A preparation process for high-yield surfactants by combined fermentation of Polygonatum sibiricum and Acinetobacter jungii

Abstract

The application provides a preparation process of a high-yield surfactant compound fermented by Polygonatum and Acinetobacter junii, and the preparation process comprises the following steps: S1, obtaining Acinetobacter junii strains; S2, performing fermentation culture on the Acinetobacter junii to obtain Acinetobacter junii secondary seed liquid; S3, inoculating the Acinetobacter junii secondary seed liquid into a fermentation culture medium containing Polygonatum micro powder, and performing semi-solid intermittent feeding fermentation to obtain surfactant fermentation liquid; S4, performing centrifugation, extraction, reduced-pressure evaporation and drying treatment on the fermentation liquid to obtain a crude extract of the surfactant fermentation liquid; and S5, performing surfactant fermentation liquid activity determination; the preparation process solves the problems of low yield of surfactant components produced by the existing Acinetobacter junii fermentation liquid, long fermentation period, low extraction yield and single increase of fermentation liquid activity.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a preparation process for high-yield surfactants by combined fermentation of Polygonatum sibiricum and Acinetobacter jungi. Background Technology

[0002] Microbial surfactants are biomolecules synthesized by microorganisms and possess the ability to reduce surface and interfacial tension. In addition to the general characteristics of surfactants, microbial surfactants also exhibit low toxicity, easy degradation, and structural specificity. Microbial surfactants have excellent application potential. In environmental pollution control, they can serve as accelerants for biodegradation by reducing interfacial tension, promoting emulsification, increasing the oil-water interface area, facilitating direct contact between cells and larger pollutant droplets, and enabling bacteria to absorb oil droplets smaller than themselves, thereby promoting pollutant degradation. Microbial surfactants are natural surfactants with high surface activity, combining hydrophilic and hydrophobic groups, obtained from microorganisms, plants, and animals through biocatalysis and biosynthesis using enzymes or microorganisms. Microbial surfactants are rising stars in the surfactant family. Compared with general chemical surfactants, they not only have the characteristics of significantly reducing surface tension, stabilizing emulsions, and having lower critical micelle concentrations, but also are non-toxic or low-toxic; have good selectivity, specificity, and biocompatibility; have diverse structures; have pharmacological effects and immune functions such as antibacterial, antiviral, and antitumor effects; are biodegradable and environmentally friendly; and can be produced from industrial waste, which is beneficial to environmental remediation.

[0003] Numerous studies have reported on various types of biosurfactants and their producing microorganisms. Surfactants produced by biological metabolism are generally classified into two main categories: biosurfactants with relatively small molecular weights and bioemulsifiers with relatively large molecular weights. Microorganisms that metabolize biosurfactants mainly include *Pseudomonas*, *Bacillus*, *Rhodococcus*, and *Nocardia*, with their main metabolic products being glycolipids and lipopeptides. Microorganisms that metabolize bioemulsifiers mainly include *Acinetobacter*, *Bacillus*, and *Geobacillus*, with their main metabolic products being glycoproteins and lipopolysaccharides. However, existing *Acinetobacter jumbo* fermentation broths produce surfactants with low yields, long fermentation cycles, low extraction rates, and limited activity in the fermentation broth. Therefore, developing new varieties of biosurfactants and their preparation processes is a pressing technical challenge that needs to be addressed. Summary of the Invention

[0004] To address the problems in the existing technology, this invention provides a preparation process for high-yield surfactants by combined fermentation of Polygonatum sibiricum and Acinetobacter juncea, aiming to solve the problems of low yield of surfactant components, long fermentation cycle, low extraction yield and single activity of the fermentation broth produced by existing Acinetobacter juncea fermentation broth.

[0005] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0006] A process for preparing high-yield surfactants by co-fermentation of Polygonatum sibiricum and Acinetobacter juncea, the process comprising the following steps:

[0007] S1. Obtain Acinetobacter juncus strain:

[0008] The collected soil samples were enriched and cultured and then screened; the selected strains were identified as Acinetobacter jungi using physiological, biochemical, and molecular biological methods.

[0009] S2. Acinetobacter jungle was fermented to obtain a secondary seed culture of Acinetobacter jungle;

[0010] S3. The secondary seed culture of Acinetobacter jumbo was inoculated into a fermentation medium containing Polygonatum sibiricum powder and semi-solid intermittent fed fermentation was carried out to obtain the surfactant fermentation broth.

[0011] S4. The fermentation broth is centrifuged, extracted, evaporated under reduced pressure and dried to obtain a crude extract of the surfactant fermentation broth.

[0012] S5. Determination of surfactant fermentation broth activity.

[0013] Step S1 of the present invention includes the following steps:

[0014] (a) Isolation and screening of bacterial strains that produce biosurfactants:

[0015] The collected soil samples were inoculated with physiological saline and cultured at 37℃ and 180 rpm for 2 hours with constant temperature shaking. After standing for 0.5 hours, the supernatant was collected and placed in YPD fermentation medium for enrichment culture at 37℃ and 180 rpm for 24 hours. The samples were then diluted and plated on blood agar plates and cultured at 37℃ for 3–7 days. Strains with large hemolysis zones were selected and purified by streaking on YPD agar medium and cultured at 37℃ for 1–3 days. Single colonies were then inoculated into YPD fermentation medium and cultured at 37℃ and 180 rpm for 3–7 days. Finally, strains were screened by measuring the size of the oil expulsion zone and surface tension.

[0016] (b) Identification of the strains that produce biosurfactants:

[0017] The selected biosurfactant-producing strains were subjected to molecular biological identification and 16S rRNA sequence analysis. Through sequence alignment, they were confirmed to be Acinetobacter junii and named Acinetobacter junii 1031. They were then deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 1.8037.

[0018] The YPD fermentation medium of this invention consists of a solute and a solvent, with water as the solvent. The solute and its mass fraction in the liquid medium are: 1% yeast extract, 2% peptone, 1% glucose, and a pH of 7.0.

[0019] The YPD agar medium of the present invention consists of a solute and a solvent, the solvent being water, and the solute and its mass fraction in the liquid medium being: 1% yeast extract, 2% peptone, 1% glucose, 2% agar powder, with a pH of 7.0.

[0020] Step S2 of the present invention includes the following steps:

[0021] (1) Primary seed culture of Acinetobacter jung: The isolated and purified Acinetobacter jung was inoculated into sterilized YPD fermentation medium and cultured in a shaker at 37°C for 8 to 16 hours. The speed of the shaker was 180 rpm and the volume of the shake flask was 20% of the total volume. When the OD value was 0.6 to 3, the primary seed culture of Acinetobacter jung was obtained.

[0022] (2) Secondary seed culture of Acinetobacter jung: The primary seed culture from step S1 was inoculated into sterilized YPD fermentation medium at an inoculation rate of 3%, and cultured in a shaker at 37°C for 5-14 hours. The shaker speed was 180 rpm, and the volume of the shake flask was 30% of the total volume. When the OD value was ≥3, the secondary seed culture of Acinetobacter jung was obtained.

[0023] The preparation process of the Polygonatum odoratum micro powder in step S3 of this invention is as follows:

[0024] Polygonatum was first-stage pulverized using a high-speed pulverizer to obtain coarse powder. The coarse powder was then passed through an 80-200 mesh sieve to remove impurities. The impurity-removed coarse powder was then second-stage pulverized using a ball mill and passed through a 400 mesh sieve to obtain micro powder with a particle size of 2-75 μm.

[0025] Step S3 of the present invention includes the following steps:

[0026] The secondary seed culture from step S2 was inoculated into a fermentation medium containing Polygonatum sibiricum powder at an inoculation rate of 0.2-20%. The pH was controlled at 5-8 and the dissolved oxygen at 15-30%. The rotation speed and dissolved oxygen were connected in series. The initial rotation speed was 60-250 r / min and the aeration rate was 0.6-1.5 vvm. After fermentation for 8-16 hours, feeding was started. When the rotation speed reached the upper limit and the dissolved oxygen rose to 50-60%, feeding was intermittently added to the feed medium. Fermentation was stopped after 30-48 hours to obtain the surfactant fermentation broth.

[0027] The fermentation medium containing Polygonatum sibiricum powder of the present invention comprises: 100 g / L Polygonatum sibiricum powder, 5 g / L glucose, 15 g / L NaNO3, 2 g / L yeast extract, 15 g / L peptone, 0.12 g / L MgSO4, 3.6 g / L KH2PO4, 1.5 g / L K2HPO4, 0.02 g / L FeSO4·7H2O, 0.5% (w / V) L-glutamate sodium, 0.00003 g / L cyanocobalamin, and pH 7.

[0028] The supplemental culture medium of the present invention comprises: 40 g / L beef extract, 100 g / L molasses, 0.3 g / L biotin, 8 g / L CuSO4, and 20 g / L ammonium sulfate.

[0029] Step S4 of the present invention includes the following steps:

[0030] The surfactant fermentation broth is centrifuged to remove cells and impurities, yielding a supernatant. An extractant is added to the supernatant in an equal proportion and mixed thoroughly. The lower layer of extract is collected and evaporated under reduced pressure at 50–60°C. The powder obtained after drying the extract is the crude extract of the fermentation broth containing surfactant.

[0031] The extractant of this invention is one or more of chloroform, petroleum ether, dichloromethane, ethyl acetate, ferulic acid, and matrine ionic liquid.

[0032] The activity determination in step S5 of this invention includes the following steps:

[0033] a. Determination of DPPH free radical scavenging ability

[0034] Weigh a certain amount of DPPH and prepare a 0.04 mg / mL DPPH solution with anhydrous ethanol; take 2 mL of solutions of different concentrations (2, 4, 6, 8 mg / mL), add 2 mL of DPPH solution, mix well, let stand at room temperature for 30 min, and then centrifuge at 5000 r / min for 10 min; take the supernatant and measure the absorbance at 517 nm, using Vc as a positive control;

[0035] The scavenging rate of the sample against DPPH free radicals was calculated using the following formula:

[0036]

[0037] A0—Absorbance value of 2ml anhydrous ethanol + 2ml DPPH solution

[0038] A1—Absorbance value of 2ml sample solution + 2ml DPPH solution

[0039] A2—Absorbance value of 2ml sample solution + 2ml anhydrous ethanol

[0040] b. Oil drain ring activity

[0041] Add 60ml of deionized water to a glass petri dish (d=9cm), then add 8ml of liquid paraffin. Let it stand until the liquid paraffin is evenly distributed on the water surface. Add 1ml of the supernatant after centrifugation to the surface and measure the diameter of the oil draining ring.

[0042] As can be seen from the above description, the present invention has the following advantages:

[0043] (1) In view of the low surfactant yield of Acinetobacter jumbo, the present invention adopts a semi-solid intermittent fed fermentation method and optimizes the fermentation medium, which significantly improves the surfactant yield of Acinetobacter jumbo from 0.02 g / L to 41.36 g / L.

[0044] (2) The present invention uses Acinetobacter juncea and Polygonatum sibiricum powder for compound fermentation, which solves the problem of single activity of Acinetobacter juncea fermentation broth and promotes Acinetobacter juncea to produce both surface activity and antioxidant capacity during fermentation. Attached Figure Description

[0045] Figure 1 shows a microscopic photograph of Acinetobacter junii 1031.

[0046] Figure 2 shows the gene sequence listing of Acinetobacter junii 1031. Detailed Implementation

[0047] The features of the present invention are further illustrated below through examples, but these examples do not limit the scope of the claims of the present invention in any way.

[0048] Example 1

[0049] (1) Isolation and screening of bacterial strains that produce biosurfactants

[0050] The screening method for microorganisms producing biosurfactants was as follows: 10g of soil samples collected from Hangzhou, Xinjiang, Jiaxing, Huzhou, and other locations were inoculated into 90mL of physiological saline and cultured at 37℃ with constant temperature shaking at 180rpm for 2 hours. After standing for 0.5 hours, 10mL of the supernatant was taken and placed in 90mL of YPD medium for enrichment culture at 37℃ and 180rpm for 24 hours. After 24 hours of culture, the supernatant was diluted and plated onto blood agar plates and cultured at 37℃ for 3 days. Strains with large hemolysis zones were selected and purified by streaking on YPD agar plates and cultured at 37℃ for 12 hours. Single colonies were inoculated into YPD fermentation medium and cultured at 37℃ and 180rpm for 3 days. The surface tension, oil expulsion zone diameter, and crude extract yield of the fermentation broth were measured, and the strain with the highest surfactant yield was selected.

[0051] In this embodiment, through preliminary screening of surfactant-producing microorganisms, and after screening of single colonies in the samples, a total of 200 single strains that produce hemolytic zones were isolated. The strain with the largest oil drainage zone diameter in the fermentation broth was selected and named 10~3~1, with an oil drainage zone diameter of 6cm. The surface tension of the 10~3~1 fermentation broth was measured using a surface tension meter, which showed that the surface tension of water could be reduced from 72.75mN / m to 35.32mN / m.

[0052] The pure strain obtained above was cultured in 100 ml YPD medium for 72 h, then centrifuged to remove bacterial cells. The pH of the fermentation supernatant was adjusted to 2 with citric acid, and the mixture was allowed to stand at 4 °C for 12 h. After centrifugation, the supernatant was discarded, and the precipitate was freeze-dried for 24 h. The precipitate was then weighed. Acinetobacter junii 1031 was fermented in YPD medium for 5 days, and the surfactant yield was 0.02 g / L.

[0053] (2) Identification of strains that produce biosurfactants

[0054] The selected strain was identified using molecular biology methods. Its 16S rRNA sequence was determined as shown in the gene sequence listing. After comparison with databases, the strain was confirmed as *Acinetobacter junii*, named *Acinetobacter junii* 1031, and deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 1.8037. Figure 1 shows a microscopic photograph of *Acinetobacter junii* 1031. Figure 2 shows the gene sequence listing of *Acinetobacter junii* 1031.

[0055] Example 2

[0056] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0057] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0058] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0059] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0060] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0061] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0062] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0063] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0064] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the ventilation rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0065] 4. Extraction of crude surfactant extract

[0066] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder remaining after drying was the crude extract of the fermentation broth containing surfactant, with a yield of 41.36 g / L and an extraction rate of 96.5%.

[0067] Comparative Example 1

[0068] 1. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0069] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0070] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0071] 2. Liquid fermentation

[0072] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of glucose 5%, NaNO3 15%, yeast extract 2%, peptone 15%, MgSO4 0.12%, KH2PO4 3.6%, K2HPO4 1.5%, FeSO4·7H2O 0.02%, L-glutamate sodium 5%, and cyanocobalamin 0.00003%, with the initial pH at rest. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. After 10 hours of fermentation, feeding was initiated. The feeding conditions were as follows: when the aeration rate reached the upper limit and the dissolved oxygen level rose to 50-60%, 200 g / L of feed medium (g / L: beef extract 40%, molasses 100%, biotin 0.3%, CuSO4 8%, ammonium sulfate 20%) was intermittently added. Fermentation was stopped after 30 hours.

[0073] 3. Extraction of crude surfactant extract

[0074] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then added to the extractant in equal proportions. The extractant used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a yield of 2.6 g / L.

[0075] Comparative Example 2

[0076] 1. Use a slicer to cut the Polygonatum rhizome into 3mm thick slices, set aside.

[0077] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0078] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0079] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0080] 3. Fermentation of Polygonatum slices mixed with Acinetobacter juncea secondary seed.

[0081] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum slices, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0082] 4. Extraction of crude surfactant extract

[0083] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a yield of 5.11 g / L.

[0084] Example 3

[0085] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0086] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0087] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0088] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0089] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0090] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0091] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0092] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0093] The secondary seed culture was inoculated into a 5L fermenter at an inoculum volume of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. Fermentation was stopped after 30 hours.

[0094] 4. Extraction of crude surfactant extract

[0095] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then added to the extractant in equal proportions. The extractant used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a yield of 15.44 g / L.

[0096] Example 4

[0097] 1. The steps for preparing traditional Chinese medicine micro powder include:

[0098] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0099] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0100] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0101] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0102] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0103] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0104] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0105] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the ventilation rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0106] 4. Extraction of crude surfactant extract

[0107] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in a 1:2 ratio (chloroform:ethyl acetate). The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant. The yield was 19.78 g / L, and the extraction rate was 48.24%.

[0108] Example 5

[0109] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0110] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0111] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0112] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0113] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0114] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0115] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0116] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0117] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the ventilation rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0118] 4. Extraction of crude surfactant extract

[0119] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacteria and impurities. Citric acid was added to the supernatant to adjust the pH to 2. The mixture was allowed to stand at 4℃ for 12 hours. After standing, the precipitate was centrifuged and freeze-dried. The crude extract containing surfactant contained 6.15g / L, with an extraction rate of 15.11%.

[0120] Example 6

[0121] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0122] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0123] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0124] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0125] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0126] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0127] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0128] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0129] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 0.2%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0130] 4. Extraction of crude surfactant extract

[0131] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a content of 10.45g / L.

[0132] Example 7

[0133] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0134] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0135] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0136] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0137] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0138] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0139] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0140] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0141] The secondary seed culture was inoculated into a 5L fermenter at a 20% inoculation rate. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0142] 4. Extraction of crude surfactant extract

[0143] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a content of 17.45g / L.

[0144] Example 8

[0145] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0146] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0147] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0148] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0149] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0150] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0151] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0152] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0153] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 5, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0154] 4. Extraction of crude surfactant extract

[0155] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a content of 8.74g / L.

[0156] Example 9

[0157] 1. The preparation steps of the traditional Chinese medicine Polygonatum odoratum powder include:

[0158] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0159] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0160] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0161] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0162] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0163] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0164] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0165] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 11, dissolved oxygen at 15-30%, and the aeration rate was 0.8 vvm, with the aeration rate connected in series with the dissolved oxygen level. The initial aeration rate was 60-250 r / min. After 10 hours of fermentation, feeding was started. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0166] 4. Extraction of crude surfactant extract

[0167] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a content of 2.45g / L.

[0168] The effects of fermentation conditions on fermentation activity are shown in the table below:

[0169] serial number DPPH activity (%) Oil drain ring diameter (cm) Blank group (water) 0 0 Example 2 61.9 6.6 Comparative Example 1 2.47 1.9 Comparative Example 2 14.24. 2.5 Example 6 13.75 0.9 Example 7 14.55 1.6 Example 8 10.01 2.2 Example 9 5.9 1.9

[0170] Example 10

[0171] 1. The steps for preparing traditional Chinese medicine micro powder include:

[0172] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0173] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0174] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0175] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0176] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0177] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0178] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0179] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g / L Polygonatum sibiricum powder, 5 g / L glucose, 15 g / L NaNO3, 2 g / L yeast extract, 15 g / L peptone, 0.1 g / L MgSO4, 3.6 g / L KH2PO4, 1.5 g / L K2HPO4, 0.0 g / L FeSO4·7H2O, and 5 g / L L-glutamate. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. After 10 hours of fermentation, feeding was started. The feeding conditions were as follows: when the aeration rate reached the upper limit and the dissolved oxygen level rose to 50-60%, 200 g / L of feed medium (g / L: 40 g / L beef extract, 100 g / L molasses, 0.3 g / L biotin, 8 g / L CuSO4, 20 g / L ammonium sulfate) was added intermittently. Fermentation was stopped after 30 hours.

[0180] 4. Extraction of crude surfactant extract

[0181] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a content of 11.25g / L.

[0182] Example 11

[0183] 1. The steps for preparing traditional Chinese medicine micro powder include:

[0184] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0185] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0186] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0187] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0188] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0189] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0190] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0191] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g / L Polygonatum sibiricum powder, 5 g / L glucose, 15 g / L NaNO3, 2 g / L yeast extract, 15 g / L peptone, 0.1 g / L MgSO4, 3.6 g / L KH2PO4, 1.5 g / L K2HPO4, 0.0 g / L FeSO4·7H2O, and 0.00003 g / L cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 5, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the aeration rate was 0.8 vvm. After 10 hours of fermentation, feeding was started. The feeding conditions were as follows: when the aeration rate reached the upper limit and the dissolved oxygen level rose to 50-60%, 200 g / L of feed medium (g / L: 40 g / L beef extract, 100 g / L molasses, 0.3 g / L biotin, 8 g / L CuSO4, and 20 g / L ammonium sulfate) was added intermittently. Fermentation was stopped after 30 hours.

[0192] 4. Extraction of crude surfactant extract

[0193] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder obtained after drying was the crude extract of the fermentation broth containing surfactant, with a content of 12.75g / L.

[0194] Example 12

[0195] 1. The steps for preparing traditional Chinese medicine micro powder include:

[0196] (1) Primary grinding: Polygonatum sibiricum is first-grinded using a high-speed grinder to obtain coarse powder of Polygonatum sibiricum.

[0197] (2) Sieving: Pass the coarse powder of Polygonatum sibiricum through an 80-mesh sieve to remove impurities and set aside.

[0198] (3) Secondary grinding: The coarse powder of Polygonatum odoratum after impurity removal in step (2) is subjected to secondary grinding using a ball mill, and the powder is passed through a 400-mesh sieve to obtain 35μm fine powder of Polygonatum odoratum, which is then set aside.

[0199] 2. Preparation of primary and secondary seed cultures of Acinetobacter juncea.

[0200] (1) Primary seed culture: The isolated and purified Acinetobacter jongii 1031 was inoculated into sterilized YPD seed culture medium and cultured in a shaker at 37°C for 8 hours to form a primary seed culture solution. The shaking speed was 180 rpm and the liquid volume in the shake flask was 20% of the total volume. When the OD value was 1, the next primary seed culture solution could be collected.

[0201] (2) Secondary seed culture: The primary seed culture solution is inoculated into the sterilized YPD seed culture medium at an inoculation rate of 3% by volume, and the secondary seed culture solution is formed by shaking and culturing at 37°C for 16 hours. The shaking speed is 180 rpm, and the liquid volume in the shake flask is 30% of the total volume. When the OD value is 3, the secondary seed solution is obtained.

[0202] 3. Semi-solid intermittent fed-batch fermentation of Polygonatum sibiricum powder and Acinetobacter juncea secondary seed.

[0203] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 13%. The fermentation medium (g / L) consisted of 100 g of Polygonatum sibiricum powder, 5 g of glucose, 15 g of NaNO3, 2 g of yeast extract, 15 g of peptone, 0.1 g of MgSO4, 3.6 g of KH2PO4, 1.5 g of K2HPO4, 0.02 g of FeSO4·7H2O, 5 g of L-glutamate, and 0.00003 g of cyanocobalamin. The initial pH was natural. During fermentation, the pH was controlled at 7, dissolved oxygen at 15-30%, and the aeration rate was set in series with the dissolved oxygen level. The initial aeration rate was 60-250 rpm, and the ventilation rate was 0.8 vvm. Feeding was started after 10 hours of fermentation. The feeding conditions are as follows: when the rotation speed reaches the upper limit and the dissolved oxygen rises to 50-60%, intermittently feed 200g / L of feeding medium (g / L: beef extract 40, molasses 100, biotin 0.3, CuSO4 8, ammonium sulfate 20), ferment for 30h, and then stop fermentation.

[0204] 4. Extraction of crude surfactant extract

[0205] The fermentation broth was centrifuged at 10,000g for 15 minutes to remove bacterial cells and impurities. The supernatant was then mixed with an extraction solvent in equal proportions. The extraction solvent used was a chloroform / ethyl acetate / ferulic acid-matrine ionic liquid in a ratio of chloroform:ethyl acetate:ferulic acid-matrine ionic liquid = 2:8:1. The mixture was thoroughly mixed, and the lower layer of extract was collected. The extract was evaporated under reduced pressure at 50℃, and the powder remaining after drying was the crude extract of the fermentation broth containing surfactant, with a yield of 41.36 g / L and an extraction rate of 96.5%.

[0206] It is understood that the above detailed description of the present invention is for illustrative purposes only and is not intended to limit the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effects; as long as the usage requirements are met, they are all within the protection scope of the present invention.

Claims

1. A preparation process for high-yield surfactant complex fermentation of Polygonatum sibiricum and Acinetobacter junii, characterized by, The preparation process comprises the following steps: S1, obtaining Acinetobacter junii species: The collected soil sample is subjected to enrichment culture and screening; the strain obtained through screening is identified through physiological and biochemical and molecular biology methods to obtain Acinetobacter junii; the preservation number of the Acinetobacter junii is CGMCC NO.1.8037; S2, fermenting and culturing the Acinetobacter junii to obtain Acinetobacter junii secondary seed liquid; S3, inoculating the Acinetobacter junii secondary seed liquid into a fermentation medium containing rhizoma polygonati powder to perform semi-solid intermittent feeding fermentation to obtain surfactant fermentation liquid; S4, performing centrifugation, extraction, reduced-pressure evaporation and drying treatment on the fermentation liquid to obtain crude extract of the surfactant fermentation liquid; S5, surfactant fermentation liquid activity determination; The preparation method of the rhizoma polygonati powder in step S3 is as follows: The rhizoma polygonati is subjected to primary crushing by using a high-speed crusher to obtain rhizoma polygonati coarse powder, the rhizoma polygonati coarse powder is screened through a 80-200 mesh sieve, impurities are removed, and the rhizoma polygonati coarse powder after the removal of impurities is subjected to secondary crushing by using a ball mill and is screened through a 400 mesh sieve to obtain rhizoma polygonati powder with a particle size of 2-75 μm; The fermentation medium containing the rhizoma polygonati powder comprises: 100 g / L of rhizoma polygonati powder, 5 g / L of glucose, 15 g / L of NaNO3, 2 g / L of yeast extract, 15 g / L of peptone, 0.12 g / L of MgSO4, 3.6 g / L of KH2PO4, 1.5 g / L of K2HPO4, 0.02 g / L of FeSO4·7H2O, 0.5% (w / V) of L-glutamic acid sodium, 0.00003 g / L of cyanocobalamin, and the pH value is 7; The extraction agent of the extraction is chloroform / ethyl acetate / asperulosidic acid matrine ionic liquid.

2. The manufacturing process according to claim 1, characterized in that, The step S1 comprises the following steps: (a) producing a surfactant-producing strain: The collected soil sample is inoculated into physiological saline, and is subjected to constant-temperature oscillation culture at 37℃ and 180 rpm for 2 h, is left to stand for 0.5 h, and then the supernatant is taken and is put into YPD fermentation medium to perform enrichment culture at 37℃ and 180 rpm for 24 h; is diluted and is coated on a blood plate, is left to stand for culture at 37℃ for 3-7 d, and then strains with large hemolytic rings are picked and are streaked on YPD agar medium to perform purification, and are cultured at 37℃ for 1-3 d; single colonies are picked and are inoculated into YPD fermentation medium, and are cultured at 37℃ and 180 rpm for 3-7 d; finally, the strains are screened through oil discharge ring size and surface tension determination methods; (b) identification of the surfactant-producing strain: The surfactant-producing strain obtained through screening is subjected to molecular biology identification and 16S rRNA sequence analysis; through sequence alignment, it is confirmed as Acinetobacter junii, is named as Acinetobacter junii 1031, and is registered and preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.1.8037; The YPD fermentation medium is composed of solutes and solvents, the solvent is water, and the mass fraction of the solutes in the liquid medium is as follows: 1% of yeast extract, 2% of peptone, 1% of glucose, and the pH value is 7.0; The YPD agar culture medium is composed of solutes and solvent, the solvent is water, and the mass fraction of the solutes in the liquid culture medium is as follows: yeast extract 1%, peptone 2%, glucose 1%, agar powder 2%, and pH value is 7.

0.

3. The manufacturing process of claim 1, wherein, The step S2 comprises the following steps: (1) Acinetobacter baumannii primary seed culture: inoculate the separated and purified Acinetobacter baumannii into the sterilized YPD fermentation culture medium, and culture at 37℃ in a shaking bed for 8-16h, the rotating speed of the shaking bed is 180rpm, the liquid volume in the shaking flask is 20% of the total volume, and when the OD value is 0.6-3, the Acinetobacter baumannii primary seed liquid is obtained; (2) Acinetobacter baumannii secondary seed culture: inoculate the primary seed liquid of step S1 into the sterilized YPD fermentation culture medium at a inoculation amount of 3%, and culture at 37℃ in a shaking bed for 5-14h, the rotating speed of the shaking bed is 180rpm, the liquid volume in the shaking flask is 30% of the total volume, and when the OD value is greater than or equal to 3, the Acinetobacter baumannii secondary seed liquid is obtained.

4. The manufacturing process of claim 1, wherein, Step S3 comprises the following steps: Inoculate the secondary seed liquid of step S2 into the fermentation culture medium containing Huangjing micro-powder at a inoculation amount of 0.2%-20%, control the pH value to be 5-8, the dissolved oxygen to be 15%-30%, the rotating speed and the dissolved oxygen are connected in series, the initial rotating speed is 60-250r / min, the aeration amount is 0.6-1.5vvm, after fermentation for 8-16h, start feeding, when the rotating speed reaches the upper limit and the dissolved oxygen rises to 50%-60%, intermittently feed the feeding medium, after fermentation for 30-48h, stop the fermentation, and the surfactant fermentation liquid is obtained.

5. The manufacturing process of claim 4, wherein, The feeding medium comprises beef extract 40g / L, molasses 100g / L, biotin 0.3g / L, CuSO4 8g / L, and ammonium sulfate 20g / L.

6. The manufacturing process of claim 1, wherein, Step S4 comprises the following steps: Centrifuge the surfactant fermentation liquid to remove the bacteria and impurities, and obtain the supernatant; add the extractant into the supernatant in an equal proportion, mix uniformly, take the lower extract, evaporate at 50-60℃ under reduced pressure, and the powder after drying the extract is the crude extract containing the surfactant.

7. The manufacturing process of claim 1, wherein, The activity determination of step S5 comprises the following steps: a. Determination of DPPH free radical scavenging ability Weigh a certain amount of DPPH, and prepare a DPPH solution with a concentration of 0.04mg / mL by using anhydrous ethanol; take 2mL of different concentrations of solution respectively, add 2mL of DPPH solution, mix uniformly, place at room temperature for 30min, and centrifuge at 5000r / min for 10min; measure the absorbance value of the supernatant at 517nm, and use Vc as a positive control; The DPPH free radical scavenging rate of the sample is calculated by the following formula: A0—2ml anhydrous ethanol+2ml DPPH solution absorbance value A1—2ml sample solution+2ml DPPH solution absorbance value A2—2ml sample solution+2ml anhydrous ethanol absorbance value b. Oil displacement ring activity In a glass dish, 60 ml of deionized water was added, and then 8 ml of liquid paraffin was added, and it was left to stand until the liquid paraffin was uniformly distributed on the water surface. On the surface thereof, 1 ml of supernatant after centrifugation was added, and the oil displacement ring diameter was measured.

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