Comparison of acid and amyloglucosidasehydrolysis for estimation of non-structuralpolysaccharides in feed samplesGilberto V Kozloski,1*† Joao BT Rocha,1 Henrique MN Ribeiro Filho2 andJuliano Perottoni11Departamento de Quımica, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, 97119-900, RS,Brasil2Centro de Ciencias Agroveterinarias-UDESC, Av Luis de Camoes 2090, Lages, SC, Brasil

Abstract: Enzymatic methods (amyloglucosidase) and methods based on acid solutions (0.1, 0.2 and

0.3M H2SO4 for 1, 2 and 3h at 100°C) for the hydrolysis of non-structural carbohydrates from different

feed samples were compared. The monomeric units resulting from the enzymatic and acid hydrolysis

were determined by the glucose oxidase and reducing sugar methods. There was a signi®cant effect of

acid concentration and of time of hydrolysis on the glucose and reducing sugar values in the

hydrolysate. Glucose values were similar for both the amyloglucosidase method and the most intense

conditions of hydrolysis (0.3M for 3h) for some samples. Under these conditions, however, the

reducing sugar values were higher. No acid hydrolysis method was found to estimate correctly the total

non-structural carbohydrates, but a-linked glucose polymers in biological samples may be determined

by sample hydrolysis with a 0.3M H2SO4 solution for 3h at 100°C since the glucose in the hydrolysate is

determined by the glucose oxidase method and the sucrose content of the sample is negligible.

# 1999 Society of Chemical Industry

Keywords: carbohydrates; amyloglucosidase; acid solutions; hydrolysis; glucose; reducing sugars

INTRODUCTIONCarbohydrates correspond to 50 to 80% of the dry

matter of forage grasses and cereals1 and represent the

primary nutritional source for the energy requirements

of animals. They may be classi®ed into two groups:

non-structural carbohydrates (NSC), which are avail-

able for plant metabolism and are represented by

sugars, starch and fructans, among others; and

structural carbohydrates, mainly represented by pec-

tins, cellulose and hemicellulose.1 The proportions of

the two groups vary with plant species and growth

stage and with plant part in the same plant. As

components of the diets of ruminants, NSC are

promptly fermented by the ruminal micro¯ora,

whereas cellulose and hemicellulose are fermented

more slowly. Pectin, although of a structural nature, is

also rapidly fermented in the rumen. Thus, depending

on the proportion of these carbohydrates in feed,

different patterns of fermentation and degrees of

digestion may occur, causing different levels of feed

assimilation by the animals. Thus, the determination

of the composition of feed in terms of these carbo-

hydrate groups permits a better understanding of the

digestive processes and a more effective prediction of

the performance of animals consuming such feeds.

Several methods have been employed for NSC

determination in feed, digesta and faecal samples. The

methods may be direct, with hydrolysis of the sample

and later determination of the resulting monosacchar-

ides, or indirect, with NSC being determined by the

following formula: NSC=100ÿ((NDF-NDF pro-

tein)�CP�EE�ash), where NDF is the ®bre in

neutral detergent, CP is the crude protein, and EE is

the ether extract.2

The direct methods differ in sample preparation by

the use of different types of extraction and hydrolysis

(with water, alcohol, acids or enzymes), and in the

determination of the monosaccharides resulting from

hydrolysis (copper-iodometric titration of Shaffer±

Somogyi, colorimetric method of Somogyi±Nelson,

glucose oxidase method or chromatography).

Among the enzymatic methods, those that seem to

be most often used are the method of Weinmann,3

which employs an enzymatic preparation denoted

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1112±1116 (1999)

* Correspondence to: Gilberto V Kozloski, Curso de Pos Graduacao em Zootecnia, Universidade Federal do Rio Grande do Sul, Av BentoGoncalves 7712, Porto Alegre, 91540-000, RS, Brasil† Present address: Curso de Pos Graduacao em Zootecnia, Universidade Federal do Rio Grande do Sul, Av Bento Goncalves 7712, PortoAlegre, 91540-000, RS, Brasil(Received 9 October 1997; revised version 15 June 1998; accepted 20 November 1998)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1112

takadiastase (mainly containing a-amylase) and later

modi®ed by Lindahl et al,4 and the method of McRae

and Armstrong,5 which involves incubation of the

sample with an amyloglucosidase (from Aspergillusniger). Herrera-Saldana and Huber,6 in turn, de-

scribed an enzymatic method in which hydrolysis is

performed in two steps, the ®rst using a thermo a-

amylase and the second a glucoamylase.

Several methods of hydrolysis with acid solutions

have been evaluated as alternatives to the use of

enzymes (Smith et al,7 Grotelueschen and Smith,8

McRae and Armstrong,5 Greub and Wedin,9 McAllan

and Smith10 and Kartchner and Theurer,11 among

others). Smith et al7 used hydrolysis with 0.1 to 0.4M

H2SO4 solutions and takadiastase hydrolysis by the

method of Weinmann3 for samples of alfalfa root and

timothy stem base. The resulting sugars were deter-

mined by the copper-iodometric titration method of

Shaffer±Somogyi. These investigators concluded that

the most adequate methods were the enzymatic

method and the method with 0.1M H2SO4, which

yielded similar results and permitted the determina-

tion of sugars and fructans in addition to starch. In our

laboratory we conducted preliminary studies in which

we analysed food samples with a high grain content,

corn silage, duodenal digesta and also faecal samples.

We used hydrolysis with 0.1M H2SO4 as proposed by

Smith et al7 and determined the resulting sugars by

Shaffer±Somogyi copper-iodometric titration. These

samples were also analysed using an amyloglucosidase.

All the results obtained by acid hydrolysis (unpub-

lished data) were signi®cantly lower than those

obtained by the enzymatic method. Some changes in

sample size and time of hydrolysis signi®cantly altered

the results, which, however, continued to be below

expected levels.

Enzymatic hydrolysis is more speci®c and seems to

be less subject to error, but it is more complex, more

time-consuming and more expensive than acid hydro-

lysis. Furthermore, with the enzymatic method, it is

recommended that samples containing signi®cant

amounts of other NSC (fructans, sucrose and others)

in addition to a-linked glucose polysaccharides, be

submitted to additional procedures in order not to

underestimate this fraction.5,8 In turn, sample pre-

paration by hydrolysis with acid solutions may cause

errors due to the partial solubilisation of structural

components such as pectins, hemicellulose and

cellulose1 or, conversely, due to incomplete solubilisa-

tion and hydrolysis of all NSC. The results would

represent overestimates in the former case and under-

estimates in the latter.

Thus, the objective of the present study was to

determine the effect of different times of hydrolysis

with sulphuric acid solutions of different molarities on

the release of glucose and reducing sugars from feed

samples varying in the proportion of concentrate for

tropical forage hay, in order to ®nd a simpler and less

expensive direct method than the enzymatic one for

the determination of NSC.

EXPERIMENTALSamples (250mg) of Cynodum dactilum cv coast cross

(bermuda grass) hay, of concentrate (18% corn, 12%

soy bran, 31% wheat bran, 37% defatted rice bran,

and 2.0% premix vit-min) and of different hay:con-

centrate proportions (25:75, 50:50 and 72:25), all

enriched with increasing proportions of corn starch

(0, 10 and 20%), as well as standard samples of corn

starch (Sigma), glucose, fructose, xylan (from birch-

wood, Sigma) and cellulose (cotton) were hydrolysed

at 100°C in a re¯ux system with 0.1, 0.2 and 0.3M

H2SO4 solutions (50ml) for 1, 2 and 3h. Hydrolysis

was carried out in triplicate, the solutions were then

®ltered through paper ®lter, and the tube was washed

with boiling distilled water. The ®ltrate was placed in

a 100ml volumetric ¯ask, and allowed to cool, and

the volume was adjusted with distilled water. A 10ml

aliquot of the content of the ¯ask was collected,

neutralised at pH 6 to 8 with 0.15M NaOH solution

and frozen for later determination of glucose and

reducing sugars. The samples were also hydrolysed

using amyloglucosidase (from Rhizopus mold, with

activity of 22000Ugÿ1, Sigma EC 3.2.1.3) incubated

with the sample for 40h at 60°C according to the

method of Kartchner and Theurer.11 The ®ltrate

resulting from the acid and enzymatic hydrolyses was

assayed for glucose concentration by the glucose

oxidase method and for reducing sugars by the

modi®ed colorimetric method of Somogy and Nelson,

according to Tuiskon.12 The standard curves for

sugar determinations by the acid and enzymatic

methods were constructed with pa glucose samples

treated like the samples (submitted to acid and

enzymatic hydrolysis), considering that glucose re-

covery after hydrolysis, ®ltration and neutralisation

was 100%. In the case of acid hydrolysis, the standard

glucose solution was prepared by subjection of 27

samples of glucose to acid hydrolysis conditions

(three for each acid hydrolysis condition), and in

the case of enzymatic hydrolysis the standard glucose

solution was prepared from 3 pa glucose samples. In

the determination of glucose and reducing sugars in

the samples submitted to acid hydrolysis, distilled

water was used as the blank and also to dilute the

standard glucose solution (standard curve). The

blank used for the samples submitted to enzymatic

hydrolysis was a solution originating from enzymatic

hydrolysis in the absence of a sample.

The results were submitted to analysis of variance

to determine the effect of the different molarities of

the acid and of the different times of hydrolysis on

the percentage of glucose and of reducing sugars in

the samples. Regressions were also done considering

the effect of these factors on the percentage of

glucose and of reducing sugars in the samples

submitted to acid hydrolysis. We also compared the

means obtained with the different methods of

hydrolysis and of determination of the sugars

resulting in the various samples by the Tukey test

(a=0.05).

J Sci Food Agric 79:1112±1116 (1999) 1113

Acid and amyloglucosidase hydrolysis for estimating polysaccharides in feed

RESULTS AND DISCUSSIONThe release of reducing sugars and glucose (deter-

mined by glucose oxidase) of all feed samples assayed,

as a function of different hydrolysis methods tested, is

presented in Figs 1 and 2. Time (P<0.01) and

molarity of the H2SO4 solution (P<0.01) for acid

hydrolysis had a signi®cant effect on reducing sugar

and/or glucose release from the samples analysed.

Under milder hydrolysis conditions, the release of

reducing sugars was lower than the release of glucose

only, a behaviour that was inverted as the intensity of

acid hydrolysis increased. It was also observed in the

hydrolysis of corn samples, but not of hay samples (Fig

3). The release of reducing sugars from hay was always

higher than the release of glucose. Furthermore, Fig 4

shows that the product of hydrolysis of standard corn

starch samples tended to be lower when determined as

reducing sugar than as glucose and this difference

tended to decrease with increasing intensity of acid

hydrolysis. Although there is no clear explanation, this

occurrence may be related to the higher sensitivity of

enzymatic method (glucose oxidase) at lower sugar

concentrations compared to the reducing sugar

method. On the other hand, the cases in which the

reducing sugar values obtained were higher than the

glucose values, under the stronger conditions of

hydrolysis, are probably related to the release of

structural carbohydrate monomers, especially from

hemicellulose and pectin, and perhaps to the hydro-

lysis of some oligosaccharides, such as sucrose. McRae

and Armstrong5 after submitting samples of various

types of feed to hydrolysis with 0.18M H2SO4 at

100°C for 8h, and McAllan and Smith10, after

submitting digesta samples to 0.5M H2SO4 hydrolysis

for 4h, observed the release of signi®cant amounts of

monosaccharides derived from hemicellulose. This

seems to occur even under milder hydrolysis condi-

tions, as observed by Grotelueschen and Smith8 after

hydrolysing alfalfa and timothy samples with 0.1M

H2SO4 at 100°C for 1h. Similarly, Greub and Wedin9

observed that, with samples containing a high propor-

tion of NSC, hydrolysis under the above conditions

Figure 1. Effect of molarity of the acidsolution on the release of reducing sugars(Y =8.91�58.66X, where n =153 pertreatment, P<0.01) and glucose(Y =15.63�32.14X, where n =144 pertreatment, P<0.01) from feed samples,compared to mean glucose concentrationobtained by the enzymatic method(amyloglucosidase, where n =51).

Figure 2. Effect of time of acid hydrolysison the release of reducing sugars(Y =10.87�10.75X, where n =153 pertreatment, P<0.01) and glucose(Y =17.87�5.31X, where n =144 pertreatment, P<0.01) from feed samples,compared to mean glucose concentrationobtained with the enzymatic method(amyloglucosidase, where n =51).

1114 J Sci Food Agric 79:1112±1116 (1999)

GV Kozloski et al

underestimated this fraction compared to the enzy-

matic method, whereas an overestimate occurred

when samples with a low NSC content were

processed. The hydrolysis of structural components

of the samples is also suggested by the release of

reducing sugars from the xylan samples, which

increased with time of hydrolysis and with normality

of the acid solution (RS=ÿ1.7�67.3M�9.2 T,

r2=0.71, P<0.01, where RS is the percent of reducing

sugars, T is the time of hydrolysis in hours andM is the

molarity of acid solution).

Also, in the present study there was no release of

glucose from the cotton samples (cellulose) and

glucose release was negligible in the hay samples, with

values not differing from zero by the t-test (P>0.05)

(Fig 3) under all hydrolysis conditions tested. McAllan

and Smith10 also observed no release of cellulose

glucose from digesta samples, even when using more

intense acid conditions.

Fructose was increasingly lost with acid hydrolysis

with increasing time and molarity of the acid solution

(FR=116.2 - 6.6T - 50.5M, r2=0.63, P<0.01, where

FR is the percent of fructose, T is the time of hydrolysis

in hours and M is the molarity of acid solution). Greub

and Wedin9 and Grotelueschen and Smith8 also

observed a loss of fructose by hydrolysis with 0.1m

H2SO4 at 100°C for 1h, or under more acidic

conditions. The latter authors, however, did not

observe this loss when they hydrolysed the samples

with 0.0025 and 0.01M H2SO4. Considering that the

samples evaluated in the present study basically

consisted of cereal grains and hay of a tropical grass,

which store energy in the form of starch, it is probable

that the effect of a possible loss of fructose was of no

importance. This may, however to cause signi®cant

errors in materials rich in fructose such as temperate

climate forage grasses, or sucrose.

Similar mean glucose concentrations were obtained

with the enzymatic hydrolysis method (amyloglucosi-

dase) and with acid hydrolysis only under conditions

of more intense acid hydrolysis (0.3m for 3h), as

shown in Figs 1 and 2. Under these conditions,

Figure 3. Effect of molarity of the acidsolution and time of hydrolysis on therelease of reducing sugars and glucosefrom hay and corn samples and meanglucose concentration in corn sampleshydrolysed with amyloglucosidase (n =3per treatment).

Figure 4. Effect of molarity of the acidsolution and time of hydrolysis on therelease of reducing sugars and glucosefrom corn starch samples, compared tomean glucose concentration obtained withthe enzymatic method (amyloglucosidase,where n =3 per treatment).

J Sci Food Agric 79:1112±1116 (1999) 1115

Acid and amyloglucosidase hydrolysis for estimating polysaccharides in feed

however, the release of reducing sugars was much

higher than the release of glucose alone. Table 1

compares the mean values of glucose and reducing

sugars of some individual samples hydrolysed enzy-

matically and with 0.3M H2SO4 for 3h.

The glucose content released by acid hydrolysis and

the reducing sugar and glucose content released by

enzymatic hydrolysis were similar in all materials

tested (P>0.05). On the other hand, except for the

starch sample, the release of reducing sugars by acid

hydrolysis was always higher than the release of

glucose only, and also higher than the release of

reducing sugars and glucose obtained by enzymatic

hydrolysis (P<0.05). The samples tested possibly do

not contain and/or contain negligible amounts of

sucrose.

Similar concentrations of reducing sugars released

by acid hydrolysis and of glucose released by enzy-

matic hydrolysis were obtained under less intense acid

hydrolysis conditions (Fig 1 and 2). Under these

conditions, however, it is probable that, on the one

hand, only part of the starch in the sample was

hydrolysed, and on the other, that hydrolysis and

release of reducing sugars from structural polysacchar-

ides occurred, as also observed by Grotelueschen and

Smith.8

CONCLUSIONSThere seems to be no de®ned limit in relation to the

intensity of acid hydrolysis of feed samples capable of

releasing the full amount of monomers derived from

NSC with minimal or no hydrolysis of structural

carbohydrates. a-linked glucose polymers of biological

samples however, can be determined by hydrolysing

the samples with 0.3M H2SO4 for 3h at 100°C, when

the glucose in the hydrolysate is determined by the

glucose oxidase method and the sucrose content of the

sample is non-signi®cant.

REFERENCES1 Van S, Nutritional Ecology of the Ruminant, 2nd edn, Cornell

University Press, Ithaca (1994).

2 Van S, Robertson JB and Lewis BA, Methods for dietary ®ber,

neutral detergent ®ber and nonstarch polysaccharides in

relation to animal nutrition. J Diary Sci 74:3583±3597 (1991).

3 Weinmann H , Determination of total available carbohydrates in

plants. Plant Physiol 22:279±290 (1947).

4 Lindahl I, Davis RE and Shepherd WO, The application of the

total available carbohydrate method to the study of carbo-

hydrate reserves of switch cane (Arundinara tecta). Plant

Physiol 24:285±294 (1949).

5 Macrae JC and Armstrong DG, Enzyme method for determina-

tion of a-linked glucose polymers in biological materials. J Sci

Food Agric. 19:578±581 (1968).

6 Herrera-Saldana R and Huber JT, In¯uence of varying protein

and starch degradabilities on performance of lactating cows. J

Dairy Sci 72:1477±1483 (1989).

7 Smith D, Paulsen GM and Raguse CA, Extraction of total

available carbohydrates from grass and legume tissue. Plant

Physiol 39:960±962 (1964).

8 Grotelueschen RD and Smith D, Determination and identi®ca-

tion of nonstructural carbohydrates removed from grass and

legume tissue by various sulfuric acid concentrations, taka-

diastase and water. J Agric Food Chem 15:1048±1051 (1967).

9 Greub LJ and Wedin WF, Determination of total available

carbohydrates in forage legume roots by extraction with

takadiastase, amyloglucosidase, or sulfuric acid. Crop Sci

9:595±598 (1969).

10 McAllan AB and Smith RH, Bacterial carbohydrates formed in

the rumen and their contribution to digesta entering the

duodenum. Br J Nutr 31:77±88 (1974).

11 Kartchner RJ and Theurer B, Comparison of hydrolysis methods

used in feed, digesta and fecal starch. J Agric Food Chem 29:8±

11 (1981).

12 Tuiskon D, DeterminacËaÄo de glicose no sangue: teste de

toleraÃncia aÁ glicose, in IntroducËaÄo aÁ BioquõÂmica: PraÂticas II,

UFRGS, Porto Alegre pp 7±13 (1970).

Table 1. Reducing sugars (RS) and glucose (G) concentrations (% ofsample) in some samples hydrolysed with acid solution (H2SO4 0.3M, during3h) or with enzyme (amyloglucosidase)*

Acid

hydrolysis

Enzymatic

hydrolysis

Sample RS G RS G SD² VC³

Hay 27.6a 3.7 3.6 0 1.78 20.0

75:25§ 26.0a 8.4 8.4 4.8 1.77 14.9

50:50§ 38.0a 18.6 18.4 12.4 2.81 12.9

25:75§ 35.7a 25.7 22.6 21.6 3.96 15.0

Corn 99.4a 73.2 84.3 72.6 7.43 9.0

Starch 105.0 102.1 108.9 110.3 12.4 11.6

* Means on same line followed by different letter differ at 5% level of

probability of type I error by Tukey test.² Standard deviation where n =3 by treatment.³ Variation coef®cient (%).§ Proportion hay:concentrate.

1116 J Sci Food Agric 79:1112±1116 (1999)

GV Kozloski et al