CN113480623A

CN113480623A - Lipopeptide composition and preparation method and application thereof

Info

Publication number: CN113480623A
Application number: CN202110907387.9A
Authority: CN
Inventors: 李泽勇; 李秋如; 吴海龙; 刘康
Original assignee: Guangzhou Tinci Materials Technology Co Ltd
Current assignee: Guangzhou Tinci Materials Technology Co Ltd
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-10-08
Anticipated expiration: 2041-08-09
Also published as: CN113480623B

Abstract

The invention belongs to the technical field of biosurfactants, and discloses a lipopeptide composition, and a preparation method and application thereof. Adding lysozyme into the fermented lipopeptide fermentation liquor, stirring for reaction, cooling to room temperature, then adding ethanol and N-acyl amino acid type surfactant, stirring and washing, adding microbial flocculant for flocculation and precipitation, centrifuging, taking supernatant, and spray drying to obtain the lipopeptide-N-acyl amino acid type surfactant composition. The method obviously improves the content of the lipopeptide in the fermentation liquor through lysozyme treatment, and can obviously improve the yield and the purity of the lipopeptide by adopting ethanol and N-acyl amino acid type surfactant for washing and desorption and microbial flocculant for precipitation and impurity removal. And the N-acyl amino acid type surfactant has the advantages of easily obtained raw materials, relatively easy synthetic method and easy biodegradation, and the obtained lipopeptide composition has the advantages of good biological safety and low cost. Completely meets the application requirements of daily chemical washing products.

Description

Lipopeptide composition and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biosurfactants, and particularly relates to a lipopeptide composition as well as a preparation method and application thereof.

Background

The biosurfactant is a degradable surfactant. The biosurfactant not only has the common properties of surfactants such as solubilization, emulsification, wetting, foaming, dispersion, surface tension reduction and the like, but also has the advantages of no toxicity, biodegradability, ecological safety, high surface activity and the like compared with other surfactants produced by a chemical synthesis method or a petroleum refining method, and has wide application prospect in the petroleum field. The biosurfactant as a biosurfactant has low toxicity, good compatibility with human bodies and environment, good characteristics of emulsification, dispersion, solubilization and the like. Has good application prospect in the fields of daily chemical products, medicines, environmental protection, petroleum industry and the like.

Lipopeptides are a biosurfactant produced primarily by bacillus subtilis. The peptide chain composed of multiple amino acids in lipopeptide molecule forms hydrophilic group, and the fat forms lipophilic group via the chain. Among them, since a hydrophilic amino acid can be bonded to a carboxyl group, a hydroxyl group, or an amino group in a chain to form a cyclic structure, a lipopeptide biosurfactant is generally a cyclic lipopeptide (cyclic lipopeptide) in which a lactone or an amide bond is bonded. Lipopeptides have strong surface activity, can increase the biodegradability of hydrophobic hydrocarbons, play an important role in bioremediation of hydrocarbon-polluted soil and oceans, can be combined with partial heavy metals to remove heavy metals in polluted soil and sediments, and have certain antibacterial activity and the like. However, low yields have limited the widespread use of lipopeptides. The yield which can be achieved by adopting a single fermentation method at present is generally 5-15 g/L. On the other hand, due to the particularity of lipopeptide production, the cost of product separation and purification is high, and therefore, it is necessary to develop a simple and inexpensive product separation and purification method.

Patent CN 101838621A discloses a preparation method of bacillus subtilis and lipopeptide biosurfactant. The fermentation broth was centrifuged to remove the cells, the pH of the supernatant was adjusted to 2.0 with HCl, the supernatant was placed in a refrigerator at 4 ℃ overnight, and the resulting product was centrifuged to obtain an acid precipitate. However, the lipopeptide product in the fermentation broth has a lower concentration, a higher solubility and a lower yield by means of acid precipitation.

Patent CN 102061333A discloses a preparation method of sodium surfactin. Heating the fermentation liquor to 30-95 ℃, keeping the temperature for 0.1-2 h to reduce the viscosity of the fermentation liquor and improve the filtration property of the fermentation liquor, then adjusting the pH value of the fermentation liquor to 2-5 by using acid to precipitate proteins which are not completely utilized by microorganisms and the surfactin, wherein sodium surfactin is still dissolved in the fermentation liquor, adjusting the pH value of the fermentation liquor to 6-7 by using alkali, the surfactin is renatured and dissolved under the condition of the pH value of 6-7, the proteins which are not completely utilized by the microorganisms cannot be renatured, and when the fermentation liquor is filtered, the thalli and the proteins which are not completely utilized by the microorganisms are removed. And then adjusting the pH value of the filtrate to 2-5 by using an isoelectric point precipitation method, standing for 12-20 h at 0-8 ℃, collecting the precipitate, and drying to obtain the crude extract of the sodium surfactin. However, the patent technology has a large influence on the yield through multiple precipitation and redissolution processes.

Patent CN104045686A discloses a method for processing bacillus subtilis antimicrobial lipopeptide fermentation broth, comprising the following steps: 1) separating the bacillus subtilis antimicrobial lipopeptide fermentation liquor by adopting a centrifugal separation method to remove bacillus subtilis thalli, and collecting clear liquid; 2) adding hydrochloric acid into the clear liquid to adjust the pH value to 3.0, stirring, fully and uniformly mixing, standing for 8 hours, and then performing centrifugal separation to keep precipitates; 3) dissolving the precipitate with pure water with the volume equal to that of the initial fermentation liquid to prepare an antibacterial lipopeptide aqueous solution, treating the antibacterial lipopeptide aqueous solution with a 100kDa polyethersulfone ultrafiltration membrane, and collecting a deep-condensed liquid; 4) adjusting the pH value of the mixed solution to 7.0, adding methanol into the deep-condensed liquid, and slowly stirring for 2 hours; 5) treating the aqueous solution by using a 100kDa polyethersulfone ultrafiltration membrane, and collecting a filtrate; 6) concentrating the filtrate, and spray drying to obtain powdered antibacterial lipopeptide. The patent technology has complex treatment steps, and the prior art usually only utilizes lipopeptide products in fermentation liquor and does not utilize partial products in thalli.

Disclosure of Invention

In view of the above disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide a method for preparing a lipopeptide composition.

Another object of the present invention is to provide a lipopeptide composition prepared by the above method.

Still another object of the present invention is to provide the use of the lipopeptide composition in daily chemical care products.

The purpose of the invention is realized by the following technical scheme:

a method for preparing a lipopeptide composition, comprising the steps of:

(1) adding lysozyme into the fermented lipopeptide fermentation liquor, and stirring for reaction at the temperature of 45-50 ℃ and the pH of 5-7;

(2) cooling the fermentation liquor treated by the bacteriolysis in the step (1) to room temperature, and then adding ethanol and N-acyl amino acid type surfactant, stirring and washing;

(3) and (3) adding a microbial flocculant into the fermentation liquor washed in the step (2) for flocculation and precipitation, centrifuging, taking the supernatant, and performing spray drying to obtain the lipopeptide-N-acyl amino acid type surfactant composition.

Further, the lipopeptide fermentation liquor in the step (1) is fermentation liquor obtained by culturing Bacillus subtilis serving as a production strain in a fermentation culture medium; the content of lipopeptide in the fermentation liquor (fermentation liquor after the thalli are removed by centrifugation) is 5-15 g/L.

Further preferably, the fermentation medium comprises a carbon source, a nitrogen source, inorganic salts and trace elements; the carbon source comprises at least one of glucose, maltose, sucrose and soluble starch, the nitrogen source comprises at least one of yeast powder, yeast fermentation liquor, peptone, nitrate and ammonium salt, the inorganic salt comprises at least one of sodium salt, potassium salt and magnesium salt, and the trace elements comprise at least one of iron elements and manganese elements.

Further, the adding amount of the lysozyme in the step (1) is 0.01-0.05% of the mass of the lipopeptide fermentation liquor.

Further, the stirring reaction time in the step (1) is 0.5-6 h.

Further, the adding amount of the ethanol in the step (2) is 0.6-1.2 times of the volume of the fermentation liquor after the bacteriolysis treatment. The invention can achieve the purposes of defoaming and promoting the elution of lipopeptide by adding ethanol. The verification proves that the adding amount of the ethanol has obvious influence on the yield and the purity of the obtained product. The promotion effect is limited due to the excessively low addition amount of the ethanol, and the yield of the product is not remarkably improved; an excessively high amount of ethanol may adversely affect the purity of the product.

Further, the N-acyl amino acid type surfactant in the step (2) is at least one of lauroyl sarcosine, lauroyl glutamic acid, cocoyl glutamic acid, myristoyl glutamic acid, cocoyl glycine, or a corresponding sodium salt or potassium salt. The adding amount of the N-acyl amino acid type surfactant is 0.5 to 1.5 percent of the mass of the fermentation liquor after the bacteriolysis treatment. The verification proves that the adding amount of the N-acyl amino acid type surfactant has obvious influence on the yield and the purity of the obtained product. Under the condition of adding amount less than 0.5%, the washing effect is poor, and the product yield is low; above 1.5% the product purity is significantly reduced.

Further, the adding amount of the microbial flocculant in the step (3) is 0.1-0.5% of the mass of the washed fermentation liquor. The verification proves that the addition amount of the microbial flocculant has obvious influence on the yield and the purity of the obtained product. Under the condition of adding amount less than 0.1%, the flocculation precipitation effect is poor, and the product purity is reduced; under the condition of adding more than 0.5 percent, partial lipopeptide and N-acyl amino acid are flocculated and precipitated, and the yield of the product is reduced.

Further, the flocculation precipitation in the step (3) is carried out under the heating condition with the temperature of 45-85 ℃. The heating process is beneficial to the coagulation of colloid components after enzymolysis treatment, improves the flocculation effect of solid components and improves the purity of products; and simultaneously, the structure of partial lipopeptide micelles is damaged, and the product yield is improved.

A lipopeptide composition is prepared by the above method.

The lipopeptide composition is applied to daily chemical washing products.

Further, the daily chemical cleaning and caring product comprises shampoo, shower gel, facial cleanser, laundry detergent, washing liquid, liquid soap, soap powder, toothpaste, facial mask liquid, cream, essence repairing liquid, toner or body lotion.

The principle of the invention is as follows: firstly, processing lipopeptide fermentation liquor by lysozyme to further dissolve out lipopeptide in thalli, then adding ethanol and N-acyl amino acid type surfactant, stirring and washing, wherein the N-acyl amino acid type surfactant has a molecular structure similar to that of the lipopeptide, and can promote the desorption of the lipopeptide and enter water phase for dissolution. The addition of ethanol can achieve a certain defoaming effect, and simultaneously destroy part of micelle structures in the fermentation broth, further promote the dissolution of lipopeptide and reduce the flocculation precipitation of the lipopeptide. The three aspects can obviously improve the yield of the lipopeptide. The lysozyme treatment can cause the thallus to be decomposed into small-particle suspended matters, protein colloid components and a small amount of soluble substances (including inorganic salts), the substances are difficult to separate and remove through simple centrifugal treatment, the invention can realize preferential selective adsorption and precipitation of the suspended matters, the protein colloids and the inorganic salt components through heating treatment and flocculation precipitation by adding a microbial flocculant, and then the separation and purification of the lipopeptide and the N-acyl amino acid type surfactant composition can be realized through centrifugal separation.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method can fully utilize the lipopeptide fermentation liquor and the lipopeptide components in the thallus, and compared with the condition of not adding lysozyme for treatment, the lipopeptide content in the fermentation liquor after treatment is improved by more than 15 percent, thereby realizing higher product yield.

(2) The invention adopts the N-acyl amino acid type surfactant with a structure similar to that of the lipopeptide to promote the desorption of the lipopeptide after the bacteriolysis treatment, and can obviously improve the yield of the lipopeptide. And the N-acyl amino acid type surfactant has the advantages of easy acquisition of raw materials, relatively easy synthesis method and easy biodegradation, and the obtained lipopeptide-N-acyl amino acid type surfactant composition has the advantages of good biological safety and low cost. Completely meets the application requirements of daily chemical washing products.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The lipopeptide fermentation broth used in the following examples is a fermentation broth obtained by culturing Bacillus subtilis (Bacillus subtilis) as a production strain in a fermentation medium (6% of maltose, 4% of soluble starch, 0.2% of yeast fermentation broth, 0.1% of peptone, 0.5% of disodium hydrogen phosphate, 1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.003% of ferric sulfate, 0.0005% of manganese sulfate, and the balance water, at a pH of 7); the lipopeptide content in the fermentation broth (after centrifugation to remove the bacteria) was 11.5g/L (the lipopeptide yield in the product of the following examples was calculated using this as the original content, and the lipopeptide content in the product was determined by high performance liquid chromatography).

Example 1

The preparation method of a lipopeptide composition of this embodiment comprises the following steps:

(1) adding lysozyme into the lipopeptide fermentation liquor, adjusting the adding amount of lysozyme to be 0.01%, 0.02%, 0.03%, 0.04% and 0.05% of the mass of the lipopeptide fermentation liquor respectively, and stirring and reacting for 3 hours under the conditions that the temperature is 45-50 ℃ and the pH value is adjusted to be 5.5-6.5 by phosphoric acid. And fermentation broth without lysozyme was used as a blank control.

(2) Cooling the fermentation liquor treated by the bacteriolysis in the step (1) to room temperature, adding ethanol with the volume 1 time of that of the fermentation liquor and sodium lauroyl sarcosinate with the mass 1.2 percent of that of the fermentation liquor, stirring and washing.

(3) And (3) heating the fermentation liquor washed in the step (2) to 75 ℃, preserving heat for 1h, then adding a microbial flocculant accounting for 0.3% of the mass of the fermentation liquor for flocculation and precipitation, centrifuging, taking the supernatant, and performing spray drying to obtain the lipopeptide-sodium lauroylsarcosine composition.

The content of lipopeptide in the fermentation broth after bacteriolysis treatment in the step (1) of this example is detected, and the results of lipopeptide content in the fermentation broth obtained under the condition of different lysozyme addition amounts and the calculation results of the yield and purity of lipopeptide in the final product are shown in the following table 1.

TABLE 1

As can be seen from the results in Table 1, with the increase of the amount of lysozyme added, the lipopeptide content and yield in the fermentation broth are both significantly improved, and the lysozyme added tends to be stable when the amount is increased to 0.04%. The lysozyme treatment enables partial lipopeptide products in the thallus to be dissolved out, thereby obviously improving the yield. The addition of lysozyme had no significant effect on the lipopeptide purity.

Example 2

(1) adding lysozyme into the lipopeptide fermentation liquor, wherein the adding amount of the lysozyme is 0.04 percent of the mass of the lipopeptide fermentation liquor, and stirring and reacting for 3 hours under the conditions that the temperature is 45-50 ℃ and the pH value is adjusted to 5.5-6.5 by phosphoric acid.

(2) Cooling the fermentation liquor treated by the bacteriolysis in the step (1) to room temperature, then respectively adding ethanol with the volume of 0.4, 0.6, 0.8, 1.0, 1.2 and 1.5 times of the fermentation liquor and sodium lauroyl sarcosine with the mass of 1.2 percent of the fermentation liquor, stirring and washing. And blank control was performed without ethanol.

The results of the calculation of the yield and purity of lipopeptide in the product obtained in this example with different amounts of ethanol added are shown in Table 2 below.

TABLE 2

Amount of ethanol added

Blank control

0.4 times of

0.6 times of

0.8 times of

1.0 times of

1.2 times of

1.5 times of

Yield of the product

67.6％

71.8％

83.4％

85.2％

86.1％

86.6％

86.9％

Purity of

86.0％

86.4％

85.8％

85.2％

84.3％

76.4％

As shown in the results in Table 2, the method can remarkably improve the yield of the lipopeptide by adding ethanol and sodium lauroyl sarcosinate for washing together. Indicating that ethanol can destroy part of lipopeptide micelle structure and reduce subsequent loss in flocculation precipitation. The purity of the lipopeptide is not greatly influenced by the adding amount of ethanol within a certain range, but the purity is obviously reduced by adding excessive ethanol (more than 1.2 times), and a large amount of soluble impurities are possibly dissolved by excessive ethanol.

Example 3

(2) Cooling the fermentation liquor treated by the bacteriolysis in the step (1) to room temperature, then adding ethanol with the volume of 1.0 time of that of the fermentation liquor, and adding sodium lauroyl sarcosinate with the mass of 0.5%, 0.75%, 1.0%, 1.2% and 1.5% of the fermentation liquor respectively, stirring and washing. And blank control was performed without addition of sodium lauroyl sarcosinate.

The results of the calculation of the yield and purity of lipopeptide in the product obtained in this example with different amounts of sodium lauroyl sarcosinate are shown in Table 3 below.

TABLE 3

Sodium lauroyl sarcosinate

Blank control

0.5％

0.75％

1.0％

1.2％

1.5％

Yield of the product

54.5％

81.3％

84.5％

85.8％

86.6％

87.0％

Purity of

85.7％

85.6％

85.1％

84.7％

84.3％

76.9％

As can be seen from the results in Table 3, the method of the invention, by adding sodium lauroyl sarcosinate for mixing and washing, has the advantages that the yield of lipopeptide is obviously improved compared with the condition of not adding sodium lauroyl sarcosinate, and the lipopeptide is in an increasing trend along with the increase of the addition amount of the sodium lauroyl sarcosinate. However, if the amount is too high, the purity of the lipopeptide may be reduced.

Example 4

(2) Cooling the fermentation liquor treated by the bacteriolysis in the step (1) to room temperature, adding ethanol with the volume 1.0 time of that of the fermentation liquor and sodium lauroyl sarcosinate with the mass 1.2 percent of that of the fermentation liquor, stirring and washing.

(3) Heating the fermentation liquor washed in the step (2) to 45 ℃, 55 ℃, 65 ℃, 75 ℃ and 85 ℃ respectively, and keeping the temperature for 1h, wherein the condition of room temperature (25 ℃) is used as a control. Then adding a microbial flocculant accounting for 0.3 percent of the mass of the fermentation liquor for flocculation and precipitation, centrifuging, taking supernatant fluid for spray drying, and obtaining the lipopeptide-sodium lauroylsarcosine composition.

The results of the calculation of the yield and purity of lipopeptide in the product obtained in this example under different flocculation precipitation temperature conditions are shown in Table 4 below.

TABLE 4

Temperature of flocculation and precipitation

25℃

45℃

55℃

65℃

75℃

85℃

Yield of the product

78.7％

82.1％

83.9％

85.7％

86.6％

87.4％

Purity of

76.2％

81.7％

83.0％

84.9％

85.8％

84.6％

As can be seen from the results in Table 4, as the flocculation temperature increases, the lipopeptide yield and purity both show an increasing trend, indicating that the heat treatment helps the flocculation of impurities and destroys the lipopeptide micelles, reducing the flocculation loss of lipopeptide.

Example 5

(3) And (3) heating the fermentation liquor washed in the step (2) to 75 ℃, preserving heat for 1h, then respectively adding microbial flocculants with the mass of 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6% of the fermentation liquor for flocculation and precipitation, taking the condition without adding the microbial flocculants as a blank control, centrifuging, taking the supernatant, and performing spray drying to obtain the lipopeptide-sodium lauroylsarcosine composition.

The results of the calculation of the yield and purity of lipopeptide in the product obtained by the present example with different amounts of microbial flocculant are shown in Table 5 below.

TABLE 5

As can be seen from the results in Table 5, as the amount of the microbial flocculant added increases, the purity of the lipopeptide tends to increase significantly, and the yield of the lipopeptide tends to decrease, indicating that a part of the lipopeptide is inevitably adsorbed by the microbial flocculant. The invention can make up the lipopeptide loss generated in the flocculation precipitation process by the modes of lysozyme treatment, ethanol and sodium lauroyl sarcosine stirring washing and flocculation precipitation under heating condition. Under the condition that the addition amount of the microbial flocculant is 0.1-0.5%, the yield and the purity of the lipopeptide can reach more than 75%.

Example 6

(2) And (2) cooling the fermentation liquor treated in the step (1) to room temperature, then adding ethanol with the volume 1.0 time of that of the fermentation liquor, and adding lauroyl sarcosine, myristoyl sodium glutamate and cocoyl potassium glycinate with the mass 1.2% of that of the fermentation liquor respectively, and stirring and washing.

The results of the calculation of the yield and purity of lipopeptide in the products obtained by adding different N-acyl amino acid surfactants are shown in Table 6 below.

TABLE 6

Surfactant species	Lauroyl sarcosine	Myristic acid monosodium glutamate	Coconut oil acyl potassium glycinate
				Yield of the product	85.7％	86.9％	87.1％
Purity of	87.2％	85.7％	86.0％

As can be seen from the results in Table 6, the lipopeptide yield and purity of the products obtained under different washing conditions with N-acyl amino acid surfactants can reach 85% or more.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for preparing a lipopeptide composition, comprising the steps of:

2. The method of claim 1, wherein the lipopeptide fermentation broth in step (1) is a fermentation broth obtained by culturing Bacillus subtilis as a production strain in a fermentation medium; the content of the lipopeptide in the fermentation liquor is 5-15 g/L.

3. The method of claim 2, wherein the fermentation medium comprises a carbon source, a nitrogen source, inorganic salts, and trace elements; the carbon source comprises at least one of glucose, maltose, sucrose and soluble starch, the nitrogen source comprises at least one of yeast powder, yeast fermentation liquor, peptone, nitrate and ammonium salt, the inorganic salt comprises at least one of sodium salt, potassium salt and magnesium salt, and the trace elements comprise at least one of iron elements and manganese elements.

4. The method for preparing a lipopeptide composition according to claim 1, wherein the lysozyme is added in the step (1) in an amount of 0.01-0.05% by mass of the lipopeptide fermentation broth; the stirring reaction time is 0.5-6 h.

5. The method according to claim 1, wherein the ethanol is added in an amount of 0.6 to 1.2 times the volume of the fermentation broth after the lysis treatment in the step (2).

6. The method of claim 1, wherein the N-acyl amino acid surfactant in step (2) is at least one of lauroyl sarcosine, lauroyl glutamic acid, cocoyl glutamic acid, myristoyl glutamic acid, cocoyl glycine, or the corresponding sodium or potassium salt; the adding amount of the N-acyl amino acid type surfactant is 0.5 to 1.5 percent of the mass of the fermentation liquor after the bacteriolysis treatment.

7. The method of claim 1, wherein the amount of the microbial flocculant added in step (3) is 0.1-0.5% by mass of the washed fermentation broth.

8. The method of claim 1, wherein the flocculation precipitation in step (3) is performed at a temperature of 45-85 ℃.

9. A lipopeptide composition prepared by the method of any one of claims 1 to 8.

10. The lipopeptide composition of claim 9, wherein the daily chemical care product comprises a shampoo, a body wash, a face wash, a laundry detergent, a hand sanitizer, a liquid detergent, a soap powder, a perfumed soap, a toothpaste, a facial mask, a cream, an essence repair liquid, a toner, or a body lotion.