SOP: Determination of Extraneous Material in Dried, Frozen and Canned Peas, Beans and Lentils

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Dried peas, beans and lentils are examined macroscopically for insect damage and for mould, by hand and with lenses and microscopes. Frozen and canned peas, beans and lentils are examined microscopically for light filth, after the seeds are worked through sieves and the light filth is recovered by heptane flotation into a trap flask and filtered onto ruled paper. Findings are classified as insect-damaged or mouldy defectives and reported as percent defectives per analytical unit, to judge compliance with Sections 4, 5 and 7 of the Food and Drugs Act.

The method covers the dried, frozen and canned products of the family Leguminosae, including but not limited to:

• Beans of the genus Phaseolus, such as French, string, snap or kidney beans (Phaseolus vulgaris L.), the scarlet runner bean (P. coccineus L.) and the lima or butter bean (P. lunatus L.). White seeded types of P. lunatus are usually grown because coloured seeds often have a relatively high cyanogenic glycoside level which can be dangerous.
• Beans of the genus Vigna such as mung beans (Vigna radiata (L.) Wilszek), from which bean sprouts are produced; the adzuki bean (Vigna angularis (Willd.) Ohwi and Ohashi), also called the aduki or azuki bean, similar but with larger seeds than the mung bean, red, cream, lilac or mottled in colour; and the large podded faba or broad bean (Vicia faba L.), also called the lupini bean.
• Soybeans and soya seeds (Glycine max (L.) Merr.)
• The true pea (Pisum sativum L.)
• Cowpeas, black-eyed peas, crowder peas, field peas and southern peas (Vigna unguiculata (L.) Walp. subsp. unguiculata)
• Yard-long beans (Vigna unguiculata subsp. sesquipedalis (L.) Verdc.)
• Pigeon peas (Cajanus cajan (L.) Millsp.)
• Chick peas, garbanzos or bengal grams (Cicer arietinum L.), rounded seeds with a characteristic beak resembling the head of a baby chicken
• Lentils (Lens culinaris Medik.)

2. Apparatus required

• Light-coloured tray
• Magnifying lens (3–5×)
• Stereoscopic microscope (10–30×)
• Absorbent paper
• Scalpel
• Forceps (No. 5 or No. 7 jeweller’s forceps or equivalent)
• Spot plate
• Compound microscope (100×)
• U.S. standard sieves No. 8 and No. 40
• Shallow pan (> 20 cm in diameter)
• 2 L beaker
• Meat or food chopper
• Trap flask or heavy-walled side-arm flask (2 L)
• Ruled filter paper (paper should be larger than the funnel)
• Suction filtration apparatus with Hirsch funnel
• Petri dishes suitably sized to fit the ruled filter paper

(The source’s materials list prints the magnifying lens (3–5×) twice; it is carried once here.)

3. Reagents

• Alcohol (70%), either ethanol or isopropanol
• To prepare: dilute 95% ethanol or 99% isopropanol with distilled water to 70% (v/v) (for example, 74 mL of 95% ethanol, or 71 mL of 99% isopropanol, made up to 100 mL).
• Isopropanol (100%)
• Glycerol–alcohol mixture (1:1)
  (The source’s materials list prints this mixture twice; it is carried once here.)
• Heptane

4. Sample collection

• A lot is the amount of the food (volume, weight, etc.) produced, stored and/or shipped under conditions as nearly uniform as possible, preferably designated by a common container code or marking, and in any event consisting of not more than one variety, grade or type of product from a single identifiable source.
• Scrutinize the entire lot for live infestation. If live infestation is found, do not sample until after fumigation or other effective treatment.
• For canned products: obtain three sample units selected at random from the lot; each sample unit should consist of whole cans to a minimum weight of 500 g. Three sample units constitute a sample.
• For frozen products: obtain three sample units selected at random from the lot, of at least 500 g each, using appropriate sampling equipment and containers. Three sample units constitute a sample.
• For dried products: obtain eight sample units selected at random from the lot, of at least 50 g each, using appropriate sampling equipment and containers. Eight sample units constitute a sample.
• Keep each sample unit separate and label them 1, 2, 3, etc. Record complete information on the lot size, the weight of individual containers, the country of origin, the exporter, the importer or domestic manufacturer, and product and lot identification; this information accompanies the sample.

5. Procedure

5.1 Macroscopic examination of dried peas, beans and lentils — insect damage

If appropriate, freeze the samples prior to analysis to minimize the possibility of a live infestation of the laboratory

↓

Prepare the analytical units: thoroughly mix a sample unit and take 100 whole peas, beans or lentils. A broken seed exceeding one half a seed counts as a whole seed; a broken piece smaller than half a seed is replaced by a whole seed. These 100 seeds constitute an analytical unit — repeat for the remaining seven sample units

↓

Transfer each seed of an analytical unit to a light-coloured tray and examine for insect damage with a magnifying lens (3–5×). Use the stereoscopic microscope only to confirm questionable material

↓

Classify as insect damaged any pea, bean or lentil containing an insect or insects, or showing evidence of tunnelling, frass or insect damage > 2 mm (longest dimension)

↓

Calculate and record the number of insect-damaged defectives for each analytical unit separately

What insect damage looks like

• In dried peas, beans or lentils, weevil egg punctures are very small. Weevil exit holes are approximately 1 mm in diameter and show little or no discoloration. Lygus sp. or aphid damage may be seen on the exterior surface but may be more extensive in the interior areas.
• Five major insect species attack dried peas, beans and lentils: the bean weevil (Acanthoscelides obtectus (Say)), the cowpea weevil (Callosobruchus maculatus (Fabricius)), the Indianmeal moth (Plodia interpunctella (Hübner)), the tobacco moth (Ephestia elutella (Hübner)) and the almond moth (Cadra cautella (Walker)).
• The bean weevil and the cowpea weevil are the most destructive. They attack beans and peas in the field, where they may become heavily infested before they reach the warehouse. The bean weevil’s eggs are smooth and white, laid singly or in clusters among or near the seeds. The eggs hatch into tiny white larvae, which eat their way into the seeds. The entrance holes are small and easily overlooked. In warm weather the larvae develop rapidly within the seeds and soon reach the pupal (resting) stage from which the adults form. The adults then chew a round opening through the seed coat and emerge. The bean weevil can develop in all varieties of common beans and cowpeas; the cowpea weevil’s life cycle is similar.
• The damage of the three moth species comes chiefly from the webbing and frass deposited among the seeds while the larvae feed. The larvae can only feed on beans and lentils that are split or have cracked seed coats.
• The lygus bug (Lygus sp.) and the aphid (Aphididae) may damage legumes by inserting their proboscis into the growing seed to inject a digestive juice and suck out plant substances such as protein. This leaves an area of dead tissue that appears as a brown spot, or in some cases a chalky pit surrounded by brown scar tissue. In some instances the damage does not penetrate the cotyledons; in others there is extensive inner damage with only a small amount detectable on the exterior.

5.2 Macroscopic examination of dried peas, beans and lentils — mould

Remix the sample unit thoroughly and take 100 whole peas, beans or lentils by the same half-seed rule as above. These 100 seeds constitute one analytical unit — repeat for the remaining seven sample units

↓

Soak the seeds 15–30 min in 70% alcohol, until the seed coat separates from the cotyledons as shown by wrinkles in the seed coat. Remove the seeds from the alcohol and place them on absorbent paper. Gently split the seeds with a scalpel, hilum side down. Separate the cotyledons and seed coat with forceps and place them into 100% isopropanol. Remove the cotyledons and seed coat and place them on a spot plate; they should dry in about 2 min. Seeds that are difficult to split by this method are first boiled in water until softened

↓

Examine the internal surface of the seed coat with a magnifying lens (3–5×) for mould hyphae. Where invasion is extensive, hyphae and fruiting bodies may be present between the cotyledons, and the cotyledons may be discoloured. Suspected hyphae and fruiting bodies may be confirmed by removing them with forceps, mounting in glycerol–alcohol mixture and examining with a compound microscope at 100–200×

↓

Classify as defective any pea, bean or lentil in which 1/4 or more of the internal surface of the cotyledons is affected by mould. Peas, beans or lentils with no visible external damage and an apparently intact seed coat (integument) may have extensive internal mould, which is why splitting is required.

5.3 Microscopic examination of frozen and canned peas, beans and lentils — light filth

Thoroughly mix a sample unit and weigh 583 g. For canned produce, use the entire contents of a can (usually 583 g) and record the weight. This amount constitutes an analytical unit — repeat for the remaining two sample units

↓

Isolate the light filth:

• For canned seeds of normal texture: pour onto a No. 8 sieve in a pan filled with enough water to stand 2–3 cm above the sieve mesh. Mash the seeds through the sieve with fingers. After as much material as possible has been worked through, remove the sieve from the pan and shake excess water back into the pan. Transfer the material retained on the sieve to a 2 L beaker. Pour the material that passed through the No. 8 sieve onto a No. 40 sieve, discarding what passes through. Let the material on the sieve drain a few minutes and shake lightly to remove free water. For unusually hard seeds or tough skins, pass the can contents through a meat or food chopper directly onto a No. 40 sieve and discard any excess water passing through. Cook frozen seeds before maceration.
• Add the material retained on the No. 40 sieve to the beaker. Add approximately 130 mL of heptane and mix thoroughly with a large spoon. Rinse any material remaining on the sieve into the beaker with water. Stir the material in the beaker and pick out any insects that rise to the top of the water layer. Repeat the stirring and searching several times until no more larvae are recovered.
• Add enough water to bring the beaker contents to within 1–2 cm of the top. Pick out any larvae visible at the surface. Stir again and let the mixture stand approximately 5 min. Skim off the heptane and the upper part of the water layer with a spoon and place in a trap or side-arm flask, previously filled to approximately 1/4 full with water. Add 90–100 mL of heptane to the material in the beaker and stir vigorously. Let stand approximately 5 additional min, skim off the heptane and upper part of the water layer as before, and add to the material already in the flask.
• Fill the flask with water. Trap off as much heptane as possible and filter onto ruled paper in a Hirsch funnel using the suction filtration apparatus. Lower the stopper into the flask; to rinse the sides of the trap flask, apply a vacuum for approximately 5 min by fitting a large rubber stopper and glass tube over the mouth of the flask (an ordinary erlenmeyer collapses under a vacuum of 50 cm of Hg (67.7 kPa), so use either less vacuum or a heavy-walled flask). Release the vacuum, add water and trap off. Add the trapped-off portion to the material already on the filter paper.
• Transfer the filter paper to a petri dish and moisten with glycerol–alcohol mixture.

↓

Examine each filter paper microscopically at 30× for whole and partial insects, mites, eggs, rodent and other hairs, and other material. Identify any insect fragments or other extraneous material if possible.

Insect damage in canned and frozen peas, beans and lentils

• Pea weevil infestation (Bruchus pisorum (L.)), by far the most commonly encountered defect in peas, occurs in the fields before harvesting. Adult weevils lay eggs on the tiny pods; after hatching, the larvae perforate the skins and enter the peas. Infestation is evidenced by small round holes on the peas, usually accompanied by discolorations around the openings. The larvae may be found under the skin, embedded in the cotyledon.
• The bean weevil (Acanthoscelides obtectus (Say)) attacks the seeds of kidney beans, lima beans and cowpeas in the field and in storage, and attacks faba beans, peas, lentils and others in storage only.
• The cowpea curculio (Chalcodermus aeneus Boheman) is a common pest in field peas and black-eyed peas. Some varieties of crowder peas and the California black-eyed pea are more susceptible than others. The greatest damage is done by the larvae feeding on the seed within the green pods. Shelled peas with curculio damage have small dark spots, which may or may not contain an egg or larva; these small dark spots are commonly called “weevil stings”. The curculio larva is legless and C-shaped; its body is pale yellow and its head is brown. The larva is less than 5 mm long when fully grown.

5.4 Recording the microscopic results

• Record the number and range in size (mm) of each type of extraneous material, separately for each analytical unit.
• If any of the insects, their fragments or other extraneous material can be identified, record that information as well.

6. Calculation

6.1 Insect-damaged and mouldy dried peas, beans and lentils

Record the percent by number of each defect category, separately for each analytical unit:

• Percent insect-damaged defectives (a) = [Total number of insect-damaged seeds ÷ Total number of seeds examined] × 100
• Percent mouldy defectives (b) = [Total number of mouldy seeds ÷ Total number of seeds examined] × 100

Combine the percent of the two categories to obtain the total percent defectives for each separate analytical unit:

Total percent defectives = (a + b)% per analytical unit

Where,
– a = percent of insect-damaged defectives in the analytical unit
– b = percent of mouldy defectives in the analytical unit

Then determine the number of analytical units in each of the following three ranges of percent defectives:

• 0–5%
• 6–10%

• 10%

6.2 Insect damage only (macroscopic)

Record the number of insect-damaged defectives, as determined in 5.1, for each analytical unit separately.

7. Precautions

• If appropriate, freeze the samples prior to analysis to minimize the possibility of a live infestation of the laboratory.

References

Health Canada, Compendium of Analytical Methods, Volume 4 — Laboratory Procedure ExFLP-15, January 2019: “Determination of Extraneous Material in Dried, Frozen and Canned Peas, Beans and Lentils” (Extraneous Matter and Food Packaging Unit, Greater Toronto Area Laboratory, Canadian Food Inspection Agency; replaces ExFLP-15 dated September 1996).

The method’s own bibliography:

• Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC), 1990, 15th ed. (cited by number and title only).
• Kvenberg, J., Brickey, Jr., P. 1984. Macroanalytical Procedures Manual, FDA Technical Bulletin No. 5, Association of Official Analytical Chemists.
• Pietrzak, E. 2009. Guidelines for the General Cleanliness of Food – An Overview. In: Volume 1. Compendium of Analytical Methods.