Dietary fibre pleads for multi-component enzyme products

Feed enzymes are mostly produced by fungi or bacteria. Those living organisms produce a wide variety of different enzyme proteins. For commercial practice, only a limited number of those activities is assayed in the laboratory. Assaying e.g. the xylanase activity obviously results in a quantification of the xylanase level, while not providing information about other activities that remain present in the fermentation product. In practice, the quantified and standardized enzyme activity is referred to as the “main” activity, while the unguaranteed activities are “side” activities.

For industrial enzymes such as feed products, removing the side activities simply is not an option. Techniques for isolation of one single protein or main activity are very expensive, while many side activities are known to be beneficial in animal nutrition. Therefore, a xylanase product mostly contains also side activities such as (amongst others) β-glucanase and vice versa.

By selection or modification of the production strains, enzyme manufacturers may increase the level of the main activity, thus changing the ratio main/side activities. Such practice has been very efficient for phytase production: in the current products, the ratio phytase/side activities are several hundred times higher compared to a few decades ago; as the side activities in current phytase products can be neglected, such phytases are considered monocomponent products.

As for fibre degrading enzymes, similar techniques also resulted in a few mono-component xylanase products. The more economical fermentation increases the margin for the enzyme producers, but the feed sector loses the benefits of the side activities.

In contrast to mono-component xylanases, for several other products more than one enzyme activity is guaranteed. Thus, the feed sector is assured to purchase several main activities above a minimal level. Certain companies achieve this by assaying more than one activity in a single fermentation product. Other companies further enlarge the scope of enzymatic activities by combining several fermentation products into a single commercial product. EU registration files show that from two up to five fermentation products are combined in single presentations.

For each of the main activities, a figure for the minimum level is guaranteed. As an example: Zympex 008 contains a guaranteed minimum level of β-xylanase, β-glucanase, β-mannanase and α-galactosidase, while the side activities remain a supplementary benefit.

Comparing the substrates clarifies why a single-component phytase is a valid option, in contrast to fibre enzymes for which multi-component products are preferred.

Phytic acid The phytase substrate is a relatively simple molecule: a six-ring to which six phosphate groups are attached. Phytase sequentially removes phosphate from the inositol-ring and (E. coli) phytase is able to hydrolyze all six phosphate groups. One single enzyme protein is able to perform all the required work!

There simply is no variation in the concerned inositol-hexakisphosphate: exactly the same phytic acid is present in all plant material. As there is no substrate variation, side activities are less meaningful for phytase (albeit combinations with fibre degrading enzymes are known to also release the phytic acid that is embedded in cell walls: Zympex Combi-P).

Steric hindrance of phytase is known to reduce its efficacy. Phytic acid forms complexes with e.g. ions such as calcium or zinc; such complex-formation blocks the access for phytase to the phosphate-inositol-bonds. Fortunately, at the low pH in the stomach, such complexes fall apart without requirement for side activities to remove the access blocking groups; it explains why phytase activity is mainly performed in the stomach.

As a mono-component phytase is able to perform all the required work, it makes sense to economize its production by selection or modification of the production strain.

Variability in e.g. Arabinoxylans In contrast to phytic acid, there is no such thing as a single, well-defined type of fibre substrate. Arabinoxylan in wheat differs from that in corn or other crops. There is variation between wheat strains, while also the climate, soil and other external conditions have an impact on arabinoxylan nature.

The backbone of arabinoxylans is invariably a chain of linked xylose-units; the length of the chain can vary from a few thousands to over half a million units. The main variation is in the nature of substitution or side chains attached to the backbone. The name arabinoxylans is derived from the fact that many arabinose-sidechains are attached to the xylose-backbone; the ratio arabinose/ xylose, an indication for frequency and length of substitution, is highly variable (0.30 - 1.40)

While β-xylanase has the highest impact on arabinoxylan degradation by splitting the backbone, the side chains may block its access to this backbone. The presence of enzymatic side activities, therefore, increases the impact of β-xylanase; it has been documented (Valls et al., 2015) that arabinofuranosidase may increase the impact by a factor 2.5 simply by preliminary removal of arabinose-sidechains. The same should be valid for other side activities, such as ‘ferulic acid esterase’, ‘acetyl esterase’ or ‘glucuronic acid esterase’.

Fibre degrading products thus benefit from the presence of side activities. Products from solid state fermentation and multi-component products therefore cope with a wider variability in fibre compositions. Such products allow a higher flexibility in feed formulation.

What’s more, the dietary fibre may well be the most variable fraction in feedstuffs. Dietary fibre can be defined as that portion of the food or feed that is not digested in the human or animal small intestine, thus excluding the portion that is fermented in the hindgut. It required 20 years of deliberation before the Codex Alimentarius was able to agree in the year 2009 on a definition for dietary fibre; there still is no agreement on whether oligosaccharides (DP 3 – 9) should be included, while the FDA still has to implement this definition in the USA.

In animal nutrition, the concerned enzymes have been described as those enzymes degrading ‘fibre’, ‘cell walls’, ‘NonStarch Polysaccharides’, ‘hemicellulose’, etc.; this highlights the variability. Although referring to similar feed fractions, those terms all cover a somewhat different scope.

Even today, quantification and characterization still remains a difficult task for which different methods are described:

• Traditionally, the ‘Proximate or Weende Analysis’ determined the Crude Fibre (C.F.); as this only represents (part) of
the cellulose and lignin, the CF is a very poor quantification of the enzyme substrate(s).

• The Van Soest method is a major improvement. The
‘Neutral Detergent Fibre’ (NDF), ‘Acid Detergent
Fibre’ (ADF) and ‘Acid Detergent Lignin’ (ADL)
allow a far more accurate substrate quantification.
Nevertheless: as the figures relate to precipitated
fractions, the correlation with the soluble substrate
fraction remains poor (although important for
viscosity aspects). Determining the ‘Neutral Detergent
Soluble Fibre’ (NDSF) is not (yet) common practice in
the feed sector.

• Certain literature refers to the ‘Water Insoluble Cell Walls’ (WICW), but shows similar limitations regarding the
soluble fractions.

• The above mentioned quantification as ‘Total dietary Fibre’ (TDF) described by the Codex is best correlated with
enzyme activity. Distinguishing ‘Insoluble’ (IDF) and ‘Soluble Dietary Fibre’ (SDF) further improves the predictability
of enzyme effects.

• Determination of the individual monosaccharides allows more specificity (albeit still imperfect as e.g. glucose can
relate as well to β-glucans as to cellulose, heteroxylans, etc.).

While a mono-component β-xylanase hydrolyzes the arabinoxylan fraction, the overview displayed in the graphic leaves no doubt that additional activities further enlarge the scope of fibre degrading products: β-glucanase, β-mannanase, α-galacosidase, ... also are cost-effective.

Apart from in vitro quantification, also the behavior in the gut of the fibre fractions is important. Gut viscosity is highly correlated to the solubility of the fibre fractions. In order to reduce the gut viscosity, the β-xylanases and β-glucanases were originally selected for their impact on the soluble fibre fractions only. While such enzymatic viscosity reduction allowed the introduction of wheat and barley in broiler diets, the impact on corn fibre remained limited as this is mostly insoluble.

The original β-xylanases and β-glucanases, with only limited activity in the insoluble fibre fraction, were not able to produce economically viable results in corn diets. This resulted in the introduction of enzymes with a more pronounced activity in the insoluble fraction. Because intact cell walls function as a cage for entrapped nutrients, those are released once enzymes break the concerned cage walls.

Apart from viscosity reduction, it is also important to break the “cell wall cages” to release extra energy and protein. Thus, today's enzyme products also produce significant effects in corn and other low viscosity diets.

As certain fibre degrading enzymes perform better on the soluble fraction, while others show a multifold activity on the insoluble fraction, it is recommended to use a combination of different xylanases and different glucanases for maximum effect in a wide range of feed compositions. This pleads for multi-component products.

Dietary fibre is fermented by the microflora in the hindgut; the volatile fatty acids that are thus produced are absorbed and used as a source of energy. Hydrolyzed fibre fractions also show an impact on the composition of the microflora. Prebiotic effects have been documented not only for mannan-oligosaccharides (MOS), but also for xylo-oligosaccharides (XOS) and galacto-oligosaccharides (GOS). Albeit more variable, such prebiotic effects are additional to effects from viscosity reduction or breaking cell wall cages.

One of the most consistent microflora effects is obtained by removing the raffinose series of oligosaccharides from vegetable protein sources, such as SBM. Although readily fermented, those oligo’s promote gas-producing bacteria, thus causing flatulence and fluctuations in feed intake. As α-galactosidase splits the raffinose oligo’s into absorbable sugars (sucrose and galactose), the animal benefits from the extra energy, while foregut bacterial growth is reduced.

Fibre in cereals differs from that in vegetable proteins. Apart from the abovementioned raffinose oligo’s, the presence of e.g. galactomannans in legume proteins also requires another type of enzymes; full hydrolysis of galactomannans is feasible only by a combination of β-mannanase and α-galactosidase. While the galactomannan level is relatively moderate in SBM, it is far more pronounced in other legumes used as vegetable proteins, while being very high in certain by-products such as copra and PKM. Therefore, enzymes have a crucial impact on the inclusion rate that can be achieved with such feedstuffs.

Multi-component enzyme products thus allow more flexibility in feed formulation and further economize on feed cost.