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28 Estimation of Nitrates and Nitrites (Wiseman and Jacobson 1965)

28 Estimation of Nitrates and Nitrites (Wiseman and Jacobson 1965)

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14.28



Estimation of Nitrates and Nitrites



369



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

distilled water.

Procedure

Sampling

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.

Analysis

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.

Calculation

Total nitrate nitrate ỵ nitriteị

Conc. of nitrate mg=mLị in final solution from standard curve



g sample taken DM basisị 10

Nitrite concentrationmg=mLị

Conc. in final solution obtained from standard curve



g sample taken ðDM basisÞ Â 10

Table 14.9 Protocol

Standard

Reagents/chemicals

Extract (mL)

Distilled water (mL)

Working standard (mL)

Conc. Of standard (mg)

Reagent (1) (mL)

Bray’s indicator (g)



Sample

1.0

0.0

0.0



9.0

0.5



Blank

0.0

1.0

0.0



9.0

0.5



1

0.0

0.0

1.0

10.0

9.0

0.5



2

0.0

0.25

0.75

7.5

9.0

0.5



3

0.0

0.5

0.5

5.0

9.0

0.5



4

0.0

0.75

0.25

2.5

9.0

0.5



370



14



14.29



Nutritional Evaluation of Forages



Estimation of Total Glucosinolates

(McGhee et al. 1965)



Principle

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.

Reagents

(a)

(b)

(c)

(d)

(e)



0.1 N AgNO3

95% Ethanol

8% (w/v) Ferric ammonium sulphate

6 N-HCl

N Potassium thiocyanate solution



Equipments

Grinder, heater, filter paper, Buchner funnel, wash bottle, beakers, and burette.

Procedure

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.

Calculation

%Glucosinolates





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



14.30



Estimation of Oxalic Acid



14.30



371



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.

Principle

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.

Reagents

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.

Procedure

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.



372



14



Nutritional Evaluation of Forages



Calculation

Oxalateg=100gị ẳ N=20 KMnO4 used mLị 0:00225 250=50 100=2

Or

Oxalate%ị ẳ N=20 KMnO4 used mLị Â 0:5625



14.31



Estimation of Trypsin Inhibitor in Forages

(Roy and Rao 1971)



Principle

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

the sample.

Reagents

1.

2.

3.

4.

5.



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



Procedure

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.

Computations

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

accurate results.



14.32



Estimation of Cyanogenic Glycosides



14.32



373



Estimation of Cyanogenic Glycosides (AOAC 1995b)



14.32.1



Qualitative Test



Reagents

1. Filter paper strips

2. Picric acid solution (1%)

3. 10% Sodium carbonate (Na2CO3)

Procedure

• 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.



14.32.2



Titrimetric Method for Quantitative Test



Acid titration method

Apparatus and Glassware

1.

2.

3.

4.

5.

6.



Micro-Kjeldahl distillation apparatus

Kjeldahl flasks

Conical flasks

Burette

Pipette

Gooch crucible



Reagents

1.

2.

3.

4.



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



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