Fermentation Extract and Toxic Plant Effect on The Physiology of ...

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20 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

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Lab. of Rumen Microbiology and Biotechnology, GSNU, Korea.


AL 757

Special Topics in Rumen Microbiology

LEC


15

Rumen Fermentation by Fungi

(Mould & Yeast)

Sung Sill Lee

Feed Additives

*
Change rumen microbial population

* Alter fermentation end products

* Increase animal production

Antibiotics


-

Selectively shift rumen microbial


population
Improve feed conversion


-

Residual problems :


Animal products and environment


-

Prohibited by law

Direct Fed Microbials (DFM)


Definition

“A source of live (viable) naturally
-
occurring
microorganisms” (FDA, 1989)


Characteristics

-

Provide a natural alternative to antibiotics

-

Consist of one or more microorganisms

Proposed Mechanism of DFM


Provide antimicrobial compounds


Compete with undesirable M/O


Promote the growth of beneficial M/O


Enzyme production


Metabolize undesirable compounds


Stimulate immune response in host

(Fuller, 1989)

Types of DFM


Bacterial DFM


Lactobacillus acidophilus, Bacillus subtilis,
Bifidobacterium
, etc.


Fungal DFM


Saccharomyces cerevisiae


Aspergillus oryzae


Mixed DFM


Mixed culture

Fungus
Aspergillus oryzae
(
AO
)


Fermented food production


Wine brewer


Industrial enzyme production


Feed additive

AO

Fermentation Extract

-

A. oryzae
cultures are grown in the absence of
nitrogen to maximize fermentation end products
and increase antimicrobial agent production.
(Kistner, 1962)


-

Consists of
AO
cells, the media it was grown in
and it’s end products sprayed onto wheat bran,
the carrier.
(Gomez
-
Alarcon, 1990)



AO secondary metabolites


Kojic acid


Antibacterial & antifungal activity


Toxicity: 4


8 mg/kg chicken feed
(Giroir et al., 1991)


Can be produced with long fermentation



Maltoryzine


Toxic to dairy cows


LC50 : 3 mg/kg
(Ciegler & Vesonder, 1987)


Aspergillus oryzae

IAM 2950

AO secondary metabolites


Cyclopiazonic acid


Natural contaminant of food


Toxicity: 0.25


50 mg/kg Dog


Cause debilitating illness in cattle and human


(Rao & Husain, 1985)



Β
-
Nitropropionic acid


Neurotoxin


LC50 : 20


50 mg/kg Rat
(Hamilton & Gould, 1987)

Is AO safe?


AO do not produce Aflatoxin


AO is not a human pathogen


Allergic reaction?


AO is not hazardous to environment



Yes,
AO is safe !

AO’s
impact on ruminant

productivity


Increase average daily gain in steers
supplemented with AO
(Church, 1989)



Decreased rectal temperature and animal
respiration rates during times of heat stress
(Huber and Higginbotham, 1985)



Increased milk production by cows
supplemented with AO
( Wallentine et al., 1986;
Gomez
-
Alarcon et al., 1991)

AO’s
impact on rumen
fermentation


Increased rumen pH
(Ollerman et al., 1990)


Decreased rumen lactate concentration
resulting from increased microbial lactate
utilization
(Nisbet and Martin, 1990)


Increase in total volatile fatty acid
production and shifts in the VFA profiles.
(Gomez
-
Alarcon, 1990; Chiquette, 1995)



Increased rumen ammonia concentration





(Frumholtz et al., 1989)


Reduced number of proteolytic microbes






(Newbold et al., 1993)


Increased number of cellulolytic microbes
and total viable microbes
(Weidmeier et al., 1987)


AO’s
impact on ruminal
fermentation (continued)

AO’s
impact on rumen microbial
population

Name

Gram

Shape

AO

Substrate

Lactobacillus ruminis

+

rod

0

CHO

Lactobacillus vitulinus

+

0

Streptococcus bovis

+(
-
)

ovoid

0

CHO

Anaerovibrio lipolytica

-

rod

0

Lipid

Butyrivibrio fibrisolvens

-

G+ cell



Fib

Fusobacterium necrophorum

-

rod

0

pathogen

Lachnospira multiparus

-

rod

0

pectin

Prevotella ruminicola

-

c
-
bacili

0

CHO

Ruminobacter amylophilus

-

ovoid

0

CHO

Ruminococcus flavefaciens

-

G+ cell

0

Veillonella parvula

-

cocci

0

lactate

Fibrobacter succinogenes

-

rod



Fib

Megarsphaera elsdenii

-

cocci



CHO

Ruminococcus albus

-

G+ cell



Fib

Selenomonas ruminantium

-

cresent



CHO

Neocallimastix frontalis

NA



Piromyces communis

NA



Sphaeromonas communis

NA



Effect of Dose Levels on Animal
Response


Three grams AO / day is a commonly used
dosage in cattle
(Gomez
-
Alarcon et al., 1990)



Cows fed 113 grams AO / day exhibited
decreased milk production and feed intake





(Van Horn, 1984)



In vitro

concentrations commonly used were
.03
-

.05 mg/ml

(Newbold et al., 1991)

Why Rumen Fungi?


Degrade various carbohydrates, &
proteins


Mechanical & enzymatic breakdown of
plant tissues


No research was performed on the
effect of
A. oryzae

on the rumen fungi

Objective

Examine the effect of
A. oryzae

on
the growth of anaerobic fungi

How AO works in fungal
culture?

Experimental Procedure

S. E. M.

Assay

Extract

Filtrate

Incubate

Effects of
A. oryzae

extract on
fungal production

*
: Not significant

Increase (%)
Strain
CMC
Glu
Pro
Mat
C2
VFA
Neo.
135.5
113.9
114.3
115.7
119.6
123.0
Pir.
120.2
115.2
125.8
118.7
113.4
119.2
Orp.
127.1
99.5*
121.7
131.0
103.0
119.5
0
10
20
30
0
48
72
96
120
144
168
Time (hours)
cellulase (IU/ml) x 10
-2
no extract
2 ul extract
10ul extract
Cellulase secretion of AO treated



N. frontalis
cultures over time

Effects of high concentration of
AO

treatment on
N. frontalis growth

0

1000

2000

3000

4000

5000

0

0.5

0.6

0.7

0.8

0.9

1

Treatment (% ,w/v)

Zoospore counts/ml

0

5

10

15

20

Sporangia Counts/ml

Zoospores

Sporangia

A

B

C

E

F

CONT

AMF7

AMF7

AMF100

AMF100

SEM result of
AO

treatment

Effects of AO powder on fungal
zoospore maturation

Type

AO

Sampling time

46

56

71

81

92

101

M

-

400

375

3775

4150

2050

2025

+

475

1025

3275

2125

1675

700

NM

-

0

75

625

800

2125

2950

+

0

300

775

600

1750

1875

G

-

225

200

2725

4475

8550

6450

+

275

400

6650

10975

10875

15275

M/G

-

1.77

1.88

1.38

0.93

0.24

0.31

+

1.72

2.56

0.49

0.19

0.15

0.05

M: Motile, NM: Non
-
motile, G: Germinated

AO supplementation: 1/20,000 (w/v)

What components in
AO

fermentation extract cause
fungal response?

Experimental Procedure

Assay

Extract

Inoculate
&
Incubate

TLC

TLC results of
AO

ether extract

13


7


6


5


4


3


2


1

12

11

10


9


8

SF

Org

-20
0
20
40
60
80
% above control
Experimental Procedure for
Bioautography Test

Agar Medium
with
B. subtilus

Stain

TLC

Diffuse

Incubate

Bioautography test results of
AO

extract

10

l

㈰2

l

㈰2

l

㈵2

l

㄰1

l

㈵2

l


-
Glucosidase Activity of

AO

extract Fraction

0
1
2
3
4
5
6
7
con2
31
56
76
96
116
136
156
176
196
216
236
256
Fraction No.
Activity (mIU/ml)
AO. extract effects on

B. subtilis

growth

How can we induce
antimicrobial compounds?

Dose (



T牥慴浥湴

T牥慴浥湴

䑯獥












㄰1









㄰1


-
N
-



-
N
-


M
-
N
-
ㄲ1

M
-
N
-


M
-
N
-


M
-
N
-


S
-
N
-
120

S
-
N
-
96

S
-
N
-
72

S
-
N
-
48

P
-
N
-
96

P
-
N
-
120

R
-
N
-
48

R
-
N
-
72

R
-
N
-
96

R
-
N
-
120

S+N
-
120

M+N
-
120

P+N
-
120

R+N
-
120

Dose dependent effects of
AO

fermentation extract
on
Bacillus subtilis

growth.

P: PDB, S: Starch Medium, R: Rice Straw Medium, M: Maltose Medium, +N:
Normal condition,
-
N: Stress condition

Bioautography test results of
AO

fermentation
extracts of different cultivation conditions

PDB

Starch

Mal.

Xylan

Cont

2

1

1

1

48

2

0

1

1

72

2

1

1

1

96

1

1

4

4

120

2

1

3

3

AN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
37
100
%
0
100
%
AO3
1: Scan ES+
329.737
2.63e5
34.21
25.06
1.46
12.38
4.18
16.21
63.23
48.95
46.91
62.04
73.35
AO3
1: Scan ES+
TIC
1.58e6
27.01
18.90
14.00
8.38
5.11
22.48
88.52
47.78
38.39
35.84
62.46
52.90
70.56
81.53
80.03
AN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
37
100
%
0
100
%
AO3
1: Scan ES+
737.991
1.18e5
8.10
0.73
18.30
12.73
29.43
24.42
74.40
63.17
48.83
35.02
36.27
47.39
50.27
65.73
77.70
82.87
87.29
AO3
1: Scan ES+
TIC
1.58e6
27.01
18.90
14.00
8.38
5.11
22.48
88.52
47.78
38.39
35.84
62.46
52.90
70.56
81.53
80.03
AN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
37
100
%
0
100
%
AO3
1: Scan ES+
764.736
1.94e5
24.27
10.02
5.74
21.74
16.56
25.90
44.64
43.32
72.29
64.28
46.58
82.50
AO3
1: Scan ES+
TIC
1.58e6
27.01
18.90
14.00
8.38
5.11
22.48
88.52
47.78
38.39
35.84
62.46
52.90
70.56
81.53
80.03
AN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
37
100
%
0
100
%
AO3
1: Scan ES+
893.153
1.18e5
8.11
2.98
65.37
27.86
19.35
15.97
58.46
40.31
29.82
56.17
44.99
68.53
81.12
74.28
89.31
AO3
1: Scan ES+
TIC
1.58e6
27.01
18.90
14.00
8.38
5.11
22.48
88.52
47.78
38.39
35.84
62.46
52.90
70.56
81.53
80.03
AN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
37
100
%
0
100
%
AO3
1: Scan ES+
903.237
1.48e5
4.72
3.43
70.47
31.10
29.43
23.68
6.93
12.90
54.26
39.00
51.73
57.00
65.49
79.18
AO3
1: Scan ES+
TIC
1.58e6
27.01
18.90
14.00
8.38
5.11
22.48
88.52
47.78
38.39
35.84
62.46
52.90
70.56
81.53
80.03
Mass Spectrum of AO extract incubated with starch
as CHO source

MN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
33
100
%
0
100
%
AO1
1: Scan ES+
829.152
1.06e5
42.81
3.37
9.36
21.57
15.31
41.64
25.47
37.43
78.96
44.95
65.96
56.51
76.50
82.83
88.02
AO1
1: Scan ES+
TIC
1.71e6
0.02
52.06
40.40
19.34
10.96
0.36
4.50
34.69
23.82
25.28
47.43
52.81
65.14
56.44
82.48
76.65
68.32
86.96
MN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
33
100
%
0
100
%
AO1
1: Scan ES+
769.637
2.66e5
38.76
28.86
16.76
11.80
7.50
22.46
52.00
70.19
AO1
1: Scan ES+
TIC
1.71e6
0.02
52.06
40.40
19.34
10.96
0.36
4.50
34.69
23.82
25.28
47.43
52.81
65.14
56.44
82.48
76.65
68.32
86.96
MN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
33
100
%
0
100
%
AO1
1: Scan ES+
637.879
2.10e5
39.78
18.80
3.96
16.37
36.75
57.86
49.77
88.83
AO1
1: Scan ES+
TIC
1.71e6
0.02
52.06
40.40
19.34
10.96
0.36
4.50
34.69
23.82
25.28
47.43
52.81
65.14
56.44
82.48
76.65
68.32
86.96
MN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
33
100
%
0
100
%
AO1
1: Scan ES+
569.836
3.60e5
10.96
0.63
52.81
29.59
32.57
51.81
AO1
1: Scan ES+
TIC
1.71e6
0.02
52.06
40.40
19.34
10.96
0.36
4.50
34.69
23.82
25.28
47.43
52.81
65.14
56.44
82.48
76.65
68.32
86.96
Mass Spectrum of AO extract incubated with
maltose as CHO source

XN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
43
100
%
0
100
%
AO4
1: Scan ES+
969.206
2.19e5
29.32
1.42
10.43
16.04
19.21
24.81
56.69
55.09
48.88
66.97
62.44
84.70
71.78
AO4
1: Scan ES+
TIC
1.40e6
32.33
20.75
9.83
7.03
14.19
30.99
23.19
45.58
34.99
40.04
56.03
51.99
67.16
63.81
88.32
77.67
72.91
79.85
XN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
43
100
%
0
100
%
AO4
1: Scan ES+
1002.822
2.01e5
35.14
9.15
4.57
26.60
12.79
44.79
41.71
88.32
57.95
49.93
71.50
AO4
1: Scan ES+
TIC
1.40e6
32.33
20.75
9.83
7.03
14.19
30.99
23.19
45.58
34.99
40.04
56.03
51.99
67.16
63.81
88.32
77.67
72.91
79.85
XN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
43
100
%
0
100
%
AO4
1: Scan ES+
917.803
1.31e5
52.07
13.43
4.39
8.94
17.03
47.28
43.47
28.18
30.01
60.81
57.29
65.55
84.63
82.48
74.71
87.96
AO4
1: Scan ES+
TIC
1.40e6
32.33
20.75
9.83
7.03
14.19
30.99
23.19
45.58
34.99
40.04
56.03
51.99
67.16
63.81
88.32
77.67
72.91
79.85
XN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
43
100
%
0
100
%
AO4
1: Scan ES+
721.328
2.02e5
29.66
6.94
4.33
19.48
14.57
46.85
45.41
38.06
79.77
77.28
81.31
AO4
1: Scan ES+
TIC
1.40e6
32.33
20.75
9.83
7.03
14.19
30.99
23.19
45.58
34.99
40.04
56.03
51.99
67.16
63.81
88.32
77.67
72.91
79.85
XN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
43
100
%
0
100
%
AO4
1: Scan ES+
969.206
2.19e5
29.32
1.42
10.43
16.04
19.21
24.81
56.69
55.09
48.88
66.97
62.44
84.70
71.78
AO4
1: Scan ES+
TIC
1.40e6
32.33
20.75
9.83
7.03
14.19
30.99
23.19
45.58
34.99
40.04
56.03
51.99
67.16
63.81
88.32
77.67
72.91
79.85
XN96
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Time
43
100
%
0
100
%
AO4
1: Scan ES+
206.806
1.72e5
48.03
24.76
20.75
15.15
9.35
40.08
38.19
26.78
43.34
55.95
57.54
67.12
79.34
77.74
AO4
1: Scan ES+
TIC
1.40e6
32.33
20.75
9.83
7.03
14.19
30.99
23.19
45.58
34.99
40.04
56.03
51.99
67.16
63.81
88.32
77.67
72.91
79.85
Mass Spectrum of AO extract incubated with rice
straw as CHO source

Summary


Nitrogen stressed

condition can induce
anti
-
microbial compounds from
AO

fermentation extract


Induced anti
-
microbial compounds from
AO

should be
more than two chemicals

Summary


AO

fermentation extract
stimulates
fungal growth, rhizoidal branching, and
enzyme production


High concentrations of
AO

fermentation
extract
inhibited fungal growth


AO

fermentation extract contains anti
-
microbial compounds ?

Hypothetical
AO

action

A. oryzae


Nutrients

2nd Metabolites

Rumen


䙩扥爠䑩朮







4
, NH
3
, Lactate Prod.



偲潴敩渠䑩D⸠

䅮業Al


䡥慴H却牥獳


䉗⁧B楮i


Milk Prod.

Stimulate

Fungi & G
-

Bacteria


Inhibit

G+ Bacteria

(Gomez
-
Alarcon et al., 1991; Huber et al., 1986; Beharka& Nagaraja, 1998)