Lactobacillus plantarum exopolysaccharide complex, and preparation method and application thereof

Abstract

The application provides a lactobacillus plantarum exopolysaccharide complex and a preparation method and application thereof, and belongs to the technical field of cosmetics. After fermentation of lactobacillus plantarum T1, crude polysaccharide is separated, and zymolytic enzyme is used to obtain a zymolytic system, the crude polysaccharide is added for treatment, modified exopolysaccharide is prepared, mixed with ferulic acid, activated, and then antibacterial peptide and amino-acetylated hyaluronic acid are added and stirred to prepare the lactobacillus plantarum exopolysaccharide complex. The lactobacillus plantarum exopolysaccharide complex prepared by the application has good moisturizing, antibacterial, anti-inflammatory, antioxidant and anti-allergic properties, so that the prepared product has good application and can be used as a safe, non-toxic and non-immunogenic effective cosmetic raw material.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to an extracellular polysaccharide complex of Lactobacillus plantarum, its preparation method, and its application. Background Technology

[0002] Lactic acid bacteria (LAB) is a general term for a class of bacteria that can ferment carbohydrates to produce large amounts of lactic acid. They are typically Gram-positive, catalase-negative, non-spore-forming, mostly non-motile, facultative or anaerobic cocci or rod-shaped bacteria. During fermentation, lactic acid bacteria produce exopolysaccharides (EPS), which are the collective term for mucopolysaccharides or capsular polysaccharides secreted outside the cells during growth and metabolism. These EPS can promote human health and have various beneficial functions, including enhancing immunity, anti-inflammation, balancing intestinal flora, and anti-tumor effects. Different lactic acid bacteria species exhibit significant differences in the yield, structure, and functional properties of their EPS. Studying the yield and functional structure of different lactic acid bacteria is of great significance for enriching information related to lactic acid bacteria EPS.

[0003] Therefore, isolating and extracting extracellular polysaccharides from strains that produce high levels of extracellular polysaccharides, and studying their functional characteristics and physiological functions, is of great significance for improving food quality and expanding the application of extracellular polysaccharides in cosmetics, food, and pharmaceuticals. Summary of the Invention

[0004] The purpose of this invention is to provide an extracellular polysaccharide complex of Lactobacillus plantarum, its preparation method and application, which has good moisturizing, antibacterial, anti-inflammatory, antioxidant and anti-allergic properties, so that the obtained product has good application and can be used as a safe, non-toxic, non-immunogenic and effective cosmetic raw material.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a method for preparing Lactobacillus plantarum extracellular polysaccharide complex. After fermentation of Lactobacillus plantarum T1, crude polysaccharide is isolated. The bacterial sludge is hydrolyzed by lysozyme to obtain a lysing system. The crude polysaccharide is added to the system to obtain modified extracellular polysaccharide. The modified extracellular polysaccharide is then mixed with ferulic acid and activated. After the activation reaction, antimicrobial peptides and amino-modified hyaluronic acid are added and the mixture is stirred to obtain Lactobacillus plantarum extracellular polysaccharide complex.

[0007] As a further improvement to the present invention, the following steps are included:

[0008] S1. Fermentation: The seed liquid of Lactobacillus plantarum T1 was inoculated into the fermentation medium, fermented, filtered, and the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate to remove protein. The filtrate was centrifuged, the supernatant was collected, ethanol was added to precipitate, the precipitate was washed, dialyzed, and freeze-dried to obtain crude extracellular polysaccharide.

[0009] S2. Preparation of the lysozyme system: Add the bacterial sludge to water, add lysozyme for enzymatic hydrolysis, filter, freeze-dry the supernatant to obtain the lysozyme system;

[0010] S3. Enzyme treatment: The crude extracellular polysaccharide was dissolved in water, added to a lysing system, heated, precipitated with ethanol, washed, dialyzed, and freeze-dried to obtain the modified crude polysaccharide.

[0011] S4. Preparation of aminated hyaluronic acid: Hyaluronic acid was dissolved in water, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and adipic acid dihydrazide were added. The pH of the solution was adjusted, and the reaction was stirred at room temperature. The pH of the solution was adjusted to neutral, dialyzed, and freeze-dried to obtain aminated hyaluronic acid.

[0012] S5. Condensation: Modified crude polysaccharide and ferulic acid were added to water, followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. After activation, antimicrobial peptides and amino-modified hyaluronic acid were added. The mixture was stirred at room temperature, dialyzed, and freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0013] As a further improvement of the present invention, the bacterial count of the *Lactobacillus plantarum* T1 seed solution in step S1 is 10. 8 -10 9 The inoculum concentration is 1-2 v / v%, and the fermentation medium formula is as follows: sucrose 10-15 g / L, glucose 12-15 g / L, urea 3-5 g / L, calcium chloride 0.5-1 g / L, magnesium chloride 0.2-0.5 g / L, potassium chloride 0.2-0.4 g / L, lysine 1-2 g / L, folic acid 0.2-0.4 g / L, vitamin B12 0.1-0.2 g / L, and vitamin C 0.5-1 g / L. The fermentation conditions are anaerobic, 36-38℃, 100-200 r / min, fermentation time 24-48 h, adding trichloroacetic acid to bring the trichloroacetic acid content to 3-5 wt%, adding ethanol to bring the ethanol content to 50-70 wt%, precipitation time 3-5 h, and dialysis time 40-48 h.

[0014] As a further improvement of the present invention, the mass ratio of bacterial mud and lysozyme in step S2 is 100:1-2, the enzymatic hydrolysis temperature is 30-35℃, and the time is 2-4h.

[0015] As a further improvement of the present invention, the mass ratio of crude extracellular polysaccharide to lysing system in step S3 is 100:0.5-1, the heating treatment temperature is 35-40℃ and the time is 1-2h, ethanol is added until the ethanol content of the system is 50-70wt%, the precipitation time is 2-4h, and the dialysis time is 40-48h.

[0016] As a further improvement of the present invention, in step S4, the mass ratio of hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and adipic acid dihydrazide is 10:4-5:12-14, the pH value of the solution is adjusted to 4.7-4.8, the stirring reaction time at room temperature is 2-3 hours, the dialysis bag used for dialysis has a pore size of 5k-8kDa, and the dialysis time is 24-48 hours.

[0017] As a further improvement of the present invention, the mass ratio of the modified crude polysaccharide, ferulic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, antimicrobial peptide and amino-modified hyaluronic acid in step S5 is 12-15:2-4:7-9:4-7:2-3:6-8, the activation reaction temperature is 0-4℃, the time is 20-40 min, the room temperature stirring reaction time is 20-24 h, the dialysis bag pore size is 5k-8kDa, and the dialysis time is 24-48 h.

[0018] As a further improvement to the present invention, the specific steps include:

[0019] S1. Fermentation: Fermenting with a bacterial count of 10... 8 -10 9 CFU / mL Lactobacillus plantarum T1 seed culture was inoculated into fermentation medium at an inoculation rate of 1-2 v / v%. Under anaerobic conditions, fermentation was carried out at 36-38℃ and 100-200 r / min for 24-48 h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 3-5 wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 50-70 wt%. After precipitation for 3-5 h, the precipitate was washed, dialyzed for 40-48 h, and freeze-dried to obtain crude extracellular polysaccharide.

[0020] The fermentation medium has the following formula: sucrose 10-15 g / L, glucose 12-15 g / L, urea 3-5 g / L, calcium chloride 0.5-1 g / L, magnesium chloride 0.2-0.5 g / L, potassium chloride 0.2-0.4 g / L, lysine 1-2 g / L, folic acid 0.2-0.4 g / L, vitamin B12 0.1-0.2 g / L, and vitamin C 0.5-1 g / L;

[0021] S2. Preparation of lysozyme system: Add 100 parts by weight of bacterial sludge to water, add 1-2 parts by weight of lysozyme, enzymatically hydrolyze at 30-35℃ for 2-4 hours, filter, freeze-dry the supernatant to obtain the lysozyme system;

[0022] S3. Enzyme treatment: Dissolve 100 parts by weight of crude extracellular polysaccharide in water, add 0.5-1 parts by weight of lysing system, heat to 35-40℃ for 1-2 hours, add ethanol until the ethanol content of the system is 50-70 wt%, precipitate for 2-4 hours, wash the precipitate, dialyze for 40-48 hours, freeze dry to obtain modified crude polysaccharide.

[0023] S4. Preparation of Aminated Hyaluronic Acid: Dissolve 10 parts by weight of hyaluronic acid in water, add 4-5 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 12-14 parts by weight of adipic acid dihydrazide, adjust the pH of the solution to 4.7-4.8, stir the reaction at room temperature for 2-3 hours, adjust the pH of the solution to neutral, dialyze using a dialysis bag with a pore size of 5k-8kDa for 24-48 hours, freeze dry to obtain aminated hyaluronic acid;

[0024] S5. Condensation: 12-15 parts by weight of modified crude polysaccharide and 2-4 parts by weight of ferulic acid were added to water, followed by 7-9 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-7 parts by weight of N-hydroxysuccinimide. After activation at 0-4℃ for 20-40 min, 2-3 parts by weight of antimicrobial peptide and 6-8 parts by weight of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 20-24 h, dialyzed through a dialysis bag with a pore size of 5k-8kDa for 24-48 h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0025] The present invention further protects an extracellular polysaccharide complex of Lactobacillus plantarum prepared by the above-described preparation method.

[0026] This invention further protects the application of the above-mentioned Lactobacillus plantarum extracellular polysaccharide complex in the preparation of moisturizing, antioxidant, anti-inflammatory, and anti-allergic cosmetics.

[0027] The present invention has the following beneficial effects:

[0028] Typically, the low yield of extracellular polysaccharides from lactic acid bacteria over multiple days, high production costs, long production cycles, and low strain stability restrict their industrial production. The *Lactobacillus plantarum* T1 strain selected in this invention is a high-yield strain of extracellular polysaccharides, producing a large amount of secondary metabolites that adhere to the cell wall or are secreted outside the cell wall during growth. Simultaneously, this invention adds a significant amount of Ca to the fermentation medium. 2+ and Mg 2+This process increases the yield of extracellular polysaccharides and alters their microstructure, leading to an increase in rhamnose content. It also upregulates the expression of genes related to rhamnose biosynthesis, such as cps4F and rfbD, as well as the expression of most proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, and ion transport. This is beneficial for extracellular polysaccharide synthesis and thus increases the yield. Furthermore, the addition of lysine and folic acid enhances the strain's stress resistance and prolongs its stationary phase, significantly improving product yield. Additionally, Ca... 2+ It is also an important component of glucan sucrase, which can catalyze the direct formation of α-glucan from substrates. The glycosides formed by catalysis can be linked to form oligosaccharides, improving the structure of extracellular polysaccharides and giving the resulting extracellular polysaccharides better antioxidant and anti-inflammatory properties.

[0029] The fermenting bacteria *Lactobacillus plantarum* still contains a rich enzyme system that can promote the synthesis of glycosidic bonds in polysaccharides. In this invention, lysozyme is used to enzymatically hydrolyze the bacterial sludge, and then an enzymatic reaction is carried out with the obtained crude extracellular polysaccharide to promote the formation of glycosidic bonds, etc., and generate modified crude polysaccharides with higher activity or smaller molecular weight that are easier to function.

[0030] Hyaluronic acid is a polysaccharide with excellent moisturizing and antioxidant properties. It is safe, non-toxic, and non-immunogenic. Through amination, it can undergo condensation reactions with modified crude polysaccharides, ferulic acid, and antimicrobial peptides, exhibiting good antioxidant activity. Acidic extracellular polysaccharides and those with negatively charged groups show stronger antioxidant activity. The negatively charged groups facilitate the hydrolysis of extracellular polysaccharides, exposing more hemiacetal hydroxyl groups, which demonstrate a strong ability to scavenge DPPH free radicals, hydroxyl radicals, and superoxide radicals, thus alleviating oxidative stress on cells. In addition, this extracellular polysaccharide complex also has good anti-inflammatory effects, promoting macrophage proliferation and the release of pro-inflammatory factors, as well as the release of tumor necrosis factor-α and interleukins.

[0031] The Lactobacillus plantarum extracellular polysaccharide complex prepared by this invention has good moisturizing, antibacterial, anti-inflammatory, antioxidant, and anti-allergic properties, thus making the prepared product have good applications and can be used as a safe, non-toxic, non-immunogenic, and effective cosmetic raw material. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Lysozyme, 20,000 U / g, purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0034] Hyaluronic acid, content >98%, purchased from Suzhou Senfida Chemical Co., Ltd.

[0035] The antimicrobial peptides, derived from Bacillus subtilis, were purchased from Wuxi Pusino Technology Co., Ltd.

[0036] Lactobacillus plantarum T1 is a strain preserved by the College of Food Science, Nanjing Agricultural University, and provided to our company for use. Preparation of the inoculum seed culture: The inoculum was inoculated into MRS medium and statically activated at 30℃ for 12-18 hours until mid-logarithmic growth, yielding a bacterial count of 10... 8 -10 9 CFU / mL Lactobacillus plantarum T1 seed culture.

[0037] Lactiplantibacillus plantarum T1, classified as Lactiplantibacillus plantarum T1, with accession number CCTCC NO: M 20231553, deposited on August 31, 2023, is deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, 430072, China.

[0038] Example 1

[0039] This embodiment provides a method for preparing an extracellular polysaccharide complex of Lactobacillus plantarum, specifically including the following steps:

[0040] S1. Fermentation: The seed culture of Lactobacillus plantarum T1 was inoculated into the fermentation medium at an inoculation amount of 1 v / v%. Under anaerobic conditions, fermentation was carried out at 36℃ and 100 r / min for 24 h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 3 wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 50 wt%. After precipitation for 3 h, the precipitate was washed, dialyzed for 40 h, and freeze-dried to obtain crude extracellular polysaccharide.

[0041] The fermentation medium has the following formula: sucrose 10g / L, glucose 12g / L, urea 3g / L, calcium chloride 0.5g / L, magnesium chloride 0.2g / L, potassium chloride 0.2g / L, lysine 1g / L, folic acid 0.2g / L, vitamin B12 0.1g / L, and vitamin C 0.5g / L.

[0042] S2. Preparation of lysozyme system: Add 100g of bacterial sludge to 500mL of water, add 1g of lysozyme, enzymatically hydrolyze at 30℃ for 2h, filter, freeze-dry the supernatant to obtain the lysozyme system;

[0043] S3. Enzyme treatment: Dissolve 100g of crude extracellular polysaccharide in 500mL of water, add 0.5g of lysing system, heat to 35℃ for 1h, add ethanol until the ethanol content of the system is 50wt%, precipitate for 2h, wash the precipitate, dialyze for 40h, freeze dry to obtain modified crude polysaccharide.

[0044] S4. Preparation of aminated hyaluronic acid: 10g of hyaluronic acid was dissolved in 500mL of water, 4g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 12g of adipate dihydrazide were added, the pH of the solution was adjusted to 4.7, the reaction was stirred at room temperature for 2h, the pH of the solution was adjusted to neutral, dialyzed with a dialysis bag with a pore size of 5kDa for 24h, and freeze-dried to obtain aminated hyaluronic acid;

[0045] S5. Condensation: 12g of modified crude polysaccharide and 2g of ferulic acid were added to 500mL of water, followed by 7g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4g of N-hydroxysuccinimide. After activation at 0℃ for 20min, 2g of antimicrobial peptide and 6g of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 20h, dialyzed through a 5kDa dialysis bag for 24h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0046] Example 2

[0047] This embodiment provides a method for preparing an extracellular polysaccharide complex of Lactobacillus plantarum, specifically including the following steps:

[0048] S1. Fermentation: The seed liquid of Lactobacillus plantarum T1 was inoculated into the fermentation medium at an inoculation amount of 2v / v%, and fermented under anaerobic conditions at 38℃ and 200r / min for 48h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 5wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 70wt%. After precipitation for 5h, the precipitate was washed, dialyzed for 48h, and freeze-dried to obtain crude extracellular polysaccharide.

[0049] The fermentation medium has the following formula: sucrose 15g / L, glucose 15g / L, urea 5g / L, calcium chloride 1g / L, magnesium chloride 0.5g / L, potassium chloride 0.4g / L, lysine 2g / L, folic acid 0.4g / L, vitamin B12 0.2g / L, and vitamin C 1g / L.

[0050] S2. Preparation of lysozyme system: Add 100g of bacterial sludge to 500mL of water, add 2g of lysozyme, enzymatically hydrolyze at 35℃ for 4h, filter, freeze-dry the supernatant to obtain the lysozyme system;

[0051] S3. Enzyme treatment: Dissolve 100g of crude extracellular polysaccharide in 500mL of water, add 1g of lysing system, heat to 40℃ for 2h, add ethanol until the ethanol content of the system is 70wt%, precipitate for 4h, wash the precipitate, dialyze for 48h, freeze dry to obtain modified crude polysaccharide.

[0052] S4. Preparation of Aminated Hyaluronic Acid: 10g of hyaluronic acid was dissolved in 500mL of water, 5g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 14g of adipate dihydrazide were added, the pH of the solution was adjusted to 4.8, the reaction was stirred at room temperature for 3h, the pH of the solution was adjusted to neutral, dialyzed using a dialysis bag with a pore size of 8kDa for 48h, and freeze-dried to obtain aminated hyaluronic acid;

[0053] S5. Condensation: 15g of modified crude polysaccharide and 4g of ferulic acid were added to 500mL of water, followed by 9g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 7g of N-hydroxysuccinimide. After activation at 4℃ for 40min, 3g of antimicrobial peptide and 8g of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 24h, dialyzed through an 8kDa dialysis bag for 48h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0054] Example 3

[0055] This embodiment provides a method for preparing an extracellular polysaccharide complex of Lactobacillus plantarum, specifically including the following steps:

[0056] S1. Fermentation: The seed culture of Lactobacillus plantarum T1 was inoculated into the fermentation medium at an inoculation amount of 1.5 v / v%. Under anaerobic conditions, fermentation was carried out at 37℃ and 150 r / min for 36 h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 4 wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 60 wt%. After precipitation for 4 h, the precipitate was washed, dialyzed for 44 h, and freeze-dried to obtain crude extracellular polysaccharide.

[0057] The fermentation medium has the following formula: sucrose 12 g / L, glucose 13.5 g / L, urea 4 g / L, calcium chloride 0.7 g / L, magnesium chloride 0.35 g / L, potassium chloride 0.3 g / L, lysine 1.5 g / L, folic acid 0.3 g / L, vitamin B12 0.15 g / L, and vitamin C 0.7 g / L;

[0058] S2. Preparation of lysozyme system: Add 100g of bacterial sludge to 500mL of water, add 1.5g of lysozyme, enzymatically hydrolyze at 32℃ for 3h, filter, freeze-dry the supernatant to obtain the lysozyme system;

[0059] S3. Enzyme treatment: Dissolve 100g of crude extracellular polysaccharide in 500mL of water, add 0.7g of lysing system, heat to 37℃ for 1.5h, add ethanol until the ethanol content of the system is 60wt%, precipitate for 3h, wash the precipitate, dialyze for 44h, freeze dry to obtain modified crude polysaccharide.

[0060] S4. Preparation of Aminated Hyaluronic Acid: 10g of hyaluronic acid was dissolved in 500mL of water, 4.5g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 13g of adipate dihydrazide were added, the pH of the solution was adjusted to 4.75, the reaction was stirred at room temperature for 2.5h, the pH of the solution was adjusted to neutral, dialyzed using a 7kDa dialysis bag for 36h, and freeze-dried to obtain aminated hyaluronic acid;

[0061] S5. Condensation: 13g of modified crude polysaccharide and 3g of ferulic acid were added to 500mL of water, along with 8g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 5.5g of N-hydroxysuccinimide. After activation at 2℃ for 30min, 2.5g of antimicrobial peptide and 7g of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 22h, dialyzed through a 7kDa dialysis bag for 36h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0062] Example 4

[0063] The difference from Example 3 is that calcium chloride and magnesium chloride were not added to the fermentation medium.

[0064] The fermentation medium formula is as follows: sucrose 12g / L, glucose 13.5g / L, urea 4g / L, potassium chloride 0.3g / L, lysine 1.5g / L, folic acid 0.3g / L, vitamin B12 0.15g / L, and vitamin C 0.7g / L.

[0065] Example 5

[0066] The difference from Example 3 is that lysine and folic acid were not added to the fermentation medium.

[0067] The fermentation medium formula is as follows: sucrose 12g / L, glucose 13.5g / L, urea 4g / L, calcium chloride 0.7g / L, magnesium chloride 0.35g / L, potassium chloride 0.3g / L, vitamin B12 0.15g / L, and vitamin C 0.7g / L.

[0068] Comparative Example 1

[0069] The difference from Example 3 is that the fermentation medium was replaced with MRS medium.

[0070] Specifically as follows:

[0071] S1. Fermentation: The seed culture of Lactobacillus plantarum T1 was inoculated into MRS medium at an inoculation rate of 1.5 v / v%. Under anaerobic conditions, fermentation was carried out at 37°C and 150 r / min for 36 h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 4 wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 60 wt%. After precipitation for 4 h, the precipitate was washed, dialyzed for 44 h, and freeze-dried to obtain crude extracellular polysaccharide.

[0072] Comparative Example 2

[0073] The difference from Example 3 is that steps S2 and S3 were not performed.

[0074] Specifically as follows:

[0075] S1. Fermentation: The seed culture of Lactobacillus plantarum T1 was inoculated into the fermentation medium at an inoculation amount of 1.5 v / v%. Under anaerobic conditions, fermentation was carried out at 37℃ and 150 r / min for 36 h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 4 wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 60 wt%. After precipitation for 4 h, the precipitate was washed, dialyzed for 44 h, and freeze-dried to obtain crude extracellular polysaccharide.

[0076] The fermentation medium has the following formula: sucrose 12 g / L, glucose 13.5 g / L, urea 4 g / L, calcium chloride 0.7 g / L, magnesium chloride 0.35 g / L, potassium chloride 0.3 g / L, lysine 1.5 g / L, folic acid 0.3 g / L, vitamin B12 0.15 g / L, and vitamin C 0.7 g / L;

[0077] S2. Preparation of Aminated Hyaluronic Acid: 10g of hyaluronic acid was dissolved in 500mL of water, and 4.5g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 13g of adipate dihydrazide were added. The pH of the solution was adjusted to 4.75, and the reaction was stirred at room temperature for 2.5h. The pH of the solution was then adjusted to neutral, and the solution was dialyzed for 36h using a dialysis bag with a pore size of 7kDa. The solution was then freeze-dried to obtain aminated hyaluronic acid.

[0078] S3. Condensation: 13g of crude extracellular polysaccharide and 3g of ferulic acid were added to 500mL of water, followed by 8g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 5.5g of N-hydroxysuccinimide. After activation at 2℃ for 30min, 2.5g of antimicrobial peptide and 7g of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 22h, dialyzed through a 7kDa dialysis bag for 36h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0079] Comparative Example 3

[0080] The difference from Example 3 is that no antimicrobial peptide was added in step S5.

[0081] Specifically as follows:

[0082] S5. Condensation: 13g of modified crude polysaccharide and 3g of ferulic acid were added to 500mL of water, along with 8g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 5.5g of N-hydroxysuccinimide. After activation at 2℃ for 30min, 9.5g of amino-modified hyaluronic acid was added. The mixture was stirred at room temperature for 22h, dialyzed through a 7kDa dialysis bag for 36h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0083] Comparative Example 4

[0084] The difference from Example 3 is that ferulic acid was not added in step S5.

[0085] Specifically as follows:

[0086] S5. Condensation: 16g of modified crude polysaccharide was added to 500mL of water, along with 8g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 5.5g of N-hydroxysuccinimide. After activation at 2℃ for 30min, 2.5g of antimicrobial peptide and 7g of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 22h, dialyzed through a 7kDa dialysis bag for 36h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0087] Comparative Example 5

[0088] The difference from Example 3 is that amino-modified hyaluronic acid was not added in step S5.

[0089] Specifically as follows:

[0090] S5. Condensation: 13g of modified crude polysaccharide and 3g of ferulic acid were added to 500mL of water, along with 8g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 5.5g of N-hydroxysuccinimide. After activation at 2℃ for 30min, 9.5g of antimicrobial peptide was added, and the mixture was stirred at room temperature for 22h. The mixture was then dialyzed for 36h using a 7kDa dialysis bag and freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

[0091] Test Example 1: Determination of NO Content in Cells

[0092] RAW264.7 macrophages in logarithmic growth phase were adjusted to a cell concentration of 4 × 10⁻⁶. 5Cells were seeded in 6-well plates at 2 mL per well. After cell adhesion, the cells were incubated with the *Lactobacillus plantarum* extracellular polysaccharide complex solution (1 mg / L) and lipopolysaccharide solution (0.5 μg / L) prepared in Examples 1-5 or Comparative Examples 1-5. The plates were incubated at 37°C and 5% CO2 for 24 h. 50 μL of cell supernatant was collected from each group and added to 96-well plates at 50 μL per well. Then, 50 μL of Griess Reagent I was added, and the plates were incubated at 37°C for 10 min. Next, 50 μL of Griess Reagent II was added to each well, and the plates were shaken to mix thoroughly. The plates were then incubated at 37°C in the dark for 10 min. The absorbance of each group was measured at 540 nm. Deionized water was used instead of supernatant in the control wells. The blank group contained no *Lactobacillus plantarum* extracellular polysaccharide complex solution or lipopolysaccharide solution, and the positive group contained dexamethasone instead of decellularized matrix material. The results are shown in Table 1.

[0093] NO content=[1-(A 给药孔 -A 对照孔 ) / A 对照孔 )]×100%

[0094] Table 1

[0095] Group NO content (μmol / L) Blank group 4.10 positive group 11.09 Example 1 22.53 Example 2 21.26 Example 3 21.07 Example 4 36.73 Example 5 42.26 Comparative Example 1 82.25 Comparative Example 2 67.83 Comparative Example 3 30.28 Comparative Example 4 72.25 Comparative Example 5 97.88

[0096] As shown in the table above, the Lactobacillus plantarum extracellular polysaccharide complexes prepared in Examples 1-3 of this invention have a good effect on inhibiting the secretion of inflammatory factors by macrophages.

[0097] Test Example 2: Inhibition of hydroxyl radicals

[0098] Add 20 μL each of FeSO4 (6 mM), the *Lactobacillus plantarum* extracellular polysaccharide complex solution (1 mg / mL) prepared in Examples 1-5 or Comparative Examples 1-5, and H2O2 (6 mmol / L) to a 96-well plate. Let stand for 10 min, then add 20 μL of 6 mmol / L salicylic acid solution and let stand for 30 min. Measure the absorbance (A) at 510 nm. Use distilled water as a blank. Perform triple replicates for each sample and calculate the hydroxyl radical scavenging rate. Ascorbic acid was used as a control group. The results are shown in Table 2.

[0099] Hydroxyl radical scavenging rate (%) = A 空 -(A 样 -A 对 ) / A 空 ×100%.

[0100] In the formula, A 空 The absorbance value A without adding the sample, A 对 The absorbance value A is the absorbance without the addition of salicylic acid.

[0101] Table 2

[0102] Group Hydroxyl radical scavenging rate (%) control group 65.2 Example 1 92.5 Example 2 93.1 Example 3 93.9 Example 4 88.5 Example 5 86.9 Comparative Example 1 80.2 Comparative Example 2 82.4 Comparative Example 3 85.9 Comparative Example 4 86.2 Comparative Example 5 78.4

[0103] As shown in the table above, the Lactobacillus plantarum extracellular polysaccharide complexes prepared in Examples 1-3 of this invention have good antioxidant effects.

[0104] Test Example 3

[0105] Test sample: Prepare an aqueous solution of 0.1 mg / mL of the Lactobacillus plantarum extracellular polysaccharide complex obtained in Examples 1-5 or Comparative Examples 1-5, which is the sample solution.

[0106] Experimental Procedure: Volunteers were randomly divided into a control group, Example 1-5 groups, and Comparative Example 1-5 groups, with 6 participants in each group. 5 mL of the sample solution was evenly applied to the flexor surfaces of the volunteers' left and right forearms. The control group received an equal volume of distilled water. Before and 7 days after application, the water content of the stratum corneum of each group was measured using a skin water content analyzer; sebum content was measured using a sebum analyzer, with three measurements taken and the average value recorded. Normal skin has a stratum corneum water content of 20%-30% and a sebum content of 5%-8%. The results are shown in Table 3.

[0107] Change rate of skin moisture content (%) = (Average skin moisture content of volunteers after the experiment - Average skin moisture content of volunteers before the experiment) / Average skin moisture content of volunteers before the experiment × 100%

[0108] Sebum content change rate (%) = (Average sebum content of volunteers' skin after the experiment - Average sebum content of volunteers' skin before the experiment) / Average sebum content of volunteers' skin before the experiment × 100%

[0109] Table 3

[0110] Group Water content of the stratum corneum (%) Sebum content (%) control group 20.24 5.48 Example 1 34.91 6.59 Example 2 34.56 6.62 Example 3 35.22 6.70 Example 4 31.29 6.51 Example 5 32.37 6.48 Comparative Example 1 30.45 6.32 Comparative Example 2 27.85 6.05 Comparative Example 3 32.68 6.14 Comparative Example 4 32.97 5.89 Comparative Example 5 29.61 6.01

[0111] As shown in the table above, the *Lactobacillus plantarum* extracellular polysaccharide complexes prepared in Examples 1-3 of this invention have good moisturizing effects and can increase sebum content to a certain extent.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an extracellular polysaccharide complex of *Lactobacillus plantarum*, characterized in that, Includes the following steps: S1. Fermentation: *Lactobacillus plantarum* T1 seed culture was inoculated into a fermentation medium, fermented, filtered, and the solid was a bacterial sludge. Trichloroacetic acid was added to the filtrate to remove protein. The filtrate was centrifuged, and the supernatant was collected. Ethanol was added to precipitate the precipitate, which was then washed, dialyzed, and freeze-dried to obtain crude extracellular polysaccharide. The fermentation medium had the following composition: sucrose 10-15 g / L, glucose 12-15 g / L, urea 3-5 g / L, calcium chloride 0.5-1 g / L, magnesium chloride 0.2-0.5 g / L, potassium chloride 0.2-0.4 g / L, lysine 1-2 g / L, folic acid 0.2-0.4 g / L, vitamin B12 0.1-0.2 g / L, and vitamin C 0.5-1 g / L. The fermentation conditions were anaerobic: 36-38℃, 100-200 r / min, for 24-48 h. S2. Preparation of the lysozyme system: Add the bacterial sludge to water, add lysozyme for enzymatic hydrolysis, filter, freeze-dry the supernatant to obtain the lysozyme system; S3. Enzyme treatment: The crude extracellular polysaccharide was dissolved in water, added to a lysing system, heated, precipitated with ethanol, washed, dialyzed, and freeze-dried to obtain the modified crude polysaccharide; S4. Preparation of aminated hyaluronic acid: Hyaluronic acid was dissolved in water, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and adipic acid dihydrazide were added. The pH of the solution was adjusted, and the reaction was stirred at room temperature. The pH of the solution was adjusted to neutral, dialyzed, and freeze-dried to obtain aminated hyaluronic acid. S5. Condensation: Modified crude polysaccharide and ferulic acid were added to water, followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. After activation, antimicrobial peptides and amino-modified hyaluronic acid were added. The mixture was stirred at room temperature, dialyzed, and freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

2. The preparation method according to claim 1, characterized in that, The bacterial count of the *Lactobacillus plantarum* T1 seed solution in step S1 is 10. 8 -10 9 The concentration of CFU / mL is 1-2 v / v%, the amount of inoculum is 1-2 v / v%, the amount of trichloroacetic acid added is 3-5 wt%, the amount of ethanol added is 50-70 wt%, the precipitation time is 3-5 h, and the dialysis time is 40-48 h.

3. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of bacterial sludge to lysozyme is 100:1-2, and the enzymatic hydrolysis temperature is 30-35℃ for 2-4 hours.

4. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of crude extracellular polysaccharide to lysing system is 100:0.5-1, the heating treatment temperature is 35-40℃, the time is 1-2h, ethanol is added until the ethanol content of the system is 50-70wt%, the precipitation time is 2-4h, and the dialysis time is 40-48h.

5. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, and adipic acid dihydrazide is 10:4-5:12-14. The pH of the solution is adjusted to 4.7-4.

8. The reaction time at room temperature is 2-3 hours. The dialysis bag used for dialysis has a pore size of 5k-8kDa. The dialysis time is 24-48 hours.

6. The preparation method according to claim 1, characterized in that, In step S5, the mass ratio of the modified crude polysaccharide, ferulic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, antimicrobial peptide, and amino-modified hyaluronic acid is 12-15:2-4:7-9:4-7:2-3:6-8. The activation reaction temperature is 0-4℃, the time is 20-40 min, the room temperature stirring reaction time is 20-24 h, the dialysis bag pore size is 5k-8kDa, and the dialysis time is 24-48 h.

7. The preparation method according to claim 1, characterized in that, Specifically, the following steps are included: S1. Fermentation: Fermenting with a bacterial count of 10... 8 -10 9 CFU / mL Lactobacillus plantarum T1 seed culture was inoculated into fermentation medium at an inoculation rate of 1-2 v / v%. Under anaerobic conditions, fermentation was carried out at 36-38℃ and 100-200 r / min for 24-48 h. After filtration, the solid was bacterial sludge. Trichloroacetic acid was added to the filtrate until the trichloroacetic acid content in the system was 3-5 wt%. After centrifugation, the supernatant was collected, and ethanol was added until the ethanol content in the system was 50-70 wt%. After precipitation for 3-5 h, the precipitate was washed, dialyzed for 40-48 h, and freeze-dried to obtain crude extracellular polysaccharide. The fermentation medium has the following formula: sucrose 10-15 g / L, glucose 12-15 g / L, urea 3-5 g / L, calcium chloride 0.5-1 g / L, magnesium chloride 0.2-0.5 g / L, potassium chloride 0.2-0.4 g / L, lysine 1-2 g / L, folic acid 0.2-0.4 g / L, vitamin B12 0.1-0.2 g / L, and vitamin C 0.5-1 g / L; S2. Preparation of lysozyme system: Add 100 parts by weight of bacterial sludge to water, add 1-2 parts by weight of lysozyme, enzymatically hydrolyze at 30-35℃ for 2-4 hours, filter, freeze-dry the supernatant to obtain the lysozyme system; S3. Enzyme treatment: Dissolve 100 parts by weight of crude extracellular polysaccharide in water, add 0.5-1 parts by weight of lysing system, heat to 35-40℃ for 1-2 hours, add ethanol until the ethanol content of the system is 50-70 wt%, precipitate for 2-4 hours, wash the precipitate, dialyze for 40-48 hours, freeze dry to obtain modified crude polysaccharide. S4. Preparation of aminated hyaluronic acid: Dissolve 10 parts by weight of hyaluronic acid in water, add 4-5 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 12-14 parts by weight of adipic acid dihydrazide, adjust the pH of the solution to 4.7-4.8, stir the reaction at room temperature for 2-3 hours, adjust the pH of the solution to neutral, dialyze using a dialysis bag with a pore size of 5k-8kDa for 24-48 hours, freeze dry to obtain aminated hyaluronic acid; S5. Condensation: 12-15 parts by weight of modified crude polysaccharide and 2-4 parts by weight of ferulic acid were added to water, followed by 7-9 parts by weight of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-7 parts by weight of N-hydroxysuccinimide. After activation at 0-4℃ for 20-40 min, 2-3 parts by weight of antimicrobial peptide and 6-8 parts by weight of amino-modified hyaluronic acid were added. The mixture was stirred at room temperature for 20-24 h, dialyzed through a dialysis bag with a pore size of 5k-8kDa for 24-48 h, and then freeze-dried to obtain the Lactobacillus plantarum extracellular polysaccharide complex.

8. An extracellular polysaccharide complex of *Lactobacillus plantarum* prepared by the method according to any one of claims 1-7.

9. The application of the *Lactobacillus plantarum* extracellular polysaccharide complex as described in claim 8 in the preparation of moisturizing, antioxidant, anti-inflammatory, and anti-allergic cosmetics.