Animal Feed Science and Technology, 32 ( 1991 ) 45-53 45

Elsevier Science Publishers B.V., Amsterdam

The influence of the Maillard reaction upon the

nutritive value of fibrous feeds

P.J. v a n Soest ~ a n d V.C. M a s o n 2"*

'Cornell University, Ithaca, NY (U.S.A.)
2.4FRC Institute for Grassland and Animal Production. Hurley, Maidenhead SL6 5LR (Gt. Britain)

ABSTRACT

Van Soest, P.J. and Mason, V.C., 1991. The influence of the Maillard reaction upon the nutritive
value of fibrous feeds. Anita. FeedSci. Technol., 32: 45-53.

Heating forages and feeds will generate Maillard products of low digestibility which are signalled
by a rise in the nitrogen (N) content of acid-detergent fibre (ADIN). Two sets of data from digestion
trials with sheep were mathematically examined by the methods of Lucas. The first set, taken from
studies of distillers grains, exhibited some digestibility of ADIN, but showed a high positive correla-
tion between fecal N and ADIN and a negative one with nitrogen digestibility and ADIN. Earlier
conclusions as to the ineffectiveness of ADIN as a method of evaluation are refuted. The second set
of data referred to ammoniated forages with their untreated controls. The treated forages are shown
to contain soluble indigestible organic matter and nitrogen in the ratio of 8.83: 1 + 0.11, agreeing with
the expected composition of Maillard polymers. With these forages the fecal N loss considerably ex-
ceeds the ADIN estimate, in contrast to the distillers grains. The excess is due in part to the presence
of indigestible soluble non-ammonia nitrogen (SNAN) that is associated with a soluble indigestible
organic fraction. The existence of indigestible soluble N and organic matter constitutes the basis for
severe criticism ofgravimetric laboratory procedures, including nylon bags, used in evaluating treated
forages and feeds, as these procedures will fail to recover the soluble indigestible components and thus
over-value treated forages. The use of ADIN as a marker for heat damage and the Maillard reaction
is validated, but in treated forages an additional examination for absorbances at 280 and 320 nm. and
SNAN is recommended.

INTRODUCTION

Plant cell walls, or their constituent fractions, have long been the basis for
predicting the nutritive value of fibrous feeds, because they generally contain
the truly indigestible residues. In most normal forages the influence of ligni-
fication is restricted to the cell wall, and non cell wall components are avail-
able for digestion (van Soest, 1982). This generalization may become re-
stricted in the case of processed by-products and treated forages to which heat
and chemical treatments have been applied. Soluble lignin-like products can

*Present address: Biocomb International Ltd., c / o 13 Nicholas Road, Henley-on-Thames, RG9

1RB, Gt. Britain.
46 P.J. VAN SOEST AND V.C. MASON

result from alkali and ammonia treatments of forages and straws through
cleavage of lignin-carbohydrate bonds (van Soest et al., 1984) and from the
Mainard reaction (Theander, 1980).
Heating is often involved in the treatment or fermentation of feeds and can
promote the Maillard reaction. This can greatly affect apparent lignin content
and cell wall characteristics (van Soest, 1965; Goering et al., 1972 ). The prin-
cipal reaction is the degradation of sugar components to phenolic products,
which catalytically destroy amino acids and polymerize with the remnants of
amino acids (Hodge, 1953; Theander, 1980). The most important carbohy-
drate sources are hemicelluloses and sucrose, and the principal products are
a synthetic artifact lignin polymer of a distinctively high nitrogen content (van
Soest, 1965), as well as soluble components of lower molecular weight (Hodge,
1953; Theander, 1980). Cell walls of heated forages and feeds are character-
istically higher in lignin and lower in hemicellulose contents than unheated
ones (Goering et al., 1973). The MaiUard polymer is lignin-like and generally
is presumed to be indigestible. Analytically a high nitrogen content of crude
lignin and acid-detergent fibre (ADIN) provides evidence of a history of
heating, and can be related to lower digestibility of protein and dry matter in
heated silages and hays (Goering et al., 1972; Yu and Thomas, 1976). Mail-
lard products are characteristic of many heat-dried feeds (Goering, 1976). It
is such a significant quality problem that measurement of ADIN is recom-
mended for heated and fermented feeds (Agricultural and Food Research
Council, 1987).
In addition to the artifact lignin polymer a variety of products may occur
in heated feeds (Theander, 1980). For example, the occurrence of the disor-
der known as "bovine bonkers" has been related to 4-methyl imidazole, a
Maillard product derived from the reaction of ammonia with sucrose (Per-
dok and Leng, 1987). Ammonia can apparently substitute for amino acids in
the Maillard reaction (Mason et al., 1990). The occurrence of indigestible
nitrogen associated with ADIN has been observed in high temperature am-
monia-treated forages (Mason et al., 1989a,b,c).
Recently, however, the indigestibility of ADIN has been challenged (Brit-
ton et al., 1986; Klopfenstein and Britton, 1987; Weiss et al., 1989) on the
basis that no significant decline in protein digestibility was observed when
heated distillers grains replaced urea in isonitrogenous diets. We have ob-
tained the data of Britton et al. (1986) and of Klopfenstein and Britton
( 1987 ) and subjected them to mathematical analysis according to the model
of Lucas (van Soest, 1967, 1982 ). The details of this analysis are published
elsewhere (van Soest, 1989).
This paper presents the results of that analysis, termed in this paper the
Nebraska Study, and compares it with digestion trials conducted at the Insti-
tute of Grassland and Animal Production, Hurley, U.K. on ammonia-treated
forages (Mason et al., 1989a,b,c). The data from the Hurley study are sub-
jected to the same mathematical treatments. The statistical analysis consisted
INFLUENCEOF MAILLARDREACTIONON VALUEOF FEEDS 47

of correlating ADIN and other dietary nitrogen fractions with fecal nitrogen
and deviations in digestibility from the regression of digestible amounts upon
dietary contents (van Soest, 1989). Estimates of true digestibility are ob-
tained from the regression slopes. The principal problem in comparing diges-
tibility values of nitrogen and soluble components is that their apparent di-
gestibility values arc confounded by metabolic and endogenous losses (Mason,
1969 ). These mathematical procedures are designed to limit the effects of the
metabolic components to obtain estimates of true digestibility or indigestibil-
ity of the respective dietary components.

THE NEBRASKA STUDY

The digestion studies undertaken at the University of Nebraska consisted

of three trials in which distillers grains, varying in heat treatment and ADIN
content, were incorporated into a basal diet composed of lucerne meal, wheat
straw and urea. The distillers grains replaced urea to form diets that were as
isonitrogenous as possible. The distillers grains formed ~ 33-40% of the net
nitrogen substitution and the net average dietary nitrogen composition was
1.9% of dry matter. It was assumed that any reduction in the apparent diges-
tibility of nitrogen would reflect upon the availability of nitrogen in distillers
grains. A total of 24 feed mixtures were investigated (three controls and 21
substituted diets) with four sheep per diet.
The coefficient of variation of nitrogen content was 4.6% which, although
small, allowed a significant correlation with nitrogen digestibility ( r = + 0.64)
and no apparent relationship between nitrogen digestibility and ADIN con-
tents. However, when fecal N is regressed on ADIN added in distillers grains
a highly significant relationship is obtained (Fig. 1 ). The slope of the regres-

J¢ Trial 1 • Y=63+O.42x
m
Trial2 o r=+0.84
Trial 4 •
"O Controls c / / ~ e • •
"6 &o

z
w
0
0
Ik
I I i

0 1 2 3
Added ADIN(glkg of dietary intake)

Fig. 1. The relationship between total fecal N and added dietary ADIN (relative to the con-
trois). Regression includes pooled data from Nebraska Trials 1, 2 and 4, with three controls and
21 diets containing distillers grains.
48 P.J. VAN SOES~ AND V.C. MASON

sion ( + 0.42 +_0.06) is an estimate of indigestibility, and indicates that whilst

there is a significant digestibility (0.58) of ADIN in distillers grains, there is
also a definite indigestible fraction that is associated with dietary ADIN. A
second analysis compared the deviations from the regression of digestible N
upon dietary N content. These deviations were regressed upon added ADIN
intake. This also resulted in a significant correlation (-0.81 ) and an esti-
mate of indigestibility of 0.37 _+0.08 for the ADIN fraction. These results re-
fute the conclusion of the Nebraska workers that ADIN is of no value in eval-
uating heated distillers grains.

THE IGAP EXPERIMENTS

Two digestion trials on ammonia-treated forages have been reported by

Mason et al. (1989a,b,c). The first consisted of the four grass species, rye-
grass ( Lolium perenne ), timothy ( Phleum pratense ) cocksfoot ( Dactylis
glomerata) and tall rescue (Festuca arundinacea) treated with ammonia by
the oven method and at ambient temperature, making four controls and eight
treated products (Mason et al., 1989a,b). The second trial investigated rye-
grass at three degrees of maturity, each sample being treated by the oven
method and providing three control and three treated materials (Mason et
al., 1989c). The combined studies provided seven control and 11 treated for-
ages. Digestion trials were with sheep. Full details of forage composition,
treatment and digestion trials are provided in the three papers. Optical den-
sity and saponification values were reported earlier (van Soest et al., 1984).
The data used here include organic matter and nitrogen digestibility; compo-
sition of the diet for total N, soluble non-ammonia N (SNAN), neutral-de-
tergent insoluble N (NDIN) and ADIN. Other fecal analyses included water-
soluble (FSN) and insoluble nitrogen (FIN). These data offer more possibil-
ities for mathematical analysis than the Nebraska experiments which are lim-
ited to nitrogen digestibility and ADIN contents of diets.
The relationship of dietary ADIN content and fecal nitrogen is shown in
Fig. 2. Overall there is no significant relationship. However, the points can be
statistically separated into two significantly differing groups. One includes
the controls and four treated forages, and the other the remaining seven treated
forages. The latter tend to be those with the highest SNAN and NDIN con-
tents. The lower group shows a significant regression in the ADIN with a slope
of 2.48, significantly greater than the unity required for fecal recovery. Clearly
there is co-association with ADIN and the NDIN and SNAN in these sam-
ples. Regression of insoluble fecal N upon ADIN produced very similar re-
sults, the same seven treated samples deviating from those that form a signif-
icant relationship with ADIN. This regression is Y= 1.30X-6. The standard
deviation of the regression coefficient is _+0.30, indicating that FIN may rep-
resent fecal recovery of ADIN. The question is whether ADIN in this set be-
INFLUENCEOF MAILLARDREACTIONON VALUEOF FEEDS 49

Controls o
0
• Treated •

i lO

0 1
i

Dietary ADIN(glkg)
Y = 2.5x ÷ 7

2
I

Fig. 2. Relationship between dietary contents of ADIN and total fecal N for diets composed of
forages treated with ammonia and their untreated controls. Calculated from the data of Mason
et al. (1989a,b,c). Values above the dashed line exceeded three standard deviations from the
mean population and have been excluded from the regression.

J¢

.¢_ Control o /
300
J¢
Y ffi 1.05x - 119 J .
o
~= 200 o°.~$o~ ."/'e"
to
o/:.; •

z lOO //~/w" " Treated •

O
Jo "/• Y ffi 0.73x - 76

O I I
._a
200 400
ND Solubles(glkg DM intake)

Fig. 3. The relationship between apparent digestible neutral-detergent solublcs (content x diges-
tion coefficient) and the neutral-detergent soluble content of the diet. Treated samples show a
lower slope of regression than unity indicating the existence of an indigestible component.

haves differently from the distillers grains in Fig. 1, which show some diges-
tibility of ADIN. The question cannot be answered clearly with these data.
More definitive chemical evidence is needed on the nature of Maillard prod-
ucts in feces and their recovery in ADIN.
A more important aspect is the existence of indigestible soluble compo-
nents in the ammonia-treated forages, which is examined in Fig. 3 by regres-
sion of apparently digestible neutral-detergent soluble organic matter upon
the organic neutral-detergent soluble contents of the forages. Regressions of
this type provide estimates of true digestibility from the slopes, and fecal en-
dogenous losses from the negative intercepts (van Soest, 1982 ). Controls and
treated samples have significantly different regression slopes. That for the
controls is not significantly different from unity ( 1.05 _+0.08 ), and that for
treated is significantly less than unity (0.73 +_0.04 ).
50 P.J. VAN SOESTAND V.C. MASON

Analogous regressions of digestible amount upon dietary contents were es-

timated for all of the nitrogen fractions. For NDIN and ADIN, it was as-
sumed that FIN represented the indigestible quantity, and FSN for SNAN,
respectively. The sum of NDIN and SNAN has also been tested as to the uni-
formity of the combination in respect to total fecal N. Estimation of the true
digestibility values for these are given in Table 1 for the controls and treated
samples.
Estimates of true digestibility of controls are near unity for total N, SNAN,
and NDIN + SNAN. That for NDIN is 0.81 and it can be expected that there
is a small cell wall fraction containing indigestible nitrogen (van Soest, 1982).
In normal forages, ADIN did not give an estimate of true digestibility owing
to too large a variance in both controls and treated forages. Some of the rea-
sons for this are apparent in Fig. 2. It is evident that ADIN does not recover
the insoluble indigestible N present in treated forage and that NDIN, al-
though fairly digestible in normal forages, may contain considerable unavail-
able N in treated ones. All of the N fractions in the treated forages except
ADIN show a true digestibility value significantly less than unity. The com-
bination of NDIN plus SNAN largely accounts for the indigestibility of N in
treated samples. Caution should be exercised in interpreting the true digesti-
bility of SNAN (0.74) in treated samples, as it is likely that some low molec-
ular weight products of the Maillard reaction could be absorbed and excreted
in urine. Whilst urine was not examined for Maillard products in these exper-
iments it should be mentioned that toxic 4-methyl imidazole was detected in
all of the ammonia-treated forages and was generally higher in those that were
oven-treated (Mason et al., 1989a).
As the treated samples show strong evidence of reduction in digestibility of
TABLE 1

Estimates of true digestibility of N fractions based on the regression of digestible components upon
dietary contents

Fraction Controls ( n = 7 ) Treated ( n = 11 )

r True SD r True SD
digestibility digestibility

Total N 0.99 1.01 ±0.05 0.98 0.67 ±0.04

SNAN l 0.98 1.01 _+0.13 0.97 0.74 _+0.07
NDIN 2 0.91 0.81 ±0.17 0.86 0.48 ±0.10
ADIN 3 0.11 NS ± 1.23 -0.23 NS _+2.01
NDIN+SNAN 0.98 1.01 _+0.04 0.97 0.52 _+0.04

'Soluble non-ammonia N.
2Neutral-detergent insoluble N.
3Acid-detergent insoluble N.
NS, no significant value.
INFLUENCE OF MAILLARD REACTION ON VALUE OF FEEDS 5I

soluble organic matter, as well as in nitrogen, a further relevant question is in

regard to the composition of that organic matter of MaiUard origin. The Mail-
lard reaction involves the reaction of sugar and amino acid at unitary ratio
and with a net loss of three molecules of water from the aromatization of the
sugar and 1 mol of CO2 from decarboxylation of the amino acid. Ammonia
can substitute for amino acid in the reaction (Hodge, 1953; Theander, 1980 ).
The resistant polymer can be expected to have an N content somewhat less
than in normal proteins. Van Soest ( 1965 ) found a ratio of 8.9 of organic
matter to N in the MaiUard product in heated forages. Regression of the un-
digested soluble organic matter in feces upon the elevation of fecal N gave a
ratio of 8.83 _+0.11, in reasonable agreement.
Previously, it had been reported that these samples contained soluble lignin
measured spectrophotometrically at 280 nm in neutral-buffer extracts (van
Soest et al., 1984). Soluble lignin is likely to be indigestible and recoverable
in feces (Neilson and Richards, 1978 ). Samples that were treated, and partic-
ularly those that were oven-treated, tended to contain more net phenolic mat-
ter than the untreated controls. The UV absorption at 280 nm is highly cor-
related with the SNAN (r= +0.92) and with fecal soluble organic matter
(r= +0.91 ). Lignin-like phenolics have the structural requirements for par-
ticipation in the Maillard reaction, and can substitute and compete with the
phenolics derived from sugar degradation (Theander, 1980). Thus it is pos-
sible that the indigestible soluble organic matter contains soluble lignin com-
ponents in addition to those phenolics derived from sugar, as well as nitrogen
in its structure.
The potential interaction of soluble lignin in the Maillard reaction could be
heterogeneous. One way is the ammoniolysis of lignin ester to produce lignin
acid amides. Indigestible amide linkages have been noted in NIR spectra of
ammonia-treated forages (F.E. Barton, personal communication, 1989). An-
other mechanism could be the condensation of phenol with sugar aldehydes,
and still another, the reaction of l-2-dihydroxyphenols in competition with
sugar-derived products with ammonia or amino acid (Theander, 1980 ). Sug-
ars could disappear via either the Maillard reaction involving ammonia, or
by aldehydic condensation with soluble phenolics released by ammoniation.
Verification of these processes will require further studies.
It would seem that the balance of component factors contributing to the
soluble indigestible fraction may vary, depending upon heat treatment. For
the low temperature treatments, soluble lignin interactions with ammonia may
dominate, whilst higher temperatures promote the conversion of sugar to
phenol with subsequent interaction with ammonia. The correlation of sugar
loss with elevation of SNAN is +0.97 (Mason et al., 1989a). In either case,
nitrogen from ammonia or other sources is associated with the soluble frac-
tion in a fairly consistent ratio.
For the phenolic-nitrogenous compounds to be both soluble and recover-
52 P.J.VANSOESTANDV.C.MASON

able in feces, molecular size cannot be too large (in order to be soluble) but
must be > 300-400 Da. Smaller components would be absorbed and proba-
bly excreted in urine. The occurrence of toxic 4-methyl imidazole is evidence
for this.
The amounts of soluble indigestible matter that can be generated in treated
forages are very considerable. The range in this study is up to 10 percentage
units of organic matter digestibility. This matter generally will be lost in lab-
oratory methods such as solubility with cellulase, lumen fluid, or in nylon
bags, all based on insoluble gravimetric residues, and this will lead to over-
estimation of the value of treated forages. Determinations of SNAN (Mason
et al., 1989a) and the measurement of optical density at 280 and 320 nm of
the soluble matter for phenolic matter (Lau and van Soest, 1981 ) can be rec-
ommended to those who intend to make a more complete laboratory evalua-
tion of treated forages.

REFERENCES

Agricultural and Food Research Council, 1987. Technical Committee on Responses to Nu-
trients, Report No. 2, Characterisation of Feedstuffs: Nitrogen. Nutr. Abstr. Rev., B, 57:
713-736.
Britton, R.A., Klopfenstein, T.J., Cleale, R., Goedeken, F. and Wilkerson, V., 1986. Methods of
estimating heat damage in protein sources. Proceedings of the Distillers Feed Conference,
16 April 1986, Cincinnati, OH, Distillers Feed Research Council, Cincinnati, OH, pp. 67-
74.
Goering, H.K., 1976. A laboratory assessment on the frequency of overheating in commercial
dehydrated alfalfa samples. J. Anita. Sci., 43: 869-872.
Goering, H.K., Gordon, C.H., Hemken, R.W., Waldo, D.R., van Soest, P.J. and Smith, L.W.,
1972. Analytical estimates of nitrogen digestibility in heat damaged forages. J. Dairy Sci.,
56: 1275-1280.
Goering, H.K., van Soest, P.J. and Hemken, R.W., 1973. Relative susceptibility of forages to
heat damage as affected by moisture, temperature and pH. J. Dairy Sci., 56: 137-143.
Hodge, J.E., 1953. Chemistry of browning reactions in model systems. J. Agric. Food Chem., 1:
928-943.
Kiopfenstein, T. and Britton, R., 1987. Heat damage - real or artifact. Proceedings of Distillers
Feed Conference, 19 April 1987, Cincinnati, OH, Distillers Feed Research Council, Cincin-
nati, OH, pp. 84-86.
Lau, M.M., and van Soest, P.J., 1981. Titrable groups and soluble phenolics as indicators of the
digestibility of chemically treated roughages. Anim. Feed Sci. Technol., 6:123-131.
Mason, V.C., 1969. Some observations on the distribution and origin of nitrogen in sheep faeces.
J. Agric. Sci., 73:99-111.
Mason, V.C., Keene, A.S., Cook, J.E., Cooper, E.M. and Hartley, R.D., 1989a. Oven and stack
ammoniation of grass hays. 1. Changes in chemical composition in relation to digestibility
in vitro and cell wall degradability. Anita. Feed Sci. Technol., 24:299-312.
Mason, V.C., Cook, J.E., Cooper, E.M., Hoadley, C.J. and Cockburn, J.E., 1989b. Oven and
stack ammoniation of grass hays. 2. Voluntary intake, digestibility and faecal nitrogen excre-
tion in sheep in relation to hay composition. Anita. Feed Sci. Technol., 24:313-326.
Mason, V.C., Cook, J.E., Dhanoa, M.S., Keene, A.S. and Hartley, R.D., 1989c. Chemical corn-
INFLUENCEOF MAILLARDREACTIONONVALUEOF FEEDS 53

position and nutritive value to sheep of untreated and oven-ammoniated ryegrass hays pre-
pared from crops of differing maturities. Anim. Feed Sci. Technol., 26: 207-220.
Mason, V.C., Cook, J.E., Dhanoa, M.S., Keene, A.S., Hoadley, C.J. and Hartley, R.D., 1990.
Chemical composition, digestibility in vitro and biodegradability of grass hays oven-treated
with different amounts of ammonia. Anita. Feed Sci. Technol., 29: 237-249.
Neilson, M.J. and Richards, G.N., 1978. The fate of the soluble lignin-carbohydrate complex
produced in the bovine rumen. J. Sci. Food Agric., 29: 573-579.
Perdok, H.B. and Leng, R.A., 1987. Hyperexcitability in cattle fed ammoniated roughages. Anim.
Feed Sci. Technol., 17: 121-144.
Theander, O., 1980. Sugars in thermal processes. In: P. Koivistoinen and I. Hyvonen (Editors),
Carbohydrate Sweeteners. Academic Press, New York, pp. 185-199.
Van Soest, P.J., 1965. Use of detergents in analysis of fibrous feeds. III. Study of effects of
heating and drying on yield of fibre and lignin in forages. J. Assoc. Off. Agric. Chem.. 48:
785-790.
Van Soest, P.J., 1967. Development of a comprehensive system of feed analysis and its appli-
cation to forages. J. Anim. Sci., 26:119-128.
Van Soest, P.J., 1982. Nutritional Ecology of the Ruminant. Comstock, Cornell University Press,
Ithaca, NY, 3rd edn.
Van Soest, P.J., 1989. On the digestibility of bound nitrogen in distillers grains: A re-analysis.
Proceedings of Cornell Nutrition Conference, 24-26 October 1989, Syracuse. NY, Cornell
University, Ithaca, NY, pp. 127-136.
Van Soest, P.J., Mascarehnas-Ferreira, A. and Hartley, R.D., 1984. Chemical properties of fibre
in relation to nutritive quality of ammonia-treated forages. Anim. Feed Sci. Technol., 10:
155-164.
Weiss, W.P., Erikson, D.O., Erickson, G.M. and Fisher, G.R., 1989. Barley distillers grains as a
protein supplement for dairy cows. J. Dairy Sci., 72: 980-987.
Yu. Y. and Thomas, J.W., 1976. Estimation of the extent of heat damage in alfalfa haylage by
laboratory measurement. J. Anim. Sci., 42: 766-774.