|
Index:
Introduction
Stability Trials in Premix(carried out at Rhodia
Food, Woodley, UK)
Stability Trial in a Premix for pigs(carried
out at Adisseo, Commentry, France)
Stability Trials in Feeds(carried out at Rhodia
Food, Woodley, UK)
Stability Trials(carried out by a Thai customer)
Stability Trials(carried out in Italy)
INTRODUCTION
The stability of Penicillium funiculosum powder enzyme
product was assayed at 30°C (86°F) for more than one
year, on concentrated powder product blend with wheat flour
meal (PF concentrated product = CPD 30) and on commercial
product ROVABIO EXCEL AP.
The results are summarized in the tables below, for the endo-1,3(4)-ß-glucanase
activity; analyses are based on DNS CMC method, and for the
endo-1,4-ß-xylanase activity, analyses are based on
birchwood xylan method.
Endo-1,3(4)-ß-glucanase activity
|
Product
|
Batch n°
|
Date of manufacture
|
Analysis Time 0
u/g
|
Date of analysis
|
Storage time
(days)
|
Analysis - storage
at 30°C
|
| (u/g) |
(%) |
|
CPD 30
|
WWP 095
|
30/06/95
|
14 476
|
22/01/97
|
572
|
15 200
|
105
|
|
CPD 30
|
WWP 364
|
26/01/96
|
14 350
|
22/01/97
|
362
|
14 560
|
101
|
|
ROVABIO EXCEL AP
|
WWP 490
|
15/08/96
|
5 426
|
02/01/98
|
505
|
5 379
|
96
|
Endo-1,4-ß-xylanase activity
|
Product
|
Batch n°
|
Date of manufacture
|
Analysis Time 0
u/g
|
Date of analysis
|
Storage time
(days)
|
Analysis - storage
at 30°C
|
| (u/g) |
(%) |
|
CPD 30
|
WWP 095
|
30/06/95
|
8 946
|
20/02/97
|
601
|
11 200
|
125
|
|
CPD 30
|
WWP 364
|
26/01/96
|
8 868
|
20/02/97
|
391
|
10 923
|
123
|
|
ROVABIO EXCEL AP
|
WWP 490
|
15/08/96
|
2 245
|
02/01/98
|
505
|
2 264
|
101
|
The endo-1,3(4)-ß-glucanase and endo-1,4-ß-xylanase
from Penicillium funiculosum blended with wheat flour
as a stabilizer systems were stable at 30°C (86° F)
over the period of 1 year.
The stability data have been used to support the following
conditions of storage and shelf life:
ROVABIO EXCEL AP
Storage: For optimum stability, store product cool,
at a temperature of < 30 °C, and dry
Shelf life: Product will maintain specified activity
for 12 months from date of analysis under optimum conditions.
The time scales of stability of the active ingredients in
premixes and feed are consistent with the time and conditions
under which such formulations are stored.
Product Guarantee
Shelf life: 12 months below 30°C
What about thermostability of powder enzymes?
The same feed was pelleted at either
- 65-70 °C
- 75-80 °C
- 85-90 °C
- 90-95 °C
% Xylanase recovery - Viscometry Method
| Temp. °C |
65-70 |
75-80 |
80-85 |
85-90 |
90-95 |
| Recovery % |
|
|
|
|
|
| Excel AP |
100
100 |
94
91 |
89
85 |
53 |
56
|
When pelleting is not a problem , Rovabio Excel AP is
the solution: application in practice
Usage rate constant: 50 g/ton - needs a dilution for a good
repartition in feeds
2 options:
- Directly with all the micro ingredients (Vits + trace
- elements) in premix.
- Via a specific premix/concentrate:
Recommendations for good mixing and recovery: an example
- Rovabio Excel AP: 100
- Calcium Carbonate: 580
- Wheat middlings: 320
- Total 1000
(Usage rate in final feed: 500 g/ton)
Rovabio Excel AP stability in vit./trace element premix
Rovabio stability in feeds during storage
Enzyme Stability Trials in Premix
(carried out at Rhodia Food, Woodley, UK)
INTRODUCTION
The objective of the trial was to determine the stability
of the various powder enzyme additives in a formulated premix
while the formulated premix was held at different temperatures.
The experiments have been carried out at different times with
three separate batches of the enzyme additives.
The full experimental conditions are given below. The concentration
of the enzyme preparations and the dosage rate in the premix
were chosen to facilitate the measurement of enzyme activity
during the course of the trial. The enzyme preparations are
characterized by activity against different substrates under
different assay systems. The enzyme preparations so characterized
are :
| ROVABIO™ BETA GLUCANASE PF P |
} endo-1,3(4)-ß-glucanase
} from Penicillium funiculosum |
| ROVABIO™EXCEL AP |
} endo-1,4-ß-xylanase
} and endo-1,3(4)-ß-glucanase
} from Penicillium funiculosum |
In the tables of results the enzyme preparations are referred
to by the major active substance and the producing micro organism
(given in the right above). The following batches of enzyme
preparations were used:
For ROVABIO™ BETA GLUCANASE
PF P |
batch numbers: WP435; WWP095; WWP181 |
| For ROVABIO™EXCEL AP |
batch numbers: WWP095; WWP181 |
PROCEDURE
- Assays were performed using the standard methods described.
Each activity measured is reported as a ratio to the major
activity for that preparation:
- endo-1,3(4)-ß-glucanase (DNS barley ß-glucan
method) for endo-1,3(4)-ß-glucanase enzyme preparations;
- endo-1,4-ß-xylanase (DNS birchwood xylan method)
for endo-1,4-ß-xylanase enzyme preparations.
- Two premixes were used in this study (Premix 1 and Premix
2). Premix 2 contained molasses, added as a spay-dried powder;
while Premix 1 did not contain molasses, the balance was
made by increasing the sodium chloride concentration. In
each case 10 % enzyme was added to give the composition
shown below.
| Component |
Premix 1 |
Premix 2 |
| % |
wt (g) |
% |
wt (g) |
Calcium carbonate
Sodium chloride
Calcium phosphate
Magnesium oxide
Choline chloride
Soya bean meal
Molasses (Dri-flo)
Enzyme |
54.5
15
4.5
4
2
10
-
10
|
327
90
27
24
12
60
-
60
|
54.5
13
4.5
4
2
10
2
10
|
327
78
27
24
12
60
12
60 |
| Total |
100 |
600 |
100 |
600 |
- Samples of Premix 1 and Premix 2 in the absence of enzyme
were weighed out and mixed and the pH of a 10 % w/v suspension
in water was measured for each premix.
- The concentration of the enzyme preparation and the dosage
rate in the premix were chosen to facilitate the measurement
of enzyme activity during the course of the trial. 90 g
aliquots of each were removed and blended with 10 g of the
powder enzyme preparations. Each of theses was divided into
3 samples for storage at the different temperatures of 20,
30 and 40 °C (68, 86 and 104°F) to enable the stability
to be measured over a period of 6 months.
- At the time indicated a 2 g sample was removed from each
test and extracted in 0.01 M sodium acetate buffer pH 5.0
for 30 minutes. The extracts were centrifuged and the supernatants,
diluted appropriately, assayed.
- For each of the powder enzyme preparations, in the first
trial, the following treatments were prepared for Premix
1 and Premix 2:
|
Time
|
Temperature (°C, °F)
|
|
20, 68
|
30, 86
|
40, 104
|
|
0
1 day
2 days
28 days
70 days
105 days
189 days
|
|
X
X
X
X
X
X
|
X
X
X
X
X
X
|
In subsequent trials (trials 2 and 3), the following treatments
were prepared for Premix 1 and Premix 2:
|
Time
|
Temperature (°C)
|
|
20
|
30
|
40
|
|
0
28 days
56 days
84 days
182 days
|
|
X
X
X
X
|
X
X
X
X
|
- One sample was prepared to correspond to the time zero
sample for each of the powder enzyme preparations.
- In the ensuing tables and graphs enzyme activities are
shown as the raw data and normalised data (expressed as
a % of the initial activity).
RESULTS AND CONCLUSIONS
- The pH values for the 10% w/v suspensions of Premix 1
and Premix 2 were as follows :
Premix 1 (no molasses) = pH 5.9 ;
Premix 2 (with molasses) = pH 6.3.
Given the pH stability of theses enzymes, it is unlikely
that the pH associated with solid premix will have an adverse
effect over short periods of time.
- The observation noted above is reflected in the short
term (over 2 days) and medium term (over 28 days) stability
of all the enzyme preparations an it can be concluded that
all are stable at temperatures up to 40°C (104°F)
over 28 days. Over 70 days there is little evidence of any
further decrease in the activities at 40°C (86°F).
- There was some variations in the activities that may
be associated in the main with the lack of complete homogeneity
in the preparations and variations in the assays. The lack
of complete homogeneity in the preparation was a function
of the method of preparation of samples in the laboratory
and does not reflect the industrial practice.
- Over a longer time period, up to 189 days (which is longer
than is representative of the use of the product) there
is no general decrease, over the three different batches,
in the activities at 40°C (86°F).
- There was no difference in results between Premix 1 and
Premix 2.
Premix 1
| Active substance |
Batch n° |
Storage temperature
(°C, °F)
|
Activity against elapsed
time (%)
(days)
|
|
0
|
1
|
2
|
28
|
70
|
105
|
189
|
|
Endo-1,3(4)-ß-glucanase
from
Penicillium funiculosum
|
WP 435 |
20, 68
30, 86
40, 104
|
100
100
100
|
103
104
109
|
103
105
99
|
98
95
99
|
96
86
84
|
87
86
80
|
84
85
74
|
| Active substance |
Batch n° |
Storage temperature
(°C, °F)
|
Activity against elapsed
time (%)
(weeks)
|
|
0
|
28
|
56
|
84
|
182
|
|
Endo-1,3(4)-ß-glucanase
from
Penicillium funiculosum
|
WWP095
WWP181 |
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
|
100
100
100
100
100
100
|
98
95
100
104
104
101
|
100
105
100
94
94
85
|
93
79
89
97
101
98
|
94
89
81
98
95
101
|
|
Endo-1,4-ß-xylanase
from
Penicillium funiculosum
|
WWP095
WWP181 |
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
|
100
100
100
100
100
100
|
106
106
93
109
104
108
|
101
104
96
114
104
97
|
102
103
103
128
111
108
|
100
101
95
100
101
106
|
Premix 2
| Active substance |
Batch n° |
Storage temperature
(°C, °F)
|
Activity against elapsed
time (%)
(days)
|
|
0
|
1
|
2
|
28
|
70
|
105
|
189
|
|
Endo-1,3(4)-ß-glucanase
from
Penicillium funiculosum
|
WP 435 |
20, 68
30, 86
40, 104
|
100
100
100
|
98
106
103
|
105
99
105
|
98
97
95
|
98
93
89
|
91
85
80
|
82
79
70
|
| Active substance |
Batch n° |
Storage temperature
(°C, °F)
|
Activity against elapsed
time (%)
(weeks)
|
|
0
|
28
|
56
|
84
|
182
|
|
Endo-1,3(4)-ß-glucanase
from
Penicillium funiculosum
|
WWP095
WWP181 |
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
|
100
100
100
100
100
100
|
120
101
117
116
112
113
|
112
116
115
89
85
77
|
114
97
106
101
89
99
|
111
107
96
101
104
99
|
|
Endo-1,4-ß-xylanase
from
Penicillium funiculosum
|
WWP095
WWP181 |
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
|
100
100
100
100
100
100
|
103
100
101
106
104
101
|
109
102
104
98
102
102
|
97
99
87
86
99
106
|
110
109
104
97
94
86
|
Enzyme Stability Trial in a Premix for
pigs
(carried out at Adisseo, Commentry, France)
| ROVABIO™ EXCEL AP |
endo-1,4-ß-xylanase and endo-1,3(4)-ß-glucanase
|
| (batch no. 2292781) |
from Penicillium funiculosum |
PROCEDURE
Assays were performed using the standard methods described.
Each activity measured is also reported as a ratio to the
major activity for that preparation:
- endo-1,4-ß-xylanase (DNS birchwood xylan method).
One type of premix for pigs was used in this study. 10% enzyme
was added to give the composition shown below:
| Component |
Pig premix
%
|
Vtiamin blend AD3EB
Traceminerals for pigs
Choline 50
Rovabio Excel AP
Sodium chloride
Calcium carbonate |
5
15
20
10
30
20
|
| Total |
100
|
RESULTS AND CONCLUSIONS
Xylanase activity, as measured by the DNS method is lower
than expected. This may be due to a lower extraction from
the premix. However, this activity is relatively stable over
a period of 6 months, at normal temperature.
|
Active substance
|
Date of analysis
|
Time
|
Activity (xyl. units)
|
Recovery
|
|
Endo-1,4-ß-xylanase
from
Penicillium funiculosum
|
23/05/00
30/06/00
09/08/00
22/06/00
23/11/00
|
0
+ 38 days
+ 78 days
+ 122 days
+ 184 days
|
181
189
188
193
168
|
100
104.4
103.8
106.6
92.8
|
Enzyme Stability Trials in Feeds
(carried out at Rhodia Food, Woodley, UK)
Several different enzyme preparation were subjected to stability
trials in a formulated feed at different temperatures. The
enzyme preparations ROVABIO™ EXCEL LC and ROVABIO™
EXCEL AP are identified in this work as endo-1,3(4)-ß-glucanase
and endo-1,4-ß-xylanase from Penicillium funiculosum.
Products were maintained at 3 different temperatures, 20,
30 and 40°C (68, 86, 104°F) to enable the stability
to be measured over a period of 6 months. Ground pelleted
feed (obtained from Sun Valley Poultry) was used in this study.
| Active substance |
Batch n° |
Storage temperature
(°C, °F)
|
Activity against elapsed
time (%)
(weeks)
|
|
0
|
4
|
9
|
13
|
26
|
|
Endo-1,3(4)-ß-glucanase
from
Penicillium funiculosum
|
WE043
WE313
MCBG/4 |
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
|
100
100
100
100
100
100
100
100
100
|
107
111
117
99
95
98
119
90
97
|
98
96
92
84
80
114
120
104
78
|
113
112
109
94
78
45
97
98
88
|
96
100
82
95
94
59
100
94
56
|
|
Endo-1,4-ß-xylanase
from
Penicillium funiculosum
|
WWP095
WE313
WWP181 |
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
20, 68
30, 86
40, 104
|
100
100
100
100
100
100
100
100
100
|
96
108
99
63
84
140
75
138
98
|
119
113
15
71
93
117
65
161
87
|
119
114
115
86
101
85
106
135
162
|
88
107
96
99
96
101
103
91
89
|
There were some variations in the activities over a 6-month
period. This was associated in the main with the lack of complete
homogeneity in the enzyme feed preparation and variations
in the assays. The lack of complete homogeneity in the enzyme
feed preparations was a function of the method of preparation
of samples in the laboratory, and does not reflect the industrial
practice. The results show different enzyme active species
to be stable. The individual results are discussed below.
| Endo-1,3(4)-ß-glucanase from Penicillium funiculosum |
The product is stable up to 40°C (104°F). There
was no significant loss of activity at 20, 30 and 40°C
(68, 86, 104°F) over 6 months. |
| Endo-1,4-ß-xylanase from Penicillium funiculosum |
The product is stable up to 40°C (104°F). There
was no significant loss of activity at 20, 30 and 40°C
(68, 86, 104°F) over 6 months. |
TRIAL REPORT: THERMAL STABILITY
TEST ON ROVABIO EXCEL AP
BY A THAI CUSTOMER
By Zhirong Jiang, Adisseo, Asia-Pacific
Objectives: To evaluate the effect of pelleting
temperatures on in-feed recovery rate of Rovabio powder.
Materials: ADISSEO provided the diluted Rovabio
Excel AP for the test.
Procedures:
Two batches of a typical broiler starter diet were manufactured
at the Customer Research Farm, one batch without Rovabio (Control),
and the other with 500 g/ton of diluted Rovabio Excel AP added
(the diluted Rovabio samples were supplied by ADISSEO Australia).
The feeds were pelleted at either low (65-70°C) (149-158°F)
or medium (75-80°C) (167-176°F) temperatures.
Representative samples were taken both before and after the
pelleting process. Samples were sent to Rhodia, UK for analysis
of in-feed Rovabio activity.
During the pelleting process, the temperatures and retention
time in the conditioning chamber, temperatures of the die
and the cooler were measured.
Results:
1. Expected activity in feed:
Specific activity (pure product) (a) 22000 - 29000* u/g
Incorporation rate (b) 500 g/ton
Theoretical activity on feed (c=axb/100) 976 u/kg
*approximate activity range for Rovabio Excel AP
2. LOW PELLETING TEMPERATURE (65-70°C) TRIAL
Conditioner temperature set at 69°C (156°F), measured
at 63°C (145°F)
Die temperature: 79°C, 174.2°F
Cooler temperature: 34°C, 93°F
Retention time in cooler: 24 secs
|
Sample reference
|
Notes
|
Analyzed activity
(units/kg)
|
Recovery
(% of expected activity)
|
Recovery
(% of activity in mash feeds)
|
|
# 0
# 1
# 2
# 3
# 4
# 5
|
Mash + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
|
892
664
704
709
688
671
|
91
68
72
73
71
69
|
100
74
79
80
77
75
|
|
Average recovery rate
after pelleting
|
77
|
3. MEDIUM PELLETING TEMPERATURE (75-80°C) (167-176°F)TRIAL
Conditioner temperature set at 79°C (174°F), measured
at: 72°C (162°F)
Die temperature: 85°C, 185°F
Cooler temperature: 34°C, 93°F
Retention time in cooler: 24 secs
|
Sample reference
|
Notes
|
Analyzed activity
(units/kg)
|
Recovery
(% of expected activity)
|
Recovery
(% of activity in mash feeds)
|
|
# 0
# 1
# 2
# 3
# 4
# 5
|
Mash + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
Pellets + Rovabio™
|
1056
690
775
741
653
636
|
108
71
79
76
67
65
|
100
65
73
70
62
60
|
|
Average recovery rate
after pelleting
|
66
|
Comments:
The average recovery rates for Rovabio Excel AP were 77%
and 66% when the feed was pelleted at low (65-70°C) (149-158°F)
or medium (75-80°C) (162-176°F) temperatures, respectively.
These recovery rates were in line with results from previous
ADISSEO trials.
Silversides and Bedford (1999) had reported previously that
the in-feed recovery of Avizyme was about 30-40% at a pelleting
temperature of 80-85°C (176-185°F). This trial therefore
demonstrated that Rovabio powder has good heat stability to
withstand low and medium pelleting temperatures.
TRIAL REPORT: THERMAL STABILITY TEST ON
ROVABIO EXCEL AP
IN ITALY
By S. Nuffer - G. Uzu, Adisseo - Europe
Objectives: To evaluate the effect of pelleting
temperatures on the in-feed recovery rates of Rovabio Excel
AP in industrial conditions. The results will be used to guide
subsequent large-scale trials with broilers in Italy.
Materials: ADISSEO provided Rovabio Excel AP
for the test.
Rovabio Excel AP was mixed in a vitamin/trace element premix.
Procedures:
The feeds were pelleted at medium (75 - 80°C) (167-176°F)
to high (80 - 85°C) (176-185°F) temperatures. A comparison
was made between 2 pellet mills, with single or double conditioner.
Representative samples were taken after the pelleting process.
Samples were sent to Rhodia, UK for analysis of in-feed Rovabio
activity.
Temperature was measured on pellets directly at the output
of the pellet mill.
Results:
|
|
Rovabio Excel recovery
(%)
|
|
Pelleting temperature
°C, °F
|
Pellet mill 1
Double conditioner
Retention time: 15"
|
Pellet mill 2
Single conditioner
Retention time: 10"
|
|
78.0, 172
80.0, 176
82.0, 180
82.7, 181
84.2, 184
84.5, 184.1
85.7, 186
86.0, 187
87.5, 190
|
88
102
97
88
54
-
65
-
-
|
94
60
56
-
-
46
-
49
47
|
Comments:
This short experiment clearly demonstrates that the critical
zone for enzyme stability is the range of temperatures from
80 to 85°C (176-185°F). Pellet mill #1 was less aggressive
to enzyme stability than Pellet mill #2, theoretically differing
only by the conditioning process. It was not possible to collect
more information to explain this difference.
Indeed, pelleting conditions cannot be described simply by
a temperature. It is more a combination of many parameters
such as conditioning time, die diameter and compression, steam
temperature and pressure.
When pelleting temperature is below 80°C (176°F),
Rovabio Excel AP can used in good conditions. Between 80 and
83°C (176 and 181°F), it is advisable to make some
tests to check the stability in the specific conditions of
the feed mill. From 83°C (181°F) and above, we recommend
to use Rovabio Excel LC for post-pelleting application.
Enzyme recovery %
Pelleting temperature °C
|
