Application of human lactoferrin mutant and combination of human lactoferrin mutant and lactobacillus in preparation of food or health care product
A human lactoferrin, mutant technology, applied in food science, resistance to vector-borne diseases, etc., can solve problems that need to be improved, and achieve the effect of improving immunity and maintaining environmental balance
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Embodiment 1
[0029] Example 1 Determination and preparation of human lactoferrin mutants
[0030] The human lactoferrin mutant is based on human lactoferrin, uses bioinformatics means and existing human lactoferrin structural analysis data to design corresponding site-specific mutations, and is obtained through molecular and cellular level experimental verification and screening. , the identified amino acid sequence of the human lactoferrin mutant is shown in SEQ ID No.1, and its nucleotide sequence is shown in SEQ ID No.2.
[0031]The human lactoferrin mutant described in the present invention was prepared by using Escherichia coli as the host bacteria. First, the upstream and downstream PCR primers were designed to amplify the human lactoferrin mutant (hereinafter referred to as mu-hLF) gene, and EcoRI and XhoI restriction sites were introduced into the upstream and downstream primers respectively; secondly, the expression vector pET28a was double digested with EcoRI and XhoI. and mu-hL...
Embodiment 2
[0032] Example 2 Effects of recombinant human lactoferrin mutants on the growth of different microorganisms
[0033] 2.1 Recombinant human lactoferrin mutants promote the growth of probiotics
[0034] Take frozen Bifidobacterium, Lactobacillus (Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus plantarum), Lactococcus strains, the above-mentioned strains are stored in the laboratory, inoculated and activated in sterile LB medium after recovery Culture for 24 hours; collect the cells by centrifugation, and use sterile LB medium to adjust the cell concentration to 1 × 10 5 CFU / mL, respectively take 1mL of the above 5 kinds of probiotics and inoculate in 20mL sterile LB medium; each probiotics are divided into three groups, respectively add 50mg / mL mu-hLF protein, 50mg / mL natural human lactoferrin (hereinafter referred to as hLF) and an equal volume of sterile water (as blank control), after culturing at 37°C on a shaker for 24 days, the OD600 value was measured us...
Embodiment 3
[0038] Example 3 Preparation of recombinant human lactoferrin and Lactobacillus reuteri composition
[0039] In this example, Lactobacillus reuteri with the strongest pro-proliferation effect in the previous experiments was selected to form a composition with human lactoferrin mutant. Proliferation and growth of Lactobacillus, so as to exert a greater immunomodulatory effect in vivo; on the other hand, the human lactoferrin mutant exhibits a certain selectivity in promoting the proliferation of Lactobacillus, which can avoid such as Lactobacillus rhamnosus, Potential pathogenic risks caused by Lactobacillus plantarum and other types of Lactobacillus improve the safety of the composition. The specific preparation process is as follows:
[0040] First, mu-hLF protein and hLF protein were prepared. For specific steps, refer to Example 1. The obtained protein was freeze-dried to obtain protein powder; secondly, Lactobacillus reuteri powder was prepared, and Lactobacillus reuteri ...
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