Novel ace inhibitory peptide in yogurt and its genetic engineering production method
A technology for inhibiting peptides and yogurt, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of restricting industrial development and practical application, high cost of chemical synthesis, difficult separation and purification, etc. , to achieve large-scale industrial production and application, low difficulty in separation and purification, and high biological activity
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Embodiment 1
[0055] Fermentation of Yogurt and Isolation and Purification of Novel ACE Inhibiting Peptides
[0056] 1. After 3 generations of lactic acid bacteria (such as L.Casei HZ1) are activated in medium such as MRS, transfer them to pure milk (sterilized at 115°C for 15 minutes) with an inoculum of 3% volume ratio, and then activate for 3 generations until the bacteria The vitality of the seed is fully restored, and it is used as a fermented seed liquid. Inoculate the fermented seed solution into pure milk with an inoculation amount of 10% by volume, and culture it anaerobically at 37°C for 24 hours to prepare yoghurt;
[0057] 2. The obtained fermented yoghurt was centrifuged at 12,000 rpm and 4°C for 20 minutes to obtain the yoghurt supernatant;
[0058] 3. After the yogurt supernatant was separated by DEAE weak anion exchange chromatography and Sephadex G-15 gel chromatography, the ACE inhibitory peptide sample obtained through rough separation was filtered through a 0.45 μm micr...
Embodiment 2
[0061] Genetic Engineering Expression and Production of Novel ACE Inhibitory Peptides
[0062] 1. Novel ACE inhibitory peptide
[0063] The host bacteria and expression plasmids used are conventional genetic engineering expression system host bacteria and expression plasmids, including Escherichia coli and food-grade genetic engineering host bacteria (lactic acid bacteria, bacillus, yeast, etc.) and their expression plasmids, etc. The construction process mainly includes the following steps :
[0064] (1) According to the amino acid sequences of two novel ACE inhibitory peptides ACEI1 and ACEI2 (see Sequence 1 and Sequence 2 for specific sequences), the coding gene sequence is optimized by using the codon preference of the host bacteria;
[0065] (2) Obtain two ACE inhibitory peptide genes by PCR or artificial synthesis, insert the genes into the multiple cloning site of the expression vector, and construct ACEI1 and ACEI2 recombinant plasmids;
[0066] (3) Transform ACEI1 a...
Embodiment 3
[0070] Product ACE inhibitory activity analysis
[0071] After Lactobacillus casei was activated for 3 generations in liquid medium (MRS), transfer it to pure milk (sterilized at 115°C for 15 minutes) with an inoculum of 3% by volume, and activate for more than 3 generations until the strain activity is fully recovered. As a fermented seed liquid, insert it into pure milk with an inoculum of 10% volume ratio, and culture it anaerobically at 37°C for 24 hours. The obtained fermentation liquid is 12000r / min, centrifuged at 4°C for 20min, and the supernatant is taken and filtered with a 0.22μm needle filter to obtain the supernatant for measuring ACEI activity.
[0072] Take 0.15mL ACE crude enzyme solution → add 0.10mL sample inhibitor (ACEI) → 5min in 37°C water bath → add 0.10mL substrate HHL (Hip-His-Leu), mix well → incubate in 37°C water bath for 60min → immediately Add 0.25mL of 1mol / L HCl to stop the reaction → let it stand for 5min → add 1.50mL ethyl acetate for extract...
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