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Enzyme activity on leguminous crops

 
Dr. Henk Ghesquiere DVM MSc

Impextraco NV

 

 

Dietary fibre is the skeletal remains of plant cells in the diet, which are resistant to hydrolysis by the digestive enzymes of man or animal.

 

This definition distinguishes endogenous and exogenous enzymes. Endogenous enzymes are those enzymes that are produced by the animal and, in the case of digestive enzymes, secreted in the gut. Amylases and proteases (such as trypsin, chymotrypsin, rennin) are endogenous enzymes, thus produced by the animal. For further improvement of feedstuff digestibility, exogenous enzymes are required: phytase, xylanase and B-glucanase are well known examples.

 

In the case of corn/soy diets, it is remarkable that many companies use products with endogenous enzymes, which are normally secreted in sufficient quantities by the animal. All the more remarkable when considering that corn/soy diets contain ample substrate for exogenous enzymes.

 

Substrate determination 

 

 

 

Developing enzyme preparations for animal nutrition is done by matching the appropriate enzymes to the substrate present in the available feedstuffs. Figure 1 shows that there are ample opportunities in legumes such as soybean meal.

 

From an economical point of view, cereals are the best digestible source of energy in animal nutrition. But in order to meet the protein requirements of monogastric animals, we have to rely on plants that are less digestible: the leguminosae. Leguminosae are plants that live in symbiosis with rhizobia what enables them to retain a very high fraction of the N (up to 100 %). Soybean meal is the most widely used protein source in animal nutrition. All leguminosae have similar characteristics as SBM, but they are quite different from cereals. While cereals accumulate starch as the storage form of energy, this is not the case in leguminosae. The energy is stored under the form of oil (most oilseeds and cakes are leguminosae) or oligosaccharides. Also, the NSP composition is quite different, with high viscosity inducing galactomannans being prominent in the NSP fraction of many leguminosae.

 

In comparison to wheat, the amount of dietary fibre in soybean meal is much higher. Soybean meal contains 30 to 35 % of carbohydrates of which only a few percent are starch. The NSP fraction ranges from 18 to 22 %, while the remaining 8 to 10 % are oligosaccharides.

The latter are easily fermented, which results in the production of gases and a bloated feeling. That is why they are called 'flatulence factors'.

 

Calculating the margin between gross energy and digestible energy reveals another interesting issue. In the table, the gross energy (G.E.) has been calculated according to the following formula, which gives a fairly accurate estimation:

 

GE = 5.72 x Crude Protein + 9.5 x Ether extract + 4.79 x Crude fibre + 4.03 x NFE

The remaining values are from the 'Centraal Veevoederbureau', the Dutch institute for animal nutrition.

 

The gross energy is the energy that becomes available at burning the concerned feedstuff in a bomcalorimeter. It gives an indication for the energy intrinsically present in the feedstuff. Since the animal is not able to digest all energy present in feedstuffs, a nutritionist relies on digestible (D.E.), metabolisable (M.E.) or net energy (N.E.). The difference between gross energy (G.E.) and digestible energy (D.E.) gives an indication for the amount of energy that is not digested, thus lost in the faeces. The bulk of the energy is absorbed in case of cereals such as wheat. But, in poultry, less than half of the energy from leguminosae is effectively absorbed, with soybean meal still being the best digestible legume.

 

Feedstuff

G.E.

O.E.pl

GE / OE

GE - OE

( MJ/kg )

( MJ/kg )

( % )

( MJ/kg )

Wheat

15.57

12.91

82.9

2.66

Soybean meal

17.65

8.78

49.7

8.87

 

Roughly spoken, a compound feed consists of 2/3 energy providing cereals and 1/3 protein providing legumes; this leaves a margin for improvement of 2.66 x 0.66 = 1.76 MJ/kg in the wheat fraction and 8.87 x 0.33 = 2.93 MJ/kg in the SBM fraction. Therefore, focusing the supplementation of enzymes on the soybean meal fraction, is worth the effort.

 

Although the amount of phytin is limited, e.g. 0.28 % in wheat and 0.39 % in SBM (on DM), phytase is very efficient in all monogastric diets. Since there is a lot more substrate available as dietary fibre, we can expect much more from fibre hydrolysing enzymes, can't we?

 

Apart from the quantitative aspect, the success of phytase in animal nutrition is mainly due to the consistency of its substrate: phytin or phytic acid. Phytic acid is a fairly simple molecule. In all plant material, exactly the same molecule is present. This consistency of its substrate is one of the main reasons for the reliability of phytase activity. The contrast with enzymes, such as xylanase, is huge, since the chemical structure of their substrate, (arabino) xylans, varies considerably with soil, season, fertilisation, cereal and even between different strains of one cereal.

 

Interestingly, also in leguminosae, a similar substrate consistency as with phytic acid is present. The flatulence factors are fairly simple molecules, which are always present under the same form. Among the NSP macromolecules, the galactomannans present in legumes are fairly simple in composition, while their composition is highly consistent within each kind of legume.

 

Flatulence factors
 

 

The raffinose series of oligosaccharides are sugars that are quite simple. Thus they are an easy substrate for the appropriate enzyme: a-galactosidase. This hydrolysis gives sucrose and one or more galactose units. Animals do not produce this enzyme. The flatulence factors are fermented in the hindgut in pigs or in the caeca in poultry, but when adding a-galactosidase to the feed they are already hydrolysed in the foregut. The sugars are then absorbed in the foregut and used by the animal as a source of energy. Thanks to the exogenous a-galactosidase, the flatulence factors escape microbial fermentation. Reducing bloat gives a better uniformity of the flock. In poultry, simply adding a-galactosidase to the feed also gives an improvement of FCR and growth rate.  In pigs, the effect is less significant, probably due to the larger microbial population in comparison to poultry. Nevertheless, absorption of these readily fermentable sugars in the foregut reduces the risk on proliferation of (possibly pathogenic) bacteria.

 

Galactomannan
 

Another well-identified fraction in the fibre of leguminous crops is the galactomannan. Galactomannan consists of a backbone of mannose units linked in a 1,4-B-manner to which galactose-side chains are attached.

 

In the food industry, guar gum is a common source of galactomannan. It is used as a thickening agent for its high viscosity inducing capacity. It is well known that a-galactosidase changes the characteristics of this thickening agent, while adding extra B-mannase eliminates the viscosity. In animal nutrition, galactomannan is known as a drawback because of its viscosity inducing capacity. Many feedstuffs are deemed unfit for use due to the presence of galactomannan, while the use of several others has been restricted (e.g. copra, palm kernel cake.

 

Even though galactomannan is a typical member of the Non-Starch Polysaccharide (NSP) fraction, it is a fairly easy one to tackle with exogenous enzymes. The backbone is exclusively a 1,4-B-mannosepolysaccharide, while only galactose side chains are attached. Within a plant species, the composition is extraordinarily consistent, while between plant species only the frequency of substitution by galactose differs. The galactose/mannose ratio is considered a characteristic for the concerned plant: e.g. 38 % galactose / 62 % mannose in guar seeds or 22 % galactose / 78 % mannose in the carob plant. A consistent substrate structure combined with the appropriate enzymes gives a reliable activity. Lack of consistency is a problem with most NSP in feedstuffs; arabinoxylans in wheat e.g. vary with breed, soil, fertilisation program, season, etc. resulting in fluctuations of the effect of added xylanases. 

 

One aspect of the galactomannan requires careful consideration. B-mannanase effectively cuts the backbone to pieces, but only on condition that it has sufficient space to adhere to this backbone (some 4 unsubstituted mannose units). For this reason, a combination of a-galactosidase and B-mannanase is required: Zympex 008. The a-galactosidase splits the galactose units from the backbone, thus liberating space for the B-mannanase to hydrolyse the backbone. This effectively reduces the viscosity.

 

Supplementing the corn/soy diet with a-galactosidase and B-mannanase gives significant improvements in FCR, growth rate, energy and ileal amino acid digestibility in broilers, layers and turkeys. Also with corn/soy diets for pigs, performance improvements above 10 % have been registered.

 

Apart from improved digestibility and reduced viscosity, the influence on the gut microflora is also important. 

 

Indeed, the concerted activity of a-galactosidase and B-mannanase gives an in vivo production of mannanoligosaccharides (MOS). Many feed mills add MOS to their diets under the form of cell walls from Saccharomyces cerevisiae for its immunity inducing effect and for eliminating some pathogenic bacteria.This consequence of enzyme activity on galactomannan is demonstrated by infection experiments with Salmonella enteritidis both in pullets and in layers. The experimental groups received enzymatically-hydrolysed galactomannan in the diet. In comparison to the control group, there was a significant reduction in Salmonella positive excreta, organs, egg shell, egg white and egg yolk.
 

 

Cofactors


For an optimal activity, the appropriate co-factors are also required. Although those co-factors remain unchanged during the enzymatic reaction, the enzyme needs them in some way before its activity can start. Co-factors are provided in Zympex 008.

 

Remaining NSP

 

In corn/soy diets, B-glucanase improves the performance of the animals. Many other enzymes are also beneficial, since the remainder of the NSP fraction of soybean meal is a heterogeneous group of different substances. Apart from pectins, also cellulose and hemicelluloses (acidic polysaccharides, arabinan, arabinogalactan) are present. Their nature is often quite complex: acidic polysaccharides e.g. are chains of galacturonic acid and galactose interspersed with rhamnose and are highly branched. A detailed description of such substances is beyond the scope of this article.

 

Impextraco has chosen to tackle this group with NSP enzymes produced by a technique different from the one used by the major industrial enzyme producers. Instead of using simple molasses and mineral nitrogen as nutrients for the enzyme producing fungi, in this technique, the fungi are grown on complex raw materials (e.g. wheat bran, complete cereals, sugar beet pulp, etc.).

 

Supplementary to the main enzyme activity, this production technique provides a wider range of additional activities. 

 

Animal nutrition continues its progress. Adding the combination of a-galactosidase and B-mannanase to the already existing battery of NSP enzymes allows feed mills to progress in the coming years.

 

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