CN110669811A - Method for improving surfactant yield - Google Patents

Abstract

The invention discloses a method for improving the yield of surfactin, which comprises the following steps: inoculating a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM618 seed with the preservation number of CGMCC No.7097 into a fermentation culture medium for culture; the fermentation medium comprises: sucrose, yeast powder, amino acid, NaNO₃，K₂HPO₄，KH₂PO₄，MgSO₄·7H₂O，FeCl₂,ZnCl₂，MnCl₂·4H₂O, natural pH and the balance of water; the amino acid is at least one of alanine, arginine, cysteine, histidine, glycine, lysine, methionine, proline, serine, ornithine and threonine. Experiments prove that the method can improve the growth of the bacillus amyloliquefaciens HM-618 with the preservation number of CGMCC 7097Yield of surfactant.

Description

Method for improving surfactant yield

Technical Field

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

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 has excellent antibacterial, antiviral, antitumor and anti-mycoplasma activities and great application potential.

At present, reports of improving the yield of the surfactant by exogenously adding non-constituent amino acids (other than valine, leucine, aspartic acid and glutamic acid) are not found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for improving the yield of the surfactant.

The technical scheme of the invention is summarized as follows:

a method for increasing the yield of surfactin, comprising the steps of: the strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 with the preservation number of CGMCC No.7097 is selected according to the 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; obtaining the surfactant.

The fermentation medium comprises the following components in g/L: 50-70 parts of cane sugar, 14-16 parts of yeast powder, 0.3-5 parts of amino acid and NaNO₃8～12，K₂HPO₄7～8，KH₂PO₄3～5，MgSO₄·7H₂O 2～4，FeCl₂0.01～0.03,ZnCl₂0.01～0.03，MnCl₂·4H₂0.01-0.03 percent of O, natural pH and the balance of water; the amino acid is at least one of alanine, arginine, cysteine, histidine, glycine, lysine, methionine, proline, serine, ornithine and threonine.

The invention has the advantages that:

experiments prove that the method can improve the yield of the surfactant produced by Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 with the preservation number of CGMCC No. 7097.

Drawings

FIG. 1 shows the effect of fermentation media containing histidine, ornithine and serine, respectively, on the production of surfactin in Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 with the collection number of CGMCC NO. 7097.

FIG. 2 is a graph showing the effect of fermentation media containing ornithine in different concentrations on the production of surfactin by Bacillus amyloliquefaciens (HM-618) with the collection number of CGMCC NO. 7097.

FIG. 3 is a graph showing the effect of fermentation media containing combinations of ornithine and serine, ornithine and threonine, and serine and threonine, respectively, on the production of surfactant from Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 with the accession number of CGMCC NO. 7097.

Detailed Description

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) related by the invention is named as Bacillus amyloliquefaciens HM-618. Is preserved in the China general microbiological culture Collection center 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. The preservation number of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 is CGMCC No.7097, which is abbreviated as HM-618.

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.

Example 1

A method for increasing the yield of surfactin, comprising the steps of:

three groups of experiments are carried out simultaneously, namely 1-1 group, 1-2 group and 1-3 group;

the seeds of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 with the preservation number of CGMCC No.7097 are selected according to the biomass OD₆₀₀Inoculating into the following fermentation culture medium at an inoculation density of 0.2, and culturing at 37 deg.C and rotation speed of 220rpm for 120 hr to obtain fermentation broth containing surfactant;

group 1-1:

the fermentation medium comprises the following components in g/L: sucrose 60, yeast powder 15, histidine (His)3, NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

1-2 groups

The fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, ornithine (Orn)3, NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

1-3 groups

The fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, 3 parts of serine (Ser) and NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

blank group HM-618 seeds were 0.2 (OD) as biomass₆₀₀) Inoculating the inoculated density into a fermentation culture medium (1-1 group of fermentation culture medium is adopted, and the medium does not contain amino acid), and culturing for 120h at 37 ℃ and at the stirring speed of 220rpm to obtain fermentation liquor containing surfactin;

the yield of the surfactant of groups 1-1 was 1.91 g/L;

the yield of the surfactant in groups 1-2 was 2.04 g/L;

the surfactant yields in groups 1-3 were 1.93 g/L.

The yields of the three groups of surfactin are shown in FIG. 1. In fig. 1: HM is blank group, His, Orn and Ser are 1-1 group, 1-2 group and 1-3 group respectively.

The yield of surfactant in the blank group was 1.61 g/L.

Example 2

A method for increasing the yield of surfactin, comprising the steps of:

four groups of experiments were performed simultaneously, 2-1, 2-2, 2-3 and 2-4 groups, respectively.

HM-618 seeds are selected according to biomass OD₆₀₀Inoculating into the following fermentation culture medium at an inoculation density of 0.2, and culturing at 37 deg.C and rotation speed of 200rpm for 120 hr; obtaining fermentation liquor containing surfactin;

2-1 group

The fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, 0.5 part of ornithine (Orn), and 0.5 part of NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

2-2 groups

The fermentation medium comprises the following components in g/L: sucrose 60, yeast powder 15, ornithine (Orn)1, NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

2-3 groups

The fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, ornithine (Orn)3, NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

2-4 groups

The fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, ornithine (Orn)5 parts, NaNO parts₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

blank group HM-618 seeds were 0.2 (OD) as biomass₆₀₀) Inoculating the strain into a fermentation culture medium (2-1 groups of fermentation culture medium without amino acid),culturing at 37 deg.C and stirring speed of 220rpm for 120h to obtain fermentation broth containing surfactant;

the yield of the surfactant in the groups 2-1 was 1.93 g/L;

the yield of the surfactant in groups 2-2 was 1.92 g/L;

the yield of the surfactant in the groups 2-3 is 2.04 g/L;

the surfactant production in groups 2-4 was 1.80 g/L.

The yields of the four groups of surfactant are shown in FIG. 2.

The yield of surfactant in the blank group was 1.61g/L

Example 3

A method for increasing the yield of surfactin, comprising the steps of:

three sets of experiments were performed simultaneously, 3-1, 3-2 and 3-3 respectively:

HM-618 seeds are selected according to biomass OD₆₀₀Inoculating into the following fermentation culture medium at an inoculation density of 0.2, and culturing at 37 deg.C and rotation speed of 220rpm for 120 hr to obtain fermentation broth containing surfactant;

group 3-1:

the fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, 3 parts of ornithine (Orn), 1 parts of serine (Ser), and NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

group 3-2:

the fermentation medium comprises the following components in g/L: 60 parts of sucrose, 15 parts of yeast powder, 3 parts of ornithine (Orn), 0.5 part of threonine (Thr) and NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

3-3 groups:

the fermentation medium comprises the following components in g/L: 60 parts of cane sugar, 15 parts of yeast powder, 1 part of serine (Ser), 0.5 part of threonine (Thr) and NaNO₃10，K₂HPO₄7.5，KH₂PO₄4，MgSO₄·7H₂O 3，FeCl₂0.02,ZnCl₂0.02，MnCl₂·4H₂O0.02, natural pH and the balance of water;

blank group HM-618 seeds were 0.2 (OD) as biomass₆₀₀) Inoculating the inoculated density into a fermentation medium (adopting 3-1 groups of fermentation medium, wherein the medium does not contain amino acid), and culturing for 120h at 37 ℃ and at a stirring speed of 220rpm to obtain fermentation liquor containing surfactin;

the yield of the surfactant in the 3-1 group was 1.86 g/L;

the yield of the group 3-2 surfactant was 1.93 g/L;

the surfactant production in groups 3-3 was 1.76 g/L. The yields of the three groups of surfactin are shown in FIG. 3.

The yield of the surfactant in the blank group is 1.61 g/L;

in fig. 3: HM is blank, Orn + Ser, Orn + Thr and Ser + Thr experimental groups.

Example 4

A method for increasing the yield of surfactin, comprising the steps of:

HM-618 seeds are selected according to biomass OD₆₀₀Inoculating into fermentation medium at an inoculation density of 0.1, and culturing at 39 deg.C and rotation speed of 100rpm for 130 hr to obtain fermentation broth containing surfactant; the yield of surfactant was 1.86 g/L.

The fermentation medium comprises the following components in g/L: 50 parts of cane sugar, 16 parts of yeast powder, 0.3 part of arginine and NaNO₃12，K₂HPO₄7，KH₂PO₄5，MgSO₄·7H₂O 2，FeCl₂0.03,ZnCl₂0.01，MnCl₂·4H₂O0.03, natural pH and the balance of water;

example 5

Three sets of experiments were performed simultaneously, 3-1, 3-2 and 3-3 respectively: and 1 group blank;

HM-618 seeds are selected according to biomass OD₆₀₀Inoculating into fermentation medium at an inoculation density of 0.3, and culturing at 35 deg.C and rotation speed of 300rpm for 110 hr to obtain product containingFermentation broth of surfactant; the yield of surfactant was 1.77 g/L.

The fermentation medium comprises the following components in g/L: sucrose 70, yeast powder 14, lysine 5, NaNO₃8，K₂HPO₄8，KH₂PO₄3，MgSO₄·7H₂O 4，FeCl₂0.01,ZnCl₂0.03，MnCl₂·4H₂O0.01, natural pH and the balance of water;

the lysine in this example was replaced by alanine, cysteine, glycine, methionine, proline, respectively, and other similar examples, the yields of surfactin obtained were, in order: 1.78g/L, 1.75g/L, 1.85g/L, 1.73g/L, 1.72 g/L.

Claims (1)

1. A method for improving the yield of surfactin is characterized by comprising the following steps: the strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HM-618 with the preservation number of CGMCC No.7097 is selected according to the 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; obtaining the surfactant.

The fermentation medium comprises the following components in g/L: 50-70 parts of cane sugar, 14-16 parts of yeast powder, 0.3-5 parts of amino acid and NaNO₃8～12，K₂HPO₄7～8，KH₂PO₄3～5，MgSO₄·7H₂O 2～4，FeCl₂0.01～0.03,ZnCl₂0.01～0.03，MnCl₂·4H₂0.01-0.03 percent of O, natural pH and the balance of water; the amino acid is at least one of alanine, arginine, cysteine, histidine, glycine, lysine, methionine, proline, serine, ornithine and threonine.

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