Discussion on common methods for identifying fishmeal adulteration

Fish meal is a high-quality animal protein raw material widely used in livestock, poultry, and aquatic feed. It has a high protein content, with a protein digestibility of about 90%; its amino acid composition is complete and balanced, with high levels of lysine and methionine; it also contains high levels of calcium and phosphorus in a favorable ratio with high utilization; it is particularly rich in minerals, vitamins, and various unknown growth factors, and has good palatability, thus playing an extremely important role in aquatic feed production. Due to its large usage and high market price, adulteration and contamination are very common.

The following introduces some conventional detection methods to qualitatively determine the adulteration and contamination of fishmeal. Commonly used methods include sensory identification, microscopic examination, near-infrared technology identification, physical identification, chemical identification, and amino acid composition analysis.

1 Sensory Identification

1.1 Color Observation

Fishmeal with uniform color and no 'carriers' such as fish bones, fish fillets, fish scales, or fish eyes is generally formulated fishmeal.

1.2 Smell

Good fishmeal has a fresh fishmeal aroma, while adulterated fishmeal has a fishy odor, ammonia smell, or rancid smell. Those mixed with plant-based lignin materials have a mixed plant odor, and those mixed with other animal-derived feed have a mixed animal odor.

1.3 Taste

Taste is the killer feature for identifying fishmeal. Good fishmeal melts in the mouth with a light fish fillet flavor, while adulterated fishmeal remains undissolved and has spicy, astringent, bitter, or rancid tastes.

1.4 Tactile Sensation

Gently rub fishmeal between thumb and index finger; good fishmeal is fine and smooth, while adulterated fishmeal is coarse with small particles.

1.5 Burning Test

Normal fishmeal emits a fishy aroma when baked on an induction cooker; if it produces an unpleasant, foul, or fragrant smell, it is adulterated fishmeal.

Discussion on common methods for identifying fishmeal adulteration

2 Microscopic Examination

Under the microscope, fish meat particles are relatively large, with a rough surface and fibrous structure, appearing yellow or yellowish-brown, translucent, resembling crushed tendons, and seemingly elastic; fish bones (including fish spines and skull bones) are semi-transparent or opaque fragments of various sizes, ranging from white to yellowish-white (some fish bone fragments are amber-colored), with a smooth surface. Fish spines are long, thin, and pointed, resembling vertebrae; upon close observation, the characteristic of a large or small end can be seen in the fish spine fragments. Fish skull bones are flaky, semi-transparent, with texture on the surface, hard and inelastic; fish scales are flat or curled, algae-like flakes, nearly transparent, with some concentric ring patterns; fish eyes are surface-cracked, milky spherical particles, semi-transparent, with dull luster and relatively hard. In fishmeal, other particles or powders that differ significantly from the above characteristics are mostly adulterants, which can be identified based on their microscopic features.

2.1 Adulteration of Plant-Based Raw Materials in Fishmeal

Fishmeal adulterated with soybean hull powder: Under a microscope, soybean hulls appear as yellow or yellow blocky substances. The hulls have concave spots, are slightly curled, and the hilum is visible. White sponge-like starch droplets can also be seen floating on the surface of the blocky substances.

Fishmeal adulterated with peanut meal: Under a microscope, peanut shells and seed coats are visible. The shells are broken, irregular, and vary in thickness, with layered structures. The inner layer is white and sponge-like with intertwined fibers, while the outer surface has raised veins forming a net-like pattern. The seed coat appears red, pink, deep purple, or brownish-yellow.

Fish meal mixed with rapeseed meal: When fish meal is adulterated with rapeseed meal, the seed coat of rapeseed meal can be seen under the microscope. The seed coat is characterized by being brown and thin, with a honeycomb-like mesh on the outer surface that is glossy, and soft, translucent white flakes attached to the inner surface. The seed coat and kernel fragments of rapeseed meal are not connected; the kernels are yellow, irregular in shape, and lack luster.

Fishmeal mixed with cottonseed meal: When fishmeal is adulterated with cottonseed meal, cotton fibers can be seen attached to the outer shell and meal particles under a microscope. The cotton fibers are white, filamentous, hollow, flat, curled, translucent, and glossy. Cottonseed hull fragments are brown or reddish-brown, relatively thick, with yellow or yellowish-brown color layers along the edges and a stepped surface. Cottonseed kernel fragments are yellow or yellowish-brown, containing many round, flat, black or reddish-brown oil glands or gossypol pigment glands.

Fishmeal mixed with rice husk powder: When fishmeal is adulterated with rice husk powder, rice husk fragments can be seen under a microscope. These fragments have a glossy surface with cross-shaped stripes and visible fuzz on the shell surface.

Fish meal mixed with wheat bran: When wheat bran is mixed into fish meal, yellow or brown flaky bran can be seen under the microscope. The outer surface of the bran has fine wrinkles, and the inner surface is adhered with many opaque white starch granules.

Fish meal mixed with sesame seed meal: When sesame seed meal is mixed into fish meal, sesame seed coats can be seen under the microscope. The sesame seed coat is thin, with tiny round protrusions on the surface, appearing black, brown, or yellowish-brown, depending on the variety.

2.2 Adulteration of animal-derived fish meal

Fish meal mixed with leather powder: When leather powder is mixed into fish meal, green, dark green, and brick-red lumps or filaments can be seen under the microscope, resembling sawdust, but not as transparent as hydrolyzed feather powder.

Fishmeal mixed with hydrolyzed feather powder: Under the microscope, semi-transparent irregular fragments can be seen, some of which are reflective. Feather shafts resembling hollow circles are also visible. Additionally, incompletely hydrolyzed raw feathers can be observed.

Fishmeal mixed with blood meal: Under the microscope, the characteristics of blood meal are visible. The particles of blood meal vary in shape, with some having sharp edges and others having rough, uneven edges. Colors range from black, resembling asphalt, to blood-red crystalline shells or small beads.

Fish meal mixed with meat and bone meal: Fish meal adulterated with meat and bone meal shows yellow to dark brown particles under the microscope, with higher fat content resulting in darker color and an oily reflective luster, and a rough surface. Under the microscope, very fine, interconnected muscle fibers can be seen. Bone fragments appear as white, gray, or light brownish-yellow blocky particles, opaque or translucent, with spots and rounded edges. Additionally, hair, hooves, and horns can be observed, often mixed with blood meal characteristics.

Fishmeal mixed with shrimp heads or shrimp meat powder: Shrimp head powder is added to fishmeal. Under the microscope, shrimp whiskers, shrimp eyes, shrimp shells, and shrimp meat can be seen. Shrimp shells resemble curled, mica-like thin flakes, semi-transparent. A small amount of shrimp meat is attached to the shrimp shells. Shrimp eyes are black spherical particles, which are relatively easy to identify in shrimp head powder. Shrimp whiskers exist as fragments under the microscope, appearing as long, tubular structures with spiral parallel lines. Shrimp legs are wide tubular fragments, semi-transparent, with or without hairs.

Fishmeal mixed with crab shell powder: Crab shell powder is added to fishmeal. Under the microscope, characteristics of crab shells can be observed. Crab shells are regular fragments, with the outer layer mostly orange-red, porous, and featuring honeycomb-like round sunspots.

Fishmeal mixed with shell powder: When shell powder is mixed into fishmeal, tiny shell particles can be seen under a microscope. Their surfaces are smooth, and the color varies greatly depending on the type of shell, ranging from white or gray to pink. Some particles have concentric or parallel textures on their outer surfaces, or dark intersecting bundles of lines, and some fragments have serrated edges.

Adulterated fishmeal aimed at increasing total nitrogen: Urea-formaldehyde polymers appear as milky yellow irregular spherical particles. When gently pressed with a probe, they disperse into crystalline forms and are insoluble in water.

3　Identification using near-infrared analysis technology

Using fishmeal as the research object, a DPLS qualitative discriminant analysis model for animal and plant adulteration components in fishmeal was established based on near-infrared reflectance spectroscopy (NIRS) technology, and the model was optimized using different variable selection methods. A PLS quantitative analysis model for animal and plant adulteration components in fishmeal was established, and the model was optimized using moving window partial least squares and different variable selection methods. A transfer method for the quantitative analysis model of adulteration components in fishmeal was established. Murray et al. conducted qualitative and quantitative analysis of meat and bone meal content in fishmeal using near-infrared spectroscopy. The results showed that near-infrared technology can effectively distinguish whether fishmeal contains meat and bone meal. Domestic related research indicates that near-infrared technology can distinguish fake fishmeal mixed with soybean meal, wheat bran, rapeseed meal, etc., from pure fishmeal and can quantitatively predict the content of adulterants.

4 Physical Identification

4.1 Water Dissolution Method

Take a small amount of the sample, place it in a clean glass or beaker, add 10 times the volume of water, stir vigorously, let it stand, and observe floating matter on the water surface and sediment at the bottom. If there are floating feather fragments or plant materials such as rice husk powder, peanut shell powder, wheat bran, etc., and sand, stones, or minerals at the bottom, it indicates the presence of hydrolyzed feather meal or plant-based adulterants.

4.2 Sieving Method

Sieve the fishmeal sample using a standard sieve with a mesh size of 2.80mm. At least 98% of standard fishmeal particles should pass through; otherwise, it indicates adulterants in the fishmeal. Different mesh sieves can be used to detect various impurities.

4.3 Odor Test Method

The authenticity of fishmeal can be determined by the smell produced when the sample is burned. If, during burning, an odor similar to burning pure hair is detected, it indicates the presence of animal adulterants; if a fragrant smell like dry-roasted grains is detected, it suggests the fishmeal contains plant-based substances. Take 20g of the sample, place it in a small flask or Erlenmeyer flask, add 10g of soybean powder and an appropriate amount of water, seal with a stopper, heat for 15 to 20 minutes, then remove the stopper. If an ammonia smell is detected, it indicates the presence of urea.

4.4　Bulk Density Method

The bulk density of pure fishmeal is generally 450g/L to 660g/L. If the bulk density is significantly higher or lower, it indicates that the fishmeal may contain impurities. The specific detection method is as follows: gently and carefully pour the fishmeal sample into a 1000ml graduated cylinder until it exactly reaches the 1000ml mark, then adjust the volume using a scraper or spoon. Note that the sample should be placed gently without shaking or tapping. Then pour out the fishmeal and weigh it (perform three parallel tests and take the average), and compare it with the bulk density of pure fishmeal.

5 Chemical Detection Methods

Chemical identification is divided into qualitative and quantitative methods.

5.1 Qualitative Identification

5.1.1 Detection of Plant Substances Mixed into Fishmeal

All plant materials contain starch and lignin. Starch can react with potassium iodide to produce a blue or blue-black compound. Under acidic conditions, lignin can react with phloroglucinol to produce a red compound. Therefore, using these two reactions, it is possible to quickly detect whether plant material has been mixed into fishmeal.

Test for starch substances mixed into fishmeal: Take 1g to 2g of fishmeal sample in a small beaker, add 10ml of water, heat and boil for 5 minutes, then cool. Add two drops of I-KI solution. If there is no color reaction, it is pure fishmeal. If the color turns blue or blue-black, it indicates the presence of starch in the sample.

Detection of lignin adulteration in fishmeal: Take 1g of fishmeal sample, place it in a test tube, moisten with phloroglucinol solution, let stand for 5 to 10 minutes, add 2 to 3 drops of concentrated hydrochloric acid, and observe the color. If the sample turns deep red, it indicates the presence of lignin.

5.1.2 Detection of blood meal adulteration in fishmeal

Blood meal contains iron, which can decompose hydrogen peroxide to release nascent oxygen, causing benzidine to oxidize into benzidine blue, resulting in a blue ring spot. Based on the presence or absence of the ring spot, it can be determined whether fish meal is adulterated with blood meal. Specific detection method: Take a small amount of the tested fish meal in a white porcelain dish, add a few drops of benzidine-glacial acetic acid mixture (1g benzidine added to 100ml glacial acetic acid, diluted with 150ml distilled water) to moisten the tested fish meal, then add one drop of 3% hydrogen peroxide solution. If the fish meal is adulterated with blood meal, the tested sample will immediately turn dark green or blue-green.

5.1.3 Detection of Non-Protein Nitrogen Compounds Adulterated in Fish Meal

5.1.3.1 Detection of Urea-Formaldehyde Polymer Adulterated in Fish Meal

Urea-formaldehyde polymer decomposes under the action of sulfuric acid to generate formaldehyde, which reacts with chromotropic acid to form a purple compound. Proteins and fats do not participate in this reaction.

5.1.3.2 Detection of Biuret Adulteration in Fishmeal

The principle of this method is that biuret can form a purple-red compound with Cu2+ under alkaline conditions. Detection method: Weigh 2g of the tested fish meal into 20ml of distilled water, stir evenly, let stand for 10 minutes, filter with dry filter paper, take 4ml of the filtrate into a test tube, add 1ml of 6mol/L NaOH solution, then add 1ml of 1.5% CuSO4 solution, shake well and observe immediately. If the solution appears purple-red, it indicates that the fish meal is adulterated with biuret; the deeper the color, the greater the amount adulterated.

5.1.3.3 Detection of urea adulteration in fish meal

Urease in raw soybean powder can decompose urea to produce ammonia, making the aqueous solution alkaline. After adding phenol red reagent, the solution turns red. The detection method is: pick out 3 to 5 suspicious particles under a microscope, place them in a 15 mL colorimetric tube, add about 0.2 g of raw soybean powder, 3 to 5 drops of phenol red indicator (1 g/L), then add 10 mL of water, quickly seal the tube, shake for a moment, and let it stand for several minutes. If the solution turns red, the sample contains urea.

5.1.3.4 Detection of ammonium salt adulteration in fishmeal

Ammonium salts rapidly decompose under the action of concentrated alkali to produce ammonia, turning pH test paper blue. The specific detection method is: take 3 to 5 particles of the suspicious substance from under the microscope, place them in a petri dish, attach a moistened pH test paper inside the dish, add about 3 ml of 30% concentrated sodium hydroxide solution to the dish, quickly cover it, and if the pH test paper immediately turns blue, the sample contains ammonium salts.

5.1.4 Detection of leather powder adulteration in fishmeal

After ashing, chromium in leather powder can partially convert to Cr6+. In a strong acid solution, Cr6+ reacts with diphenylcarbazide to form a purple-red water-soluble chromium-diphenylthiocarbazone compound. This reaction is highly sensitive, allowing detection of trace amounts of chromium. The detection method is: take 1g to 2g of the tested fish meal into a porcelain crucible, carbonize and ash it. After cooling, moisten the ash with a small amount of distilled water, add 10ml of (NH4)2SO4 to acidify the solution, then add a few drops of diphenylcarbazide solution (0.2g to 0.5g of diphenylcarbazide dissolved in 100ml of 90% ethanol). If a purple-red color appears shortly after, it indicates the presence of leather powder.

5.2 Quantitative Identification

5.2.1 Detection of Crude Protein

The national industry standard uses crude protein content as the main indicator for evaluating the quality and grading of fish meal, so crude protein should be tested first. The crude protein content is determined according to the national standard method. Generally, the higher the crude protein content, the better the quality of the fish meal. However, this is not always the case, because crude protein content is obtained by measuring total nitrogen content, which cannot exclude interference from non-protein substances.

5.2.2 Inspection of True Protein

It is relatively important to determine the true protein content of fishmeal, which has also been proven in production practice. Although the method for determining true protein content has not yet been included in national standards, it is currently widely used to determine whether high-nitrogen compounds have been added, in order to eliminate interference from non-protein substances. According to available data, the ratio of true to crude protein content in fishmeal is generally: over 80% for imported fishmeal and over 75% for domestic fishmeal. The author has tested fishmeal produced by some manufacturers, where the crude protein content reached 55%–60%, but the true protein content was only about 10%. Such fishmeal is of low quality or adulterated.

5.2.3 Determination of pepsin digestibility

Pepsin digestibility refers to the ratio of protein in fish meal that can be decomposed by pepsin to crude protein. For qualified fish meal, its pepsin digestibility should not be less than 85%. Measuring pepsin digestibility can identify whether adulterants in fish meal are high-protein but difficult-to-absorb raw materials, such as feather meal, leather meal, etc. The measurement is carried out according to the national standard method. Sometimes, when testing fish meal samples, the crude protein and true protein content are very high, but the pepsin digestibility is low, generally only 50% to 60%, or even only 30% to 40%. Such fish meal is also of poor quality or adulterated.

5.2.4 By determining the amino acid content of fishmeal, adulteration can be effectively identified. When using amino acid indicators to assess fishmeal quality, one should not only refer to methionine and lysine levels but should consider multiple amino acid indicators comprehensively, with special attention to serine, histidine, leucine, isoleucine, and others. In practical work, amino acid indicators should be combined with routine indicators, sensory inspection, and microscopic examination for comprehensive judgment.

In addition, besides analyzing the above four indicators, depending on the actual situation, other indicators such as crude ash, crude fiber, total chromium, calcium, and phosphorus should also be analyzed.

6 Conclusion

Select the identification method and determine the identification sequence. Sensory identification is a primitive but important, simple, and low-cost method, and other identification methods cannot function without its coordination. Near-infrared (NIR) technology is based on extensive physicochemical testing and is convenient and fast to use. Physical identification is chosen when sensory identification fails to detect adulterated fishmeal. Chemical identification is used when both sensory and physical methods struggle to determine the authenticity and quality of fishmeal, especially quantitative chemical identification, which is an essential method for assessing fishmeal quality. Generally, in the actual testing process, identification plans and schemes should be designed based on specific needs.