Gellan Gum (E418) — Method of Analysis

1. Scope and Regulatory Basis

1.1 Purpose and scope

This document is the Method of Analysis (MOA) for gellan gum manufactured by Cinogel Biotech. It defines, in full, the analytical procedures used to demonstrate that a production lot conforms to the product specification and to the applicable monographs.

It covers the four functional (rheological and optical) methods that customers most often ask to see — gel strength of the low acyl grade, gel strength of the high acyl grade, viscosity of the high acyl grade, and transmittance of the low acyl grade — and extends them into a complete release-testing package: identification, physicochemical determinations, elemental impurities, residual solvents and microbiological examination. Each method is written to be reproducible without further guidance: principle, apparatus, reagents, instrument parameters, procedure, calculation, acceptance criteria and critical control points.

1.2 Product forms covered

Gel strength and transmittance are grade-specific because the two grades gel through different mechanisms and are tested at different concentrations. Testing an LA sample by the HA method, or the reverse, will give a meaningless number.

1.3 Regulatory and monograph basis

The specification limits referenced throughout this document are taken from three sources, cross-tabulated in Appendix B:

1.4 How to use this document

• Testing against a specification: read the method chapter, then confirm the limit in Appendix B.
• Setting up a laboratory: Chapter 4 and Appendix A list the apparatus; Chapter 3 lists every reagent and solution.
• Troubleshooting: Appendix C maps each symptom to its likely cause and corrective action.
• Comparing with an earlier issue: Appendix D records exactly what changed and why.
• Numbers in monospace are fixed parameters; changing one changes the result and must be recorded as a method revision.

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2. Definitions, Abbreviations and Grades

2.1 Key definitions

2.2 Abbreviations and symbols

2.3 Structural basis of the two grades

Gellan gum is a high molecular mass anionic polysaccharide produced by pure-culture fermentation of a carbohydrate by Sphingomonas elodea (formerly Pseudomonas elodea), recovered with propan-2-ol or ethanol, dried and milled. Its repeating tetrasaccharide unit is one rhamnose, one glucuronic acid and two glucose units, neutralised to a mixed potassium, sodium, calcium and magnesium salt; relative molecular mass ≈ 500 000.

These structural differences are why the two grades cannot share a single gel strength method.

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3. Reagents, Solutions and Reference Materials

3.1 Water and general reagents

Use distilled water or water of at least grade 3 of ISO 3696 (equivalent to the water specified in GB/T 6682-2008) unless a method states otherwise. Reagents are of analytical reagent grade; volumetric, standard and impurity solutions are prepared per GB/T 601, GB/T 602 and GB/T 603, as GB 25535-2010 A.1 requires. Water quality matters more than usual here: gel strength is ion-sensitive, and calcium and magnesium carried in with tap or hard water will raise the apparent gel strength.

3.2 Prepared solutions

% (w/v) = g per 100 mL of solution; % (w/w) = g per 100 g of mixture — the convention for all test media in this document.

3.3 Solution preparation notes

• Weighing accuracy: sample weights to 0.001 g, water weights to 0.01 g; weighing error propagates directly into the result.
• Water top-up: every gel method requires the flask to be re-weighed after heating and the evaporated water restored; omitting this inflates the gel strength.
• Sample conditioning: bring the container to room temperature and mix before weighing — gellan gum is hygroscopic and stratifies in transit.

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4. Apparatus, Instrumentation and Sampling

4.1 General laboratory apparatus

4.2 Gel strength instrumentation

Gel strength is measured on a Brookfield texture analyser or an equivalent instrument capable of driving a cylindrical probe into a gel at a constant, low speed while recording force against distance. The probe geometry is the single most important instrument variable, because the result is normalised to the probe face area.

The low acyl probe has a face area of exactly 1.00 cm², so the instrument reading in grams is already the gel strength in g/cm² and no division is required. The high acyl probe is smaller, so its reading must be divided by the area factor. This is why the two calculation steps look different but express the same quantity.

4.3 Instrument set-up and calibration

1. Daily verification. Before each series, run a check gel of known acceptance value, or verify the load cell against a certified mass. Record the outcome in the instrument log.
2. Probe fit. Confirm the probe is clean, dry and fully seated. A film of dried gel on the face changes the contact area and the rupture pattern.
3. Trigger setting. Set the trigger force low—1.0 g is typical—so that the probe begins recording at first contact with the gel surface without pre-loading it.
4. Test speed. Set the test speed to 0.1 mm/s (6 mm/min). Gellan gels are strain-rate sensitive; a faster probe reads a higher force and gives a different number.
5. Penetration distance. Fix a penetration distance sufficient to pass the rupture point—10 mm is typical for a 0.5 % low acyl gel. Record it in the instrument method file and do not change it between samples of the same series.
6. Stage levelling. Level the instrument stage. A tilted gel surface makes the probe contact on one edge and under-reads.
7. Temperature. Equilibrate the instrument and the gel to the test temperature stated in the method (20 °C for low acyl, 25 °C for high acyl) before measuring.

4.4 Sampling and sub-sampling

1. Take a representative laboratory sample from the lot in its original sealed packaging. For bulk or multi-bag lots, sample from more than one position.
2. Record lot number, grade, packaging date, and the condition of the packaging on receipt.
3. Before withdrawing a test portion, mix the container contents thoroughly by inverting and rotating. Gellan gum powder segregates by particle size during transport, and particle size correlates with hydration rate and therefore with gel strength.
4. Withdraw the test portion with a clean, dry scoop and immediately close the container. Do not return unused portions to the stock container.
5. Store the laboratory sample in a sealed container in a cool, dry place. Determine loss on drying first if the sample has been exposed to air, since moisture uptake changes the effective solids content of every subsequent weighing.
6. For microbiological testing, take the test portion aseptically with a sterile scoop into a sterile container, separately from the chemical sub-sample.

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5. Gel Strength — Low Acyl Gellan Gum

5.1 Principle

A 0.50 % (w/w) low acyl gellan solution is hydrated at 80 °C, gelled by the addition of a controlled amount of calcium chloride solution, and poured into shallow aluminium boxes. The gel is allowed to set and is then conditioned for 20 h at 20 °C. A cylindrical probe of 1.00 cm² face area is driven into the gel at 0.1 mm/s and the peak force is recorded and expressed as g/cm².

The divalent calcium ion cross-links the glucuronic acid residues of the deacylated backbone, forming the firm, brittle gel characteristic of the low acyl grade. Because the gel strength is directly controlled by the calcium dose, the volume and freshness of the calcium chloride solution are critical method parameters, not incidental details.

5.2 Instrument and test conditions

5.3 Reagents

• Distilled water, 120 g, weighed to 0.01 g
• Calcium chloride solution, 2.7 % (w/v), 2 mL, prepared fresh on the day of use
• Boiling distilled water, for restoring evaporated water

5.4 Procedure

1. Prepare the water charge. Weigh 120 g of distilled water to the nearest 0.01 g into a 250 mL Erlenmeyer flask containing a magnetic stirring bar.
2. Add the sample. Weigh 0.6 g of the mixed sample on clean, dry weighing paper to the nearest 0.001 g. Start the stirrer and add the sample slowly and evenly across the surface of the water so that it disperses without forming lumps. Weigh and record the gross weight of the flask.
3. Hydrate. Place the flask on the magnetic stirrer and heat with stirring. When the temperature reaches 80 °C, start timing and hold with stirring at 380 r/min for 10 min. The solution must be clear and free of undissolved particles before proceeding.
4. Add the calcium chloride. Add 2 mL of fresh 2.7 % (w/v) calcium chloride solution dropwise into the stirred solution, then continue stirring for a further 1 min. Add slowly: localised excess calcium produces premature gel particles that do not redissolve.
5. Restore the water. Re-weigh the flask, record the weight difference, and add boiling distilled water from a pipette to restore the contents to the original gross weight. Stir for 1 min to homogenise.
6. Cast the gel. Quickly pour the hot solution into the two clean, dry 50 × 30 mm aluminium boxes, approximately 50 g per box. Avoid drawing bubbles into the box.
7. Set. Place the boxes in an insulated turnover tray at ambient temperature for 30 min to gel.
8. Condition. Cover the boxes with their lids and transfer to a 20 °C constant temperature chamber for 20 h before measurement.
9. Measure. Place the gel, still in its box, on the texture analyser stage. Position the probe at the centre of the gel surface and measure under the conditions of 5.2.
10. Repeat. Carry out two parallel determinations and average the results.

5.5 Measurement and calculation

The instrument reading at the peak of the force–distance curve is the gel strength value. Because the probe face area is 1.00 cm², the reading is reported directly in g/cm².

Read the peak of the curve, not the value at the end of the run. For a brittle low acyl gel the force rises to a sharp maximum and then falls away as the gel fractures; the maximum is the gel strength, and the force beyond the peak describes the fracture, not the gel.

5.6 Acceptance criteria and reporting

• The gel strength limit is that declared on the product Certificate of Analysis for the grade and specification concerned. Gel strength is a functional, supplier-defined parameter and is not limited by EU E 418, JECFA or GB 25535-2010.
• Report the mean of the two parallel determinations in g/cm², together with the individual results.
• Report the test conditions with the result: probe face area, probe speed, penetration distance, calcium chloride concentration and volume, conditioning time and temperature, and test temperature.
• Record the instrument method file name or number on the worksheet so the parameter set can be traced.

5.7 Critical control points

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6. Gel Strength — High Acyl Gellan Gum

6.1 Principle

A 1.00 % (w/w) high acyl gellan solution is hydrated at 90 °C, gelled with calcium chloride solution, cast into a 70 × 35 mm aluminium box and conditioned for 20 h at 20 °C. A ø6 mm cylindrical probe is driven into the gel at 0.1 mm/s at 25 °C and the peak force, divided by the probe face area, is reported as g/cm².

High acyl gellan needs a higher temperature and a longer hydration time than the low acyl grade because the retained glyceryl and acetyl groups make the polymer more difficult to wet out and dissolve. It is also tested at a higher concentration and a higher temperature, reflecting the softer, more elastic gel it forms.

6.2 Instrument and test conditions

6.3 Reagents

• Distilled water, 120 g, weighed to 0.01 g
• Calcium chloride solution, 2.7 % (w/v), 2 mL, prepared fresh on the day of use
• Boiling distilled water, for restoring evaporated water

6.4 Procedure

1. Prepare the water charge. Weigh 120 g of distilled water to the nearest 0.01 g into a 250 mL Erlenmeyer flask containing a magnetic stirring bar.
2. Add the sample. Weigh 1.2 g of the mixed sample to the nearest 0.001 g. Start the stirrer and add the sample slowly and evenly to disperse it in the water. Weigh and record the gross weight of the flask.
3. Preheat and hydrate. Preheat the flask, then place it on the magnetic stirrer and heat with stirring at 600 r/min. When the temperature reaches 90 °C, start timing and hold for 20 min until the sample is completely dissolved. Throughout this stage no undissolved material may settle on the bottom of the flask and the solution must not overflow.
4. Add the calcium chloride. Add 2 mL of fresh 2.7 % (w/v) calcium chloride solution dropwise into the stirred solution and continue stirring for 1 min.
5. Restore the water. Re-weigh the flask, record the weight difference, and restore the contents to the original gross weight with boiling distilled water. Stir for 1 min.
6. Cast the gel. Quickly pour the hot solution into the clean, dry 70 × 35 mm aluminium box.
7. Set. Place the box in an insulated turnover tray at ambient temperature for 30 min to gel.
8. Condition. Cover the box with its lid and transfer to a 20 °C constant temperature chamber for 20 h.
9. Measure. Bring the gel to 25 °C, place it on the texture analyser stage, centre the probe, and measure under the conditions of 6.2.
10. Repeat. Carry out two parallel determinations and average the results.

6.5 Measurement and calculation

The instrument reading is divided by the probe face area, 0.2826 cm², to give the gel strength in g/cm². This normalisation is what allows the high acyl result to be compared with the low acyl result on the same basis.

6.6 Acceptance criteria and reporting

• The gel strength limit is that declared on the product Certificate of Analysis for the grade and specification concerned. As with the low acyl grade, this parameter is not limited by the food-additive monographs.
• Report the mean of the two parallel determinations in g/cm², together with the individual results, and state the probe diameter and face area factor used.
• Report the conditioning time and temperature, the test temperature and the calcium chloride charge.

6.7 Critical control points

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7. Viscosity — High Acyl Gellan Gum

7.1 Principle

High acyl gellan gum is a thickening agent as well as a gelling agent, and its solution viscosity is a release parameter in its own right. A dilute solution of the high acyl grade is prepared in a defined medium containing sodium chloride and sucrose, boiled to complete dissolution, cooled to 25 °C, and its apparent viscosity is read on a rotational viscometer using a small-sample spindle.

The sodium chloride and sucrose are not incidental. The salt screens the electrostatic repulsion between the anionic polymer chains and the sucrose raises the refractive index and modifies the solvent quality, so the medium reproduces the ionic strength and solids environment of a typical beverage or dairy application. A viscosity measured in plain water will not agree with a customer's application data.

7.2 Test medium composition

The test medium is defined by mass fraction in distilled water. The quantities below give approximately 400 g of medium, sufficient for a single determination with rinsing.

600 µL of 20 % (w/v) NaCl contains 0.12 g NaCl; 0.12 / 400 = 0.030 %. The designations 0.045 %, 0.03 % and 4 % are therefore the masses of gellan, NaCl and sucrose expressed as a percentage of the 400 g water charge, which is the basis on which the published method states them. Referred to the total mass of the prepared medium (about 416 g) the same figures become 0.043 %, 0.029 % and 3.84 %; state the basis whenever a result is compared between laboratories.

7.3 Instrument and conditions

7.4 Procedure

1. Weigh the medium. Weigh 400 g of distilled water into a 500 mL beaker.
2. Add the sample. Weigh 0.18 g of the sample to the nearest 0.001 g and disperse it in the water with stirring using a glass rod.
3. Add the salt. Add 600 µL of 20 % (w/v) sodium chloride solution.
4. Add the sucrose. Add 16 g of sucrose.
5. Dissolve. Place the beaker on a hot plate, bring to the boil and stir continuously with a glass rod until the sample is completely dissolved. The solution must be free of undissolved particles and of any gel-like specks on the walls or base of the beaker.
6. Cool. Remove from the heat and cool to room temperature, 25 °C. Do not force-cool in an ice bath; rapid cooling traps bubbles and leaves the solution un-equilibrated.
7. Load the adapter. Transfer the solution to the small-sample adapter, taking care not to introduce air. Fill to the level mark required by the spindle.
8. Equilibrate. Hold at 25 °C for 5 min until the temperature of the sample is uniform.
9. Measure. Start the spindle and allow the reading to stabilise, then record the viscosity. Read a stable value only; a drifting reading means the sample is not yet at temperature or contains bubbles.
10. Repeat. Carry out a second determination and average the results.

7.5 Calculation and reporting

With a defined spindle, speed and adapter geometry, the instrument displays viscosity directly in mPa·s (equivalent to centipoise). Report:

• The mean of the two determinations in mPa·s, and the individual results.
• Spindle model, speed and the viscometer model, since these determine the conversion factor.
• The medium composition and the test temperature, 25 °C.
• The equilibration time before reading, 5 min.

7.6 Critical control points

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8. Transmittance (Clarity) — Low Acyl Gellan Gum

8.1 Principle

A 0.50 % (w/w) low acyl gellan solution is prepared, gelled with calcium chloride, filled into a 1 × 1 cm spectrophotometer cell and allowed to set at 20 °C. The transmittance of the set gel is measured at 497 nm against distilled water as the blank.

Transmittance is the optical expression of gel clarity and is the parameter customers in beverage, confectionery and plant-tissue-culture applications look at most closely: a hazy gel is visible in the finished product. Clarity depends on the completeness of dissolution, on the amount of insoluble residue carried through from fermentation and recovery, and on the absence of entrapped air.

8.2 Instrument and conditions

8.3 Procedure

1. Prepare the water charge. Measure 120 mL of distilled water into a 250 mL Erlenmeyer flask containing a magnetic stirring bar.
2. Add the sample. Weigh 0.6 g of the sample to the nearest 0.001 g on clean, dry weighing paper. Start the stirrer and add the sample slowly and evenly to the water to disperse and dissolve it. Weigh and record the gross weight of the flask.
3. Hydrate. Place the flask on the magnetic stirrer and heat. When the temperature reaches 80 °C, start timing and stir for 30 min. The solution must be completely clear—any residual haze at this point will be read as low transmittance.
4. Add the calcium chloride. Add 2 mL of fresh 2.7 % (w/v) calcium chloride solution and continue stirring uniformly on the magnetic stirrer.
5. Restore the water. Remove the flask from the stirrer, re-weigh, and add distilled water to restore the contents to the original weight. Stir to homogenise.
6. Fill the cell. Fill the 1 × 1 cm cell with the hot solution. Take care to avoid bubbles; if bubbles form, allow the cell to stand briefly at 20 °C before the gel sets, or draw the solution through a warm pipette.
7. Set. Transfer the filled cell to the 20 °C incubator and hold until the gel has set.
8. Zero the instrument. Set 100 % transmittance with distilled water in a matched cell at 497 nm.
9. Measure. Remove the cell from the incubator, wipe the optical faces dry, and read the transmittance of the gel at 497 nm. Handle the cell by the frosted faces only.
10. Repeat. Carry out three parallel determinations and average the results.

8.4 Measurement and reporting

• Report the mean of three parallel determinations as transmittance, % T, at 497 nm.
• Report the cell path length, the wavelength, the gel concentration and the setting temperature.
• The transmittance limit is that declared on the product Certificate of Analysis. It is a functional, supplier-defined parameter and is not limited by EU E 418, JECFA or GB 25535-2010.

8.5 Critical control points

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9. Identification Tests

The identification tests below are monograph tests. They confirm the identity of the material as gellan gum by its solubility and by the two characteristic ion-triggered gel reactions. They are qualitative, and both gel tests must be passed.

9.1 Solubility

Disperse a small portion of the sample in water with stirring and warm gently; a viscous solution forms. Add a portion to ethanol; the material does not dissolve and precipitates or remains dispersed as a separate phase. Solubility in water with the characteristic viscous, slightly stringy solution is the first identity check.

9.2 Gel test with calcium ion

1. Add 1.0 g of the sample to 99 mL of water.
2. Stir for about 2 h using a motorised stirrer fitted with a propeller-type blade. The resulting solution is 1 % (w/v).
3. Draw a small amount of this solution into a wide-bore pipette.
4. Transfer it into a 10 % (w/v) calcium chloride solution.

Expected result. A tough, worm-like gel forms immediately as the solution enters the calcium chloride.

A wide-bore pipette is specified because a narrow tip shears the viscous solution and gives an irregular, stringy transfer. The immediate, tough, coherent gel strand is the positive criterion; a soft or slow-forming gel is a fail.

9.3 Gel test with sodium ion

1. To the 1 % (w/v) solution prepared in 9.2, add 0.50 g of sodium chloride.
2. Heat to 80 °C with stirring and hold at 80 °C for 1 min.
3. Allow the solution to cool to room temperature.

Expected result. A firm gel forms on cooling.

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10. Physicochemical Determinations

These methods determine the parameters that appear on every Certificate of Analysis and that are limited by one or more monographs: moisture, nitrogen, gellan content, pH, ash and particle size.

10.1 Loss on drying

1. Dry a clean weighing dish with its lid at 105 °C, cool in a desiccator and weigh to the nearest 0.001 g.
2. Weigh about 2 g of the mixed sample into the dish to the nearest 0.001 g and record the weight.
3. Place the uncovered dish in the oven at 105 °C and dry for 2.5 h.
4. Cover the dish, transfer to a desiccator and cool to room temperature.
5. Weigh and calculate the loss in mass as a percentage of the sample weight.
6. Carry out two determinations in parallel and average.

Gellan gum is hygroscopic: weigh the test portion promptly and keep the container closed between weighings. A high loss on drying result is often simply a sample that has been standing open.

10.2 Nitrogen content

1. Weigh about 0.5 g of the sample to the nearest 0.001 g into a digestion tube.
2. Add catalyst and concentrated sulfuric acid, and digest until the mixture is clear and colourless.
3. Cool, dilute, and make the digest alkaline.
4. Distil the liberated ammonia into a receiver of standard acid.
5. Titrate the excess acid and calculate the nitrogen content.
6. Run a blank determination alongside the sample and subtract it.

Gellan gum produced by fermentation can carry a small amount of nitrogen-containing material from the fermentation broth. The monograph limits nitrogen to not more than 3 % as a purity and process-control check rather than for any toxicological reason.

10.3 Gellan gum content (assay)

1. Prepare the filter bed. Weigh about 1.0 g of chromatographic-grade diatomaceous earth into a glass filter crucible and spread it evenly. Dry at 105 °C for 5 h, cool in a desiccator and weigh accurately.
2. Dry the test portion. Dry the sample at 105 °C for 2.5 h as in 10.1.
3. Weigh the test portion. Weigh about 0.2 g of the dried sample to the nearest 0.001 g.
4. Dissolve. Add 50 mL of water and stir in a water bath at about 80 °C for 30 min until completely dissolved.
5. Precipitate. Add 200 mL of anhydrous ethanol pre-heated to 60–70 °C, mix well and allow to stand for 12 h.
6. Filter and wash. Filter through the prepared crucible, then wash the residue with ethanol solution (78 + 22) five times: 20 mL for each of the first three washes and 10 mL for each of the last two.
7. Dry and weigh. Dry the residue at 105 °C for 5 h, cool in a desiccator and weigh accurately.
8. Calculate. Express the mass of the residue as a percentage of the mass of the dried test portion taken.

The test portion is dried before weighing, so the result is already on the dried basis. If the sample is weighed as received instead, correct the result for the loss on drying determined in 10.1; the two routes are equivalent provided the correction is made, and an uncorrected as-received result would under-read by the moisture content, which may be up to 15 %.

10.4 Carbon dioxide yield

The sample is treated under the conditions specified in the monograph and the carbon dioxide released is collected and determined. Because the method responds to the uronic acid content of the polymer, it is both an assay and an identity confirmation: material that is not a uronic-acid polysaccharide does not give the expected yield.

10.5 pH

1. Disperse 1.0 g of the sample in 100 g of distilled water with stirring.
2. Calibrate the pH meter with standard buffers and confirm the slope is within the manufacturer's acceptance range.
3. Bring the dispersion to 25 °C and measure the pH with the electrode immersed under gentle stirring.
4. Allow the reading to stabilise before recording. Gellan dispersions are viscous and the electrode response is slower than in a buffer.
5. Carry out two determinations and average; report to one decimal place.

The pH value of gellan gum depends on the mixed cation form of the glucuronic acid and therefore on the recovery and ion-exchange history of the lot. Because it is not fixed by the monographs, the acceptance range is agreed with the customer and stated on the product specification.

10.6 Ash content

1. Ignite a clean crucible at 550 °C, cool in a desiccator and weigh.
2. Weigh about 2 g of the sample into the crucible and record the weight.
3. Char carefully, then incinerate at 550 °C until the ash is free of carbon.
4. Cool in a desiccator and weigh. Calculate the ash as a percentage of the sample weight, on the dried basis.

Ash reflects the mixed potassium, sodium, calcium and magnesium counter-ions of the polymer and is therefore a useful lot-to-lot consistency indicator, but it is not a monograph limit for gellan gum.

10.7 Particle size / mesh

1. Dry the sample if necessary and mix thoroughly.
2. Weigh 100 g of the sample to the nearest 0.01 g onto the top sieve of a stacked certified sieve set.
3. Shake for the validated time, or run the air-jet sieve for the validated period, until the mass on each sieve is constant.
4. Weigh the fraction retained on each sieve and calculate the cumulative percentage passing the declared mesh.
5. Report the mesh size, the sieving method and the percentage passing.

Particle size is declared by mesh grade (for example 80 mesh, 100 mesh, 200 mesh) and is closely linked to dispersion behaviour: a finer powder hydrates faster and disperses less readily, and the two together determine how the customer must handle the product. Report the sieve standard used, because mesh designations differ between the ISO and US systems.

10.8 Gel point and melting point

1. Prepare a 1 % (w/w) solution of the sample at the defined ionic environment and load it onto the rheometer in the sol state.
2. Cool from above the melting temperature at 1 °C/min while measuring the storage modulus G′ at 1 Hz. Record the temperature at which G′ rises sharply as the gel point.
3. Heat the formed gel at the same rate and record the temperature at which the network breaks down as the melting point.
4. Report both temperatures and the ionic environment.

Gellan gum shows pronounced thermal hysteresis: the melting point is substantially higher than the gel point. Low acyl gels set strongly and remain set on reheating; high acyl gels are thermally reversible within a lower window. These two temperatures are useful when developing or verifying a customer application. This determination is optional and is not part of routine lot release.

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11. Impurities and Residual Solvents

11.1 Lead

1. Prepare a sample solution as directed for organic compounds in the limit test, or by validated microwave digestion.
2. Calibrate the instrument across the working range with certified lead standards, including a matrix-matched blank.
3. Verify calibration with an independent check standard and, where available, a certified reference material.
4. Determine the lead content and express the result in mg/kg of the sample as received.

Lead is the single elemental impurity limited by all three of the monographs applied by this document, which makes it the most frequently requested elemental result on a gellan gum Certificate of Analysis. Report the method used, since AAS and ICP-MS can differ slightly at low levels.

11.2 Arsenic, mercury and cadmium

1. Digest the sample by a validated closed-vessel microwave procedure.
2. Determine arsenic, mercury and cadmium by the validated instrumental method, each with its own calibration line and check standard.
3. Run a reagent blank with every batch and subtract it.
4. Report each element separately in mg/kg. Do not report a single combined total.

11.3 Heavy metals (as Pb)

Where a customer specification still calls for "heavy metals, as Pb", carry out the monograph limit test: treat the sample under the prescribed conditions, compare the colour produced with that of a standard lead solution, and report pass or fail against the limit. Because the current EU monograph and GB 25535-2010 control the individual elements, this test is reported only when a specification requires it.

11.4 Residual isopropanol

Isopropanol is the recovery solvent used to precipitate the polysaccharide from the fermentation broth, so its residue is a direct measure of how completely the product was washed and dried. Both JECFA and GB 25535-2010 (Appendix B) give the same procedure: the sample is dispersed in water containing an antifoam emulsion, shaken, distilled, and the distillate examined by gas chromatography against isopropanol and tert-butyl alcohol standard solutions, tert-butyl alcohol serving as the internal standard.

1. Prepare the flask. Disperse 1 mL of a suitable antifoam emulsion, such as Dow Corning G-10 or equivalent, in 200 mL of water in a 1000 mL round-bottom distilling flask.
2. Add the sample. Add about 5 g of the sample, accurately weighed, and shake the flask for 1 h.
3. Distil. Connect a fractionating column and collect about 100 mL of distillate, adjusting the heat so that foam does not enter the column.
4. Add the internal standard. Add 4.0 mL of tert-butyl alcohol standard solution to the distillate.
5. Chromatograph. Inject 5 µL of the mixed standard solution and, separately, of the sample preparation, under the conditions below.
6. Quantify. Calculate the relative response factor from the standard (f = A_IPA / A_TBA), apply it to the sample peak areas and report the isopropanol content in mg/kg.

11.5 Residual ethanol

The current EU monograph permits recovery with either propan-2-ol or ethanol but limits only propan-2-ol; the JECFA compendium adds an ethanol limit of 50 mg/kg. Where the lot was recovered with ethanol, determine and report residual ethanol by headspace gas chromatography in the same way as isopropanol, with an ethanol calibration and a suitable internal standard.

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12. Microbiological Examination

Gellan gum is a non-sterile food additive. The microbiological criteria are hygiene criteria: they demonstrate that the product was manufactured, dried and packed under adequate process control.

12.1 Sample preparation and neutralisation

1. Take the test portion aseptically and weigh 25 g into a sterile container.
2. Add 225 mL of sterile diluent and blend or stomache to give a homogeneous 1:10 primary suspension.
3. Allow the suspension to hydrate and settle. Gellan gum forms a viscous, gelling suspension that can clog pipettes and mask colonies; use wide-bore pipettes and prepare serial dilutions promptly.
4. Where the suspension inhibits growth, add a validated neutralising agent such as lecithin with polysorbate 80, or use membrane filtration, and document the neutralisation in the method record.
5. Plate or inoculate the dilutions within the validated holding time.

12.2 Total (aerobic) plate count

Inoculate plate count agar with the prepared dilutions, incubate aerobically and count the colonies. Under GB 4789.2 the plates are incubated at 36 °C ± 1 °C for 48 h ± 2 h; under ISO 4833-1 the temperature is 30 °C for 72 h. Report in CFU/g. Select the dilution giving a countable number of colonies and, where two dilutions are countable, apply the calculation rules of the standard.

12.3 Yeasts and moulds

Inoculate the appropriate selective agar and count yeasts and moulds separately where the method requires it. Under GB 4789.15 the plates are incubated at 28 °C ± 1 °C and read at 5 days; under ISO 21527-1/-2 the temperature is 25 °C. Examine the plates daily—moulds grow more slowly than bacteria and a plate read before day 5 will under-count. Report the total in CFU/g.

12.4 Escherichia coli

Carry out the enrichment and selective isolation steps of the chosen standard on a 5 g test portion. Confirm any presumptive colonies biochemically or by an equivalent validated method. Report as negative or positive per 5 g.

12.5 Salmonella spp.

1. Pre-enrich a 25 g test portion in buffered peptone water.
2. Transfer to selective enrichment broths and incubate as specified.
3. Sub-culture onto selective agars and examine for typical colonies.
4. Confirm presumptive colonies biochemically, and serologically if the standard requires it.
5. Report as negative per 25 g, which also satisfies the EU requirement of negative in 10 g.

12.6 Coliforms

Determine coliforms by the most probable number technique on the prepared dilutions and report in MPN/100 g. Coliforms are a GB parameter; where a lot is destined for export only, confirm whether the customer specification requires it before reporting.

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13. Data Handling, Acceptance Criteria and Reporting

13.1 Replicates and averaging

Each parallel determination must be an independent preparation from the sub-sample: separately weighed, separately hydrated, separately cast and separately conditioned. Re-reading the same gel twice is not a parallel determination and does not provide any information about method variability.

13.2 Precision and repeatability limits

13.3 Rounding and significant figures

• Gel strength: report to the nearest 1 g/cm².
• Viscosity: report to the nearest 1 mPa·s below 100, and to the nearest 5 mPa·s above.
• Transmittance: report to 0.1 % T.
• Loss on drying, nitrogen, ash and gellan content: report to 0.1 %.
• pH: report to 0.1 pH units.
• Elemental impurities: report in mg/kg to the same number of significant figures as the specification limit.

Round only the final reported value. Do not round intermediate values before averaging—rounding each parallel result and then averaging introduces a bias that is visible when results sit close to a specification limit.

13.4 Out-of-specification handling

1. Confirm the result is real. Check the raw data: weighing records, temperature logs, instrument calibration status, reagent preparation dates and the flask gross weights.
2. Check the obvious laboratory causes first. Work through Appendix C for the parameter concerned. Calcium chloride solution age, water top-up, incomplete hydration and incorrect probe geometry account for the majority of anomalous gel strength results.
3. Repeat on a fresh sub-sample. If a laboratory cause is identified and documented, repeat the determination from a new test portion and record both the original and the repeat, with the reason for repeating.
4. If the result stands. Quarantine the lot, raise an OOS record, and route it through investigation, retest and final disposition procedures. Do not average an out-of-specification result with a compliant one to produce a passing mean.
5. Customer notification. Where a lot has already been shipped and an OOS result is confirmed, notify the customer with the data and the disposition.

13.5 Certificate of Analysis

The Certificate of Analysis reports, for each parameter: the test item, the specification limit, the method reference, and the result. For the method-dependent functional parameters, the Certificate also states the conditions, because the number has no meaning without them. A complete gel strength entry therefore reads as follows.

A customer receiving a Certificate on which the method conditions are stated can reproduce the result. A Certificate that lists only "Gel Strength: 680" invites a dispute that neither party can resolve.

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14. Method Verification and Transfer

14.1 Verification parameters

Not every parameter of a full validation needs to be repeated for these methods. The following verification set is sufficient to demonstrate that a laboratory can perform the method as written and obtain acceptable results.

Robustness testing is what justifies the critical control points: it is how the laboratory establishes, empirically, that calcium chloride freshness and the water top-up step matter and that a small variation in the ambient setting stage does not.

14.2 Verification frequency

• On adoption: the full verification set in 14.1 before the method is used for release testing.
• Annual review: re-examine the precision data accumulated over the year against the limits in 13.2, and update those limits where the data support it.
• On change: re-verify whenever a critical instrument, probe, balance, incubator or reagent supplier is changed, or whenever any fixed parameter in a method is modified.
• Ongoing: include a control sample of known value in each batch of routine determinations and chart the results.

14.3 Method transfer to customer laboratories

Gel strength and viscosity numbers are only comparable when the conditions are identical. When a customer laboratory will verify incoming lots, agree the following in writing before the first shipment:

• The exact probe geometry and face area, and whether the area factor is applied.
• Probe speed, trigger force and penetration distance.
• Test temperature, and the conditioning time and temperature of the gel.
• The calcium chloride concentration, the volume added, and the requirement that it be prepared fresh.
• For viscosity: the viscometer model, the spindle, the speed and the small-sample adapter geometry.
• For transmittance: the wavelength, the cell path length and the blank convention.
• The replicate count and the acceptance limits.

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A. Apparatus, Instrument and Reagent Checklist

Use this list when setting up or auditing a laboratory to run the methods in this document.

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B. Specification Limits Cross-Reference

Limits for gellan gum as set by the three principal authorities applied to this product, with the method that determines each one. Where authorities differ, the stricter limit is the default under this MOA.

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C. Troubleshooting Guide

Symptoms observed in the functional and physicochemical methods, with the likely cause and the corrective action. Work through the laboratory causes before concluding that a lot is genuinely out of specification.

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D. Revision Notes — Changes from the Published Version

This issue supersedes the methods previously published on the company website. The changes are listed here so that a customer or auditor comparing the two documents can see exactly what was altered and why. No test principle was changed; the substance of the change is that missing parameters have been supplied, ambiguous wording has been resolved and the four published methods now sit inside a complete release-testing package.

D.1 Corrections to the four published methods

D.2 Additions — scope extended to a complete release package

The published version covered four methods only. A Certificate of Analysis cannot be supported by four methods, so this issue adds the following, each written in the same format as the four functional methods.

D.3 Open items for the next issue

• Viscosity spindle speed. Confirm the S62 setting as r/min rather than revolutions and align the viscometer method file.
• Probe area factor. Standardise on the theoretical 0.2827 cm² or the in-house 0.2826 cm², and state the choice on the Certificate of Analysis.
• Declared limits. Insert the grade-specific limits for gel strength, viscosity, transmittance, pH, ash and particle size into the specification table.
• Penetration distance and trigger force. Record the validated values for each gel type in the texture analyser method file and reproduce them in the next issue.
• Neutralisation for microbiology. Validate the neutralisation step for the viscous suspension and record the diluent.

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E. References

1. Commission Regulation (EU) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 — entry E 418 Gellan Gum.
2. JECFA (FAO/WHO). Gellan Gum, INS No. 418. Prepared at the 49th JECFA (1997), published in FNP 52 Addendum 5, superseding the specifications prepared at the 46th JECFA (1996); compendium edition 2014.
3. Food Chemicals Codex (FCC). Gellan Gum monograph.
4. GB 25535-2010. National Food Safety Standard of the People's Republic of China — Food Additive: Gellan Gum.
5. GB 5009.3. National Food Safety Standard — Determination of moisture in foods (direct drying method).
6. GB/T 6682-2008. Water for analytical laboratory use — Specification and test methods; GB/T 601, GB/T 602 and GB/T 603 — Preparation of standard volumetric solutions, standard solutions for impurity determination, and preparations and products for chemical analysis.
7. GB 5009.11, GB 5009.12, GB 5009.15, GB 5009.17. National Food Safety Standards — Determination of arsenic, lead, cadmium and mercury in foods.
8. GB 4789.2, GB 4789.3, GB 4789.4, GB 4789.15. National Food Safety Standards — Microbiological examination of foods: aerobic plate count, coliforms, Salmonella, yeasts and moulds.
9. ISO 4833-1. Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — Colony count at 30 °C.
10. ISO 21527-1 / ISO 21527-2. Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and moulds.
11. ISO 6579-1. Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella.
12. ISO 7251 and ISO 16649. Microbiology of food and animal feeding stuffs — Enumeration of presumptive Escherichia coli.
13. ISO 3310-1. Test sieves — Technical requirements and testing — Test sieves of metal wire cloth.
14. ISO 3696. Water for analytical laboratory use — Specification and test methods.
15. 21 CFR 172.665. Gellan gum. United States Code of Federal Regulations, food additives permitted for direct addition to food for human consumption.
16. EFSA Panel on Food Additives and Nutrient Sources added to Food. Re-evaluation of gellan gum (E 418) as a food additive. EFSA Journal 2018;16(6):5296.
17. Cinogel Biotech. Product specification sheet for gellan gum (E 418), low acyl and high acyl grades. www.cinogel.com.

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