A method for screening different polyphenols for probiotic and antioxidant capacity enhancement of lactobacillus plantarum

Abstract

The application discloses a method for screening different polyphenols for probiotic and antioxidation capacity improvement of lactobacillus plantarum, and belongs to the technical field of probiotics. Lactiplantibacillus plantarum The application takes lactobacillus plantarum L15 (L15) as a test strain, adds a polyphenol unified solubilization pretreatment step to eliminate test errors caused by water solubility differences of different polyphenols, and is cultured in MRS culture medium with different concentration gradients of tea polyphenols, quercetin, curcumin and resveratrol respectively, so as to detect multiple indexes of the strain, establish a standardized comprehensive evaluation system, objectively compare the synergistic effects of polyphenols, and comprehensively evaluate the probiotic effects and antioxidation capacity improvement of each polyphenol. The method is simple, systematic and repeatable, and provides a screening basis for development of polyphenol-probiotic functional products.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a method for screening different polyphenols to promote the beneficial effects of Lactobacillus plantarum and enhance its antioxidant capacity. Background Technology

[0002] With increasing health awareness among residents, supplementing the diet with probiotics to maintain gut microbiota balance and promote overall health has become a common strategy. However, probiotics face a core challenge in practical application: during food processing, storage, and human gastrointestinal digestion, adverse factors such as high temperatures, stomach acid, and bile salts can easily lead to a sharp decline in the number of live bacteria, making it difficult to ensure that sufficient live probiotics successfully reach the colon and colonize to exert their effects.

[0003] In recent years, polyphenols have received widespread attention as a novel type of "selective prebiotic." Polyphenols are secondary metabolites widely found in plants. They not only possess excellent antioxidant and anti-inflammatory physiological activities, but more importantly, they can provide multiple forms of protection for probiotics. For example, they can alleviate oxidative stress during processing and storage, help resist damage from gastric acid and bile salts, and promote the adhesion and colonization of probiotics in the gut, thereby significantly improving the survival rate and antioxidant capacity of probiotics.

[0004] Current research mainly focuses on the probiotic effects of one or more prebiotics on probiotics. While low concentrations of polyphenols not only have probiotic effects on probiotics but also enhance their antioxidant capacity, few studies systematically compare the effects of different polyphenols on the probiotic and antioxidant capabilities of probiotics. Existing techniques typically involve screening probiotic strains with antioxidant capabilities and adding antioxidants such as selenium, vitamins, and polyphenols to create a composite with overall antioxidant activity. However, in this approach, polyphenols function independently as exogenous antioxidants without substantially exploring the probiotics' own stress resistance and antioxidant capacity (such as their tolerance to gastric acid and bile salts and their free radical scavenging ability). Furthermore, the mechanisms by which polyphenols with different structures affect the same probiotic strain are unclear, and a systematic screening method is lacking. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for screening different polyphenols for their probiotic and antioxidant effects on Lactobacillus plantarum includes the following steps: (1) Add water to tea polyphenols, quercetin, curcumin and resveratrol at a mass ratio of 1:4 to 1:6 and stir for 2 hours. Filter to remove insoluble solids and prepare a homogeneous polyphenol stock solution. After activating Lactobacillus plantarum L15, inoculate it into MRS liquid medium containing polyphenols of different concentration gradients and incubate at 37°C. (2) Detect the viable number, surface hydrophobicity, acid resistance survival rate, bile salt resistance survival rate, DPPH free radical scavenging rate and ABTS free radical scavenging rate of the cultured bacterial solution; (3) Based on the test results, the growth viability, gastrointestinal tolerance, and in vitro antioxidant activity of the strains are comprehensively evaluated. A comprehensive evaluation is completed by comparing the data of each group of indicators, and the optimal polyphenol species and suitable concentration are screened out. The purpose of this invention is to improve the antioxidant capacity of probiotics and to provide an antioxidant combination. Further, the polyphenols to be tested are selected from one or more of tea polyphenols, quercetin, curcumin, and resveratrol.

[0006] Further, the concentration gradient of the tea polyphenols is 1, 2, 3, 4, 5 mg / mL; the concentration gradient of the quercetin is 0.02, 0.04, 0.06, 0.08, 0.1 mg / mL; the concentration gradient of the curcumin is 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL; and the concentration gradient of the resveratrol is 0.05, 0.10, 0.15, 0.20, 0.25 mg / mL.

[0007] Further, the activated Lactobacillus plantarum L15 described in step (1) is inoculated at an inoculum size of 3% (v / v); the culture time is 24 h.

[0008] Furthermore, the method for determining the surface hydrophobicity in step (2) is as follows: collect bacterial cells, resuspend them in PBS to adjust the OD. 600 =0.6±0.05, take the bacterial suspension and mix it with xylene and shake it. After standing and separating the layers, measure the OD600 of the aqueous phase. Calculate according to the formula: Surface hydrophobicity (%)=(1-A / A0)×100.

[0009] Further, the method for determining the acid resistance survival rate in step (2) is as follows: the cultured bacterial solution is inoculated into MRS medium at pH 3.0, anaerobic cultured at 37°C for 2 hours, and the survival rate is counted and calculated.

[0010] Further, the method for determining the bile salt tolerance survival rate in step (2) is as follows: the cultured bacterial solution is inoculated into MRS medium containing 0.3% bovine bile salt, anaerobically cultured at 37°C for 4 hours, and the survival rate is counted and calculated.

[0011] Further, the method for determining the DPPH free radical scavenging rate in step (2) is as follows: take the bacterial suspension and mix it with 0.2 mmol / L DPPH anhydrous ethanol solution, react in the dark for 30 min, and then measure the absorbance at 517 nm after centrifugation.

[0012] Further, the method for determining the ABTS free radical scavenging rate in step (2) is as follows: take the bacterial suspension and mix it with the ABTS cation free radical solution, react in the dark for 30 min, and then measure the absorbance at 734 nm after centrifugation.

[0013] The present invention also includes the application of polyphenol-Lactobacillus plantarum combination obtained by screening through the above method, wherein the polyphenol is tea polyphenol, the optimal concentration of which is 3 mg / mL, and after co-culturing with Lactobacillus plantarum L15, it is used to prepare functional foods, probiotic supplements or antioxidant products. Attached Figure Description

[0014] Figure 1 The effect of different types and concentrations of polyphenols on the viable count of L15 *Lactobacillus plantarum* (A: tea polyphenols; B: quercetin; C: curcumin; D: resveratrol).

[0015] Figure 2 The figure shows the effect of different types and concentrations of polyphenols on the surface hydrophobicity of Lactobacillus plantarum L15.

[0016] Figure 3 The figure shows the effect of different concentrations of polyphenols on the acid tolerance of Lactobacillus plantarum L15 (pH 3.0 treatment for 2 h).

[0017] Figure 4 The figure shows the effect of different concentrations of polyphenols on the bile salt tolerance of Lactobacillus plantarum L15 (0.3% bile salt treatment for 4 h).

[0018] Figure 5 The figure shows the effect of different concentrations of polyphenols on the DPPH free radical scavenging rate of Lactobacillus plantarum L15 suspension.

[0019] Figure 6 The figure shows the effect of different concentrations of polyphenols on the ABTS free radical scavenging rate of Lactobacillus plantarum L15 suspension. Detailed Implementation

[0020] Lactobacillus plantarum L15 ( Lactobacillus plantarum L 15) Sourced from the Northeast Agricultural University Microbial Culture Collection Tea polyphenols (purity ≥98%), quercetin (purity ≥98%), curcumin (purity ≥98%), and resveratrol (purity ≥98%): all are commercially available.

[0021] MRS culture medium: purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0022] Example 1: Effects of different concentrations of polyphenols on the viable count and surface hydrophobicity of *Lactobacillus plantarum*

[0023] (1) Viable bacteria count determination Tea polyphenols, quercetin, curcumin, and resveratrol were weighed separately and added to deionized water at a polyphenol:β-cyclodextrin mass ratio of 1:5. The mixture was stirred and embedded at room temperature for 2 hours. Undissolved solids were removed by filtration through a 0.22 μm filter membrane, and the corresponding polyphenol stock solutions were prepared for later use. The activated Lactobacillus plantarum L15 second-generation bacteria were inoculated at a 3% (v / v) inoculation rate into MRS liquid medium containing different concentrations of polyphenols (tea polyphenols: 1~5 mg / mL; quercetin: 0.02~0.1 mg / mL; curcumin: 0.1~0.5 mg / mL; resveratrol: 0.05~0.25 mg / mL) and incubated at 37℃ for 24 hours. The culture medium of Lactobacillus plantarum L15 was diluted 10-fold with PBS phosphate buffer to a suitable concentration gradient. 100 μL of the culture medium was inoculated into MRS solid medium. The plate was spread evenly with a glass rod and then inverted and incubated in a 37°C incubator for 48 h.

[0024] (2) Surface hydrophobicity determination Overnight cultured *Lactobacillus plantarum* L15 cells at different polyphenol concentrations were centrifuged at 5000 rpm for 10 min to collect bacterial cells, which were then washed twice with sterile PBS buffer to obtain a bacterial suspension. Subsequently, the obtained bacterial cells were resuspended in sterile PBS and A was adjusted. 600 To 0.6 ± 0.05 (A0). Mix 2400 μL of bacterial suspension with 800 μL of xylene by shaking, let stand at room temperature for 1 h to separate the layers, carefully aspirate the lower aqueous phase, and measure the absorbance A. 600 Value (A). The calculation formula is as follows:

[0025] Where A0 represents the initial absorbance of the bacterial cells; A is the absorbance of the lower aqueous phase after xylene treatment.

[0026] The results are as follows Figure 1 As shown, all four polyphenols exhibited a bidirectional regulatory effect, promoting the growth of *Lactobacillus plantarum* at low concentrations and inhibiting its growth at high concentrations. Tea polyphenols (3 mg / mL) and quercetin (0.04 mg / mL) showed the highest viable bacterial counts, both increasing by 0.18 log CFU / mL compared to the control group. Curcumin (0.2 mg / mL) and resveratrol (0.15 mg / mL) also showed the highest viable bacterial counts, increasing by 0.16 log CFU / mL compared to the control group. The differences in optimal concentrations among these four polyphenols are essentially due to their different molecular structures, antibacterial activity strengths, and bidirectional regulatory effects on *Lactobacillus plantarum*.

[0027] like Figure 2As shown, the surface hydrophobicity of *Lactobacillus plantarum* bacterial suspension initially increased and then decreased with increasing concentrations of the four polyphenols. The highest surface hydrophobicity (39.59%) was observed with a polyphenol concentration of 3 mg / mL, a 1.05-fold increase compared to the control group without added polyphenols. The second highest was the bacterial suspension with added resveratrol (0.15 mg / mL). Increased surface hydrophobicity facilitates the adhesion of probiotics to intestinal epithelial cells or mucosal surfaces, thereby enhancing their colonization and survival capabilities in the intestine.

[0028] Example 2: Effects of optimal polyphenol concentrations on the acid and bile salt tolerance and antioxidant activity of *Lactobacillus plantarum*

[0029] (1) Determination of acid resistance 6 mol / L HCl solution was added to culture media containing 3 mg / ml tea polyphenols, 0.04 mg / ml quercetin, 0.2 mg / ml curcumin, 0.15 mg / ml resveratrol, and no polyphenols, respectively, and the pH was adjusted to 3.0. The cultured bacterial strains (10... 9 CFU / mL was inoculated at a rate of 3% and transferred to both acidified and unacidified control media. The media were incubated at 37°C for 2 hours. After incubation, serial dilutions were performed, and colony counting was performed using the MRS agar pour plate method.

[0030]

[0031] (2) Test of bile salt tolerance 0.3% ox bile salt was added to culture media containing 3 mg / ml tea polyphenols, 0.04 mg / ml quercetin, 0.2 mg / ml curcumin, 0.15 mg / ml resveratrol, and no polyphenols, respectively. The cultured strains (10...) 9 The inoculum (CFU / mL) was transferred at a 3% inoculum to a medium containing 0.3% ox bile salt and a control without ox bile salt. The medium was then anaerobically cultured at 37°C for 4 hours. After the culture was terminated, the medium was serially diluted and colony counts were performed using the MRS agar pour plate method.

[0032]

[0033] (3) Antioxidant activity assay a. DPPH scavenging ability determination Mix 1 mL of sample with 1 mL of DPPH solution (0.2 mmol / L) dissolved in anhydrous ethanol, react at room temperature in the dark for 30 minutes, then centrifuge at 5000 r / min for 10 minutes. Measure the absorbance of the supernatant at 517 nm. The clearance rate is calculated using the following formula: In the blank group, anhydrous ethanol was used instead of DPPH solution, and in the control group, PBS was used instead of the sample.

[0034] b.ABTS Equal volumes of 4.95 mM potassium persulfate solution and 7 mM ABTS solution were mixed and reacted at room temperature in the dark for 12–14 h to prepare an ABTS cationic radical solution. Before use, the ABTS cationic radical solution was diluted with anhydrous ethanol to ensure that the absorbance of the diluted solution was 0.70 ± 0.02 at 734 nm.

[0035] Add 1 ml of sample solution and 3 ml of ABTS cation radical solution to a 10 ml centrifuge tube, mix well, and react at room temperature in the dark for 30 min. Then centrifuge at 5000 r / min for 10 min, and measure the absorbance of the supernatant at 734 nm. To eliminate the influence of the sample itself on the test results, use an equal volume of deionized water instead of ABTS cation radical solution as the sample control group, and use an equal volume of deionized water instead of sample solution as the negative control group.

[0036]

[0037] In the formula, A1 - Absorbance of the negative group, the negative group is deionized water + ABTS cationic free radicals; B1 - Absorbance of the sample group, where the sample group consists of sample + ABTS cation radicals; B2 - Absorbance of the sample control group, which consists of the sample and deionized water.

[0038] The result is Figures 3 to 6 It was found that the acid resistance of *Lactobacillus plantarum* strains with the optimal concentration of polyphenols increased, with the strain containing 3 mg / ml tea polyphenols showing the best acid resistance (90.85%). The bile salt resistance of *Lactobacillus plantarum* strains with the optimal concentration of polyphenols also significantly increased, with the strain containing 3 mg / ml tea polyphenols showing the best and most significant increase. The DPPH free radical scavenging capacity of *Lactobacillus plantarum* strains with 3 mg / ml tea polyphenols and 0.04 ml / ml quercetin increased, but the DPPH free radical scavenging capacity of strains with 0.2 mg / ml curcumin and 0.15 mg / ml resveratrol decreased. This indicates that different polyphenols have different effects on DPPH scavenging capacity, with tea polyphenols and quercetin showing better performance in this indicator. The ABTS radical scavenging capacity of *Lactobacillus plantarum* bacterial suspensions with the addition of optimal concentrations of tea polyphenols, quercetin, and resveratrol all increased, but the ABTS radical scavenging rate of the bacterial suspension with the addition of curcumin did not change significantly. Among them, the ABTS radical scavenging capacity of the bacterial suspension with the addition of the optimal concentration of tea polyphenols increased most significantly.

[0039] Example 3: Comprehensive Screening Results and Application Examples

[0040] Based on the above indicators, 3 mg / mL tea polyphenols showed the best performance in terms of viable bacteria count, surface hydrophobicity, acid and bile salt resistance, and ABTS scavenging rate, and were therefore selected as the best polyphenol type and the best concentration.

[0041] A bacterial suspension obtained by co-culturing 3 mg / mL tea polyphenols with *Lactobacillus plantarum* L15 for 24 hours can be used directly as a liquid probiotic preparation or freeze-dried into a lyophilized powder. Maltodextrin, inulin, and fructooligosaccharides can be added as preservatives during lyophilization to improve the survival rate of the strains during room temperature storage. Testing showed that the survival rate of this bacterial suspension in simulated gastrointestinal fluid was significantly higher than that of the control group, and both DPPH and ABTS clearance rates were increased. It can be used to prepare functional foods, probiotic supplements, or antioxidant products.

[0042] The above description is merely 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 screening different polyphenols to promote the probiotic effect and enhance the antioxidant capacity of Lactobacillus plantarum, characterized in that, Includes the following steps: (1) β-cyclodextrin was used to encapsulate and solubilize tea polyphenols, quercetin, curcumin and resveratrol to unify the solubility system. After activating L15 plant lactobacillus, it was inoculated into MRS liquid medium containing different concentration gradients of the polyphenols to be tested and incubated at 37℃. (2) Detect the viable number, surface hydrophobicity, acid and bile salt resistance survival rate, DPPH free radical scavenging rate and ABTS free radical scavenging rate of the cultured bacterial solution; (3) Based on the test results, comprehensively evaluate the probiotic effects and antioxidant capacity enhancement of each polyphenol, and screen out the best polyphenol types and the best concentrations.

2. The method according to claim 1, characterized in that, The concentration of Lactobacillus plantarum L15 was 10. 9 CFU / mL.

3. The method according to claim 1, characterized in that, The polyphenols to be tested are selected from one or more of tea polyphenols, quercetin, curcumin, and resveratrol.

4. The method according to claim 2, characterized in that, The concentration gradients of the tea polyphenols are 1, 2, 3, 4, and 5 mg / mL; the concentration gradients of the quercetin are 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL; the concentration gradients of the curcumin are 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL; and the concentration gradients of the resveratrol are 0.05, 0.10, 0.15, 0.20, and 0.25 mg / mL.

5. The method according to any one of claims 1 to 4, characterized in that, The optimal polyphenol mentioned in step (3) is tea polyphenol, with an optimal concentration of 3 mg / mL.

6. The application of the method according to any one of claims 1 to 5 in screening food-grade polyphenol-probiotic complex functional compositions.

7. A polyphenol-Lactobacillus plantarum composition obtained by screening using the method according to any one of claims 1 to 5, characterized in that, The composition comprises tea polyphenols and Lactobacillus plantarum L15, wherein the concentration of tea polyphenols is 3 mg / mL.

8. The composition according to claim 7, characterized in that, The dosage form of the composition is any one of liquid bacterial agent, lyophilized powder, tablet, capsule, granule or powder. During the preparation of the dry powder, one or more of maltodextrin, inulin and fructooligosaccharides can be added as a protective agent to improve the storage stability of the strain.

9. A method for improving the probiotic and antioxidant capacity of Lactobacillus plantarum, characterized in that, After pre-encapsulating tea polyphenols with β-cyclodextrin, the tea polyphenols were added to the culture medium of Lactobacillus plantarum L15 at a concentration of 3 mg / mL for co-culture.

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