Effects of BLFs on the growth performance of broilers
Table 2 showed that BLF supplementation significantly improved (p < 0.01) the average daily gain (ADG) and average daily feed intake (ADFI) of broilers than CON treatment from d 1–42. Although no significant difference was observed in the BLF and antibiotic administration, these treatment significantly decreased (p < 0.01) the feed:gain ratio and mortality rate of birds compared with the CON treatment.
Table 2
Effects of BLFs supplementation on growth performance of broilers Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH).
Item | Stage | CON | ANT | BLFL | BLFH | SEM | P-val |
ADG, g/d | d 1–21 | 41.0 ± 1.39 | 38.4 ± 2.97 | 39.8 ± 2.42 | 38.3 ± 1.97 | 2.39 | 0.159 |
d 21–42 | 91.7 ± 7.60b | 100.5 ± 6.36a | 103.3 ± 8.23a | 103.2 ± 6.79a | 8.35 | 0.037 |
d 1–42 | 80.4 ± 4.28b | 97.6 ± 4.74a | 98.5 ± 2.35a | 97.7 ± 5.29a | 8.75 | < 0.001 |
ADFI, g/d | d 1–42 | 135.1 ± 4.16b | 142.2 ± 0.92a | 147.2 ± 5.12a | 147.4 ± 7.95a | 7.03 | 0.005 |
F:G | d 1–42 | 1.64 ± 0.04a | 1.54 ± 0.04b | 1.56 ± 0.04b | 1.55 ± 0.04b | 0.05 | 0.001 |
Mortality rate, % | d 1–42 | 3.01 ± 0.63a | 1.50 ± 0.22b | 0.83 ± 0.09b | 1.00 ± 0.09b | 0.29 | 0.003 |
Note: All measurements were expressed as Mean ± SD (n=6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH).
Effects of BLFs on carcass yield of broilers
As shown in Table 3, dietary BLFH supplementation significantly increased (p < 0.01) the leg meat percentage of broilers compared to CON and ANT supplementation, even though there was no significant difference in carcass yield between CON and BLF pretreatment. In addition, the breast percentage of BLFH birds was higher (p = 0.088) than that of CON birds.
Table 3
Effects of BLFs supplementation on carcass yield of broilers Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH).
Item | CON | ANT | BLFL | BLFH | SEM | P-val |
Carcass yield, % | 87.0 ± 1.56 | 89.1 ± 1.35 | 88.8 ± 1.82 | 88.6 ± 1.70 | 1.74 | 0.072 |
Breast meat percentage, % | 16.6 ± 0.53 | 17.1 ± 0.89 | 17.3 ± 0.45 | 17.4 ± 0.69 | 0.70 | 0.088 |
Leg meat percentage, % | 13.1 ± 0.53b | 13.2 ± 0.54b | 13.5 ± 0.64ab | 14.2 ± 0.62a | 0.72 | 0.003 |
Note: All measurements were expressed as Mean ± SD (n=6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH).
Effects of BLFs on meat texture profile of broilers
Brittleness
Neither BLF nor ANT treatment influenced the brittleness of the leg and breast meat compared to that of the CON group (p > 0.05), although BLFL and BLFH increased the brittleness compared to that of the CON and ANT groups (Tables 4 and 5).
Table 4
Effects of BLFs supplementation on texture profile of broilers’ leg meat
Item | CON | ANT | BLFL | BLFH | SEM | P-val |
Brittleness | 179.7 ± 46.00 | 183.5 ± 42.87 | 213.1 ± 34.10 | 205.7 ± 34.70 | 40.44 | 0.275 |
Elasticity | 0.30 ± 0.03 | 0.33 ± 0.04 | 0.32 ± 0.04 | 0.33 ± 0.04 | 0.04 | 0.332 |
Resilience | 0.14 ± 0.02b | 0.19 ± 0.04a | 0.19 ± 0.03a | 0.20 ± 0.03a | 0.04 | 0.007 |
Gumminess | 52.13 ± 9.10c | 75.2 ± 12.56b | 86.8 ± 12.58ab | 93.7 ± 12.59a | 19.61 | < 0.001 |
Chewiness | 43.68 ± 7.36b | 49.2 ± 6.68ab | 47.0 ± 7.22ab | 52.1 ± 9.41a | 7.90 | 0.210 |
Cohesiveness | 0.26 ± 0.03 | 0.27 ± 0.05 | 0.27 ± 0.04 | 0.26 ± 0.03 | 0.04 | 0.744 |
Hardness | 204.9 ± 32.96a | 210.6 ± 29.47a | 159.9 ± 53.64b | 174.5 ± 32.89ab | 42.30 | 0.039 |
Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Table 5
Effects of BLFs supplementation on texture profile of broilers’ breast meat
Item | CON | ANT | BLFL | BLFH | SEM | P-val |
Brittleness | 207.3 ± 42.86 | 226.8 ± 48.46 | 201.1 ± 45.08 | 214.9 ± 30.65 | 30.65 | 0.654 |
Elasticity | 0.26 ± 0.02b | 0.29 ± 0.04b | 0.30 ± 0.03a | 0.30 ± 0.03a | 0.03 | 0.031 |
Resilience | 0.14 ± 0.53b | 0.15 ± 0.54ab | 0.17 ± 0.64ab | 0.19 ± 0.62a | 0.03 | 0.014 |
Gumminess | 47.13 ± 7.82b | 60.9 ± 14.55ab | 81.1 ± 8.81a | 80.2 ± 13.86a | 18.18 | < 0.001 |
Chewiness | 34.85 ± 4.88b | 47.48 ± 9.33a | 42.4 ± 8.70ab | 48.3 ± 12.18a | 10.25 | 0.025 |
Cohesiveness | 0.26 ± 0.03 | 0.27 ± 0.05 | 0.27 ± 0.04 | 0.26 ± 0.03 | 0.03 | 0.744 |
Hardness | 243.3 ± 53.0a | 182.7 ± 25.4b | 156.2 ± 37.1b | 177.1 ± 39.5b | 50.33 | 0.001 |
Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Chewiness
Compared to that of the CON group, dietary BLF supplementation increased the chewiness of broiler breast and leg meat, and a significant difference was observed only in the BLFH treatment group (p < 0.05; Tables 4 and 5). Moreover, ANT treatment significantly increased the chewiness of broiler breast meat compared to the CON treatment (p < 0.05).
Cohesiveness
Feeding low amount of BLFs significantly decreased the cohesiveness of broiler leg meat compared to the CON treatment (p < 0.05; Table 4). Meanwhile, both BLFL and BLFH broilers had significantly lower cohesiveness of leg meat than ANT birds (p < 0.01, p < 0.05). Feeding with antibiotics or BLFs did not affect the cohesiveness of breast meat in comparison with that of the CON group (Table 5).
Elasticity
Both BLFL and BLFH treatments improved broiler breast and leg meat elasticity in comparison with the CON treatment (Tables 4 and 5), while significant differences were only observed in breast meat (p < 0.05).
Gumminess
BLF supplementation remarkably enhanced (p < 0.001) the gumminess of bird breast and leg meat compared to the CON treatment (Tables 4 and 5). Moreover, BLFH treatment significantly increased the gumminess of leg meat compared with that of the ANT birds (p < 0.01). In addition, either BLFL or BLFH birds had a higher gumminess of breast meat in comparison with that of the ANT birds (p < 0.01, p < 0.05).
Hardness
BLFL feeding significantly decreased the hardness of the leg muscles of birds compared to the CON and ANT treatments (p < 0.05; Table 4). BLFH also decreased (p = 0.071) the hardness of leg muscles compared to the ANT supplementation. Both ANT and BLF supplementation significantly decreased the hardness of the breast meat of birds in comparison with the CON treatment (p < 0.05; Table 5).
Resilience
Feeding ANT, BLFL, and BLFH significantly increased the resilience of leg meat compared to the CON supplementation (p < 0.05; Table 4). Moreover, BLFL and BLFH supplementation enhanced the resilience of breast meat compared to the CON treatment (p = 0.085 and p < 0.05, respectively; Table 5).
Effects of BLFs on Aw and pH of broilers’ meat
There was no significant influence on the Aw of breast meat when fed with ANT or BLFs compared with CON (Table 6). Although BLF supplementation increased the Aw of bird leg meat compared to the CON treatment, no significant change was observed (p > 0.05; Table 6). Meanwhile, BLFL, BLFH, and CON pretreatment dramatically increased the Aw of leg meat compared with the ANT treatment (p < 0.01). Neither BLF nor ANT supplementation affected the pH of breast and leg meat compared to the CON treatment (p > 0.05; Table 6).
Table 6
Effects of BLFs on Aw and pH of broilers’ meat
Item | CON | ANT | BLFL | BLFH | SEM | P-val |
Breast meat PH | 6.10 ± 0.13 | 6.14 ± 0.12 | 6.07 ± 0.15 | 6.14 ± 0.96 | 0.12 | 0.687 |
Breast meat Aw | 0.97 ± 0.01 | 0.98 ± 0.01 | 0.98 ± 0.01 | 0.98 ± 0.01 | 0.01 | 0.827 |
Leg meat pH | 6.33 ± 0.12 | 6.24 ± 0.06 | 6.26 ± 0.12 | 6.28 ± 0.08 | 0.10 | 0.296 |
Leg meat pH | 0.98 ± 0.01a | 0.97 ± 0.01b | 0.98 ± 0.01a | 0.98 ± 0.01a | 0.01 | 0.001 |
Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Effects of BLFs on the sensory color of broilers’ meat
There was no significant difference in L*, a*, and b* of sensory color of broilers’ leg meat among the treatment groups (Table 7). Supplementation with BLFs dramatically increased (p < 0.05) the c* value of leg meat color compared to CON and ANT supplementations. Although BLF feeding did not affect (p > 0.05) the L* value of the birds’ breast meat compared with CON treatment (Table 8), it significantly increased a* and c* values. No significant differences were found in the values of leg and breast meat between the two BLF treatment groups.
Table 7
Effects of BLFs supplementation on broilers’ leg meat colour
Item | L* | a* | b* | c* |
CON | 51.02 ± 1.23 | 5.28 ± 0.75 | 10.76 ± 1.26 | 13.05 ± 0.86b |
ANT | 51.51 ± 1.22 | 5.11 ± 0.09 | 9.96 ± 0.43 | 14.49 ± 0.59b |
BLFL | 52.58 ± 1.48 | 5.31 ± 0.13 | 10.61 ± 0.71 | 16.00 ± 0.71a |
BLFH | 55.41 ± 1.64 | 5.83 ± 0.12 | 11.31 ± 0.37 | 15.88 ± 0.52a |
SEM | 0.595 | 0.309 | 0.383 | 0.388 |
P-val | 0.484 | 0.367 | 0.479 | 0.015 |
Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Table 8
Effects of BLFs supplementation on broilers’ breast meat colour
Item | L* | a* | b* | c* |
CON | 51.73 ± 1.21 | 5.25 ± 0.23b | 8.65 ± 0.62b | 10.03 ± 0.45b |
ANT | 53.95 ± 1.87 | 5.23 ± 0.35b | 9.35 ± 0.65b | 11.91 ± 0.73ab |
BLFL | 53.08 ± 1.64 | 6.60 ± 0.31a | 10.06 ± 0.58ab | 12.03 ± 0.61a |
BLFH | 53.60 ± 2.58 | 6.90 ± 0.26a | 11.98 ± 0.63a | 12.71 ± 0.85a |
SEM | 0.910 | 0.172 | 0.369 | 0.398 |
P-val | 0.850 | 0.007 | 0.005 | 0.017 |
Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Effects of BLFs on the microstructure of broilers’ breast meat
The BLF groups had less intramuscular fat than the CON and ANT groups, and the BLFH group had the least intramuscular fat (Fig. 1a–d). Compared with that in the CON and ANT groups, intact myofibrils with distinct A-bands, I-bands, and Z-lines were more clearly visible in the pectoral muscle of the BLFL and BLFH groups (Fig. 1e–1h). In addition, the breast muscle fibers in the BLF-treated birds were arranged in a more orderly manner, and there was less connective tissue present.
Effects of BLFs on protein secondary structure of broilers’ breast meat
Data showed that BLF supplementation significantly decreased the β-sheet ratio and increased the β-turn ratio compared to the CON treatment (p > 0.05; Table 9). No significant difference was found in the ratio of α-helices and random coils among all treatment groups.
Table 9
Effects of BLFs supplementation on the secondary structures of broilers’ breast meat protein Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH).
Item | β-sheet | α-helix | β-turn | random coil |
CON | 44.31 ± 1.245a | 2.21 ± 0.836 | 52.41 ± 0.981b | 1.07 ± 0.523 |
ANT | 44.01 ± 0.831a | 2.73 ± 0.586 | 51.87 ± 0.819c | 1.39 ± 0.597 |
BLFL | 41.59 ± 1.658b | 1.95 ± 0.963 | 55.35 ± 1.810a | 1.11 ± 0.506 |
BLFH | 41.39 ± 1.036b | 2.78 ± 0.276 | 54.76 ± 0.961a | 1.08 ± 0.536 |
SEM | 1.797 | 0.768 | 1.897 | 0.532 |
P-val | 0.001 | 0.073 | 0.001 | 0.601 |
Note: All measurements were expressed as Mean ± SD (n=6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH).
Effects of BLFs on the antioxidative capacity of broilers’ serum and breast meat
No significant difference was noted in SOD and GSH-Px contents in broilers’ serum among the treatment groups (Table 10). The supplementation of BLFs significantly increased (p < 0.05) the content of serum total antioxidant capacity and catalase, whereas BLFH significantly decreased (p < 0.05) serum MDA content. Interestingly, high SOD and GSH-Px contents in breast meat were found in BLF-supplemented broilers.
Table 10
Effects of BLFs on antioxidative capacity of broilers’ serum and breast meat
Item | CON | ANT | BLFL | BLFH | SEM | P-val |
Serum | T-AOC | 12.7 ± 1.30b | 13.2 ± 0.90ab | 14.8 ± 1.38a | 14.7 ± 0.79a | 1.41 | 0.110 |
MDA | 10.3 ± 0.45a | 9.8 ± 0.95a | 9.1 ± 1.26ab | 7.8 ± 0.90b | 1.30 | 0.178 |
SOD | 87.3 ± 15.17 | 94.7 ± 12.23 | 96.2 ± 11.60 | 98.3 ± 10.93 | 12.47 | 0.448 |
GSH-Px | 9.9 ± 0.56 | 10.1 ± 0.52 | 10.5 ± 0.82 | 10.5 ± 0.91 | 0.74 | 0.402 |
CAT | 6.2 ± 0.58b | 7.1 ± 1.07a | 7.2 ± 0.89a | 7.2 ± 0.72a | 0.89 | 0.196 |
Breast meat | MDA | 10.6 ± 2.62a | 8.6 ± 0.88a | 7.7 ± 0.96ab | 8.0 ± 1.04b | 1.87 | 0.131 |
SOD | 47.2 ± 5.67b | 52.8 ± 6.43ab | 63.0 ± 8.00a | 62.7 ± 6.31a | 9.25 | 0.068 |
GSH-Px | 0.7 ± 0.10b | 0.9 ± 0.13ab | 1.0 ± 0.15a | 1.0 ± 0.20a | 0.20 | 0.332 |
Note: All measurements were expressed as Mean ± SD (n = 6). Different letters in the same rows are different significantly (p < 0.05). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Effects of BLFs on the untargeted metabolomes of broilers’ breast meat
The Venn diagram shows the 808 metabolites identified in all experimental groups, whereas 88 and 29 unique metabolites were found in the BLFH and BLFL groups, respectively (Fig. 2a). The distribution of the principal component analysis scores in the BLFH and BLFL groups was significantly different from that in the CON group. The samples from the BLFH and BLFL groups were mainly concentrated in the fourth quadrant; those from the CON group were mainly concentrated in the second quadrant; and those from the ANT group were mainly concentrated in the first quadrant. In addition, the BLFL group showed proper aggregation, whereas the CON and ANT groups showed significant outliers (Fig. 2b). Meanwhile, orthogonal partial least squares discriminant analysis showed significant differences between BLF treatments (BLFL and BLFH groups) and control treatments (CON and ANT groups).
The contents of pumiloside, citranaxanthin (flavonoids), amino acids (N-gamma-glutamyl-s-propylcysteine, 2-amino-isobutyric acid, and isoleucyl-valine), and organic acids (cinnavalininate and 3-hydroxy−3-methyl-glutaric acid) in the BLF groups were significantly higher than those in the CON group (Table 11). In addition, the levels of the phenolic compound Kanzonol W, aromatic compound 3-p-coumaroyl−1,5-quinolactone, and biliverdin IX were also significantly upregulated by BLF treatment.
Table 11
Comparison of the transportation of fourteen metabolites across the broilers’ breast meat
No | Compounds | Formula | Related Category | RT (min) | Mass (m/z) | BLFL VS Control | | BLFH VS Control | | Anti VS Control |
trend | p value | | trend | p value | | trend | p value |
1 | Pumiloside | C26 H28 N2 O9 | Alkaloids | 20.407 | 572.2 | up | 0.000 | | up | 0.000 | | up | 0.007 |
2 | Psychosine sulfate | C24 H47 N O10 S | Glycosides | 13.734 | 601.3159 | up | 0.000 | | up | 0.000 | | up | 0.006 |
3 | N-gamma-Glutamyl-S-propylcysteine | C11 H20 N2 O5 S | Amino acid | 13.032 | 352.1322 | up | 0.001 | | up | 0.000 | | up | 0.047 |
4 | Isoleucyl-Valine | C11 H22 N2 O3 | Amino acid | 4.488 | 230.1627 | up | 0.000 | | up | 0.000 | | up | 0.006 |
5 | 2-amino-isobutyric acid | C4 H9 N O2 | Amino acid | 0.758 | 103.0632 | up | 0.000 | | up | 0.000 | | up | 0.006 |
6 | 1-Methylhistidine | C7 H11 N3 O2 | Amino acid | 0.749 | 169.0848 | up | 0.000 | | / | 0.000 | | up | 0.007 |
7 | Kanzonol W | C20 H16 O5 | Phenolic compounds | 10.935 | 336.1018 | up | 0.000 | | up | 0.002 | | up | 0.016 |
8 | Citranaxanthin | C33 H44 O | Flavonoid | 20.591 | 516.3593 | up | 0.000 | | up | 0.000 | | up | 0.023 |
9 | Cinnavalininate | C14 H8 N2 O6 | Organic acid | 0.739 | 300.0403 | up | 0.000 | | up | 0.000 | | up | 0.006 |
10 | 3-Hydroxy-3-methyl-glutaric acid | C6 H10 O5 | Organic acid | 0.744 | 162.0526 | up | 0.010 | | up | 0.017 | | up | 0.042 |
11 | (+)-Chebulic acid | C14 H12 O11 | Organic acid | 5.291 | 356.039 | up | 0.000 | | / | 0.000 | | up | 0.006 |
12 | 3-p-Coumaroyl-1,5- quinolactone | C16 H16 O7 | Others (Aromatic compounds) | 7.871 | 412.1006 | up | 0.020 | | up | 0.020 | | up | 0.037 |
13 | Biliverdin IX | C33 H34 N4 O6 | others | 11.638 | 582.2478 | up | 0.028 | | up | 0.000 | | up | / |
14 | 4-Acetamido-2-aminobutanoic acid | C6 H12 N2 O3 | others | 0.775 | 160.0842 | up | 0.000 | | up | 0.000 | | up | 0.006 |
Note: up represents the distinguished metabolites was up-regulated compared to the control group. / represents no statistical difference compared to the control group (n = 6). Birds supplemented with basal diet (Con), supplemented with 20 mg bacitracin/kg (ANT), supplemented with 50 mg BLFs/kg (BLFL), supplemented with 250 mg BLFs/kg (BLFH). |
Correlations between texture profile, protein secondary structure, and changed metabolites of broilers’ breast meat
Correlation heatmap analysis was performed to investigate the relationship between texture profile, protein secondary structure, and changed metabolites of broilers’ breast meat (Fig. 3). Metabolites that had changed significantly, except biliverdin IX, 3-hydroxy−3-methyl-glutaric acid, and 3-p-coumaroyl−1,5-quinolactone, were positively correlated with resilience, gumminess, cohesiveness, and α-helix, showing a correlation index value of approximately 0.3 to 0.7. However, a remarkable negative correlation was observed between hardness and significant altered metabolites except biliverdin IX, for which the value of the correlation index was lower than−0.5. In addition, the random coil of protein secondary structure was also negatively correlated with the significantly changed metabolites except for 3-hydroxy−3-methyl-glutaric acid, and 3-p-coumaroyl−1,5-quinolactone, showing a correlation index value of−0.2 to−0.6.