Qian Wu

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.

The Gram-negative strain of Citrobacter freundii, YNLX, has the ability to degrade hyaluronic acid. In this study, we expressed a C. freundii hyaluronic acid lyase, from polysaccharide lyase family 8, in Escherichia coli. The purified recombinant enzyme (rHynACF8) showed a substantially higher cleavage activity of hyaluronic acid than chondroitin sulfate. We found that its optimal pH and temperature are 5.5 and 35 °C, respectively. In addition, the enzyme activity was not notably affected by most metal ions. K_m and k_cat of rHynACF8 towards HA were 1.5 ± 0.01 mg/mL and 30.9 ± 0.5 /s, respectively. rHynACF8 is an endo-acting enzyme. Its cleavage products had dramatically increased antioxidant activity than hyaluronic acid in vitro (p < 0.001). As the molecular weight of hyaluronic acid decreased, the intramolecular interactions among antioxidant functional groups were removed; in the process of the cracking reaction, new double bonds formed and conjugated with the carbonyl group. We presumed that the structural change is the critical factor influencing antioxidant capacity. Overall, we found that rHynACF8 from Gram-negative bacteria with metal ion resistance, indicated the relationship between the function and structure of its antioxidant cleavage product.

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.