Isolation of LAB
Colonies surrounded by an obvious calcium-dissolving circle were picked from a plate of Lactic acid bacteria culture medium (MRS) solid medium (added the (w/v) 1% Caco3), representing strains with an ability to produce acid. Single colonies with different morphologies were streaked on solid MRS medium, and three rounds of this purification were performed. Select the single colonies that form milky white colonies on MRS medium, with neat edges, smooth surface and the surrounding characteristics of dissolved calcium carbonate. The PCR products were sequenced, and the sequences was subjected to a BLAST analysis at the GenBank database. In this way, 23 strains of lactic acid bacteria were selected (Table 1).
Analysis Of Tolerance
The survival rates of the lactic acid bacteria after treatment with bile salts at concentrations of 0.1%, 0.3%, or 0.5% are shown in Table 2. At a bile salt concentration of 0.5%, the survival rates for strains L1, L8, L51, and L52 were < 10% and those of the other strains were > 10%. Table 3 shows the survival rates of lactic acid bacteria after treatment with artificial gastric juice for 4 h. The survival rates of E1, L1, L3, L4, L5, L531, E5, and L621 were > 60% after they were treated with artificial gastric juice for 4 h, showing strong tolerance for artificial gastric juice; Strains such as E2, L7, L8, E5, L411, L522, L623, L624 and L623 also showed a certain degree of tolerance for artificial gastric juice, with survival rates of > 40%.
The various strains differed greatly in heat tolerance (as shown in Fig. 1). Among the seven resistant strains, only strains E5 and L522 had survival rates of < 30% after treatment at 42°C for 10 min. The survival rates of the remaining five strains after treatment at 42°C were > 30%. With heat treatment at 60°C for 10 min, the survival rate of L3 was almost 0%, and the survival rates of E5 and L522 were < 30%. In a comprehensive comparison, the three strains with the best heat tolerance were two strains of L. salivarius (L621 and L4) and one strain of Enterococcus faecalis (E1).
Analysis Of Surface Hydrophobicity, Auto-aggregation, And Co-aggregation With Pathogens
The surface hydrophobicity results for the seven selected strains of lactic acid bacteria are shown in Fig. 2-A. The surface hydrophobicity of the different strains varied greatly (P < 0.05). L4 had the highest surface hydrophobicity at 46.65%, followed by L621 (40.46%). The remaining five strains, E5, L522, L531, L3, and E1, had surface hydrophobicities of 25.5%, 28.67%, 27.93%, 32.34%, and 27.33%, respectively.
The auto-aggregation ability of the seven lactic acid bacterial strains are shown in Fig. 2-B. These differed greatly among the strains, in the range of 21.12% – 60.18% (P < 0.05). L621 had the highest auto-aggregation ability at 60.18%, followed by L4, at 58.48%. The auto-aggregation ability of the four strains L531, E5, L3, and E1 were 53.32%, 35.34%, 46.79%, and 35.67%, respectively. The surface auto-aggregation ability of L522 was worst, at 21.12%.
The abilities of the seven lactic acid bacteria to co-aggregate with pathogenic bacteria are shown in Fig. 2-C. There was little difference in the ability of different strains to co-aggregate with pathogenic bacteria, which ranged between 54.61% and 59.77% (P > 0.05). L621 had the strongest ability to co-aggregate with pathogenic bacteria, at 59.77%, followed by L4, at 58.29%. The surface hydrophobicity of strain E5 was 54.61%, that of strain L522 was 55.84%, that of strain L531 was 56.25%, that of strain L3 was 55.30% and that of strain E1 was 54.93%.
Antibacterial Activity Of Lactic Acid Bacteria Against Pathogenic Bacteria
The antibacterial activity of the seven strains of lactic acid bacteria against two strains of pathogens differed significantly (P < 0.05), and the antibacterial effects against E. coli AE17 was better than that against S. aureus ATCC 25923. The antibacterial activities of L3 and L4 against E. coli AE17 was significantly greater than those of the other five strains, followed those of by L531 and L621. Among the seven strains, L4 has significant antibacterial activity against S. aureus ATCC 25923 (P < 0.05); there was no significant differences in the antibacterial activities of L3, E5, L531, L621, and E1; and L522 had the weakest antibacterial activity against S. aureus ATCC 25923 (Fig. 3).
Preparation Of Lactic Acid Bacteria Compound
Based on the data for heat resistance, hydrophobicity, surface auto-aggregation ability, coacervation ability with pathogenic bacteria and antagonistic antibacterial activity in vitro, two strains with the strongest activities, L. salivarius L4 and L. salivarius L621, were screened and compounded in proportion with the strain of L. casei 1.2435 (L. casei 1.2435 is a gift from Professor Zhang Ming, School of life sciences, Anhui Agricultural University, and the research group has previously proved that L.s casei 1.2435 has a preventive effect on pathogenic bacteria of duck intestinal infection.) (Shi et al. 2020) existing in the early stage of the laboratory to prepare the compound preparation of lactic acid bacteria.
The optimal combination was determined by measuring the diameter of the antibacterial circles produced. The antibacterial activity against E. coli was strongest when the ratio of 1.2435: L621: L4 was 1:1:2, its antibacterial activity against E. coli was the strongest, and the diameter of antibacterial agent was 18.28mm, which was significantly different from other combinations (P < 0.05). Therefore, the ratio of components in the composite preparation was 1:1:2, and the minimum inhibitory concentration (It refers to the lowest concentration that can inhibit the growth of pathogenic bacteria in the culture medium after 18 hours of culture in vitro) was 1.5 × 104 CFU/mL (as shown in Fig. 4).
At the optimal ratio of L. casei 1.2435: L. salivarius L621: L. salivarius L4 of 1:1:2, the antibacterial activities were determined when different amounts of inoculum were used. The differences in the antibacterial activities achieved with different amounts of inoculum was not significant (P > 0.05). However, 1% inoculum had the strongest antibacterial activity and was the best inoculum, as shown in Table 4.
With the optimal compound ratio for L. casei 1.2435: L. salivarius L621: L. salivarius L4 (1:1:2) and the optimal amount of inoculum (1%), the antibacterial activities after different cell free fermentation supernatant of lactic acid bacteria times were determined, as shown in Table 5. The antibacterial activity was the strongest at 14 h, and the differences between the periods of fermentation were significant (P < 0.05). Thus, the optimal fermentation time was 14 h.
Effects Of Lactic Acid Bacterial Compound On Growth Performance Of Cherry Valley Ducks Brood
period
During the brooding period, the average daily weight gain in the experimental group (EG) increased by 6.93% compared with that in the control group (CG), the average daily feed intake increased by 6.42%, and the feed-to-weight ratio decreased by 0.89%. These data show that the lactic acid bacterial compound tended to improve the growth performance of Cherry Valley ducks, as shown in Table 6.
Effect of lactic acid bacterial compound on the structure of the cecal intestinal flora of Cherry Valley ducks
A Venn diagram was constructed at the level of bacterial operational taxonomic units (OTUs), and the results are shown in Fig. 5. The number of annotated OTUs only detected in CG was 330, the number detected in EG was 350, the number detected in both CG and EG was 315, and the number of unique OTUs in CG group is 15, and the number of unique OTUs in EG group is 35.
Species annotation at the phylum level showed that the compositions of the intestinal flora of the Cherry Valley ducks in CG and EG were basically the same, with Firmicutes and Bacteroidetes the dominant phyla. However, there were differences in their relative abundances. In EG, the relative abundance of Firmicutes was 5.58% higher than in CG, as shown in Fig. 6.
The species annotation at the genus level is shown in Fig. 7. The relative abundances of Parabacteroides (relative abundance in CG, 17.0%; relative abundance in CG, 11.5%), [Ruminococcus]_torques_group (CG, 8.3%; CG, 4.5%), and Enterococcus (CG, 6.3%; CG, 1.6%) were significantly higher in CG than in EG (P < 0.05). The relative abundances of Lactobacillus (CG, 5.9%; CG, 14.4%) and Blautia (CG, 3.3%; CG, 8.9%) were significantly higher in EG than in CG (P < 0.05).
The 15 most abundant genera in GG and EG samples were compared and analyzed by Fisher's exact test is shown in Fig. 8. The abundance of Lactobacillus (P < 0.001)、unclassified_f__Lachnospiraceae (P < 0.001) 、Parabacteroides (P < 0.001)、 [Ruminococcus]_torques_group (P < 0.001) 、Faecalibacterium (P < 0.001) 、Ruminococcaceae_UCG-014 (P < 0.001) and unclassified_f__Ruminococcaceae (P < 0.001) in EG group was significantly higher than that in CG group. The abundance of Bacteroides (P < 0.001)、Enterococcus (P < 0.001)、Blautia (P < 0.001)、norank_f__Ruminococcaceae (P < 0.001)、Butyricicoccus (P < 0.001)、Anaerotruncus (P < 0.001)、Subdoligranulum (P < 0.001) and Erysipelatoclostridium (P < 0.001) in CG group was significantly higher than that in EG group.