Thailand

Workshop

Control of Dextran at the Factory

with Dextranase

1. Control of dextran with dextranase

2. Pullulanase
 Measurement of Dextran
to Check if Dextranase is Working
 Mannitol versus Dextran Measurement

 Mannitol cannot be broken down in the factory or

refinery. It is very stable.

 Up to date, mannitol is a qualitative measure of dextran

in juice and a quantitative measure of Leuconostoc
deterioration in juice. If mannitol is present in juice
then dextran is definitely present

 Mannitol measurements cannot be used to measure

how much dextran has been hydrolyzed by dextranase

 Antibody dextran is best factory measurement of

dextranase action but is expensive and kit needed
 ASI Enzymatic Antibody Haze
Dextran Dextran Dextran

High MW Dextran High MW Dextran High MW Dextran

Medium MW Dextran Medium MW Dextran

Low MW Dextran Indigenous Cane Polysacch.

Oligosaccharides
e.g., kestoses
Bagacillos

Sucrose

Long and Expensive Not Specific and

Complicated and Imprecise Difficult to Interpret
(False Positives)
 Application of Dextranase
to Control Dextran in the Factory
 Control of Dextran with Dextranase
• Background information on commercial dextranases:
 Principle of dextranase action

 Problem associated with different activity units for commercial

dextranases

 New ICUMSA tentative method to measure dextranase activity at the

factory
• Industrial conditions that affect the action of dextranases

• Dextranase factory studies

 Adding as a working solution in the factory

 Measuring how much dextran has been hydrolyzed by dextranase

 Effect of heating juice

 Basic Chemical Structure of Dextran (a-(1→6)-a-D-glucan)

(1→3)-D-glycoside branching is shown in the figure

Glucose molecules are linked by (1→6)-D-glycoside bonds. Approximately
5% branching occurs in cane dextrans, mostly through (1→4), and (1→3),
and to a lesser extent (1→2)-D-glycoside linkages.
 Principle of Dextranase Action
Dextranase hydrolyzes(1→6)-D-Glycoside Linkages in Random “Endo” Sites of
HMW Dextran
High MW dextran
(>1000 KDa)

H 2O dextranase
Med MW dextran
(~100 – 1000 KDa)

H 2O dextranase
Lower MW dextran
(~45-100 KDa)

H 2O dextranase
Oligosaccharides (2 to 10 degrees of polymerization), i.e., isomaltotriose, isomaltose

H 2O dextranase
Reaction times are not represented glucose*
Dots and connecting lines represent chains of glucose molecules linked by (1→6) glycosidic
bonds in the dextran molecule
* Rarely occurs in factory From: Eggleston et al (2005). Process. Biochem.
 Commercial Dextranases
• Most are produced from fungi – Chaetomium gracile and
Chaetomium erraticum, and Penicillium (not allowed in the
U.S.)

• “Band-Aid” enzyme – only used when there is processing

problem or when a processing problem is expected

• Limited “Research + Development”

• High price per lb (or kg) compared to other industrial enzymes

• For the forseeable future, will not be tailored for the sugar
industry (too small a market)
 Problem
• At the present time, the activities or strengths of commercial
dextranases as vendors use numerous methods with different
units of activity to measure and quote the activity:

For Example
Current Different Units of Strength or Activity
u/g
Du/g
U/mL
Du/mL
kDu-A/g

• The dextranase market is very dynamic – activities and prices

can change regularly
 A Large Range Exists in the Activities of Commercial
Dextranases Available Worldwide to the Sugar Industry

Commercial Dextranase Activity DU/mL Classification

Dextranase 2003 2004 2008 2009

A 52000
For Example
51920 52000 52000 “Concentrated”

B 5499 6500 2500 nd “Non-Concentrated”

C 4786 2750 “Non-Concentrated”

D 5X 8000 “Non-Concentrated”

D 3000 “Non-Concentrated”

Eggleston Classification:

“Concentrated” Dextranases 25,000 - 58,000 DU/mL

“Non-Concentrated” Dextranases <25,000 DU/mL
From: Eggleston et al. (2007) ACS Book Chapter.
 Activity per Unit Dollar Varies Enormously Among
Commercial Dextranases

Commercial Dextranase Activity Classification

Dextranase DU/ml/US$
2003 2004 2008 2009

A 2832.2 2827.9 2813.9 2813.9 “Concentrated”

B 916.5 583.3 416.6 “Non-Concentrated”

D 5X 490.5 “Non-Concentrated”

D 735.3 “Non-Concentrated”
Activities Can Be Checked Using Ion Chromatography
Profiles: Research Tool

40 ̊C
Conc. Dextranase 4.6X dil
Conc dex tranase 4.6X dilution 86F

Non-Conc. dextranase
Midland 86F
isomaltose
Dextran with no dextranase
Dextran c ontrol

isomaltotriose added
uC

0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Minutes

From: Eggleston et al. (2005). Process. Biochem.

A very simple titration method to measure dextranase was
identified and modified for use at the factory

simple burette
 Principle

Reaction End
Color
(a) K3Fe(CN)6 + Reducing Sugar → Na2CO3 → K4Fe(CN)6 yellow

(b) 2K3Fe(CN)6 + 2KI → Acetic acid → 2K4Fe(CN)6 + I2 orange

(c) 2K4Fe(CN)6 + 3ZnSO4 → K2Zn[Fe(CN)6]2. + 3K2SO4 orange

(d) I2 + starch indicator → starch-I2 complex dark blue

(e) Starch-I2 complex + 2NaS2O3 → Na2S4O6 + 2NaI white

 Measuring Dextranase Activity Using Simple Equipment at the Factory

Incubation at 37oC
Boiling

Adding reagents Simple Titration

 Accuracy

Spectrophotometric method DU/g

Comparison of Two Different Dextranase Activity Methods

70
60
50
Thousands 40
30
20
10
0
0 10 20 30 40 50 60
Simple Titration Method DU/mL
Thousands

R-square = 1 # pts = 4
y = 944 + 1.24x

 The excellent correlation between the two methods, confirms the

accuracy of the simple titration method.

 ICUMSA Method GS7-8 (2010): “Standard Measurement of the Activity

of Dextranases at Sugarcane or Sugar Beet Factories using a Simple
Titration Method” – Tentative Status
Storage Characteristics at Room Temperature (~25 oC) and
Refrigerated Temperature (4 oC)
50
Conc. dextranase activity D U/mL

40

“Concentrated” Dextranase
Thousands

30

20

10 Conc at room temp

Conc at refrig. temp

N o n -c o n c . d e x tra n a s e a c tiv ity D U /m L

0 10 20 30 40 50 60 70 80 90 100
Storage time (days)

5
Thousands
4

Non-conc at room temp

“Non-Concentrated” 2
Non-conc at refrig. temp

Dextranase 1

0
0 25 50 75 100 125 150
Storage time (days)

From: Eggleston and Monge (2005) Proc. Biochem.

 Three Important Reasons to Measure the Dextranase
Activity at the Factory:

1. Economically compare activities of different commercial

dextranases (vendor dextranase activity units differ, and
activities and prices change regularly)

2. Monitor the changing activities of dextranase on

factory storage

3. Measure the activity of delivered batches.(some non-

concentrated dextranases have been known to arrive with no
activity)
Industrial Conditions That Affect
the Action of Dextranases
R e l . D e x tr a n a s e A c ti v i ty (i s o m a l to tr i o s e p e a k h t
Effect of Temperature
140

120

100
Pure Dextran T2000TM
2000 ppm
Thousands

80
50 ppm dextranase
Conc 4.6X dil
Non-conc. A

pH 5.4
60 Non-Conc. B

40 25 min
20

0
20 30 40 50 60 70

Temperature oC
R e l. D e x tr a n a s e A c tiv ity (is o m a lto tr io s e p e a k h t )

80

Juice
70
Addition
Dextran in Juice
60
Evaporator
Syrup
3177ppm/Brix
50
100ppm dextranase
Tho usan ds

Addition Conc. 4.6X dil

40 Non-conc. A
Non-conc. B
pH 5.4
30 25 min
20

10

0
20 30 40 50 60 70

Temperature oC
 Effect of Brix
2500
1000ppm Dextran
Dextran 1000 ppm
100
100 ppm
ppm dextranase
dextranase
Rel. Dextranase Activity (isomaltotriose peak ht)

pH
pH 5.4
5.4
2000 Evaporator 25 min
25 min
application 120ooF
50 C
Evaporator
Thousands

1500
Syrups Conc 4.6X dil
Non-conc. A
1000 Non-conc B

Juices
500
Juice
application
0
10 20 30 40 50 60 70
Brix

 pH optimum is between 5 and 6

Effect of Dextran Concentrations on Dextranase Action

100

80
% dextran breakdown

60

40

20

0
0 2000 4000 6000 8000
Antibody dextran (ppm/Brix)

 It is easier to breakdown large amounts of dextran than

smaller amounts with dextranase
 Importance of Contact Between
Dextran (Substrate) and Dextranase (Enzyme)

Contact .......
........ Low Contact
..
Low Dextran Dextranase

.......
........ High Contact
..

High Dextran Dextranase

 “Concentrated” Dextranase (52000 DU/ml) on Cane Juice
(3380 ppm/Brix); Application: ppm on juice
% d e x t r a n r e m a in in g
120 (32.2 oC)
110
100 0
90 4
80 8
70 10
60 20
50 40
40 80
30
20
0 5 10 15 20 25 30
Residence time (mins)

120
48.9 oC
% d e x tr a n r e m a i n i n g

110
100
0
90
4
80 8
70 10
60 20
40
50
80
40
30
20
0 5 10 15 20 25 30
Residence time (mins)
 Laboratory Studies
Cost of Application to Juice versus Syrup
“Non-concentrated” Dextranase based on US $6 per lb
“Concentrated” Dextranase based on US $18.36 per lb

“Non-concentrated” Dextranase (5999 DU/ml) Application

ppm/ % Dextran Equivalent
Conditions
juice breakdown Cost

Juice 32.2 ˚C 5 min 4 4.7 % US$1.00

Syrup 50.0 ˚C 20 min 90 7.4 % US$14.29

“Concentrated” Dextranase (52000 DU/ml) Application

ppm/ % Dextran Equivalent
Conditions
juice breakdown Cost

Juice 32.2 ˚C 5 min 4 57.3 % US$1.00

Syrup 50.0 ˚C 20 min 90 29.6 % US$11.62

Dextranase Cane Factory Studies

 Louisiana USA sugarcane factories

 Factory 1
 Installed a 5 min retention time mixed juice tank to improve the
application of dextranase
 Factory 1
“Non- concentrated” dextranase: 2750 DU/ml; Normal temp ~32 ˚C

8000
2ppm Factory
Antibody dextran (ppm/Brix)

Data
6000

10.7ppm In
4000 Out

2ppm 4.2ppm
2000

0
0 1 2 3 4 5

Sample No.

 From these results the staff at Factory 1 decided to use a

“concentrated” dextranase
 Contact Between
Dextran (Substrate) and Dextranase (Enzyme)

“Concentrated” Dextranase: Factory Working Solutions

Low Contact
.............. “Conc”
Dextran
. .. dextranase-
No dilution

. ...
. . .. .
.. .. . . ... .
Same ppm
1:1 dilution of
.. . dextranase
to juice

.. ..
. . .
. . . .. 1:4 dilution
. . .
High
Contact
. .
 “Concentrated” Dextranase: Storage of Factory Working Solutions
100

80
% activity

60

40

20 1:1 dilution in tap water

0
0 20 40 60 80 100 120 140
Time (hours)

120

100

80

% activity
60

40

20
1:4 dilution in tap water
0
0 20 40 60 80 100 120 140
100 Time (hours)

80
% activity

60

40

20
1:4 dilution in 24 Brix raw sugar
(no dextran)
0
0 20 40 60 80 100 120 140 160
Time (hours)

 12 hour maximum storage of working solutions are recommended

 Factory 1
6 ppm “concentrated” dextranase (52000 Du/ml); 1:1 dilution;
Normal temp ~32.2 ˚C
800
Antibody dextran (ppm/Brix)

600

In
400
Out

200

0
0 1 2 3 4 5 6 7 8

Sample number

 Average drop in antibody dextran of 94%

 Factory 1
6 ppm “concentrated” dextranase (52,000 Du/ml); 1:4 dilution
Normal temp ~32.2 ˚C

1600
Antibody dextran (ppm/Brix)

1200

In
800 Out

400

0
0 1 2 3 4 5 6 7 8

Sample number

 Average drop in antibody dextran of 85%

R e l . D e x tr a n a s e A c ti v i ty (i s o m a l to tr i o s e p e a k h t
Effect of Temperature
140

120

100
Pure Dextran T2000TM
2000 ppm
Thousands

80
50 ppm dextranase
Conc 4.6X dil
Non-conc. A

pH 5.4
60 Non-Conc. B

40 25 min
20

0
20 30 40 50 60 70

Temperature oC
R e l. D e x tr a n a s e A c tiv ity (is o m a lto tr io s e p e a k h t )

80

Juice
70
Addition
Dextran in Juice
60
Evaporator
Syrup
3177ppm/Brix
50
100ppm dextranase
Tho usan ds

Addition Conc. 4.6X dil

40 Non-conc. A
Non-conc. B
pH 5.4
30 25 min
20

10

0
20 30 40 50 60 70

Temperature oC
By Heating the Juice Temperature by only 10 ̊C to 37 ̊C More
Dextran Broken Down By Dextranase

Antibody Dextran (ppm/Brix)

Sample % Dextran
numbera TankIN TankOut †
Hydrolysis
  Juice Temperature ~27 ̊C  
1 5686 1159 79.6
2 5728 3248 43.3
3 6266 2920 53.4
4 3862 2188 43.4
5 4210 1174 72.1
6 4280 3723 13.0
Average: 5005 2402 50.8
Juice Temperature ~37 ̊C*
7 5985 1044 82.6
8 4942 2628 46.7
9 2169 175 91.9
10 3292 469 85.8
11 4748 846 98.2
12 5445 119 97.8
Average: 4430 753 83.8

†
5 min retention time in tank * Recycled heated juice into tank
 Final recommendation: juice tank with 5 min retention time at no greater than
50 ̊C
Different Dextran Measurements give Different
Results of Dextranase Action
 Factory 2
“Conc” dextranase (52000Du/ml); 1:4 dilution; Temp ~51 ˚C; Ret. Time 12 min
3000 Haze
Haze dextran (ppm/Brix)

Dextran
2000
In
-11% -32% -3%
+2% -19% Out
1000 -39%
-42%

0
1 2 3 4 5 6 7
Sample No.
Av. -21%
Same samples measured using the antibody dextran method:
250 Antibody
Antibody
Antibody Dextran (ppm/Brix)

200 Dextran
Dextran
150 In
100 +2% +35% Out
50 -71% -24% -100% -100% -44%
0
1 2 3 4 5 6 7

Sample No. Av. -43%

 Antibody Dextran Haze Dextran

High Molecular Weight (MW) High Molecular Weight (MW)

Dextran Dextran

Medium MW Dextran

Oligosaccharides

Protein (if TCA is not used)

Other Polysaccharides

Other (fibrous microparticles)

& sucrose
Effect of Biocide (Carbamate) on Dextranase Action
10ppm conc. dextranase on juice at 49 oC and pH 5.4

110
100 Conc.10ppm
% dextran rem aining

Conc.10ppm +Biocide 10ppm

90 Conc. 10ppm +Biocide 20ppm
80
70
60
50
40
30
20
0 5 10 15 20
Residence time (mins)

 Dextranase can be used in the presence of carbamate biocide

 Final Recommendations for Adding Dextranase

 Only add dextranase if dextran is present. Haze dextran does not always
indicate that dextran is present. If mannitol is present then dextran is
present

 Optimum addition of dextranase: Concentrated dextranase added to

juice at 50 ˚C

 Mannitol cannot be used to check if dextranase is working. Best check is

to use antibody dextran

NOTE: USDA is currently trying to develop an inexpensive, fast, easy, and

reliable factory dextran method
 Application of Pullulanase
to Control Dextran in the Factory

 Pullulanase is a starch degrading enzyme

 Pullulanse specifically breaks down α-1,6 linkages in amylopectin
(a branched starch polysaccharide)
 Pullulanase is known as a debranching enzyme
 Used in production of High Fructose Corn Syrup (saccharification)
 Ion Chromatography Fingerprint Profiles

50 ˚C

nC
5 ppm Dextranase + 5 ppm Pullulanase

5 ppm Pullulanase

5 ppm Dextranase

Dextran with no enzyme

min
 Application of Pullulanase
to Control Dextran in the Factory

 Still needs to be optimized at the sugarcane factory

 Sold in higher volumes than dextranase

 Could be used if dextranase is not available

 Cost?