SOP: Determination of Extraneous Material in Soy Sauce and Fermented Soybean Sauces
GIFSQ — Global Institute for Food Safety & Quality
1. Principle
Light filth — insects, insect fragments, mites, hairs, feather barbules and other extraneous material — is separated from soy sauce, oyster flavoured sauce and fermented soybean sauces by sieving, digestion and oil or heptane flotation in a Wildman trap flask, filtered onto ruled filter paper, and counted under a stereoscopic microscope. Heavy filth is isolated from all three sauces by sedimentation and decantation, filtered onto preweighed ashless filter paper, dried and weighed by difference, with ashing when the residue is appreciable. The method applies to the sampling and examination of soy sauce and fermented soybean sauces for all extraneous material to determine compliance with Sections 4, 5 and 7 of the Food and Drugs Act.
2. Apparatus required
- Balance
- 1 L beakers
- Smooth magnetic stirring bar, Teflon coated (1 × 5 cm)
- Stirrer hot plate
- U.S. standard No. 230 sieve
- Wildman 2 L trap flask
- Rubber policeman attached to a glass rod
- Wash bottles
- Suction filtration apparatus with Buchner or Hirsch funnel (5–7 cm perforated plate)
- Ruled filter paper, larger than the funnel plate (7–9 cm)
- Petri dishes to suit the size of filter paper used
- Stereoscopic microscope (30–70×)
- Ashless filter paper, larger than the funnel plate (7–9 cm)
- Drying oven (40–60°C)
- Crucible
- Desiccator
- Muffle oven
3. Reagents
- Isopropanol, 99% and 40%
- 99% isopropanol — use as supplied.
- 40% isopropanol — to prepare: dilute 99% isopropanol with distilled water to 40% (v/v) (for example, 40 mL of 99% isopropanol made up to 100 mL).
- Alcohol, 95% and 60%
- 95% alcohol — use as supplied.
- 60% alcohol — to prepare: dilute 95% ethanol with distilled water to 60% (v/v) (for example, 63 mL of 95% ethanol made up to 100 mL).
- Heptane
- Tween 80–40% isopropanol solution — to 40 mL of polysorbate 80, add 210 mL of 40% isopropanol, and mix
- Tetrasodium EDTA isopropanol solution — dissolve 5 g of Na₄EDTA in 100 mL of H₂O, add 150 mL of 99% isopropanol, and mix
- Tween 80–40% isopropanol and tetrasodium EDTA isopropanol solution, 1:1 (v/v) — mix just before use
- To prepare: mix equal volumes of the Tween 80–40% isopropanol solution and the tetrasodium EDTA isopropanol solution just before use.
- Flotation oil — 85 mL of paraffin oil, white, light, 125/135 Saybolt universal viscosity, plus 15 mL of heptane
- Glycerol–95% alcohol mixture (1:1)
- To prepare: mix equal volumes of glycerol and 95% alcohol (for example, 50 mL of each).
- 15% hot (45–55°C) NaCl solution
- To prepare: dissolve 15 g of NaCl in distilled water and make up to 100 mL; heat to 45–55°C before use.
4. Sample preparation
A lot is that amount of the food — by volume, weight or otherwise — produced, stored and/or shipped under conditions as nearly uniform as possible, preferably designated by a common container code or marking, and in any case consisting of not more than one variety, grade or type of product from a single identifiable source.
- Scrutinize the entire lot for any evidence of live infestation. Do not sample until after fumigation or other effective treatment if live infestation is found.
- Obtain three sample units for each of the light and heavy filth analyses, selected at random from the lot, using appropriate sampling equipment and containers. Each sample unit for light filth analysis is a whole container. Sample units for both light and heavy filth are at least 100 g each. Three sample units constitute a sample.
- Keep each sample unit separate and label it 1, 2 or 3. Record the lot size, weight of individual containers, country of origin, exporter, importer or domestic manufacturer, and product and lot identification, to accompany the sample.
5. Procedure
Light filth — soy sauce
Take the entire contents of the container as the analytical unit for light filth
↓
Wash the entire contents of the bottle through the No. 230 sieve with a forceful stream of hot (45–55°C) tap water
↓
Rinse the inside of the bottle or container with water and shake to dislodge any remaining particles; pour the contents onto the No. 230 sieve; repeat the bottle rinsing at least three more times; wash the sieve contents until the wash water is clear
↓
Quantitatively transfer the contents of the sieve to a beaker and filter through ruled filter paper
↓
Repeat the isolation for the remaining two analytical units
Light filth — oyster flavored sauce
Take the entire contents of the container as the analytical unit for light filth
↓
Transfer the entire contents of the bottle or container to a No. 230 sieve; wash the contents with a forceful stream of hot (45–55°C) tap water until the wash water is clear
↓
Quantitatively transfer the sieve contents to the 2 L trap flask with water; dilute to 800 mL with water
↓
Add the magnetic stirring bar and 50 mL of heptane
↓
Slowly bring the stirrer to a maximum speed that does not produce audible or visible splashing or a permanent emulsion — all of the material should be in suspension and a portion of the stirring bar should be visible at the bottom of the vortex; stir for 5 min
↓
Slowly add deaerated water down the trap rod, keeping the top of the stopper just above the heptane layer, until the bottom of the heptane layer is about 1 cm above the top of the fully raised stopper
↓
Swirl the stopper through the lower portion of the trap flask to suspend the settlings
↓
Let the mixture stand for 30 min, with intermittent stirring of the bottom layer during the first 20 min
↓
Spin the rod slowly to remove sediment that may be on top of the stopper while gently raising the rod to its highest position; at that position, rinse the rod and the neck of the flask with water; hold the stopper in place and trap off by pouring the liquid into a beaker
↓
Rinse the material on the rod and in the neck of the flask with water and add it to the beaker
↓
Lower the rod gently without losing any liquid — sudden splashing can occur, especially when the stopper sticks in the neck of the flask
↓
Filter the contents of the beaker onto ruled filter paper with the suction filtration apparatus
↓
Add 35 mL of heptane to the trap flask; hand stir with a vigorous rotary motion for 1 min
↓
Let the mixture stand for 30 min, with intermittent stirring of the bottom layer during the first 20 min
↓
Trap off into a beaker, rinsing the neck of the flask with water followed by 95% alcohol
↓
Filter the contents quantitatively onto a second ruled filter paper
↓
Repeat the isolation for the remaining analytical units
↓
Transfer the filter paper or papers to petri dishes and moisten with the glycerol–ethanol mixture
Light filth — fermented soybean sauces
Take the entire contents of the container as the analytical unit for light filth
↓
Empty the entire bottle or container into a beaker of appropriate size; add 1 volume of water and 1/2 volume (of the original sample volume) of 99% isopropanol, and add a magnetic stirring bar
↓
Place the beaker on the stirrer hot plate and boil for 10 min with continuous magnetic stirring
↓
Wet sieve the contents of the beaker until the wash water is clear
↓
Transfer the contents of the wet sieve to the 2 L trap flask with 60% alcohol; rinse the sides of the flask with 60% alcohol and bring the volume to 800 mL with 60% alcohol
↓
Add 100 mL of the 1:1 Tween 80–40% isopropanol and tetrasodium EDTA isopropanol solution slowly down the trap rod, holding the top of the stopper just below the top of the 60% alcohol level; hand stir gently for 1 min
↓
Let stand for 1 min
↓
Add 50 mL of flotation oil slowly down the trap rod, holding the top of the stopper just above the top of the 60% alcohol level
↓
Slowly bring the stirrer to a maximum speed that does not produce audible or visible splashing or a permanent emulsion — all of the material should be in suspension and a portion of the stirring bar should be visible at the bottom of the vortex; stir for 5 min
↓
Slowly add deaerated 60% alcohol down the trap rod, keeping the top of the stopper just above the oil layer; when the stopper is almost fully raised, continue adding alcohol until the oil layer passes the stopper and the bottom of the oil layer is about 1 cm above the top of the fully raised stopper
↓
Swirl the stopper through the lower portion of the trap flask so as to suspend the settlings
↓
Let the mixture stand for 30 min, with intermittent stirring of the bottom layer during the first 20 min
↓
Continue as for the oyster flavoured sauce isolation, from the trap-off step through the glycerol–ethanol moistening — at the second extraction, add 35 mL of flotation oil instead of the heptane
Examination of the light-filth papers
Examine each filter paper with the stereoscopic microscope at 30× magnification for all types of extraneous material; use a higher magnification (70×) only to confirm questionable particles
↓
Record the number and range in size of all types of light filth extraneous material elements detected, for each analytical unit separately
Heavy filth — all sauces
Shake the container to distribute the contents evenly; weigh a 100 g analytical unit for heavy filth, and repeat for the remaining two sample units
↓
Transfer the analytical unit into a 1 L beaker; add 200 mL of water and hand stir vigorously with the rubber policeman for 30 s; let the suspension stand undisturbed for 5 min
↓
Decant the suspended food material without disturbing the heavy filth at the bottom
↓
Add 200 mL of water and repeat the stirring; let settle for 5 min and decant as before
↓
Add 200 mL of hot (45–55°C) 15% NaCl; let stand for 5 min, then decant without disturbing the bottom layer
↓
Repeat the hot 15% NaCl step once more
↓
Remove the NaCl and remaining plant tissue by washing the heavy material several times in water
↓
Filter the contents of the beaker onto an oven-dried, preweighed ashless paper; transfer the filter paper to a petri dish
↓
Examine the filter paper at 30× magnification with the stereoscopic microscope for rodent pellets and insect excreta
↓
Dry the filter paper in the oven at 40–60°C and weigh it; determine the weight of heavy filth by difference
↓
If there is an appreciable amount of residue, transfer the filter paper with its contents to a preweighed crucible and ash in the muffle oven at about 500°C for 2 h; cool the material in the desiccator and weigh it; determine the ashed weight by difference
↓
Repeat the isolation for the remaining analytical units
↓
Record the amount (mg) of heavy filth found
6. Calculation
Heavy filth (mg) = W₂ − W₁
Ashed weight (mg) = W₄ − W₃
- Where, W₁ = oven-dried, preweighed ashless filter paper; W₂ = the same paper plus the dried heavy-filth residue; W₃ = preweighed crucible; W₄ = crucible plus the ashed material, cooled in the desiccator.
7. Precautions
- If appropriate, freeze the samples before analysis to minimize the possibility of a live infestation of the laboratory.
- If live infestation is found in the lot, do not sample until after fumigation or other effective treatment.
References
Health Canada, Compendium of Analytical Methods, Volume 4 — Laboratory Procedure ExFLP-10, January 2019: “Determination of Extraneous Material in Soy Sauce and Fermented Soybean Sauces.” (Replaces ExFLP-10, April 1995.)
Pietrzak, E. 2009. Guidelines for the General Cleanliness of Food — An Overview. In: Volume 1. Compendium of Analytical Methods.