1、BS ISO 12824:2016Royal jelly SpecificationsBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS ISO 12824:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 12824:2016.The UK participation in its preparation was entrusted to Tec
2、hnical Committee AW/-/2, Food Technical Committee Chairmen.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The
3、 British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 88352 1 ICS 67.180.10 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 3
4、0 September 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 12824:2016 ISO 2016Royal jelly SpecificationsGele royale SpcificationsINTERNATIONAL STANDARDISO12824First edition2016-09-15Reference numberISO 12824:2016(E)BS ISO 12824:2016ISO 12824:2016(E)ii ISO 2016
5、All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the inte
6、rnet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyr
7、ightiso.orgwww.iso.orgBS ISO 12824:2016ISO 12824:2016(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Requirements 24.1 Description . 24.2 Odour and taste 24.3 Chemical requirements 24.4 Hygienic requirements . 25 Test methods . 35.1 General . 35.2 Sample collection 35.3
8、 Test methods of chemical requirements . 36 Packaging, marking, storage and transportation . 36.1 Packaging . 36.2 Marking . 36.3 Storage and transportation 4Annex A (normative) Determination of moisture content 5Annex B (normative) Determination of 10-HDA 8Annex C (normative) Determination of prote
9、in.13Annex D (normative) Determination of sugar .20Annex E (normative) Determination of total acidity 26Annex F (normative) Determination of total lipid 27Annex G (normative) Determination of C13 isotopic ratio 29Annex H (informative) Determination of furosine 30Annex I (informative) Pollen screenin
10、g 32Bibliography .35 ISO 2016 All rights reserved iiiContents PageBS ISO 12824:2016ISO 12824:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally c
11、arried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the wor
12、k. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the diffe
13、rent approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of this document may
14、 be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patent
15、s).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles
16、 in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information.The committee responsible for this document is ISO/TC 34, Food products.iv ISO 2016 All rights reservedBS ISO 12824:2016INTERNATIONAL STANDARD ISO 12824:2016(E)Royal jelly Specifications1 ScopeThis
17、International Standard specifies the production and sanitary requirements for royal jelly and establishes a series of organoleptic and chemical test methods to control royal jelly quality. It also specifies the requirements of transport, storage, packaging and marking for royal jelly. This Internati
18、onal Standard applies to the royal jelly production (collecting, preliminary processing and packaging) and trade links. This International Standard is not applicable to royal jelly products in which other foods are mixed.2 Normative referencesThe following documents, in whole or in part, are normati
19、vely referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 4833-1, Microbiology of the food chain Horizontal method for
20、the enumeration of microorganisms Part 1: Colony count at 30 C by the pour plate techniqueISO 21528-2, Microbiology of food and animal feeding stuffs Horizontal methods for the detection and enumeration of Enterobacteriaceae Part 2: Colony-count methodISO 6579, Microbiology of food and animal feedin
21、g stuffs Horizontal method for the detection of Salmonella spp.3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1royal jellymixture of secretions from hypopharyngeal and mandibular glands of worker bees, free from any additiveNote 1 to entry: It i
22、s the food of larval and adult queens. It is a raw and natural food, unprocessed except for filtration which does not undergo addition of substances. The colour, the taste and the chemical composition of royal jelly are determined by absorption and transformation by the bees fed with the following t
23、wo types of foods during the royal jelly production time: type 1: only bees natural foods (pollen, nectar and honey); type 2: bees natural food and other nutrients (proteins, carbohydrates, etc.).3.210-HDA10-hydroxy-2-decenoic acidcharacteristic material of royal jelly ISO 2016 All rights reserved 1
24、BS ISO 12824:2016ISO 12824:2016(E)4 Requirements4.1 DescriptionRoyal jelly is milky white, pale yellow, with luster. It is pasty or jelly-like at room temperature with fluidity and shall be free from bubbles and foreign substances. Minor crystallization phenomena can occur naturally in royal jelly d
25、uring storage.4.2 Odour and tasteIt is pungent, unfermented and shall not be rancescent. It is acerb, spicy and it brings acrid taste to palate and throat.4.3 Chemical requirementsRoyal jelly shall comply with the requirements given in Table 1.Table 1 Chemical requirements of royal jellyCharacterist
26、ic Requirement Analysis methodType 1 Type 2Moisture content (%) min.max.62,068,5Annex A10-HDA(%) min. 1,4 Annex BProtein % min.max.1118Annex CTotal sugar % min.max.718Annex DFructose % 29 Annex DGlucose % 29 Annex DSucrose % 0,99.Standard dilutions for calibration curve:Standard 10-HDA 1,0 g/100 ml:
27、c = 5 g/ml(corresponds to 1,0 g/100 g in sample)Pipette (x * 385) l 10-HDA-S0standard stock solution into a 10 ml measuring flask and dilute to volume with sample solventStandard 10-HDA 1,5 g/100 ml:c = 7,5 g/ml(corresponds to 1,5 g/100 g in sample)Pipette (x * 578) l 10-HDA-S0standard stock solutio
28、n into a 10 ml measuring flask and dilute to volume with sample solventStandard 10-HDA 2,0 g/100 ml:c = 10 g/ml(corresponds to 2,0 g/100 g in sample)Pipette (x * 770) l 10-HDA-S0standard stock solution into a 10 ml measuring flask and dilute to volume with sample solventStandard 10-HDA 2,5 g/100 ml:
29、c = 12,5 g/ml(corresponds to 2,5 g/100 g in sample)Pipette (x * 963) l 10-HDA-S0standard stock solution into a 10 ml measuring flask and dilute to volume with sample solventx = factor of 10-HDA-S0stock solution (see B.1.1.3) ISO 2016 All rights reserved 9BS ISO 12824:2016ISO 12824:2016(E)B.1.3.4 Sam
30、ple determinationInject 20 l into the chromatograph.Measure filtered (diluted) extract by HPLC at 216 nm against an external standard calibration curve.B.1.4 Calculation1) Standard calibration curveDetermine the equation of the straight line for a plot of peak area versus purity corrected concentrat
31、ion g/ml of the 10-HDA standard solutions of the form:y = ax + b (B.1)wherey is the area of the 10-HDA peak;a is the slope of the standard curve;x is the purity corrected concentration of the standard;b is the y-intercept of the standard calibration curve.2) Using the 10-HDA peak area from the sampl
32、e, calculate the amount of 10-HDA in the measuring solution from the calibration curve as follows:x = (y b) / a (B.2)wherex is the concentration g/ml of 10-HDA in the measuring solution of the sample;y is the area of the 10-HDA peak in the sample.The 10-HDA content (C10-HDA) in royal jelly (sample i
33、n g/100 g) is given by Formula (B.3):C10-HDA= x 40/m (B.3)wherex is the calculated concentration g/ml of 10-HDA in the measuring solution of the sample;40 is the dilution factor considering the extraction volume of 40 ml, the pipette volume used for dilution (1ml) and the volume of the measuring fla
34、sk used for dilution (10 ml);m is the actual mass of the royal jelly sample, in mg.B.1.5 PrecisionRelative deviation of parallel experiments shall not be more than 2,0 % (lyophylisates) and 5,0 % (fresh royal jelly).10 ISO 2016 All rights reservedBS ISO 12824:2016ISO 12824:2016(E)B.2 HPLC-UV interna
35、l standard (Alternative method)B.2.1 ReagentsUse only double distilled water.B.2.1.1 Methanol, UV purity or analytical purity with light transmittance above 30 % at detection wavelength (210 nm).B.2.1.2 Anhydrous alcohol, GR.B.2.1.3 Internal standard substance, trans-2-hexenoic acid, of 99,0 % purit
36、y.B.2.1.4 10-HDA standard, of purity above 99,0 %. Decompress and dry for 24 h in the vacuum drying oven or desiccator with concentrated sulfuric acid before it is used.B.2.1.5 10-HDA standard solution. Weigh approximately 12,5 mg dried 10-HDA standard sample, weigh accurately, dissolve with anhydro
37、us alcohol and transfer it to a 25 ml volumetric flask, dilute to the mark with anhydrous alcohol and mix evenly.The concentration of 10-HDA obtained in solution is 0,5 mg/ml.B.2.1.6 Internal standard solution. Weigh approximately 650 mg of trans-2-hexenoic acid, weigh accurately, dissolve with anhy
38、drous alcohol and transfer it to a 1 000 ml volumetric flask, dilute to the mark with anhydrous alcohol and mix evenly.The concentration of internal standard solution obtained in solution is 0,65 mg/ml.B.2.1.7 Hydrochloric acid (c = 0,03 mol/l). Take 100 ml of 0,1 mol/l hydrochloric acid, add 200 ml
39、 double distilled water.B.2.1.8 Mobile phase, (CH3OH + 0,03 mol/l HCl+H2O) = 55 + 10 + 35 or (CH3OH + 25 mmol/l phosphate buffer pH 2,5) = 55 + 45.B.2.2 ApparatusB.2.2.1 HPLC, with ultraviolet detector, recorder or microprocessor.B.2.2.2 Chromatographic column, 4,6 mm 250 mm stainless steel, fill am
40、orphous silica gel with C18 bonded stationary phase, of 5 m or 10 m particle size.B.2.2.3 Ultrasonic cleaner.B.2.2.4 Mixer, Vortex mixer or equivalent.B.2.2.5 Analytical balance, capable of weighing to the nearest 0,000 01 g.B.2.3 ProcedureB.2.3.1 Sample treatmentDefreeze the sample to the room temp
41、erature and stir evenly with glass rod, weigh approximately 0,5 g, and put it in a 50 ml volumetric flask that has been weighed already, weigh accurately, add 1 ml of 0,03 mol/l hydrochloric acid and 2 ml water, put it on the vortex mixer and mix to dissolve the sample, ISO 2016 All rights reserved
42、11BS ISO 12824:2016ISO 12824:2016(E)add anhydrous alcohol 30 ml add while shaking lightly, add 10 ml internal standard solution accurately, dilute to the mark with anhydrous alcohol and mix evenly, put it in the ultrasonic bath for 15 min immediately, or put it on the vortex mixer and shake for 15 m
43、in, take out, test after centrifugation for 10 min at 3 000 r/min and filtration with 0,45 m membrane filter, if necessary. Put it in a refrigerator if it shall not be tested immediately.B.2.3.2 Chromatography conditionTest wavelength: 210 nm; column temperature: 35 C; mobile phase velocity of flow:
44、 1 ml/min.B.2.3.3 Determination of correction factorWeigh 10-HDA standard solution 0,5, 1,0, 2,0, 3,0, 4,0, 5,0 mL separately and transfer them to respective 10 ml volumetric flasks. Add accurately 2 ml internal standard solution, dilute to the mark with anhydrous alcohol, and mix evenly. Weigh resp
45、ectively 10 l of these solutions, inject it into the chromatograph, plot the mass ratio of 10-HDA per internal standard against the peak area ratio of that, and draw a linear calibration curve. Calculate the correction factor, F, which is the slope of the calibration curve.B.2.3.4 Sample determinati
46、onWeigh 10 l sample solution, inject it into the chromatograph, and correct the measurement by “internal standard method”.B.2.4 CalculationThe 10-HDA content in royal jelly, is given by Formula (B.4):XFAAmm2100= issi(B.4)whereX2is the 10-HDA content in royal jelly, %;F is the correction factor;Aiis
47、the peak area of tested group in sample;Asis the peak area of internal standard in sample;msis the mass of the internal standard, in grams;miis the mass of sample, in grams.B.2.5 PrecisionRelative deviation of parallel experiments shall not be more than 2,0 %.12 ISO 2016 All rights reservedBS ISO 12
48、824:2016ISO 12824:2016(E)Annex C (normative) Determination of proteinC.1 Kjeldahl method (automatic) (Reference method)C.1.1 ReagentsC.1.1.1 Concentrated sulfuric acid, 95 % to 98 %, reagent grade.C.1.1.2 Catalyst. Weigh 7,0 g potassium sulfate and 0,4 g copper sulfate.C.1.1.3 Mixed indicator. Disso
49、lve 100 mg methyl red in 100 ml methanol and 100 mg bromocresol green in 100 ml methanol.When potentiometric titration is used, no indicator is required.C.1.1.4 Boric acid solution, 4 % (w/v). Dissolve 400 g boric acid in 5 to 6 l hot deionized water. Mix and add more hot deionized water to a volume of about 9 l.Cool to room temperature, add 100 ml bromocresol green solution and 70 ml methyl red solution, and dilute to a final volume of 10 l. Adjust the pH of the boric acid solution to
