CN103849667A - Cecropin antibacterial peptide production process - Google Patents

Abstract

The invention discloses a cecropin antibacterial peptide production process. The process comprises: (1) a step of rejuvenating a bacillus subtilis genetically engineered bacterium carrying the cecropin antibacterial peptide, namely, a step of taking out the bacillus subtilis strain carrying the cecropin antibacterial peptide stored at -70 DEG C in a state of freeze-dried powder, reactivating the strain in a slant culture manner, inoculating the strain to a slant culture medium and allowing the strain to grow under the slant growth conditions; (2) a step of seed culture and amplification, namely, a step of inoculating the strain activated in the slant into a conical flask having a volume of 250 mL, filling 30 mL of a shake flask culture medium, and culturing under shake-flask culture conditions; and (3) a step of fermenting by the bacillus subtilis. The production process is simple, shortened and convenient, saves more than 40% of material consumption, largely reduces the consumption of raw materials, and saves the cost.

Description

A kind of cecropin antimicrobial peptides production technique

Technical field

The present invention relates to a kind of cecropin antimicrobial peptides production technique.

Background technology

Cecropin (cecropins) is first found Antibacterial Peptide From Animals, 1980, is separated and obtains from sky silkworm chrysalis by Boman etc.Such peptide antibiotics generally contains 37～39 amino-acid residues, not containing halfcystine, its N end regions has strong basicity, can form parents' spirane structure of almost Perfect, and can form hydrophobic spiral at C end regions, the hinge area that has glycine and proline(Pro) to form between the two, the C end of most polypeptide is amidated, and amidation has vital role to its anti-microbial activity.

After this, people have been separated to cecropins class antibacterial peptide in succession from silkworm, tussah, fruit bat, sarcophagid.1989, the people such as Lee divided high to cecropin P1 from chitterlings, had illustrated that cecropins may extensively exist in animal.Cecropin (cecropins) has very strong lethality to gram-positive microorganism, part Gram-negative bacteria, and fungi and eukaryotic cell are not had to toxicity.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of cecropin antimicrobial peptides production technique.

Technical scheme of the present invention is: a kind of cecropin antimicrobial peptides production technique, and its step is as follows:

(1) carry the subtilis genetic engineering bacterium rejuvenation of cecropin antimicrobial peptides: take out from-70 ℃ of refrigerators the bacillus subtilis bacterial classification that carries cecropin antimicrobial peptides that lyophilized powders are preserved, bring back to life bacterial classification in slant culture mode, be inoculated on slant medium, under inclined-plane growth conditions, grow;

(2) seed culture and expansion: the bacterial classification having activated on inclined-plane is inoculated on the Erlenmeyer flask of 250 ml, fills 30 ml shake-flask culture bases, cultivate under shake-flask culture condition;

(3) fermentation of subtilis: from seed culture to last 30M ³ferment tank is cultivated needs 3 grades of seeds to expand, and first order seed expands: seed good shake-flask culture is expanded respectively in the Erlenmeyer flask of 3 500 ml by 1:100, fill 50 ml seed culture mediums, under seed culture condition, cultivate; Secondary seed expands: the seed that first order seed is expanded expands in 30 L seed fermentation tanks in 1:100 ratio, fills 15 L seed culture mediums, under seed culture condition, cultivates; Three grades of seeds expand: the seed that secondary is expanded expands in 3 M by 1:100 ³seed fermentation tank, fills 1.5 M ³seed culture medium, cultivates under seed culture condition, and the seed that secondary seed enlarged culturing is good is inoculated in 30M by 1:20 ³fermentor tank, dress 21M ³fermention medium, cultivates under fermentation culture conditions.

The invention has the beneficial effects as follows: technique of the present invention is simple, the technical process of producing the antibacterial skin of cecropin is shortened, easy, save feed consumption rate more than 40%, greatly reduce raw-material consumption, save cost.

Embodiment

A kind of cecropin antimicrobial peptides production technique, its step is as follows:

(1) carry the subtilis genetic engineering bacterium rejuvenation of cecropin antimicrobial peptides: take out from-70 ℃ of refrigerators the bacillus subtilis bacterial classification that carries cecropin antimicrobial peptides that lyophilized powders are preserved, bring back to life bacterial classification in slant culture mode, be inoculated on slant medium, under inclined-plane growth conditions, grow;

(2) seed culture and expansion: the bacterial classification having activated on inclined-plane is inoculated on the Erlenmeyer flask of 250 ml, fills 30 ml shake-flask culture bases, cultivate under shake-flask culture condition;

(3) fermentation of subtilis: from seed culture to last 30M ³ferment tank is cultivated needs 3 grades of seeds to expand, and first order seed expands: seed good shake-flask culture is expanded respectively in the Erlenmeyer flask of 3 500 ml by 1:100, fill 50 ml seed culture mediums, under seed culture condition, cultivate; Secondary seed expands: the seed that first order seed is expanded expands in 30 L seed fermentation tanks in 1:100 ratio, fills 15 L seed culture mediums, under seed culture condition, cultivates; Three grades of seeds expand: the seed that secondary is expanded expands in 3 M by 1:100 ³seed fermentation tank, fills 1.5 M ³seed culture medium, cultivates under seed culture condition, and the seed that secondary seed enlarged culturing is good is inoculated in 30M by 1:20 ³fermentor tank, dress 21M ³fermention medium, cultivates under fermentation culture conditions.

Claims (1)

1. a cecropin antimicrobial peptides production technique, is characterized in that, its step is as follows:

(1) carry the subtilis genetic engineering bacterium rejuvenation of cecropin antimicrobial peptides: take out from-70 ℃ of refrigerators the bacillus subtilis bacterial classification that carries cecropin antimicrobial peptides that lyophilized powders are preserved, bring back to life bacterial classification in slant culture mode, be inoculated on slant medium, under inclined-plane growth conditions, grow;

(2) seed culture and expansion: the bacterial classification having activated on inclined-plane is inoculated on the Erlenmeyer flask of 250 ml, fills 30 ml shake-flask culture bases, cultivate under shake-flask culture condition;

(3) fermentation of subtilis: from seed culture to last 30M ³ferment tank is cultivated needs 3 grades of seeds to expand, and first order seed expands: seed good shake-flask culture is expanded respectively in the Erlenmeyer flask of 3 500 ml by 1:100, fill 50 ml seed culture mediums, under seed culture condition, cultivate; Secondary seed expands: the seed that first order seed is expanded expands in 30 L seed fermentation tanks in 1:100 ratio, fills 15 L seed culture mediums, under seed culture condition, cultivates; Three grades of seeds expand: the seed that secondary is expanded expands in 3 M by 1:100 ³seed fermentation tank, fills 1.5 M ³seed culture medium, cultivates under seed culture condition, and the seed that secondary seed enlarged culturing is good is inoculated in 30M by 1:20 ³fermentor tank, dress 21M ³fermention medium, cultivates under fermentation culture conditions.