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28 Estimation of Nitrates and Nitrites (Wiseman and Jacobson 1965)
Estimation of Nitrates and Nitrites
5. Standard nitrate solution:
Stock solution (1 mg/mL): 1.37 g sodium nitrate in 1 L distilled water and
working solution (10 mg/mL); dilute 1 mL of stock solution to 100 mL with
Collect samples from different places in the field. Moist sample (silage) should be
kept frozen until analysed. Green may be dried immediately in an oven at 60 C.
1. Take 1 g air dry material or 3–5 g moist sample (nitrate content ranging between
1 and 8 mg) in a conical flask of 150 mL capacity.
2. To above add 100 mL of 0.1 N HCl and place for 1 h with occasional shaking.
3. If the extract is strongly coloured, decolourize with activated charcoal and then
filter through Whatman filter paper (no. 1).
4. The sample extract, blank and standard are run as given below (Table 14.9).
5. Shake the above solutions avoiding exposure from light.
6. Centrifuge at 3,000 rpm for 5 min.
7. Remove any film on top and collect the clear red supernatant.
8. Read at 520 nm and find out concentration of nitrate in sample from the standard
curve drawn taking OD against concentration.
Total nitrate nitrate ỵ nitriteị
Conc. of nitrate mg=mLị in final solution from standard curve
g sample taken DM basisị 10
Conc. in final solution obtained from standard curve
g sample taken ðDM basisÞ Â 10
Table 14.9 Protocol
Distilled water (mL)
Working standard (mL)
Conc. Of standard (mg)
Reagent (1) (mL)
Bray’s indicator (g)
Nutritional Evaluation of Forages
Estimation of Total Glucosinolates
(McGhee et al. 1965)
The glucosinolates (GSL) of sample are collected in water. It is then treated with
0.1 N AgNO3 and 95% ethanol. The solution with ferric ammonium sulphate in
acidic medium is titrated against N/100 potassium thiocyanate.
0.1 N AgNO3
8% (w/v) Ferric ammonium sulphate
N Potassium thiocyanate solution
Grinder, heater, filter paper, Buchner funnel, wash bottle, beakers, and burette.
Put 10 g of ground sample into 250 mL boiling water for enzyme deactivation for
5 min. The enzyme deactivation step is normally not allowed to prolong 5 min to
avoid the breakdown of glucosinolates. Filter the contents on a Buchner funnel.
Wash the residue with 50 mL hot water and filter again. Make the volume of filtrate
to 500 mL. Take 25 mL aliquot in a beaker and add 10 mL of 0.1 N AgNO3 solution
and 25 mL ethanol (95%). Reflux the contents on a water bath for 45 min. Cool
to room temperature and make volume to 100 mL with distilled water and filtered
through ordinary filter paper. Take 25 mL of supernatant in a 125-mL flask
containing 2 mL of 6 N-NHO3 and 6 mL of 8% (w/v) ferric ammonium sulphate
solution. Titrate this homogenous mixture against 0.01 N potassium thiocynate till
a pale salmon colour is obtained. Run parallel blank with each determination.
Blank titrationị 4:0 Â 0:01 Â Mol. Wt. of GSL Â Total volume
1; 000 Â 2:0 Â Sample Wt: Â 25:0
Total volume ¼ 500 mL
Mol. wt. of GSL ¼ 411
Estimation of Oxalic Acid
Estimation of Oxalic Acid (Abaza et al. 1968)
Oxalates are present in free and salt forms in the feed, fodder and forages. Beet and
spinach are rich in oxalates, and among straw/stover, paddy straw contains substantial amount of oxalic acid. Conversely, its low levels are present in peas, beans and
brassicas, etc. In monogastric animals such as pigs, poultry and rabbits, oxalates
containing diets reduce growth as well as calcium retention. However, ruminants are
able to decompose oxalic acid with the help of rumen micro-organisms.
The sample is extracted in hydrochloric acid (HCl) and precipitated in the form of
calcium oxalate by adding calcium chloride. The precipitate is then washed and
titrated with N/20 potassium permanganet (KMnO4) in the presence of dilute
sulphuric acid (H2SO4) at 70 C. 1 mL of N/20 potassium permanganet is equivalent
to 2.25 mg of oxalate.
1. 6 N hydrochloric acid – Add equal amounts of conc. HCl and distilled water to
prepare 6 N HCl.
2. Calcium chloride solution (5%) – Dissolve 5 g of calcium chloride anhydrous
(CaCl2) in 100 mL distilled water.
3. Sulphuric acid (H2SO4) in water (1:4 ratio) – A volume of sulphuric acid is
slowly poured into four volumes of distilled water.
4. Concentrated ammonia (NH4OH).
5. Methyl red indicator.
1. To weigh 2 g of sample in a 250-mL volumetric flask, add 190 mL of distilled water
and 10 mL of 6 N HCl. Boil the contents on a water bath. Make up the volume after
cooling and filter the supernatant through Whatman (no. 41) filter paper.
2. Pipette out 50 mL of supernatant in a beaker and add 20 mL of 6 N HCl.
Evaporate to about half of its volume and filter. The precipitate is washed in
between to make the volume to about 125 mL.
3. Add 3–4 drops of methyl red to the filtrate followed by concentration ammonia
till the solution turns faint yellow. Heat the contents to 90–100 C and filter (using
Whatman no. 41 filter) after cooling to remove the precipitated impurities, if any.
Add 10 mL of 5% CaCl2 with constant stirring and allow to stand for 24 h.
4. Filter through Whatman no. 41 filter paper and wash the precipitate several times
with hot water to make it free from calcium ions. Transfer the precipitate to the
original beaker by washing with distilled water. Then add dilute sulphuric acid
solution (1:4) till the precipitate is completely dissolved.
5. Warm the contents to 70 C and titrate with N/20 KMnO4 near to end point and
stir the filter paper to remove to the side contents of the beaker, wash with hot
distilled water and complete the titration.
Nutritional Evaluation of Forages
Oxalateg=100gị ẳ N=20 KMnO4 used mLị 0:00225 250=50 100=2
Oxalate%ị ẳ N=20 KMnO4 used mLị Â 0:5625
Estimation of Trypsin Inhibitor in Forages
(Roy and Rao 1971)
The casein is used as a substrate for assaying the activity of the trypsin enzyme. The
inhibition of the trypsin enzyme activity is measured in the extract prepared from
5% Trichloroacetic acid (TCA) solution
2% Casein solution in phosphate buffer
0.1 M Sodium phosphate buffer (pH – 7.5)
Trypsin solution (5 mg/mL)
0.001 M Hydrochloric acid
Put 4 g of the finely ground deffated material feed in stoppered 250-mL conical
flask and treat with 40 mL of 0.10 M sodium phosphate buffer (pH 7.5) and 40 mL
of distilled water. Shake the suspension in the flask for 3 h on shaker and centrifuge
at 700 Â g for 30 min at 15 C. Dilute the supernatant so that 40–60% activity of
control enzyme is inhibited. Prepare incubation mixture containing 0.5 mL trypsin
solution, 2.0 mL casein solution (2%), 1.0 mL 0.1 M sodium phosphate buffer,
0.4 mL HCl (0.001 M) and 0.1 mL extract or supernatant. Make the volume of
incubation mixture to 4.0 mL. Incubate the mixture at 37 C for 20 min. Add 6.0 mL
5% TCA to stop the reaction. Run parallel blank. Record the absorbance on the
spectrophotometer at 280 nm.
One trypsin unit is defined as an increase of 0.01 absorbance unit at 280 nm in
20 min for 10 mL reaction mixture and the trypsin inhibitory activity as the number
of trypsin unit inhibits (TUI) is expressed per mg of protein. To express the value of
specific activities, the total TUI expressed per mg of protein. The value for trypsin
inhibitory activity must have about 40–60% inhibition to have reproducible and
Estimation of Cyanogenic Glycosides
Estimation of Cyanogenic Glycosides (AOAC 1995b)
1. Filter paper strips
2. Picric acid solution (1%)
3. 10% Sodium carbonate (Na2CO3)
• Dip filter paper strips in 1% picric acid solution and after drying, further dip into
10% Na2CO3 solution and dry again. Store the strips in stoppered bottle.
• Place the sample of plant material in test tube. Insert a piece of moistened
sodium picrate paper in tube while taking care that it does not come in contact
with the sample. Add few drops of chloroform and stopper tube hermetically.
The sodium picrate paper gradually turns orange and then brick red if plant tissue
contains cyanogenic glycosides. The rapidity of change in colour depends upon
amount of free HCN present.
• This test works well with fresh plant materials, but relatively dry substances
particularly seeds of various plants should be ground and moistened with H2O
and allowed to hydrolyse in stoppered test tube containing sodium picrate paper.
Titrimetric Method for Quantitative Test
Acid titration method
Apparatus and Glassware
Micro-Kjeldahl distillation apparatus
Silver nitrate (0.01 N)
Nitric acid concentrate
0.02 N Potassium cyanide (KHCN)
Ferric alum indicator
Place about 10–20 g finely ground sample (sieve no. 20) in 800-mL Kjeldahl
flask. Add 100 mL H2O and macerate at room temperature for 2 h. Further add