Aspartame-acesulfame salt | 7 |
| |
Beeswax | 11 |
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Calcium L-5-methyltetrahydrofolate | 15 |
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Candelilla wax | 21 |
| |
Ethyl maltol | 23 |
| |
Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae | 25 |
| |
Maltol | 29 |
| |
Phospholipase A1 from Fusarium venenatum expressed in Aspergillus oryzae | 31 |
| |
Pullulan | 35 |
| |
Quillaia extract (Type 1) | 39 |
| |
Quillaia extract (Type 2) | 43 |
| |
Sucrose esters of fatty acids | 47 |
| Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 55th JECFA (2000) and published in FNP 52 Add 8 (2000). The ADI for aspartame (0-40 mg/kg bw) established at the 25th JECFA (1981) and the ADI for acesulfame K (0-15 mg/kg bw) established at the 37th JECFA (1990) cover the aspartame and acesulfame moieties of the salt. | |
| | |
SYNONYMS | Aspartame-acesulfame, INS No. 962 | |
| | |
DEFINITION | The salt is prepared by heating an approximately 2:1 ratio (w:w) of aspartame and acesulfame K in solution at acidic pH and allowing crystallization to occur. The potassium and moisture are eliminated. The product is more stable than aspartame alone. | |
| | |
Chemical names | 6-methyl-1,2,3-oxathiazine-4(3H)-one-2,2-dioxide salt of L-phenylalanyl-2-methyl-L-á-aspartic acid. | |
| | |
C.A.S. number | 106372-55-8 | |
| | |
Chemical formula | C18H23O9N3S | |
| | |
Structural formula | ||
| | |
Formula weight | 457.46 | |
| | |
Assay | 63.0% to 66.0% aspartame (dried basis) and 34.0% to 37.0% acesulfame (acid form on a dried basis). | |
| | |
DESCRIPTION | A white, odourless, crystalline powder | |
| | |
FUNCTIONAL USES | Sweetening agent, flavour enhancer | |
| | |
CHARACTERISTICS | | |
| | |
IDENTIFICATION | | |
| | |
Solubility (FNP 5) | Sparingly soluble in water, and slightly soluble in ethanol. | |
| | |
PURITY | | |
| | |
Loss on drying (FNP 5) | No more than 0.5% (105°, 4h) | |
| | |
Transmittance (FNP 5) | The transmittance of a 1% solution in water determined in a 1 cm cell at 430 nm with a suitable spectrophotometer using water as a reference, is not less than 0.95, equivalent to an absorbance of not more than approximately 0.022. | |
| | |
Specific rotation (FNP 5) | [a] D 20+14.5 to +16.5. | |
| | |
5-Benzyl-3,6-dioxo-2-piperazineacetic acid | Not more than 0.5% | |
| | |
Lead (FNP 5) | Not more than 1 mg/kg. | |
| | |
TESTS | | |
| | |
PURITY TESTS | | |
| | |
5-Benzyl-3,6-dioxo- 2-piperazine acetic acid | Principle Apparatus Mobile phase Standard Sample Procedure % = 1000(AUCS)/(ASWu) Where AU and AS are the peak areas of 5-benzyl-3,6-dioxo-2-piperazine acetic acid in the sample and standard, respectively, CS is the concentration of 5-benzyl-3,6-dioxo-2-piperazine acetic acid in the standard in mg/ml and Wu is the weight, in mg, of aspartame-acesulfame salt taken in the sample preparation. | |
| | |
METHOD OF ASSAY | Principle Apparatus Standard solution F = [(WSx 1000)/(122 x (VS-VO))] | |
| | |
| Where: | WS = weight of primary benzoic acid (g) |
| | |
| Procedure Acesulfame content (% m/m) = [(V1-VB) x N x 163/(10 x W)] | |
| | |
| Where: | W = Weight of sample (g) |
| Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 39th JECFA (1992) and published in FNP 52 Add 1 (1992), and incorporating the decisions on the metals and arsenic specifications agreed at the 63rd JECFA (2004) and published in FNP 52 Add 12 (2004). The 65th JECFA (2005) considered the additive to be of no toxicological concern for the functional uses listed. |
| |
SYNONYMS | INS No. 901 |
| |
DEFINITION | Beeswax is obtained from the honeycombs of bees (Fam. Apidae,e.g. Apis melliferaL) after the honey has been removed by draining or centrifuging. The combs are melted with hot water, steam or solar heat; the melted product is filtered and cast into cakes of yellow beeswax. White beeswax is obtained by bleaching the yellow beeswax with oxidizing agents (e.g. hydrogen peroxide, sulfuric acid) or sunlight. Beeswax consists of a mixture of esters of fatty acids and fatty alcohols, hydro-carbons and free fatty acids; minor amounts of free fatty alcohols are also present. |
| |
C.A.S. number | 8006-40-4 (yellow beeswax) |
| |
DESCRIPTION | Yellow beeswax: yellow or light-brown solid that is somewhat brittle when cold and presents a dull, granular, non-crystalline fracture when broken; it becomes pliable at about 35°. It has a characteristic odour of honey. White beeswax: white or yellowish white solid (thin layers are translucent) having a faint and characteristic odour of honey |
| |
FUNCTIONAL USES | Glazing agent; release agent; stabilizer; texturizer for chewing gum base; carrier for food additives (including flavours and colours); clouding agent |
| |
CHARACTERISTICS | |
| |
IDENTIFICATION | |
| |
Solubility (FNP 5) | Insoluble in water; sparingly soluble in alcohol; very soluble in ether |
| |
PURITY | |
| |
Melting range (FNP 5) | 62 - 65° |
| |
Acid value (FNP 5) | 17 - 24 |
| |
Peroxide value | Not more than 5 |
| |
Saponification value (FNP 5) | 87 -104 |
| |
Carnauba wax | Passes test |
| |
Ceresin, paraffins, and certain other waxes | Passes test |
| |
Fats, Japan wax, rosin and soap | Passes test |
| |
Glycerol and other polyols | Not more than 0.5 % (calculated as glycerol) |
| |
Lead (FNP 5) | Not more than 2 mg/kg |
| |
TESTS | |
| |
PURITY TESTS | |
| |
Peroxide value | Weigh accurately 5 g of the sample into a 200-ml conical flask. Add 30 ml of a 2:3 solution of chloroform and acetic acid TS and close the flask with a stopper. Heat with warm water and swirl to dissolve the sample. Cool to room temperature and add 0.5 ml of saturated potassium iodide solution. Close the flask with the stopper and shake vigorously for 60±5 sec. Add 30 ml of water and titrate imme- diately with 0.01 N sodium thiosulfate using starch TS as indicator. Carry out a blank determination. |
| |
| Peroxide value = (a-b) x N x 1000/W |
| |
| where |
| |
| a = volume (ml) of sodium thiosulfate used for the sample |
| |
Carnauba wax | Transfer 100 mg of the sample into a test tube, and add 20 ml of n-butanol. Immerse the test tube in boiling water, and shake the mixture gently until the sample dissolves completely. Transfer the test tube to a beaker of water at 60°, and allow the water to cool to room temperature. A loose mass of fine, needle- like crystals separates from clear mother liquor. Under the microscope, the crystals appear as loose needles or stellate clusters, and no amorphous masses are observed, indicating the absence of carnauba wax. |
| |
Ceresins, paraffins and certain other waxes | Transfer 3.0 g of the sample to a 100 ml round-bottomed flask, add 30 ml of a 4% w/v solution of potassium hydroxide in aldehyde-free ethanol and boil gently under a reflux condenser for 2h. Remove the condenser and immediately insert a thermometer. Place the flask in water at 80° and allow to cool, swirling the solution continuously. No precipitate is formed before the temperature reaches65°, although the solution may be opalescent. |
| |
Fats, Japan wax, rosin and soap | Boil 1 g of the sample for 30 min with 35 ml of a 1 in 7 solution of sodium hydroxide, maintaining the volume by the occasional addition of water, and cool the mixture. The wax separates and the liquid remains clear. Filter the cold mixture and acidify the filtrate with hydrochloric acid. No precipitate is formed. |
| |
Glycerol and other polyols | To 0.20 g of the sample in a round-bottom flask, add 10 ml of ethanolic potassium hydroxide TS, attach a reflux condenser to the flask and heat in a water bath for 30 min. Add 50 ml of dilute sulfuric acid TS, cool and filter. Rinse the flask and filter with dilute sulfuric acid TS. Combine the filtrate and washings and dilute to 100.0 ml with dilute sulfuric acid TS. Place 1.0 ml of the solution in a tube, add 0.5 ml of a 1.07 % (w/v) solution of sodium periodate, mix and allow to stand for 5 min. Add 1.0 ml of decolourized fuchsin solution (see below) and mix. Any precipitate disappears. Place the tube in a beaker containing water at 40°. Allow to cool while observing for 10 to 15 min. Any bluish-violet colour in the solution is not more intense than a standard prepared at the same time in the same manner using 1.0 ml of a 0.001 % (w/v) solution of glycerol in dilute sulfuric acid TS. Decolourized fuchsin solution: Dissolve 0.1 g of basic fuchsin in 60 ml of water. Add a solution of 1 g of anhydrous sodium sulfite (Reagent grade) in 10 ml of water. Slowly and with continuous shaking of the solution add 2 ml of hydrochloric acid. Dilute to 100 ml with water. Allow to stand protected from light for at least 12 h, decolourize with activated charcoal and filter. If the solution becomes cloudy, filter before use. If on standing the solution becomes violet, decolourize again by adding activated charcoal. Store protected from light. |
| New specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005). At the 65th JECFA (2005) the Committee had no safety concerns for the use of the substance in dry crystalline or microencapsulated form as an alternative to folic acid used in dietary supplements, foods for special dietary uses and other foods. | |||||||
| | |||||||
SYNONYMS | L-5-Methyltetrahydrofolic acid, calcium salt | |||||||
| | |||||||
DEFINITION | Calcium L-5-methyltetrahydrofolate is the calcium salt of L-5-methyltetrahydrofolic acid, which is the predominant, naturally occurring form of folate. It is synthesized by reduction of folic acid to tetrahydrofolic acid followed by methylation and diastereoselective crystallization (in water) of L-5-methyltetrahydrofolic acid as its calcium salt. The product contains variable amounts of water of crystallization. | |||||||
| | |||||||
Chemical name | N-{4-[[((6S)-2-amino-3,4,5,6,7,8-hexahydro-5-methyl-4-oxo-6-pteridinyl)methyl]amino]benzoyl}-L-glutamic acid, calcium salt | |||||||
| | |||||||
C.A.S. number | 151533-22-1 | |||||||
| | |||||||
Chemical formula | C20H23CaN7O6 (anhydrous form) | |||||||
| | |||||||
Structural formula | (anhydrous form) | |||||||
| | |||||||
Formula weight | 497.5 (anhydrous form) | |||||||
| | |||||||
Assay | 95.0 - 102.0% (anhydrous basis) | |||||||
| | |||||||
| | |||||||
DESCRIPTION | White to light yellowish, almost odourless, crystalline powder | |||||||
| | |||||||
FUNCTIONAL USES | Nutritional supplement | |||||||
| | |||||||
CHARACTERISTICS | | |||||||
| | |||||||
IDENTIFICATION | | |||||||
| | |||||||
Solubility (FNP 5) | Sparingly soluble in water and very slightly soluble or insoluble in most organic solvents; soluble in alkaline solutions | |||||||
| | |||||||
Infrared absorption | The infrared absorption spectrum of a potassium bromide dispersion of the sample corresponds to that of a Calcium L-5-methyltetrahydrofolate standard (see Appendix). | |||||||
| | |||||||
Calcium | Dilute 30 g of acetic acid (glacial) to 100 ml with water. Dissolve 5.3 g of K4Fe(CN)6 in 100 ml of water. To 5 ml of the acetic acid solution, add 20 mg of the sample and then 0.5 ml of the potassium ferrocyanide solution. Mix and add 50 mg of ammonium chloride. A white crystalline precipitate is formed. | |||||||
| | |||||||
Liquid chromatography | Retention time matches that of a reference standard (see under TESTS) | |||||||
| | |||||||
PURITY | | |||||||
| | |||||||
Water (FNP 5) | Not more than 17.0% (Karl Fischer method) | |||||||
| | |||||||
Calcium | 7.0 - 8.5% (anhydrous basis) | |||||||
| | |||||||
| calcium content on the anhydrous basis. | |||||||
Other folates and related substances | Not more than 2.5% | |||||||
| | |||||||
D-5-Methylfolate | Not more than 1.0% | |||||||
| See description under TESTS | |||||||
Total viable aerobic count (FNP 5) | Not more than 1000 CFU/g | |||||||
| | |||||||
Lead (FNP 5) | Not more than 2 mg/kg | |||||||
| | |||||||
TESTS | | |||||||
| | |||||||
PURITY TESTS | | |||||||
| | |||||||
Other folates and related substances | Principle Reference standard solution Sample solution Mobile phase solutions | |||||||
| | |||||||
| Chromatography conditions | |||||||
| Column: | Hypersil-ODS, 5 µm; 250 x 4 mm (Thermo Hypersil Keystone or equivalent) | ||||||
| Flow rate: | 1.1 ml/min | ||||||
| Gradient: | Time (min) | % Mobile | % Mobile | Remark | |||
| | | phase A | phase B | | |||
| | 0 | 100 | 0 | Start | |||
| | 0 - 14 | 100 - 45 | 0 - 55 | Linear gradient | |||
| | 14 - 17 | 45 - 0 | 100 | Linear gradient | |||
| | 17 - 22 | 0 | 100 | Hold | |||
| | 22 - 31 | 100 | 0 | Reconditioning | |||
| | |||||||
| Temperature: Room temperature | |||||||
| | |||||||
| Retention times given below are approximate: | |||||||
| | |||||||
| Folates and related substances | Retention time (min) | ||||||
| 4-Aminobenzoylglutamic acid (ABGA) | 3.1 | ||||||
| 4a-Hydroxy-5-methyltetrahydrofolic acid (HOMeTHFA) | 4.3 | ||||||
| D-Pyrazino-s-triazine derivative (D-Mefox) | 6.1 | ||||||
| L-Pyrazino-s-triazine derivative (L-Mefox) | 6.3 | ||||||
| Tetrahydrofolic acid (THFA) | 8.5 | ||||||
| 7,8-Dihydrofolic acid (DHFA) | 11.2 | ||||||
| Folic acid (FA) | 11.4 | ||||||
| 5,10-Methylenetetrahydrofolic acid (CH2THFA) | 11.7 | ||||||
| 5-Methyltetrahydrofolic acid (5-MTHF) | 13.6 | ||||||
| 5-Methyltetrahydropteroic acid (MeTHPA) | 15.1 | ||||||
| N2-Methylamino-5-methyltetrahydrofolic acid (DiMeTHFA) | 17.6 | ||||||
| | |||||||
| Sample analysis Xi (%) = Ai ×WS × S × (RF)i/AS × W | |||||||
| | |||||||
| where Ai = the peak area for each folate (other than 5-MTHF) and related substance | |||||||
| | |||||||
| Folates and related substances | RF | ||||||
| ABGA | 0.93 | ||||||
| HOMeTHFA | 1.11 | ||||||
| L-Mefox and D-Mefox | 1.11 | ||||||
| DHFA | 0.98 | ||||||
| FA | 0.86 | ||||||
| MeTHPA | 0.68 | ||||||
| THFA | 1.00 | ||||||
| CH2THFA | 1.00 | ||||||
| DiMeTHFA | 1.00 | ||||||
| If there are any unidentified impurities, apply a RF of 1.00 Calculate the total amount of "Other folates and related substances" by summing the Xi for all impurities. System suitability test System suitability test solution (SST solution): Weigh accurately 50 mg of a L-5-MTHF-Ca sample containing DiMeTHFA into a 100-ml volumetric flask. (Available from Merck Eprova AG). Add 1 ml of the Mixed folates solution and a small quantity of water to dissolve, mix and dilute to volume with water. Procedure: Inject 10 µl of the SST solution immediately. The resolution between L-5-MTHF and MeTHPA must be at least 5. | |||||||
| | |||||||
D-5-Methylfolate | Principle Sample preparation Mobile phase | |||||||
| | |||||||
| Chromatography Conditions | |||||||
| Column: | Chiral Protein HSA, 5 µm, 150 x 4 mm (ChromTech or equivalent) | ||||||
| Flow rate: | 1 ml/min | ||||||
| Temperature: | 40° | ||||||
| Injection volume: | 10 µl | ||||||
| Detection: | UV (280 nm) | ||||||
| Run time: | 22 min | ||||||
| Solvent: | Water | ||||||
| | |||||||
| Sample analysis Calculation D-5-MTHF (%) = 100AD/AT System suitability test | |||||||
| | |||||||
METHOD OF ASSAY | Calculate the percentage of L-5-MTHF-Ca in the sample from the content of 5-MTHF-Ca (L- and D-diastereoisomers), determined in the test for "Other folates and related substances", and the content of D-5-MTHF-Ca, determined in the test for D-5-Methylfolate, and correcting for water content, as follows: L-5-MTHF-Ca (%) =100 × AT× WS× S × (100 - D) × 1.083/AS × W × (100 - %H2O) | |||||||
| | |||||||
| where | |||||||
| AT is taken from the calculation for the D-5-Methylfolate analysis |
| Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 39th JECFA (1992) and published in FNP 52 Add 1 (1992), and incorporating the decisions on the metals and arsenic specifications agreed at the 63rd JECFA (2004) and published in FNP 52 Add 12 (2004). The 65th JECFA (2005) considered the additive to be of no toxicological concern for the functional uses listed. |
| |
SYNONYMS | INS no. 902 |
| |
DEFINITION | Crude candelilla wax is obtained by first boiling the dried stalks of the candelilla plant (Euphorbia antisyphilitica) in water acidified with sulfuric acid to release the wax. The molten wax is then skimmed off and allowed to solidify and refined by further treatment with sulfuric acid and subsequent passage through filter-presses. |
| |
C.A.S. number | 8006-44-8 |
| |
DESCRIPTION | Yellowish-brown hard, brittle, lustrous solid with an aromatic odour when heated |
| |
FUNCTIONAL USES | Glazing agent, texturizer for chewing gum base, surface-finishing agent, carrier for food additives (including flavours and colours), clouding agent |
| |
CHARACTERISTICS | |
| |
IDENTIFICATION | |
| |
Solubility (FNP 5) | Insoluble in water; soluble in toluene |
| |
Infrared absorption | The infrared spectrum of the sample, melted and prepared for analysis on a potassium bromide plate, corresponds to that of a candelilla wax standard (see Appendix). |
| |
PURITY | |
| |
Melting range (FNP 5) | 68.5 - 72.5° |
| |
Acid value (FNP 5) | Between 12 and 22 |
| |
Saponification value (FNP 5) |
Between 43 and 65 |
| |
Lead (FNP 5) | Not more than 2 mg/kg |
APPENDIX
Infrared spectrum of candelilla wax
| (Tentative) Information on functional uses and method of assay required Revised tentative specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2006), superseding specifications prepared at the 14th JECFA (1970) and published in NMRS 48B (1971) and in FNP 52 (1992), and incorporating the decisions on metals and arsenic specifications agreed at the 57th JECFA (2001) and published in FNP 52 Add 9 (2001). An ADI of 0-2 mg/kg bw was established at the 18th JECFA (1974) |
| |
SYNONYMS | INS No. 637 |
| |
DEFINITION | |
| |
Chemical names | 2-Ethyl-3-hydroxy-4-pyrone |
| |
C.A.S. number | 4940-11-8 |
| |
Chemical formula | C7H8O3 |
| |
Structural formula | |
| |
Formula weight | 140.14 |
| |
Assay | Not less than 98% |
| |
DESCRIPTION | White, crystalline powder having a characteristic odour |
| |
FUNCTIONAL USES | Flavour enhancer, stabilizer, flavouring agent (See 'Flavouring agents' monograph No. 1481) |
| |
CHARACTERISTICS | |
| |
IDENTIFICATION | |
| |
Solubility | Sparingly soluble in water; soluble in alcohol |
| |
Melting range | 89 - 93° |
| |
PURITY | |
| |
Water (FNP 5) | Not more than 0.5 % w/w (Karl Fischer Method) |
| |
Sulfated ash (FNP5) | Not more than 0.2 % w/w |
| |
Lead (FNP 5) | Not more than 1mg/kg Determine using an atomic absorption technique appropriate to the specified level. The selection of sample size and method of sample preparation may be based on the principles of the methods described in FNP 5, "Instrumental Methods". |
| |
METHOD OF ASSAY | Prepare a solution of ethyl maltol in 0.1 N hydrochloric acid containing 10 µg/ml, and determine the extinction at 276 mm. E (1%, 1 cm): 276 nm: 655-675 Calculation Ethyl maltol (%) = 100 x E(sample)/E(standard) where E = E (1%, 1 cm) |
| Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11 (2003). An ADI "not specified" was established at the 61st JECFA (2003). | ||||
| | ||||
SOURCES | Produced by submerged fed-batch pure culture fermentation of a genetically modified strain of Aspergillus oryzaecontaining the laccase gene derived from Myceliophthora thermophila,using recombinant DNA techniques and traditional mutagenesis. The enzyme is isolated from the fermentation broth by filtration to remove the biomass and concentrated by ultrafiltration and/or evaporation. Residual production microorganisms are removed from the enzyme concentrate by germ filtration. The final product is formulated using food-grade stabilizing and preserving agents. | ||||
| | ||||
ACTIVE PRINCIPLES | Laccase (synonyms: urishiol oxidase; p-diphenol oxidase) | ||||
| | ||||
SYSTEMATIC NAMES AND NUMBERS | Benzenediol:oxygen oxidoreductase; EC 1.10.3.2; CAS No. 80498-15-3 | ||||
| | ||||
REACTIONS CATALYSED | Oxidation of a range of phenolic substances with concomitant reduction of oxygen to water. | ||||
| | ||||
DESCRIPTION | Brown liquid | ||||
| | ||||
FUNCTIONAL USES | Enzyme preparation. Used in the brewing of beer to prevent the formation of off-flavour compounds such as trans-2-nonenal. Scavenges oxygen that otherwise would react with fatty acids, amino acids, proteins, and alcohols to form off-flavour precursors. | ||||
| | ||||
GENERAL SPECIFICATIONS | Must conform to the latest edition of the JECFA General Specifications and Considerations for Enzyme Preparations used in Food Processing, | ||||
| | ||||
CHARACTERISTICS | | ||||
| | ||||
IDENTIFICATION | | ||||
| | ||||
Laccase activity | The sample shows laccase activity See description under TESTS | ||||
| | ||||
TESTS | | ||||
| | ||||
Laccase activity | Principle Apparatus Reagents Reagent Solutions Maleic acid, 1 M solution: Dissolve 23.2 g of maleic acid in approximately 150 ml water in a 200-ml volumetric flask. Make to volume and mix. Triton X-100, 10% stock solution: In a beaker, add 25.0 g of Triton X-100 to approximately 200 ml water; stir to dissolve. Transfer to a 250-ml volumetric flask and make up to volume with water. TRIS buffer, 25 mM (pH 7.50): Add 5 ml of 1 M TRIS, 2 ml of 1 M maleic acid solution, and 1 ml of Triton X-100 10% solution to a 200-ml volumetric flask. Add 150 ml water and adjust pH to 7.50±0.05 using 1 M maleic acid solution. Make up to volume with water. (Note: do not adjust the pH with hydrochloric acid because chloride inhibits laccase activity.) PEG 6000, 50 g/l solution: Weigh 250 g of PEG 6000 in a beaker, transfer to a 5000 ml volumetric flask, add water and stir until dissolved. Add water to volume. Syringaldazine, 0.56 mM stock solution: Rinse a 50-ml volumetric flask with water and ethanol to remove any soapy residues. Weigh 10.0 mg of syringaldazine in a weighing boat and transfer to the volumetric flask. Add 96% ethanol to the mark and stir until the syringaldazine is dissolved (approximately 3 h). The solution must be stored in a dark bottle in a refrigerator. Syringaldazine, 0.22 mM working solution: Rinse a 10-ml volumetric flask with water and ethanol to remove any soapy residues. Transfer 4.0 ml of syringaldazine stock solution to the flask and add water to volume. The solution can be kept in a dark bottle for up to two hours at room temperature. Glycine buffer, 1.5%: Dissolve 75 g glycine, 150 g glucose, and 250 g PEG 6000 in approximately 4.5 litre water in a 5-l volumetric flask. Adjust pH to 9.20±0.05 using NaOH or 1 M maleic acid. Add water to volume. (Note: Do not adjust pH with hydrochloric acid because chloride inhibits laccase activity.) Standard and sample solutions Laccase working standard solutions (for the construction of the standard curve): Prepare six solutions by diluting the laccase stock solution with PEG 6000 solution as shown in the table below. Use the diluter and vials compatible with the centrifugal analyser. | ||||
| | ||||
| Sample No. | Dilution factor | Laccase stock solution (µl) | PEG 6000 solution (µl) | Activity, LAMU/ml |
| 1 | 30 | 20 | 580 | 0.01167 |
| 2 | 24 | 25 | 575 | 0.01458 |
| 3 | 20 | 30 | 570 | 0.01750 |
| 4 | 15 | 40 | 560 | 0.02333 |
| 5 | 12 | 50 | 550 | 0.02917 |
| 6 | 10 | 60 | 540 | 0.03500 |
| | ||||
| Laccase control: Use a laccase preparation with known activity. Accurately weigh the amount of the preparation sufficient to obtain laccase activity of approximately 0.70 LAMU/ml in a 200-ml volumetric flask. Place the preparation in the flask and add the PEG 6000 solution to volume. Stir on the magnetic stirrer for 15 min. This is a stock solution. It should be prepared daily. Dilute the stock solution with the PEG 6000 solution 30 times using diluter. Place the diluted solution in a vial. Test samples: Dilute test samples on the basis of the anticipated enzyme content to obtain activity between 0.0117 and 0.0350 LAMU/ml. Procedure Analysis Calculations Where A = DAbs/min converted to activity (LAMU/ml) |
| (Tentative) Revised specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add13 (2005), superseding specifications prepared at the 25th JECFA (1981) and published in FNP 52 (1992). An ADI of 0-1 mg/kg bw was established at the 25th JECFA (1981) |
| |
SYNONYMS | INS No. 636 |
| |
DEFINITION | |
| |
Chemical names | 3-Hydroxy-2-methyl-4-pyrone |
| |
C.A.S. number | 118-71-8 |
| |
Chemical formula | C6H6O3 |
| |
Structural formula | |
| |
Formula weight | 126.11 |
| |
Assay | Not less than 99% |
| |
DESCRIPTION | White to off-white crystalline powder having a characteristic caramel-butterscotch odour |
| |
FUNCTIONAL USES CHARACTERISTICS |
Flavour enhancer, stabilizer, flavouring agent (see Flavouring agents monograph No. 1480) |
| |
IDENTIFICATION | |
| |
Solubility (FNP 5) | Soluble in water and ethanol |
| |
Melting range (FNP 5) | 160 - 164° |
| |
Test for phenol | Dissolve 0.1 g of the sample in 10 ml of ethanol and add 3 drops of ferric chloride TS. A reddish violet colour is produced. |
| |
Precipitation test | Dissolve 0.5 g of the sample in 10 ml of sodium hydroxide TS and pass carbon dioxide through the solution. White crystals are formed; collect and recrystallize from dilute ethanol. The crystals melt between 160 - 164° |
| |
Iodoform reaction | Dissolve 0.1 g of the sample in 5 ml dioxane, add 1 ml of sodium hydroxide TS, and add sufficiently iodine-potassium iodide TS (Iodine TS) with shaking until the colour remains. Heat on a water bath for 5 min. Yellow crystals are formed. |
| |
PURITY | |
Lead (FNP 5) | Not more than 1 mg/kg |
| |
METHOD OF ASSAY | Standard Solution Assay Solution Procedure Calculate the percent of Maltol in the sample by the formula: % of Maltol = 100 × WS × AA/AS × WA where AA = absorbance of the Assay Solution |
| New specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005). The 65th JECFA (2005) was unable to assess the safety of the additive. | ||||
| | ||||
SYNONYMS | Phospholipase A1 | ||||
| | ||||
SOURCES | Produced by submerged fed-batch pure culture fermentation of a genetically modified strain of Aspergillus oryzaecontaining the phospholipase A1 gene derived from Fusarium venenatum. The enzyme is isolated from the fermentation broth by filtration to remove the biomass and concentrated by ultrafiltration and/or evaporation. Residual production microorganisms are removed from the enzyme concentrate by germ filtration. The final product is formulated using food-grade stabilizing and preserving agents. | ||||
| | ||||
ACTIVE PRINCIPLES | Phospholipase A1 | ||||
| | ||||
SYSTEMATIC NAMES AND NUMBERS | Phosphatidylcholine 1-acylhydrolase; EC 3.1.1.32; CAS No. 9043-29-2 | ||||
| | ||||
REACTIONS CATALYSED | Hydrolysis of the sn-1 ester bond of diacylphospholipids to form 2-acyl-1-lysophospholipids and free fatty acids | ||||
| | ||||
DESCRIPTION | Brown liquid. | ||||
| | ||||
FUNCTIONAL USES | Enzyme preparation. Used in cheese production to reduce the loss of fat and milk solids and increase cheese yield. | ||||
| | ||||
GENERAL SPECIFICATIONS | Must conform to the latest edition of the JECFA General Specifications and Considerations for Enzyme Preparations used in Food Processing | ||||
| | ||||
CHARACTERISTICS | | ||||
| | ||||
IDENTIFICATION | | ||||
| | ||||
Phospholipase A1 activity | The sample shows phospholipase A1 activity See description under TESTS | ||||
| | ||||
TESTS | | ||||
| | ||||
Phospholipase A1 activity | Principle 1 LEU is defined as the amount of enzyme that under standard conditions (pH=8.0; 40° ±0.5) results in the same rate of sodium hydroxide consumption (in microeq/min) as the Lecitase standard diluted to a nominal activity of 1 LEU/g. The quantification limit of the method is approximately 1.5 LEU/ml. (Note: The method can be carried out using either an automated system or standard laboratory equipment for carrying out titration experiments. Procedures and calculations for both the automated and manual versions are described.) | ||||
| | ||||
| Automated method - PHM290 pH-Stat Controller (Radiometer) Reagents and solutions Calcium chloride, 0.32 M: Weigh 4.70 g calcium chloride (CaCl2 2H2O, Merck 2382 or equivalent). Dissolve in water in a 100 ml volumetric flask. Add water to volume and mix. The solution is stable for up to one week at room temperature. Sodium deoxycholate, 0.016 M: Weigh 6.7 g sodium deoxycholate (C24H39NaO4). Dissolve in water in a 1000-ml volumetric flask. Add water to volume and mix. The solution is stable for up to one week at room temperature. Lecithin substrate: Use lecithin (L-á-phosphatidylcholine) Sigma P-5638 or equivalent. Mix lecithin with a spoon and weigh exactly 20.0g. Transfer to a 1000-ml beaker, add 400 ml of water and stir until the lecithin is dissolved. Add 20 ml of 0.32 M calcium chloride and stir for 1-2 min until calcium chloride is dissolved. Add 200 ml of 0.016 M sodium deoxycholate and 400 ml of water. Stir for about 0.5 hour and homogenize for 10 min on a Silverson L4R homogenizer. The solution is stable for up to one day at room temperature. Hydrochloric acid, 1 N: Use HCl solution standardized for preparation of 1000 ml of 1 N HCl, for example, one ampoule of Merck Titrisol No. 1.09970 or equivalent. Transfer the HCl solution quantitatively to a 1000-ml volumetric flask. Add water to volume. The solution is stable for up to 6 months at room temperature. Hydrochloric acid, 0.001 N: Transfer 1 ml of 1 N HCl to a 1000-ml volumetric flask. Add water to volume. The solution is stable for up to one week at room temperature. Standard and sample solutions Standard working solutions: Transfer quantitatively 5 ml of the standard stock solution to a 200-ml volumetric flask. Add 0.001 N HCl to volume. This solution contains approximately 7.5 LEU/ml and is referred to as 100% standard. Using the 100% standard, prepare additional standard solutions in 25-ml volumetric flasks according to the following table: | ||||
| | ||||
| Standard solution | Approximate strength (LEU/ml) | Volume of 100% standard (ml) | Volume of 0.001 N HCl | |
| 80% | 6.0 | 20 | 5 | |
| 60% | 4.5 | 15 | 10 | |
| 40% | 3.0 | 10 | 15 | |
| 20% | 1.5 | 5 | 20 | |
| The standard solutions are used by the automated system to calculate a 2nd degree polynomial standard curve. An example of the standard (calibration) curve is shown below Calibration Curve Control sample: A phospholipase product with known activity, for example, 10500 LEU/ml, is used to prepare the control sample. Transfer 1 ml of the control solution to a 50-ml volumetric flask and add 0.001 HCl to volume to obtain a stock solution. Transfer 1 ml of the stock solution to a 50-ml volumetric flask and add 0.001 N HCl to volume. Analyse the control sample in each run. A result within 10 percent of the nominal activity is acceptable. Blank sample: Use 0.001 N HCl as the blank sample. Test samples: Remove the test sample from a refrigerator or freezer and keep it at room temperature for approximately 2 h before analysis. Accurately weigh the sample into a measuring flask and add 0.001 N HCl to volume. Repeat dilution if necessary to obtain the activity of approximately 6 LEU/ml. The dilution should be performed within one hour of the analysis. Enter the sample weight (in grams) and the dilution volume (in milliliters) into the calculation program for the automated method or into the calculation formula for the manual method. Procedure Calculations Manual method Calculation The activity of the test samples in LEU/g is calculated according to the following equation: | ||||
| where: | W(g) is the sample weight and |
| New specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005). An ADI "not specified" was established at the 65th JECFA (2005). | |
| | |
SYNONYMS | INS No. 1204 | |
| | |
DEFINITION | Linear, neutral glucan consisting mainly of maltotriose units connected by á-1,6 glycosidic bonds. It is produced by fermentation from a food grade hydrolysed starch using a non-toxin producing strain of Aureobasidium pullulans. After completion of the fermentation, the fungal cells are removed by microfiltration, the filtrate is heat-sterilized and pigments and other impurities are removed by adsorption and ion exchange chromatography. | |
| | |
C.A.S. number | 9057-02-7 | |
| | |
Chemical formula | (C6H10O5)x | |
| | |
Structural formula | ||
| | |
Assay | Not less than 90% of glucan on the dried basis | |
| | |
DESCRIPTION | White to off-white odourless powder | |
| | |
FUNCTIONAL USES | Glazing agent, film-forming agent, thickener | |
| | |
CHARACTERISTICS | | |
| | |
IDENTIFICATION | | |
| | |
Solubility (FNP 5) | Soluble in water, practically insoluble in ethanol | |
| | |
pH (FNP 5) | 5.0 - 7.0 (10% solution) | |
| | |
Precipitation with polyethylene glycol 600 | Add 2 ml of polyethylene glycol 600 to 10 ml of a 2% aqueous solution of pullulan. A white precipitate is formed. | |
| | |
Depolymerisation with pullulanase | Prepare two test tubes each with 10 ml of a 10% pullulan solution. Add 0.1 ml pullulanase solution having activity 10 units/g (refer to pullulanase activity, under Methods for enzyme preparations in FNP 5) to one test tube, and 0.1 ml water to the other. After incubation at about 25° for 20 min, the viscosity of the pullulanase-treated solution is visibly lower than that of the untreated solution. | |
| | |
PURITY | | |
| | |
Loss on drying (FNP 5) | Not more than 6% (90°, pressure not more than 50 mm Hg, 6h) | |
| | |
Mono-, di- and oligosaccharides | Not more than 10% (expressed as glucose) See description under TESTS | |
| | |
Viscosity | 100-180 mm2/s (10% w/w aqueous solution at 30°) | |
| | |
Lead (FNP 5) | Not more than 1 mg/kg | |
| | |
Microbiological criteria (FNP 5) | Yeast and moulds: | Not more than 100 CFU/g |
| Coliforms: | Negative in 25 g |
| Salmonella: | Negative in 25 g |
| | |
TESTS | | |
| | |
PURITY TESTS | | |
| | |
Mono-, di- and oligosaccharides | Principle Equipment Procedure Measurement of mono-, di- and oligosaccharides: Weigh accurately 0.8 g sample and dissolve in water to make 100 ml (stock solution). Place 1 ml of the stock solution in a centrifuge tube. Add 0.1 ml saturated potassium chloride solution. Add 3 ml methanol and mix vigorously for 20 sec. Centrifuge at 11000 rpm for10 min. Add 0.2 ml of the supernatant to 5 ml modified anthrone solution (0.2 g anthrone in 100 g 75% (v/v) sulfuric acid, freshly prepared). Add 0.2 ml of glucose standard solution and 0.2 ml water (blank control) to separate 5 ml portions of modified anthrone solution. Mix rapidly. Place samples in a 90º water bath and incubate for 15 min. Measure absorbance of the test solution at 620 nm. Calculate the percent of mono-, di- and oligosaccharides expressed as glucose, C, in the sample: C (%)= [(At - Ab) × 0.41 × G × 100]/(As - Ab)×W where At is absorbance of the test solution | |
| | |
Viscosity | Dry the sample for 6 h at 90º under reduced pressure (50 mm Hg). Weigh 10.0 g of the sample and dissolve in water to yield 100 g of solution. Use an Ubbelohde-type (falling-ball) viscometer. Charge the viscometer with sample in the manner dictated by the design of the instrument. Immerse the viscometer vertically in the thermostatic tank at 30 ± 0.1º and allow to stand for 20 min so that the sample equilibrates with the temperature in the tank. Adjust the meniscus of the column of liquid in the capillary tube to a position about 5 mm above of the first mark. With the sample flowing freely, measure, in seconds, the time required for the meniscus to pass from the first to the second mark. Calculate the viscosity, V: V(mm2/s) = C× t where C = calibration constant of the viscometer (mm2/s2) | |
| | |
METHOD OF ASSAY | Calculate the percentage of pullulan on dried basis, P, as the difference between100% and the sum of the percentages of known impurities (mono-, di- and oligosaccharides and water). P(%) = 100 - (L+C) where L is loss on drying |
| Specifications prepared at the 61st JECFA (2003), published in FNP 52 Add 11 (2003) and republished in FNP 52 Add 13 (2005). A group ADI of 0.1 mg quillaia saponins per kg bw was established at the 65th JECFA (2005) for Quillaia Extract (Type 1) and Quillaia Extract (Type 2). | |||
| | |||
SYNONYMS | Quillaja extract, Soapbark extract, Quillay bark extract, Bois de Panama, Panama bark extract, Quillai extract; INS No. 999 | |||
| | |||
DEFINITION | Quillaia extract (Type 1) is obtained by aqueous extraction of the milled inner bark or of the wood of pruned stems and branches of Quillaja saponaria Molina (family Rosaceae). It contains triterpenoid saponins (quillaia saponins, QS) consisting predominantly of glycosides of quillaic acid. Polyphenols and tannins are major components and some sugars and calcium oxalate will be present. Quillaia extract (Type 1) is available commercially as liquid product or as spray-dried powder that may contain carriers such as lactose, maltitol or maltodextrin. The liquid product is usually preserved with sodium benzoate or ethanol. | |||
| | |||
C.A.S. number | 68990-67-0 | |||
| | |||
Formula weight | Monomeric saponins range from ca. 1800 to ca. 2300, consistent with a triterpene with 8-10 monosaccharide residues | |||
| | |||
ASSAY | Saponin content: not less than 20 % and not more than 26 % on the dried basis | |||
| | |||
DESCRIPTION | Red-brownish liquid or light brown powder with a pink tinge | |||
| | |||
FUNCTIONAL USES | Emulsifier, foaming agent | |||
| | |||
CHARACTERISTICS | | |||
| | |||
IDENTIFICATION | | |||
| | |||
Solubility (FNP 5) | Very soluble in water, insoluble in ethanol, acetone, methanol and butanol | |||
| | |||
Foam | Dissolve 0.5 g of powder extract in 9.5 g of water or 1 ml of liquid extract in 9 ml of water. Add 1 ml of this mixture to 350 ml of water in a 1000-ml graduated cylinder. Cover the cylinder, vigorously shake it 30 times, and allow settling. Record the foam level (ml) after 30 min. Typical values are 150 ml of foam | |||
| | |||
Chromatography | Determine as in METHOD OF ASSAY. The retention time of major peak of the sample corresponds to the major saponin peak (QS-18) of the standard. | |||
| | |||
Colour and turbidity | Powder form only: Dissolve 0.5 g in 9.5 g of water. The solution is not turbid. Determine the absorbance of the solution against water at 520 nm. The absorbance is less than 1.2. | |||
| | |||
PURITY | | |||
| | |||
Water (FNP 5) | Powder form: not more than 6% (Karl Fischer Method) | |||
| | |||
Loss on drying (FNP 5) | Liquid form: 50 to 80% (2 g, 105°, 5h) | |||
| | |||
pH (FNP 5) | 3.7 - 5.5 (4 % solution) | |||
| | |||
Ash (FNP 5) Tannins | Not more than 14% on a dried basis (use 1.0 g for powder samples; for liquid samples, use the residue from loss on drying) Not more than 8% on a dried basis See description under TESTS | |||
| | |||
Lead (FNP 5) | Not more 2 mg/kg. | |||
| | |||
TESTS | | |||
| | |||
PURITY TESTS | | |||
| | |||
Tannins | Weigh either 3.0 g of the powder form or an equivalent amount of liquid sample, accounting for solids content determined from loss on drying. Dissolve in 250 ml of water. Adjust the pH to 3.5 with acetic acid. Dry 25 ml of this solution at 105° for 5 h and determine the weight of the dried solid, in g (Wi). Mix 50 ml of the solution with 360 mg of polyvinyl polypyrrolidone. Stir the solution for 30 min at room temperature; then centrifuge at 800 ×g. Recover the supernatant and dry this solution at 105o (5h).Weigh the recovered solid (Wf, ing). The percentage of tannins in the sample is: % tannins (dried basis) = 100 × (Wi - Wf/2)/Wi | |||
| | |||
METHOD OF ASSAY | Principle Sample preparation Standard preparation High performance liquid chromatography (HPLC) | |||
| | |||
| Column: | Vydac 214TP54 (4.6 x 250 mm length, 5 µm pore) or equivalent | ||
| Column temperature: | room temperature | ||
| Pump: | gradient | ||
| Solvent A: | 0.15% trifluoroacetic acid in HPLC-grade water. | ||
| Solvent B: | 0.15% trifluoroacetic acid in HPLC-grade acetonitrile. | ||
| Gradient: | Time(min) 0 | % solvent A 70 | % solvent B 30 |
| | 40 | 55 | 45 |
| | 45 | 70 | 30 |
| Flow rate: | 1 ml/min | ||
| Detection wavelength: | 220 nm | ||
| Injection volume: | 20 µl | ||
| | |||
| Calculation Csap= (Asample/Astandard) x CStandard where CStandard (mg/ml) is the saponins concentration of the standard injected (e.g., CStandard = 13.5 mg/ml if the saponin content of 1.5 g of standard sample is 90 %) and Asample and Astandard are the sums of the peak areas attributed to the four principle saponins in the sample preparation and in the standard preparation, respectively, as noted in the figure. (Tannins and Polyphenols will elute before the saponins. The peaks due to the saponins will appear after the major peak due to the polyphenols - see figure.) The percentage of saponins in the test sample is: % Saponins = 100 × Csap/(0.1Wsample) where Wsample is the weight of the sample (mg) taken for the sample preparation and 0.1 is the inverse of the sample volume, 10 ml. |
Appendix
Chromatogram of Standard (15 mg solids/ml equivalent to 13.5 mg saponins/ml).
Chromatogram of Quillaia extract (Type 1) (55 mg solids/ml)
| Revised specifications prepared at the 65th JECFA and published in FNP 52 Add 13 (2005), superseding specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11 (2003). A group ADI of 0.1 mg quillaia saponins per kg bw was established at the 65th JECFA (2005) for Quillaia Extract (Type 1) and Quillaia Extract (Type 2). | |||
| | |||
SYNONYMS | Quillaja extract, Soapbark extract, Quillay bark extract, Bois de Panama, Panama bark extract, Quillai extract | |||
| | |||
DEFINITION | Quillaia extract (Type 2) is obtained either by chromatographic separation or ultrafiltration of the aqueous extraction of the milled inner bark or of the wood of pruned stems and branches of Quillaja saponariaMolina (family Rosaceae). It contains triterpenoid saponins (quillaia saponins, QS) consisting predominantly of glycosides of quillaic acid. Polyphenols and tannins are minor components. Some sugars and calcium oxalate will also be present. | |||
| | |||
C.A.S. number | 68990-67-0 | |||
| | |||
Formula weight | Monomeric saponins range from ca. 1800 to ca. 2300, consistent with a triterpene with 8-10 monosaccharide residues | |||
| | |||
Assay | Saponin content: not less than 65% and not more than 90% on the dried basis | |||
| | |||
DESCRIPTION | Light red-brownish liquid or powder | |||
| | |||
FUNCTIONAL USES | Emulsifier, foaming agent | |||
| | |||
CHARACTERISTICS | | |||
| | |||
IDENTIFICATION | | |||
| | |||
Solubility (FNP 5) | Very soluble in water, insoluble in ethanol, acetone, methanol, and butanol | |||
| | |||
Foam | Dissolve 0.5 g of the powder form in 9.5 ml of water or 1 ml of the liquid form in 9 ml of water. Add 1 ml of this solution to 350 ml of water in a 1000-ml graduated cylinder. Cover the cylinder, vigorously shake it 30 times, and allow settling. Record the foam volume (ml) after 30 min. Typical volumes are about 260 ml. | |||
| | |||
Chromatography | Determine as in METHOD OF ASSAY. The retention time of major sample peak corresponds to the major saponin peak (QS-18) of the standard. | |||
| | |||
Colour and turbidity | Powder form only: Dissolve 0.5 g in 9.5 ml of water. The solution shall not turbid Determine the absorbance of the solution against water at 520 nm. The absorbance shall be less than 0.7. | |||
| | |||
PURITY | | |||
| | |||
Water (FNP 5) | Powder form: not more than 6% (Karl Fischer Method) | |||
| | |||
Loss on drying (FNP 5) | Liquid form: 50 to 80% (2 g, 105°, 5h) | |||
| | |||
pH (FNP 5) | 3.7 - 5.5 (4 % solution) | |||
| | |||
Ash (FNP 5) | Not more than 5% on a dried basis (use 1.0 g for powder samples; for liquid samples, use the residue from Loss on drying) | |||
| | |||
Tannins | Not more than 8% on a dried basis | |||
| | |||
Lead (FNP 5) | Not more 2 mg/kg. | |||
| | |||
TESTS | | |||
| | |||
PURITY TESTS | | |||
| | |||
Tannins | Weigh either 3.0 g of the powder form or an equivalent amount of liquid sample, accounting for solids content determined from loss on drying. Dissolve in 250 ml of water. Adjust the pH to 3.5 with acetic acid. Dry 25 ml of this solution at 105° for 5 h and determine the weight of the dried solid, in g (Wi). Mix 50 ml of the solution with 360 mg of polyvinyl polypyrrolidone. Stir the solution for 30 min at room temperature; then centrifuge at 800 ×g. Recover the supernatant and dry this solution at 105o(5h).Weigh the recovered solid (Wf, ing). The percentage of tannins in the sample is: % tannins (dried basis) = 100 × (Wi - Wf/2)/Wi | |||
| | |||
METHOD OF ASSAY | Principle: Sample preparation: Aqueous extracts (~ 550 mg solids/ml):Weigh 1 g of sample and dilute with 9 ml of water. Filter through a 0.2 µm filter. Standard preparation: High performance liquid chromatography (HPLC): | |||
| | |||
| Column: | Vydac 214TP54 (4.6 x 250 mm length, 5 µm particle size) or equivalent | ||
| Column temperature: | Room temperature | ||
| Pump: | Gradient | ||
| Solvent A: | 0.15% trifluoroacetic acid in HPLC-grade water. | ||
| Solvent B: | 0.15% trifluoroacetic acid in HPLC-grade acetonitrile. | ||
| Gradient: | Time(min) | % solvent A | % solvent B |
| | 0 | 70 | 30 |
| | 40 | 55 | 45 |
| | 45 | 70 | 30 |
| Flow rate: | 1 ml/min | ||
| Detection wavelength: | 220 nm | ||
| Injection volume: | 20 µl | ||
| | |||
| Calculation: Csap= (Asample/Astandard)CStandard where CStandard (mg/ml) is the saponins concentration of the standard injected (e.g., CStandard = 13.5 mg/ml if the saponin content of 1.5 g of standard sample is 90 %) and Asample and Astandard are the sums of the peak areas attributed to the four principle saponins in the sample preparation and in the standard preparation, respectively, as noted in the figure. (Tannins and polyphenols will elute before the saponins. The peaks corresponding to the saponins will appear after the major peak corresponding to the polyphenols) The percentage of saponins in the test sample is: % Saponins = 100 × Csap/(0.1Wsample) where Wsample is the weight of the sample (mg) taken for the sample preparation and 0.1 is the inverse of the sample volume, 10 ml. |
Appendix
Chromatogram of standard (15 mg solids/ml equivalent to 13.5 mg saponins/ml).
| (Tentative) Information required on
Note: The tentative specifications will be withdrawn unless the requested information is received before the end of the year 2006. Tentative specifications prepared at the 65th JECFA (2005) and published in FNP 52 Add 13 (2005), superseding the specifications prepared at the 61st JECFA (2003) and published in FNP 52 Add 11 (2003). An ADI of 0-30 mg/kg bw for this substance together with sucroglycerides was established at the 49th JECFA (1997). | ||||||
| | ||||||
SYNONYMS | Sucrose fatty acid esters, INS No. 473 | ||||||
| | ||||||
DEFINITION | Mono-, di- and tri-esters of sucrose with food fatty acids, prepared from sucrose and methyl and ethyl esters of food fatty acids by esterification in the presence of a catalyst or by extraction from sucroglycerides. Only the following solvents may be used for the production: dimethylformamide, dimethyl sulfoxide, ethyl acetate, isopropanol, propylene glycol, isobutanol and methyl ethyl ketone. | ||||||
| | ||||||
Assay | Not less than 80% of sucrose esters | ||||||
| | ||||||
DESCRIPTION | Stiff gels, soft solids or white to slightly greyish white powders | ||||||
| | ||||||
FUNCTIONAL USES | Emulsifier | ||||||
| | ||||||
CHARACTERISTICS | | ||||||
| | ||||||
IDENTIFICATION | | ||||||
| | ||||||
Solubility (FNP 5) | Sparingly soluble in water, soluble in ethanol | ||||||
| | ||||||
Fatty acids | Add 1 ml of ethanol to 0.1 g of the sample, dissolve by warming, add 5 ml of dilute sulfuric acid TS, heat in a water bath for 30 min and cool. A yellowish white solid or oil is formed, which has no odour of isobutyric acid, and which dissolves when 3 ml of diethyl ether are added. Use the aqueous layer separated from the diethyl ether in the Test for sugars. | ||||||
| | ||||||
Sugars | To 2 ml of the aqueous layer separated from the diethyl ether in the test for fatty acids, carefully add 1 ml of anthrone TS down the inside of a test tube; the boundary surface of the two layers turns blue or green. | ||||||
| | ||||||
PURITY | | ||||||
| | ||||||
Sulfated ash (FNP 5) | Not more than 2% | ||||||
| | ||||||
Acid value (FNP 5) | Not more than 6 | ||||||
| | ||||||
Free sucrose | Not more than 5% | ||||||
| | ||||||
Dimethylformamide | Not more than 1 mg/kg | ||||||
| | ||||||
Dimethyl sulfoxide | Not more than 2 mg/kg | ||||||
| | ||||||
Ethyl acetate, isopropanol and propylene glycol | Not more than 350 mg/kg, singly or in combination | ||||||
| | ||||||
Isobutanol | Not more than 10 mg/kg | ||||||
| | ||||||
Methanol | Not more than 10 mg/kg | ||||||
| | ||||||
Methyl ethyl ketone | Not more than 10 mg/kg | ||||||
| | ||||||
Lead (FNP 5) | Not more than 2 mg/kg | ||||||
| | ||||||
TESTS | | ||||||
| | ||||||
PURITY TESTS | | ||||||
| | ||||||
Free sucrose | Determine by gas liquid chromatography (FNP 5). Standard solutions Internal standard solution Chromatography conditions | ||||||
| Column: | 2% Dexsil 300GC on Uniport HP 80/100 mesh (slightly polar, 2.1 m x 3.2 mmi.d.) or equivalent | |||||
| Carrier gas: | Nitrogen | |||||
| Flow rate: | 40 ml/min | |||||
| Detector: | FID | |||||
| Temperatures: | - injection: | 280° | ||||
| | - column: | Hold for 1 min at 160°, then 160-300° at15°/min, hold for 60 min at 300° | ||||
| | - detector: | 320° | ||||
| The retention times of free sucrose and octacosane measured under the above conditions are approx. 8.2 and 9.8 min, respectively. Procedure Standard curve Measure the peak areas for sucrose and internal standard. Calculate the ratio of their peak areas, and obtain the amount of sucrose in sample from the standard curve. Calculate the percentage of free sucrose from: | ||||||
| | ||||||
Dimethylformamide | Determine by gas liquid chromatography (FNP 5). Standard solutions Chromatography conditions | ||||||
| Column: | Polyethylene glycol (30 m x 0.32 mmi.d. with a 0.5 µm film) | |||||
| Carrier gas: | Helium | |||||
| Pressure: | 150 kPa (constant pressure) | |||||
| Detector: | Nitrogen phosphorus detector (NPD) (synonym: Flame thermoionic detector (FTD)) | |||||
| Temperatures: | - injection: | 180° | ||||
| | - column: | Hold for 2 min at 40°, then 40-160° at 20°/min, hold for 2 min at 160° | ||||
| | - detector: | 325° | ||||
| Injection method: | Splitless injection of 1.0 µl with auto-injector, followed by start of purge after 1.0 min. | |||||
| | ||||||
| The retention time of dimethylformamide measured under the above conditions is approx. 6.4 min. Procedure Standard curve Calculate the concentration CDFA of dimethylformamide from: CDFA (mg/kg) = [C (µg/ml) x 20 (ml)]/W (g) where C= dimethylformamide concentration detected (µg/ml) Note: The nitrogen phosphorus detector is insensitive to components that do not contain nitrogen or phosphorus. As a consequence, the capillary column can become obstructed with compounds of low volatility, although the baseline of the chromatogram is stable. Accordingly, the column must be reconditioned frequently. Overnight reconditioning (flow carrier gas in the reverse direction at 180°) is required after about every 15 samples. | ||||||
| | ||||||
Dimethyl sulfoxide | Determine by gas liquid chromatography (FNP 5). Standard solutions Chromatography conditions | ||||||
| Column: | 10% PEG 20M and 3% KOH on Gas Chrom Z (2 m x 3 mmi.d.) or equivalent. Raise the oven temperature to 180° at a rate of10º/min and let stabilize for 24 to 48 h with 30 to 40 ml/min of nitrogen for conditioning | |||||
| Carrier gas: | Nitrogen | |||||
| Flow rate: | 50 ml/min | |||||
| Detector: | Flame photometric detector (using 394 nm sulfur filter) | |||||
| Temperatures | - injection: | 210° | ||||
| | - column: | 160° | ||||
| | ||||||
| The retention time of dimethyl sulfoxide measured under the above conditions is approx. 3.4 min. Procedure Standard curve Calculate the concentration CDMSO of dimethyl sulfoxide in mg/kg from: CDMSO (mg/kg) = [C (µg/ml) x 25 (ml)]/W (g) where C = dimethyl sulfoxide concentration determined (µg/ml) | ||||||
| | ||||||
Propylene glycol | Determine by gas liquid chromatography (FNP 5). Internal standard solution Standard solution Chromatography conditions | ||||||
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| Column: | Polydimethylsiloxane (30 m x 0.32 mmi.d. with 0.25 µm film) | |||||
| Carrier gas: | Helium | |||||
| Flow rate: | 1.5 ml/min (Constant flow) | |||||
| Detector: | FID | |||||
| Temperatures | - injection: | 230° | ||||
| | - column: | Hold for 5 min at 60°, then 60-250° at 20°/min, hold for 5 min at 250° | ||||
| | - detector: | 250° | ||||
| The retention times of ethylene glycol and propylene glycol derivatives are approx. 7.7 min and 7.9 min, respectively. | ||||||
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| Procedure Calculate the concentration CPG of propylene glycol in mg/kg from: where CPG (mg/kg) = [C (µg/ml) x 10 (ml)]/W (g) Note: It will be necessary to clean the injection port and to recondition column at300° after about every 20 samples, because of contamination of the column. | ||||||
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Methanol, isopropanol, isobutanol, ethyl acetate and methyl ethyl ketone | Determined by gas chromatography with a head space sampler. Standard solutions Procedure | ||||||
| Column: | 100% Polydimethylsiloxane (30 m x 0.53 mmi.d. with 1.5 µm film, for example DB-1 manufactured by J&W Co. Ltd.) | |||||
| Carrier gas: | Nitrogen | |||||
| Flow rate: | 3.5 ml/min | |||||
| Detector: | FID | |||||
| Temperatures | - injection: | 110° | ||||
| | - column: | 40° | ||||
| | - detector: | 110° | ||||
| Head space sampler: | | |||||
| | - sample heat insulating temperature: | 80° | ||||
| | - sample heat insulating period: | 40 min | ||||
| | - syringe temperature: | 85° | ||||
| | - sample gas injection: | 1.0 ml | ||||
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| Calculation
where
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METHOD OF ASSAY | Determine by HPLC using the following conditions: Procedure Chromatography conditions | ||||||
| Column: | Styrene-divinylbenzene copolymer for gel permeation chromatography (TSK-GEL G2000 (Tosoh) or equivalent) | |||||
| Mobile phase: | HPLC-grade degassed tetrahydrofuran | |||||
| Flow rate: | 0.7 ml/min | |||||
| Detector: | RI | |||||
| Temperatures: | - Column: | 38° | ||||
| | - Detector: | 38° | ||||
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| Record the chromatogram for about 90 min. Calculate the percentage of sucrose ester content in the sample from: % sucrose ester = 100 A/T where A = the sum of peak areas for the three main components, the mono-, di- and tri-esters, eluting at about 65, 68 and 73 min, respectively T = he sum of all peak areas eluting within 90 min |