A preparation method and application of lactobacillus plantarum ferment based on environmental stress
By preparing Lactobacillus plantarum ferment under sodium dodecyl sulfate stress, the problem of insufficient anti-inflammatory peptide content in natural strains was solved, achieving a highly efficient anti-inflammatory effect, which is suitable for use in cosmetics.
Patent Information
- Application Number
- CN202511600663.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-02-13
- Estimated Expiration
- 2045-11-04
AI Technical Summary
In existing technologies, the content of anti-inflammatory peptides synthesized by natural strains is limited, and the effect of stressor selection on the enhancement of anti-inflammatory activity of fermentation products is unclear, making it difficult to improve the anti-inflammatory activity of fermentation products.
Using sodium dodecyl sulfate as a stressor, *Lactobacillus plantarum* was induced to grow in a culture medium containing sodium dodecyl sulfate. By combining enzymatic hydrolysis and fermentation processes, *Lactobacillus plantarum* fermentation products containing anti-inflammatory active peptides were prepared.
It significantly enhances the anti-inflammatory activity of Lactobacillus plantarum ferment, effectively inhibits the expression of the inflammatory factor IL-6, and promotes the expression of the human defensin HBD2. The product is safe with no residue risk and is suitable for industrial production.
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Figure CN121046495B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of microbial engineering and biological fermentation application, and particularly relates to a preparation method and application of Lactobacillus plantarum fermentation product based on environmental stress. BACKGROUND
[0002] Skin inflammatory response is the core inducement of sensitive skin, barrier damage and other problems, so the market urgently needs safe and efficient natural anti-inflammatory skin care raw materials. Anti-inflammatory peptides generated by Lactobacillus plantarum fermented milk are concerned for their safety and biological activity, and the current industrial production uses natural strains, and a stable fermentation process has been formed. However, the content of anti-inflammatory peptides synthesized by natural strains still has room for improvement.
[0003] Stress breeding is a classic strategy for directional strengthening of microorganisms. Moderate stress can activate the stress metabolic pathway of microorganisms and directionally enrich target products. However, the choice of stressor directly determines the technical feasibility. It is not clear how different stressors affect the gain effect of anti-inflammatory activity of fermentation products.
[0004] Therefore, selecting an appropriate stressor to improve the anti-inflammatory activity of fermentation products is a technical problem to be solved by those skilled in the art. SUMMARY
[0005] The purpose of this section is to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments.
[0006] The purpose of the present application is to overcome the deficiencies in the prior art and provide a preparation method of Lactobacillus plantarum fermentation product based on environmental stress, characterized by comprising,
[0007] Step 1: activating Lactobacillus plantarum;
[0008] Step 2: inducing and culturing Lactobacillus plantarum in a culture medium containing sodium dodecyl sulfate to obtain Lactobacillus plantarum under environmental stress;
[0009] Step 3: dissolving milk powder in deionized water, adding alkaline protease, adjusting the pH to 7-10, and performing enzymolysis to obtain an enzymolysis solution;
[0010] Step 4: mixing the enzymolysis solution obtained in step 3 with sugar to obtain a fermentation substrate;
[0011] Step 5: inoculating the Lactobacillus plantarum obtained in step 2 into the fermentation substrate obtained in step 4, and after fermentation culture, centrifuging to collect the supernatant, sterilizing, and obtaining the Lactobacillus plantarum fermentation product under environmental stress.
[0012] As a preferred scheme of the preparation method of the Lactobacillus plantarum fermentum based on environmental stress: the Lactobacillus plantarum has a preservation number of CCTCC No: M 20232264.
[0013] As a preferred scheme of the preparation method of the Lactobacillus plantarum fermentum based on environmental stress: in step 3, the milk powder is cow milk powder, the mass concentration of the milk powder solution is 5-15%, and the mass concentration of the alkaline protease is 0.05-1%; the enzymolysis temperature is 20-60 DEG C, and the enzymolysis time is 1.5-6 hours.
[0014] As a preferred scheme of the preparation method of the Lactobacillus plantarum fermentum based on environmental stress: in step 4, the sugar includes one or more of glucose, galactose oligosaccharide and fructose oligosaccharide, and the mass concentration is 0.5-2%.
[0015] As a preferred scheme of the preparation method of the Lactobacillus plantarum fermentum based on environmental stress: in step 5, the inoculation amount of the Lactobacillus plantarum is 2-10%; wherein the inoculated Lactobacillus plantarum is in the logarithmic growth phase, and the strain concentration reaches more than 10 10 CFU / mL.
[0016] As a preferred scheme of the preparation method of the Lactobacillus plantarum fermentum based on environmental stress: in step 5, the fermentation culture temperature is 35-37 DEG C, and the time is 12-48 hours.
[0017] As a preferred scheme of the preparation method of the Lactobacillus plantarum fermentum based on environmental stress: in step 2, the concentration of sodium dodecyl sulfate in the medium containing sodium dodecyl sulfate is 0.4-0.5 mg / mL.
[0018] The application further provides the Lactobacillus plantarum fermentum prepared by the preparation method of the Lactobacillus plantarum fermentum based on environmental stress, and the Lactobacillus plantarum fermentum contains anti-inflammatory active peptides, and the amino acid sequence of the anti-inflammatory active peptides is DQPFFHYN.
[0019] The application further provides application of the Lactobacillus plantarum fermentum in cosmetics.
[0020] The Lactobacillus plantarum fermentum can inhibit expression of inflammatory factor IL-6 and promote expression of human defensin HBD2.
[0021] The present application has the following beneficial effects: the present application first discovers that sodium dodecyl sulfate stress can promote the synthesis of Lactobacillus plantarum anti-inflammatory peptide, and breaks through the efficacy threshold of a natural strain. The sodium dodecyl sulfate used in the present application is a quasi-component for cosmetics, and there is no residual risk after stress, and the subsequent fermentation substrate is milk, and the obtained product is green and safe, and has zero burden on the skin. The method of the present application has mild conditions and simple operation, is conducive to the realization of industrial production, and fully guarantees the stability of product quality. BRIEF DESCRIPTION OF DRAWINGS
[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor. Among them:
[0023] Figure 1 is a column chart of the influence of Example 4, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Comparative Example 7 on the LPS-induced secretion level of inflammatory factor IL-6 of RAW264.7 cells.
[0024] Figure 2 Influence of Example 4, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Comparative Example 7 on the secretion level of HBD2 of HaCat cells. DETAILED DESCRIPTION
[0025] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below.
[0026] The Lactobacillus plantarum used in the following examples is a strain disclosed in patent CN118146346A, which was preserved in China Center for Type Culture Collection on November 17, 2023, and the preservation number is CCTCC No: M 20232264.
[0027] In the following examples, the formula of the MRS liquid medium is: casein peptone 10.0 g, beef infusion powder 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, Tween 80 1.0 g, sodium acetate 5.0 g, triammonium citrate 2.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.05 g, distilled water 1000.0 mL, pH 6.2 ~ 6.5. The preparation method of the MRS solid medium is to add 20 g of agar to 1000.0 mL of the liquid medium.
[0028] The main experimental materials of the present application are shown in Table 1.
[0029] Table 1
[0030]
[0031] Example 1:
[0032] Anti-inflammatory performance test:
[0033] RAW 264.7 was cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cells in the logarithmic growth phase were collected. The cell suspension was adjusted to a concentration of 1×10 5 6 / mL, 500 μL was taken and inoculated in a 24-well plate, and cultured for 24 h. The original culture solution was removed, and washed twice with PBS. 500 μL of DMEM solution containing 1 μg / mL LPS and 5 mg / mL milk peptide fermentate was added, and incubated for 24 h. The level of pro-inflammatory cytokine IL-6 in the supernatant of macrophages was determined according to the instructions of the commercially available ELISA kit (Signalway Antibody, USA). The cells at the bottom of the well were washed twice with PBS, 500 μL of 0.5 mg / mL MTT solution was added, and incubated in the dark for 4 h. The supernatant was removed, 500 μL of dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance at 490 nm was detected.
[0034] Example 2:
[0035] Human beta defensin 2 (HBD2) content test:
[0036] HaCat cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cells in the logarithmic growth phase were collected. The cell suspension was adjusted to a concentration of 3×10 5 6 / mL, 2 mL was taken and inoculated in a 6-well plate, and cultured for 24 h. The original culture solution was removed, and washed twice with PBS. 2 mL of DMEM solution containing different concentrations of Lactobacillus plantarum fermentate was added, and incubated for 24 h. The cells and supernatant were collected, and ultrasonicated in an ice bath for 15 min, and then repeatedly frozen and thawed 4 times. After centrifugation at 1500 g, 4 ℃ for 10 min, the supernatant was taken. The level of human defensin HBD2 in the cell supernatant was determined according to the instructions of the commercially available HBD2 kit (Signalway Antibody, USA).
[0037] Example 3:
[0038] Preparation of Lactobacillus plantarum resistant to sodium dodecyl sulfate stress:
[0039] Lactobacillus plantarum was inoculated in MRS liquid medium, and after 20 h of incubation at 37 ℃ and 200 r / min, the bacterial solution was diluted and spread on MRS solid medium, and after 24 h of incubation at 37 ℃, a single colony was picked and inoculated in MRS liquid medium containing 0.4 mg / mL sodium dodecyl sulfate, and shake flask culture was carried out at 37 ℃ and 200 rpm. After the bacterial solution became turbid, it was diluted and spread on MRS solid medium containing 0.4 mg / mL sodium dodecyl sulfate. After the colonies grew, Lactobacillus plantarum resistant to sodium dodecyl sulfate was obtained.
[0040] Example 4:
[0041] Preparation method of milk peptide fermentation product:
[0042] 1) Preparation of milk peptide:
[0043] Milk powder was added to deionized water at a solid-liquid ratio of 7:50 g / mL; alkaline protease was added to deionized water at a solid-liquid ratio of 1:200 g / mL, and it was fully dissolved in a 50 ℃ water bath;
[0044] The milk aqueous solution and the alkaline protease solution were mixed at a ratio of 6:1 (v / v), the pH was adjusted to 8.0, and the mixture was incubated in a 50 ℃ water bath for 3.5 h.
[0045] After enzyme hydrolysis, the temperature was raised to 90 ℃, and the mixture was incubated in a water bath for 10 min to inactivate the enzyme. Then the mixture was cooled to room temperature, and the pH was adjusted to 6.5 to obtain the milk peptide.
[0046] 2) Preparation of milk peptide fermentation product:
[0047] 2.8 g of galactooligosaccharide was dissolved in 40 mL of deionized water. The galactooligosaccharide solution and 60 mL of milk peptide solution containing 350 mg of yeast powder were separately placed in a high-pressure steam sterilization pot and sterilized at 115 ℃ for 15 min. Then 10 mL of the sterilized galactooligosaccharide solution was added to the 60 mL of milk peptide solution to obtain the fermentation substrate.
[0048] The single colony of Lactobacillus plantarum resistant to sodium dodecyl sulfate after stress treatment in Example 3 was inoculated in MRS liquid medium, and incubated at 37 ℃ and 200 rpm for 20 h to the logarithmic growth phase (bacterial concentration reached 10 10 CFU / mL). 3.5 mL of bacterial suspension was inoculated in the fermentation substrate prepared above, and fermentation was carried out at 37 ℃ and 200 rpm for 30 h. After fermentation, the fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The bacterial cells and impurities were removed by filtration with a 0.22 μm filter membrane, and the milk peptide fermentation product was obtained by freeze-drying.
[0049] The obtained milk peptide ferment was subjected to anti-inflammatory performance test and HBD2 content test according to the method of Example 1.
[0050] The milk peptide ferment was subjected to omics analysis, and the obtained peptide segment was subjected to anti-inflammatory activity analysis, and it was found that the milk peptide ferment contained an anti-inflammatory active peptide segment: DQPFFHYN.
[0051] The 369 peptide segment sequences in the obtained milk peptide ferment were analyzed using the Biopep database to explore potential biological activity. The results showed that the peptide segment with the amino acid sequence DQPFFHYN had good anti-inflammatory activity.
[0052] Comparative Example 1:
[0053] Preparation of Lactobacillus plantarum resistant to ammonia water stress:
[0054] The difference between this comparative example and Example 3 is only that the stress used is 0.049 vol% ammonia water solution.
[0055] Comparative Example 2:
[0056] Preparation of Lactobacillus plantarum resistant to phenoxyethanol stress:
[0057] The difference between this comparative example and Example 3 is only that the stress used is 0.8 vol% phenoxyethanol solution.
[0058] Comparative Example 3:
[0059] Preparation of Lactobacillus plantarum resistant to Staphylococcus aureus stress:
[0060] Staphylococcus aureus and Lactobacillus plantarum single colonies were respectively picked and inoculated in a co-culture medium (beef extract 5 g, peptone 10 g, sodium chloride 5 g, glucose 10 g, yeast powder 4 g, potassium phosphate dibasic 2 g, deionized water 1000 mL, pH 6.8-7.0), and after culture to the logarithmic growth phase, the number of Staphylococcus aureus colonies was adjusted to 10 6 CFU / mL, and the number of Lactobacillus plantarum colonies was adjusted to 10 8 CFU / mL, and the two were mixed at a volume ratio of 1:1, and after 12 h of shaking culture at 37 °C, the mixed solution was diluted and spread on MRS solid medium, and after the growth of colonies, Lactobacillus plantarum resistant to Staphylococcus aureus stress was obtained.
[0061] Comparative Example 4:
[0062] The only difference between this comparative example and Example 4 is that the *Lactobacillus plantarum* used was not subjected to stress before fermentation. The obtained samples were then dissolved in sterile DMEM medium to prepare sample solutions of different concentrations. After being filtered and sterilized using a 0.22 μm aqueous filter membrane, anti-inflammatory experiments and HBD2 content tests were conducted.
[0063] Comparative Example 5:
[0064] The only difference between this comparative example and Example 4 is that the fermentation strain used is the ammonia-resistant Lactobacillus plantarum obtained in Comparative Example 1. The obtained sample was then dissolved in sterile DMEM medium to prepare sample solutions of different concentrations. After filtration and sterilization using a 0.22 μm aqueous filter membrane, anti-inflammatory experiments and HBD2 content tests were conducted.
[0065] Comparative Example 6:
[0066] The only difference between this comparative example and Example 4 is that the fermentation strain used is the phenoxyethanol-resistant Lactobacillus plantarum obtained in Comparative Example 2. The obtained samples were then dissolved in sterile DMEM medium to prepare sample solutions of different concentrations. After filtration and sterilization using a 0.22 μm aqueous filter membrane, anti-inflammatory experiments and HBD2 content tests were conducted.
[0067] Comparative Example 7:
[0068] The only difference between this comparative example and Example 4 is that the fermentation strain used is the *Lactobacillus plantarum* strain resistant to *Staphylococcus aureus* stress obtained in Comparative Example 3. The obtained samples were then dissolved in sterile DMEM medium to prepare sample solutions of different concentrations. After sterilization by filtration through a 0.22 μm aqueous filter membrane, anti-inflammatory experiments and HBD2 content tests were conducted.
[0069] Depend on Figure 1 It was found that the level of the inflammatory factor IL-6 was significantly increased in cells treated with LPS. In contrast, Example 4 significantly reduced IL-6 secretion levels at a concentration of 5 mg / mL, and the level of IL-6 was negatively correlated with the sample concentration, that is, the level of IL-6 measured in RAW 264.7 cells gradually decreased as the sample concentration increased.
[0070] Depend on Figure 2 It can be seen that Example 4, at a concentration of 0.2 mg / mL, can significantly increase the amount of HBD2 secreted by cells, while Comparative Examples 4-7 cannot increase the amount of HBD2 secreted by cells at the corresponding concentrations.
[0071] In summary, the milk peptide fermentation product prepared from *Lactobacillus plantarum* resistant to sodium dodecyl sulfate stress used in this invention exhibits strong anti-inflammatory effects and better skin barrier repair function. It can alleviate skin problems and is a promising bioactive substance for application in cosmetics.
[0072] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the present application.
Claims
1. A method for the preparation of an environmental stress-based Lactobacillus plantarum fermentate, characterized by: Comprising, Step 1 : Activation of Lactobacillus plantarum (Lp) by heat treatment Lactobacillus plantarum ) Step 2: inducing culture of Lactobacillus plantarum in a culture medium containing sodium dodecyl sulfate to obtain environmental stress Lactobacillus plantarum; Step 3: dissolving milk powder in deionized water, adding alkaline protease, adjusting pH to 7-10, and performing enzymolysis to obtain an enzymolysis solution; Step 4: mixing the enzymolysis solution obtained in step 3 with sugar to obtain a fermentation substrate; Step 5: inoculating the Lactobacillus plantarum obtained in step 2 into the fermentation substrate obtained in step 4, and performing fermentation culture, centrifuging to collect supernatant, sterilizing, and obtaining an environmental stress Lactobacillus plantarum fermentation product; The milk powder is cow milk powder. The Lactobacillus plantarum has a preservation number of CCTCC No: M 20232264.
2. The process for the preparation of an environmental stress-based Lactobacillus plantarum fermentate according to claim 1, characterized in that: In step 3, the milk powder is dissolved in deionized water, and the mass concentration of the milk powder is 5-15%, and the mass concentration of the alkaline protease is 0.05-1%; the enzymolysis temperature is 20-60 ℃, and the enzymolysis time is 1.5-6 hours.
3. The method for the preparation of an environmental stress based Lactobacillus plantarum fermentate according to claim 1 or 2, characterized in that: In step 4, the sugar includes one or more of glucose, galactose oligosaccharide, and fructose oligosaccharide, and the mass concentration is 0.5-2%.
4. The method for the preparation of an environmental stress based Lactobacillus plantarum fermentate according to claim 1 or 2, characterized in that: In step 5, the inoculation amount of Lactobacillus plantarum is 2-10%; wherein the inoculated Lactobacillus plantarum is in logarithmic growth phase, and the strain concentration reaches 10 10 CFU / mL or more.
5. The method for the preparation of an environmental stress based Lactobacillus plantarum fermentate according to claim 1 or 2, characterized in that: The fermentation culture temperature in step 5 is 35-37 ℃, and the time is 12-48 hours.
6. The method for the preparation of an environmental stress based Lactobacillus plantarum fermentate according to claim 1 or 2, characterized in that: In step 2, the concentration of sodium dodecyl sulfate in the culture medium containing sodium dodecyl sulfate is 0.4-0.5 mg / mL.