Fibre lignin cellulose nitrogenfree extract and starch

Fibre can mean many things. Crude fibre is an attempt to measure the roughage material in a feedstuff that is indigestible as far as the animal is concerned. It is an attempt to approximate the effect on the feedstuff of the digestive processes within the digestive tract by the use of inorganic chemicals, in this case, boiling dilute sulphuric acid, then boiling dilute sodium hydroxide, and the weight loss on ignition (which corrects for mineral ash content) of the residue is the fibre content.

There are many modifications of this method, they may be to make the process more representative of the ruminant digestive system, or the desired residue may be just the plant cell walls. It is not always possible to say that one procedure is better than another, therefore the chosen procedure may be that which has been used by workers involved in animal nutrition over a number of years in a certain geographical area. The decision may be to use the usual procedure favoured by the referees for research papers in a particular journal.

For several decades one of the leading authorities on the extraction of fibre from feedstuffs with particular reference to ruminant nutrition has been Professor P.J. Van Soest. His book, Nutritional Ecology of the Ruminant, has many helpful details (Van Soest, 1982, 1994). Various detergents are used to fractionate forage matter into its components. Neutral detergent is useful for separating the insoluble plant cell wall fraction, which is only partially digested by ruminal microorganisms (Van Soest and Wine, 1967). He considered that rather than the crude fibre (which entails the loss of soluble fibre components and variable amounts of the hemicelluloses and lignin), it is the proportion of plant cell wall and its degree of lignification that best determines the character and nutritive value of feeds and forage. The cellular contents determine the proportion of completely available nutrients, and consist of the bulk of the protein, starch, sugars, lipids, organic acids and soluble ash. The various processes are given in Table 4.1.

Table 4.1. Effect of detergents and reagents in forage analysis.

Residue type

Reagent

Process

Products

Acid detergent fibre (ADF)a

Cetyl trimethylammonium bromide in 0.5 M H2SO4

Boil for 1 h

Lignocellulose + insoluble mineral

Neutral detergent fibre (NDF)

Sodium lauryl sulphate, EDTA, pH 7.0

Boil for 1 h

Herbage cell wall minus pectins

Unavailable N

Acid detergent

Kjeldahl nitrogen on ADF residue

Maillard products plus lignified N

Cellulosea

None required

Ash from lignin step

By weight loss

Lignina

72% H2SO4 on ADF

3 h @ 20°C

Crude lignin

Hemicellulose

Not required

Calculate NDF-ADF Hemicellulose by difference

Silica (SiO2)

Conc. HBr treatment of ADF ash

Add dropwise to ash,1 h @ 25°C

SiO2 residue

aVan Soest and Wine (1968).

aVan Soest and Wine (1968).

In the NDF method, the sodium lauryl sulphate (sodium dodecyl sulphate) forms strong protein complexes which are soluble under the right conditions. The EDTA-disodium salt complexes with any Ca or Mg which would otherwise be included with the cell walls. The addition of sodium sulphite to cleave disulphide bridges in any added animal protein (e.g. keratin) in the feed is usually omitted unless major quantities of such substances are present. This is because the sulphite will also dissolve cell wall lignin, reducing its recovery (Moir, 1982; Van Soest et al., 1991). The reagent 2-ethoxyethanol, which aids solution of starches, is toxic and has been replaced by triethylene glycol (Cherney, 2000). Van Soest later recommended omission of anti-foaming decahydronaphthalene (decalin) because it greatly slowed the filtration step (Van Soest, 1973).

The ADF method has tended to replace the crude fibre procedure, especially when further fractionating the feed into lignin and cellulose. However, for an improved correlation between acid detergent fibre and ruminant digestibility, the modified acid detergent fibre (MADF) method of Clancy and Wilson was developed in Ireland (Clancy and Wilson, 1966). Although NIRS is currently the preferred technique to predict OMD (organic matter digestibility) which is then converted to a ME (metabolizable energy) value, this expensive procedure is rarely available to smaller laboratories. Prediction equations were developed for ME from MADF values, and although somewhat less accurate, they may give a working basis for ration formulation. Some examples are given below:

Fresh grass: ME (MJ kg-1 DM) = 16.20 - 0.0185[MADF] (Givens et al, 1990) Grass hays: ME = 15.86 - 0.0189[MADF] (Moss and Givens, 1990)

Grass silage: ME = 15.0 - 0.0140[MADF] (Givens et al, 1989)

This subject is discussed in depth in Chapter 4, 'Feed evaluation and diet formulation' in the Agriculture and Food Research Council (AFRC) advisory manual Energy and Protein Requirements of Ruminants (Alderman and Cottrill, 1993), and more recently by Coleman et al. (1999).

With non-ruminants, the only fibre determination required is by neutral detergent. Ruminants and other herbivores, which can partially digest fibre, will need the ADF or MADF methods.

Lignin and cellulose

Lignin, like fibre, is a complex substance. Lignins are phenolic polymers that occur in plant cell walls, and they impart, with cellulose, rigidity to stems. There are several molecular building blocks in lignin. When oxidized with nitrobenzene, lignin from angiosperms (grasses, herbs and flowers) yield p-hydroxybenzaldehyde, vanillaldehyde (from coniferyl alcohol component) and syringaldehyde. Lignin from gymnosperms (coniferous trees), however, lacks the syringyl group (Harborne, 1984). A typical lignin structural unit is shown in Fig. 4.2.

Estimation of lignin is complicated by the presence of strongly bound proteins. Other contaminants are carbohydrates, chemically bonded cinnamic acids, cutins and tannins. A partial loss of lignin may also occur in the determination, and it is not yet possible to prepare a pure analytical lignin fraction. The relative merits of about 15 procedures are reviewed by Cherney (2000). In the procedure described by Van Soest and Wine (1968), the crucible plus residue from the ADF method is left to stand for 1.5 h in a buffered potassium permanganate solution to dissolve the lignin. The cellulose residue is reacted with demineralizing solution until white, washed successively with 80% ethanol and acetone, dried overnight at 100°C, cooled and weighed. The loss in weight is equal to the lignin content. The crucible may be ashed for 3 h at 500°C and the loss in weight is the cellulose content.

Nitrogen-free extract (Nifext)

This is obtained by subtracting the sum of the percentages of water, protein, fat, fibre and ash from 100. It represents the starch, gums, sugars and organic acids (all N-free), which may be extracted by water or diastase from cleaned, dried and defatted foods. As it is mainly starch, it will be high in the case of cereal grains and lower with seeds containing more oil and protein.

Coniferyl group

Syringyl group ch2oh ch—

choh

Coniferyl group

Syringyl group

p-hydroxybenzyl section

Fig. 4.2. Typical lignin structural unit.

p-hydroxybenzyl section

Fig. 4.2. Typical lignin structural unit.

Starch

Starch may be determined using specific enzymes such as amyloglucosidase, but the extraction and hydrolysis stage is slow, enzyme activity can vary, reagents are expensive and complete hydrolysis is difficult. Although acid-hydrolysis lacks specificity, with the simple case of starch in potatoes, it becomes an ideal procedure (Faithfull, 1990). The freeze-dried, milled sample is washed with 10% v/v ethanol/water to remove sugars, dextrins and tannins which can amount to about 12%. Note: The use of 80% v/v ethanol/water is recommended for pre-extraction with enzyme methods, followed by heat treatment to gelatinize the starch; 90% v/v ethanol/water tends to make the starch resistant to enzymatic hydrolysis (Hall et al., 2001). The suspension is centrifuged, washed into McCartney bottles using 1 M HCl and heated at 106°C for 40 min. After adjusting the pH to 3.0, it is diluted to 100 ml, and a further dilution with saturated benzoic acid solution provides the solution for analysis of the products of starch hydrolysis. Starch has been given the formula C36H62O31.12H2O, with residue units of C6H10O5. The hydrolysis is to units of glucose, C6H12O6, so when using the weight of glucose to determine the initial weight of starch, a correction factor of x0.9 is required. Although one would anticipate the hydrolysis to yield only glucose, some of the glucose is subsequently converted by the hot acid to fructose and 5-hydroxymethylfurfural which produce more colour with the anthrone reagent than glucose itself. The use of fructose solutions as standards corrects for this effect, otherwise a correction factor of x0.8 is used with glucose standards. Partial hydrolysis of potato cell walls to chromogenic products led to a further correction factor of x0.98, and a correction for any moisture must be made.

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