Viscosity reduction by three feed enzymes was measured in a high throughput pressure sensing system using commercial dosages on purified polysaccharides (arabinoxylan and ß-glucan). Fluorescence and antibody microscopy techniques were also used to visualize effects on the solubilisation of endosperm cell walls of wheat and barley. Viscosity data clearly demonstrated depolymerisation of mixed-linked ß-glucans by the two multicomponent enzymes. The two multicomponent enzymes also reduced the viscosity of the arabinoxylan solution, while the monocomponent xylanase most effectively depolymerised the arabinoxylan. Microscopy data revealed that the multicomponent enzyme chosen for the studies could solubilize barley cell walls by targeting both the ß-glucan strutures as well as the arabinoxylans. The xylanase could effectively solubilise barley cell walls by merely attacking the arabinoxylans.

1. The results obtained in this study demonstrates the strong hydrolytic capacity of xylanases and ß-
glucanases that results in efficient viscosity reduction of soluble xylans and ß-glucans. By using
microscopy techniques it was also possible to demonstrate the cell wall degrading efficacy of
commercial feed enzymes commonly used in animal production. A notable effect demonstrated was
the capacity of a monocomponent xylanase to degrade the cell wall constituents of barley. This
finding indicates that a ß-glucanase is not necessarily essential for releasing nutrients encapsulated
by insoluble cell walls partly made up by mixed-linked ß-glucans. However, in situations where high
concentrations of soluble ß-glucans increase digesta viscosity to levels detrimental for nutrient
absorption a dedicated ß-glucanase may provide an efficient solution.
The methods employed in this study may be used as a valuable in vitro methodology to demonstrate
the complexity of feed ingredient degradation and solubilisation by commercial feed enzymes.
Coupling of such results with actual animal performance data may provide an important tool for
better understanding the relationship between cell wall degradation and viscosity reduction as
predictors of the productive value of feed enzymes
Solubilisation and viscosity reduction effects on cereal cell walls
mediated by xylanase and ß-glucanase
Jonas Ravn1, Helle Juel Martens2, Dan Pettersson1,
Ninfa Rangel Pedersen1
1Novozymes A/S, Feed Applications, Krogshoejvej 36, 2880 Bagsvaerd, Denmark
2University of Copenhagen, Dept of Plant and Environmental Sciences, Thorvaldsensvej 40, 1871 Fredriksberg, Denmark
Viscosity reduction by three feed enzymes was measured in a high throughput pressure sensing
system using commercial dosages on purified polysaccharides (arabinoxylan and ß-glucan).
Fluorescence and antibody microscopy techniques were also used to visualize effects on the
solubilisation of endosperm cell walls of wheat and barley.
Viscosity data clearly demonstrated depolymerisation of mixed-linked ß-glucans by the two
multicomponent enzymes. The two multicomponent enzymes also reduced the viscosity of the
arabinoxylan solution, while the monocomponent xylanase most effectively depolymerised the
arabinoxylan. Microscopy data revealed that the multicomponent enzyme chosen for the studies
could solubilize barley cell walls by targeting both the ß-glucan strutures as well as the
arabinoxylans. The xylanase could effectively solubilise barley cell walls by merely attacking the
arabinoxylans.
Three commercial enzymes; RONOZYME WX (monocomponent xylanase), RONOZYME MultiGrain (a
multienzyme product containing mainly xylanase and ß-glucanase) and RONOZYME VP (a
commercial multicomponent enzyme containing over 40 different activities mainly targeting other
components besides arabinoxylan and mixed-linked ß-glucan) were tested. Substrates used for the
viscosity study, arabinoxylan and ß-glucan, were obtained from Megazymes, Ireland.
Viscosity studies were conducted using a Hamilton pipetting workstation with eight microprocessor-
controlled pipetting channels that are analogous to manual pipettes. The viscosity pressure (ViPr)
assay displays a linear correlation between pressure measured (Pa) and kinematic viscosity (ABEL
& PETTERSSON, 2011). During the complete aspiration and dispensing process the air
displacement is continuously monitored in intervals of 10 ms by a pressure sensor to verify an
uninterrupted substrate flow. Pressure measured in this work is defined as viscosity determined in
relation to the ViPr assay.
Microscopy studies using antibody label were carried out as described below
Figure 2. Antibody labelling of wheat and barley.
Sections of barley and wheat (10 µm) were treated for 3h with RONOZYME MultiGrain or
RONOZYME WX. Enzyme or buffer droplets were applied directly on the glass slide.
Autofluorescence, as well as histochemical staining, was used to visualize the endosperm
structures. Fluorescence microscopy was performed using a Nikon Eclipse 80i Microscope (Nikon
Corporation, Japan) to visualize structures in both wheat (data not shown)and barley using
Calcofluor white.
Within 30 minutes a distinct viscosity reduction was observed when adding the commercial
products to a viscous arabinoxylan solution (Fig. 3). There was no noticeable viscosity reduction
observed for the ß-glucan solution when adding the monocomponent xylanase, as expected,
while the two multicomponent products (RONOZYME Multigrain and VP) caused a marked
viscosity reduction (Fig. 3) due to their ß-glucanase activity.
Figure 3. Viscosity reduction of an arabinoxylan solution 1g/100 mL (left figure) and a ß-glucan solution 0.5 g/100
mL (right picture) when adding commercially related dosages of the respective enzymes.
Staining with calcoflour white revealed the mixed linked ß-glucan structures present in the cell walls of
barley and on enzyme addition with the Ronozyme Multigrain the cell wall structures were disrupted
and no longer visible (Fig. 4). A similar effect was not observed for the monocomponent xylanase (data
not shown). Both the multicomponent and the monocomponent xylanase product efficiently degraded
the arabinoxylan present in the cell walls (Fig.5), as indicated by immuno-labeling with the LM 11 label
targeting arabinoxylans (disapppearance of red colour).
Introduction
Even after milling of feed ingredients some nutrients will remain within intact cell walls built up by
indigestible fibre components. Commercial exogenous feed enzymes, such as mixed-linked ß-
glucanases or arabinoxylanases, are used to degrade fibre components in cereals by de-
polymerising soluble fibres. This may reduce digesta viscosity and thereby facilitate nutrient
absorption. Exogenous microbial enzymes may also mediate solubilisation of the insoluble cell wall
matrix, thereby releasing captured nutrients.
ABEL, G., PETTERSSON, D. 2011. Viscosity pressure assay. Patent WO 2011107472 A1.
Figure 4. Barley stained with calcoflour white,
left picture (control), and the same sample
after 3 h of enzyme incubation with
RONOZYME Multigrain.
Figure 5. Barley labelled with the LM 11 immuno label (left picture)
and after 3 h when treated with RONOZYME Multigrain (middle
picture) or RONOZYME WX (right picture).
3h Buffer 3h enzyme 3h Buffer 3h enzyme 3h enzyme
7 µm section
3 hours, 39 °C
pH5, stirring.
2. Labeling 3. Confocal microscopy
Alexa-555
Secondary ab α-rat
Primary ab (LM)
1. Treatment
Conclusions
Figure 1. Iodine stained starch
in milled maize endosperm
particles showing intact cells
despite the milling process.
Abstract
Methods
References
Results