Application of a cyclic dipeptide in promoting the growth of Lactobacillus plantarum

Abstract

This invention belongs to the field of bioactive peptides, specifically relating to the application of a cyclic dipeptide in promoting the growth of *Lactobacillus plantarum*. This invention provides an active cyclic dipeptide, Cyclo(Leu-Pro), which promotes the growth of *Lactobacillus plantarum*, shortens the lag phase of *Lactobacillus plantarum* in harsh environments, accelerates its growth rate, and enhances the stress resistance of *Lactobacillus plantarum*.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptides, specifically relating to the application of a cyclic dipeptide in promoting the growth of Lactobacillus plantarum. Background Technology

[0002] Lactobacillus plantarum is a Gram-positive bacterium belonging to the genus Lactobacillus. Its optimal growth temperature is 30–37°C, and its optimal pH is around 6.5. This bacterium is widely distributed in dairy products, meat products, and the gastrointestinal environment. It has been reported that Lactobacillus plantarum possesses numerous beneficial functions, including antibacterial, anti-inflammatory, antioxidant, cholesterol-lowering, nutrient digestion and absorption promotion, host immune regulation, and maintenance of intestinal flora homeostasis.

[0003] Cyclodipeptides (CDPs), also known as 2,5-diketopiperazines, are a class of relatively simple compounds formed by the cyclization of dipeptides and their amides. They are secondary functional metabolites or byproducts of protein metabolism in bacteria, fungi, and animals. Related studies have shown that CDPs possess potent biological activities such as anticancer, antileukemia, antiviral, and antibacterial activity. Furthermore, some CDPs, as diffusion molecules involved in intercellular communication, may constitute a new type of quorum sensing signal or interspecies signaling mechanism in bacteria, regulating the life activities of the bacterial community or host.

[0004] Cyclo (Leu-Pro) is a product formed by the cyclization of leucine and proline. It appears as white or colorless crystals or powder, with a molecular weight of 210.3 Da and a melting point between 163-165℃. Its CAS number is 2873-36-1. Cyclo (Leu-Pro) contains a stable six-membered ring structure with certain conformational constraints, making it more stable than other peptide compounds. It has been reported that this compound, as a signaling molecule for intercellular communication, can activate or inhibit quorum sensing in bacterial communities. It also possesses antibacterial, antifungal, and antiviral effects.

[0005] However, no literature has yet reported on the bioactivity of Cyclo(Leu-Pro) in promoting the growth of Lactobacillus plantarum and enhancing its stress resistance. Summary of the Invention

[0006] To address the above problems, the present invention discloses the following technical solution:

[0007] The use of cyclic (leucine-proline) in any of the following (1)-(8):

[0008] (1) Application in improving the growth of Lactobacillus plantarum;

[0009] (2) Application in improving the resistance of Lactobacillus plantarum to temperature stress;

[0010] (3) Application in improving the salt stress resistance of Lactobacillus plantarum;

[0011] (4) Application in improving the acid stress resistance of Lactobacillus plantarum;

[0012] (5) Application in the preparation of formulations that enhance the growth of Lactobacillus plantarum;

[0013] (6) Application in the preparation of formulations that enhance the resistance of Lactobacillus plantarum to temperature stress;

[0014] (7) Application in the preparation of formulations that enhance the salt stress resistance of Lactobacillus plantarum;

[0015] (8) Application in the preparation of formulations that enhance the acid stress resistance of Lactobacillus plantarum.

[0016] Preferably, the concentration of the cyclic (leucine-proline) is 0.05-10 μg / mL.

[0017] Preferably, the concentration of the cyclic (leucine-proline) is 1 μg / mL.

[0018] The beneficial effects of this invention are:

[0019] (1) Cyclo(Leu-Pro) has a significant promoting effect on the growth of Lactobacillus plantarum, and can significantly increase its viable count and biomass;

[0020] (2) Cyclo (Leu-Pro) can shorten the lag phase of Lactobacillus plantarum under stress conditions, increase the growth rate, and enhance the stress resistance of Lactobacillus plantarum. Attached Figure Description

[0021] Figure 1 Effects of different concentrations of Cyclo(Leu-Pro) on the growth curves of Lactobacillus plantarum;

[0022] Figure 2 Effects of different concentrations of Cyclo(Leu-Pro) on the OD value of Lactobacillus plantarum;

[0023] Figure 3 The effect of different concentrations of Cyclo(Leu-Pro) on the viable count of Lactobacillus plantarum;

[0024] Figure 4 Effect of different concentrations of Cyclo(Leu-Pro) on the viable count of Lactobacillus plantarum;

[0025] Figure 5 The effect of Cyclo (Leu-Pro) on the temperature stress resistance of Lactobacillus plantarum;

[0026] Figure 6 The effect of Cyclo (Leu-Pro) on the salt stress resistance of Lactobacillus plantarum;

[0027] Figure 7 Effects of Cyclo (Leu-Pro) on the acid stress resistance of Lactobacillus plantarum. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0029] The cyclic dipeptide Cyclo(Leu-Pro) used in the experiment was purchased from Nanjing Yuanpeptide Biotechnology Co., Ltd., batch number: Yuanpeptide-603009, peptide sequence Cyclo(-Leu-Pro), purity 95%, molecular weight 210.3 Da, CAS number: 2873-36-1.

[0030] Example 1: Effect of Cyclo (Leu-Pro) on the growth of Lactobacillus plantarum

[0031] ① Take out the Lactobacillus plantarum stored in glycerol at -80℃ and thaw it at room temperature. Inoculate it into MRS broth medium at an inoculation rate of 1% and incubate it at 37℃ for 18 hours. Activate it for two generations to restore the cell viability.

[0032] ② Prepare a 10 mg / mL stock solution of Cyclo (Leu-Pro) with ultrapure water, and then dilute it to solutions of 1, 5, 10, 50, 100, 500, and 1000 μg / mL. Store the solutions at 4°C for later use.

[0033] ③ The activated second-generation bacterial culture was inoculated into MRS broth medium at a 1% inoculum. The control group received 1% ultrapure water, while the experimental groups received 1% Cyclo (Leu-Pro) solution of different concentrations. After mixing, the cultures were incubated at 37℃ for 18 hours. 200 μL of each culture was injected into 96-well plates, and the absorbance of *Lactobacillus plantarum* at 600 nm was dynamically monitored using an automated microplate reader. A growth curve of *Lactobacillus plantarum* was plotted with incubation time on the x-axis and absorbance on the y-axis. The OD value and viable cell count of *Lactobacillus plantarum* in both the control and experimental groups were measured.

[0034] ④ Results Analysis: The results are as follows Figure 1-4As shown, Cyclo(Leu-Pro) exhibited a significant growth-promoting effect on *Lactobacillus plantarum* after 12 hours of culture; at 18 hours of culture, the OD values ​​of the control group and each experimental group (concentration from low to high) were significantly higher. 600 The nm values ​​were 1.44, 1.42, 1.49, 1.53, 1.57, 1.52, and 1.52, respectively. The biomass of the experimental groups increased by -1.62%, 3.61%, 6.41%, 9.20%, 5.34%, and 5.83% compared to the control group, respectively. The highest biomass of *Lactobacillus plantarum* was observed when the Cyclo(Leu-Pro) concentration was 1 μg / mL. Similar results were observed when determining the viable cell count. Compared to the control group, the viable cell count in each experimental group significantly increased, with the highest viable cell count (8.73 × 10⁻⁶) achieved when the Cyclo(Leu-Pro) concentration was 1 μg / mL. 10 CFU / mL. This indicates that Cyclo(Leu-Pro) can increase the proliferation rate of Lactobacillus plantarum and maintain its viable cell count at a high level.

[0035] Example 2: Effect of Cyclo (Leu-Pro) on the stress resistance of Lactobacillus plantarum

[0036] ① Prepare a 100 μg / mL solution of cyclic dipeptide Cyclo (Leu-Pro) with ultrapure water and store it at 4°C for later use.

[0037] ② The activated second-generation bacterial culture was inoculated into MRS broth medium at a 1% inoculum. The control group received 1% ultrapure water, while the experimental groups received 1% of 100 μg / mL Cyclo (Leu-Pro) solution. After mixing, the cultures were incubated statically at different temperatures (28℃, 37℃, 42℃) for 18 hours. Growth curves, OD values, and viable cell counts were measured. Results are as follows: Figure 5 As shown, when cultured at 28℃, the addition of Cyclo(Leu-Pro) accelerated the growth rate of *Lactobacillus plantarum*, shortened the lag phase, and accelerated its entry into the logarithmic phase. When cultured at 37℃ and 42℃, the addition of Cyclo(Leu-Pro) further accelerated the growth rate of *Lactobacillus plantarum* in the logarithmic phase, resulting in higher biomass in both the logarithmic and stationary phases compared to the control group. At 18 hours of culture, the OD values ​​of each control group (from lowest to highest temperature) were... 600 The nm values ​​were 1.55, 1.54, and 1.31, while the OD600 nm values ​​of the experimental groups were 1.59, 1.69, and 1.41, respectively. The biomass of the experimental groups increased by 2.95%, 9.43%, and 7.53% compared to the control group, respectively. The logarithmic viable counts of the control groups were 9.15, 9.46, and 8.92, respectively, while the logarithmic viable counts of the experimental groups were 9.73, 10.12, and 9.29, respectively. Notably, at 37℃, the viable count of *Lactobacillus plantarum* increased from 2.90 × 10⁻⁶ to 1.55.9 CFU / mL increased to 1.33 × 10⁻⁶ 10 CFU / mL. In summary, at 28–37℃, the growth rate of *Lactobacillus plantarum* accelerated, and the viable cell count and OD value significantly increased (P<0.05). This indicates that Cyclo(Leu-Pro) enhances the resistance of *Lactobacillus plantarum* to temperature stress.

[0038] ③ The activated second-generation bacterial suspension was inoculated at a 1% inoculum into MRS broth containing 2%, 4%, 6%, and 8% NaCl, respectively. The control group received 1% ultrapure water, while the experimental groups received 1% of 100 μg / mL Cyclo (Leu-Pro) solution. After mixing, the cultures were incubated at 37°C for 18 hours. Growth curves, OD values, and viable cell counts were measured. Results are as follows: Figure 6 As shown, with increasing salt concentration, the growth and metabolism of *Lactobacillus plantarum* were inhibited to varying degrees, and its OD... 600 The nm value showed a decreasing trend. However, during the logarithmic and stationary phases, the OD of the experimental group... 600 The OD600nm values ​​were consistently higher than those of the control group. The addition of Cyclo(Leu-Pro) shortened the lag phase of *Lactobacillus plantarum*, allowing it to enter the logarithmic phase earlier, resulting in significantly higher biomass in both the logarithmic and stationary phases compared to the control group. Cyclo(Leu-Pro) showed the most significant effect on *Lactobacillus plantarum* growth at a salt concentration of 6%. After 18 hours of culture, the OD600nm values ​​of the control groups were 1.50, 1.30, 0.22, and 0.18, respectively, while the OD600nm values ​​of the experimental groups were significantly higher. 600 The nm values ​​were 1.59, 1.45, 0.76, and 0.22, respectively. The biomass of the experimental groups increased by 6.00%, 11.54%, 245%, and 22.22% compared to the control groups, respectively. The logarithmic viable counts of the control groups were 9.88, 9.79, 9.18, and 8.07, respectively, while the logarithmic viable counts of the experimental groups were 10.08, 10.24, 9.38, and 8.15, respectively. At a salt concentration of 4%, the viable count of *Lactobacillus plantarum* increased from 6.13 × 10⁻⁶ to 10⁻⁶. 9 CFU / mL increased to 1.73 × 10⁻⁶ 10 CFU / mL. In summary, when the salt concentration was 2%–6%, the lag phase of *Lactobacillus plantarum* was shortened, and the OD values ​​and viable cell counts of each experimental group were significantly higher than those of the control group (P<0.05). This indicates that Cyclo(Leu-Pro) can help *Lactobacillus plantarum* adapt quickly to harsh environments, promote its growth and metabolism, and enhance its resistance to salt stress.

[0039] ④ The activated second-generation bacterial suspension was inoculated at a 1% inoculum into MRS broth medium at pH 3, 4, 5, and 6, respectively. The control group received 1% ultrapure water, while the experimental groups received 1% of 100 μg / mL Cyclo (Leu-Pro) solution. After mixing, the cultures were incubated at 37°C for 18 hours. Growth curves, OD values, and viable cell counts were then measured. Results are as follows: Figure 7 As shown, the growth of *Lactobacillus plantarum* was inhibited to varying degrees as the pH decreased, and its OD... 600 The nm value showed a decreasing trend. However, during the logarithmic and stationary phases, the OD of the experimental group... 600 The OD600 nm values ​​were consistently higher than those of the control group. At pH 4–6, the addition of Cyclo(Leu-Pro) accelerated the growth rate of *Lactobacillus plantarum*, resulting in higher biomass during both the logarithmic and stationary phases compared to the control group. The effect of Cyclo(Leu-Pro) on *Lactobacillus plantarum* growth was most significant at pH 4; however, at pH 3, there was no significant difference between the experimental and control groups. After 18 hours of culture, the OD600 nm values ​​of the control groups (pH from low to high) were 0.16, 0.61, 1.42, and 1.56, respectively, while the OD600 nm values ​​of the experimental groups were significantly higher. 600 The nm values ​​were 0.19, 1.09, 1.52, and 1.61, respectively. The biomass of the experimental groups increased by 18.75%, 78.69%, 7.04%, and 3.21% compared to the control groups, respectively. The logarithmic viable counts of the control groups were 6.89, 8.79, 9.80, and 9.91, respectively, while the logarithmic viable counts of the experimental groups were 6.96, 8.98, 10.05, and 10.30, respectively. At pH 6, the viable count of *Lactobacillus plantarum* increased from 8.07 × 10⁻⁶ to 10⁻⁶. 9 CFU / mL increased to 2.01 × 10⁻⁶ 10 CFU / mL. In summary, when the pH is 4–6, the logarithmic growth rate of Lactobacillus plantarum is accelerated, and the OD value and viable cell count of each experimental group are significantly higher than those of the control group (P<0.05). This indicates that Cyclo(Leu-Pro) enhances the acid stress resistance of Lactobacillus plantarum.

Claims

1. Use of cyclo(leucine-proline) in any one of (1)-(4) below: (1) use in improving salt stress resistance of Lactobacillus plantarum; (2) use in improving acid stress resistance of Lactobacillus plantarum; (3) use in preparing a preparation for improving salt stress resistance of Lactobacillus plantarum; (4) use in preparing a preparation for improving acid stress resistance of Lactobacillus plantarum.

2. Use according to claim 1, characterized in that, The concentration of the cyclo(leucine-proline) is 0.05-10 μg / mL.

3. Use according to claim 2, characterized in that, The concentration of the cyclo(leucine-proline) is 1 μg / mL.

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