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. 2023 Nov 9;12(22):4068.
doi: 10.3390/foods12224068.

Optimum Fermentation Conditions for Bovine Lactoferricin-Lactoferrampin-Encoding LimosiLactobacillus reuteri and Regulation of Intestinal Inflammation

Weichun Xie  1 , Xueying Wang  1 , Jiyao Cai  1 , Huitao Bai  1 , Yilan Shao  1 , Zhuoran Li  1 , Limeng Cai  1 , Senhao Zhang  1 , Jiaxuan Li  1 , Wen Cui  1 , Yanping Jiang  1 , Lijie Tang  1   2
Affiliations

Optimum Fermentation Conditions for Bovine Lactoferricin-Lactoferrampin-Encoding LimosiLactobacillus reuteri and Regulation of Intestinal Inflammation

Weichun Xie et al. Foods. .

Abstract

The multifunctional antibacterial peptide lactoferricin-lactoferrampin (LFCA) is derived from bovine lactoferrin. Optimization of the fermentation process should be studied since different microorganisms have their own favorable conditions and processes for growth and the production of metabolites. In this study, the culture conditions of a recombinant strain, pPG-LFCA-E/LR-CO21 (LR-LFCA), expressing LFCA was optimized, utilizing the high-density fermentation process to augment the biomass of LimosiLactobacillus reuteri and the expression of LFCA. Furthermore, an assessment of the protective effect of LR-LFCA on intestinal inflammation induced by lipopolysaccharide (LPS) was conducted to evaluate the impact of LR-LFCA on the disease resistance of piglets. The findings of this study indicate that LR-LFCA fermentation conditions optimally include 2% inoculation volume, 36.5 °C fermentation temperature, 9% dissolved oxygen concentration, 200 revolutions/minute stirring speed, pH 6, 10 mL/h glucose flow, and 50% glucose concentration. The inclusion of fermented LR-LFCA in the diet resulted in an elevation of immunoglobulin levels, significant upregulation of tight junction proteins ZO-1 and occludin, reinforcement of the intestinal barrier function, and significant amelioration of the aberrant alterations in blood physiological parameters induced by LPS. These results offer a theoretical framework for the implementation of this micro-ecological preparation in the field of piglet production to enhance intestinal well-being.

Keywords: LimosiLactobacillus reuteri; high-density fermentation; intestinal inflammation; lactoferricin-lactoferrampin (LFCA); protein expression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The biological characteristics of LR-LFCA and single-factor optimization of fermentation conditions. (A) The growth curve of LR-LFCA. (B) Identification of expressed LFCA by Western blot. M. Marker; 1. LR-LFCA cell precipitation; 2. LR-LFCA cell supernatant; 3. LR-CON cell precipitation; 4. LR-CON cell supernatant. (C) Effects of different inoculation volumes on LR-LFCA density and LFCA expression. (D) Effects of different culture temperatures on LR-LFCA density and LFCA expression. (E) Effects of different stirring speeds on LR-LFCA density and LFCA expression. (F) Effects of different dissolved oxygen on LR-LFCA density and LFCA expression. (G) Effects of different pH on LR-LFCA density and LFCA expression.
Figure 2
Figure 2
Response surface and contour map of pH, dissolved oxygen, and temperature affecting bacterial biomass. (A) PH and temperature. (B) PH and oxygen content (%). (C) Temperature and oxygen content (%).
Figure 3
Figure 3
Response surface and contour map of pH, dissolved oxygen, and temperature affecting bacterial protein concentration. (A) PH and temperature. (B) PH and oxygen content (%). (C) Temperature and oxygen content (%).
Figure 4
Figure 4
MIC50 and inhibition zone diameter of LR-LFCA fermentation supernatant. (A) Bacteriostatic curve of chloramphenicol against tested bacteria. (B) Bacteriostatic curve of bovine lactoferrin peptide standard against test bacteria. (C) Bacteriostatic curve of LFCA against test bacteria. (D) The diameter of inhibition zone of LR-LFCA before and after optimization.
Figure 5
Figure 5
Effects of optimized LR-LFCA on serum antibody, intestinal mucosal antibody, and intestinal inflammatory factor mRNA expression of LPS-treated piglets. (A) Changes of IgM and IgG in serum of LPS-treated piglets. (B) Changes of sIgA content in intestinal mucus of LPS-treated piglets. (C) Changes of inflammatory factor mRNA expression in jejunum of LPS-treated piglets. (D) mRNA expression of inflammatory cytokines in ileum of LPS-treated piglets. ** p < 0.01; * p < 0.05; ns, not significantly different.
Figure 6
Figure 6
Effects of optimized LR-LFCA on intestinal barrier function and intestinal tissue morphology of LPS-treated piglets. (A) Changes of endotoxin in serum of piglets. (B) Relative mRNA expression of ZO-1 and occludin in jejunum and ileum of piglets were detected by real-time PCR. (C) The proteins expression levels of ZO-1 and occludin in jejunum and ileum of piglets were detected by ELISA kit. (D) Pathological changes and pathological sections of intestine (100×). * p < 0.05; ** p < 0.01 vs. CON; # p < 0.05; ## p < 0.01 vs. LPS + LR-LFCA.
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