Heat-Killed Lactobacillus acidophilus Promotes Growth by Modulating the Gut Microbiota Composition and Fecal Metabolites of Piglets

Abstract

Probiotics can improve animal growth performance and intestinal health. However, understanding the effects of paraprobiotics on the growth performance and gut microbiota of piglets and how the paraprobiotics exert their impact are still limited. The present study was conducted to investigate the effects of heat-killed Lactobacillus acidophilus IFFI 6005 supplementation on the growth performance, intestinal microbiota, and fecal metabolites of piglets. First, a feed-additive sample of heat-killed Lactobacillus acidophilus IFFI 6005 was prepared by culture. Second, 96 (initial BW = 14.38 ± 0.67 kg, weaning age of 40 days) healthy piglets were selected and randomized into four treatment groups. Each treatment group consisted of three replicates (n = 8). Pigs were fed a basal diet (NC), basal diet plus antibiotics (PC), basal diet plus Lactobacillus acidophilus IFFI 6005 at 600 g/t (LA, 1.0 × 10¹⁰ cfu/g), and basal diet plus heat-killed Lactobacillus acidophilus IFFI 6005 at 600 g/t (HKLA), respectively; the trial lasted for 30 days. The results showed that the ratios of feed to gain (F:G) and diarrhea rate of both the HKLA and PC groups were significantly lower compared with the NC and LA groups (p < 0.05); however, there was no significant difference between the HKLA and PC group (p > 0.05). In addition, the average daily weight gain (ADG) of the HKLA group was significantly higher (p < 0.05) than that of the other three groups in terms of growth performance. Finally, 16S rRNA sequencing and metabolome analysis based on fecal samples further elaborated that the addition of heat-killed Lactobacillus acidophilus IFFI 6005 to the feed improved the intestinal microbial diversity and abundance (p < 0.05) and reduced the abundance of pathogenic bacteria (p < 0.05), but it did not affect the abundance of Lactobacillus (p > 0.05). Through the comparison of microbial abundance and metabolite content between the two groups (NC_vs_HKLA), the largest differences were found in six microorganisms and 10 metabolites in the intestine (p < 0.05). These differential metabolites were involved in the digestion, absorption and utilization of protein and starch, as well as in oxidative stress. In summary, addition of heat-killed Lactobacillus acidophilus IFFI 6005 as a new feed additive in piglets has beneficial effects on the growth performance, intestinal bacteria and metabolites, and can be used as an alternative to antibiotics.

Keywords: antibiotics; gut microbiome; metabolite; paraprobiotics; pig nutrition.

Figure 1

Culture and preparation of heat-killed Lactobacillus acidophilus. (a) High-density fermentation results of Lactobacillus acidophilus IFFI 6005 in a 50-L fermenter. To determine the biomass and pH, culture samples were collected every 4 h from 0 to 48 h. Biomass and pH are represented by squares and triangles, respectively. (b) Antibacterial activity of the fermentation concentrate with heat-killed Lactobacillus acidophilus IFFI 6005. The test method for antibacterial activity was the agar well diffusion method. CK represents the diameter of the inhibition zone measured by adding an equal amount of blank medium, indicated by a red histogram. The results represent the mean ± SD.

Figure 2

Alpha diversity in intestinal microorganisms from each treatment group (n = 6 in each treatment). (a) Sob index; (b) PD-tree index; (c) Chao1 index; abbreviations: NC = control group; HKLA = test group. * and ** represent p < 0.05 and p < 0.01, respectively, using a t-test.

Figure 3

Differential analysis of the microbiota in weaned piglet fecal samples (n = 6 in each treatment). (a) PCoA of the gut bacterial communities between two groups; (b) Venn analysis at the OTU level; (c) analysis of species composition of differential operational taxonomic units at the genus level; (d) statistical analysis of differential species of abundance; (e) the prediction of the function for specific OTUs in the Greengene database. (f) Comparison of the contents of the top 5 Lactobacillus in the two groups. Abbreviations: NC = control group; HKLA = test group. PCoA = intergroup principal coordinate analysis; PCo1 = the principal coordinate components that explain the largest possible variation in the data; PCo2 = the principal coordinate components that explain the largest proportion of the remaining variability. The mean (SD) values of each group were determined using a Student’s t-test. Median (IQR) values were determined using a Mann–Whitney U test. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively, using t-tests.

Figure 4

Metabolites in weaned piglet fecal samples (n =6 in each treatment). (a) Score plot of the PLS-DA model; (b) histogram of differential metabolites with p < 0.05 and |log₂(FC)| > 2 between two groups; (c) enrichment of differential metabolites in metabolic pathways; (d) The key differential metabolite load map; (e) top 10 metabolite and bacterial boxplots showed differences between two groups; abbreviations: NC = control group; HKLA = test group. The mean (SD) values of each group were determined using a Student’s t-test. Median (IQR) values were determined using a Mann–Whitney U test.

Figure 5

Association analysis of differential microorganisms and metabolites (n = 6 in each treatment). (a) Correlation analysis of key fecal differential microorganisms with metabolites; (b) correlation analysis of the differential metabolite chenodeoxycholate with the top 10 differential microorganisms. The legend shows the magnitude of Spearman’s correlation coefficient; red indicates a positive correlation, and blue indicates a negative correlation. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively, using t-tests.