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