म नक भवन, 9 बह दरश ह दन क तकन क सम त एफ ए ड 03 मह दय/ मह दय, आपक अवल कन ह त न न ल खत भ रत य म नक क मस द स ल न ह : मस द स य

Transcription

1 म नक भवन, 9 बह दरश ह ज़फर म ग, नई द ल Manak Bhavan, 9 Bahadur Shah Zafar Marg, New Delhi दरभ ष Website: त र: म नकस थ Phones fad@bis.org.in Grams: Manaksanstha य पक प रच लन मस द ल ख षण स पन स दभ दन क तकन क सम त एफ ए ड 03 षत एफ ए ड 03/ट ए पअ र उ ग स म त, एफ ए ड 3 क सम त सद य 2 ख एव क ष वभ ग प रषद (एफ ए ड स ) क सद य 3 च रखन व ल सभ नक य मह दय/ मह दय, आपक अवल कन ह त न न ल खत भ रत य म नक क मस द स ल न ह : क.स. मस द स य व षय 1 एफएड 02(2231)स भ रत य म नक मस द - र यल ज ल - व श क पय इस मस द क अवल कन कर अपन स म तय यह बत त हए भ ज क य द अ तत: यह म नक मस द र य म नक क प म क शत ह ज ए त इन पर अमल करन म आपक यवस य अथव क र ब र म य क ठन इय आ सकत ह स म तय क पय स ल न प म अध -ह त र क भ ज स म तय म भ जन क अ तम त थ : य द क ई स म त त नह ह त ह अथव स म त म क वल भ ष स ब ध ट हई त उपर त ल ख क यथ वत अ तम प दय ज य ग य द क ई स म त तकन क क त क हई त वषय स म त क अ क पर मश स अथव उनक इ छ पर आग क क य व ह क लए वषय स म त क भ ज ज न क ब द ल ख क अ तम प द दय ज य ग ध यव द य व द, भवद य त उप र ल खत ( ड. आर क बज ज ) व नक एफ एव म ख (ख एव क ष वभ ग) ई-म ल : fad@bis.org.in

2 म नक भवन, 9 बह दरश ह ज़फर म ग, नई द ल Manak Bhavan, 9 Bahadur Shah Zafar Marg, New Delhi दरभ ष Website: त र: म नकस थ Phones fad@bis.org.in Grams: Manaksanstha DRAFT(S) IN WIDE CIRCULATION TECHNICAL COMMITTEE: FAD 3 DOCUMENT DISPATCH ADVICE Our Ref: Date FAD 3/T ADDRESSED TO: 1. All Members of Apiary Industry Sectional Committee, FAD 3 2. Selected members of FADC 3. All interested Dear Madam/Sir(s), Please find enclosed the following document: Sl Doc No. No. Title 1. FAD (2231) C Draft Indian Standard Royal jelly Specification Kindly examine the draft Indian Standard and forward your views stating any difficulties which you are likely to experience in your business or profession, if this is finally adopted as Indian Standard. Last date for comments: Comments if any may please be made in the format attached and mailed to the undersigned at the above address. P.S - It is requested to kindly give your specific comments/inputs, on Annex J (Page no-12) method for detection of total lipids in royal jelly by soxhlet extraction of lipids (as provided by Dr P.K.Chhuneja, PAU), if any. Thanking you, Encl: As above. Yours faithfully, (Dr.R.K.Bajaj) Scientist F & Head (Food & Agri) fad@bis.org.in

3 Draft Indian Standard ROYAL JELLY SPECIFICATION Not to be reproduced without the permission of BIS or used as a Standard Last date for receipt of comments is Apiary Industry Sectional Committee, FAD 3 FOREWORD (Adoption clause will be added later on) Royal jelly is a thick fluid secreted by worker bee and it contains remarkable amounts of proteins, lipids, vitamins, sugars, and specific vital factors that act as biocatalysts in cell regeneration processes within the human body. Recently, both beekeeping and harvesting of products are carried out under technically advanced conditions and the apiculture has developed continuously. Royal jelly, the food of queen bee and Bee larvae, is a milky white, pale yellow or shallow orange thick fluid synthesized by worker bee s subpharyngeal gland and mandibular gland. The desired characteristics of royal jelly depend upon many factors including the source, collection time, storage condition and the processing techniques. This draft Indian Standard specifies the requirements, method of sampling and test for royal jelly. In the preparation of this standard, due consideration has been given to the Legal Metrology (Packaged Commodities) Rules, 2011 & Food safety and standards act 2006 & rules framed there under. The standard is however subject to restrictions imposed under these rules wherever applicable. For the purpose of deciding whether a particular requirement of this standard is complied with, the final value, observed or calculated, expressing the result of a test or analysis, shall be rounded off in accordance with IS 2 : 1960 Rules for rounding off numerical values (revised). The number of significant places retained in the rounded off value should be the same as that of the specified value in this standard. 1

4 Draft Indian Standard ROYAL JELLY SPECIFICATION 1 SCOPE This standard prescribes the requirements and the method of sampling and test for royal jelly. 2 REFERENCES The standards listed in Annex A contain provisions, which through reference in this text, constitute provisions of this standard. At the time of publication, the editions indicated were valid. All standards are subject to revision and parties to agreements based on this standard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated at Annex A. 3 TERMS AND DEFINITIONS For the purposes of this document, the following term and definition shall apply. 3.1 Royal jelly: Royal jelly is the natural food of larvae destined to be queen bees and adult queen bees. It is milky white, pale yellow or shallow orange, thick fluid produced by nurse honey bees as a mixture of secretions from their hypopharyngeal and mandibular glands mixed in a ratio of 1:1. It contains proteins, lipids, vitamins, sugars and other nutritious substances. 4 REQUIREMENTS 4.1 General characteristics Royal jelly is a natural product which is milky white, pale yellow or shallow orange, with luster. It is pasty at normal temperature with fluidity, and shall be free from the bubbles and any foreign substances. 4.2 Odour and taste It is pungent, unleavened and shall not be rancescent. It is acerb, spicy with sweet taste, and it brings acrid taste to palate and throat. It shall meet requirements given in Table Chemical & microbiological requirements: Royal jelly shall comply with the requirements given in Table 2. 5 SAMPLING 5.1 Sample preparation Sample collector shall use food grade plastic bar, tube or spoon. Put the sample into the sample bottle, stir sufficiently in order to mix it evenly, and put it aside as the sample to be tested. Each sample shall not be less than 20 g. The sample shall be tested immediately and it shall be stored in the fridge below C if it is not tested in time. 6 TEST METHODS 2

5 6.1 Tests shall be carried out as prescribed in the appropriate Annexes specified in col 4 & 5 of table Quality of Reagents Unless specified otherwise, pure chemicals shall be employed in tests and reagent grade water (see IS1070:1999) shall be used, where the use of water as a reagent is intended. NOTE - Pure chemicals shall mean chemicals that don t contain impurities which affect the results of analysis. 7 PACKING 7.1 Royal jelly shall be packed in clean, sound and dry containers made of food grade but non-metallic material which do not affect the product and have to be fit for food contact. 7.2 Storage The temperature for storage shall be below -18 o C. Royal jelly produced in different areas and times should be stored separately. (In bottle or in box). 7.3 Transportation It shall be transported at low temperature, and shall not be stored and transported with toxic; corrosive material or material with peculiar smell or that might produce pollution. 8 MARKING The following information shall be marked on each package or on a label: a) Name of the product, and trade name or brand name, if any; b) Name and address of the producer or packer; c) Batch or code number; d) Net quantity and tare; e) Date of packing; f) Best before date, g) Freezing date and h) Any other details required under the under the Legal Metrology (Packaged commodities) Rules, 2011 and Food Safety & Standards (Packaging and Labelling) regulation, BIS Certification Marking The product may also be marked with the Standard Mark. The use of the Standard Mark is governed by the provisions of the Bureau of Indian Standards Act, 1986 and the Rules and Regulations made there under. The details of conditions under which the license for the use of Standard Mark may be granted to manufacturers or producers may be obtained from the Bureau of Indian Standards. 3

6 Sl No. (1) Table 1 Organoleptic Requirements for royal jelly (Clause 4.2) Characteristic Requirements (2) (3) i) Colour Milky white, light yellow or shallow orange, with gloss ii) Odour Pungent scent, unleavened and must not be rancescent iii) Flavour and taste Acerb, spicy and sweet taste, feeling acrid in palate and throat iv) Appearance/Texture Pasty at normal temperature with fluidity must not have the bubble and the impurity. Table 2 Requirements for Royal jelly (Clause 4.3) Sl No Characteristic Requirements Method of Test, Ref to Annex of this standard Clause of relevant IS (1) (2) (3) (4) (5) i) Moisture percent by B - mass (m/m) ii) 10- Hydroxy Decanoic Acid percent by mass (m/m), Min 1.4 C - iii) Protein, percent by mass (m/m) iv) Total sugar, percent by mass (m/m) v) Ash, percent by mass (m/m), Max vi) Acidity [(1mol NaOH) ml/100g] D E F G - vii) Starch To pass test H - viii) Lipids, percent by mass J - ix) Fructose, percent of total sugars Annex C of 4941:1994 x) Glucose, percent of total sugars Annex C of 4941:1994 xi) Sucrose, percent of total sugars Annex C of 4941:1994 xii) Thiamine, mg/kg :1969 4

7 xiii) Riboflavin, mg/kg :1969 xiv) Niacin, mg/kg :1969 xv) Folic acid, mg/kg :1974 xvi) Total plate count < :2012 (CFU/g) xvii) Coliforms/g Not detected :2012 xvii) Salmonella/g Not detected (Part 3):1999 xix) Fungi, mould, spores/g, Max :1999 ANNEX A (Clause 2) IS No. Title 1070: 1992 Reagent grade water (third revision). 5398: 1969 Methods for estimation of Thiamine (vitamin B1) in foodstuffs 5399:1969 Methods for estimation of Riboflavin (vitamin B2) in foodstuffs 5400:1969 Methods for estimation of Nicotinic acid (Niacin) in foodstuffs 5401(Part 1):2012 Microbiology of food and animal feeding Stuffs horizontal method /ISO 4832: 2006 for the detection and enumeration of coliforms part 1 colony-count technique (Second Revision) 5402:2012 Microbiology of food and animal feeding Stuffs horizontal method /ISO 4833:2003 for the enumeration of micro-organisms colony-count technique at 30 c (Second Revision) 5403: 1999 Method for yeast and mould count of Foodstuffs and animal feeds (first revision) 5887(Part 3):1999 General guidance on methods for the detection of salmonella /IS0 6579: 1993 (Second revision) 7234: 1974 Methods for estimation of Folic acid in foodstuffs ANNEX B [Table 2,Sl.No (i)] DETERMINATION OF MOISTURE CONTENT B.1 Apparatus B-1.1 Vacuum drying oven; B-1.2 Weighing bottle. B-1.3 Analytical balance of least count of g B.2 Procedure Weigh approximately 0.5 g of the royal jelly sample, put it in the weighing bottle which has already been dried to the constant weight, spread evenly, weigh accurately and put it in the vacuum drying oven, dry for 4 h at 70 0 C and under the pressure between MPa and 0.10 M Pa (730 mmhg~760 mmhg), take out the weighing bottle and put it in the drying oven, weigh until it has been cooled for 30 min, dry repeatedly until the weight difference between two consecutive times is not more than 2 mg, namely, until the constant weight is achieved. B.3 Calculation 5

8 The moisture content in royal jelly, X, expressed as a percentage by mass, is given as under m1 m2 X = 100 m1 m3 where X is the moisture content in royal jelly, percent; m 1 is the mass of the weighing bottle and the sample, in grams; m 2 is the mass of weighing bottle and the sample that is dried until the constant weight is achieved, in grams; m 3 is the mass of the weighing bottle, in grams. B.4 Precision Relative deviation of parallel experiments shall not be more than 0.8 percent. ANNEX C [Table 2, Sl.No (ii)] DETERMINATION OF 10 - HYDROXY DECANOIC ACID C.1 Reagents All water used in this experiment is double distilled water. C-1.1 Methanol: UV purity or analytical purity with light transmittance above 30percent at detection wavelength. C-1.2 Anhydrous alcohol C-1.3 Internal standard substance: methyl-p-hydroxybenzoate, of 99.0 percent purity. C HDA standard: of purity above 99.0 percent. Decompress and dry for 24 h in the vacuum drying oven with concentrated sulphuric acid before it is used. C HDA standard solution: Take approximately 25 mg dried 10-HDA standard sample, weigh accurately, dissolve with anhydrous 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 1 mg/ml. C-1.6 Internal standard solution: Take approximately 650 mg of dried methyl-phydroxybenzoate, weigh accurately, dissolve with anhydrous alcohol and transfer it to a 1000 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. C-1.7 Hydrochloric acid(c=0.03 mol/l): take 100 ml of 0.1mol/l hydrochloric acid, add 200 ml double distilled water. C-1.8 Mobile phase (CH 3 OH+0.03mol/L HCL+H 2 O) = C.2 Apparatus C-2.1 HPLC: with ultraviolet detector, recorder or microprocessor; C-2.2 Chromatographic column: 4.6 mm 250 mm stainless steel, fill amorphous silica gel with C 18 bonded stationary phase, of 5 µm or 10 µm particle size; C-2.3 Ultrasonic cleaner; C-2.4 Vortex mixer C-2.5 Analytical balance of least count of g C.3 Procedure C.3.1 Sample treatment Defreeze the sample to the room temperature and stir evenly with glass rod, weigh approximately 0.5 g, and put it in a 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 6

9 sample, add anhydrous alcohol 30 ml, add while shake 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 min, take out, test after centrifugation for 10min at 3000 r/min. Put it in the fridge if it shall not be tested immediately. C.3.2 Chromatography condition Test wavelength: 210 mm; column temperature: 35 0 C; mobile phase velocity of flow: 1 ml/min. C.3.3 Determination of correction factor Weigh 10-HDA standard solution 0.5, 1, 2, 3, 4, 5 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 respectively 2 µl of these solutions, inject it into the chromatograph, and calculate by peak area ratio, the value shall be linear, calculate the correction factor F. C.3.4 Sample determination Weigh 4 µl sample solution, inject it into the chromatograph, fix the quality by internal standard method. C.4 Calculation The 10-HDA content in royal jelly is calculated as give under A X =F i A m s s m 100 i Where X is the 10-HDA content in royal jelly, percent; F is the correction factor; A i is the peak area of tested group in sample; A s is the peak area of internal standard in sample; m s is the mass of the international standard, in grams; m i is the mass of sample, in grams. C.5 Precision Relative deviation of parallel experiments shall not be more than 2.0. ANNEX D [Table 2, Sl.No (iii)] DETERMINATION OF PROTEIN D.1 Reagents D-1.1 Concentrated sulphuric acid (ω=95~98 percent) D-1.2 Mixed reagent of copper sulfate and potassium sulfate: weigh 1 g copper sulfate and 10 g potassium sulfate, put it in the mortar and mix evenly, grind finely to use. D-1.3 Mixed indicator: weigh two samples of methyl red ethanol solution (ρ=1 g/l), three samples of bromocresol green ethanol solution (ρ=2 g/l), mix evenly; D-1.4 Boric acid absorption solution (ρ=20 g/l): weigh 2,0 g boric acid, put it in the 100 ml measuring cylinder, add 20 ml ethanol, dilute to the mark with distilled water, shake until the boric acid is dissolved, put it aside for later use; D-1.5 Sodium hydroxide solution (ρ=400 g/l): weigh 40 g sodium hydroxide, dilute to 100 ml with distilled water; 7

10 D-1.6 Dilute hydrochloric acid: weigh 5, 7 ml concentrated sulphuric acid, dilute to 100 ml with distilled water; D-1.7 Hydrochloric acid standard solution (0, 1 mol/l): dilute to 10 times before using. D.2 Apparatus D-2.1 Kjeldahl nitrogen determination method digestion equipment, 50 ml Kjeldahl flask (If far infrared digesting electric furnace is used, a 50 ml digesting tube and retort funnel shall be collocated); D ml acid burette; D-2.3 Analytical balance of least count of g D-2.4 Semi micro method distillation unit (see Figure 1) A 1000 ml round bottom flask B safety bottle C distiller connected with the ball for nitrogen D funnel E condenser tube F 100ml conical flask G H nip for rubber tube I Safety tube Figure 1-Semimicro Method Distilled Unit for Determining Protein Content D.3 Procedure D.3.1 Cleaning of distillation unit Link distillation unit, add proper amount of distilled water and a few drops of methyl red indicator in bottle A, add dilute sulphuric acid to make it acidic, add a few granules of glass beads and zeolites, add 50 ml distilled water from funnel D, close nip G, open condensate water, boil the distilled water in bottle A. When the vapor comes from the top of the condenser tube, remove the fire, close nip H, make the distilled water in bottle C flow reversely to bottle B. Open nip G, discharge the distilled water in bottle B, close nip B and G. Immerse the top of the condenser tube in approximate 50 ml distilled water, make the distilled water flow reversely to bottle C from the 8

11 top of the condenser tube, and then flow to bottle B, discharge the distilled water with the above method. Clean the apparatus twice or three times like this. D.3.2 Digestion Weigh approximately 1 g of royal jelly sample, put it on a filter paper that is weighed, pack it well after being weighed accurately, put it in a Kjeldahl flask or a digesting tube. Add 2 g of mixed reagent of copper sulfate and potassium sulfate, add 10 ml concentrated sulphuric acid slowly along the bottle wall, mix sufficiently, put a small funnel at the bottle mouth, make the flask lean at 45, heat slowly at comparative low temperature at first, keep the temperature of the solution below the boiling point, increase the electric power gradually until the boiling is stopped. When the digestion solution is boiling, maintain this state and watch out that the solution shall not overflow, heat another 30 min until the solution becomes clear green, transfer to a 100 ml volumetric flask after it is cooled, dilute to the mark with distilled water, and shake evenly for later use. D.3.3 Distillation Weigh 10 ml boric acid of 20 g/l, put it in a 100 ml conical flask, add five drops of mixed indicator, immerge the top of the condenser tube in the solution, take 5 ml of the above digestion solution accurately, move to reaction tube through funnel D, then add 10 ml sodium hydroxide of 400 g/l, clean the funnel D repeated with a little distilled water, close nip G, add a few milliliters of distilled water in funnel D for the purpose of closing tube. Heat bottle A (dilute sulphuric acid shall be added drop by drop into the distilled water in the bottle so as to keep its acidity), distill the vapor, when the boric solution starts to become cyan from wine red, keep distilling for 10 min, lift the top of the condenser tube from the solution, make the vapor continue to wash for 1 min, drip-washing the top with a little distilled water, stop distillation. D.3.4 Titration The absorption solution shall be titrated with 0.01 mol/l hydrochloric acid standard solution. When the color changes from cyan to grey purple, the endpoint has been reached. D.4 Calculation The protein content in royal jelly is given by Equation below X = (V 1 V 0 ) c m 0.05 Where X is the protein content in royal jelly, given by mass fraction, percent; V 1 is the volume of 0.01 mol/l hydrochloric acid standard solution consumed When the sample is titrated, in milliliters; V 0 is the volume of 0.01 mol/l hydrochloric acid standard solution consumed When blank titration is made, in milliliter; c 1 is the concentration of hydrochloric acid standard solution, in mol/l.; is the millimol mass of nitrogen, in grams; m 4 is the mass of sample, in grams; 6.25 is the coefficient of protein conversed from nitrogen. D.5 Precision Relative deviation of parallel experiments shall not be more than 3.0 percent. 9

12 ANNEX E [Table 2, Sl.No (iv)] DETERMINATION OF TOTAL SUGAR E.1 Reagents E-1.1 Glucose standard solution: weigh accurately g pure glucose (specific rotation is +52,5 Z~+53 Z) with constant weight after it is dried at the temperature from 98 0 C to C, dissolve with distilled water and add 5ml hydrochloric acid, dilute to 1000 ml with distilled water, every milliliter of this solution equals to 1mg glucose. E-1.2 Alkaline Cupric Tartrate test Solution A: dissolve 15 g copper sulfate (CuSO 4 5H 2 O) and 0.05 g methylene blue, in 1000 ml water, store in a tightly stoppered bottle. E-1.3 Alkaline Cupric Tartrate test Solution B: weigh 50 g potassium sodium tartrate and 75 g sodium hydroxide, dissolve with distilled water, add 4 g potassium ferrocyanide, dilute to 1000 ml with distilled water when it is dissolved completely, store in a tightly stoppered polyethylene plastic bottle. E-1.4 Calibration of alkaline cupric tartrate Test solution: weigh accurately 5 ml respectively from alkaline cupric tartrate Test solution A and B, put them in 150 ml conical bottles, add 10 ml distilled water, add approximately 9 ml glucose standard solution from burette, heat to the boiling point within 2 min, keep adding glucose standard solution at the speed of one drop per 2 s when it is boiling, the endpoint is reached until the blue color of the solution has just faded, record the total volume of the glucose standard solution consumed, operate three times parallely at the same time, take the mean value, calculate per 10 ml (5 ml respectively from solution A and B) of alkaline cupric tartrate Test solution equals to the mass (mg) of the glucose. E-1.5 Zinc acetate solution (ρ=219 g/l): weigh 21,9 g zinc acetate, add 3 ml acetic acid, dissolve with distilled water and dilute to 100 ml. E-1.6 Potassium sodium tartrate solution (ρ=106 g/l). E-1.7 Concentrated hydrochloric acid (ω=36%~38%). E-1.8 Hydrochloric acid (c = 6 mol/l): weigh 50 ml hydrochloric acid, add distilled water and dilute to 100 ml. E-1.9 Sodium hydroxide solution (ρ=200 g/l). E-1.10 Methyl red indicator (ρ=1 g/l,ethanol solution). E.2 Apparatus E-2.1 Electric-heated thermostatic water bath: temperature fluctuation ±1 0 C; E-2.2 Analytical balance, with least count of g or electronic balance. E.3 Procedure E.3.1 Sample treatment Weight approximately 4 g of royal jelly sample, put it in a 100 ml volumetric flask, add 50 ml distilled water, shake and dissolve the sample, then add 5 ml zinc acetate solution and potassium sodium tartrate respectively and slowly, dilute to the mark with distilled water and mix evenly. Allow it to stand for 30 min and filtrate with dried filter paper, discard a few milliliters of initial filtrate, the filtrate is for later use. Take accurately 50 ml of the above filtrate, put it in a 100ml volumetric flask, add 10ml hydrochloric acid (c=6 mol/l), mix evenly, put it in a electric-heated thermostatic water bath, hydrolyze for 10min at the temperature from 68 0 C to 70 0 C, leave it to room temperature by cooling with flowing water, add two drops of methyl red indicator and mix evenly, neutralize with sodium hydroxide (ρ=200 g/l) until the solution becomes yellow, dilute to the mark with distilled water and mix evenly, which serves as sample solution and is prepared for later use. 10

13 E.3.2 Sample solution titration Weigh accurately 5 ml of Alkaline Cupric Tartrate test Solution A and B respectively, put them in 150 ml conical bottles, heat to the boiling point within 2 min, at a speed that is fast at first and slow later, add sample solution drop by drop from the burette, and keep the solution in boiling state, when the solution color starts to lose, titrate at the speed of one drop per 2 s, the endpoint is reached until the color blue has just faded, record the volume of the sample solution consumed. E.4 Calculation The total sugar content in royal jelly is calculated as below X = T 100 m V 2 / Where X is the total sugar content (counted by glucose), given by mass fraction, percent; T is the titer value of Alkaline Cupric Tartrate TS, the mass of which 10 ml Alkaline Cupric Tartrate TS (5 ml respectively from solution A and B) equals to glucose, in milligrams; M is the mass of the sample, in grams; V is the volume of sample solution consumed in titration, in milliliters. E.5 Precision Relative deviation of parallel experiments shall not be more than 3.0 percent. ANNEX F [Table 2, Sl.No (v)] DETERMINATION OF ASH F.1 Reagents F-1.1 Concentrated sulphuric acid (ω=95 percent~98 percent) F.2 Apparatus F-2.1 Analytical balance of least count of g F-2.2 Quartz or porcelain crucible: 30 ml; F-2.3 Dessicator: with silica gel desiccant inside; F-2.4 High temperature furnace. F.3 Procedure Weigh approximately 1.5 g royal jelly sample, put it in a crucible that it dried to the constant weight, heat with small fire until the sample carbonize completely and become smokeless. Leave it to room temperature, add 0.5 ml ~ 1 ml concentrated sulphuric acid, and moisten the sample. Heat at low temperature and evacuate the sulphuric acid completely, put it in a high temperature furnace, incandesce at C to C until it has no carbon granule, namely, it achieves complete ashing. Take it out until the temperature drops below C, put it in a dessicator, leave it to room temperature and weigh. Incandesce repeatedly until the weight difference between two consecutive times is no more 0.3 g, namely, until the constant weight is achieved F.4 Calculation The ash content in royal jelly is given by Equation X = m 1 - m m 3 - m 2 where X is the ash content in royal jelly, given by mass fraction, percent; 11

14 M 1 is the mass of crucible and ash, in grams; M2 is the mass of crucible, in grams; M 3 is the mass of crucible and sample, in grams. F.5 Precision Relative deviation of parallel experiments shall not be more than 2.0%. ANNEX G [Table 2, Sl.No (vi)] DETERMINATION OF ACIDITY G.1 Reagents G-1.1 Sodium hydroxide (0.1 N) G.2 Appartus G-2.1 Acidimeter: ph value, to the nearest 0.1; G-2.2 Burette: 10 ml G-2.3 Analytical balance of least count of g G.3 Procedure Weigh 1.00 g royal jelly sample, put it in a 100 ml beaker, and 75 ml boiled and cooled distilled water, titrate with sodium hydroxide standard solution (c=0,1 mol/l), the end point is achieved until the acidimeter indicates at ph 8.3. G.4 Calculation The milliliter quantity of sodium hydroxide standard solution consumed in titration multiplies concentration value (mol/l), and then multiplies 100, namely, the acidity of sample is achieved. G.5 Precision Relative deviation of parallel experiments shall not be more than 5.0 percent. ANNEX H [Table 2, Sl.No (vii)] DETERMINATION OF STARCH H.1 Reagents H-1.1 Iodine solution (ρ=13 g/l): weigh 1.3 g iodine, 3.6 g potassium iodide, put them in a 200 ml beaker, add 30 ml distilled water, then add 1 drop of concentrated hydrochloric acid, dissolve and dilute to 100 ml with distilled water, mix evenly, put it in a brown volumetric flask, stopper tightly for later use. H.2 Procedure Weigh approximately 0.2 g of royal jelly sample, put it in a 50 ml beaker, add 10 ml distilled water, mix evenly, heat to the boiling point, add a few drops of iodine solution (ρ=13 g/l) until it is cooled to room temperature, it must not show blue. ANNEX J [Table 2, Sl.No (viii)] TOTAL LIPIDS IN ROYAL JELLY BY SOXHLET EXTRACTION OF LIPIDS. J -1. Apparatus and Reagents J-1.1 Soxhlet apparatus, NaOH (1N), NaHCO 3, H 2 SO 4 and separation funnels. J-2. Procedure 12

15 J-2.1 Extract the lipid and defatted fractions from Royal jelly samples with diethyl ether for 24 h using Soxhlet apparatus. Extract the lipid fraction to separate acidic fraction, phenolic fraction and neutral fraction. J Extraction of neutral fraction -Treat the lipid extracts in diethyl ether with 1N NaOH, decant the ether phase separately containing neutral compounds. J Extraction of Phenolic fraction - Acidify the aqueous phase containing alkali soluble compounds with dil. H 2 SO 4 and then alkalize with NaHCO 3, extract the phase with diethyl ether. The ether phase is separated out containing phenolic compounds. J Extraction of acidic fraction - Acidify the aqueous phase containing organic acids again with dil. H 2 SO 4 & extract the acidified phase with diethyl ether. The ether phase is separated containing acidic compounds where as water soluble compound are discarded. Dissolve the total lipid content obtained as per J-2.1.1, J & J-2.1.3, in diethyl ether and after evaporation, express lipid content as per cent lipid constituent. 13

16 FORMAT FOR SENDING COMMENTS ON BIS DOCUMENTS (Please use A4 size sheet of paper only and type within fields indicated. Comments on each clauses/sub-clauses/table/fig. etc be started on a fresh box. Information in Column 4 should include reasons for the comments and suggestions for modified wording of the clauses when the existing text is found not acceptable. Adherence to this format facilitates Secretariat s work) Doc. No.: TITLE: LAST DATE OF COMMENTS: NAME OF THE COMMENTATOR/ORGANIZATION: Sl. No. Clause/Subclause/ para/table/fig. No. commented Commentator/ Organization/ Abbreviation Type of Comments (General/Editorial/ Technical) Justification Proposed change