Abstract
Lactoferrin (LF) is a multifunctional protein found in mammals, and it shows broad-spectrum antimicrobial activity. To improve the functional properties of specific probiotics in order to provide both the beneficial characteristics of lactic acid bacteria and the biological activity of LF, cDNAs of bovine LF (BLF), human LF (HLF), or porcine LF (PLF) were cloned into a nisin-inducible plasmid. These were then transformed into the selected eight probiotics, which are LF-resistant hosts. Expression of recombinant LFs (rLFs) was analyzed via SDS-PAGE and Western blot analysis. Although the selected host strains may not contain the nisRK genes (NisK, the sensor kinase; NisR, the regulator protein), the components of autoregulation, a low level of LFs expression can be successfully induced by using nisin within bacterial cells in a time-dependent manner in three engineered clones, including Lactobacillus delbrueckii/HLF, L. delbrueckii/BLF, and L. gasseri/BLF. Lactobacillus delbrueckii and Lactobacillus gasseri originate from yogurt and human milk, respectively, and both strains are functional probiotic strains. Therefore, we further compared the antibacterial activities of disrupted recombinant probiotic clones, conventional strains (host control), and vector control ones by using agar diffusion and broth inhibition analysis, and the expression of rLFs in the above three clones considerately improved their antibacterial efficacies against four important food-borne pathogens, namely, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Salmonella enterica. In conclusion, this study provides a simple strategy for the production of functional LFs (BLF and HLF) in both functional and LF-resistant hosts for applications in the field.
Highlights
Lactoferrin (LF) is an 80 kDa iron-binding protein present mostly in the milk and exocrine fluids of mammals, and it has been shown to display extensive biological effects, including antimicrobial, anti-inflammatory, and immune-modulating properties
We previously demonstrated that the combination of natural bovine LF (BLF) and specific probiotics can lead to synergistic antibacterial activity in vitro [23,24]
We have previously shown that the combination of purified BLF and specific probiotics can lead to synergistic antibacterial activity in vitro [23,24], and in this study, our results reveal that the strategy of a combination of recombinant LFs and specific probiotics can be obtained by our recombinant L. delbrueckii/BLF and L. delbrueckii/human LF (HLF) and L. gasseri/BLF
Summary
Lactoferrin (LF) is an 80 kDa iron-binding protein present mostly in the milk and exocrine fluids of mammals, and it has been shown to display extensive biological effects, including antimicrobial, anti-inflammatory, and immune-modulating properties. LF possesses antimicrobial activity against a wide spectrum of pathogens, it exerts minor antibacterial activities on specific probiotic strains, and it even possesses prebiotic activity on specific probiotic strains [4,5,6]. There is evidence indicating that oral LF could be effective in the prevention of sepsis and necrotizing enterocolitis in preterm neonates, this is not yet fully established [7]. In animal models, LF supplementation is effective in preventing preterm delivery and intrauterine infections [8,9]. Early-life LF administration can enhance growth performance and decrease diarrhea incidence in suckling piglets by promoting the development of intestinal functions and by modifying the microbiota in the small intestine [10]. The simultaneous combination of LF preparations and specific lactobacilli strains can improve women’s health through probiotic and prebiotic input [11]
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