CN110699408A - Method for improving surfactant yield by mixed fermentation - Google Patents

Abstract

The invention discloses a method for improving the yield of surfactant by mixed fermentation, which comprises the following steps: respectively using seeds of Bacillus amyloliquefaciens HM-618 and CGMCC No.7097 and seeds of Bacillus subtilis and CGMCC1.12939 according to biomass OD₆₀₀Inoculating the strain into a fermentation medium at an inoculation density of 0.1-0.3, and culturing at 35-39 ℃ and a rotation speed of 100-300 rpm for 110-130 h to obtain surfactant; the co-culture of Bacillus amyloliquefaciens HM-618 (CGMCC No. 7097) and Bacillus subtilis (CGMCC 1.12939) can improve the yield of the surfactin.

Description

Method for improving surfactant yield by mixed fermentation

Technical Field

The invention belongs to the technical field of fermentation engineering, and relates to a method for improving the yield of surfactant by using mixed bacteria.

Background

The surfactant is composed of four amino acids (valine, leucine, aspartic acid and glutamic acid) and fatty acid chain, has high surface activity, and has the characteristics of no toxicity or low toxicity, biodegradability and no environmental pollution compared with chemical surfactants. It also has good antibacterial, antiviral, antitumor and anti-mycoplasma activities, and has great application potential.

The wild strain discovered at present almost has milligram-level surfactant yield, and the production, separation and purification costs are extremely high.

The co-culture fermentation can improve the yield of microbial products and obtain products with higher quality, but the research on improving the yield of the surfactant by co-culture microorganisms is not reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for improving the yield of surfactin by mixed fermentation.

A method for improving the yield of surfactin by mixed fermentation comprises the following steps: respectively using seeds of Bacillus amyloliquefaciens HM-618 and CGMCC No.7097 and seeds of Bacillus subtilis and CGMCC1.12939 according to biomass OD₆₀₀Inoculating the strain into a fermentation medium at an inoculation density of 0.1-0.3, and culturing at 35-39 ℃ and a rotation speed of 100-300 rpm for 110-130 h to obtain surfactant;

the fermentation medium comprises 50-70 g/L of glucose, 4-6 g/L of yeast powder and NaNO₃8～12g/L，K₂HPO₄7～8g/L,KH₂PO₄3～5g/L,MgSO₄·7H₂O 2～4g/L,FeCl₂0.01～0.03g/L,ZnCl₂0.01～0.03g/L,MnCl₂·4H₂0.01-0.03 g/L of O, the balance of water and natural pH.

THE ADVANTAGES OF THE PRESENT INVENTION

The co-culture of Bacillus amyloliquefaciens HM-618 (CGMCC No. 7097) and Bacillus subtilis (CGMCC 1.12939) can improve the yield of the surfactin.

Detailed Description

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is named as Bacillus amyloliquefaciens HM-618 and is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 7097. The preservation address is microbial research institute of China academy of sciences, No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation time is 2013, 1 month and 8 days, and the product survives.

In the following examples and comparative examples, the preservation number of HM-618 of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is CGMCC No.7097 and abbreviated as HM-618.

Bacillus subtilis, CGMCC1.12939, is purchased from China general microbiological culture Collection center (CGMCC) in 2016 (11 months) in China, and is hereinafter referred to as Bacillus subtilis CGMCC 1.12939.

The HM-618 seeds of each example were obtained by culturing HM-618 in HM-618 seed medium.

HM-618 seed Medium (g/L): 60 parts of glucose, 10 parts of yeast powder, 10 parts of peptone, 5 parts of NaCl, 10 parts of beef extract, natural pH and the balance of water.

The Bacillus subtilis CGMCC1.12939 seed of each embodiment is obtained by culturing Bacillus subtilis CGMCC1.12939 in a Bacillus subtilis CGMCC1.12939 seed culture medium.

Bacillus subtilis CGMCC1.12939 seed culture medium (LB culture medium) (g/L): tryptone 10, Yeast extract 5, NaCl 10)

The present invention will be further described with reference to the following examples.

Example 1

A method for improving the yield of surfactin by mixed fermentation comprises the following steps:

respectively using HM-618 seeds and Bacillus subtilis CGMCC1.12939 seeds according to biomass OD₆₀₀Inoculating into fermentation medium at an inoculation density of 0.2, and culturing at 37 deg.C and rotation speed of 200rpm for 120 hr to obtain fermentation broth containing surfactant;

the fermentation medium formula comprises 60g/L of glucose, 5g/L of yeast powder and NaNO₃10g/L，K₂HPO₄7.5g/L,KH₂PO₄4g/L,MgSO₄·7H₂O 3g/L,FeCl₂0.02g/L,ZnCl₂0.02g/L,MnCl₂·4H₂O0.02 g/L, and the balance of water, wherein the pH is natural.

The yield of the surfactant was 0.995 g/L.

Control group 1

Seeds of HM-618 were expressed as OD biomass₆₀₀The cells were inoculated into the fermentation medium of example 1 at an inoculation density of 0.2, and cultured at 37 ℃ and a rotation speed of 200rpm for 120 hours to obtain a fermentation broth containing surfactin.

The surfactant yield was 0.674 g/L.

Example 2

A method for improving the yield of surfactin by mixed fermentation comprises the following steps:

respectively using HM-618 seeds and Bacillus subtilis CGMCC1.12939 seeds according to biomass OD₆₀₀Inoculating into fermentation medium at an inoculation density of 0.1, and culturing at 35 deg.C and rotation speed of 100rpm for 130 hr to obtain fermentation liquid containing surfactin.

The fermentation medium comprises 50g/L of glucose, 4g/L of yeast powder and NaNO₃8g/L，K₂HPO₄7g/L,KH₂PO₄3g/L,MgSO₄·7H₂O 2g/L,FeCl₂0.01g/L,ZnCl₂0.01g/L,MnCl₂·4H₂O0.01 g/L, and the balance of water, and the pH is natural.

Yield of surfactant 0.873g/L

Control group 2

Seeds of HM-618 were expressed as OD biomass₆₀₀The cells were inoculated into the fermentation medium of example 2 at an inoculation density of 0.1, and cultured at 35 ℃ and a rotation speed of 100rpm for 130 hours to obtain a fermentation broth containing surfactin.

The yield of surfactant was 0.631g/L

Example 3

A method for improving the yield of surfactin by mixed fermentation comprises the following steps:

respectively using HM-618 seeds and Bacillus subtilis CGMCC1.12939 seeds according to biomass OD₆₀₀Inoculating into fermentation medium at an inoculation density of 0.3, and culturing at 39 deg.C and rotation speed of 300rpm for 110 hr to obtain fermentation liquid containing surfactin.

The fermentation medium comprises 70g/L of glucose, 6g/L of yeast powder and NaNO₃12g/L，K₂HPO₄8g/L,KH₂PO₄5g/L,MgSO₄·7H₂O 4g/L,FeCl₂0.03g/L,ZnCl₂0.03g/L,MnCl₂·4H₂O0.03 g/L, and the balance of water, wherein the pH is natural.

The yield of the surfactant is 0.890 g/L.

Control group 3

Seeds of HM-618 were expressed as OD biomass₆₀₀The cells were inoculated into the fermentation medium of example 3 at an inoculation density of 0.3, and cultured at 39 ℃ and a rotation speed of 300rpm for 110 hours to obtain a fermentation broth containing surfactin.

The surfactant yield was 0.665 g/L.

Claims (1)

1. A method for improving the yield of surfactin by mixed fermentation is characterized by comprising the following steps: respectively using seeds of Bacillus amyloliquefaciens HM-618 and CGMCC No.7097 and seeds of Bacillus subtilis and CGMCC1.12939 according to biomass OD₆₀₀Inoculating the strain into a fermentation medium at an inoculation density of 0.1-0.3, and culturing at 35-39 ℃ and a rotation speed of 100-300 rpm for 110-130 h to obtain surfactant;

the fermentation medium comprises 50-70 g/L of glucose, 4-6 g/L of yeast powder and NaNO₃8～12g/L，K₂HPO₄7～8g/L,KH₂PO₄3～5g/L,MgSO₄·7H₂O 2～4g/L,FeCl₂0.01～0.03g/L,ZnCl₂0.01～0.03g/L,MnCl₂·4H₂0.01-0.03 g/L of O, the balance of water and natural pH.