These techniques include some methods applicable in determining the quality of ingredients and of finished products based on the amount of micronutrients present such as digestible fibre, minerals, vitamins, enzyme activity, etc. In addition, the most widely used method for quantifying chromic oxide during evaluation of diet digestibility is given.
This method gives an approximate degree of fibre digestibility in feed. The sample is digested with cetyl-trimethyl-ammonia in sulphuric acid and the residue is considered indigestible fibre.
Reagents
Material and equipment
Method
Grind the sample in a hammer mill so that it will go through the 1 mm screen.
Take a subsample and dry overnight in a kiln at 105°C.
Cool sample in dryer.
Weigh out 1 g of dry sample to within milligrams and place in a 500 ml Erlenmeyer flask.
Add 100 ml acid detergent solution and 2 ml antifoam.
Place flask in the heating jacket with the condenser attached.
Bring to boil rapidly (3 to 5 minutes) and continue heating for 2 hours.
Filter contents of flask by gravity in a preweighed crucible.
Rinse flask with hot distilled water and filter.
Rinse the contents of the crucible with 300 ml hot distilled water. Use weak suction (vacuum).
Rinse residue with acetone and dry.
Place crucible in kiln at 105°C for 12 hours.
Cool sample in dryer and weigh immediately.
Calculations
Where:
W1 = weight of sample (g)
W2 = weight of residue (g)
Figure 7. Determination of fiber content by the acid detergent method
This method is useful for determining vegetable fibres in feed. Apparently it is able to separate soluble nutritional components from those that cannot be utilized completely or which depend on prior biological fermentation. This method has limited accuracy when protein values are very high and fibre values low.
Reagents
Material and equipment
Method
Weigh out approximately 1g of sample ground to pass through a 1mm screen and place in a balloon flask to begin reflux.
Add 100 ml neutral detergent at room temperature and 2 ml amylase per sample, in this order.
Heat so that the solution boils in 5 to 10 minutes. When boiling point is reached, reduce temperature to avoid formation of foam. Adjust temperature so that the solution boils gently and maintain reflux for 60 minutes counted from when boiling point is reached.
Shake the flask to suspend the sample and decant into a preweighed crucible prepared by vacuum suction. Start with slight vacuum and increase as necessary. Transfer all the sample to the crucible, using a minimum of hot water (80°C) to wash out the flask. Release the vacuum, carefully loosen the layer of sample in the bottom of the crucible without removing it, and add hot water. Repeat this wash several times. Finally, rinse the sample with acetone without removing it from the filter and dry under vacuum.
Dry the crucibles at 105°C for 12 hours and weigh while hot (this should not take more than 30 seconds). If the crucibles cannot be weighed immediately, cool them in a dryer with phosphorus pentoxide (P2O5) as desiccant.
Record the fibre residue recovered in terms of cell walls.
Calculate the cellular content (soluble material) subtracting this from 100.
NOTE:
In the case of grains and mill by-products a gelatinous substance may form deriving from starches and protein that blocks the filter and hinders washing. To improve filtration, force air backwards through the filter. At the same time, prevent the sample from adhering to the bottom of the flask while bringing a boil by heating rapidly and stirring constantly.
Calculations
Cell walls (%) on “dry basis” or “as is”:
The percentage of cellular content is calculated by subtracting the % of cell walls from 100: % cellular content + 100 - cell walls
(Van Soest, P.J. and R.H. Wine, 1967, J. Assoc. Official Anal. Chem., 50:50).
Figure 8. Determination of fiber content by the neutral detergent method
This method determines the amount of mineral substances insoluble in chlorhydric acid that are contained in feedstuffs. Depending on the mineral content of the sample, apply method A (for feed with high organic material content) or B, recommended for feed with high mineral content, including those containing over 1% of insoluble ash as previously evaluated by method A.
Reagents
Material and equipment
Method A
Calcine the sample following the method for determining ash. Transfer the residue to a 250–400 ml beaker using 75 ml 3N chlorhydric acid and evaporate until dry; continue to heat for 1 hour more to dehydrate any silica present. Cool, add 75 ml HCI 3N solution and gently heat to boiling for 15 minutes. While hot, filter the solution through an ash-free filter paper and wash the residue with hot water until the filtrate is no longer acid.
Dry the filter paper containing the residue and calcine at 550–700°C in a preweighed platinum crucible. Cool in dryer and weigh.
Figure 9. Determination of ash insoluble in chlorhydric acid-Method A
Method B
Weigh out 5 g of sample to within 0.001 g and transfer to a 250–400 ml beaker. Add 25 ml 3N chlorhydric acid solution. Stir carefully and wait until no further gas is given off.
Add another 50 ml chlorhydric acid and wait until no further gas is given off then place the beaker in a boiling water-bath for 30 minutes to hydrolyze the starches present. Filter the solution while still hot through an ash-free filter paper then wash with 50 ml warm water until the filtrate is no longer acid (see note). Place filter paper containing residue in a preweighed platinum crucible, dry and calcine the sample at 550–700°C. Transfer the ashes to a beaker using 75 ml 3N chlorhydric acid and continue as in the above method from the stage when the sample is heated gently for 15 minutes.
NOTE:
If filtering becomes difficult, repeat the analysis replacing the 50 ml of 3N chlorhydric acid with 50 ml of trichloroacetic acid and rinse the filter with warm B solution.
Calculations
Measure the weight of residue and express the result as a percentage (%) of the sample.
Figure 10. Determination of ash insoluble in chlorhydric acid - Method B
This method determines the total amount of carbohydrates, based on hydrolyzable starch and soluble sugar content.
Reagents
Material and equipment
Method
Extraction:
Weigh out 1.0 g of dry or 2.5 g of wet sample to within 0.001 g containing approximately 60 to 300 mg of total available carbohydrates.
Transfer quantitatively to a stoppered 100 ml graduated cylinder.
Add 10 ml water and stir with glass rod to disperse the sample.
Add 13 ml of the perchloric acid solution. Stir constantly with glass rod for 20 min.
Rinse rod with distilled water and bring volume to 100 ml. Mix and filter into a 250 ml volumetric flask.
Rinse the graduated cylinder with distilled water and add this to the volumetric flask. Calibrate the flask with distilled water and shake.
Determination:
Dilute 10 ml of the extract to 100 ml with distilled water. Use a pipette to transfer 1 ml of diluted filtrate to a test-tube
Using a pipette take two 1 mg samples of distilled water for duplicate blanks and put each one in a test-tube.
Take two 1 ml duplicate blanks using the dilute glucose solution.
Quickly add freshly prepared anthrone reagent to all the tubes. Cap the tubes and mix vigorously. Place in a water-bath and heat for 12 minutes.
Cool rapidly to room temperature. Transfer the solution to 1 cm spectrophometer cells. The green colour is stable for only 2 hours.
Read the absorption rate at 630 nm against the blank.
Calculations (Clegg, K.M. (1956). J. Sci. Food Agric. 7, 40).
Where:
W = weight of sample (g)
a = absorption of diluted standard(1)
Figure 11. Determination of total carbohydrates available in feed
This technique allows the proportion of nitrogen of protein and non-protein origin in material to be identified. It can be applied to all types of materials.
Reagents
Material and equipment
Method
Weigh out 2 g of sample with under 25% crude protein, 1 g for ranges between 25 and 50% protein and 0.5 g of materials containing over 50% crude protein. Transfer to a Kjeldahl flak and add approximately 50 ml distilled water, a few granules of antiboil and one or two drops of antifoam.
Heat to boiling point for 30 minutes (taking care that it does not dry out). While still hot, add 2 ml of aluminium and potassium sulphate solution, mix thoroughly and heat to boiling point; add 50 ml copper acetate solution, mixing in well, and let cool. Filter under vacuum. Wash the flask and precipitate with 50 ml cold distilled water.
To evaluate protein nitrogen, analyze the residue by the Kjeldahl total nitrogen process. Non-protein nitrogen is analyzed by the same technique using the filtered liquid. The results obtained from solids and liquids by the Kjeldahl method will give direct data for protein nitrogen (PN) and non-protein nitrogen (NPN) respectively.
Figure 12. Determination of protein and non-protein nitrogen in feed
The most accurate method for determining protein concentrations is probably that of acid hydrolysis. Most other methods are sensitive to the amino-acid content of proteins and so absolute concentrations cannot be obtained. Lowry's method is no exception but it is significantly constant from protein to protein. This has led to it's being widely used and to the results being accepted completely, with absolute precision in all determinations for almost all cases where mixtures of proteins or crude extracts are involved.
Reagents
| PREPARATION OF PROTEIN STANDARDS | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Stock solution (μl) | 0.00 | 1.25 | 2.50 | 6.25 | 12.50 | 25.00 | 62.50 | 125.00 | 250.00 |
| Water (μl) | 500 | 499 | 498 | 494 | 488 | 475 | 438 | 375 | 250 |
| Protein concentration (μg/ml) | 0 | 10 | 20 | 50 | 100 | 200 | 500 | 1,000 | 2,000 |
Method
To 0.1 ml of sample or standard sol. add 0.1 ml NaOH 2N sol. Heat to 100°C in boiling water-bath for 10 minutes to hydrolyze the sample.
Let cool to room temperature and add 1 ml of freshly prepared complex reagent. Leave to rest 10 minutes at room temperature.
Add 0.1 ml Folin reagent, agitate in a vortex mixer and leave to rest at room temperature for 30–60 minutes (do not exceed this time).
Read the absorption at 750 nm if the protein concentration is less than 500 μg/ml, or at 550 nm if between 100 and 2,000 μg/ml.
Draw a standard absorption curve as a function of initial protein concentration and use this to determine the protein content of the sample.
Figure 13. Determination of protein in feed by Lowry's method
NOTES:
If the sample appears as a precipitate, dissolve in NaOH 2N and hydrolyze as in step (i). Use 0.2 aliquots of the hydrolyzed solution for step (ii).
All the cells or other complex samples may need pre-treatment as described by Burton (1984) for DNA.
Mix rapidly once the Folin reagent is added; this is important for proper reproducibility.
A group of standards is necessary for each test, preferably in duplicate or triplicate.
The urea content of feed ingredients can be quantified with this technique.
Reagents
Material and equipment
Method
Weigh approximately 2 g of sample to within 0.001 g or an amount expected to contain 50–200 mg urea and place in a 500 ml volumetric flask. Add 150 ml chlorhydric acid 0.02N, agitate for 30 minutes then stir in 10 ml of the sodium acetate solution. Add 1g active charcoal, shake thoroughly and let the mixture rest for 15 minutes. Add 5 ml Carrez solution I, followed by 5 ml Carrez solution II, stirring well between the two additions. Calibrate with distilled water and mix well. Filter one part through a dry filter paper into a clean, dry 250 ml beaker.
Determination
Transfer 10 ml of filtrate to a test-tube with a ground glass stopper. Add 10 ml 4-DMAB solution. Stir; rest for 15 minutes. Measure the absorption of the solution as indicated above against a reference solution prepared with 10 ml 4-DMAB solution and 10 ml water. Draw a graph relating absorption to the amount of urea present.
Expression of results
Determine the quantity of urea in the sample referring to the calibration curve drawn. Express results as a percentage of the sample: % urea × 0.4665 = % N urea
NOTE:
If the sample is highly coloured, the amount of active charcoal should be increased to above 5 g. The final solution after filtering should be colourless.
Figure 14. Determination of urea contents in feed ingredients
This method can determine uric acid content and its salts in both chicken manure and in feed and ingredients.
Reagents
Sodium hydroxide solution. Dissolve 50 g NaOH in 50 ml water, stir well and store solution in a plastic container.
Neutral formaldehyde solution:
Check the concentration of the solution available by mixing 30 ml of it with 50 ml NaOH IN solution and 25 ml hydrogen peroxide solution (20 volumes). Heat in water-bath until effervescence stops. Cool and titrate with NaCI IN using phenophthalein indicator. Perform a blank titration using 3 ml water instead of formaldehyde and calculate the concentration as follows:
1ml NaOH 1N solution = 0.03 g formaldehyde.
| Where | B = blank titration |
| T = titration of sample |
Prepare a neutral solution with 17.5 g of formaldehyde, 250 ml water and 500 ml ethanol. Adjust the pH to 7.0 with a solution of NaOH 0.1N. Dilute to 1,000 ml with water, stir and adjust pH again if necessary.
Buffer solution of succinate. Heating, dissolve 29.5 g succinic acid in 750 ml water and 20 ml of the NaOH solution. Let cool and add a sufficient amount of formol solution containing 17.5 g formaldehyde, stir well and adjust pH to 6.0 with NaOH solution. Dilute to 1,000 ml with distilled water, stir and adjust pH if necessary.
Sodium thiosulphate solution. Dissolve 25g Na2S2O3 • 5H2O in 1,000 ml distilled water.
Actol solution. Heating, dissolve 3 g actol in 50 ml distilled water and 1 ml lactic acid. Dilute to 100 ml with water, filter and store in a dark bottle. Do not expose to direct light.
Ammoniated magnesium solution. Dissolve 8.75 g of MgSO4 • 7H2O and 17.5 g NH4 Cl in 50 ml water. Add 30 ml NH4OH (d = 0.88 g/ml), stir well and dilute to 100 ml with water.
Benedict and Hitchcock's reagent. Mix 35 ml actol solution with 15 ml ammoniated magnesium solution. Add 50 ml ammonia hydroxide (d = 0.88 g/ml). Prepare immediately before using.
Standard uric acid solution. Weigh 250 mg uric acid to within 0.1 mg and transfer to a round-bottomed flask coupled to a reflux condenser. Add 100 ml of the ethyl formaldehyde solution and heat by reflux in a water-bath for 30 min, shaking frequently. Cool and transfer to a 250 ml volumetric flask, washing the round flask with ethyl formaldehyde sol. calibrate with this solution and mix. 1 ml contains 1 mg uric acid.
Petroleum ether, boiling point 40–60°C.
Material and equipment
Extraction of uric acid:
From chicken manure: weigh some 0.4 g dry manure to within 0.001 g and place in a 150 ml round-bottomed flask. Add 60 ml neutral formaldehyde solution, connect to a reflux condenser and heat in water-bath for one hour. Cool and filter through a crucible (porosity 4) into a 100 ml volumetric flask. Rinse the flask 3 times consecutively with 10 ml portions of the ethyl formaldehyde solution, filtering each quantity through the crucible into the volumetric flask. Calibrate with the same solution and shake.
From feed: weigh 4 to 5 g of sample to within 0.001 g and extract fat with petroleum ether. Transfer the defatted sample quantitatively to a round-bottomed flask and remove the remaining solvent with a gentle air current. Continue the analysis as above, beginning with the addition of 60 ml ethyl formaldehyde solution.
Determination
Using a pipette, transfer 20 ml preweighed extract of sample according to the methods described, to a 50 ml centrifuge tube. Add 10 ml Benedict and Hitchcock's reagent, mix well and leave in dark to rest one hour. Centrifuge at 2,000 rpm for 15 min. Remove supernatant and leave to drain for 10 min. Carefully clean away any remaining liquid without disturbing the precipitate and add 20 ml sodium thiosulphate. Dissolve the precipitate, stirring with a thin glass rod. Using a pipette transfer 5 ml of this solution to a 200 ml volumetric flask containing 40ml succinate buffer solution, calibrate with distilled water and mix well. Measure the absorption of the solution at 294 nm in 10 mm silica cells compared against a solution prepared by mixing 5 ml sodium thiosulphate solution with 40 ml succinate buffer solution brought to 200 ml with distilled water. Determine the amount of uric acid by means of a calibration curve.
Calibration curve
Using a pipette, transfer 2, 4, 6, 8, 10 and 12 ml standard uric acid solution (equivalent to similar amounts of uric acid in mg) into a series of 50 ml centrifuge tubes and bring them up to 20 ml with the ethyl formaldehyde solution. Add 10 ml Benedict and Hitchcock's reagent to each tube, stir well and leave in darkness for one hour. Continue the method as for determination, from the centrifuge stage. Measure the absorption of the solution and draw the calibration curve, plotting absorption (y) against the corresponding amount of uric acid in mg (x).
Expression of results
The N content of the uric acid as a % of the sample is given by the formula:
| Where | A = mg of uric acid (in the aliquot of extract of sample) determined by photometric measurement. |
| W = weight of sample in g. |
Figure 15. Determination of uric acid in chicken manure and feed ingredients
This method gives an estimate of the availability of minerals on the basis of digestibility in acid.
Reagents, material and equipment
Method
Use the residue from the determination of ash. Carefully heat 25ml chlorhydric acid to avoid spattering; filter through paper and rinse this with hot water until it is acid free. Place the filter paper and residue in a dry, preweighed porcelain crucible and place in crucible furnace at 600°C for 2 hours or until it is free of carbon.
Figure 16. Determination of ash soluble and insoluble in chlorhydric acid
Calculation
The evaluation of calcium in feed is very important, since imbalance with phosphorous or other minerals will lead to reduced growth.
Reagents
Material and equipment
Figure 17. Determination of calcium in feed and feed ingredients
Method
In a porcelain crucible, calcine 2.5 g finely ground sample. Add 40 ml HCl and a few drops of NHO3 to the residue; heat the crucible to boiling, cool and transfer to a 250 ml volumetric flask, calibrate and mix. Transfer 100 ml sol. for cereals or feedstuffs containing cereals or 25 ml for feed containing minerals into a beaker. Dilute to 100 ml and add 2 drops methyl red. Add NH4OH drop by drop until the mixture turns dark orange, then add 2 drops HCl to give a pink colour. Dilute with 50 ml water, boil and shake in 10 ml 4.2% sol. of ammonia oxalate. Adjust the pH with acid if necessary to bring back the pink colour.
Let rest, filter and wash the precipitate with the NH4OH (1.5%) solution. Place the filter paper with the precipitate in a beaker, add a mixture of 125 ml water and 5 ml H2SO4, heat to 70°C and titrate with the permanganate solution; calculate:
Like calcium, this mineral is essential for the proper growth of farmed organisms; sufficient amounts must be present in diets to meet the animals metabolic needs. In proteins of vegetable origin, this element may be partly in the form of phytates, therefore availability may be limited and so the phytic acid content should be determined before such materials are used.
Reagents
Molybdate reagent. Add 40 g ammonia molybdate 4•H2O to 400 ml hot water and cool. Dissolve 2 g ammonia meta-vanadate in 250 ml hot water, cool and add 450 ml perchloric acid at 70%. Gradually shake the molybdate sol into the vanadate sol and dilute to 21.
Standard phosphorous solution. Prepare a stock solution by dissolving 8.788 g di-hydrogen potassium orthophosphate in water and bring to 11. Prepare the working solution by diluting the stock sol to 1 in 20 (Working conc. 0.1 mg P/ml).
Material and equipment
Method
Place an aliquot as for the determination of Ca in a 100 ml flask and add 20 ml molybdovanadate reagent. Calibrate, mix and let rest for 10 min.
Transfer aliquots of the standard working sol containing 0.5, 0.8, 1.0 and 1.5 mg of P into 100 ml flasks and process as above.
Read the sample at 400 nm, adjusting the 0.5 standard to 100% transmission.
Determine mg of phosphorous in the sample using a standard curve.
Figure 18. Determination of phosphorus in diets and feed ingredients
This method is to determine the amount of common salt present in fish meal and other ingredients.
Reagents
Method.
Weigh 2 g of sample in a 250 ml Erlenmeyer flask; moisten with 20 ml water, add 15 ml of the silver nitrate solution with a pipette and stir well.
Add 20 ml concentrated nitric acid and 10 ml potassium permanganate solution and mix. Heat the mixture continuously until the liquid is clear and the nitrous vapours disappear. Cool.
Add 10 ml urea solution and leave to rest 10 min.
Add 10 ml acetone and 5 ml ferric indicator and titrate the excess silver nitrate with the thiocyanate solution to a final reddish-brown.
Calculations
Calculate results as NaCl:
Figure 19. Determination of sodium chloride in fish meal and other ingredients
Reagents
Material and equipment
Method
Heat 2 g of sample in a silica crucible at 100°C to eliminate moisture. Add a few drops of olive oil and heat over flame until flames are no longer produced.
Calcine at 500°C in crucible furnace for 24 hours, cool and add 2 ml concentrated HCl to dissolve residue.
Bring up to 100 ml with distilled water; take 1ml of the solution and dilute again to 100 ml with distilled water.
Adjust the flame photometer to give a reading of 100 with the standard 10 ppm solution and read the sample solution.
If the reading does not fall between 50 and 100, immediately dilute to give the required reading.
Figure 20. Determination of potassium content in feed ingredients
Chromic oxide is the substance most commonly considered in evaluating the digestibility of experimental fish diets. The following method is a modification of the one proposed by Furukawa and Tsukahara so as to handle microsamples in determining the chromic oxide content of feed and faeces.
Reagents
Material and equipment
Method
Grind the feed and faeces finely, having priorly eliminated flakes and any other foreign material; keep dry. Weigh 50 to 100 mg sample to within 0.0001 g, place in a 100 ml Kjeldahl flask and weigh the scale pan again to adjust the weight of the sample. Add 5 ml HNO3 and digest at a gentle boil for at least 30 min or until yellowish vapours stop rising. If the quantity of liquid reduces significantly and nitrous vapour is still given off add 5ml more nitric acid and continue with the digestion. Finally, the solution should be clear, greenish and should not give off nitrous vapours. Leave to cool.
Once the solution is cool, carefully slide down the flask sides 3 ml perchloric acid. Do this very cautiously under an extraction hood because if the digestion is not complete there may be an explosive reaction. Place the flask in the digester again and continue boiling until the solution turns from green to lemon yellow; turn off the digester and leave to cool. When cold there should be a reddish ring around the edge of the liquid; if not, or if the liquid turns green again, repeat digestion until the change is permanent.
Transfer the cold liquid to a 25 ml volumetric flask, washing the Kjeldahl flask several times with distilled water, and calibrate. Adjust the spectrophotometer to 0 with a blank reagent and read at 350 nm. The blank should be prepared at the same time as the sample, using only the acids and distilled water.
Calculations
Calculate the amount of chromic oxide (mg) present in the sample:
Where: Y = absorption 0.0032 and 0.2089 are constants.
Calculate the % of chromic oxide in the sample:
| Where: | X = weight of chromic oxide |
| A = weight of sample. |
Figure 21. Determination of chromic oxide in faeces and feeds
The method is used to determine the amount of free lysine that reacts with 1-dinitric fluorobenzene.
Reagents
Stock solution of mono E N diphenylnitrate-lysine •HCl (p.m. 366.77, 39.85% lysine). Dissolve 314 mg in 250 ml concentrated HCl; it dissolves slowly, so preparation should be started the day before.
Standard dilute solution of DPN-lysine. Dilute 10 ml of stock solution in 100 ml distilled water to make the standards. After this, 2 ml of the solution contains around 0.1 mg of lysine, and dissolved to 100 ml it gives a net Absorption of around 0–4 to 435 nm in a 1 cm cell.
Solution of 1-fluoride 2,4 dinitrobenzene (FDNB). Handle with disposable plastic gloves. If the FDNB is solid, place in a flask of hot water to melt; take up approximately 0.4 ml with an automatic pipette and dissolve in 15 ml of ethyl alcohol per sample. This must be prepared daily.
Methoxycarbonil chloride (methyl chloroformate). MCC is unstable and must be kept under refrigeration. Handle with caution.
Sodium bicarbonate solution at 8% in distilled water.
Sodium hydroxide solution at 12%. Used to increase the pH to 8.5 during the reaction with MCC. Must be stored in a plastic container. Have a dropper with HCl 1M at hand to correct any excess.
Sodium carbonate buffer, pH 8.5. Dissolve 19.5 g of NaHCO3 and 1g Na2CO3 in 250 ml distilled water; adjust pH if necessary. Store in a well filled and thoroughly sealed bottle to prevent CO2 loss.
Concentrated chlorhydric acid
Material and equipment
Method
Mix the sample so that it will go through the 5 mm mesh. The amount and sample used should contain about 12 mg of available lysine, equivalent to 0.3–2 g of the sample. Transfer to a round-bottomed flask. Add 2–3 glass beads and 10 ml NaHCO3, shake again so that the sample does not adhere to the sides.
Add 15 ml FDNB and agitate gently for 2 hours. Make sure that the sample does not stay on the sides. Evaporate in ethanol, except for the water; in a water-bath, the flask should lose approximately 12.5 g.
Cool and add 30 ml HCl.1M to neutralize the NaHCO3. The addition of the water present will bring the volume to 40 ml and the acid concentration to 6M. Reflux gently for 16 hours.
Disconnect the flask and wash the refrigerant with a little water. Filter while still hot into a 250 ml volumetric flask. Rinse the flask and the filter several times with distilled water and collect in the volumetric flask; calibrate when cold with distilled water. Sometimes a dinitrophenyl precipitate forms that can be extracted with a pipette once precipitated or else eliminated with ether.
Place 2 ml of the filtrate in two sealed test-tubes and label them A and B. Add 5 ml ether to tube B and shake. Extract the ether (upper layer) with an automatic pipette. Place the test-tube in a water-bath at 90°C until all the ether is driven off and cool.
Add one drop phenophthalein to the tube and add the NaOH solution drop by drop until a slight pink appears; add 2 ml carbonate buffer and in an extraction hood add 0.5 ml methylchloroformate. Seal tube and shake vigorously, freeing pressure carefully. After 5 or 10 minutes, add 0.75 ml HCl drop by drop, shaking to prevent foam from forming. The solution is extracted with ether four times as described above. Eliminate residual ether in a water-bath, cool and bring the volume to 10 ml with distilled water.
In the pauses during the processes with tube B, extract tube A with ether three times; eliminate the residual ether and bring to 10 ml with HCl. Read the absorption of both tubes to 435 nm in the spectrophotometer with distilled water. The reading of tube A minus that of tube B (blank) is the absorption of the DNP-Lysine.
Absorption of standard DNP-Lysine
Place 2 ml diluted DNP-L in two test-tubes, A and B, following the process described above. Repeat until confident of the processes. The net absorption will be used to calculate problem samples. Blank B usually has an absorption of 0.01; if the figure is much higher, check the procedure, especially the, the pH of the carbonate buffer, the wavelength being read and the peroxide-free ether.
Precautions with feedstuffs of vegetable origin
With feed of vegetable origin, complete hydrolysis is not achieved after the 16 hours, and since a longer time will decompose the DNP-L, a second digestion must be carried out in the case of unknown materials. For this, after digestion it is filtered and the filtrate is processed as described. Place the residue in a round-bottomed flask with HCl 6 M and a few glass beads (the residue from the replicas may be added); the volume of HCl should be less than 30 ml. Leave in a reflux apparatus for 16 hours, filter while hot and bring to 100 ml following the method described. If the result is very small it is eliminated, if not, the corrections necessary for the material are made.
To determine the loss of DNP-L, a correction factor must be calculated; this varies according to material. For how to calculate consult Makade, M. L. and Liener, I. E., 1969. Anal. Biochem., 27:271.
Calculations
| Where: | C = g of lysine/16g of N |
| Ws = weight of standard expressed as mg of Lysine in 2 ml; 0.1 if prepared as indicated. | |
| Wu = Weight of sample in mg. | |
| As = net absorption of standard. | |
| Au = Net absorption of unknown. | |
| V = Volume of filtered hydrolyzed material; 250 ml is recommended (less for second digestion). | |
| a = aliquot of filtrate; 2ml recommended. | |
| CP = Crude protein, 6.25 × g N/100 mg material. |
To express the result as g lysine/kg of PC, change one 100 for 10 in the formula.
NOTE: glass equipment may take on a yellow tinge due to the dinitrophenyl. Hydrolyates and DNP-L solutions must be protected from light, especially if they have alkaline pH.
Figure 22. Determination of lysine available in feed ingredients
Fish, depending on the species, have different capacity for using carbohydrates in their diet, therefore it is important to have a rapid method for determining total sugars available.
Reagents
Material and equipment
Method
Dissolve 8 g molasses and bring to 500 ml with distilled water (filter). Hydrolyze 100 ml of filtrate with 5 ml HCl (Sp. g 1:18) and leave for 24 hours. Neutralize with NaOH at 20% using phenophthalein as indicator and dilute to 200 ml.
Standardization of Soxhlet solution: measure 10 ml of Soxhlet solution (a) and (b) with a graduated pipette and empty into a Erlenmeyer flask, mix and add 30 ml water. Using a burette, add a volume of standard working solution almost enough to reduce the copper in the Soxhlet solution. Bring to boiling and boil for 2 min. Add 4 drops methylene blue and rapidly complete titration while still boiling until a brilliant orange. Repeat several times and determine the volume of solution required to completely reduce 20 ml of Soxhlet solution.
Titration of sample: first do an approximate titration; place 10 ml each of solutions (a) and (b) into a flask, add 10 ml aliquots of sample solution. Add 40 ml water and bring to boil. If the blue colour persists, titrate with a standard working solution and calculate the approximate sugar content of the sample. To determine sugar content precisely, put 10ml of Soxhlet solutions (a) and (b) into a flask and add an aliquot of the sample solution. The volume of the sample used will depend on its sugar content. Add water as shown in the table, mix and boil. While boiling, add standard working solution with a burette until titration is almost complete. Add methylene blue and complete titration.
| VOLUME OF SAMPLE USED IN SOXHLET TITRATION (AOAC, 1970) | |||
| ml of H2O | ml of sample | g of sample in aliquot | Total as invert sugar (%) |
| 40 | 10 | 0.08 | 73 |
| 35 | 15 | 0.12 | 82–58 |
| 30 | 20 | 0.16 | 61–41 |
| 25 | 25 | 0.20 | 49–35 |
| 20 | 30 | 0.24 | 49–29 |
Calculation
| Where: | F = volume of standard necessary to reduce 20 ml Soxhlet sol. |
| M = volume of standard sugar sol. to complete titration. | |
| I = weight of invert sugar in 1 ml standard working sol. | |
| W = weight of sample in the aliquot used. |
Figure 23. Determination of total available sugars in molasses
