CN102899375A - Method for increasing titer of bacitracin in fermentation liquid by using oxygen carrier - Google Patents

Abstract

The invention relates to a method for increasing titer of bacitracin in fermentation liquid by using oxygen carrier. A certain amount of oxygen carrier is added into a fermentation medium to improve the oxygen dissolution condition and greatly increase titer of bacitracin fermentation liquid, without changing other fermentation conditions. The method has simple process; the oxygen carrier in the production process can be recycled, so as to reduce production cost; therefore, the method has an important guiding significance for antibiotic production.

Description

A method for improving the titer of bacitracin in fermentation broth by using oxygen carrier

technical field

The invention belongs to the field of microorganisms, and relates to an optimized application technology in a microorganism fermentation process, in particular to a method for increasing the titer of bacitracin in a fermentation liquid by using an oxygen carrier.

Background technique

Bacitracin, white or off-white powder in appearance, odorless or slightly smelly, bitter in taste, hygroscopic, soluble in water, methanol and ethanol, insoluble in acetone, ether and chlorine. Bacitracin is a broad-spectrum antibiotic that has a strong inhibitory effect on Gram-positive bacteria, especially pathogenic bacteria such as Clostridium, Staphylococcus, Streptococcus, Corynebacterium, and Neisseria. It is sensitive and has a strong inhibitory effect on penicillin-resistant Staphylococcus aureus. It is suitable for the treatment of abscesses, acne, purulent skin diseases, amoebic dysentery, gonorrhea and meningococcus and other infectious diseases, and its toxicity to the kidneys is relatively high. big.

At present, the research on the production of bacitracin by microbial fermentation is mainly the optimization of fermentation conditions, such as the optimization of the medium and the optimization of the fermentation mode, which has not achieved good results; some factories have also screened high-yield bacitracin through traditional microbial mutation breeding strains, the disadvantage of this method is that the randomness is too large, and the long-term mutagenesis will continuously reduce the viability of the strains.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a method for improving the titer of bacitracin in the fermentation liquid by using oxygen carrier, by adding oxygen carrier in the fermentation liquid, the dissolved oxygen condition of the fermentation liquid is improved, and the bacterial growth rate is promoted. The growth of the body, the synthesis of more bacitracin, increase the titer of the fermentation broth, and the oxygen carrier in the fermentation process can be recycled, saving more costs.

The technical scheme that the present invention realizes purpose is as follows:

A method for increasing the titer of bacitracin in a fermentation broth by using an oxygen carrier, adding an oxygen carrier to a fermentation medium for microbial fermentation to produce bacitracin, and the oxygen carrier is one of n-dodecane, oleic acid or soybean oil or a mixture of two or more.

Moreover, the added amount of the oxygen carrier is 0.5-10% of the volume of the fermentation medium.

And, described microorganism is Bacillus licheniformis, is preserved by China General Microorganism Culture Collection Center, and its number is CGMCC 1.105.

Moreover, the specific fermentation steps are as follows:

⑴ Bacteria culture

Inoculate Bacillus licheniformis into the seed medium, and culture at 30-40°C, 150-200rpm, for 20-30 hours at a constant temperature;

⑵Fermentation culture

Inoculate the seed culture solution obtained in step (1) into the fermentation medium, 30-40°C, 200-300rpm, add sterile oxygen carrier in the fermenter, the amount of oxygen carrier added is 0.5-10% of the volume of the fermentation medium Cultivate at constant temperature for 40-45 hours, and ferment to obtain bacitracin.

Moreover, the seed culture medium is: beef extract 5-10g/L, peptone 2-5g/L, sodium chloride 5-10gL, pH7.2.

Moreover, the fermentation medium is: corn flour 20-40g/L, sucrose 10-20gL, ammonium sulfate 1-10g/L, calcium carbonate 1-10g/L, corn steep liquor 20-40g/L.

Beneficial effects of the present invention:

In the fermentation process of the present invention, oxygen carriers are added to the fermentation liquid, which can increase the dissolved amount of oxygen in the fermentation liquid, increase the transfer rate of oxygen in the fermentation liquid, and reduce energy consumption in the fermentation process. The oxygen carrier used in the experiment The dosage is small, and it is water-insoluble. It can be recycled after fermentation. It can increase the output of bacitracin without increasing the production cost, and has certain guiding significance for other fermentations that require more oxygen.

Detailed ways

Below in conjunction with the examples, a kind of high-salt dilute black soybean soy sauce of the present invention and preparation method thereof are further described, the following examples are illustrative, not limiting, and cannot limit the protection scope of the present invention with the following examples.

The present invention improves the dissolved oxygen condition of the fermented liquid by adding an oxygen carrier into the fermented liquid, promotes the growth of bacteria, synthesizes more bacitracin, improves the titer of the fermented liquid, and the oxygen carrier in the fermentation process can be recycled, Save more cost. The types of oxygen carriers are n-dodecane, oleic acid and soybean oil. The volume of the oxygen carrier added to the fermentation liquid is 0.5-10%. The oxygen carrier can increase the dissolved oxygen in the fermentation liquid, which is beneficial to the growth of bacteria and bacitracin Synthesis.

The Bacillus licheniformis used in the following examples is preserved in China General Microorganism Culture Collection Center, and its number is CGMCC 1.105. The method of the present invention is not limited to this single bacterial species, any bacitracin that can be produced by fermentation can be used, and the amount of oxygen carrier added will vary according to the different bacterial species and fermentation broth, but this is a matter for those skilled in the art. reasonably known.

The method for measuring the titer of bacitracin in the fermentation broth is: the titer of the bacitracin in the fermentation broth is detected by a microorganism two-dose method.

Example 1

A method for improving the titer of bacitracin in a fermentation broth by using an oxygen carrier, the steps are as follows:

(1) First-level seed cultivation: Inoculate Bacillus licheniformis as a production strain into a seed medium with a pH of 7.2 from a slant, and cultivate it on a rotary shaker at 160r/min at a temperature of 37°C for 24 hours to obtain a seed suitable for inoculation. The seed culture medium contains: beef extract 10g/L, peptone 5g/L and sodium chloride 10g/L.

⑵Secondary fermentation culture: Inoculate the seed culture solution into the fermentation medium at an inoculum volume of 5% (v/v), and add filter-sterilized 1% (v/v) n-dodecane before inoculation. The fermentation medium contains: corn steep liquor 35g/L, sucrose 12g/L, ammonium sulfate 3g/L, calcium carbonate 2g/L, corn flour 25g/L, pH=7.2.

(3) The fermentation culture method is carried out on a 7L fermenter with a liquid volume of 3.5L, 250rpm, and 37°C for 45 hours.

In this example, the titer of bacitracin in the fermentation broth was 594.8u/ml, which was 13.86% higher than the titer of 522.4u/ml in the culture medium without oxygen carrier.

Example 2

A method for improving the titer of bacitracin in a fermentation broth by using an oxygen carrier, the steps are as follows:

(1) First-level seed cultivation: Same as (1) in Implementation Case 1.

⑵Secondary fermentation culture: Inoculate the seed culture solution into the fermentation medium at an inoculum volume of 5% (v/v), and add filter-sterilized 2% (v/v) oleic acid before inoculation. The fermentation medium contains: corn steep liquor 30g/L, sucrose 15g/L, ammonium sulfate 2g/L, calcium carbonate 3g/L, corn flour 20g/L, pH=7.2.

(3) The fermentation culture method is carried out on a 7L fermenter, and the liquid volume of the fermenter is 3.5L. The fermentation was carried out for 46 hours under the conditions of 250 rpm and 37° C. temperature.

In this example, the titer of bacitracin in the fermentation broth was 562.7u/ml, which was 7.71% higher than the titer of 522.4u/ml in the medium without oxygen carrier.

Example 3

A method for improving the titer of bacitracin in a fermentation broth by using an oxygen carrier, the steps are as follows:

(1) First-level seed cultivation: Same as (1) in Implementation Case 1.

(2) Fermentation culture: Inoculate the seed culture solution into the fermentation medium at an inoculum volume of 5% (v/v), and add filter-sterilized 5% (v/v) soybean oil before inoculation. The fermentation medium contains: corn steep liquor 30g/L, sucrose 13g/L, ammonium sulfate 4g/L, calcium carbonate 2g/L, corn flour 25g/L, pH=7.2.

(3) The fermentation culture method is carried out on a 7L fermenter, and the liquid volume of the fermenter is 3.5L. The fermentation was carried out for 42 hours under the conditions of 200 rpm and 37° C. temperature.

In this example, the titer of bacitracin in the fermentation broth was 547.1u/ml, which was 4.73% higher than the titer of 522.4u/ml in the culture medium without oxygen carrier.

Claims (6)

1. one kind is utilized oxygen carrier to improve the method that bacitracin is tired in the fermented liquid, it is characterized in that: add in microbial fermentation is produced the fermention medium of bacitracin and add oxygen carrier, described oxygen carrier is one or more mixture of n-dodecane, oleic acid or soybean oil.

2. the oxygen carrier that utilizes according to claim 1 improves the method that bacitracin is tired in the fermented liquid, and it is characterized in that: the add-on of described oxygen carrier is the 0.5-10% of fermention medium volume.

3. the oxygen carrier that utilizes according to claim 1 improves the method that bacitracin is tired in the fermented liquid, and it is characterized in that: described microorganism is Bacillus licheniformis, is preserved by Chinese common micro-organisms DSMZ, and it is numbered CGMCC 1.105.

4. the oxygen carrier that utilizes according to claim 3 improves the method that bacitracin is tired in the fermented liquid, and it is characterized in that: concrete fermentation step is as follows:

⑴ spawn culture

Bacillus licheniformis is inoculated in the seed culture medium, 30-40 ℃, 150-200rpm constant temperature culture 20-30 hour;

⑵ fermentation culture

The seed culture fluid that step ⑴ is obtained is inoculated in the fermention medium, 30-40 ℃, 200-300rpm adds aseptic oxygen carrier in fermentor tank, the add-on of oxygen carrier is 0.5-10% constant temperature culture 40-45 hour of fermention medium volume, and fermentation obtains bacitracin.

5. the oxygen carrier that utilizes according to claim 4 improves the method that bacitracin is tired in the fermented liquid, and it is characterized in that: described seed culture medium is: extractum carnis 5-10gL, peptone 2-5gL, sodium-chlor 5-10gL, pH7.2.

6. the oxygen carrier that utilizes according to claim 4 improves the method that bacitracin is tired in the fermented liquid, and it is characterized in that: described fermention medium is: Semen Maydis powder 20-40gL, sucrose 10-20g/L, ammonium sulfate 1-10g/L, calcium carbonate 1-10gL, corn steep liquor 20-40g/L.