CZ277686B6 - Method of determining feeding stuffs digestibility by making use of enzymes - Google Patents

Abstract

The invention involves animal nutrition, in particular the evaluation of nutritional value of feedstuffs for farm animals. It consists of determining the digestibility of feedstuffs by an enzymatic method using an enzymatic cellulolytic complex with minimum activity of xylanase enzyme at a level of 300 μg of xylose/h per mg of enzyme, the minimum activity of β-glucosidase at a level of 1825 mg of glucose/h per mg of enzyme and minimum activity of pectinase at a level of 280 μg of glucose/h per mg of enzyme.

Description

The invention relates to a method for determining the digestibility of feedstuffs by the enzymatic method.

Determination of feed digestibility is an important nutritional criterion and a prerequisite for the application of new, improved feed assessment systems. It is commonly carried out on sheep or cattle in balance trials, in which the intake of nutrients and the amount of excreted nutrients are determined when feeding the test feed. These experiments can be performed only in specialized workplaces (research institutes) equipped with experimental equipment for animal housing.

The disadvantage of this method is high laboriousness, low seriality, cost and time. This method cannot be used in conventional laboratories. Also, in vitro methods, which are based on incubation of feed samples with rumen fluid, are not applied because of the dependence on rumen fluid source.

Another known procedure does not require the use of animals since it is a laboratory method applicable in conventional laboratories. Digestion fluid digestion of the feed sample is replaced by enzymatic digestion with a complex of enzymes obtained by fermenting the fungus Trichoderma viride (JL De Boever, et al., Animal Ferd Sei Techol., 19, (1988), p. 247). In this case, the cellulose enzyme in the dry state is used, the criterion being the activity referred to as <2 _χ . The introduction of this method is not tied to special equipment. Multiple feed samples may be incubated simultaneously. The prerequisite for obtaining digestibility results corresponding to in vitro values is to use the correct method and appropriate enzyme complex of the desired properties. Given the variability of the enzymatic spectrum of commercially available celluloses, which also affects the digestibility result, it is important to define enzyme quality criteria in order to obtain digestibility values correlating with in vivo values.

Feeds represent a highly heterogeneous substrate including cellulose, hemicellulose, pectin and lignin. The proportion of individual groups in forage crops varies considerably with respect to species, vegetation phase, mowing order, climatic conditions.

The cellulose activity indicator C _x (Trichoderma viride) used so far does not sufficiently characterize the enzyme quality to determine the digestibility of feed. It has been found that xylanase, β-glucosidase and pectinase activities must also be evaluated for a perfect enzyme evaluation. In the absence of these activities or at their lower values, the results of the determination do not correspond to the real values measured in vivo.

The dried feed sample is incubated first with a solution of pepsin in 0.1 M HCl, followed by incubation with the enzyme complex in acetate buffer. The undigested residue of the feed is determined by weighing and the digestibility of the feed is calculated.

This method is refined by the determination of other enzyme activities according to the invention, which is based on the fact that the cellular complex contains a minimum xylanase enzyme activity of 300 μg xylose / h / mg enzyme, a minimum β-glucosidase activity of 1825 μg glucose / h / mg enzyme and pectidase activity of 280 μg glucose / h / mg. For starch-containing feed, a sample of feed with lemon buffer and amylase enzyme is incubated for 1 h at 70-80 ° C prior to incubation with the cellulose complex.

The following is an example of a method for determining the digestibility of feed by the enzymatic method.

Example

A 0.5 g feed sample (ground to a particle size of mm) is weighed in triplicate on an analytical balance (0.0001 g accuracy) into a S1 fritted filter cup of 40 mm diameter. Seal the crucibles from below with a rubber stopper, fill with 40 ml of a 0.2% pepsin solution in 0.1 M HCl and incubate for 24 hours at 39 ° C. This is followed by heating for 30 min at 80 ° C. After removal, the liquid fraction is sucked off and the residue of the feed is rinsed. Seal the crucibles again, fill with enzyme complex acetate buffer and continue incubation for 24 hours at 39 ° C. After the incubation is complete, the liquid is aspirated and the undigested residue is repeatedly rinsed with water. This is followed by drying at 105 ° C for 4 hours, weighing on an analytical balance, ashing in a muffle furnace for 2 hours at 640 ° C and weighing.

Used solutions and chemicals:

0.2% pepsin in 0.1 M HCl

Octated buffer:

1.36 g sodium acetate

0.60 ml acetic acid} make up to 1 l H ₂ 0 g cellulase / 1000 ml buffer + 0,1 g chloramefenicol

Calculation

The digestibility of organic matter (SOH) is calculated from the undigested amount of feed, from the amount of ash and from the weight according to the formula:

SOH (wt.%) = 1 / W - W ₂ / n. (1 - 0.001 p / .S.0,001). 100 = drying crucible weight and undigested residue W ₂ = crucible weight in muffle furnace n = sample weight (g) p = sample ash (g / dry) s = sample dry weight (g)

For a specific example, if

W _x = 47.9997 g

W ₂ = 47.8043 g

η = 0.5 g. p = 94.9 g s = 910.9 g the digestibility value of organic feed material is SOH = 52.5990 wt.

For starch-rich feed, incubation with the enzyme amylase should be performed after incubation with pepsin and heating. The undigested feed is incubated at 80 ° C for one hour with citrate buffer and enzyme amylase (2 g enzyme. ¹ buffer). The digestibility results obtained are corrected for in vivo values by the digestibility of the standard.

Claims (2)

Method for determining the digestibility of feeds by the enzymatic method based on the activities of a mixture of enzymes produced by the fungus Trichoderma viride, in which the sample is first incubated in a solution of 0.2% by weight of pepsin in 0.1 M hydrochloric acid followed by heating at 80 ° C for 30 minutes and further incubation with acetate buffer and cellulase complex, after which the indigestible food residue is dried, cremated and the digestibility of the organic matter is calculated, characterized in that the cellulase complex contains minimum xylanase enzymes of 300 μg xylose / h / mg enzyme, minimal activity β-glucosidase of 1825 mg glucose / h / mg enzyme and a minimum pectinase activity of 280 μg glucose / h / mg enzyme. 2. Method of determining digestibility according to claim 1, characterized in that, for starch-containing feedingstuffs, a sample with citrate buffer and amylase enzyme is incubated for 1 hour at 70 to 80 ° C prior to incubation with the cellulase complex.