detection of urine on Grains (method 963.28)(method 963.28)

The magnesium uranyl acetate test is applied to determine the presence of urine on grains. A sample of the grain is sprayed with magnesium uranyl acetate to cause a greenish fluorescence when viewed under UV light (AOAC 2005).

Urea is detected with urease-bromothymol blue paper and is confirmed through its reaction with xanthydrol to form dix-anthylurea crystals, which are detected microscopically.

A. Samples, Ingredients, and Reagents

• Grain samples

1. Prepare the following reagents:

a. Magnesium uranyl acetate solution. Dissolve 100 g of magnesium uranyl in 60 mL of glacial acetic acid and dilute to 500 mL.

Dissolve in 330 g of magnesium acetate [Mg(C2H3O2)2 · 4H2O] in 60 mL of glacial acetic acid and dilute to 200 mL. Heat solu-tion to the boiling point until clear, pour the magnesium solution into the uranyl solution, cool and dilute to 1 L in a volumetric flask.

The working magnesium uranyl acetate tion is prepared by mixing the previous solu-tion with the urease solusolu-tion described next in 1/10 amounts with the posterior addition of 22 mL glycerol. The working solution is mixed and filtered through filter paper.

b. Urease solution. Wet 0.2 g of urease pow-der with a small amount of water, stir into a paste and dilute to 10 mL with distilled water.

c. Bromothymol blue solution. Dissolve 0.15 g bromothymol blue powder in mortar with 2.4 mL 0.1 N NaOH solution. After dissolv-ing, wash mortar and pestle with distilled water and dilute to 50 mL.

d. Urease-bromothymol blue test paper. Mix 10 mL of the indicator solution c with 10 mL of the urease solution b. Pour mix-ture onto watch glass and use clean twee-zers to dip pieces of filter paper (Whatman no. 5) into the solution. Hang orange paper to dry at room temperature. Store dry paper in well-stoppered dark glass bottle.

2. Weigh 50 g of grain and place it on a shallow tray in a hood or ventilated area.

3. Spray with the magnesium uranyl acetate solu-tion, making several sweeps horizontally and vertically across the sample. Let stand for 1 to 3 minutes and examine under short-wave UV light.

4. Wearing laboratory gloves and with clean twee-zers, transfer kernels that showed greenish fluo-rescence to spot plate.

5. Add one to four drops of distilled water to each suspect kernel on spot plate. Let stand for 3 to 5 minutes.

122 Cereal Grains: Laboratory Reference and Procedures Manual 6. Place strip of urease-bromothymol paper on

microscope slide and transfer drop of the aque-ous extract of step 5 to paper with stirring rod and cover with a second slide.

7. The development of blue spots within 4 minutes indicate the presence of urea.

8. For a confirmatory test, transfer one to two drops of extract of step 5 to microscope slide and evaporate to dryness. Add drop of a mixture of two parts acetic acid and one part water and a very small amount of xanthydrol crystals.

9. If urea is present, dixanthylurea crystals will quickly form and are visible at 60× or lower with wide-field stereoscopic microscope.

4.3.5 rEsEarcH suGGEstions

1. Compare a sound lot of maize with damaged maize produced according to procedure in Section 4.2.1.2.

Treat one lot with the recommended amount of phostoxin or phosphine. After 2 months of stor-age, determine grain damage in terms of dry matter loss and physical properties (test and 1000 kernel weights) and the number of insects. In addition, mill subsamples into a flour and determine the number of insect fragments.

2. Prepare a lot of the same type of sound maize according to procedure in Section 4.2.1.2 (effect of temperature and grain moisture content on grain sta-bility). Treat one lot with the 0.2% propionic acid that acts as an antimold agent. After 2 months of storage, determine grain damage in terms of physical prop-erties (test and 1000 kernel weights) and aflatoxins using the enzyme immunoadsorbent ELISA assay.

3. Determine the amount of aflatoxins associated with a contaminated lot of maize following the TLC, enzyme immunoadsorbent (AflaTest), and HPLC assays.

4. Determine the presence of mammalian feces by the alkaline phosphatase detection method (AOAC 2005, Method 986.28) and compare results with the AACC method (AACC 2000, Method 28-50).

4.3.6 rEsEarcH quEstions

1. Define the following terms:

a. Extraneous material b. Heavy and light filth c. Berlese funnel

d. Good Manufacturing Practices e. Detection action levels f. Cast skins

g. Pheromone

2. List at least three reasons for conducting analysis for extraneous material in cereal-based products and foods.

3. What are the main characteristics of insects belong-ing to the Lepidoptera or Coleoptera orders? What environmental factors and intrinsic grain character-istics favor insect growth and infestation?

4. What are the differences between primary and sec-ondary grain insects?

5. How are insects and rodents usually controlled in grain elevators?

6. Besides direct losses, what other sorts of damage do insects usually cause to cereal-based products?

7. Compare environmental minimum and optimum temperature and grain moisture or water activity requirements for insects and molds.

8. Why is a lot of grain with a high amount of dockage or extraneous material more prone to deterioration compared with a clean counterpart?

9. Investigate the use of X-ray and NIRA for the detec-tion of internal insect infestadetec-tion or insect pests.

10. What is the principle of the insect fragment assay?

Why is this test considered as one of the most criti-cal for food trade and an indicator of sanitation?

11. What are the main insecticides used on contact sur-faces and to treat stored grains? What are the advan-tages or disadvanadvan-tages of these insecticides?

12. What are the main toxic effects to insects and humans of organochloride, organophosphate, and carbamate insecticides?

13. What are the differences between an insecti-cide and a fumigant? List at least three impor-tant insecticides and fumigants applied in grain elevators.

14. Why do most regulatory agencies monitor the amount of residual pesticides in processed grain products? What are the toxic effects of methyl bro-mide, malathion, and pyrethrines to insects and

17. Investigate methods to commercially detoxify mycotoxin-contaminated kernels.

18. Why do most mycotoxins fluoresce when exposed to UV light?

19. What are the advantages and disadvantages of deter-mining aflatoxins with HPLC and the AflaTest?

20. What are the principles of urine and feces analy-ses? Why are these tests important for regulatory agencies?

21. What are the morphological differences among the three major rodents that infest grain elevators? How can you detect if the grain elevator or processing industries have rodent infestations?

123 Storage of Cereal Grains and Detrimental Effects of Pests

22. What are the main preventive control measurements for rodents in grain storage facilities or elevators?

23. What are the main toxic effects of methyl bromide, warfarin, fluoroacetamide, and arsenic trioxide?

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5 Dry-Milling Processes and Quality