Nutrient parameters, fermentation parameters and mycotoxin contents of fresh whole-crop corn at milk and dough stages are shown in Table 2. The contents of dry matter (DM), starch, lactic acid and deoxynivalenol (DON) at the milk stage were significantly higher than those at the dough stage (P < 0.01). However, the contents of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), pH, acetic acid and Aflatoxin B1 (AFB1) at the milk stage were lower than those at the dough stage, although the difference was not significant.
Table 2
Nutritional parameters, fermentation parameters and mycotoxin contents of fresh whole-crop corn
Item
|
Fresh whole-crop corn
|
Milk stage
|
Dough stage
|
Nutritional parameters
|
DM %
|
29.74 ± 0.71a
|
37.52 ± 1.07b
|
CP %
|
7.86 ± 0.11
|
7.65 ± 0.30
|
Starch %
|
30.24 ± 0.54a
|
33.53 ± 1.12b
|
NDF %
|
43.62 ± 0.96
|
42.25 ± 1.26
|
ADF %
|
23.83 ± 1.64
|
22.99 ± 1.98
|
Fermentation parameters
|
pH
|
4.55 ± 0.09
|
4.39 ± 0.08
|
Lactic acid %
|
0.36 ± 0.05a
|
0.67 ± 0.04b
|
Acetic acid %
|
0.40 ± 0.04
|
0.37 ± 0.13
|
Propionic acid %
|
< 0.03
|
< 0.03
|
Butyric acid %
|
< 0.03
|
< 0.03
|
NH3-N %
|
0.31 ± 0.15
|
0.31 ± 0.02
|
Mycotoxins
|
DON mg/kg
|
0.40 ± 0.17a
|
2.27 ± 0.25b
|
AFB1 µg/kg
|
1.29 ± 0.06
|
1.05 ± 0.23
|
ZEN µg/kg
|
< 50
|
< 50
|
3.1. Effects of Lactobacillus plantarum on nutrition parameters
The effects of the harvest stage and the dose of L. plantarum added, as well as their interaction, on nutrition parameters in whole-crop corn silage are shown in Tables 3 and 4. Overall, all nutrition parameters (DM, starch, NDF, ADF, and CP) decreased with ensiling. DM, starch and NDF concentrations at the milk stage were significantly lower than those at the dough stage for the whole ensiling period. Starch concentration in the MM group was significantly higher than that in the ZM group only on day 42 of silage fermentation (P > 0.05). At the dough stage, addition of 1×1010 cfu/kg L. plantarum reduced DM loss compared with DM loss without the addition of L. plantarum. Harvest stage and the addition of L. plantarum, as well as their interaction, did not significantly affect CP concentrations of whole-crop corn silage for the whole fermentation period (P > 0.05). On day 7 of fermentation, the NDF concentration in the 1×1011 cfu/kg L. plantarum group was significantly high than that in no addition and 1×1019 cfu/kg L. plantarum groups (P > 0.05). Harvest stage and the dose of L. plantarum added, as well as their interaction, significantly affected ADF in the whole-crop corn silage on day 42 (P < 0.05).
Table 3
Effects of harvest period, dose of Lactobacillus plantarum and their interactions on DM, starch and CP concentrations
Ensiling
days
|
Harvest stage
|
Dose of L.p2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
Milk stage
|
Dough stage
|
0
|
1×109
|
1×1010
|
1×1011
|
ZM
|
LM
|
MM
|
HM
|
ZD
|
LD
|
MD
|
HD
|
Harvest stage
|
Dose of L.p2
|
Harvest stage×
dose of L.p2
|
DM (%)
|
3
|
28.90a
|
37.01b
|
32.69
|
33.36
|
32.68
|
33.10
|
28.92a
|
28.73a
|
28.80a
|
29.16a
|
36.46b
|
37.98b
|
36.55b
|
37.03b
|
0.86
|
< 0.01
|
0.55
|
0.42
|
7
|
28.59a
|
37.26b
|
32.42
|
32.89
|
33.42
|
32.96
|
28.46a
|
28.46a
|
29.19a
|
28.22a
|
36.38b
|
37.31b
|
37.65b
|
37.70b
|
0.93
|
< 0.01
|
0.42
|
0.60
|
21
|
27.91a
|
36.26b
|
32.26
|
31.43
|
32.50
|
32.14
|
28.18a
|
27.13a
|
27.94a
|
28.38a
|
36.34b
|
35.73b
|
37.06b
|
35.89b
|
0.96
|
< 0.01
|
0.86
|
0.94
|
42
|
27.60a
|
34.96b
|
30.79
|
30.72
|
31.66
|
31.97
|
27.29a
|
27.24a
|
27.87a
|
28.01a
|
34.29b
|
34.19b
|
35.44b
|
35.92b
|
0.82
|
< 0.01
|
0.38
|
0.93
|
Starch (%)
|
3
|
29.21a
|
32.55b
|
30.47
|
30.85
|
31.40
|
30.79
|
28.80a
|
29.37a
|
29.72a
|
28.94a
|
32.14b
|
32.32b
|
33.09b
|
32.64b
|
0.21
|
< 0.01
|
0.47
|
0.94
|
7
|
28.41a
|
31.62b
|
29.61
|
30.27
|
30.92
|
29.27
|
27.65a
|
28.80ab
|
29.31ab
|
27.89a
|
31.57bc
|
31.75b
|
32.53c
|
30.64abc
|
0.33
|
< 0.01
|
0.34
|
0.93
|
21
|
28.09a
|
30.62b
|
28.74a
|
29.65ab
|
30.00b
|
29.03ab
|
27.12a
|
28.42ab
|
29.13b
|
27.70ab
|
30.36c
|
30.8c
|
30.88c
|
30.36c
|
0.19
|
< 0.01
|
< 0.05
|
0.60
|
42
|
27.08a
|
29.31b
|
27.77
|
27.92
|
28.78
|
28.31
|
26.85a
|
26.91ab
|
27.24ab
|
27.32ab
|
28.68bc
|
28.92c
|
30.32c
|
29.30c
|
0.19
|
< 0.01
|
0.25
|
0.63
|
CP (%)
|
3
|
7.53
|
7.52
|
7.58
|
7.39
|
7.56
|
7.56
|
7.62
|
7.35
|
7.58
|
7.56
|
7.54
|
7.42
|
7.54
|
7.57
|
0.06
|
0.92
|
0.68
|
0.98
|
7
|
7.37
|
7.29
|
7.34
|
7.24
|
7.41
|
7.33
|
7.44
|
7.31
|
7.37
|
7.35
|
7.24
|
7.18
|
7.45
|
7.30
|
0.04
|
0.41
|
0.63
|
0.72
|
21
|
7.23
|
7.12
|
7.28
|
7.10
|
7.25
|
7.08
|
7.34
|
7.19
|
7.29
|
7.12
|
7.21
|
7.01
|
7.21
|
7.05
|
0.06
|
0.32
|
0.50
|
0.98
|
42
|
7.05
|
7.05
|
7.19
|
6.92
|
7.08
|
7.00
|
7.24
|
6.91
|
6.98
|
7.06
|
7.13
|
6.94
|
7.19
|
6.94
|
0.06
|
0.97
|
0.49
|
0.76
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; LM: 1×109 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; HM: 1×1011cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; LD: 1×109 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage; HD: 1×1011 cfu/kg of L. plantarum + dough stage |
2L.p: L. plantarum |
Table 4
Effects of harvest period, dose of Lactobacillus plantarum and their interactions on NDF and ADF concentrations
Ensiling
days
|
Harvest stage
|
Dose of L.p2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
Milk stage
|
Dough stage
|
0
|
1×109
|
1×1010
|
1×1011
|
ZM
|
LM
|
MM
|
HM
|
ZD
|
LD
|
MD
|
HD
|
Harvest stage
|
Dose of L.p2
|
Harvest stage×
dose of L.p2
|
NDF (%)
|
3
|
42.57a
|
40.43b
|
41.29
|
41.69
|
41.09
|
41.92
|
42.74
|
42.86
|
42.31
|
42.36
|
39.85
|
40.52
|
39.88
|
41.48
|
0.36
|
< 0.01
|
0.84
|
0.77
|
7
|
41.65a
|
39.98b
|
40.12a
|
40.42a
|
40.77ab
|
41.96b
|
40.53ab
|
41.00abc
|
42.23bc
|
42.84c
|
39.71a
|
39.84
|
39.31a
|
41.08abc
|
0.24
|
< 0.01
|
< 0.05
|
0.45
|
21
|
41.33a
|
39.41b
|
39.90
|
40.26
|
40.52
|
40.81
|
40.48
|
40.98
|
42.03
|
41.81
|
39.32
|
39.53
|
39.00
|
39.80
|
0.35
|
< 0.05
|
0.82
|
0.79
|
42
|
40.77a
|
38.99b
|
39.73
|
39.84
|
40.07
|
39.90
|
40.24
|
40.83
|
41.22
|
40.80
|
39.22
|
38.85
|
38.92
|
38.99
|
0.35
|
< 0.05
|
0.99
|
0.93
|
ADF (%)
|
3
|
22.88
|
21.75
|
21.69
|
22.64
|
22.25
|
22.68
|
22.40
|
23.33
|
23.35
|
22.46
|
20.99
|
21.95
|
21.16
|
22.90
|
0.35
|
0.13
|
0.74
|
0.61
|
7
|
21.93
|
20.83
|
20.71
|
20.97
|
21.54
|
22.28
|
21.37
|
21.44
|
22.56
|
22.34
|
20.04
|
20.51
|
20.53
|
22.22
|
0.33
|
0.12
|
0.38
|
0.78
|
21
|
21.81
|
20.58
|
20.86
|
20.72
|
21.37
|
21.82
|
21.93
|
21.37
|
22.26
|
21.67
|
19.78
|
20.08
|
20.49
|
21.97
|
0.31
|
0.06
|
0.58
|
0.53
|
42
|
21.74a
|
20.01b
|
20.26a
|
21.37b
|
20.37a
|
21.51b
|
21.13b
|
22.67c
|
21.66b
|
21.51b
|
19.39a
|
20.07a
|
19.08ab
|
21.50b
|
0.12
|
< 0.01
|
< 0.01
|
< 0.01
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; LM: 1×109 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; HM: 1×1011cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; LD: 1×109 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage; HD: 1×1011 cfu/kg of L. plantarum + dough stage |
2L.p: L. plantarum |
3.2. Effects of Lactobacillus plantarum on fermentation parameters
The important effects of fermentation parameters after 3–42 days of ensiling are presented in Tables 5 and 6. The interaction effects of harvest stage × the dose of L. plantarum added on pH on day 21 of fermentation and on lactic acid on days 21–42 of fermentation were significant (P < 0.05). Whole-crop corn silage at the dough stage had a significantly higher pH compared with that at the milk stage on days 7–42 (P < 0.01). The dose of L. plantarum added exerted a significant effect on pH on silage fermentation days 21–42 (P < 0.01). Harvest stages (days 3 and 42) as well as the dose of L. plantarum added exerted a significant effect on lactic acid concentration on days 3 and 21–42 of fermentation (P < 0.05). The pH in the MM and HM groups at the milk stage were significantly lower than that in the ZM and LM groups on day 21, while pH in the MM group was significantly lower than that in the other three groups on day 42 (P < 0.01). Lactic acid concentration in the silage of MM groups on day 21 was significantly higher compared to that in the other three groups, while pH values in the MM and HM groups on day 42 were significantly higher than those in the ZM and LM groups on day 42 (P < 0.01). Harvest stage × dose of L. plantarum exerted a significant interaction effect on NH3-N concentration on day 3 of fermentation (P < 0.05). The dose of L. plantarum exerted a significant effect on the concentrations of both NH3-N and acetic acid in silage on days 3–7 (P < 0.01). At the milk stage, the NH3-N concentration in the MD group as well as in the HD group was significantly lower than that in the ZD group on days 3–7 (P < 0.01). However, acetic acid concentration in MD as well as in HD was significantly higher than that in ZD on days 3–7 (P < 0.01). At the dough stage, the NH3-N concentration in LD as well as in MD was significantly lower than that in ZD on day 3 (P < 0.01).
3.3. Effects of Lactobacillus plantarum on mycotoxins
The harvest stage and the dose of L. plantarum and their interaction effects on the mycotoxin content of the whole-crop corn silage are presented in Table 5. During the whole ensiling process, the DON content at the milk stage was always significantly lower than that at the dough stage. On day 3 of ensiling, harvest stage × dose of L. plantarum exerted a significant interaction effect on DON content (P < 0.01). Furthermore, the DON content in the 1×1010 cfu/kg group was significantly lower than that in the 1×109 cfu/kg and 1×1011 cfu/kg groups (P < 0.01). At the dough stage, the DON content in MD as well as in HD was significantly lower than that in ZD on day 42 of ensiling (P < 0.01). As far as AFB1 content was concerned, the AFB1 content in milk stage was significantly higher than that in the dough stage on days 7–42 of ensiling (P < 0.01). At the dough stage, the AFB1 content in MD as well as in HD was significantly lower than that in ZD as well as in LD on day 3 of ensiling (P < 0.01). The zearalenone (ZEN) content in each group was lower than the minimum limit of the kit, so it was not described.
Table 5
Effects of harvest period, dose of Lactobacillus plantarum and their interaction on pH and lactic acid concentrations
Ensiling
days
|
Harvest stage
|
Dose of L.p2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
Milk stage
|
Dough stage
|
0
|
1×109
|
1×1010
|
1×1011
|
ZM
|
LM
|
MM
|
HM
|
ZD
|
LD
|
MD
|
HD
|
Harvest stage
|
Dose of L.p2
|
Harvest stage×
dose of L.p2
|
pH
|
3
|
3.92
|
3.95
|
3.96
|
3.94
|
3.92
|
3.92
|
3.95
|
3.92
|
3.90
|
3.89
|
3.97
|
3.96
|
3.93
|
3.94
|
0.01
|
0.14
|
0.43
|
0.97
|
7
|
3.82a
|
3.91b
|
3.89
|
3.87
|
3.85
|
3.85
|
3.84ab
|
3.83ab
|
3.81a
|
3.80a
|
3.93c
|
3.90bc
|
3.89bc
|
3.90bc
|
0.01
|
< 0.01
|
0.31
|
0.86
|
21
|
3.75a
|
3.89b
|
3.89d
|
3.83c
|
3.76a
|
3.81b
|
3.85d
|
3.77c
|
3.63a
|
3.73b
|
3.92e
|
3.89e
|
3.88de
|
3.88de
|
0.02
|
< 0.01
|
< 0.01
|
< 0.01
|
42
|
3.71a
|
3.86b
|
3.83b
|
3.82b
|
3.73a
|
3.78b
|
3.76b
|
3.75b
|
3.61a
|
3.71b
|
3.89c
|
3.87c
|
3.84c
|
3.85c
|
0.02
|
< 0.01
|
< 0.01
|
0.09
|
Lactic acid (%)
|
3
|
5.24a
|
4.85b
|
4.75a
|
4.85a
|
5.37b
|
5.21ab
|
4.89ab
|
4.97ab
|
5.55b
|
5.53b
|
4.61a
|
4.73a
|
5.19ab
|
4.88ab
|
0.08
|
< 0.05
|
< 0.05
|
0.79
|
7
|
5.73
|
5.87
|
5.39
|
5.74
|
6.02
|
6.06
|
5.13
|
5.68
|
5.99
|
6.13
|
5.65
|
5.81
|
6.04
|
5.98
|
0.14
|
0.62
|
0.31
|
0.85
|
21
|
6.55
|
6.03
|
5.42a
|
6.21ab
|
7.10b
|
6.42b
|
4.95a
|
6.49b
|
8.07c
|
6.69b
|
5.89ab
|
5.93ab
|
6.13ab
|
6.16ab
|
0.16
|
0.11
|
< 0.05
|
< 0.05
|
42
|
7.49a
|
6.27b
|
6.15a
|
6.29a
|
7.94c
|
7.12b
|
6.27a
|
6.41a
|
9.38c
|
7.89b
|
6.04a
|
6.18a
|
6.50a
|
6.35a
|
0.10
|
< 0.01
|
< 0.01
|
< 0.01
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; LM: 1×109 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; HM: 1×1011cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; LD: 1×109 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage; HD: 1×1011 cfu/kg of L. plantarum + dough stage |
2L.p: L. plantarum |
Table 6
Effects of harvest period, dose of Lactobacillus plantarum and their interaction on Acetic acid and NH3-N concentrations
Ensiling
days
|
Harvest stage
|
Dose of L.p2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
milk stage
|
dough stage
|
0
|
1×109
|
1×1010
|
1×1011
|
ZM
|
LM
|
MM
|
HM
|
ZD
|
LD
|
MD
|
HD
|
Harvest stage
|
dose of L.p2
|
Harvest stage×
dose of L.p2
|
Acetic acid (%)
|
3
|
1.39a
|
1.22b
|
1.10a
|
1.31ab
|
1.44b
|
1.39b
|
1.10a
|
1.39ab
|
1.49bc
|
1.62c
|
1.08a
|
1.26ab
|
1.39ab
|
1.17ab
|
0.05
|
< 0.05
|
< 0.05
|
0.21
|
7
|
1.50
|
1.41
|
1.17a
|
1.52b
|
1.51b
|
1.63b
|
1.02a
|
1.65bc
|
1.53bc
|
1.76c
|
1.32ab
|
1.37ab
|
1.51bc
|
1.46bc
|
0.06
|
0.33
|
< 0.0
|
0.06
|
21
|
1.60
|
1.78
|
1.43
|
1.65
|
1.75
|
1.93
|
1.36
|
1.39
|
1.60
|
1.90
|
1.44
|
1.91
|
1.83
|
1.95
|
0.07
|
0.16
|
0.07
|
0.48
|
42
|
1.95
|
2.24
|
2.00
|
2.06
|
2.02
|
2.31
|
1.73
|
1.76
|
1.88
|
2.26
|
2.18
|
2.31
|
2.16
|
2.31
|
0.10
|
0.20
|
0.71
|
0.87
|
NH3-N (%)
|
3
|
0.82
|
0.82
|
1.13b
|
0.70a
|
0.66a
|
0.79a
|
1.19c
|
0.84abc
|
0.70ab
|
0.55a
|
1.07bc
|
0.57a
|
0.63a
|
1.02bc
|
0.06
|
0.98
|
< 0.01
|
< 0.05
|
7
|
1.28
|
1.15
|
1.47b
|
1.19a
|
1.03a
|
1.14a
|
1.59b
|
1.36ab
|
1.08a
|
1.07a
|
1.36ab
|
1.03a
|
0.99a
|
1.21ab
|
0.06
|
0.17
|
< 0.05
|
0.31
|
21
|
1.87
|
1.64
|
1.99
|
1.78
|
1.60
|
1.65
|
2.10
|
1.83
|
1.65
|
1.92
|
1.88
|
1.73
|
1.56
|
1.38
|
0.08
|
0.15
|
0.36
|
0.72
|
42
|
2.24
|
1.93
|
2.43
|
2.13
|
1.75
|
2.03
|
2.43
|
2.41
|
1.98
|
2.14
|
2.44
|
1.85
|
1.53
|
1.92
|
0.10
|
0.14
|
0.16
|
0.41
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; LM: 1×109 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; HM: 1×1011cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; LD: 1×109 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage; HD: 1×1011 cfu/kg of L. plantarum + dough stage |
2L.p: L. plantarum |
Table 7
Effects of corn silage harvest period, dose of Lactobacillus plantarum and their interaction on mycotoxin concentrations
Ensiling
days
|
Harvest stage
|
Dose of L.p2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
Milk stage
|
Dough stage
|
0
|
1×109
|
1×1010
|
1×1011
|
ZM
|
LM
|
MM
|
HM
|
ZD
|
LD
|
MD
|
HD
|
Harvest stage
|
Dose of L.p2
|
Harvest stage×
dose of L.p2
|
DON (mg/kg)
|
3
|
0.42a
|
2.80b
|
1.40ab
|
1.64b
|
1.14a
|
2.26c
|
0.48a
|
0.37a
|
0.39a
|
0.45a
|
2.32c
|
2.92d
|
1.89b
|
2.07c
|
0.28
|
< 0.01
|
< 0.01
|
< 0.01
|
7
|
0.41a
|
4.01b
|
2.43
|
1.94
|
2.12
|
2.35
|
0.47a
|
0.37a
|
0.38a
|
0.43a
|
4.40b
|
3.50b
|
3.85b
|
2.28b
|
0.39
|
< 0.01
|
0.37
|
0.55
|
21
|
0.43a
|
2.45b
|
1.78
|
1.32
|
1.20
|
1.45
|
0.46a
|
0.46a
|
0.37a
|
0.44a
|
3.11b
|
2.18b
|
2.04ab
|
2.47b
|
0.27
|
< 0.01
|
0.72
|
0.77
|
42
|
0.38a
|
2.40b
|
1.70
|
1.44
|
1.11
|
1.32
|
0.39a
|
0.37a
|
0.37a
|
0.39a
|
3.00c
|
2.51bc
|
1.86b
|
2.25b
|
0.23
|
< 0.01
|
0.12
|
0.13
|
AFB1 (µg/kg)
|
3
|
1.59
|
1.37
|
1.71
|
1.57
|
1.33
|
1.31
|
1.68ab
|
1.38ab
|
1.63ab
|
1.67ab
|
1.74ab
|
1.77a
|
1.02bc
|
0.95d
|
0.09
|
0.17
|
0.23
|
0.06
|
7
|
1.87a
|
1.30b
|
1.65
|
1.55
|
1.60
|
1.54
|
1.87a
|
1.76ab
|
1.83a
|
2.04a
|
1.43ab
|
1.34ab
|
1.37ab
|
1.04b
|
0.10
|
< 0.01
|
0.96
|
0.56
|
21
|
1.79a
|
1.13b
|
1.59
|
1.42
|
1.35
|
1.49
|
1.83a
|
1.75a
|
1.66a
|
1.92a
|
1.34b
|
1.09b
|
1.03b
|
1.06b
|
0.08
|
< 0.01
|
0.18
|
0.48
|
42
|
1.71a
|
1.02b
|
1.54
|
1.34
|
1.22
|
1.36
|
1.87a
|
1.64a
|
1.60ab
|
1.72a
|
1.20bc
|
1.03c
|
0.84c
|
0.99c
|
0.09
|
< 0.01
|
0.18
|
0.94
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; LM: 1×109 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; HM: 1×1011cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; LD: 1×109 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage; HD: 1×1011 cfu/kg of L. plantarum + dough stage |
2L.p: L. plantarum |
3.4. Effects of Lactobacillus plantarum on bacterial community
A total of 3,996,296 valid sequences and 2,430,268 high-quality sequences (nonchimeric) were detected. A sequencing depth of 20,000 sequences (97%) was reached, indicating that the sequencing was sufficient to reflect the diversity in the samples (Fig. 1). Diversity and richness were represented by the Chao1 index and the Simpson index, respectively in Fig. 2. The Chao1 index among groups did not indicate a difference in either the harvest stage or the L. plantarum treatment. The Simpson index of the ZD group was significantly higher than that of the ZM group (P < 0.01), indicating that bacterial diversity at the dough stage was higher than that at the milk stage. Addition of L. plantarum to silage reduced the Simpson index indicating that the bacterial diversity was reduced, however the difference was not significant. These results showed that microbial community diversity was greater during the dough harvest, and that addition of 1×1010 cfu/kg L. plantarum reduced diversity in the microbial community.
Abundance of the top 5 bacteria at the phylum level and the top 18 bacteria at the genus level of corn silage for 42 days of fermentation are presented in Fig. 3. Firmicutes was the dominant phylum, its relative abundance accounting for more than 77% in each group (Table 8). The relative abundances of both Firmicutes and Proteobacteria were influenced by harvest stage and the dose of L. plantarum. Addition of L. plantarum significantly increased the abundance of Firmicutes, but decreased the abundance of Proteobacteria. Lactobacillus was the dominant genus in all silage, its relative abundance reaching more than 74% in each group. The relative abundances of Lactobacillus, Weissella, Enterobacter, Gluconobacter, Lactococcus, Sphingomonas, Pantoea, Asaia, Serratia, Stenotrophomonas, Cedecea and Methylobacterium were significantly affected by harvest stage in Table 9 (P < 0.01). Addition of 1×1010 cfu/kg L. plantarum to the silage significantly reduced the relative abundances of Weissella, Klebsiella, Leuconostoc, Enterobacter, Gluconobacter, Pantoea and Asaia compared with no addition of L. plantarum (P < 0.01). The relative abundances of Weissella, Klebsiella, Gluconobacter, Asaia and Pediococcus in the silage were significantly affected (P < 0.01) by the interaction between harvest stage and the dose of L. plantarum.
Table 8
Effects of corn silage harvest period, dose of Lactobacillus plantarum and their interaction on the relative abundance of bacteria on the phylum level
Phylum
|
Harvest stage
|
Dose of L.p2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
Milk stage
|
Dough stage
|
0
|
1×1010
|
ZM
|
MM
|
ZD
|
MD
|
Harvest stage
|
dose of L.p2
|
Harvest stage×
dose of L.p2
|
Firmicutes
|
79.38b
|
90.57a
|
82.50a
|
87.46b
|
77.19a
|
81.58a
|
87.80b
|
93.34b
|
7.84
|
< 0.01
|
< 0.05
|
0.78
|
Proteobacteria
|
18.79b
|
8.72a
|
16.26b
|
11.26a
|
21.29d
|
16.29c
|
11.22b
|
6.22a
|
6.73
|
< 0.01
|
< 0.01
|
1.00
|
Bacteroidetes
|
0.88
|
0.41
|
0.77
|
0.52
|
0.95
|
0.80
|
0.59
|
0.23
|
0.68
|
0.11
|
0.37
|
0.70
|
Actinobacteria
|
0.50
|
0.18
|
0.30
|
0.38
|
0.37
|
0.63
|
0.22
|
0.14
|
0.44
|
0.08
|
0.63
|
0.34
|
Patescibacteria
|
0.21
|
0.01
|
0.01
|
0.20
|
0.02
|
0.39
|
0.01
|
0.01
|
0.47
|
0.31
|
0.33
|
0.35
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage |
2L.p: L. plantarum |
Table 9
Effects of corn silage harvest period, dose of Lactobacillus plantarum and their interaction on the relative abundance of bacteria on the genus level
Genus
|
Harvest stage
|
Dose of p.l2 (cfu/kg)
|
Treatment1
|
SEM
|
P-value
|
Milk stage
|
Dough stage
|
0
|
1010
|
ZM
|
MM
|
ZD
|
MD
|
Harvest stage
|
Dose of p.l2
|
Harvest stage
×dose of p.l2
|
Lactobacillus
|
75.97a
|
86.67b
|
78.77
|
83.85
|
74.10a
|
77.84ab
|
83.45bc
|
89.87c
|
8.76
|
< 0.01
|
0.08
|
0.63
|
Weissella
|
1.28a
|
2.51b
|
2.24a
|
1.55b
|
1.21a
|
1.34a
|
3.26b
|
1.76a
|
1.00
|
< 0.01
|
< 0.01
|
< 0.01
|
Klebsiella
|
1.03
|
1.40
|
1.47a
|
0.95b
|
0.96a
|
1.09a
|
1.98b
|
0.82a
|
0.65
|
0.07
|
< 0.05
|
< 0.01
|
Leuconostoc
|
0..21
|
0.13
|
0.23
|
0.11
|
0.27b
|
0.16ab
|
0.20b
|
0.06a
|
0.12
|
0.06
|
< 0.01
|
0.78
|
Enterobacter
|
0.84a
|
0.52b
|
0.82a
|
0.53b
|
0.94b
|
0.73b
|
0.71b
|
0.33a
|
0.31
|
< 0.01
|
< 0.01
|
0.39
|
Gluconobacter
|
0.92a
|
0.25b
|
0.93a
|
0.24b
|
1.43c
|
0.40b
|
0.42b
|
0.09a
|
0.56
|
< 0.01
|
< 0.01
|
< 0.01
|
Lactococcus
|
0.04a
|
0.01b
|
0.03
|
0.03
|
0.04b
|
0.04b
|
0.02a
|
0.01a
|
0.02
|
< 0.01
|
0.35
|
0.78
|
Sphingomonas
|
0.44
|
0.11
|
0.25
|
0.31
|
0.41b
|
0.47b
|
0.08a
|
0.14a
|
0.20
|
< 0.01
|
0.20
|
1.00
|
Pantoea
|
0.79a
|
0.19b
|
0.65a
|
0.34b
|
1.02d
|
0.57c
|
0.27b
|
0.11a
|
0.39
|
< 0.01
|
< 0.01
|
0.07
|
Aquabacterium
|
0.67
|
0.47
|
0.65
|
0.48
|
0.65
|
0.68
|
0.65
|
0.29
|
0.34
|
0.15
|
0.22
|
0.14
|
Acetobacter
|
0.42
|
1.01
|
0.52
|
0.91
|
0.47
|
0.37
|
0.56
|
1.45
|
1.13
|
0.21
|
0.40
|
0.30
|
Asaia
|
1.13a
|
0.07b
|
0.99a
|
0.21b
|
1.87b
|
0.39a
|
0.11a
|
0.03a
|
0.94
|
< 0.01
|
< 0.01
|
< 0.01
|
Acinetobacter
|
0.27
|
0.21
|
0.26
|
0.21
|
0.29
|
0.25
|
0.23
|
0.18
|
0.09
|
0.09
|
0.18
|
0.87
|
Serratia
|
0.71
|
0.30
|
0.58
|
0.43
|
0.72c
|
0.70c
|
0.44b
|
0.16a
|
0.30
|
< 0.01
|
0.08
|
0.12
|
Stenotrophomonas
|
0.31
|
0.10
|
0.25
|
0.17
|
0.39b
|
0.23ab
|
0.11a
|
0.10a
|
0.19
|
< 0.01
|
0.21
|
0.25
|
Methylobacterium
|
0.11
|
0.06
|
0.09
|
0.08
|
0.10b
|
0.11b
|
0.08b
|
0.04a
|
0.04
|
< 0.01
|
0.29
|
0.07
|
Pediococcus
|
0.37
|
0.5
|
0.36
|
0.51
|
0.57
|
0.16
|
0.15
|
0.86
|
0.65
|
0.60
|
0.57
|
< 0.05
|
Cedecea
|
0.66
|
0.13
|
0.46
|
0.33
|
0.74b
|
0.57b
|
0.18a
|
0.09a
|
0.32
|
< 0.01
|
0.10
|
0.58
|
a,b,cValues with different superscripts in the same row within each trial factor in the same row (harvest stage, L. plantarum addition and each treatment group) indicate a significant difference (P < 0.05). |
1ZM: 0 cfu/kg of L. plantarum + milk stage; MM: 1×1010 cfu/kg of L. plantarum + milk stage; ZD: 0 cfu/kg of L. plantarum + dough stage; MD: 1×1010 cfu/kg of L. plantarum + dough stage; |
2L.p: L. plantarum |