1/28

INTERNATIONAL HONEY COMMISSION MELISSOPALYNOLOGY WORKING GROUP

ALTIPARMAK GIDA San. ve Tic. A.Ş. APILAB

REPORT

Proficiency Testing Scheme for Royal Jelly RJ-2014/01

2/28

Organized by Tuğçe Daştan İlknur Coşkun Altıparmak Gıda San. ve Tic. A.Ş. Apilab Çavusbaşı Cad. Çekmeköy No:70 34782 İstanbul/Türkiye Tel.:+90 216 643 02 02 Fax:+90 216 641 30 30 E-mail: tugce.dastan@altiparmak.com.tr ilknur.coskun@altiparmak.com.tr info@apilab.com.tr

ACKNOWLEDGEMENTS We would like to thank participants for their cooperation, Günay Başdoğan and Gizem Mergen who kindly provided and dispatched the royal jelly samples.

mailto:tugce.dastan@altiparmak.com.trmailto:ilknur.coskun@altiparmak.com.trmailto:info@apilab.com.tr

3/28

SUMMARY

1. The test material for royal jelly proficiency test, RJ-2014/01 was dispatched in

February 2014. Royal jelly test material was analyzed for qualitative pollen, moisture,

pH, free acidity, sugar profile (Fructose, Glucose, Saccharose, Maltose, Erlose,

Melezitose) and 10-HDA content. For this proficiency test, 15 laboratories were

participated from 8 countries.

2. The assigned value (X) for each parameter was calculated from the median value of

results which were submitted by the participants (1, 2, and 3).

3. For pollen analysis, the target standard deviation was calculated from robust

statistics Algorithm A (1,2). For other physicochemical analyses, the target standard

deviation was calculated using relative standard deviation obtained from validation

trials. This value was used for the calculation of z-scores. Z-Scores were considered

satisfactory if |z|≤2.

4. Following results were given for this proficiency test.

Analyte Assigned value X

Number of satisfactory scores |z|≤ 2

Total number of scores

Satisfactory %

Po

llen

an

alys

is

Brassicaceae 65% 12 14 86

Vicia spp.

19%

13

14

93

Morus spp. 3% 13 14 93

Ph

ysic

och

emic

al a

nal

ysis

Moisture

67,1 g/100g

6

6

100

Acidity

367 meq/kg

4

5

80

pH

3,99

7

7

100

Fructose

6,2 g/100g

6

6

100

Glucose

7,1 g/100g

5

6

83

10-HDA

1,98 g/100g

5

5

100

5. Data of this report shall not be used without the permission of Altıparmak Gıda San. ve Tic. A. Ş.

4/28

CONTENTS Acknowledgements ...........................................................................................................2

Summary ...........................................................................................................................3

Contents ............................................................................................................................4

1. INTRODUCTION..............................................................................................................5

1.1. Proficiency Testing………………………………………………………………………………………………..5

2. TEST MATERIAL…………………………………………………………………………………………………………….5

2.1. Preparation of Samples………………………………………………………………………………………...5

2.2. Homogeneity of Samples………………………………………………………………………………………5

2.3. Distribution of Samples…………………………………………………………………………………………5

Table1. Homogeneity Data for Royal jelly Test Material......................................................6

3. MATERIALS AND METHODS………………………………………………………………………………………….7

4. STATISTICAL EVALUATION OF RESULTS…………………………………………………………………………7

5. RESULTS AND DISCUSSION……………………………………………………………………………………………8

Table2. Participated analyses and the analysis methods ………………...……………………………….9

Table3. Geographical origin results of the royal jelly and declared information……………..10

Table4. The statistical data of laboratory results……………………………………………………………..10

5.1. Qualitative Pollen Analysis…………………………………………………………………………………11

Table5. Results and z-scores of the pollen analysis in Royal jelly sample…………………….…..11

Table6. Number and Percentage of Satisfactory z- Scores……………………………………………….12

5.2. Geographical origin……………………………………………………………………………………………..12

Figure2. Z-score histogram for Brassicaceae pollen in Royal jelly Test Material……………….13

Figure3. Z-score histogram for Vicia spp. pollen in Royal jelly Test Material………..…………..14

Figure4. Z-score histogram for Morus spp. pollen in Royal jelly Test Material………..………..15

5.3.Physicochemical Analyses…………………………………………………………………………………...16

Table7. Results and z-scores of the physicochemical analysis in Royal jelly sample………...16

Table 8. Results of Saccharose, Maltose, Erlose and Melezitose analysis in Royal jelly sample………………………………………………………………………………………………………..16

Figure5. Z-score histogram for Moisture in Royal jelly Test Material..……………………………..17

Figure6. Z-score histogram for Acidity in Royal jelly Test Material……..……………………………18

Figure7. Z-score histogram for pH in Royal jelly Test Material………………………………………….19

Figure8. Z-score histogram for Fructose in Royal jelly Test Material…………………………………20

Figure9. Z-score histogram for Glucose in Royal jelly Test Material………………………………….21

Figure10. Z-score histogram for 10-HDA in Royal jelly Test Material………………………………..22

1. REFERANCES……………………………………………………………………………………………………………….23

2. APPENDIX 1 Participant Laboratories …………………………………………………………………………24

3. APPENDIX 2 Result Form …………………………………………………………………………………………….25

4. APPENDIX 3 Methods and References…………………………………………………………………………27

5. APPENDIX 4 Comments…………………….……………………………………………………………….……..28

5/28

1. INTRODUCTION

1.1. Proficiency Testing

Laboratory proficiency/comparison tests are an important tool for determining the

laboratory performance. And also provide comparison of the laboratory's results with other

laboratories. Hence, it is a requirement for ISO 17025 accreditation (4).

This proficiency test was organized with the aim of determine the performance of analytical

measurements in laboratories. This report includes the results of inter-laboratory tests for

the qualitative pollen and some other physicochemical analyses in royal jelly.

2. TEST MATERIAL

2.1. Preparation of Samples Royal jelly sample was homogenized by stirring with a mechanical mixer for 20 minutes.

After stirring, royal jelly samples were dispensed about 30 grams into suitable containers

(falcon tube) without waiting and then were labeled. Packaged and labeled samples were

stored at -18 ± 3 °C until the date of delivery.

2.2. Homogeneity of Samples

12 randomly selected test samples were analyzed in duplicate within the same day for the

detection of homogeneity. Results were evaluated according to ISO-13528 homogeneity test

(1, 5). Firstly, data were checked by Cochran's test, and no outlier was detected. And then its

homogeneity was determined according to the statistical analysis of samples. Samples were

sent to laboratories.

Statistical evaluation results were given in Table 1. The results showed that the samples were homogeneous.

2.3. Distribution of Samples

The dispatch date was 04 February 2014. Each participant received an individually labeled

royal jelly sample packed with cooling blocks in a nylon bag.

6/28

Table1. Homogeneity Data for Royal jelly Test Material

Sample ID Analyte

Moisture %

Acidity meq/kg

pH Fructose %

Glucose %

10-HDA %

rep. 1 rep. 2 rep. 1 rep. 2 rep. 1 rep. 2 rep. 1 rep. 2 rep. 1 rep. 2 rep. 1 rep. 2

1

69,8 69,3 375 375 3,82 3,88 5,8 6,0 7,5 7,5 1,94 1,96

2

68,7 68,8 370 370 3,85 3,89 5,8 5,9 7,5 7,6 1,95 1,99

3

69,4 68,3 370 370 3,87 3,91 5,7 5,7 7,5 7,4 2,06 2,11

4

69,8 69,2 370 360 3,87 3,89 5,7 5,7 7,5 7,6 2,00 2,10

5

68,7 69,9 375 375 3,88 3,90 5,7 5,6 7,6 7,6 2,06 2,09

6

68,8 69,4 375 370 3,87 3,89 5,7 5,6 7,6 7,5 2,10 2,17

7

68,2 68,3 375 370 3,93 3,92 5,8 6,0 7,6 7,4 1,96 2,04

8

68,4 68,2 375 375 3,92 3,91 5,8 5,9 7,3 7,1 1,92 1,91

9

69,2 68,4 375 370 3,90 3,89 5,7 5,7 7,5 7,2 1,95 2,04

10

68,6 69,4 375 370 3,92 3,91 5,7 5,7 7,6 7,4 1,98 2,05

11

69,2 69,3 375 370 3,91 3,91 5,7 5,6 7,5 7,4 1,88 1,91

12

69,3 68,3 375 370 3,91 3,90 5,7 5,6 7,5 7,3 1,95 1,99

Mean, n 68,9 24 372 24 3,9 24 5,7 24 7,5 24 2,0 24

*RSDR% 5,0

2,5

4,5

10,0

10,0

12,0 Target sd (σ) 3,45

9,30

0,18

0,57

0,75

0,24

Sx 0,407

2,787

0,022

0,102

0,109

0,071 Sw 0,511

3,227

0,019

0,076

0,112

0,042

Ss 0,187

1,599

0,017

0,086

0,076

0,065 critical (0,3x σ) 1,034

2,791

0,053

0,172

0,224

0,072

Ss ≤ critical? Accept Accept Accept Accept Accept Accept

* Obtained from validation trials

Sx : the standard deviation of sample averages Sw : the within-samples standard deviation Ss : the between-samples standard deviation

7/28

3. MATERIALS AND METHODS

A total of 15 laboratories from Austria, Germany, Greece, Italy, Switzerland, Spain and

Turkey participated in this test (see Appendix 1). Participated analyses and methods were

given in Table 2. All samples were delivered to the laboratories as about 30 g packages with

cooling blocks. Laboratory code of each participant was labeled on the samples. It was

proposed to store at -18 ± 2 °C until analysis date to all participants and analyze using

routine analytical methods. The dispatch date was 04 February 2014. At last, test materials

have reached to all laboratories on 05/07 February 2014. And also the submission forms (see

Appendix 2) for recording the results was sent to all participants by e-mail. We requested to

send results of analysis from all participants until 7 March 2014. Further comments made by

the participants are mentioned in Appendix 3.

4. STATISTICAL EVALUATION OF RESULTS

According to ISO 13528:2013 following parameters were calculated: mean value, standard

deviation, median value, robust standard deviation, assigned value, standard uncertainty of

the assigned value, target standard deviation, relative standard deviation of reproducibility

and z-score(1). The assigned value for each parameter was specified as median value. The

median value was calculated from submitted results.

For pollen analysis, target standart deviation was calculated using the robust statistics (1,2).

With robust statistics, outliers in the data was corrected and all results were evaluated. The

outliers were checked by Grubbs’ test (5,6). And z-scores were calculated from the target

standart deviation.

For physicochemical analyses, target standart deviation was calculated using relative

standard deviation obtained from validation trials. As there is not much collaborative studies

on these tests, validation trials were preferred for calculation (fitness for purpose) (3). The

target standard deviation was calculated from robust statistic for acidity. The outliers were

checked by Grubbs’ test (5,6). And z-scores were calculated from the target standart

deviation.

σ = X * RSDR u(x)=1,25*s/

Where

x: reported results

X: the assigned value

σ: the target standard deviation

RSDR: the relative standard deviation

u(x): standard uncertainty of the assigned value

s: robust standard deviation

8/28

p: number of participants

Z: z-Score, is an indication of the performance of a participants.

Results are considered satisfactory if |z|≤2.

Results are considered to give action signal if 2

9/28

Table2. Participated analyses and the analysis methods

Lab Code

Analyses Methods

Pollen Moisture Acidity pH Sugar 10-HDA Pollen Moisture Acidity pH Sugar profile 10-

HDA

RJ.01 X Microscopic method:

http://www.apimondia.com/apiacta/articles/2006/micro_char_rj.pdf http://www.tandfonline.com/doi/pdf/10.1080/00173130701393874

RJ.02 X Pollen analysis of royal Jelly: M,Lucia Piana, Paola belligoli, Livia Persano Oddo, Sara Piperno

RJ.03 X X X Microscopy

http://www.apimondia.com/apiacta/articles/2006/micro_char_rj.pdf Infrared

dessication pHmeter

RJ.04 X X X X X X Louveaux et al.,1978 / DIN 10760 gravimetric method

titrimetric / potentiometric

method

Potentiometric method

Fructose, Glucose, Saccharose and maltose, HPLC-

RID

HPLC-UV

RJ.05 X X X X X

RJ.06 X X X X X X http://www.apimondia.com/apiacta/articles/2006/micro_char_rj.pdf gravimetric

method

titrimetric / potentiometric

method

Potentiometric method

Fructose, Glucose, Saccharose,

Melezitose and Maltose, HPLC-

RID

HPLC-UV

RJ.07 X

RJ.08 X Microscopic method:

Ricciardelli D'Albore G.L., Battaglini Bernardini M., 1978 - Origine geographique de la gelée royale. Apidologie 9(1): 1-17

Karl Fischer titration method

gc method gc method

RJ.09 X Ricciardelli d'Albore, G. et Battaglini Bernardini M. (1978): Origine géographique de la Gelée Royale; Apidologie 9 (1) 1-17

RJ.10 X X X X X X http://www.apimondia.com/apiacta/articles/2006/micro_char_rj.pdf Gravimetry Potentiometric method

HPLC/RID HPLC/UV

RJ.11 X X X X

RJ.12 X

RJ.13 X

RJ.14 X X

RJ.15 X X X X X X

Microscopic method:

http://www.apimondia.com/apiacta/articles/2006/micro_char_rj.pdf http://www.tandfonline.com/doi/pdf/10.1080/00173130701393874

Moisture and other volatiles:

gravimetric method

titrimetric / potentiometric

method

Potentiometric method

Fructose, Glucose, Saccharose and maltose, HPLC-

RID

HPLC-PDA

10/28

Table 3. Geographical origin results of the royal jelly and declared information.

Lab Code Number of samples

per year Geographical

origin

Accreditation

Pollen Physicochemical

RJ.01 - China no -

RJ.02

11/28

5.1. Qualitative Pollen Analysis

Most of the participants presented results about the qualitative pollen analysis according to

“Pollen analysis of royal jelly: contribution to analytical methods and characterization” (8).

The rest used different methods were mentioned above Table 2. The statistical data showed

that the percentage of pollen and the relative standard deviation (RSD%) was inversely

correlated. These results are similar with pollen analysis in honey (7). The results and z-

scores of the qualitative pollen analysis per participant are presented in Table 5.

Table 5. Results and z-scores of the pollen analysis in Royal jelly sample.

Lab Code

Pollen analysis

Brassicaceae Vicia spp. Morus spp.

Assigned value: 65 Assigned value:18,5 Assigned value: 3

% z-score % z-score % z-score

RJ.01 40 -2,8* 21 0,5 4 0,7

RJ.02 68 0,3 24 1,2 0,012 -2,0

RJ.03 64 -0,1 16 -0,5 n.a -

RJ.04 72 0,8 19 0,1 3 0,0

RJ.05 53 -1,4 14 -1,0 4 0,7

RJ.06 82 1,9 72 -2,5* 5 1,3

RJ.07 64 -0,1 25 1,4 3 0,0

RJ.08 70 0,6 14 -1,0 0.05).

*z-score value 2

12/28

Table 6. Number and Percentage of Satisfactory z- Scores

Analyte Assigned value X

Number of satisfactory scores |z|≤ 2

Total number of scores

Satisfactory %

Po

llen

an

alys

is

Brassicaceae 65% 12 14 86

Vicia spp.

19%

13

14

93

Morus spp. 3% 13 14 93

Ph

ysic

och

emic

al a

nal

ysis

Moisture

67,1 g/100g

6

6

100

Acidity

367 meq/kg

4

5

80

pH

3,99

7

7

100

Fructose

6,2 g/100g

6

6

100

Glucose

7,1 g/100g

5

6

83

10-HDA

1,98 g/100g

5

5

100

5.2. Geographical origin

Microscopic analysis of royal jelly were performed according to the basic principles of

melissopalynology. Identification of pollen grains that contain royal jelly allows to determine

the geographical origin of royal jelly. This royal jelly was produced in China. In total, 11

laboratories reported result of geographical origin. Almost all of participants stated correct

predictions. References and methods used was given in Appendix 3. Geographical origin

results were shown in Table 3.

13/28

Figure 2. z-score histogram for Brassicaceae pollen in Royal jelly Test Material.

-6,1

-2,8

-1,4

-0,5

-0,1 -0,1 -0,1 0,1 0,1 0,3 0,5

0,6 0,8

1,9

-7,0

-6,0

-5,0

-4,0

-3,0

-2,0

-1,0

0,0

1,0

2,0

3,0

RJ.13 RJ.01 RJ.05 RJ.12 RJ.03 RJ.07 RJ.09 RJ.11 RJ.15 RJ.02 RJ.10 RJ.08 RJ.04 RJ.06

Z-sc

ore

Laboratory code

Brassicaceae

14/28

Figure 3. z-score histogram for Vicia spp. pollen in Royal jelly Test Material.

-2,5

-1,0 -1,0

-0,5 -0,5 -0,5

-0,1 0,1 0,1 0,3

0,5

1,2 1,2 1,4

-3,0

-2,0

-1,0

0,0

1,0

2,0

3,0

RJ.06 RJ.08 RJ.05 RJ.03 RJ.12 RJ.13 RJ.09 RJ.04 RJ.10 RJ.15 RJ.01 RJ.02 RJ.11 RJ.07

Z-sc

ore

Laboratory code

Vicia spp.

15/28

Figure 4. z-score histogram for Morus spp. pollen in Royal jelly Test Material.

-2,0 -1,9

-1,3

-0,7

0,0 0,0 0,0 0,0

0,7 0,7 0,7 0,7

1,3

-3,0

-2,0

-1,0

0,0

1,0

2,0

3,0

RJ.02 RJ.08 RJ.11 RJ.10 RJ.04 RJ.07 RJ.13 RJ.15 RJ.01 RJ.09 RJ.05 RJ.12 RJ.06 Z-sc

ore

Laboratory code

Morus spp.

16/28

5.3. Physicochemical Analyses

Methods used in physicochemical analyses were given in Table 2. 6 laboratories were

reported results for moisture and sugar profile analysis, 5 laboratories for acidity and 10-

HDA analysis. 7 laboratories were reported results for pH analysis. Statistical assessment was

performed. No outlier was found in moisture analysis. According to z-score value, 100% of

the results were satisfactory. One outlier was found in acidity analysis. According to z-score

value, 80% of the results were satisfactory. No outlier was found in pH analysis. According to

z-score value, 100% of the results were satisfactory. No outlier was found in Fructose.

According to z-score value, 100% of the results were satisfactory. No outlier was found in

glucose. According to z-score value, 83% of the results were satisfactory (Table 6 and 7). In

Saccharose, Maltose, Erlose and Melezitose results were under limit of quantification (Table

8). Hence statistical assesment was not performed. Z-scores histograms of physcochemical

analysis were shown in Figure 5, 6, 7, 8, 9 and 10.

Table 7. Results and z-scores of the physicochemical analysis in Royal jelly sample.

Lab Code

Moisture Acidity pH Sugar

10-HDA Fructose Glucose

Assigned value: 67,1

Assigned value: 367,0

Assigned value: 3,99

Assigned value: 6,2

Assigned value: 7,1

Assigned value: 1,98

% z-score meq/kg z-score

z-score % z-score % z-score % z-score

RJ.03 66,1 -0,3 43,51 -14,0** 4,05 0,3

RJ.04 67,7 0,2 353 -1,5 4,10 0,6 6,2 -0,1 7,5 0,6 1,90 -0,3

RJ.05 64,92 -0,6

3,802 -1,1 6,3 0,1 7,0 -0,1 2,00 0,1

RJ.06 68,1 0,3 370 0,3 3,99 0,0 5,42 -1,3 5,8 -1,8 2,401 1,8

RJ.10 66,5 -0,2 367 0,0 4,01 0,1 6,7 0,7 5,22 -2,7* 1,97 0,0

RJ.14

3,97 -0,1 6,3 0,1 7,2 0,1

RJ.15 68,6 0,4 371 0,4 3,89 -0,6 5,7 -0,8 7,5 0,6 1,98 0,0 1 Significant outlier. P < 0.05 2 Furthest from the rest, but not a significant outlier (P > 0.05). *z-score value 2

17/28

Figure 5. z-score histogram for Moisture in Royal jelly Test Material.

-0,7

-0,3 -0,2

0,2 0,3

0,5

-3,0

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

3,0

RJ.05 RJ.03 RJ.10 RJ.04 RJ.06 RJ.15 Z-sc

ore

Laboratory code

Moisture

18/28

Figure 6. Z-score histogram for Acidity in Royaljelly Test Material.

-14,0

-1,7 -0,2 0,3 0,3

-14

-13

-12

-11

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

RJ.03 RJ.04 RJ.10 RJ.07 RJ.15

Z-sc

ore

Laboratory code

Acidity

19/28

Figure 7. z-score histogram for pH in Royal jelly Test Material.

-1,1

-0,6 -0,1

0,0 0,1

0,3

0,6

-3,0

-2,0

-1,0

0,0

1,0

2,0

3,0

RJ.05 RJ.15 RJ.14 RJ.06 RJ.10 RJ.03 RJ.04

Z-sc

ore

Laboratory code

pH

20/28

Figure 8. z-score histogram for Fructose in Royal jelly Test Material.

-1,3

-0,8

-0,1

0,1 0,1

0,7

-3,0

-2,0

-1,0

0,0

1,0

2,0

3,0

RJ.06 RJ.15 RJ.04 RJ.05 RJ.14 RJ.10

Z-sc

ore

Laboratory code

Sugar Profile-Fructose

21/28

Figure 9. z-score histogram for Glucose in Royal jelly Test Material.

-2,7

-1,8

-0,1

0,1

0,6 0,6

-3,0

-2,0

-1,0

0,0

1,0

2,0

3,0

RJ.10 RJ.06 RJ.05 RJ.14 RJ.15 RJ.04

Z-sc

ore

Laboratory code

Sugar Profile-Glucose

22/28

Figure 10. z-score histogram for 10-HDA in Royal jelly Test Material.

-0,3

-0,04 0,0 0,1

1,8

-3

-2

-1

0

1

2

3

RJ.04 RJ.10 RJ.15 RJ.05 RJ.06

Z-sc

ore

Laboratory code

10-HDA

23/28

6. REFERANCES

1- ISO 13528:2013, Statistical methods for the use of proficiency testing by inter-

laboratory comparisons.

2- Analytical Methods Committee, No.6, 2001, Robust Statistics: a method of coping

with outliers.

3- Pure Appl. Chem., Vol. 78, No.1, 2006, The International Harmonized Protocol For

The Proficiency Testing of Analytical Chemistry Laboratories.

4- ISO/IEC 17025:2012, General requirements for the competence of testing and

calibration laboratories

5- International Journal of Education and Research, Vol. 1 No. 5, 2013, Statistical

Analysis/Methods Of Detecting Outliers In a Univariate Data In A Regression Analysis

Model

6- Ann. Inst. Statist. Math., Vol. 46, No. 4, 1994, On The Joint Distribution Of Grubbs'

Statistics

7- Von der Ohe W., Persano Oddo L., Piana M.L., Morlot M., Martin P. (2004)

Harmonized methods of melissopalynology, Apidologie (Suppl. 1): 18-25.

8- Apiacta 41 (2006), Pollen analysis of royal jelly: contribution to analytical methods

and characterization.

24/28

APPENDIX 1

Participant Laboratories

Country Laboratories

AUSTRIA Institut für Saat- und Pflanzgut, flanzenschutzdienst und Bienen

FRANCE Naturalim France Miel Laboratoire d'Expertise des Miels

FRANCE Famille Michaud Apiculteurs

GERMANY Quality Services International GmbH

GERMANY Breitsamer + Ulrich GmBH &Co KG

GERMANY Intertek Food Services GmbH

GERMANY Chemisches- und Veterinäruntersuchungsamt

GREECE Apiculture-Sericultue Laboratory School of Agriculture

ITALY Istituto Zooprofilattico delle Regioni Lazio

ITALY Centro Agrochimico Regionale

ITALY CRA-API Honey Bee and Silkworm Unit of the Italian Agricultural research Council

ITALY Piana Ricerca e Consulenza srl a socio unico

SPAIN Casa de la Miel. Cabildo de Tenerife

SWITZERLAND Biologisches Institut für Pollenanalyse

TURKEY Altıparmak Gıda San. ve Tic. A.Ş

25/28

APPENDIX 2

Result Form

1 4

LABORATORY CODE NUMBER ACCREDITATION2

DATE OF RECEIPT NUMBER OF SAMPLES ANALYSED PER YEAR

3 500 5

STATE OF SAMPLE

SAMPLE CODE

METHODOLOGY 6

1 QUALITATIVE POLLEN ANALYSISReference:

Other (please specify)…………………………………………………………………

RCF7

minutes

all pollen

types

Value(%)

1 Brassicaceae Brassicaceae

2 Leguminosae Vicia

3 Moraceae Morus

4

0

6

2 GEOGRAPHICAL ORIGIN Reference: (please specify)…………………………………………………………………

10

Royal jelly from:

3 OTHER POLLEN TYPES AND COMMENTS

1

2

3

4

5

6

7

8

9

10

Pollen analysis of royal jelly: contribution to analytical methods and

characterization (Apiacta 41 (2006) p:28-43)

Sum

Please write the code number of your laboratory

Other

8

Fill in the the presence (%) of the specific pollen forms in the royal jelly sample.To calculate their percentage INCLUDE also all nectarless taxa.Use NUMERICAL data

with no decimals.

Please write the relative centrifugal force (for more information see Pendleton, Grana, 2006)

This field is optional

This field is automatically filled to sum 100%

…………………………………………………………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………….....................................................................

Please write the date when you received the Royal jelly sample

Please describe the state of the delivered sample e.g. good, damaged, liquid, freezed e.t.c. If the sample is inappropriate for analysis please contact us

Please mark if your laboratory is accredited for Royal jelly pollen analysis

Please mark the approximate number of royal jelly samples that you perform pollen analysis per year

Please mark or write the method you use for pollen anaysis / geographical determination

9

INTERNATIONAL HONEY COMMISSION

Pollen FormFamily

RJ-2014/01

MELISSOPALYNOLOGY WORKING GROUP

INTER-LABORATORY RING TEST FOR ROYAL JELLY QUALITATIVE

MELISSOPALYNOLOGICAL ANALYSIS

26/28

1

LABORATORY CODE NUMBER2

DATE OF RECEIPT NUMBER OF SAMPLES ANALYSED PER YEAR

3 500 4

STATE OF SAMPLE

SAMPLE CODE

METHODOLOGY

1 MOISTURE CONTENT Result Accreditation 5

1 Gravimetric %

2 Karl Fischer titration %

3 Other method (please specify)…………………………………………………………………

METHODOLOGY (How do you performed the measurement of acidity ?)

2 ACIDITY Result Accreditation5

1 Titrimetric meq.kg-1

2 Potentiometric

3 Other method (please specify)..................................................................................................................

METHODOLOGY (How do you performed the measurement of pH ?)

3 pH Result Accreditation5

1 Potentiometric

METHODOLOGY

4 SUGARS Result Accreditation5

Fructose % HPLC-RID6

Other method (please specify)………….....

Glucose % HPLC-RID Other method (please specify)………….....

Saccharose % HPLC-RID Other method (please specify)………….....

Maltose % HPLC-RID Other method (please specify)………….....

Erlose % HPLC-RID Other method (please specify)………….....

Melezitose % HPLC-RID Other method (please specify)………….....

Other % (please specify)…………………………………………………………………

METHODOLOGY

5 10-HDA Result Accreditation5

1 HPLC-PDA/UV %

2 Other method (please specify)..................................................................................................................

6 COMMENTS

1

2

3

4

5

6

INTERNATIONAL HONEY COMMISSION

MELISSOPALYNOLOGY WORKING GROUP

INTER-LABORATORY RING TEST FOR ROYAL JELLY

PHYSICOCHEMICAL ANALYSIS

RJ-2014/01

Please mark if your laboratory is applied this method for Royal jelly samples

……………………………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………….....................................................................

Please mark the approximate number of royal jelly samples that you perform per year

Please write the code number of your laboratory

Please write the date when you received the Royal jelly sample

Please describe the state of the delivered sample e.g. good, damaged, liquid, freezed e.t.c. If the sample is inappropriate for analysis please contact us

Please mark if your laboratory is accredited for Royal jelly analysis

27/28

APPENDIX 3

Methods and Referances

Methods used by participants. Qualitative Pollen Analysis

Methods Laboratory number

Piana M. L., Belligoli P., Persano Oddo L., Piperno S., Pollen analysis of royal jelly: contribution to analytical methods and characterization, Apiacta 41 (2006)

01 02 04 05 07 08 09 10 11 12 14 15

Ricciardelli D'Albore G.L., Battaglini Bernardini M., - Origine geographique de la gelée royale. Apidologie 9(1): 1-17, 1978

08 09 11 15

Louveaux et al.,1978 / DIN 10760 04

Dimou, M., Goras, G. and Thrasyvoulou, A. Pollen analysis as a means to determine the geographical origin of royal jelly. Grana 46: 118-122, (2007).

11 15

Barth, O.M., Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil (2005). J.Apicult. Res., 44, 78-81

11

Geographical origin

Methods Laboratory number

Piana M. L., Belligoli P., Persano Oddo L., Piperno S., Pollen analysis of royal jelly: contribution to analytical methods and characterization, Apiacta 41 (2006)

01 02 09 11 15

Ricciardelli D'Albore G.L., Battaglini Bernardini M., - Origine geographique de la gelée royale. Apidologie 9(1): 1-17, 1978

08 11 15

Ricciardelli D´Albore G., Textbook of Melisopalynology. Apimondia, Bucharest, 1997.

11 15

28/28

APPENDIX 4

Comments

Further information about the melissopalynological analysis stated by participants at the 3rd paragraph of the report form.

Laboratory Code Other Pollen types and Comments

RJ.01

Presence of abundant yeast, presence of Quercus robur gr., Lamiaceae forma M tipo Lavandula, Salix, Tilia, Apiaceae, Ligustrum, Pyracantha, Rosaceae, Alnus, Eriogonium, Echium, Acanthaceae, Corylaceae, Asteraceae forma J

RJ.02

Camellia (theacea), poaceae, juglandaceae, trifolium, trema, fagopyrum, caryophyllaceae, sapindacea,sophora astragalus sinicus.This sample of royal jelly has a very low pollen counts (350 pollens for 1g)

RJ.03

Pinus, Rosaceae, Asteraceae T type, Asteraceae S type, Polygonum…, Euphorbiaceae, avorted pollen

RJ.04

Brassicaceae (Kreuzblütler, Crucifers); Brassica napus (Raps, Rape); Salix sp. (Weiden, Willow); Vicia (Wicken, Vetch) -Type; Trifolium/Melilotus (Kleearten, Clover-Types); Trifolium repens (Weißklee, White Clover); Poaceae (Süßgräser, Grasses) (P); Anthriscus (Kerbel, Chervil) -Type; overrepresented small pollen - types; Aster-Type; unidentified Pollen; Restpollen;

RJ.05 -

RJ.06 -

RJ.07 -

RJ.08 Indeterminated pollens 4% - Other pollen types: Astragalus sinicus, Castanea, Pterocarya

RJ.09

Quercus, Rosaceae, Ulmus, Astragalus, Fagopyrum, Asteraceae-HA-Type, Castanea sativa, Artemisia, Pinaceae, Poaceae

RJ.10

Acanthaceae, Compositae, Atremisia, Helianthus-t, Cyperaceae, Zea, Leguminosae, Fagopyrum, Rumex, Rosaceae, Tilia, Pollen unknown

RJ.11

There are two pollen forms included into Brassicaceae family: form 1; reticulate, with a P/E = 1.30; and form 2; fine reticulate with a P/E = 0.95. Many aborted or broken pollen

RJ.12 -

RJ.13

One type of pollen with reticulate exine and one aperture (colpat) was detected very frequently, but not identified as belonging to family of Brassicacae.

RJ.14 -

RJ.15

Other pollen types: Rosaceae, Compositae, Betulaceae, Rhus, Trifolium, Astragalus (chinese type), Poaceae, Salix, Ulmaceae, Quercus, Zea mays, Caryophyllaceae, Polygonaceae