QUICK QUALITY TESTS FOR PROTEIN MEALS

Dr. Jowaman Khajarern and Dr. Sarote Khajarern

Department of Animal Science, Faculty of Agriculture
Khon Kaen University, Thailand

INTRODUCTION

Since the early 1970s, the feed industry in Southeast Asia has made tremendous expansion. High
quality materials especially protein meals (fish meal and soybean meal) are often in short supply and
also exhibit a variation in the quality aspects of nutrient availability. Since protein sources especially
fish meal and soybean meal generally have high unit cost, the company must establish written
ingredient quality standards for purchasing, but the methods for examining the physical qualities,
especially for foreign materials and evidence of mold, must be fast, accurate and practical by the
operators at the receiving plant. The operators must be trained to recognize and understand the
quality of raw material to perform their visual and other quick physical and chemical examinations,
and in the proper methods of sampling. The objective of this paper is to highlight the most important
method of quick tests for protein meals used for the purchasing of the raw materials.

Sources of Protein Meals for Non-ruminants and Aquaculture

Sources Plant Protein Animal Protein

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Conventional Major Soybean meal Fish meal
Fullfat soybean Meat meal
Sunflower meal Meat and bone meal
Sesame meal Milk products
Peanut meal Poultry byproducts
Canola and Rapeseed meal
Corn gluten meal
Palm kernel meal
Copra meal
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Minor Soya protein isolate Feather meal
Wheat gluten meal Blood meal
Mung bean Marine soluble products
Potato protein Shrimp products
Cotton seed meal Squid products
Kapok seed meal Yeast

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Non-conventional Rubber seed meal Leather meal
Lupins Silk worm pupa meal
Safflower meal Crab products
Linseed meal Lard pulp
Field peas Brewer's byproducts
Mustard meal Distiller's byproducts
Cocoa seed meal

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Quality Control in Different Protein Meals

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Ingredient Quality Physical characteristics (analyst's skills): color, texture, odor and
(Qualitative) taste, particle size (screen analysis), shape, adulteration, damage
and deterioration, bulk density, spot and quick chemical tests.
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Ingredient Quality Proximate analysis: moisture, CP, CF, EE, NFE, ash, silica or
(Quantitative) sand, salts etc.
Protein quality: protein solubility or dispersibility, bitrogen
solubility, mailard reaction product, biogenic amines, trypsin
inhibitor activity, urease activity, dye binding, pepsin
digestibility, urea and nonprotein nitrogen.
Amino acid: composition, digestibility, availability
Anti-nutritional factors:
- Extrinsic (contaminants): mycotoxins, insects, weeds,
insecticide, herbicides, fungicides.
- Intrinsic: allergins, lectins, phytoestrogens,
glucosinolate, saponins, tannins, ricin, sinapine,
gossypol, lipoxygenase.
Decomposition and rancidity test: acid value, peroxide value, etc.
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Quick Test for Fish Meal Quality Control

Basically, fish meal is produced from two types of raw materials, fish wastes from human food
industry and whole fish. Differences in composition of fish meal may be attributed to many causes:
variation in raw materials, differences in processes, contamination of raw materials with some waste
products or with sand, impurities, excessive salts and/or fat content or excessive moisture,
adulteration with other sources of meal, meat and bone meal, plant proteins, etc. Fish meal is also
prone to contamination with biogenic amine and gizzarosine found during processing and storage that
have been allowed to spoil or putrefy and have dramatic impact on the quality and nutritive
composition of fish meal. The main aspects of quality control for consideration are:

Raw material type: whole fish or trimmings

Processing temperature: low temperature (LT) high temperature (HT) on digestibility of
protein and over-heated by using racemization of aspartic acid (Luzanna et al., 1996) to
indicate digestibility of protein and a simple dilute pepsin test (strength 0.0002 %) at 450C will
identify overheated fish meal.
Freshness of raw material: Fish spoils protein breakdown to amines (histamine, cadaverine
putrescine and tyramine). The sum of the four should be less than 2,000 ppm or predominant
in anchovy, mackerel, sardine, etc. or cadaverine predominant in capelin, sandeel, sprat, etc).
Lipid quality: Oxidation of fat to free fatty acids given a crude measure of fish freshness.
Oxidation of fat can reduce growth of animal and aquaculture. Ethoxyquin has been found to
be the most effective antioxidant. Free fatty acid (FFA) peroxide value (PV) and quick test for
rancidity should be assessed for quality control.
Microbial standards: Fish meal should be free of salmonella and mycotoxin. Because
carbohydrate or starch is absent, mold growth generally does not occur if fish meal is properly
stored in dry conditions and stabilized with antioxidants.

Quick Test for Quality Grade of Fish Meal

1. Place 10g of fish meal in a 100 ml tall form beaker and pour 80 ml carbon tetrachloride into
the beaker, stir and allow to settle (5 min.).
2. Scrape off the floating organic fraction with stainless steel spoon into filter paper. Clean off
the side of the beaker and spoon and then pour off the liquid to separate floating and the
submerged fraction into the filter paper.
3. Place the two fractions into a 1100C oven for 10 min. then allow to cool and weigh each
fraction.
4. The approximate percentage of organic (fish flesh) and inorganic (fish bone) may be
calculated.

Shrimp and Salmon Grade Inorganic Fraction

Fish meal Grade A (CP>70%) <12%
Fish meal Grade B (CP>65%) <16%
 Fish meal Grade C (CP>62%) <20%

Animal Grade Inorganic Fraction

Fish meal Grade A (CP>58%) <30%
Fish meal Grade B (CP>35-58%) <35%
Fish meal Grade C (CP>50-55%) <40%
Fish and bone meal (CP 35-50%) >40%

Quick Test for Quality Grade of Fish Meal

Principle:

Most adulterants such as (ammonium sulfate, ammonium phosphate, ammonium nitrate, etc) are
non-protein nitrogen and are soluble in water. When they react with mercuric potassium iodide
alkaline solution mixture, a heavy orange precipitate color occurs.

Reagent:

1.25g mercuric chloride is made into a thin paste with a little water, 3.5 g potassium iodide is added
and solution takes place. 12 g sodium hydroxide is dissolved in 50 ml. Water is added and the whole
made up to 100 ml. The rather turbid solution is allowed to stand for several days, decanted and
stored in a brown bottle.

Procedure:

1. Put 2-3 g of test sample in a 100 ml beaker and add 10-15 ml distilled water, then stir and let
stand for 2-3 min.
2. Put 3-5 drops of test sample into white porcelain spot plates and add 2-3 drops of mercuric-
potassium iodide alkaline solution mixture.
3. If non-protein nitrogen is present, a heavy orange precipitation color appears. The intensity of
orange precipitation depends on the amount of non-protein nitrogen present.

Quick Test of Hydrolyzed Feather Meal from Fish Meal

Principle:

Hydrolyzed feather meal contains a high percentage of cystine (6-7%). When the sample is digested
with sodium hydroxide, the cystine and cysteine are liberated and reaction with lead acetate gives the
dark brown black color on the surface of the particle.

Reagents:

Solution A: Sodium hydroxide 10%

Solution B: Dissolve 50 g of lead acetate in 800 ml water, then add 20 ml glacial acetic acid, stir and
pour 20 ml glucerol, shake and dilute with water to 1,000 ml.

Procedure:

1. Place one teaspoon of well mixed standard hydrolyzed feather meal and test sample of fish
meal into two sets of petri dishes.
2. Add 10-15 ml of solution A into all the two sets of each test sample. Swirl gently to spread
samples evenly in each dish and let them stand for 10 min.
 3. Add 10-15 ml of solution B into each first set of petri dishes and into the second set add 10-15
ml distilled water. Mix gently by turning around each petri dish and let them stand again for 10
min.
4. During standing, a visible browning reaction color develops until black colored particles
appear in the first set of petri dish for the standard hydrolyzed feather meal. When compared
to the second set (without adding solution B), no visible brown color develops after 10 mins.
5. Compare the test fish meal sample with the standard hydrolyzed feather meal sample and
also the visible browning color between the first and second set of each test sample. If the
color of these two sets differs, the fish meal is adulterated with hydrolyzed feather meal.

Quick Test of Hydrolyzed Leather Meal from Fish Meal

Leather meal is waste by-products from hide trimmings which contain chromium salts. When a sample
is ashed, the visible dark green chromium salts develops. Diphenyl carbazide is an excellent reagent
to react with chromium in dilute sulfuric acid (0.1 MH2SO4) which produces a violet color and will fade
within 10-15 mins.

Reagents:

Solution A: (0.1 MH2SO4

Solution B: Dissolve 1-2 g diphenyl carbazide in 100 ml distilled water.

Procedure:

1. Ash fish meal test sample at 6000C for 2 hrs and examine for the presence of dark green
chromium salts. Dark green particle of ash is a conducive evidence of the adulteration of
leather meal in the sample.
2. To confirm the presence of chromium, place a small amount of ash into a white spot plate.
Add 2-3 drops of solution A and then add 2-3 drops of solution B.
3. A red-violet color indicates the presence of chromium.

Decomposition Test for Animal and Marine Products

Principle:

As animal and marine products spoil, protein breaks down to amines. The residue of these biogenic
amine can indicate the freshness or decomposition of the sample. If the sample is badly decomposed,
the test sample will darken quickly with saturated lead acetate paper and it is not suitable for feeding.

Reagents:

1. Dilute sulfuric acid: 5 ml of conc. H2SO4 to 45 ml H2O

2. Saturated lead acetate solution.

Procedure:

1. Put 5 g of test sample into 250 ml erlenmeyer flask.

2. Prepare a cork, which fits tightly, with a 2" x " strip of white filter paper pinned to the bottom,
moistened with saturated lead acetate.
3. Add 50 ml dilute sulfuric acid into the sample then immediately insert the cork and let it stand
in a warm room for 16 hrs.
4. If the sample is badly decomposed, the test paper will darken quickly.

Quick Test for Identification of Plant Protein and Animal Protein

Principle:

Carbohydrates from plants contain starch and cellulose. When it reacts with iodine and chlor-zinc
iodine solution, the starchy tissue releases a blue color and the plant fiber or cellulose develops a
purple-brown color when examined under a microscope.

Reagents:

1. Iodine solution: 0.5 g I and 1.5 g KI dissolved in 25 ml distilled water.

2. Chlor-zinc-iodine solution: Dissolve 100 g ZnCl2 in 60 ml water, then add 20 g KI and 0.5 g I.

Procedure:

1. Mix 1-2 g test sample with 100 ml boiling water or boil the mixture for 2-3 min. Place a few ml
of the cooled mixture on a spot plate or test tube and add 5-6 drops of iodine solution. If
starch is present, the mixture turns blue.
2. Spread 1-2 g test sample into a petri dish. Add 5-6 drops of chlor-zinc iodine solution and let
stand for 10 min. A purple brown color indicates the presence of plant fiber, whereas yellow
indicates animal fiber (protein) using a microscopic examination.

Quality Considerations for Soybean Meal

Soybean meal is a major plant protein source for poutlry, livestock and aquaculture. Soybean meal
used in Asian countries exists in several forms, with solvent extracted material containing hulls being
the most common. Many locations use imported soybean meal from USA, Brazil, Argentina and India.
Full fat soybean is also available, produced by extrusion or dry roasting in small scale plants. The
following are specifications for soybean meal and dehulled soybean meal (Hi-Pro).

Bulk Density Range: 57-64 g/100 cc for both soybean meal and soybean (Hi-Pro).
Screen Analysis: 95-100% through US #10 screen; 40-60% through US #20, 6% maximum
through US #80 for both soybean and soybean (Hi-Pro).
Desired Physical Properties for both soybean and soybean (Hi-Pro).
o Color: Light tan to light brown
o Odor: Fresh, not musty, not sour or burned
o Texture: Homogeneous, free flowing, no lumps or cakes, without coarse particles or
dusty
o Taste: Bland and free of any beany or burned taste
Urease actvity: 0.05-0.20 pH unit change for both soybean meal and soybean (Hi-Pro).
Moisture (maximum): 12% for both soybean meal and soybean (Hi-Pro).
Protein solubility 0.2% KOH: 73-85% for both soybean meal and soybean (Hi-Pro).
Protein Dispersibility Index: 15-30% for both soybean meal and soybean (Hi-Pro).
Nitrogen Solubility Index: 15-30% for both soybean meal and soybean (Hi-Pro).
Contaminants: Particularly check for urea, non-protein nitrogen or ammonia for both soybean
meal and soybean (Hi-Pro).
Protein (minimum): 44.0% for soybean meal; 47.5-49% for soybean (Hi-Pro).
Fat (minimum): 0.5% for both soybean meal and soybean (Hi-Pro).
Fiber (maximum): 7.0% for soybean meal; 3.3-3.5% for soybean (Hi-Pro).
Ash (maximum): 6% for both soybean meal and soybean (Hi-Pro).
Silica (maximum): 2% for both soybean meal and soybean (Hi-Pro).
ME ((kcal/kg): Approximately 2,375 for soybean meal; 2,525 for soybean (Hi-Pro).

Rancidity test for Animal Products and Oilseed Meal

Method I

Reagents:
 1. Acetic chloroform mixture (6:4): Glacial acetic acid 60 ml and 40 ml chloroform.
2. Saturated potassium iodide.
3. Starch indicator.

Procedure:

1. Put 5g test sample into 250 ml Erlenmeyer flask.

2. Add 50 ml of acetic chloroform mixture and 1 ml saturated potassium iodide and shake.
3. Add 50 ml distilled water and starch indicator, then shake again.

The development of blue color indicates rancidity.

Method II

Reagents:

1. 0.1% Phloroglucinal solution in ether.

2. Kerosene.

Procedure:

1. Shake 10 ml of oil or melted fat sample with 10 ml of 0.1% phloroglucinal solution and 10 ml
of conc. Hydrochloric acid for 20 sec. A pink color indicates incipient rancidity.
2. If oil is diluted 1 in 20 kerosene and the test is still positive, the rancidity will probably be
evident to the taste and smell.

Quick Test for Urea (Qualitative Test)

Reagents:

1. Urease solution: 0.2 g urease stir into 10 ml H2O.

2. Bromothymol blue solution: Rub 0.15 g Bromothymol blue in mortar with 2.4 ml 0.1 N NaOH,
wash mortar and pestle with H2O and dil. To 50 ml H2O.
3. Test paper A: Mix 10 ml urease solution 1 and 10 ml indicator soln. 2. Pour mixture into watch
glass, dip pieces of filter paper (Whatman No. 5) in soln. And hang paper to dry. Store dry
paper (Orange color) in well-stoppered dark glass bottle in a cool place.
4. Test paper B: Dilute indicator soln. 2 with equal portion with H2O. Dip pieces of filter paper
(same kind used for test paper A) in indicator soln. And hang to dry as in 3.

Procedure:

1. Stir 2-3 g of test sample in 50 ml H2O and let it stand for 2-3 min.
2. Placing 2-3 drops of test sample on dry test paper A, the appearance of blue or green spot
after a few minutes of incubation at room temperature indicates urea.
3. For detection of urea in a very small, dry particle, dip both test paper A and B H 2O and then
shake the papers to remove excess H2O by using clean tweezers. Place the papers on a
clean flat piece of glass. Place the sample on the papers and cover with another clean flat
piece of glass by pressing down gently. Blue spots on the test paper A indicate urea (30-60
sec.). Spots continue to develop and enlarge for 10-20 min. and then fade gradually. Time
varies inversely with urea concentration. If blue spots develop on both paper A and B, this
indicates alkaline particles.
Microscopic Identification of Protein Meals

Feed microscopy experts must be fast and accurate to obtain quality assurance. Adulterants and
contaminants in both ingredients and finished feeds are best detected microscopically. Special
training on description and characteristics of feed ingredients are the keys for rapid microscopic
identification.

Preparation of sample

If the feed is mashed, use about a teaspoonful of feed for sieving. Rub the feed in a mortar with a
pestle if the feed is pelleted or crumbled. When the sample is reduced to a mash-like consistency, it is
divided in three sets of sieves (20, 40, 60 mesh), and thus end up with four fractions of the feeds.
Each fraction is put in a glass petri dish cover. The sample is now ready for microscopic examination.
Using a stereomicroscope, scan each particle in the first and second dishes. The feed in the third and
fourth dishes is used for high power compound microscope and also for quick chemical spot test. For
a clearer observation of plant histology and microscopic appearance, the feed is heated with 8% KOH
steam bath for 30-45 min. If this treatment is not satisfactory, treat the fresh portion for a short time by
gently heating with acidified chloral hydrate glycerol solution.

Precise characteristics on Microscopic Identification of Protein Meals

(Contact us for a copy of the annex picture).

Crab Products: Orange pigmentation, segmented antennae with calcareous shell and will
effervesce in diluted HCI, honey combed round cells on the outer layer of shell.
Fish Products: Curved scales with concentric rays, bone sexhibit lacunae with well defined
canaliculi, milky glass beads with broken surface eye lenses.
Shrimp Meal: Segmented leg and antennae, thin shell with mica-like and in some areas, may
appear cross-hatch type of marking, feathery delicate gill tissue, amber colored cells of
compound eyes.
Squid Products: Mottled body fragment with black pigment spots, tentacles or sucker pieces
present, no lacunae or surface lines on internal shell fragments.
Blood Meal: Spherical particle, smooth surface with glass when rubbed, dark red to almost
black in color.
Meat Meal and Meat and Bone Meal: Strong greasy odor, consist of hoof, horn, hair, fluff
and vegetable fiber, cylindrical rods smooth muscle with alternative dark and light striated
muscle.
Soybean Meal: Yellow to brown oval hilium with a clear slit, pox-marked outer surface hull,
hourglass and palisade cells from the hull are the major cellular keys for soybeans and also
elongated cells below the peripheral cells of the cotyledon.
Peanut Meal: Thin skin with copper to red color, higly pitted cell of pod fragment and the lack
of palisade cells in the testa, elongated pite in hypodermal stone cells and unique cross fiber
cell of pod.
Sunflower Meal: Striped or all black varieties for the hull, leathery hull with a paper-like lining.
Twin hairs, united almost to their tips on the outer surface of the cypsela, unbroken pericarp
fragment may appear as broken pieces in the medium, the outer epiderm of transversely
elongated cells with zigziag walls.
Rapeseed Meal: Many species of rape are lumped together and are difficult to identify
separately by structured features. For all practical purposes, the examination of the seed coat
or testa for degree of reticulation is important. The inner surface seed coat has a delicate
semi-transparent, white sheet adhering to the surface.
Sesame Meal: Seed coat or outer epidermal cells contain calcium oxalate crystals with black
brown or yellow brown colored and granular surface.
Cottonseed Meal: Long, flat and twisted fibers adhering to the hulls, kernel fragments are
yellow to brown containing many round, red, brown gossypol glands. The hull edge has a light
brown layer with stairstep facets. The epidermal cells are heavy walled with dark pigmented
interiors. Palisade cells can also be used for identification.
Copra Meal: Irregularly shaped flaky fragment with large, colorless, straight, thin walled cells
of endosperm containing oil globules.
REFERENCES:

1. Association of Official Analytical Chemists, 1984. Official Methods of Analysis. 12 th Edition,

AOAC, Box 540, Benjamin Franklin Station, Washington DC 20044.
2. Khajarern, J., D. Sinchermsiri, and A. Hanbunchong, 1987. Manual Feed Microcopy and
Quality Control. Dhornhvaj Co., Ltd. Bangkok, Thailand.
3. Luzzana, U., T. Mentasti, V. Moretli, A. Albertini, and E. Valfre, 1996. Aspartic acid
raccimization in fish meal as induced by thermal treatment. Aquac. Nutr. 2: 95-99.
4. Manual of Microscopic Analysis of Feeding Stuffs, 1966. The American Association of Feed
Microscopists.
5. Manual of of Microscopial Analysis of Feedstuff, 1992. 3rd Edition, The American Association
of Feed Microscopists.
6. Peason, D., 1970. The Chemical Analysis of Foods, 6th Edition. J & A Churchill, 164
Gloucester Place, London.

AMERICAN SOYBEAN ASSOCIATION

541 Orchard Road #11-03 Liat Towers
Singapore 238881.
TEL: (65) 6737-6233, FAX: (65) 6737-5849.

URL: http://www.asasea.com

MITA (P) NO. 096/11/97 (Vol. FT45-1998)