Method for improving lactobacillus plantarum metabolite through illumination pretreatment
By pretreatment with alternating red and blue light, the enzyme activity and extracellular polysaccharide production of Lactobacillus plantarum were enhanced, solving the problems of activity and stability of Lactobacillus plantarum under environmental stress and achieving efficient enhancement of metabolites.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENYANG AGRI UNIV
- Filing Date
- 2026-02-26
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, the activity and stability of Lactobacillus plantarum during industrial production, storage, and passage through the gastrointestinal tract are affected by environmental stress, leading to a decrease in viable bacterial count and weakened function. Existing methods also suffer from problems such as long strain selection cycles, high costs, complex operations, and the potential introduction of exogenous components.
Before the activation culture stage of Lactobacillus plantarum, a light pretreatment of alternating red and blue light for 20-100 minutes is used to enhance bacterial activity and metabolites. The red light wavelength is 620nm and the blue light wavelength is 450nm.
It significantly improved the enzyme activity, extracellular polysaccharide yield, and antioxidant capacity of Lactobacillus plantarum, and enhanced the yield and activity of its metabolites. The operation is simple and does not require the addition of exogenous substances.
Smart Images
Figure CN122012269A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of microbial fermentation, and specifically to a method for enhancing the metabolites of Lactobacillus plantarum through light pretreatment. Background Technology
[0002] Lactobacillus plantarum is an important lactic acid bacterium widely used in food fermentation, probiotic preparations, and animal husbandry. It possesses various beneficial functions, including regulating intestinal flora, enhancing immunity, and lowering cholesterol. Its activity and metabolites (such as various enzymes and extracellular polysaccharides) directly determine its application effects. Enzyme activity affects its metabolic capacity and functional characteristics, while extracellular polysaccharides are closely related to its probiotic properties and the texture of fermentation products. Cell concentration is a key indicator for measuring its growth efficiency and biomass. However, during industrial production, storage, and passage through the gastrointestinal tract, the activity and stability of Lactobacillus plantarum are affected by various environmental stresses, leading to a decrease in viable cell count and weakened function, which significantly limits its application effectiveness.
[0003] Currently, methods to improve the activity and metabolites of Lactobacillus plantarum mainly focus on optimizing culture medium composition (such as adding carbon sources, nitrogen sources, and growth factors), altering physical conditions (such as temperature and pH), or conducting strain selection. While these methods are effective, they also have some limitations: strain selection is time-consuming and highly uncertain; adding chemicals to the culture medium may increase costs and potentially introduce exogenous components into the final product, making them unsuitable for foods pursuing "clean labels," and they also suffer from high costs and complex operations.
[0004] While light, as a green and physical regulatory tool, has been applied in the cultivation of microalgae and some photosynthetic bacteria, its systemic effects on non-photosynthetic lactic acid bacteria, particularly *Lactobacillus plantarum*, and its potential for targeted enhancement of bacterial activity and metabolites, remain largely unexplored. Existing research on the effects of light on lactic acid bacteria primarily focuses on the fermentation process, with inconsistent results; some reports even suggest that light may generate free radicals that could damage the bacteria. Current techniques do not utilize light as a pretreatment method during the critical "pre-activation" phase to systematically enhance various properties of *Lactobacillus plantarum* (including enzyme activity, antioxidant capacity, and extracellular polysaccharides). Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a method for enhancing the metabolites of Lactobacillus plantarum through light treatment, comprising the following steps: pre-treating the Lactobacillus plantarum strain with light before the activation culture stage.
[0006] Furthermore, before activating Lactobacillus plantarum, it was irradiated with alternating red and blue light for 20-100 minutes.
[0007] Furthermore, the wavelength of red light is 620nm and the wavelength of blue light is 450nm, and the red and blue light are alternately irradiated for 40 minutes.
[0008] Furthermore, the strain used for *Lactobacillus plantarum* is P-8. Furthermore, the object of the light treatment is a liquid bacterial suspension.
[0009] Furthermore, the concentration of the liquid bacterial suspension is 10. 6 -10 9 CFU / mL.
[0010] Furthermore, the light pretreatment specifically involves taking 1% of the activated Lactobacillus plantarum solution into a sterilized, reactivated culture medium, and then evenly distributing the solution into identical 6-well plates. Before the reactivation, the plates containing the solution are placed in a dark environment with the light source positioned at the top, and irradiated with red and blue light for 20 minutes each.
[0011] Furthermore, the "before" stage of activation culture of Lactobacillus plantarum specifically refers to the period after the first generation activation of the strain and before the second generation activation.
[0012] Furthermore, the first-generation activation is carried out at a temperature of 37°C for 18-24 hours, preferably 24 hours; the second activation is carried out at a temperature of 37°C for 14-24 hours, preferably 16 hours.
[0013] Furthermore, the activation culture is carried out in MRS liquid medium at a temperature of 37°C for 16 hours, with the first generation activation time being 24 hours and the second activation time being 16 hours.
[0014] The technical solution of this invention has the following advantages: This invention provides a method for enhancing the metabolites of *Lactobacillus plantarum* through light treatment. This method is a simple, low-cost physical treatment that requires no exogenous substances. By applying light to *Lactobacillus plantarum* during a specific culture stage, the activity of some enzymes and the yield of extracellular polysaccharides can be significantly increased. Furthermore, this method can effectively enhance the antioxidant capacity of *Lactobacillus plantarum*, thereby improving the overall yield and activity of its metabolites. Attached Figure Description
[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0016] Figure 1 middle, Figure 1 (A) shows the growth curve of Lactobacillus plantarum; Figure 1 (B) The bacterial cell concentration (OD) of *Lactobacillus plantarum* in the untreated group (CK) and the treated group (L) at 8, 16, and 24 h after activation. 600 ); Figure 2 The relative activities of L-α-rhamnosidase in the fermentation supernatant of Lactobacillus plantarum in the CK and L groups were compared. Figure 3 The relative activities of β-glucosidase in the fermentation supernatant of Lactobacillus plantarum in the CK and L groups were compared. Figure 4 The relative activities of β-galactosidase in the fermentation supernatant of Lactobacillus plantarum in the CK and L groups were compared. Figure 5 The malondialdehyde content in the fermentation supernatant of Lactobacillus plantarum in the CK and L groups; Figure 6 The content of extracellular polysaccharides in the fermentation supernatant of Lactobacillus plantarum in the CK and L groups; Figure 7 The antioxidant activity of Lactobacillus plantarum fermentation supernatant in CK and L groups was measured. Detailed Implementation
[0017] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.
[0018] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. Example 1
[0019] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0020] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0021] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0022] S4: The optical density (OD) of the sample is determined using the optical density method at a wavelength of 600 nm using spectrophotometry. 600 The concentration of Lactobacillus plantarum cells at different activation time points before and after light treatment was calculated.
[0023] To illustrate the effects of the present invention, this embodiment compares the method of the present invention with the cell concentration of *Lactobacillus plantarum* that has not been exposed to light: The comparison results are as follows Figure 1 As shown. Figure 1 The figure shows the cell concentration (OD600) of Lactobacillus plantarum at 8, 16, and 24 hours after activation. The overall OD value shows a trend of first increasing and then decreasing. In summary, the light group (L group) and the control group (CK group) reached their maximum values at 16 hours after activation, indicating that a certain amount of light does not reduce the cell concentration of Lactobacillus plantarum. Example 2
[0024] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0025] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0026] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0027] S4: To determine the relative enzyme activity of α-L-rhamnoside in *Lactobacillus plantarum*, the activated *Lactobacillus plantarum* solution was first centrifuged, then sonicated at 0°C for 15 min, and centrifuged again. The supernatant was the enzyme solution. The enzyme solution was preheated at 60°C for 5 min. A certain mass of the enzyme solution was added to the reaction mixture prepared with phosphate-buffered saline (PBS, pH 6.5) and α-L-rhamnoside (pNPR). The reaction was carried out at 60°C for 10 min. Sodium carbonate was then added. The reaction was terminated with a 2M solution. The reaction solution was centrifuged (10,000 rpm, 10 min), and the absorbance of the supernatant was measured at 410 nm.
[0028] like Figure 2 As shown, the relative enzyme activity first increased and then decreased over time. At different time points, the relative enzyme activity of rhamnosidase in group L was higher than that in group CK. The maximum value in group L reached 100%, while the relative enzyme activity in group CK was around 95% at the same time point. In subsequent fermentation, the stronger enzyme activity of rhamnosidase in group L will have a more significant effect on improving the flavor and aroma of the juice. Example 3
[0029] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0030] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0031] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0032] S4: To determine the relative enzyme activity of β-glucosidase in *Lactobacillus plantarum*, the activated *Lactobacillus plantarum* solution was first centrifuged, then sonicated at 0℃ for 15 min, and centrifuged again. The supernatant was the enzyme solution. A certain amount of the enzyme solution was taken and disodium hydrogen phosphate-citric acid solution (pH 6.0) was added. The mixture was preheated in a 40℃ water bath for 5 min, and then 1.6 mmol of p-nitrophenol-β-D-glucosidase (pNPG) solution, which had been preheated for 10 min, was added. After reacting for 30 min, 0.5 mol / L Na₂CO₃ solution was immediately added, and the mixture was placed at room temperature. The absorbance was measured at 400 nm.
[0033] like Figure 3 As shown, the relative activity of β-glucosidase in *Lactobacillus plantarum* gradually decreased over time, with the maximum value reaching 100% in group L, while the enzyme activity in group CK was only 93% at the same time point. β-glucosidase can improve the bitterness of *Schisandra chinensis* pomace, and the higher relative enzyme activity in group L may indicate that it significantly improves the bitterness of *Schisandra chinensis* pomace in subsequent experiments. Example 4
[0034] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0035] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0036] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0037] S4: To determine the relative enzyme activity of β-galactosidase in *Lactobacillus plantarum*, the activated *Lactobacillus plantarum* solution was first centrifuged, then sonicated at 0℃ for 15 min, and centrifuged again. The supernatant was the enzyme solution. A certain amount of the enzyme solution was taken and added to a β-galactosidase solution that had been preheated for 10 min. After reacting at 37℃ for 10 min, 4 ml of [unspecified ingredient] was immediately added. The solution was placed at room temperature, and the absorbance was measured at 400 nm.
[0038] like Figure 4 As shown, the relative activity of β-galactosidase in *Lactobacillus plantarum* gradually decreased over time, with the maximum value reaching 100% in group L, while the enzyme activity in group CK was only about 92% at the same time point. The higher relative enzyme activity of β-galactosidase in group L has a significant effect on improving the digestibility of dairy products. Example 5
[0039] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0040] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0041] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0042] S4: Take 1 ml of the centrifuged sample and add 10% trichloroacetic acid (TCA) solution. Centrifuge and collect the supernatant. Add 0.67 g of malondialdehyde (TBA) solution to the supernatant, centrifuge and boil for 20 min, cool to 25°C and centrifuge again. Measure the absorbance at 450 nm, 532 nm and 600 nm.
[0043] like Figure 5 As shown, the malondialdehyde (MDA) content in *Lactobacillus plantarum* was slightly higher after light exposure than in the control group. This indicates that light pretreatment does not affect the integrity of the cell membrane structure. Example 6
[0044] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0045] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0046] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0047] S4: The extracellular polysaccharide (EPS) content in *Lactobacillus plantarum* was determined by alcohol precipitation. 50 ml of twice-activated liquid culture medium was centrifuged at 6000 rpm for 30 min. 15 ml of the supernatant was collected, and 30 ml of 96% ethanol solution was added. The mixture was placed in a 4℃ refrigerator for 24 h to allow EPS precipitation. Afterward, it was centrifuged again at 6000 rpm for 30 min, the precipitate was collected, eluted with ethanol solution, and centrifuged again. The precipitate was dried at -80℃ for 2 h, cooled, and weighed to calculate the EPS content.
[0048] like Figure 6 As shown, the EPS content in the CK group was 370.21 mg / L, while after light exposure, the EPS content in *Lactobacillus plantarum* reached 390.13 mg / L, which was approximately 20 mg / L higher than that in the CK group. This suggests that the L group may have better utilized the carbon source in the culture medium, preventing excessive lactic acid production and oxidizing it to CO2. Example 7
[0049] S1: Take Lactobacillus plantarum powder frozen in the refrigerator and culture it on solid culture medium for 48 hours at a temperature of 37°C.
[0050] S2: The solid-activated Lactobacillus plantarum was subjected to first-generation liquid culture for 24 hours at a temperature of 37°C.
[0051] S3: Take 1% of the activated liquid culture medium containing bacteria and add it to the sterilized liquid culture medium (the above operations are performed in a clean bench). After sealing the culture medium, treat it with light.
[0052] S4: Take an appropriate amount of Lactobacillus plantarum fermentation broth, centrifuge, and collect the supernatant. Use the FRAP kit to determine the antioxidant activity of the CK group and the L group.
[0053] like Figure 7 As shown, the FRAP content of group L was 514 mmol / g, while the FRAP content of group CK was only 191 mmol / g. The FRAP content of Lactobacillus plantarum increased by nearly 2.7 times after light treatment, which can enhance the antioxidant activity of Lactobacillus plantarum cells.
[0054] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for enhancing the metabolites of Lactobacillus plantarum through light treatment, characterized in that, Includes the following steps: Before the activation culture stage of Lactobacillus plantarum, the Lactobacillus plantarum strain is subjected to light pretreatment.
2. The method according to claim 1, characterized in that, Before activating Lactobacillus plantarum, irradiate it with alternating red and blue light for 20-100 minutes.
3. The method according to claim 2, characterized in that, The wavelength of red light is 620nm and the wavelength of blue light is 450nm. The red and blue light are alternately irradiated for 40 minutes.
4. The method according to claim 1, characterized in that, The strain used for Lactobacillus plantarum is P-8.
5. The method according to claim 1, characterized in that, The object subjected to the light treatment is a liquid bacterial suspension.
6. The method according to claim 5, characterized in that, The concentration of the liquid bacterial suspension is 10. 6 -10 9 CFU / mL.
7. The method according to claim 1, characterized in that, The light pretreatment specifically involves taking 1% of the activated Lactobacillus plantarum solution and adding it to a sterilized, reactivated culture medium. The solution is then evenly distributed into identical 6-well plates. Before the reactivation, the plates containing the solution are placed in a dark environment with the light source positioned at the top, and irradiated with red and blue light for 20 minutes each.
8. The method according to any one of claims 1-6, characterized in that, Specifically, the period before the activation culture stage of Lactobacillus plantarum refers to the period after the first generation activation of the strain and before the second generation activation.
9. The method according to claim 8, characterized in that, The first activation was performed at 37°C for 18-24 hours; the second activation was performed at 37°C for 14-24 hours.
10. The method according to claim 8, characterized in that, The activation culture was carried out in MRS liquid medium at a temperature of 37°C for 16 hours, with the first generation activation time being 24 hours and the second activation time being 16 hours.