Index:
From grain to nutrients ...
Chemical Composition of Grains
NSP Contents of Raw Materials
Arabinoxylan structure
Xylanase action
Penicillium funiculosum
Rovabio Excel profile
Cellulose structure
Mixed-linked ß-glucan structure
Glucanase and cellulase action
Rovabio Excel profile
Pectin structure
Pectinase Action
Rovabio Excel profile
Multi-enzyme preparation advantages
Released oligosaccharides by xylanase
Released oligosaccharides by multi-enzyme preparation
Different Xylanases secreted by P. funiculosum
Different profiles of activity...
Conclusions

From grain to nutrients ...

A grain contains germ (the embryo), reserves (storage tissues: endosperm and aleurone layer) for embryo growth and maturation and protection tissues (envelopes to protect against external aggression; i.e., pericarp or bran). In practice, bran often contains the aleurone layer.

Polysaccharides differ between protection tissues and storage tissues: more complex (i.e., more branched) in the bran than in the endosperm.

Cells in the endosperm and in the bran are protected by cell walls, which contain either arabinoxylans and b-glucans or heteroxylan and cellulose.

Chemical Composition of Grains

Grains mainly contain starch (a glucose polymer in a-1,4, a-1,6): about 2/3. Surprisingly, Non Starch Polysaccharides (NSPs) are as abundant as proteins. Vegetable protein sources (soybean meal) contain even more NSPs. NSPs also differ between materials: soluble and insoluble proportions as well as type of NSPs (pentosans, etc.)

Composition of raw materials (% DM):

*Literature review, 2002

For the NSPs, differences between raw materials are:
- Content of water-soluble NSPs
- Nature of main NSP (pentosans, mixed-linked ?-glucan, cellulose or pectins)

NSP Contents of Raw Materials

Wheat and rye do not contain soluble b-glucans. They mainly contain pentosans, either soluble or insoluble. Whereas rye and wheat show the same content of total pentosans, rye contains more soluble pentosans than wheat.

For pentosans, heteroxylans are always insoluble, while arabinoxylans and arabinogalactans can be either soluble or insoluble.

Corn does not contain b-glucans, either soluble or insoluble. Its pentosans are mainly insoluble.

Barley is rich in b-glucans, mainly soluble. It also contains pentosans.

Most of the NSPs from vegetable protein sources are pectins of different types. At least a third of NSP’s are of soluble form, which will be responsible for limitation of digestibility.

Arabinoxylan structure

Arabinoxylan stands for arabinose residues branched on a xylose backbone.
The xylose backbone is composed of xylose pyranosyl (Xylp), whereas the arabinose residues are arabinose furanosyl (Araf). These two sugars are composed of 6 carbons, but their structures are different: two different cycles for pentoses.
Xylopyranose residues may be branched by only one arabinofuranose or by two arabinofuranoses. Mono or disubstitution will then affect the susceptibility to exogenous enzymes.

Arabinose residues might even be substituted by ferulic acid.

Xylanase action

• Endo-1,4-b-xylanase cuts the linear chain of b-1,4-xylose units. It cuts the poor substituted regions, but there are different b-xylanases with different actions.
• Feruloyl esterase releases the feruloyl acid.
• a-arabinofuranosidase cuts the bond between arabinose and xylose, but only when xylose is mono-substituted.
• b-xylosidase cuts the linear chain of 2,3 or 4 b-1,4-xylose units to release xylose.

Penicillium funiculosum

Penicillium funiculosum is a filamentous fungus which secrets protein into the medium. From the natural cocktail produced we guarantee xylanase, b-glucanase, and cellulase activities.

Rovabio Excel profile

All enzymes required to breakdown arabinoxylans are present in Rovabio Excel. Rovabio also contains arabinanase activities allowing to breakdown arabinose links.

Collaboration: S. Kermasha, L. L’Hocine McGill Univ Montreal, CAN
µmol/min/mg prot
4 batches Excel LC

Cellulose structure

Cellulose are composed of linear chains of glucopyranose units and it ’s a microfibril type of structure with hydrogen bond between fibrils.

Mixed-linked ß-glucan structure

As cellulose, mixed-linked b-glucan is a linear chain of glucopyranose units, but with different bonds : b-1,4 bonds and b-1,3 bonds.

In barley, the ß -glucans contain approximately 70 % of ß-(1-4) linkages, in which segments of two to five ß-(1,4) bonds are separated by single ß-(1,3) linkages.
(Parrish et al., 1960)

Glucanase and cellulase action

• Endo-1,3(4)-b-glucanase cuts the b-1,4 bonds between the glucose units located just after the b-1,3 bonds.
• Endo-1,4-b-glucanase cuts the b-1,4 bonds between the glucose units when there are only b-1,4 bonds in the linear chain of glucose.
• Cellobio-hydrolase cuts the cellobiose. Cellobiose is two glucose units linked by b-1,4 bond.
• b-glucosidase cuts the linear chain of 2,3 or 4 b-1,4-glucose units to release glucose.

Rovabio Excel profile

All enzymes required to break down mixed-linked b-glucan and cellulose are present in Rovabio Excel. Rovabio also contains laminarinase activities, allowing to breakdown linear chains of b-1,3 glucose units.

Collaboration: S. Kermasha, L. L’Hocine McGill Univ Montreal, CAN
µmol/min/mg prot
4 batches Excel LC

Pectin structure

Pectin is composed of two regions : a rhamnogalacturonan region (alternate Rhamnose -Rha- and Galacturonic Acid -GalA- linear chain substituted by Arabinose -Ara- chain) and an homogalacturonan region. Soybean does not contain homogalacturonan regions. Galacturonic acid units might also be substituted by Methyl (Me) or Acetyl (Ac) groups.

Pectinase Action

Rhamnogalacturonase cuts the bonds between galacturonic acid and rhamnose in the rhamnogalacturonan region. Polygalacturonase cuts the linear chain of galacturonic acid in the homogalacturonan region. Pectin esterase releases the methyl residue linked to the galacturonic acid. Pectin acetylesterase releases the acetyl residue linked to the galacturonic acid.

Rovabio Excel profile

Pectin degrading activities are also present in the Rovabio with important pectinase and polygalacturonase activities. Rovabio Excel also contains an endo-1,4 b-mannanase and other residuals activities such as protease and b-mannosidase.

Collaboration: S. Kermasha, L. L’Hocine McGill Univ Montreal, CAN
µmol/min/mg prot
4 batches Excel LC

Multi-enzyme preparation advantages

The experimental design is :

• control : with no enzyme added
• xylanase (pure from Megazyme)
• b-glucanase (pure from Megazyme)
• mix of these two pure enzymes
• multi-enzyme preparation = natural cocktail of enzymes (with also the side activities (debranching ....))

This experiment shows that endo b-1,4 xylanase reduces the viscosity of the wheat and maize, whereas b-1,3(4)-glucanase has no action. The addition of the mix of these two enzymes doesn’t reduce more the viscosity of these cereals than with only xylanase. This observation may be explained by a low level of b-glucan in these two cereals.
However, when enzymes other than endo b-1,4 xylanase and b-1,3(4) glucanase are added on these two cereals, the viscosity is more reduced than with only endo b-1,4 xylanase (cf multi-enzyme).

So, to break down the cell walls of cereals, a wide range of enzymes other than endo b-1,4 xylanase are required.

Multi-enzyme preparation advantages

The experimental design is:

• control: Barley with no enzyme added
• xylanase (pure from Megazyme)
• b-glucanase (pure from Megazyme)
• mix of these two pure enzymes

This experiment shows that endo b-1,4 xylanase reduce the viscosity of barley. The addition of b-1,3(4)-glucanase reduces more the viscosity of this cereal and also the addition of the mix of these two enzymes. This observation may be explained by the NSPs composition of this cereal.

Released oligosaccharides by xylanase

Analysis of the released oligosaccharides also demonstrates the importance of the range of enzyme activities to allow the maximum of liberation of the monomers. When only xylanase are added to the arabinoxylans, low quantities of xylose, xylobiose and xylotriose are released with different oligosaccharides. No arabinose is released.

Released oligosaccharides by multi-enzyme preparation

When a multi-enzyme preparation is added to arabinoxylans, many arabinose, xylose, xylobiose and xylotriose units are released. This preparation approximately released 3 times more xylose units and 2 times more xylobiose and xylotriose units than the endo-b 1,4 xylanase.

Different Xylanases secreted by P. funiculosum

Rovabio Excel contains not only a range of enzyme activities, but also different xylanase proteins, which have different molecular sizes and enzymatic characteristics allowing to work on the different wheat NSPs, particularly soluble and all along the digestive tract.

Collaboration IFR-Norwich : C. Furniss & G. Williamson

Different profiles of activity...

The different xylanases also have different optimum pH levels, which allow the Rovabio Excel to work at different levels of the digestive tract.

Collaboration Rhodia (N. Fish) and IFR-Norwich (C. Furniss, G. Williamson)

Conclusions

• Non Starch Polysaccharides have complex structures.
• Rovabio™ Excel : The Versatile Solution!
  • A natural multi-enzyme combination : to allow efficient breakdown of the undigestible cell wall structures present in a wide range of raw materials, to support the extreme variation in physiological conditions of the digestive tract.