Before we turn to xylanases, their inhibitors and how the inhibitors affect the biotechnological application of these enzymes, we need to look at the substrate for the xylanases – arabinoxylan. If we take a look at wheat, the main constituents are starch, protein, non-starch polysaccharides - including arabinoxylan – and lipids Arabinoxylan is a complex carbohydrate polymer with a beta-1,4-xylosyl main chain. This backbone is substituted in the C2 or C3 position – or both – with arabinose. The average of A over X is approx. 0.55. Finally, arabinose can be substituted with hydroxycinnamates – like ferulic acid – via ester linkages. The arabinoxylan is located in the endosperm cell wall, where it is reported to account for up to 70% of the wall material, and in the pericarp. Depending on the fractionation of the flour, the arabinoxylan concentration may vary from 2-5%. The more bran in the flour – the higher the arabinoxylan concentration.
• Viscosity increase• Hydrocolloid effect• Less sticky dough• Dough stability
• Viscosity decrease• Loss of hydrocolloid effect• Stickier dough• Dough stability?
AX Populations:
WU-AX HMW S-AX andWE-AX
LMW S-AX andWE-AX
Xyl Xyl
Presenter
Presentation Notes
Even though arabinoxylan is a minor constituent, it has a huge impact on the technological properties of wheat flour. If we look at what I will call the functionality of arabinoxylan and, of course, the functionality of xylanases in baking applications – arabinoxylan can be divided into three populations: Water unextractable AX: WU-AX High molecular weight, solubilised and water extractable AX: HMW WE-AX Low molecular weight, solubilised and water extractable AX: LMW WE-AX When processing the flour – either by mixing or stirring – the AX population will be more soluble and have a lower molecular weight. This is due to mechanical or enzymatic solubilisation and depolymerisation of the arabinoxylan. If we evaluate the functionality of the different populations of arabinoxylan, then we find that the water unextractable arabinoxylan has a negative effect on gluten strength and gluten yield. High molecular weight, water extractable arabinoxylan increases the viscosity of the dough system, mimicking a hydrocolloid effect. This gives a less sticky and more stable dough. The low molecular weight, water extractable arabinoxylan population reduces the dough liquid viscosity and tends towards increased dough stickiness. However, it will still bring stability to the dough. So, when applying a microbial xylanase to the system, the xylanase has several populations of substrate. Ideally, the xylanase will solubilise all the water unextractable arabinoxylan and leave it as high molecular weight arabinoxylan.
Functionality of AX and xylanase in baking
Effect of xylanase treatment on dough. Dough stained with calcofluor white
Xylanase treated dough45 minutes, 34°C
Control dough45 minutes, 34°C
Presenter
Presentation Notes
This slide shows two pictures of dough systems which have been stained by calcoflour white. Calcoflour white stains beta-glucan a light blue/white colour. So what we see is a staining of cell wall fragments – fragments that contain up to 70% arabinoxylan. Looking at the first picture it is easy to imagine that these fragments have a negative influence on the gluten matrix. The second picture shows dough which has been treated with xylanase for 45 min at 30°C. Here it can be seen that the cell wall fragments have either been removed or are significantly reduced in number or size. People could argue that it is easy to show a picture that verifies theory. However, our measurements of the stained particles show that they decrease in size and number as a function of xylanase activity. So these two pictures are representative for the dough systems.
Functionality of arabinoxylan and xylanase
Upgrading gluten quality by the removal of WU-AX
Creation of hydrocolloid in dough – leading to an improved gluten matrix
Xylanases facilitate the upgrading of weak flour to baking quality flour and minimise batch to batch variation
Xylanases add stability to the dough system and the production process in general
Xylanases improve the final product (structure, shape and volume)
7
Presenter
Presentation Notes
A short summary of the functionality of arabinoxylan and the effect of xylanases.
8
Volume response in crusty rolls
Control 100ppm 150ppm 200ppm
Medium European flour, 70 min
GRINDAMYL® POWERBake 900
Presenter
Presentation Notes
This slide shows the performance of GRINDAMYL™ POWERBake 900 in a crusty roll recipe – with a fermentation time of 70 min, which is longer than that used in a normal procedure. The main reason for extending the fermentation time is to stress the system and, thereby, make it more unstable. This is normally evident from the more uneven crust and crumb structure.
Shock test on Tweedy bread, UK flour
9
100ppm GRINDAMYL® POWERBake 900
Control systemwithout xylanase
Normal: 4.5ccm/g
Shocked: 4.1ccm/g
Normal: 4.7ccm/g
Shocked: 4.5ccm/g
Presenter
Presentation Notes
In a Chorleywood procedure (Tweedy) with high-speed mixing, the addition of GRINDAMYL™ POWERBAKE 900 results in a much more stable system compared to a control system to which no xylanases have been added. Compared to other commercial enzymes, the effect of GRINDAMYL™ POWERBake 900 is also outstanding for the dough stability it provides – as can be seen on the next slide.
Protein 14,3 10,2 13,2 12,9Wet gluten 30,8 26,7 33,8 30,9Falling number 681 434 298 392Optimal ascorbic acid 0 30 10 40Extensibility short very ext. very ext. very ext.
Flour analysis - Farinograph
Application tests of selected flours
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2002048 2002063 2000122 2002007
Non-optimised flour – from previous slide
Optimised flour + base of 0.2% PANODAN® A2020
200ppm GRINDAMYL® XV 100ppm GRINDAMYL® XV 100ppm GRINDAMYL® XV 100ppm GRINDAMYL® XV
Application tests of selected flours
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2002048 2002063 2000122 2002007
Non-optimised flour
Optimised flour + base of 0.2% PANODAN® A2020
200ppm GRINDAMYL® XV 100ppm GRINDAMYL® XV 100ppm GRINDAMYL® XV 100ppm GRINDAMYL® XV
Application tests of selected flours
Page 12
Effect of xylanases on High top loaves
5,85
6,13
6,32
6,49
6,42
5,4
5,6
5,8
6
6,2
6,4
6,6
Control Market standard @ 30 ppm POWERBake® 950 @ 30 ppm
POWERBake® 950 @ 50 ppm
GRINDAMYL® H 460 @50 ppm
Spec
ific
volu
me,
ccm
/g
Effect of xylanases on high ash content flour - High top loaves
4,9
5,23
5,12
5,49
5,42 5,42
4,6
4,7
4,8
4,9
5
5,1
5,2
5,3
5,4
5,5
5,6
Control TS-E 1731 @ 50ppm TS-E 1732 @ 50ppm TS-E 1731 @ 60ppm TS-E 1732 @ 60ppm Regular flour: 10.5% protein flour with market sample @
This slide shows the main lipids found within the three categories. Triglycerides and mono-di glycerides, which are non-polar lipids and the glycolipids and phospholipids, where the most important ones are digalactosyl diglycerides and monogalactosyl diglycerides as well as lysophosphatidyl cholines (lyso-lecithin) and phosphatidyl cholines (lecithin). Here it is important to notice that a relatively high part of the phospholipids are starch-bounded lipids – and are therefore less available to enzymes like lipases.
In this presentation we will concentrate on the functionality of a lipase with glycolipase activity as these lipids are some of the more available lipids in flour. So this slide shows an overview of some of the most important components found within the category of glycolipids. It is a well-known fact that the glycolipids are highly surface active components and that e.g. DGMG is one of the most active/polar components.
Hydrolysis of digalactosyldiglyceride, DGDG
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GlycolipaseOH2+
+
FFA
O
OH
H
H
HO
H
O
OHHH
OH
O
OH
H
H
HO
H
OHHH
O
O
O
O
O
O
OH
H
H
HO
H
O
OHHH
OH
O
OH
H
H
HO
H
OHHH
O
O
OH
O
HO
O
DGMG
DGDG
Presenter
Presentation Notes
This slide gives an example of one of the main reactions taking place when using a glycolipase, where the di-galactosyl-diglyceride is hydrolysed to di-galactosyl-monoglyceride and free fatty acid. This is - as just mentioned - one of the most surface active components in flour. This reaction releases a potential emulsifying effect naturally present in the flour.
Polar lipids
Gas Cell Inter phase
Stabilisation of gas cells
Presenter
Presentation Notes
This schematic drawing simply shows the stabilisation of air cells/bubbles by the use of polar lipids – stabilisation of the inter phase between lipids and protein.
Based on the baking trial just shown lipid analyses have been made. For making these lipid analyses we use the fully fermented dough. This slide shows the amount of DGDG and DGMG found in each of the trials. It is seen that use of the GRINDAMYL™ POWERBake 4100 results in a modification of the DGDG as this lipid decreases by increased dosage. At the same time the amount of DGMG is increased. The results is in percent, based on dry weight as the extraction is made on basis of freeze dried dough. For this analysis GLC (gas liquid chromatography) has been used). It is important to notice that a higher amount of lipid is extracted from the dough. Backup infomation: The lipid analysis is made on basis of a fully fermented dough system (dough just before it goes into the oven) which has been freeze-dried. After freeze drying the lipids are extracted by use of water saturated butanol (WSB) for making the GLC analysis – reflecting the non-polar and galactolipids. The freeze dried dough can also be extracted by use of a blend of iso-propanole and water (87.5%:12.5%), which is when used for HLPC/MA analysis – reflecting the phospholipids. This method is used for the lipid analysis shown on the next slide.
Looking at the modification of the phospholipids – phosphatidyl cholines and lysophosphatidyl cholines – we can see that when using POWERBake 4100, we also modify the PC to LPC. However, the amount of lipid is much less than the glycolipids shown on the previous slide due to the fact that more of the phospholipids are bound to the starch. Looking at the Y axis we can see that the percentage of PC and LPC is approx. 10 times lower than the DGDG and DGMG. Here it is also a known fact that LPC is more surface active than PC so the use of POWERBake 4100 releases more surface active components.
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20
30
40
50
60
70
80
0,0 0,3 0,6 1,0 1,3 1,6 1,9
Sur
face
tens
ion,
mN
/m
Microgram lipid/ml
Control Glycolipase DATEM
Surface tension (pH 5.5, 25ºC)
Presenter
Presentation Notes
Besides the lipid analysis we have also been looking at some physical studies. Here we have been looking at the effect of dough lipids extracted from a dough, which has been treated with the glycolipase. As a control system we have a dough without addition of lipase. We have also compared the effect of the lipase treated dough with a dough which have been treated with DATEM. It is seen that the surface tension is reduced by addition of lipids - especially lipids from the dough treated by use of glycolipase or DATEM – compared to a control system. Reduction in surface tension means an increase in surface activity and thereby a stabilisation of the air bubbles in the dough system. The slide indicates that the stabilisation obtained by use of the glycolipase is similar to the well-known effect obtained by use of DATEM.
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Confocal laser scanning microscopy (CLSM)
After mixing the dough is stained with FITC and Nile red. Proteins, yeast cells and starch granules appear green and lipids appear red.
Dough is resting at 34°C, 45 min.
Measurements are made 10 µm into the dough.
Pictures are captured each min. The image size is 375x375 µm
Control dough Lipase-treated dough
Evaluation of dough fermentation by CLSM
Presenter
Presentation Notes
Here we have pictures or small video films of a control dough and a dough which has been treated by use of glycolipase. The light green continuous system is the gluten network and the bigger round particles are the starch granules. The very small green dots are yeast cells. The red dots are the lipids here surrounding the air cells. By clicking the picture a small video film will run and show the dough development during the fermentation. Looking at the control system it is seen that the air cells are growing and that the gluten firm is not stable enough to stabilise the air bubbles – so bigger and more in-homogeneous air cells/structure is formed. By use of glycolipase a good stability of the air cells are obtained as the protein/lipid interphase surrounding the air cells are stabilised.
Evaluation of dough fermentation by CLSM
Page 24
Control dough Lipase-treated dough
0 min 15 min 0 min 15 min
30 min 45 min 30 min 45 min
Presenter
Presentation Notes
Here we have pictures of a control dough and a dough which has been treated by use of glycolipase. The light green continuous system is the gluten network and the bigger round particles are the starch granules. The very small green dots are yeast cells. The red dots are the lipids here surrounding the air cells. (By clicking the picture a small video film will run and show the dough development during the fermentation.) Looking at the control system it is seen that the air cells are growing and that the gluten firm is not stable enough to stabilise the air bubbles – so bigger and more in-homogeneous air cells/structure is formed. Bigger areas with no stable gluten network appear. By use of glycolipase a good stability of the air cells are obtained as the protein/lipid interphase surrounding the air cells are stabilised. The gluten film/network is maintained and does not change a lot during fermentation. It is seen that the pictures captured during fermentation are quite similar – only small differences appear – indicating a very stable system.
GRINDAMYL® POWERBake 4100 in crusty rolls
Specific volume g/ccm: 85 ppm GRINDAMYL® POWERBake 4100
7.53
0.3%DATEM
7.63
Presenter
Presentation Notes
Here we see the effect of GRINDAMYL™ POWERBake 4100 in a dosage of 85 ppm tested versus a standard DATEM in a dosage of 0.3%. When testing the POWERBake™ 4100 a nice volume and crumb structure is obtained and the result is quite similar to the effect obtained by use of DATEM. In this test the volume is a bit higher when using the DATEM.
Toast bread – straight dough
0.3%DATEM
85 ppm POWERBake® 4100
0.15% PANODAN® A2020
15 ppm POWERBake® 4100
Presenter
Presentation Notes
Testing the POWERBake™ 4100 in toast bread shows that it is possible to get quite a good result with regards to the volume response compared to a standard dosage of 0.3% DATEM. Here the POWERBake™ 4100 is also tested versus PANODAN® 663, which is an optimised emulsifier (DATEM)/enzyme solution also containing glycolipase. Use of the pure enzyme solution does have a bit smaller volume compared to the PANODAN® 663 solution. But one thing is to make a test bake in the laboratory, where less dough stability is needed. The next slide shows the effect in a shock-treated system.
Shock-treated toast bread – straight dough
0.3%DATEM
85 ppm POWERBake® 4100
0.15% PANODAN® A2020
15 ppm POWERBake® 4100
Presenter
Presentation Notes
If we shock-treat the fully fermented dough of the same test system as shown on the previous slide, it is clear that use of the pure enzyme solution lacks dough stability, which is given by use of the systems containing DATEM. This means that in baking procedures where a high dough stability is needed, it is necessary to add emulsifiers to the dough system in order to get this stability. In small bread items like crusty rolls, etc. it is often sufficient to use an optimised enzyme complex, but in bigger bread items like pan bread, a higher dough stability is needed.
French bread
Control
200 ppm GRINDAMYL® H XV
40 ppm GRINDAMYL® POWERBake 4100
+ extra xylanase
French bread
200 ppm GRINDAMYL® XV 40 ppm GRINDAMYL® POWERBake 4100 + extra xylanase
Conclusion / Product Overview
Functionality Benefits Products
Modification of Arabino xylan
• Improve dough handling • Improved tolerance towards variations in raw material and process conditions• Increase volume
• GRINDAMYL® H 460 • GRINDAMYL® H 490• POWERBake® 950
Modification of Arabino xylan and starch
• Improve dough handling • Improve tolerance towards variations in raw material and process conditions• Increase volume
• GRINDAMYL® S 100• GRINDAMYL® XV• POWERBake® 920
Modification of flour lipids
• Stabilise gas cell resulting in an improved crumb structure• Increase dough stability• Increase volume• Cost attractive emulsifier replacer
