Innovative Probiotic Lactic Yeast for the pharmaceutical, nutritional and agricultural industries

Trial#79: Effects of Turval B0399 in the diet of the weaned piglet; Tolerance test and effects on the intestine microorganism.

Head of the test: Prof. Paolo Bosi (DIPROVAL –UNIVERSITY OF BOLOGNA)
Dr. Claudia Silvestro (agronomist Turval Lab.)
Dr. Paolo Valles (biologist Turval Lab.)

Summary
The test was conducted at the Experimental Zootechnical Structure of the DIPROVAL – University of Bologna, on 63 weaned piglets (crosses Large White x Landrace), 21 days old, taken from 12 litters. Sex was registered.
Two dosage of Turval B0399:
(recommended dosage) 1,5 g/kg and (overdose: tolerance test) 15 g/kg of feed were used.
All the subjects were slaughtered at the end of the test.
During the experimentation the following parameters were evaluated:
-          growth in post-weaning period,
-          conversion index,
-          values of intestinal pH (duodenum, ileum, colon and cecum),
-          concentration of total coliforms
-          lactobacilli in the cecum.
-          Kluyveromyces in the colon
 


 
0- EXPERIMENTAL CONDITIONS AND GENERALITIES.
 
Objective
Effects of recommended dosage and over-dosage (tolerance test) of Turval B0399 in the diet of the weaned piglet on:
growth in post-weaning period,
conversion index,
value of intestinal pH,
concentration of total E. Coli in the intestine (cecum)
presence of kluyveomyces in the colon.
 
Methodology
The test was conducted at the Experimental Zootechnical Structure of the DIPROVAL – University of Bologna, on 63 weaned piglets (crosses Large White x Landrace), 21 days old, taken from 12 litters. Sex was registered. The trial lasted 28 days on average.
All the subjects were slaughtered at the end of the test.
 
1 Species, breed and sex of animals and method of their identification
 
Breed:                        Large White x Landrace
Age:                           21 day old
Sex:                            mixed, registered for statistical analysis
 
Method of identification:
 Through auricular numbering from No. 1 to No. 63 of the subjects tested.
All of the subjects were tattooed on the ear with the number.
033RE 109 indicated the stock room from which they came.
 
 
 
2 Number of groups tested and statistical aspects.
 
2.1 Groups tested
·      Number of groups:                                                         3
·      Number of groups treated with TurvalB0399: 2
Turval B0399, 1,5 g/kg of feed (T1)
Turval B0399, 15 g/kg of feed (T2 overdose: tolerance test)
 
·      Number of groups of control (C):                                  1
·      Number of animals in each group:                               21
·      Total number of animals (piglets):                                63
·      12 litters were weaned at 21 days on average and groups were formed as follows.
From each litter, 3 to 11 subjects were collected.
From each litter, triplet of subjects similar for the live weight were created and one subject per triplet was assigned at random to each dietary treatment. After this, for litters where residual subjects were present, these piglets were randomly assigned to one or two of the dietary treatments, in order to equalize the total number of subjects. In any case all the litter were represented in each treatment. The overall distribution is reported in the data annex.
The weight and sex was registered.
 
2.2-Statistical analysis of the data:
All individual data was elaborated through analysis of the variance factor, utilizing the procedure GLM of the SAS package (SAS/STAT User’s Guide, Vers.6, 4th ed., Vol.2, Cary, NC, SAS Institute Inc., 1989.). Diet, sex and litter were considered in the model. The number of subjects chosen was statistically sufficient. 
The 2 degrees of liberty of the “diet” treatment were divided in the orthogonal contrasts “Control vs Turval B0399 diets” and “Between Turval B0399 diets”. In substance, in the first comparison the general effect of Turval B0300 was compared with a normal standard diet. With the second contrast, the differences between the 2 dietary levels of the products were tested. However for better information of the reader, 2 other contrasts are displayed: Control vsT1 and Control vsT2, i.e. both the effect of the proposed dose of the products and of the ten times dose are compared to the standard diet.
For the recovery of Turval B0399 in the cecum, the effect of dietary treatment was pested by the Likelihood Ratio Chi-Square Test.
 
3 Concentration of the additive in the feed
 
3.1- Dosage:
Concentration of Turval B0399 in the feed for testing
 
T1: recommended dosage 1,5 g of additive /kg of feed = (5 x 10 6 x CFU /g x 1,5 /1000 = 7,5 x 10 3  CFU Kluyveromices B0399/g of feed
 
T2: over dosage (10 x T1) 15 g of additive /kg of feed = 75 x 10 3 CFU Kluyveromices B0399/g of feed
 
3.2-Identification of testing feeds :
·      C   = Control group: feed without additive
·      T1 = Treated group: feed with recommended dosage:
·      T2 = Treated group: feed with overdose (tolerance test).
 


 
3.3-Characteristics of the feed
The nutritional composition of the daily ration in qualitative and quantitative terms in shown in Table 2.3.B.1.
No auxinic type additive was added.
 
Table 2.3.B1. Ingredients of the base diet (%) utilized in test 79
 
Components
%
 
 
Barley flakes dehulled
20.00
Rice flakes
37.00
Soya flour extract (48%)
15.00
Extruded soybean
10.00
Lactose
2.50
Milk whey spray powder
12.00
Monocalcium phosphate
1.50
Lysine hydrochloride
0.50
DL methionine
0.25
L threonine
0.20
L tryptophan
0.025
Vitamin - mineral supplement
1.00
Appetizer
0.025
 


3.4-Characteristics of the additive
The composition of the additive in qualitative and quantitative terms is shown in the following Tables.
 
Batch number : 10-7.0402
 
 
Table 1.3.2 - CFU of Kluyveromyces B0399 in the additive.
(cfr: analysis Chemio n. 25700)
Description
UFC/g
Batch n
 
 
 
Cells of Kluyveromyces fragilis B0399 in the additive
>5 x 106
10-7.0402
 
 
The analysis of the additive is shown in Table 1.3.1
 
Table 1.3.1 Analysis of additive
Parameter
Percentage value % on additive as is
Humidity
10,0%
Total fats
4,0%
Proteinse
22,0%
Raw fiber
4,5%
Ashes
11,0%
 
 
 
 


 
4.- Experimental facilities utilized and operative conditions

·      Experimental facility: DIPROVAL Univ. of Bologna, in Reggio Emilia, Italy 
The subjects were reared in individual cages with slattened floor.
The internal temperature was automatically controlled and set at 28 °C (starting day) to 21 °C (final day).
In the 1st week infrared light lamps placed over each subject supplied additional heating.
·      State of health of the animals at the beginning of the experiment: good
·      Conditions of feeding and breeding: normal
·      Control of feeding and measures taken to prevent the contamination of the groups of reference (especially through cross contamination of the foods caused by microorganisms):
The diets were prepared from the same feed. The feed was produced in a single phase and in a quantity sufficient for sustaining the entire test for all the animals. To obtain the diets with Turval B0399, the basic diet in the quantity of 10 kg at a time, was supplemented with 15 g and 150 g of the additive, respectively for T1 and T2, thoroughly mixed in a plastic container and labeled with the name of the dietetic treatment (name of the lots).
 
 
5 - Date, duration and nature of the tests

5.1-Duration of the testing
·      Beginning of test: 11 April 2002
·      Total Duration : 28 days
·      Final weight of reference: 11 kg
·      At the end of testing the animals were slaughtered.

5.2-Nature of the tests conducted on live animals:
-          Recording weight at the beginning and at the end of the test.
-          The general health of the subjects was also observed.
5.3-Nature of the tests conducted on slaughtered animals:
-          Determination of the pH in various parts of the intestine: duodenum, ileum, cecum, colon.
-          Collection of content of the colon for the total E.Coli and Lactobacilli count.
 
 
6 Unwanted effects and other drawbacks verified during the testing
(cf. Table 2.3.B.2)
 
Seven subjects died during the trial, respectively 2 for control group, 1 for T1 and 4 for T2. All the subjects died suddenly and at the necroscopy showed signs of enteritis and of colibacillosis. In 5 cases an Escherichia coli hemolytic strain was isolated; in one case from kidney, liver and brain a Sterptococcus suis was isolated.


 
7- Results and discussion
 
7.1-Nutritional effectiveness:
-          Growth in post-weaning period
-          Conversion index
In Par 9 Annex the individual data and results of the subjects are shown.
In table 2.3.B.2 the results regarding the growth of the subjects are shown. Firstly the average starting weights of all the subjects are displayed. Secondly the average starting weights of the subjects that were in trial till the end of the experiment are presented. In this case, due to the high number of subjects that died in the overdose dietary treatment and to their individual starting weights, a statistically significant difference was observed between T1 and T2 treatments, in favor of the latter. In any case, the diets did not significantly modify the final live weight and the rate of growth, which remained low, on the average 173 g per day,-- a common phenomenon in the first post-weaning week.
The feed intake was not affected neither the feed conversion was influenced by the diet.
The data on growth performance should be evaluated considering the negative health condition of the overdose group. In this case the live weight gain was not affected in the residual subjects, bur, overall, 4 of 21 subjects died for intestinal pathology.
For the standard does group at the contrary no negative drawback was observed and in this group the mortality was the lowest (1 of 21).
In the similar trial 51 B executed by this Department in 1999, a positive effect on growth performance was observed.
The absence of a evident effect of Turval B0399 in this parameter of the present trial, could be attributed to differences in the base diet, that could have affect also the presence of other bacteria aspects not cultivated or not cultivable.
This could have consequently contributed to maintain a similar results between the different diets.
 


 
Table 2.3.B.2 – Effect of the addition of Turval B0399 on the growth of piglets weaned at 21 days of ageon growth performance and tolerance test.
 
 
 
Control group
(C)
Treated group
(T1)
Tolerance test group
(T2)
Orthogonal comparisons
Other comparisons
SEM
 
u.m.
 
 
 
C vs
(T1+T2)
T1 vs T2
C vs T1
C vs T2
 
Subjects
N
21
21
21
 
 
 
 
 
Initial live weight
g
6063
5958
6227
ns
ns
ns
ns
258
Subjects till the end of the trial
g
19
20
17
 
 
 
 
 
Initial live weight
g
6322
6108
6734
ns
*
ns
ns
295
Days in trial
N
28.9
28.6
28.4
 
 
 
 
 
Final live weight
g
11616
10874
11796
ns
ns
ns
ns
648
Daily weight gain
g/d
180
164
175
ns
ns
ns
ns
20
Daily feed consumption
g/d
275
268
283
ns
ns
ns
ns
12
Conversion index a
g/g
1.92
1.90
2.37
ns
ns
ns
ns
0.74
 
 
 
 
 
 
 
 
 
 
 
*=P <0.05 ; Ns = P > 0,05.
 
aThe average values obtained with the variance analysis don’t necessarily coincide with those obtained by calculating the relationship between the average values of consumption of feed and those of growth.


 
7.2-Effects on the pH of the intestine.
 
The effect of the addition of Turval B0399 in the piglets’ diet on pH in the different parts of the intestine is presented in table 2.3.B.3.
No pH was affected in a statistically significant manner.
In the similar trial 51 B executed by this Department in 1999, a positive effect on ph was observed.
The absence of a evident effect of Turval B0399 in this parameter of the present trial, could be attributed to differences in the base diet, that could have affect also the presence of other bacteria aspects not cultivated or not cultivable.
This could have consequently contributed to maintain a similar pH between the different diets.
 
 
Table 2.3.B.3 – Effect of the addition of Turval B0399 in the diet of piglets weaned at 21 days of age on pH of the different parts of the intestine.
 
 
Control group
(C)
Treated group
(T1)
Tolerance test group
(T2)
Orthogonal comparisons
Other comparisons
SEM
 
pH
pH
pH
C vs
(T1+T2)
T1 vs T2
C vs T1
C vs T2
 
Duodenum
5.15
5.06
5.37
ns
ns
ns
ns
0,43
Ileum
7.10
7.10
7.22
ns
ns
ns
ns
0,17
Colon
6.01
6.25
6,13
ns
ns
ns
ns
0,23
Cecum
6.25
6.25
6.13
ns
ns
ns
ns
0,14
 
 
 
 
 
 
 
 
 
Ns = P > 0,05.
 


 
 
7.3-Concentration of total E.Coli and Lactobacilli in colon
 
In table 2.3.B.4, the effect of the dietary addition of Turval B0399 on the presence of total E.Coli and Lactobacilli in the colon content is presented.
The number of total coliforms found in the colon was reduced. The content of Lactobacilli was increased by Turval B0399 supplementation.
The ratio coliforms/lactobacilli was reduced by both dosages in a statistically significant manner (P<0.01 for T2 and P<0.05 for T1).
In table 2.3.B.5, the effect of the dietary addition of Turval B0399 on the recovery of Turval B0399in the colon content is presented.
With the standard dosage and overdose of the additive, the recovery of Kluyveromyces B0399 was 36.8% and 47.1 % respectively. In the control diet no subject was found positive for the presence of Kluyveromyces B0399. This difference against supplemented diets was statistically significant (Likelihood Ratio Chi-Square Test: P=0.001).
 
 
Table 2.3.B4 – Effect of the addition of Turval B0399 in the diet of piglets weaned at 21 days of age, on the presence of total E.Coli and lactobacilli in the colon (ln CFU/g))
 
 
 
Control group
(C)
Treated group
(T1)
Tolerance test group
(T2)
Orthogonal comparisons
Other comparisons
SEM
 
U.M.
 
 
 
C vs
(T1+T2)
T1 vs T2
C vs T1
C vs T2
 
Total E.Coli
Ln
18.5
 
17.8
16.7
**
*
ns
**
0.59
Lactobacilli
Ln
14.9
15.5
15.2
Ns
ns
ns
ns
0.64
Total E.Coli/
Lactobacilli
Ln
1.24
1.15
1.11
**
ns
*
**
0.36
** = P<0.01 ; * = P<0.05 ; NS = P>0.05.
 
 
Table 2.3.B5 – Effect of the addition of Turval B0399 in the diet of piglets weaned at 21 days of age, on the recovery of Kluyveromyces B0399 in the colon.
 
 
Subjects
Control group
(C)
Treated group
(T1)
Tolerance test group
(T2)
Positive
0
36.8%
47.1 %
Negative
100 %
63.2%
52.9 %
 
Likelihood Ratio Chi-Square Test: P=0.001.


 
8- General discussion on the results of the test
 
The trial showed that the recommended dose dietary addition of Turval B0399 did not negatively affect the growth performance of weaned piglets, the health and the pH of the different intestinal tracts. This supplementation positively improved the ratio between total Coli and Lactobacilli; this presumably contributed to the maintenance of a good health of the subjects.
In the similar trial 51 B executed by this Department in 1999, a positive effect on the growth performance and pH was observed.
The absence of a evident effect of Turval B0399 in this parameter of the present trial, could be attributed to differences in the base diet, that could have affect also the presence of other bacteria species not cultivated or not cultivable.
This could have consequently contributed to maintain a similar performances results between the different diets.
In the case of the dietary overdose (tolerance test) with Turval B0399 the growth performance of weaned piglets was not influenced, but the health was impaired, at least in the 4 subjects that died for intestinal pathologies. In the other subjects the pH in the gut were nod modified.
The balance between Coli and Lactobacilli was sensibly improved. Presumably this observation would not have been found in the subjects that died, however the data do not permit to identify the reasons why these four subjects performed differently.
 
It is interesting to note that in 1/3 (Diet T1) to 2/3 (Diet T2: tolerance test) of the subjects fed the additive, the Turval B0399 was recovered still cultivable in the colon content. Furthermore the data show that apparently no contamination was present between the different groups.


9- Annex - Individual data.
 
number
litter
treatment
nmac
sex
starting LW
final LW
feed intake
 
 
 
 
 
 
 
 
1
1
T2
4
m
8020
14585
9518
2
1
T1
4
m
8060
13691
7965
3
1
C
1
m
7300
11126
8585
4
2
T2
1
m
6540
10041
7553
5
2
T1
3
m
6540
8562
5612
6
2
C
3
m
4900
11043
8578
7
3
T2
.
m
4900
7020
2750
8
3
T1
1
m
6760
9329
7965
9
3
C
1
m
7020
10730
7159
10
3
T2
4
m
6860
10781
7553
11
3
T1
2
m
5520
9707
6807
12
3
C
3
m
5280
11986
7126
13
4
T2
2
m
6560
11269
7553
14
4
T1
1
m
5000
8151
5612
15
4
C
1
m
6140
8905
7377
16
4
T2
2
m
5880
10930
6616
17
4
T1
3
m
5980
9747
7665
18
4
C
1
m
4640
8233
7123
19
5
T2
3
m
6080
9373
5273
20
5
T1
3
f
6420
13287
6807
21
5
C
2
m
7020
10451
7159
22
5
T2
3
m
5400
10888
6616
23
5
T1
4
m
6620
10446
7743
24
5
C
3
m
6560
12780
7377
25
5
T2
4
m
7740
14757
9518
26
5
T1
2
m
7500
12808
7965
27
5
C
4
m
5820
11620
7159
28
5
T2
1
m
6060
9218
5273
29
5
T1
4
m
6360
14790
7604
30
6
C
4
f
7140
14887
8585
31
6
T2
.
f
5480
12560
6935
32
6
T1
3
m
6760
11710
7665
33
6
C
3
m
6060
10712
8585
34
6
T2
1
f
8360
12270
9518
35
6
T1
2
f
6520
10371
7743
36
6
C
2
m
7150
12080
7123
37
7
T2
2
m
6000
11681
8012
38
7
T1
.
f
5900
8010
4717
39
7
C
4
m
6260
12804
8578
40
8
T2
3
m
5780
11780
8012
41
8
T1
1
m
4040
6658
6807
42
8
C
4
m
5880
9745
8578
43
9
T2
3
m
6920
12200
8487
44
9
T1
2
m
4580
9136
7527


 
 
 
number
litter
treatment
nmac
sex
starting LW
final LW
feed intake
45
9
C
4
m
5000
11832
7123
46
10
T2
.
f
5920
6150
2150
47
10
T1
1
m
6680
8053
7604
48
10
C
3
m
6620
11708
6948
49
10
T2
.
m
4730
6348
4173
50
10
T1
4
m
6080
10707
7665
51
11
C
3
m
6620
7987
6948
52
11
T2
3
m
5300
10016
8487
53
11
T1
3
m
4300
8314
7527
54
11
C
.
m
4300
.
.
55
11
T2
4
m
6140
11893
8487
56
11
T1
2
m
4080
7284
5612
57
11
C
4
m
5620
11742
7377
58
12
T2
1
m
7320
7809
6935
59
12
T1
4
m
5540
10832
7527
60
12
C
.
m
5850
5620
3703
61
12
T2
2
m
5460
10769
6616
62
12
T1
3
m
6580
11624
7743
63
12
C
2
m
6300
9112
7126
 


 
Number
Ph ileum
pH duodenum
pH cecum
pH colon
days
DLWG
AV.FEED INTAKE
FEED CONVERSION
 
 
 
 
 
 
 
 
 
1
7,42
4,42
5,88
5,95
31
211,774
307,032
1,44981
2
7,16
4,78
6,28
6,43
31
181,645
256,935
1,41449
3
7,62
.
6,45
6,72
26
147,154
330,192
2,24386
4
8,09
.
6,24
6,24
26
134,654
290,5
2,15738
5
.
.
6,76
6,9
30
67,4
187,067
2,77547
6
7,69
5,14
6,31
6,28
30
204,767
285,933
1,39639
7
.
.
.
.
.
.
.
.
8
.
.
6,17
6,54
26
98,808
306,346
3,10043
9
7,61
.
6,63
6,59
26
142,692
275,346
1,92965
10
7,18
4,91
6,86
6,47
31
126,484
243,645
1,92629
11
7,32
5,38
5,72
6,03
27
155,074
252,111
1,62575
12
7,41
4,71
5,48
5,59
30
223,533
237,533
1,06263
13
7,72
5,09
5,56
5,68
27
174,407
279,741
1,60395
14
7,65
7,92
6,74
6,68
26
121,192
215,846
1,78102
15
7,43
.
6,13
6,75
26
106,346
283,731
2,66799
16
7,1
3,81
5,56
5,81
27
187,037
245,037
1,3101
17
7,58
.
7,27
7,24
30
125,567
255,5
2,03478
18
7,38
4,4
5,86
6,61
26
138,192
273,962
1,98247
19
7,58
6,51
7,02
6,86
30
109,767
175,767
1,60128
20
6,75
4,9
5,93
6,23
30
228,9
226,9
0,99126
21
7,64
5,32
6,08
6,32
27
127,074
265,148
2,08656
22
7,21
5,94
5,74
6,24
30
182,933
220,533
1,20554
23
7,26
4,67
6,05
6,15
31
123,419
249,774
2,02378
24
7,39
5,06
5,74
6,01
30
207,333
245,9
1,18601
25
7,88
5,48
5,62
5,88
31
226,355
307,032
1,35642
26
.
5,28
5,62
5,67
27
196,593
295
1,50057
27
7,51
5,81
5,56
5,8
31
187,097
230,935
1,23431
28
6,98
5,55
6,99
7,1
26
121,462
202,808
1,66973
29
7,37
3,98
5,63
5,84
31
271,935
245,29
0,90202
30
7,17
5,06
5,21
5,33
31
249,903
276,935
1,10817
31
.
.
.
.
.
.
.
.
32
7,61
.
6,62
6,48
30
165
255,5
1,54848
33
7,81
5,03
5,45
6,15
30
155,067
286,167
1,84544
34
7,4
7
6,74
7,13
26
150,385
366,077
2,43427
35
7,46
.
6,02
6,02
27
142,63
286,778
2,01065
36
7,92
6,67
5,81
6,04
27
182,593
263,815
1,44483
37
7,56
4
5,81
6,12
27
210,407
296,741
1,4103
38
.
.
.
.
.
.
.
.
39
7,38
4,57
6,8
6,64
31
211,097
276,71
1,3108
40
6,44
5,88
5,54
6,12
30
200
267,067
1,3353
41
7,49
.
6,71
6,75
26
100,692
261,808
2,6001
42
7,25
5,21
6,74
6,8
31
124,677
276,71
2,2194
43
7,39
6,94
6,2
6,36
30
176
282,9
1,6074
44
7,3
5,12
5,53
6,5
27
168,741
278,778
1,6521


 
 
 
Number
Ph ileum
pH duodenum
pH cecum
pH colon
days
DLWG
AV.FEED INTAKE
FEED CONVERSION
45
7,4
5,58
5,7
5,79
31
220,387
229,774
1,0426
46
.
.
.
.
.
.
.
.
47
7,61
4,21
6,83
7,13
26
52,808
292,462
5,5382
48
6,56
5,46
5,86
6,5
30
169,6
231,6
1,3656
49
.
.
.
.
.
.
.
.
50
7,57
5,73
5,82
6,08
31
149,258
247,258
1,6566
51
7,37
4,88
7,32
.
30
45,567
231,6
5,0827
52
7,05
5,13
5,9
5,93
30
157,2
282,9
1,7996
53
7,66
.
6,61
7,39
30
133,8
250,9
1,8752
54
.
.
.
.
.
.
.
.
55
7,18
5,2
6,47
6,48
31
185,581
273,774
1,4752
56
6,47
5,35
5,75
6,08
27
118,667
207,852
1,7516
57
7,07
5,9
5,63
6,4
31
197,484
237,968
1,205
58
7,57
6,45
6,78
6,78
26
18,808
266,731
14,182
59
7,71
4,32
5,83
6,21
31
170,71
242,806
1,4223
60
.
.
.
.
.
.
.
.
61
7,41
5
5,49
5,8
27
196,63
245,037
1,2462
62
7,73
.
6,97
6,91
30
168,133
258,1
1,5351
63
6,61
6,7
5,59
6,13
27
104,148
263,926
2,5341
 

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