of butyric acid and stigmasterol		— ethyl ester of butyric acid — stigmasterol: Annex VIII	Note 2
		— ethyl ester of butyric acid and triglycerides of enanthic acid		— ethyl ester of butyric acid — triglycerides of enanthic acid: Annex V	Note 2
		lecithin (E 322)	Up to 0,5 % m/m		Note 2
		NaC1	Up to 0,75 % m/m	ISO 15648:2004|IDF 179:2004
		Fat acidity	1,2 mmole/100 g of fat	ISO 1740:2004|IDF 6:2004
		PV (max.)	Up to 0,5 meq. oxygen/1 000  g fat	ISO 3976:2006|IDF 74:2006	Note 1
		Flavour	Clean
		Smell	Absence of foreign odours
		Other	Absence of neutralising agents, anti-oxidants and preservatives
Regulation (EC) No 1898/2005 chapter IV	Unsalted butter	Fat	Min. 82 % m/m	ISO 17189:2003|IDF 194:2003
		Water	Up to 16 % m/m	ISO 3727-1:2001|IDF 80-1:2001
		SNF	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
Regulation (EC) No 1898/2005 chapter IV	Salted butter	Fat	Min. 80 % m/m	ISO 17189:2003|IDF 194:2003
		Water	Up to 16 % m/m	ISO 3727-1:2001|IDF 80-1:2001
		SNF (excluding salt)	Up to 2 % m/m	ISO 3727-2:2001|IDF 80-2:2001
		Salt	Up to 2 % m/m	ISO 15648:2004|IDF 179:2004
Article 9 and Title II of Regulation (EC) No 1255/1999	Cheese made from ewes’ and/or goats’ milk	Cows’ milk	< 1 % m/m	Annex IX
Regulation (EEC) No 2921/90	Annex I — Acid casein	Water	Up to 12,00 % m/m	ISO 5550:2006|IDF 78:2006
		Fat	Up to 1,75 % m/m	ISO 5543:2004|IDF127:2004
		Free acidity	Up to 0,30 ml of 0,1 N NaOH solution/g	ISO 5547:1978|IDF 91:1979
Regulation (EEC) No 2921/90	Annex I — Rennet- casein	Water	Up to 12,00 % m/m	ISO 5550:2006|IDF 78:2006
		Fat	Up to 1,00 % m/m	ISO 5543:2004|IDF 127:2004
		Ash	Min. 7,50 % m/m	ISO 5545:1978|IDF 90:1979
Regulation (EEC) No 2921/90	Annex I — Caseinates	Water	Up to 6,00 % m/m	ISO 5550:2006|IDF 78:2006
		Milk protein	Min. 88,00 % m/m	ISO 5549:1978|IDF 92:1979
		Fat and ash	Up to 6,00 % m/m	ISO 5543:2004|IDF 127:2004
		Fixed ash		ISO 5544:1978|IDF 89:1979
		Ash		ISO 5545:1978|IDF 90:1979
Regulation (EEC) No 2921/90	Annex II — Acid casein	Water	Up to 10,00 % m/m	ISO 5550:2006|IDF 78:2006
		Fat	Up to 1,50 % m/m	ISO 5543:2004|IDF 127:2004
		Free acidity	Up to 0,20 ml of 0,1 N NaOH solution/g	ISO 5547:1978|IDF 91:1979
		TBC (max.)	30 000 / g	ISO 4833:2003	Note 3
		Coliforms	Absence in 0,1 g	Annex X	Note 3
		Therm. (max.)	5 000 / g	ISO 4833:2003	Notes 3 and 4
Regulation (EEC) No 2921/90	Annex II — Rennet-casein	Water	Up to 8,00 % m/m	ISO 5550:2006|IDF 78:2006
		Fat	Up to 1,00 % m/m	ISO 5543:2004|IDF 127:2004
		Ash	Min. 7,50 % m/m	ISO 5545:1978|IDF 90:1979
		TBC (max.)	30 000 / g	ISO 4833:2003	Note 3
		Coliforms	Absence in 0,1 g	Annex X	Note 3
		Therm. (max.)	5 000 / g	ISO 4833:2003	Notes 3 and 4
Regulation (EEC) No 2921/90	Annex II — Caseinates	Water	Up to 6,00 % m/m	ISO 5550:2006|IDF 78:2006
		Milk protein	Min. 88,00 % m/m	ISO 5549:1978|IDF 92:1979
		Fat and ash	Up to 6,00 % m/m	ISO 5543:2004|IDF 127:2004 ISO 5544:1978|IDF 89:1979 or ISO 5545:1978|IDF 90:1979
		TBC (max.)	30 000 / g	ISO 4833:2003	Note 3
		Coliforms	Absence in 0,1 g	Annex X	Note 3
		Therm. (max.)	5 000 / g	ISO 4833:2003	Notes 3 and 4
Regulation (EEC) No 2921/90	Annex III — Caseinates	Water	Up to 6,00 % m/m	ISO 5550:2006|IDF 78:2006
		Milk protein	Min. 85,00 % m/m	ISO 5549:1978|IDF 92:1979
		Fat	Up to 1,50 % m/m	ISO 5543:2004|IDF 127:2004
		Lactose	Up to 1,00 % m/m	ISO 5548:2004|IDF 106:2004
		Ash	Up to 6,50 % m/m	ISO 5544:1978|IDF 89:1979 or ISO 5545:1978|IDF 90:1979
		TBC (max.)	30 000 / g	ISO 4833:2003	Note 3
		Coliforms	Absence in 0,1 g	Annex X	Note 3
		Therm. (max.)	5 000 / g	ISO 4833:2003	Notes 3 and 4
Regulation (EC) No 2799/1999	Compound feedingstuffs and skimmed-milk powder (SMP) (for use in feedingstuffs)	Water (acid buttermilk powder)	Up to 5 % m/m	Annex XIX
		Protein	31,4 % m/m (min.) of the non-fat dry matter	ISO 8968-1|2|3:2001|IDF 20-1|2|3:2001
		Water (SMP)	Up to 5 % m/m	ISO 5537:2004|IDF 26:2004
		Fats (SMP)	Up to 11 % m/m	ISO 1736:2000|IDF 9C:1987
		Rennet whey (SMP)	Absence	Annex XIII	Note 6
		Starch (SMP)	Absence	Annex XVII
		Water (mixtures)	Up to 5 % m/m of the non-fat matter	ISO 5537:2004|IDF 26:2004
		Fat (mixtures)		Commission Directive 84/4/EEC (OJ L 15, 18.1.1984, p. 29)
		Rennet whey (mixtures)	Absence	Annex XIII
		SMP content (of end product)	Min. 50 % m/m	Annex XVI
		Fat (in end product)	Min. 2,5 % m/m or 5 % m/m	Commission Directive 84/4/EEC (OJ L 15, 18.1.1984, p. 29)	Note 7
		Starch (in end product)	Min. 2 % m/m	Annex XVII	Note 8
		Copper (in end product)	25 ppm	Commission Directive 78/633/EEC (OJ L 206, 26.7.1987, p. 43)
Regulation (EC) No 214/2001	SMP (spray)	Fat	Up to 1,0 % m/m	ISO 1736:2000|IDF 9C:1987
		Protein	31,4 % (2) m/m (min.) of the non-fat dry matter	ISO 8968-1/2:2001|IDF 20-1/2:2001
		Water	Up to 3,5 % m/m	ISO 5537:2004|IDF 26:2004
		Acidity	Up to 19,5 ml, 0,1 N NaOH, 10 g solids non-fat	ISO 6091:1980|IDF 86:1981
		Lactates	Up to 150 mg/100 g solids non-fat	ISO 8069:2005|IDF 69:2005
		Phosphatase	Negative	ISO 11816-1:2006|IDF 155-1:2006
		Insolubility index	Up to 0,5 ml at 24 °C	ISO 8156:2005|IDF 129:2005
		Scorched particles	Disc A or B (15,0 mg)	ADPI (1990)
		TBC	40 000 / g	ISO 4833:2003	Note 3
		Coliforms	Negative/0,1 g	Annex X	Note 3
		Buttermilk	Negative	Annex XIV
		Rennet whey	Negative	Annex XII
		Acid whey	Negative		Note 2
		Anti-microbial agents		Annex XV

PART B

The reference methods listed in Part B may be used for analysing products covered by any of the Regulations listed in column 1.

Commission Regulation	Product	CN code	Parameter	Limit	Reference method	Remark
Regulation (EEC) No 2658/87 Regulation (EC) No 2535/2001 Regulation (EC) No 1282/2006	Milk and cream, not concentrated nor containing added sugar or other sweetening matter	0401	Fat (≤ 6 % m/m)	The limits are those specified in the description of the CN code for the specific product, further specified, where applicable, in Commission Regulation (EEC) No 3846/87 (OJ L 366, 24.12.1987, p. 1), Part 9 of the export nomenclature or Regulation (EC) No 2535/2001 (OJ L 341, 22.12.2001, p. 29)	ISO 1211:2001|IDF 1D:1996
			Fat (> 6 % m/m)		ISO 2450:1999|IDF 16C:1987
	Milk and cream, concentrated or containing added sugar or other sweetening matter	0402	Fat (liquid form)		ISO 1737:1999|IDF 13C:1987
			Fat (solid form)		ISO 1736:2000|IDF 9C:1987
			Protein		ISO 8968-1|2|3:2001|IDF 20-1|2|3:2001
			Sucrose (normal content)		ISO 2911:2004|IDF 35:2004
			Sucrose (low content)			Note 2
			Solids (SCM)		ISO 6734:1989|IDF 15B:1991
			Solids (EMC)		ISO 6731:1989|IDF 21B:1987
			Water (milk powder)		ISO 5537:2004|IDF 26:2004
			Water (cream powder)		Annex XVIII
	Buttermilk, fermented or acidified milk and cream, concentrated or not concentrated, containing added sugar or other sweetening matter	0403	Fat		ISO 1211:2001|IDF 1D:1996 ISO 1736:2000|IDF 9C:1987 ISO 2450:1999|IDF 16 C:1987 ISO 7208:1999|IDF 22B:1987 ISO 8262-3:2005|IDF 124-3:2005
			Protein		ISO 8968-1|2|3:2001|IDF 20-1|2|3:2001
			Sucrose (normal content)		ISO 2911:2004|IDF 35:2004
			Sucrose (low content)			Note 2
			Water (acid buttermilk powder)		Annex XIX
			Water (sweet buttermilk powder)		ISO 5537:2004|IDF26:2004
			Solids (other products)		Methods approved by the competent authority
	Whey, whether or not concentrated or containing added sugar or other sweetening matter; products consisting of natural milk constituents	0404	Fat		ISO 1736:2000|IDF 9C:1987 ISO 2450:1999|IDF 16C:1987 ISO 7208:1999|IDF 22B:1987
			Protein		ISO 8968-1|2|3:2001|IDF 20-1|2|3:2001
			Sucrose (normal content)		ISO 2911:2004|IDF 35:2004
			Sucrose (low content)			Note 2
		0404 90	Protein		ISO 8968 1/2 2001|IDF 20-1/2:2001
			Water		IDF 21B:1987
			Solids		ISO 6734:1989|IDF 15B:1991
			(Concentrated products)		ISO 6731:1989|IDF 21B:1987
	Butter and other fats derived from milk; dairy spreads	0405	Fat (if ≤ 85 % m/m)		ISO 17189:2003|IDF 194:2003
		Butter	Water		ISO 3727-1:2001|IDF 80-1:2001
			SNF		ISO 3727-2:2001|IDF 80-2:2001
			NaCl		ISO 15648:2004|IDF 179:2004
			Fat (if > 99 % m/m)		IDF 24:1964
	Butteroil		Water (if fat < 99 % m/m)		ISO 5536:2002|IDF 23:2002
	Cheese and curd	0406	Fat		ISO 1735:2004|IDF 5:2004
			Solids		ISO 5534:2004|IDF 4:2004
			Solids (Ricotta)		ISO 2920:2004|IDF 58:2004
			NaCl		ISO 5943:2006|IDF 88:2006
			Lactose		ISO 5765-1/2:2002|IDF 79-1/2:2002
Regulation (EEC) No 2658/87	Compound feedingstuffs	2309	Lactose		Annex XI

Notes to list of European Union reference methods:

Note 1: Milk fat isolation as described in ISO 1740:1991 (protection from light).

Note 2: No reference method has been established. Methods approved by the competent authority.

Note 3: Sample to be prepared according to ISO 8261:2001|IDF 122:2001.

Note 4: Incubation for 48 hours at a temperature of 55 °C, care should be taken to prevent the culture medium from drying out.

Note 5: % m/m SNF = % m/m solids — % m/m fat.

Note 6: Commission Directive 84/4/EEC.

Note 7: Commission Regulation (EC) No 2799/1999 (OJ L 340, 31.12.1999, p. 3-27).

Note 8: Commission Directive 78/633/EEC.

---

(1)  Without prejudice to the requirements in the specific Regulation

(2)  The minimum protein content would be 34 % per 1 September 2009.

ANNEX II

(Article 3)

EVALUATION OF COMPLIANCE OF A CONSIGNMENT WITH THE LEGAL LIMIT

1.   PRINCIPLE

In cases where detailed sampling procedures are given by the relevant legislation, these procedures are followed. In all other cases a sample of at least 3 sample units taken randomly from the consignment submitted to control is used. A composite sample may be prepared. The result obtained is 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 must 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 must be carried out. Internal repeatability standard deviation sir and the internal reproducibility standard deviation siR have to be used instead of σr and σ R , resp., in the formulae for the computation of the expanded uncertainty U.

The decision rules are as under (1). However, if the consignment is judged to be not in compliance with the legal limit the measurements must be repeated with the method specified in this Regulation and the decision reached according to (1).

ANNEX III

(Article 4)

EVALUATION OF ASSESSORS AND THE RELIABILITY OF RESULTS IN SENSORY ANALYSES

The following procedures are applicable if scoring methods are used (IDF Standard 99C:1997).

A.   DETERMINATION OF THE ‘REPEATABILITY INDEX’

At least ten samples will be analysed as blind duplicates by an assessor within a period of 12 months. This will usually happen in several sessions. The results for individual product characteristics are evaluated using the following formula:

where:

w1	:	repeatability index
xi1	:	score for the first evaluation of sample xi
xi2	:	score for the second evaluation of sample xi
n	:	number of samples

The samples to be evaluated should reflect a broad quality range. wI should not exceed 1,5 (5-point scales).

B.   DETERMINING THE ‘DEVIATION INDEX’

This index should be used to check whether an assessor uses the same scale for quality evaluation as an experienced group of assessors. The scores obtained by the assessor are compared with the average of the scores obtained by the assessor group.

The following formula is used for the evaluation of results:

where:

xi1; xi2	:	see section (A)
;	:	average score of the assessor group for the first and second evaluation respectively of sample xi
n	:	number of samples (at least 10 per 12 months).

The samples to be evaluated should reflect a broad quality range. DI should not exceed 1,5 (5-point scales).

Member States must notify any difficulties encountered when applying this procedure.

Where individual assessors are found to exceed the 1,5 limit for Deviation or Repeatability indices, the Official authority expert/s must perform one or more random ‘Re-performance’ checks on samples graded by them over the next few weeks, or perform one or more ‘Accompanied’ checks with those assessors. Close monitoring is necessary to decide whether to retain their services. Findings should be documented and retained as proof of follow up action.

C.   COMPARISON OF THE RESULTS OBTAINED IN DIFFERENT REGIONS OF A MEMBER STATE AND IN DIFFERENT MEMBER STATES

Where applicable, a test must be organised at least once per year to compare the results obtained by assessors from different regions. If significant differences are observed, the necessary steps should be taken to identify the reasons and arrive at comparable results.

Member States may organise tests to compare the results obtained by their own assessors and by assessors from neighbouring Member States. Significant differences should lead to an in-depth investigation with the aim of arriving at comparable results.

Member States should notify the Commission of the results of these comparisons.

ANNEX IV

(Article 4)

SENSORY EVALUATION OF BUTTER

1.   SCOPE

The purpose of this procedure for sensory evaluation of butter is to provide a uniform method applicable in all Member States.

Refer to the current IDF International Standard for Milk and Milk Products, IDF 99 — Parts 1, 2, 3 on Sensory Evaluation, for further detail.

2.   DEFINITIONS

‘Sensory evaluation’ (assessment) means the examination of the attributes of a product by the sense organs.

‘Panel’ means a group of selected assessors working, during the assessment, without intercommunication, and without influencing one another.

‘Assessor’ is defined as someone chosen for his/her ability to perform a sensory test. This type of assessor may have limited experience.

‘Expert Assessor’ is defined as someone with a high degree of sensory sensitivity and experience of sensory methodology, who is able to make consistent and reliable sensory assessments of various products. This type of assessor will have a good long term sensory memory.

‘Scoring’ means sensory evaluation by a panel, using a numerical scale. A nomenclature of defects must be used.

‘Grading’ means a quality classification which is performed on the basis of scoring.

‘Control documents’: documents used to record the individual scores for each attribute and the final grade of the product. (This document may also be used to record chemical composition.)

3.   TEST ROOM

Refer to ISO 8589 and ISO/DIS 22935-2 | IDF 99-2 par 7 for more details.

Precautions must be taken in order that the assessors in the test room are not influenced by external factors.

The test room must be free from foreign odours and easy to clean. The walls must be of a light colour and non reflective.

The test room and its lighting must be such that the properties of the products to be scored are not affected.

The room must be equipped with appropriate thermostatic control to enable a constant temperature of butter to be maintained. Butter should have a temperature of 12 °C (±2 °C) at the time of grading.

4.   SELECTION OF ASSESSORS

An assessor must be familiar with butter products and be competent to carry out sensory grading. His/her competence should be monitored on a regular basis (at least once a year) by the competent authority.

4.1.   ISO/DIS 22935-1 | IDF 99-1 par 4 (recruitment) and par 5.1 should be consulted for details on general requirements and screening tests which may be used prior to official use of a new assessor.

It is essential that training is ongoing and general sessions should be held on a regular basis. Refer to ISO 8586-1 for information on panel training.

4.2.   Initial training should cover the following:

—	general theory and practical importance of sensory evaluation,

—	methods, scales and description of sensory impressions,

—	detection and recognition of sensory attributes and specific sensory terms,

—	background training on the manufacture of butter,

—	validated references and samples to help the assessor to identify specific flavours and flavour intensity within the product.

5.   REQUIREMENTS FOR THE PANEL

The number of assessors in the panel should be uneven, the minimum number being three. The majority must be employees of the competent authority or authorised persons not employed by the dairy industry.

A panel Leader shall be responsible for the entire procedure and may participate in the panel.

A number of factors must be taken into account before evaluation in order to obtain optimal performances from the subjects:

—	subjects must not be suffering from an illness which could affect their performance. In such a case, the assessor concerned should be replaced on the panel by another,

—	subjects must be on time to take part in the evaluation and make sure that they have enough time to make their evaluation,

—	subjects must not use strong-smelling substances like perfume, after-shave lotion, deodorant, etc. and should avoid eating strong-flavoured (e.g. highly spiced) food, etc.,

—	subjects may not smoke, eat or drink anything other than water during the half hour before the evaluation.

6.   PERFORMANCE

All Assessors should participate in regular sensory evaluation panels to maintain their competence. The frequency will depend on the volume and throughput of butter, and where possible, should be at least one panel per month.

Senior Assessors should also participate in a number of panels each year, and where possible, at least once per quarter.

7.   SAMPLING AND PREPARATION OF THE SAMPLE

It is essential that the identity of the samples is concealed during the assessment so that any possible bias is avoided. The samples should be coded.

This should be organized prior to the evaluation. Requirement for temperature of butter during its transportation to the test room should be set (6 °C ± 2 °C).

When the sensory evaluation is carried out at a cold store, the sample is taken using a butter trier. If the sensory evaluation is carried out at another location other than the cold store, then at least a 500 g sample should be taken. During the evaluation, the butter should have the temperature of 12 °C (±2 °C) (refer: in ISO/DIS 22935-2 | IDF 99-2 the evaluation temperature of butter is 14 °C ± 2 °C). Large deviations should be avoided at all cost.

8.   ASSESSMENT OF THE VALUE OF EACH ATTRIBUTE

8.1.   The sensory evaluation is to be carried out in relation to the following three attributes: appearance, consistency and flavour:

‘Appearance’ involves the following features: colour, visible purity, absence of physical contamination, absence of mould growth and uniformity of water dispersion. Water dispersion is tested according to IDF-Standard 112A/1989.

‘Consistency’ involves the following features: Body, texture and firmness. Spreadability may be monitored using physical means should an Individual Member State so wish in order to satisfy customer requirements. The Commission may decide to harmonise methodology in the future.

‘Body’ is the term which refers to the cohesiveness of the product as it is being consumed. It is normally associated with firmness and spreadability, and should be uniform throughout the product. It is closely related to texture and is the ability of the product to stand up under its own weight. It is indicated by resistance during cutting and can be measured mechanically and by mouthfeel and fingerfeel.

‘Flavour’ is the characteristic as perceived in the mouth, predominantly by the taste buds of the tongue.

‘Aroma’ is the characteristic as perceived by the nose and sense of smell.

A significant deviation from the recommended temperature prevents a reliable evaluation of consistency and flavour. The temperature is of paramount importance.

Grading of butter must be deferred if the temperature is outside the recommended band.

8.2.   Each attribute has to be sensory evaluated separately. The scoring has to be done according to table 1.

8.3.   It may be desirable for the assessors to score together, before starting the assessment, one or more reference samples for appearance, consistency and flavour, in order to achieve uniformity.

8.4.   Scoring for acceptance is as follows:

Refer to part 7 — Nomenclature, and description of criteria applicable to points, when scoring.

	Maximum	Required
Appearance	5	4
Consistency	5	4
Flavour/aroma	5	4

—	Where the required score is not obtained, a description of the defect has to be given.

—	The score given by each assessor for each attribute must be recorded in the control document.

—	The product is accepted or rejected on the basis of a majority decision.

—	Cases where differences between the individual scoring for each attribute are wider than adjacent points should not occur frequently (not more than once per 20 samples). Otherwise the competence of the panel should be checked by the panel leader.

9.   SUPERVISION

A panel leader who must be an official employee of the competent authority and may be a member of the panel must be generally responsible for the entire procedure. He/she must record the individual scores for each attribute in the control document and certify whether the product is accepted or rejected.

10.   NOMENCLATURE

Refer to the appended table 2.

11.   REFERENCE

FIL-IDF 99C:1997 Sensory evaluation of dairy products by scoring — Reference method

ISO/DIS 22935 | IDF 99 International Standard for Milk and Milk Products — Sensory analysis — Parts 1-3

ISO 8586-1 Sensory analysis — General guidance for selection, training and monitoring of assessors — Part 1

ISO 8589 Sensory analysis — General guidance for the design of test rooms

FIL-IDF 112A:1989 Butter — Determination of water dispersion value

Table 1

Butter scoring

Appearance			Consistency			Flavour + aroma
Points	No (1)	Remarks	Points (quality class)	No (1)	Remarks	Points (quality class)	No (1)	Remarks
5		Very good ideal type highest quality (equal dry)	5		Very good ideal type highest quality (well spreadable)	5		Very good ideal type highest quality (absolutely pure finest aroma)
4		Good  (2) no evident defects	4	17 18	Good  (2) hard soft	4		Good  (2) no evident defects
3	1 2 3 4 5 6 7 8	Fair (slight defects) loose (free), moisture not uniform, two coloured streaky mottled, marbled speckled oil separation overcoloured weak, open texture	3	14 15 16 17 18	Fair (slight defects) short, brittle, crumbly pasty, doughy, greasy sticky hard soft	3	21 22 25 27 33 34 35	Fair (slight defects) unclean foreign flavour acid cooked flavour, scorched flavour feed flavour coarse, bitter oversalted
2	1 3 4 5 6 10 11 12	Poor (evident defects) loose (free) moisture streaky mottled, marbled speckled oil separation foreign matter mouldy undissolved salt	2	14 15 16 17 18	Poor (evident defects) short, brittle, crumbly pasty, doughy, greasy sticky hard soft	2	21 22 23 25 32 33 34 35 36 38	Poor (evident defects) unclean foreign flavour stale acid oxidized flavour, metallic flavour feed flavour coarse, bitter oversalted musty-flat, putrid chemical flavour
1	1 3 4 5 6 7 9 10 11 12	Very poor (strong defects) loose (free) moisture streaky mottled, marbled speckled oil separation overcoloured granular foreign matter mouldy undissolved salt	1	14 15 16 17 18	Very poor (strong defects) short, brittle, crumbly pasty, doughy, greasy sticky hard soft	1	22 24 25 26 28 29 30 31 32 34 35 36 37 38	Very poor (strong defects) foreign flavour cheesy, lactic cheese flavour acid yeasty mould flavour rancid oily, fishy tallowy oxidized flavour, metallic flavour coarse, bitter oversalted musty-flat, putrid malty chemical flavour

Table 2

Table of butter defects

I. Appearance

1. loose (free), moisture

2. not uniform, two coloured

3. streaky

4. mottled, marbled

5. speckled

6. oil separation

7. overcoloured

8. weak (open texture)

9. granular

10. foreign matter

11. mouldy

12. undissolved salt

II. Consistency

14. short, brittle, crumbly

15. pasty, doughy, greasy

16. sticky

17. hard

18. soft

III. Flavour and aroma

20. without flavour

21. unclean (3)

22. foreign flavour

23. stale

24. cheesy, lactic cheese flavour

25. acid

26. yeasty

27. (a) cooked flavour

(b) scorched flavour

28. mouldy flavour

29. rancid

30. oily, fishy

31. tallowy

32. (a) oxidized flavour

(b) metallic flavour

33. feed flavour

34. coarse, bitter

35. oversalted

36. musty-flat, putrid

37. malty

38. chemical flavour

---

(1)  Table 2.

(2)  The defects mentioned under ‘good’ are only very small deviations from the ideal type.

(3)  This designation should be used as seldom as possible and only when the defect cannot be described more accurately.

ANNEX V

(Article 5)

DETERMINATION OF THE CONTENT OF ENANTHIC ACID TRIGLYCERIDE IN BUTTER, BUTTER-OIL AND CREAM BY GAS CHROMATOGRAPHIC ANALYSIS OF TRIGLYCERIDES

1.   SCOPE

This method lays down a method for the determination of the content of the triglyceride of enanthic acid in butter-oil, butter and cream.

2.   TERMS AND DEFINITION

Enanthic acid content: content of the triglyceride of enanthic acid determined by the procedure specified in this method.

Note: The enanthic acid content is expressed in kg per ton of product for butter-oil and butter, and it is expressed in kg per ton of milk fat for cream.

3.   PRINCIPLE

Milk fat is extracted from the different products according to ISO 14156 | IDF 172:2001. The quantitative determination of the content of the triglyceride of enanthic acid in the extracted fat is determined by capillary gas chromatography (GC). The result obtained for the sample is evaluated by reference to the triglyceride of caproic acid as internal standard.

Note: Tributyrin has also been found to be a satisfactory internal standard.

4.   REAGENTS

Use only reagents of recognized analytical grade.

4.1.   n-Hexane

4.2.   Standard triglyceride of caproic acid, at least 99 % pure

4.3.   Standard triglyceride of enanthic acid, at least 99 % pure

4.4.   Anhydrous sodium sulfate (Na2SO4).

5.   APPARATUS

Usual laboratory equipment and particularly the following:

5.1.   Analytical balance precise at 1 mg

5.2.   Volumetric flasks, of capacities 10 ml and 20 ml

5.3.   Tubes for centrifuge, of capacity 30 ml

5.4.   Rotary evaporator

5.5.   Oven, capable of being maintained at a temperature of 50 °C ± 5 °C

5.6.   Filter paper, medium porosity, of diameter about 15 cm

Gas chromatography equipment

5.7.1.   Gas chromatograph equipped with a split/splitless or on-column injector and a flame ionization detector (FID)

GC column, with a stationary phase which has successfully employed to perform triglyceride separation (100 % dimethylpolysiloxane or 5 % phenyl-95 % methylpolysiloxane). Select the stationary phase, the column length (between 4 m and 15 m), the internal diameter (between 0,22 mm and 0,50 mm) and the film thickness (0,12 μm or more) taking into account the laboratory experience and the injection system applied. In any case the selected column shall produce both a complete separation between the solvent peak and the triglyceride of caproic acid and a baseline resolution between triglyceride of caproic and enanthic acid peaks. Examples of applicable conditions are listed below.

5.7.2.1.   Example of applicable conditions using a split injector:

—	Carrier gas: helium

—	Column head pressure: 100 KPa

—	Column: 12 m length, 0,5 mm internal diameter, 0,1 μm film thickness fused silica column

—	Stationary phase: 100 % dimethylpolysiloxane or 5 % phenyl-95 % dimethylpolysiloxane (for ex. HT5)

—	Column temperature: initial temperature of 130 °C, maintained for 1 min, raised at a rate of 20 °C/min up to 260 °C and then raised at a rate of 30 °C/min up to 360 °C; maintain 10 mn at 360 °C

—	Detector temperature: 370 °C

—	Injector temperature: 350 °C

—	Split ratio 1:30

—	Amount of sample injected: 1 μl.

5.7.2.2.   Example of applicable conditions using an on-column injector:

—	Carrier gas: hydrogen (constant flow system)

—	Column head pressure: 89 kPa

—	Column: 4 m length, 0,32 mm internal diameter, 0,25 μm film thickness, fused silica column

—	Stationary phase: 5 % phenyl, 95 % dimethylpolysiloxane

—	Column temperature: initial temperature of 60 °C, maintained for 2 min, raised at a rate of 35 °C/min up to 340 °C, maintained at this temperature for 5 min

—	Detector temperature: 350 °C

—	Amount of sample injected: 1 μl

5.8.   Injection syringe, of capacity 5 μl.

6.   SAMPLING

It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage.

Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in IDF: standard 50C:1995 or ISO 707-1997 — Milk and milk products — Methods of sampling.

7.   PROCEDURE

7.1.   Preparation of the test sample and test portion

Proceed according to ISO 14156 | IDF 172:2001

7.1.1.   Butter-oil, Butter

7.1.1.1.   Melt 50 g to 100 g of test sample in the oven (5.5)

7.1.1.2.   Place 0,5 g to 1,0 g of anhydrous sodium sulfate (5.4) in a folded filter paper

7.1.1.3.   Filter the fat through the filter paper containing anhydrous sodium sulfate collecting the filtrate in a beaker maintained in the oven (5.5). When decanting the melted butter onto the filter paper, take care that no serum is transferred

7.1.2.   Cream

7.1.2.1.   Bring the test sample to a temperature of 20 °C ± 2 °C

7.1.2.2.   Mix or stir the sample thoroughly

7.1.2.3.   Dilute a suitable amount of test sample so as to obtain 100 ml of test portion with a mass fraction of fat of approximately 4 %

7.1.2.4.   Proceed as with raw milk and homogenized milk (see ISO 14156 | IDF 172:2001, §8.3) to extract the fat from the cream

7.1.2.5.   Weigh in a 10 ml volumetric flask (5.2), to the nearest 1 mg, 1 g of the extracted fat. Add 1 ml of the solution 7.2.2. Complete to 10 ml with n-hexane (4.1) and homogenise

7.1.2.6.   Introduce 1 ml of the solution 7.1.1.2 in a 10 ml volumetric flask (5.2) and dilute to 10 ml with n-hexane (4.1)

7.2.   Preparation of the calibration standards

7.2.1.   Dissolve 100 mg of the triglyceride of enanthic acid (4.3) in 10 ml of n-hexane (4.1)

7.2.2.   Dissolve 100 mg of the triglyceride of caproic acid (4.2) in 10 ml of n-hexane (4.1)

7.2.3.   Introduce 1 ml of the solution 7.2.2 in a 10 ml volumetric flask (5.2). Complete to 10ml with n-hexane (4.1)

7.2.4.   Introduce 1 ml of the solution 7.2.1 and 1 ml of the solution 7.2.2 in a 10 ml volumetric flask (5.2). Complete to 10 ml with n-hexane (4.1)

7.2.5.   Introduce 1 ml of the solution 7.2.4 in a 10 ml volumetric flask (5.2) and complete to 10 ml with n-hexane (4.1)

7.3.   Chromatographic determination

7.3.1.   Inject 1 μl of the standard solution 7.2.5 twice

7.3.2.   Inject 1 μl of each sample solution

Note: If the on column injector system is adopted an increased dilution should be applied both to the standard and sample solutions.

7.3.3.   Repeat the operation 7.3.1 every 3 samples in order to bracket samples between duplicate standard injections. Results are based upon the mean average response factors from the standard chromatograms.

8.   CALCULATION OF RESULTS

For each chromatogram, integrate the area of the peaks associated with the triglycerides of enanthic acid and caproic acid.

Follow those instructions for each bracketed sequence i.e. for a set of bracketed samples, the standard injected twice immediately before them is STD1 and the standard injected twice immediately after them is STD2.

8.1.   Calibration

8.1.1.   Calculate the response factor for each duplicate of STD1, Rf1(a) and Rf1(b)

Rf1 (a) or (b) = (Peak area for caproic acid triglyceride/Peak area for enanthic acid triglyceride) × 100

Calculate the mean average response factor, Rf1

Rf1 = (Rf1(a) + Rf1(b)) / 2

8.1.2.   Similarly, calculate the mean average response factor STD2, Rf2

8.1.3.   Calculate the mean average response factor, Rf

Rf = (Rf1 + Rf2) /2

8.2.   Test samples

For each sample chromatogram obtained between STD1 and STD2, calculate the enanthic acid content, C (kg/t):

C = (Peak area for enanthic acid triglyceride × Rf × 100)/(Peak area for caproic acid triglyceride × Wt × 1 000)

where:

—	Wt = weight of fat taken (g),

—	100 = dilution volume for sample,

—	1 000 = conversion factor (for μg/g to kg/t)

For butter samples, take the fat content of butter into account and calculate a corrected concentration value, Cbutter (kg/t of butter)

Cbutter = Cfat × F

where F is the fat content of butter.

9.   PRECISION

Details of an interlaboratory test on butter in accordance with ISO 5725-1 and ISO 5725-2 on the precision method are shown in (12.).

The values for repeatability and reproducibility limit are expressed for the 95 % probability level and may not be applicable to concentration ranges and matrices other than those given.

9.1.   Repeatability

The absolute differences between two individual single test results, obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than 0,35 kg/t.

9.2.   Reproducibility

The absolute differences between two individual single test results, obtained with the same method on identical test material in different laboratories with different operators using different equipment will in not more than 5 % of cases be greater than 0,66 kg/t.

10.   TOLERANCE LIMITS: LOWER LIMITS (CASE OF INSUFFICIENT QUANTITIES)

10.1.    Three samples must be taken from the traced product in order to check on the correct tracing of the product

10.2.   Butter and concentrated butter

10.2.1.   The incorporation rate is 11 kg of at least 95 % pure enanthic acid triglyceride per tonne of butter, i.e. 10,45 kg/t

10.2.2.   The results of three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

—	9,51 kg/t (95 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination),

—	6,89 kg/t (70 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination),

—	The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation respectively between 9,51 kg/t and 6,89 kg/t.

10.3.   Cream

10.3.1.   The incorporation is 10 kg of at least 95 % pure enanthic acid triglyceride per tonne of milk fat, i.e. 9,50 kg/t traced milk fat

10.3.2.   The results of the three samples obtained from the analysis of the product are used to check the rate of the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

—	8,60 kg/t (95 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination),

—	6,23 kg/t (70 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination),

—	The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation respectively between 8,60 kg/t and 6,23 kg/t.

11.   TOLERANCE LIMITS: UPPER LIMITS (CASE OF EXCEEDING QUANTITY BY MORE THAN 20 %)

11.1.    Three samples must be taken from the traced product in order to check on the correct tracing of the product

11.2.   Butter and concentrated butter

11.2.1.   The results of three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the mean of these results is compared with the following limits:

—	Upper limit is 12,96 kg/t

11.3.   Cream

11.3.1.   The results of three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the mean of these results is compared with the following limits:

—	Upper limit is 11,82 kg/t.

12.   ADDITIONAL INFORMATION: STATISTICAL ANALYSIS OF RESULTS ON THE DETERMINATION OF TRIENANTOATE IN BUTTERFAT BY TRIGLYCERIDE ANALYSIS

Four collaborative trials have been carried out to determine the trienantoate content in traced butter.

Nine laboratories participated to the 1st ring test and no specifications were provided about the analytical methods to use:

10 laboratories participated to the 2nd ring test and 4 different methods were applied:

—	Quantification of methylheptanoate by using n-nonane or methylnonanoate as internal standard

—	Quantification of trienantoate by using tricaproate as internal standard

—	Quantification of methylheptanoate by using a calibration sample/mixture

—	Quantification of methylheptanoate by using a calibration mixture.

Moreover, if FAME were analysed, two different methylation procedures were used (De Francesco and Christopherson & Glass).

Due to the results obtained, two methods were chosen to perform the 3rd ring test:

—	Quantification of methylheptanoate by using n-nonane or methylnonanoate as internal standard

—	Quantification of trienantoate by using tricaproate as internal standard.

The results of 7 labs showed that the FAME method produced a higher variability and consequently it was decided to use only the determination of trienantoate as triglyceride following the procedure of the q Quantification of trienantoate by using tricaproate as internal standard. Moreover the triglyceride analysis has to be carried out by capillary column.

In the 4th ring test four samples (A, B, C, D) were circulated and nine laboratories provided results (Tables 1-2).

Two laboratories (DE and UE) analysed the samples by using FAME method.

Due to the reduced number of laboratories, the Statistical calculation has been performed both on the complete set (Figures 1-2) of data including FAME results and on the data obtained from TG analysis.

Tests for outliers:

—	sample A. Dixon, Cochran and Grubbs tests at levels 1 and 5 %, showed one laboratory outlier.

—	sample B. Grubbs test at level 5 % showed one laboratory outlier.

—	sample C. Dixon and Grubbs tests at levels 1 and 5 %, showed one laboratory outlier.

—	sample D. Dixon and Grubbs tests at levels 1 end 5 %, showed one laboratory outlier.

The outlier has been excluded from the calculation.

It is worth noting that the results obtained by FAME method were never considered as outliers by the tests applied.

Precision parameters

Tables 1 and 2 report the results of all the laboratories and the precision parameters calculated on an acceptable number (8) of labs but, unfortunately not deriving from the same analytical method.

Tables 3 and 4 report the results deriving only from TG method and the corresponding precision parameters. The acceptance of these parameters is subjected to the acceptance of the low number of laboratories (6).

Figures 2 and 3 show the trend of Sr and SR calculated on the 4 samples of the 2 data set described above.

Table 5 reports the Sr and SR values together with the corresponding pooled values and overall r and R parameters.

Finally the Critical Difference at 95 % of probability level has been calculated.

Table 1

Statistical Results of TG + FAME* methods

Sample A		R1	R2	Mean	N. of labs retained after eliminating outliers	8
RENNES	FR1	11,0	11,1	11,1	N. of outliers	1
RIKILT	NL	11,2	11,2	11,2	Outliers	DК
ZPLA	DE*	11,6	11,8	11,7	Mean value	11,3
ADAS	GB	11,4	11,2	11,3	True value	11,0
CNEVA	FR2	11,4	11,4	11,4	Repeatability standard deviation (Sr)	0,09
LODI	IT	11,1	11,3	11,2	Repeatability relative sd (RSDr%)	0,80
EELA	FI	11,3	11,2	11,3	Repeatability r (95 %)	0,26
ISPRA	UE*	11,0	11,0	11,0	Relative Repeatability r %	2,24
D.V.F.A.	DK	13,3	11,8	12,6	Reproducibility standard deviation (SR)	0,23
					Reproducibility relative sd (RSDR%)	2,04
					Reproducibility R (95 %)	0,84
					Relative Reproducibility R %	5,71
Sample B		R1	R2	Mean	N. of labs retained after eliminating outliers	8
RENNES	FR1	12,7	12,8	12,8	N, of outliers	1
RIKILT	NL	13,5	13,3	13,4	Outliers	DK
ZPLA	DE*	14,0	13,8	13,9	Mean value	13,4
ADAS	GB	13,4	13,5	13,5	True value	13,5
CNEVA	FR2	13,3	13,4	13,4	Repeatability standard deviation (Sr)	0,14
LODI	IT	13,9	13,5	13,7	Repeatability relative sd (RSDr%)	1,04
EELA	FI	13,4	13,2	13,3	Repeatability r (95 %)	0,40
ISPRA	UE*	13,2	13,3	13,3	Relative Repeatability r %	2,91
D.V.F.A.	DK	14,1	14,8	14,5	Reproducibility standard deviation (SR)	0,35
					Reproducibility relative sd (RSDR%)	2,61
					Reproducibility R (95 %)	0,99
					Relative reproducibility R %	7,31

Table 2

Statistical Results of TG + FAME* methods

Sample C		R1	R2	Mean	N. of labs retained after eliminating outliers	8
RENNES	FR1	8,9	9,2	9,1	N. of outliers	1
RIKILT	NL	9,2	9,3	9,3	Outliers	DK
ZPLA	DE*	9,2	9,4	9,3	Mean value	9,3
ADAS	GB	9,5	9,3	9,4	True value	9,3
CNEVA	FR2	9,4	9,4	9,4	Repeatability standard deviation (Sr)	0,14
LODI	IT	9,2	9,5	9,4	Repeatability relative sd (RSDr%)	1,50
EELA	FI	9,4	9,6	9,5	Repeatability r (95 %)	0,40
ISPRA	UE*	9,4	9,3	9,4	Relative Repeatability r %	4,20
D.V.F.A.	DK	10,7	10,9	10,8	Reproducibility standard deviation (SR)	0,17
					Reproducibility relative sd (RSDR%)	1,82
					Reproducibility R (95 %)	0,47
					Relative Reproducibility R %	5,10
Sample D		R1	R2	Mean	N. of labs retained after eliminating outliers	8
RENNES	R1	1,6	1,6	1,6	N. of outliers	1
RIKILT	NL	2,1	2,1	2,1	Outliers	DK
ZPLA	DE*	2,3	2,3	2,3	Mean value	2,1
ADAS	GB	2,1	2,2	2,2	True value	2,1
CNEVA	FR2	2,1	2,1	2,1	Repeatability standard deviation (Sr)	0,08
LODI	IT	2,2	1,9	2,1	Repeatability relative sd (RSDr%)	3,81
EELA	FI	2,3	2,3	2,3	Repeatability r (95 %)	0,22
ISPRA	UE*	2,3	2,3	2,3	Relative Repeatability r %	10,67
D.V.F.A.	DK	3,4	2,9	3,2	Reproducibility standard deviation (SR)	0,24
					Reproducibility relative sd (RSDR%)	11,43
					Reproducibility R (95 %)	0,67
					Relative Reproducibility R %	32,00

Table 3

Statistical Results of TG + FAME* methods

Sample A		R1	R2	Mean	N. of labs retained after eliminating outliers	6
RENNES	FR1	11,0	11,1	11,1	N. of outliers	1
RIKILT	NL	11,2	11,2	11,2	Outliers	DК
ADAS	GB	11,4	11,2	11,3	Mean value	11,2
CNEVA	FR2	11,4	11,4	11,4	True value	11,0
LODI	IT	11,1	11,3	11,2	Repeatability standard deviation (Sr)	0,09
EELA	FI	11,3	11,2	11,3	Repeatability relative sd (RSDr%)	0,80
D.V.F.A.	DK	13,3	11,8	12,6	Repeatability r (95 %)	0,25
					Relative Repeatability r %	2,24
					Reproducibility standard deviation (SR)	0,13
					Reproducibility relative sd (RSDR%)	1,16
					Reproducibility R (95 %)	0,36
					Relative Reproducibility R %	3,25
Sample B		R1	R2	Mean	N. of labs retained after eliminating outliers	6
RENNES	FR1	12,7	12,8	12,8	N. of outliers	1
RIKILT	NL	13,5	13,3	13,4	Outliers	DК
ADAS	GB	13,4	13,5	13,5	Mean value	13,3
CNEVA	FR2	13,3	13,4	13,4	True value	13,5
LODI	IT	13,9	13,5	13,7	Repeatability standard deviation (Sr)	0,15
EELA	FI	13,4	13,2	13,3	Repeatability relative sd (RSDr%)	1,13
D.V.F.A.	DK	14,1	14,8	14,5	Repeatability r (95 %)	0,42
					Relative Repeatability r %	3,16
					Reproducibility standard deviation (SR)	0,33
					Reproducibility relative sd (RSDR%)	2,48
					Reproducibility R (95 %)	0,93
					Relative Reproducibility R %	6,94

Table 4

Statistical Results of TG method

Sample C		R1	R2	Mean	N. of labs retained after eliminating outliers	6
RENNES	FR1	8,9	9,2	9,1	N. of outliers	1
RIKILT	NL	9,2	9,3	9,3	Outliers	DК
ADAS	GB	9,5	9,3	9,4	Mean value	9,3
CNEVA	FR2	9,4	9,4	9,4	True value	9,3
LODI	IT	9,2	9,5	9,4	Repeatability standard deviation (Sr)	0,15
EELA	FI	9,4	9,6	9,5	Repeatability relative sd (RSDr%)	1,61
D.V.F.A.	DK	10,7	10,9	10,8	Repeatability r (95 %)	0,42
					Relative Repeatability r %	4,51
					Reproducibility standard deviation (SR)	0,19
					Reproducibility relative sd (RSDR%)	2,04
					Reproducibility R (95 %)	0,53
					Relative Reproducibility R %	5,71
Sample D		R1	R2	Mean	N, of labs retained after eliminating outliers	6
RENNES	FR1	1,6	1,6	1,6	N. of outliers	1
RIKILT	NL	2,1	2,1	2,1	Outliers	DK
					Mean Value	2,1
ADAS	GB	2,1	2,2	2,2	True value	2,1
CNEVA	FR2	2,1	2,1	2,1	Repeatability standard deviation (Sr)	0,09
LODI	IT	2,2	1,9	2,1	Repeatability relative sd (RSDr%)	4,29
EELA	FI	2,3	2,3	2,3	Repeatability r (95 %)	0,26
D.V.F.A.	DK	3,4	2,9	3,2	Relative Repeatability r %	12,01
					Reproducibility standard deviation (SR)	0,25
					Reproducibility relative sd (RSDR%]	11,90
					Reproducibility R (95 %)	0,69
					Relative Reproducibility R %	33,32

Table 5

Repeatability and reproducibility (with FAME)

	No of labs	Outlier	Repeatability Sr (95 %)	Reproducibility SR (95 %)
Sample A	8	1	0,09	0,23
Sample Β	8	1	0,14	0,35
Sample C	8	1	0,14	0,17
Sample D	8	1	0,08	0,24
Pooled value			0,116	0,256
			r	R
Pooled value* 2,8			0,324	0,716
CrD95 =0,40 Minimum purity stated for trienantoate = 95 % Minimum limit stated for trienantoate in butterfat = 11 kg/t Taking the Critical Difference for a 95 % probability level into consideration, the mean of the two results shall not be less than:   in the case of incorporation of 95 % pure trienantoate 10,05 kg/t

Repeatability and reproducibility (without FAME)

	No of labs	Outlier	Repeatability Sr (95 %)	Reproducibility SR (95 %)
Sample A	6	1	0,09	0,13
Sample B	6	1	0,15	0,33
Sample C	6	1	0,15	0,19
Sample D	6	1	0,09	0,25
Pooled value			0,124	0,237
			r	R
Pooled value * 2,8			0,347	0,663
CrD95 = 0,36 Minimum purity stated for trienantoate = 95 % Minimum limit stated for trienantoate in butterfat = 11 kg/t Taking the Critical Difference for a 95 % probability level into consideration, the mean of the two results shall not be less than:   in the case of incorporation of 95 % pure trienantoate 10,09 kg/t.

Figure 1 (*1)

Experimental results: Sample A

14,0

outlier

13,5

13,0

12,5

kg/t

R1

12,0

outlier

R2

mean

true

11,5

11,0

10,5

10,0

FR1

NL

DE (*)

GB

FR2

IT

FI

UE (*)

DK

labs

Experimental results: Sample B

16,0

15,5

outlier

15,0

outlier

14,5

kg/t

R1

14,0

R2

mean

13,5

true

13,0

12,5

12,0

FR1

NL

DE (*)

GB

FR2

IT

FI

UE (*)

DK

labs

Experimental results: Sample C

12,0

11,5

outlier

11,0

outlier

10,5

kg/t

R1

R2

10,0

mean

true

9,5

9,0

8,5

8,0

FR1

NL

DE (*)

GB

FR2

IT

FI

UE (*)

DK

labs

Experimental results: Sample D

4,0

outlier

3,5

outlier

3,0

2,5

kg/t

R1

2,0

R2

mean

1,5

true

1,0

0,5

0,0

FR1

NL

DE (*)

GB

FR2

IT

FI

UE (*)

DK

labs

Figure 2

Repeatability and Reproducibility standard deviation at different levels (TG+FAME)

0,4

0,3

S(r,R)

0,2

Sr

SR

0,1

0

0

2

4

6

8

10

12

14

Mean values (kg/t)

Figure 3

Repeatability and Reproducibility standard deviation at different levels (TG)

0,35

0,3

0,25

S(r,R)

0,2

Sr

0,15

SR

0,1

0,05

0

0

2

4

6

8

10

12

14

Mean values (kg/t)

Figure 4

Example using an on-column injector

mV

Caproic acid triglyceride

Enanthic acid triglyceride

2

3

4

5

6

7

8

9

10

Time

min

---

(*1)  = FAME method.

ANNEX VI

(Article 5)

DETERMINING THE VANILLIN CONTENT IN CONCENTRATED BUTTER, BUTTER OR CREAM BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1.   SCOPE AND FIELD OF APPLICATION

The method describes a procedure for the quantitative determination of vanillin in concentrated butter, butter or cream.

2.   PRINCIPLE

Extraction of a known quantity of sample with a mixture of isopropanol/ethanol/acetonitrile (1:1:2). Precipitation of the majority of fat by cooling between –15 °C and –20 °C, followed by centrifugation.

After dilution with water, determination of the vanillin content by high-performance liquid chromatography (HPLC).

3.   APPARATUS

Usual laboratory apparatus and, in particular, the following:

3.1.   Freezer, operating in the temperature range –15 °C to –20 °C

3.2.   Syringes, disposable of 2 ml capacity

3.3.   Membrane microfilters of 0,45 μm pore size, resistant to a solution containing 5 % extraction solution (4.4)

3.4.   Liquid chromatography system consisting of a pump (flow of 1,0 ml/min), an injector (20 μl injection, automatic or manual), an UV detector (operated at 306 nm, 0,01 Å full scale), a recorder or integrator and a column thermostat operating at 25 °C

3.5.   Analytical column (250 mm × 4,6 mm ID) packed with LiChrospher RP 18 (Merck, 5 μm) or equivalent

3.6.   Guard column (ca. 20 mm × 3 mm ID) dry-packed with LiChrospher RP 18 (5 to 10 μm) or equivalent

3.7.   Centrifuge operating at 2 000 rpm.

4.   REAGENTS

All reagents used must be of recognised analytical quality.

4.1.   Isopropanol

4.2.   Ethanol 96 % (v/v)

4.3.   Acetonitrile

4.4.   Extraction solution

Mix isopropanol (4.1), ethanol (4.2) and acetonitrile (4.3) in the ratio of 1:1:2 (v/v).

Vanillin (4-hydroxy-3-methoxybenzaldehyde) ≥ 98 %

4.5.1.   Vanillin stock solution (= 500 μg/ml)

Weigh accurately to 0,1 mg about 50 mg (CM mg) vanillin (4.5) in a 100 ml volumetric flask, add 25 ml extraction solution (4.4) and make up with water.

4.5.2.   Vanillin standard solution (= 10 μg/ml)

Pipette 5 ml of the vanillin stock solution (4.5.1) into a volumetric flask of 250 ml and make up with water.

4.5.3.    Methanol, HPLC quality

4.5.4.    Acetic acid, glacial

4.5.5.    Water, HPLC quality

4.5.6.   HPLC mobile phase

Mix 300 ml methanol (4.5.3) with about 500 ml water (4.5.5) and 20 ml acetic acid (4.5.4) in a volumetric flask of 1 000 ml and make up with water (4.5.5). Filter through 0,45 μm filter (3.3).

5.   PROCEDURE

5.1.   Preparation of the test sample

5.1.1.   Butter

Heat the sample until melting starts. Avoid local overheating at about 30 °C. The butter may not separate in two phases, in any case. When the sample becomes sufficiently plastic, homogenise it by shaking. Stir the butter for 15 s before taking a sample. Weigh, to the nearest 1 mg, about 5 g (SM g) of butter into a 100 ml volumetric flask.

5.1.2.   Concentrated butter

Immediately before sampling place the container, with concentrated butter, into an oven at 40 to 50 °C until it is melted completely. Mix the sample by swirling or stirring, avoiding formation of air bubbles by too vigorous stirring. Weigh, to the nearest 1 mg, about 4 g (SM g) of concentrated butter into a 100 ml volumetric flask.

5.1.3.   Cream

Heat the sample in a waterbath or incubator at a temperature of 35 to 40 °C. Distribute the fat homogeneously by swirling and, if necessary, by stirring. Cool the sample quickly to 20 ± 2 °C. The sample should look homogenous; otherwise the procedure should be repeated. Weigh, to the nearest 1 mg, about 10 g (SM g) of cream into a 100 ml volumetric flask.

5.2.   Preparation of the test solution

Add about 75 ml extraction solution (4.4) to the test portion (5.1.1, 5.1.2 or 5.1.3), stir, or shake vigorously, for about 15 minutes and make up with extraction solution (4.4). Transfer about 10 ml of this extract to a test tube fitted with stopper. Place the test tube in the freezer (3.1) and allow it to stand for about 30 minutes. Centrifuge the cold extract for 5 minutes at approximately 2 000 rpm and decant immediately. Allow the decanted solution to adjust to room temperature. Pipette 5 ml of the decanted solution into a 100 ml volumetric flask and make up with water. Filter an aliquot through a membrane microfilter (3.3) using a syringue (3.2). The filtrate is ready for determination by HPLC.

5.3.   Calibration

Pipette 5 ml of the vanillin standard solution (4.5.2) into a 100 ml volumetric flask. Add 5 ml extraction solution (4.4) and make up to the mark with water. This solution contains 0,5 μg/ml of vanillin.

5.4.   Determination by HPLC

Allow the chromatographic system to stabilise for about 30 minutes. Inject the standard solution (5.3). Repeat this until the difference in peak area or peak height between two successive injections is less than 2 %. Under the conditions described the retention time of vanillin is about 9 minutes. Analyse the standard solution (5.3) in duplicate by injecting 20 μl. Inject 20 μl of the test solutions (5.2). Determine the area or height of the vanillin peak obtained. Repeat the duplicate injection of the standard solution (5.3) after 10 injections of test samples (5.2).

6.   CALCULATION OF THE RESULTS

Calculate the average peak area (or height) (AC), of the vanillin peaks associated with the bracketing duplicate injections for each batch of test solutions (four areas or heights in total).

Calculate the response factor (R):

where CM is the mass of vanillin in mg (4.5.1).

The content (mg/kg) of vanillin I in the test sample is given by:

where:

AS	=	peak area or height of the vanillin peak of the test sample
SM	=	mass of test sample in g (5.1.1, 5.1.2 or 5.1.3).

Note: Where cream is analysed for vanillin, the tracer concentration has to be expressed as mg tracer/kg milk fat. This is done by multiplying C by 100/f. f is the fat content of the cream in percent (m/m).

20	=	factor which takes into account the dilutions of the standard and the test sample
0,96	=	correction factor for the fat content in first dilution of the test sample

Note: Instead of peak area, peak heights can be used (see 8.3).

7.   ACCURACY OF THE METHOD

7.1.   Repeatability (r)

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 16 mg/kg.

7.2.   Reproducibility (R)

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 27 mg/kg.

8.   TOLERANCE LIMITS

8.1.   Three samples must be taken from the traced product in order to check homogeneity

Tracer obtained either from vanilla or from synthetic vanillin

8.2.1.   The incorporation rate for 4-hydroxy-3-methoxybenzaldehyde is 250 g per tonne of concentrated butter or butter. Where cream is traced, the incorporation rate is 250 g per tonne of milk fat

8.2.2.   The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

—	220,8 mg/kg (95 % of the minimum incorporation rate),

—	158,3 mg/kg (70 % of the minimum incorporation rate).

The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 220,8 mg/kg and 158,3 mg/kg.

Tracer obtained exclusively from vanilla beans or integral extracts thereof:

8.3.1.   The incorporation rate for 4-hydroxy-3-methoxybenzaldehyde is 100 g per tonne of concentrated butter or butter. Where cream is traced, the incorporation rate is 100 g per tonne of milk fat.

8.3.2.   The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

—	78,3 mg/kg (95 % of the minimum incorporation rate),

—	53,3 mg/kg (70 % of the minimum incorporation rate).

The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 78,3 mg/kg and 53,3 mg/kg.

9.   NOTES

9.1.   Recovery of added vanillin at a level of 250 mg/kg butteroil varies from 97,0 to 103,8. The average content found was 99,9 % with a standard deviation of 2,7 %.

9.2.   The standard solution contains 5 % extraction solution to compensate for peak broadening caused by the presence of 5 % of the extraction solution of the test samples. This enables quantification by peak height.

9.3.   The analysis is based on a linear calibration line with a zero intercept.

9.4.   By using appropriate dilutions of the standard solution (4.5.2), the linearity should be checked the first time the analysis is carried out and then at regular intervals and after changes in or repair of the HPLC equipment. Vanillin may be degraded to vanillin acid, divanillin and other compounds by activity of intrinsic enzymes in unpasteurised cream or products thereof.

ANNEX VII

(Article 5)

DETERMINING THE ETHYL ESTER OF BETA-APO-8'-CAROTENIC ACID IN CONCENTRATED BUTTER AND BUTTER BY SPECTROMETRY

1.   SCOPE AND FIELD OF APPLICATION

The method describes a procedure for the quantitative determination of the ethyl ester of beta-apo-8'-carotenic acid (apo-carotenic ester) in concentrated butter and butter. The apo-carotenic ester is the sum of all substances present in an extract of samples obtained under the conditions described in the method which absorb light at 440 nm.

2.   PRINCIPLE

The butterfat is dissolved in light petroleum and the absorbance measured at 440 nm. The apo-carotenic ester content is determined by reference to an external standard.

3.   APPARATUS

3.1.   Pipettes — graduated, of capacity 0,25, 0,50, 0,75 and 1,0 ml

3.2.   Spectrophotometer — suitable for use at 440 nm (and 447-449 nm) and equipped with cells of optical path length 1 cm

3.3.   Volumetric flasks, 20 ml and 100 ml

3.4.   Analytical balance, sensitivity 0,1 mg capable of weighing to the nearest 1 mg, with a readability of 0,1 mg

3.5.   Oven, 45 °C ± 1 °C

3.6.   Fast-filtering ashless filters.

4.   REAGENTS

All reagents must be of recognised analytical grade.

Apo-carotenic ester suspension (approximately 20 %)

4.1.1.   Establish the content of the suspension as follows:

Warm the suspension between 45 °C and 50 °C and homogenize in the unopened original container. Weigh about 400 mg in a volumetric flask (100 ml), dissolve in 20 ml chloroform (4.4) and make up the volume with cyclohexane (4.5). Dilute 5 ml of this solution to 100 ml with cyclohexane (solution A). Dilute 5 ml of solution A to 100 ml with cyclohexane. Measure the absorbance at 447-449 nm (measure the maximum against cyclohexane as a blank using cells with 1 cm optical path length).

Apo-carotenic ester content P (%) = (Absmax × 40 000) / (Msusp × 2 550) or develop: (Absmax / 2 550) × (100 / 5) × (100 / 5) × (100 / Msusp)

Absmax	=	absorbance of the measuring solution at the maximum
Msusp	=	mass of suspension (g)
2 550	=	reference Abs (1 %, 1 cm) value
P	=	Purity (content) of the suspension (%)

Note: Apo-carotenic ester suspension is sensitive to air, heat and light. In the unopened, original container (sealed under nitrogen) and in a cool place it can be stored for about 12 months. After opening the contents should be used within a short period.

4.1.2.   Apo-carotenic ester standard solution, approx. 0,2 mg/ml

Weigh to the nearest 1 mg about 0,100 g of apo-carotenic ester suspension (4.1.1) (W), dissolve in petroleum spirit (4.2), transfer quantitatively into a volumetric flask of capacity 100 ml, and make up to the mark with petroleum spirit.

This solution contains (W × P) / 10 mg/ml of apo-carotenic ester.

Note: The solution must be stored in a cool place in the dark. Discard unused solution after one month.

4.2.   Petroleum spirit (40-60 °C)

4.3.   Sodium sulphate, anhydrous, granular, previously dried at 102 °C for two hours

4.4.   Chloroform

4.5.   Cyclohexane

5.   PROCEDURE

5.1.   Preparation of the test sample

5.1.1.   Concentrated butter

Melt the sample in an oven at approximately 45 °C.

5.1.2.   Butter

Melt the sample in an oven at approximately 45 °C and filter a representative portion through a filter containing about 10 g of anhydrous sodium sulphate (4.3) in an environment shielded from strong natural and artificial light and maintained at 45 °C. Collect a suitable amount of butterfat.

5.2.   Determination

Weigh, to the nearest 1 mg approximately 1 g of concentrated butter (or extracted butterfat (5.1.2)), (M). Transfer quantitatively to a 20 ml (V) volumetric flask using petroleum spirit (4.2), make up to the mark and mix thoroughly.

Transfer an aliquot to a 1 cm cell and measure the absorbance at 440 nm, against a petroleum spirit blank. Obtain the concentration of apo-carotenic ester in the solution by referring to the obtained standard curve (C μ/ml).

5.3.   Calibration

Pipette 0, 0,25, 0,5, 0,75 and 1,0 ml of apo-carotenic ester standard solution (4.1.2) into five 100 ml volumetric flasks. Dilute to volume with petroleum spirit (4.2) and mix.

The approximate concentrations of the solutions range from 0 to 2 μg/ml and are calculated accurately by reference to the concentration of the standard solution (4.1.2) (W × P) / 10 mg/ml. Measure the absorbances at 440 nm against a petroleum spirit (4.2) blank.

Plot the values of absorbance on the y axis against apo-carotenic ester concentration on the x axis. Calculate the equation of the standard curve.

6.   CALCULATION OF THE RESULTS

6.1.   Apo-carotenic ester content, expressed as mg/kg product, is given by:

Concentrated butter: (C × V)/M

Butter: 0,82 (C × V)M

where:

C	=	apo-carotenic ester content, μg/ml, read from the calibration graph (5.3)
V	=	volume (ml) of the test solution (5.2)
M	=	mass (g) of the test portion (5.2)
0,82	=	a correction factor for the butterfat content of butter.

7.   ACCURACY OF THE METHOD

7.1.   Repeatability

7.1.1.   Butter analysis

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 1,4 mg/kg.

7.1.2.   Concentrated butter analysis

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 1,6 mg/kg.

7.2.   Reproducibility

7.2.1.   Butter analysis

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 4,7 mg/kg.

7.3.   Concentrated butter analysis

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 5,3 mg/kg.

7.4.   Source of precision data

The precision data were determined from an experiment conducted in 1995 involving 11 laboratories and 12 traced samples (six blind duplicates) for butter and 12 traced (six blind duplicates) for concentrated butter.

8.   TOLERANCE LIMITS

8.1.   Three samples must be taken from the traced product in order to check on the correct tracing of the product.

8.2.   Butter

8.2.1.   The incorporation rate for butter, taking into account background absorbance, is 22 mg/kg.

8.2.2.   The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

—	17,7 mg/kg (95 % of the minimum incorporation rate),

—	12,2 mg/kg (70 % of the minimum incorporation rate).

The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 17,7 mg/kg and 12,2 mg/kg.

8.3.   Concentrated butter

8.3.1.   The incorporation rate for concentrated butter, taking into account background absorbance, is 24 mg/kg

The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits:

—	19,2 mg/kg (95 % of the minimum incorporation rate),

—	13,2 mg/kg (70 % of the minimum incorporation rate).

The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 19,2 mg/kg and 13,2 mg/kg.