A kind of method for preparing high f value oligopeptide using chlorella powder as raw material
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A chlorella and oligopeptide technology, applied in the field of protein preparation, can solve the problems of imperfect process, easy formation of inclusion bodies, short synthetic sequence, etc., and achieves the effects of good solubility, reduced production cost and low cost
Active Publication Date: 2020-06-30
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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Problems solved by technology
The method of separating and extracting natural active peptides has the characteristics of high efficiency, low toxicity, and no pollution. However, the content of biologically active peptides in organisms is generally a small amount, and the cost of separating and purifying active peptides from natural organisms is relatively high and the process is difficult. not perfect
As a new preparation method of biologically active peptides, chemical synthesis is still immature, and still has the disadvantages of short synthetic sequence, low efficiency, low purity, high cost and high toxicity.
Some scholars have also proposed the method of gene recombination to prepare active peptides, but this method is not perfect, and it is prone to unfavorable situations such as difficulty in modification after translation, formation of inclusion bodies, etc.
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Embodiment 1
[0032] Example 1 Obtaining of recombinant carboxypeptidase (CPA)
[0033] 1. Cloning of Aspergillus niger M00988 CPA and construction of expression vector
[0034] According to the carboxypeptidase gene sequence of NCBI Aspergillus niger, two pairs of primers CPA-F / CPA-R were designed, and the sequences are shown in Table 1.
[0035] Table 1 The sequences of primers for carboxypeptidase gene cloning
[0036]
[0037] The DNA of Aspergillus niger M00988 (CGMCC No. 18828) was extracted as a template, and the sequence is shown in SEQ ID NO: 6. The target gene fragment CPA was obtained by PCR amplification with CPA-F / CPA-R primers. The PCR amplification conditions are: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 68°C for 1 minute, extension at 72°C for 1 minute, 35 cycles, and extension at 72°C for 10 minutes to obtain a fragment with a size of about 1500 bp, which was detected by agarose electrophoresis Such as figure 1 s...
Embodiment 2
[0050] Example 2 Preparation of high F value oligopeptide
[0051] 1. Chlorella protein extraction
[0052] Accurately weigh a certain amount of chlorella powder, put it in a 250mL Erlenmeyer flask, add deionized water according to the appropriate amount of material-to-liquid ratio g / mL, stir, put it in a shaker, and swell at room temperature at 200rpm for 12 hours. Add an appropriate amount of solid sodium hydroxide to the swollen chlorella suspension, add slowly and keep stirring during the addition process. After completion, sonicate at a certain temperature and time. Next, use 0.1mol / L sodium hydroxide solution and hydrochloric acid solution to adjust the pH=7, centrifuge at 5000rpm 25°C for 10min, take the supernatant, measure the volume with a graduated cylinder and record it, then take 10mL from it into the Erlenmeyer flask to 1:4 (V / V) solid-liquid ratio, add 95% ethanol solution at 4°C, stir for 1min, and mix thoroughly. Centrifuge again at 5000 rpm at 25° C. for 1...
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Abstract
The invention provides a method for preparing high-F-value oligopeptide by taking chlorella powder as a raw material. The method comprises the following steps: adding trypsin into chlorella protein according to 70000-90000 U / mg protein under the conditions that the pH is 8 and the temperature is 27-52 DEG C to carry out first enzymolysis; then carrying out second enzymolysis by using recombinant carboxypeptidase derived from aspergillus niger, wherein the enzymolysis conditions are as follows: the pH is 6.5-7.5, the enzyme addition amount is 3-7% of the protein amount and the enzymolysis temperature is 27-40 DEG C; and then adjusting the pH to 4.5, adding activated carbon according to a solid-to-liquid ratio of 1 to 10 to remove impurities, and conducting filtering to obtain a high-F-valueoligopeptide solution. According to the method for preparing the high-F-value oligopeptide, chlorella is taken as the raw material, the protein content is high, the cost is low and the yield is high.The F value of the product is easy to elevate, the production cost of an enzyme method is reduced, the energy consumption in a later purification process is reduced, environmental pollution is reduced, the high-F-value oligopeptide is effectively prepared, and industrial production and application of the high-F-value oligopeptide are promoted.
Description
technical field [0001] The invention belongs to the field of protein preparation, and in particular relates to a method for preparing oligopeptides with high F value. Background technique [0002] As the most productive chlorella in the world's microalgae industry ( Chlorella vulgaris ), has the characteristics of wide distribution, large biomass, and easy acquisition. It is a high-protein marine organism with a variety of high-benefit use values that have not yet been developed. Common Chlorella vulgaris in my country ( C. vulgaris ), Chlorella pyrenoidosa ( Chlorella pyrenidosa ), Chlorella ellipsoides ( C. ellipsoidea )Wait. The protein content of Chlorella pyrenoidosa powder is as high as 63.36%-63.98%. Contains 8 kinds of essential amino acids (EAA), and the content reaches 23.35%, and the amino acid score (AAS) is 64.3, which is better than the general plant protein source; the average food intake rate of this microorganism has reached the standard of FAO / WHO...
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