Application of proline in enhancing the osmotic stress resistance of Lactobacillus rhamnosus

Abstract

This invention discloses the application of proline in improving the osmotic stress resistance of Lactobacillus rhamnosus, belonging to the field of microbial technology; this invention uses Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus Using *Lactobacillus rhamnosus* as the research subject, a suitable osmotic stress environment was provided, and the osmotic stress resistance of *Lactobacillus rhamnosus* was improved by exogenously adding proline; the growth (OD) of *Lactobacillus rhamnosus* under osmotic stress conditions with and without proline was measured. 600 The study further analyzed the cell membrane response of *Lactobacillus rhamnosus* under osmotic stress. The results showed that under osmotic stress, exogenous proline addition can significantly improve the growth performance and survival rate of *Lactobacillus rhamnosus* under osmotic stress, maintain the integrity of the cell structure, reduce the damage to the cells caused by osmotic stress, and enhance the resistance of the cells to osmotic stress. This provides a theoretical basis and evidence for improving the fermentation performance of lactic acid bacteria under stress.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of proline in improving the osmotic stress resistance of Lactobacillus rhamnosus. Background Technology

[0002] Lactic acid bacteria (LAB) are Gram-positive, catalase-negative, and facultative anaerobic bacteria. They have attracted widespread attention due to their unique physiological functions, such as promoting digestion and absorption by producing organic acids, digestive enzymes, and various bioactive substances; resisting the invasion of pathogens and maintaining the balance of intestinal flora; enhancing the body's innate immunity by regulating host-specific and non-specific immunity; increasing the activity of antioxidant enzymes to regulate and alleviate oxidative stress and protect cells from damage caused by oxidative stress; and breaking down different organic compounds in the food matrix, expressing several other metabolic activities, such as flavor, astringency, and color.

[0003] Lactic acid bacteria are widely used in the food and medical industries. Through continuous development and exploration, more and more lactic acid bacteria products are appearing in the form of dairy products, fermented foods, dietary supplements, starter cultures, and enzyme preparations. However, during the production, storage, and distribution of food and the use of drugs, lactic acid bacteria inevitably face various environmental stresses, such as osmotic pressure, acid, oxidation, starvation, and bile osmotic pressure stress. These stresses reduce the number of live bacteria reaching the human intestine, and limit their growth performance and metabolic capacity to a certain extent, thus preventing them from performing their physiological functions effectively and seriously affecting the industrial application of lactic acid bacteria.

[0004] Lactobacillus rhamnosus exhibits good adhesion to the intestines and tolerance to gastrointestinal conditions, as well as strong adhesion to the intestinal mucosa. It plays a crucial role in resisting intestinal pathogens and regulating the intestinal microbial environment, a role currently irreplaceable by other probiotic strains. Lactobacillus rhamnosus is also frequently used in fermented foods, which are characterized by high salt content. During fermentation, changes in external osmotic pressure can cause bacterial cell volume to expand or shrink, disrupting intracellular physiological metabolism and even leading to cell death. Osmotic stress caused by high salt concentrations can trigger intracellular water outflow, resulting in structural and physiological damage to the cells, leading to reduced bacterial growth rate, decreased survival rate, and impaired metabolic activity of the probiotics. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this invention provides a method for improving the effect of proline on Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus Its application in osmotic stress resistance, specifically in the cultivation of Lactobacillus rhamnosus. Lacticaseibacillus rhamnosus Add proline to the culture medium.

[0006] Preferably, the concentration of proline in the culture medium of the present invention is 1.2-1.6 g / L.

[0007] Preferably, the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus The vaccination rate was 2%.

[0008] Beneficial effects of this invention: (1) Proline enhances the activity of Lactobacillus rhamnosus (Lactobacillus casei) Lacticaseibacillus rhamnosus It plays a significant role in osmotic stress resistance and can effectively maintain the resistance of Lactobacillus rhamnosus (Lactobacillus) under osmotic stress. Lacticaseibacillus rhamnosus The number of viable bacteria, and under osmotic pressure stress, Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The number of live bacteria in the product was twice that of the product without proline.

[0009] (2) Exogenous proline can be transported into the cells of lactic acid bacteria, maintaining intracellular osmotic pressure balance and reducing damage to the cell membrane; this indicates that exogenous proline addition is an effective strategy that can be used to optimize Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus This study examines the stress tolerance of lactic acid bacteria in scenarios such as food processing and probiotic preparation production, and provides a theoretical basis and technical reference for improving the stress resistance of other lactic acid bacteria. Attached Figure Description

[0010] Figure 1 The *Lactobacillus rhamnosus* in Examples 1 and 2 of this invention (… Lacticaseibacillus rhamnosus Growth curves under MRS broth culture, sodium lactate stress, and sodium lactate stress with proline addition.

[0011] Figure 2 It is the Lactobacillus rhamnosus in Example 1 of this invention ( Lacticaseibacillus rhamnosus The number of viable bacteria (CFU / mL) under sodium lactate stress and under sodium lactate stress with proline (different letters indicate significant differences between groups). p <0.05).

[0012] Figure 3 It is the Lactobacillus rhamnosus in Example 4 of this invention ( Lacticaseibacillus rhamnosus Scanning electron microscope images of the morphology and structure; among which Figure 3 (a) is due to sodium lactate stress. Figure 3 (b) Adding proline under sodium lactate stress.

[0013] Figure 4 It is the Lactobacillus rhamnosus in Example 3 of this invention ( Lacticaseibacillus rhamnosus Lactate dehydrogenase activity assay results (U / gprot) (different letters indicate significant differences between groups) p <0.05).

[0014] Figure 5 It is the Lactobacillus rhamnosus in Example 3 of this invention ( Lacticaseibacillus rhamnosus Dynamic analysis of cell membrane fluidity parameters, quantitative detection results of polarization degree (P), anisotropy (r), and microviscosity (η).

[0015] Figure 6 This is the flow cytometry detection of Lactobacillus rhamnosus in Example 3 of the present invention. Lacticaseibacillus rhamnosus Changes in cell membrane permeability (marked by SYTO 9 and PI double staining); among which, Figure 6 (a) Lactobacillus rhamnosus without proline ( Lacticaseibacillus rhamnosus Changes in cell membrane permeability; Figure 6 (b) Lactobacillus rhamnosus after proline addition ( Lacticaseibacillus rhamnosus Changes in cell membrane permeability; quadrant Q1 represents dead cells, quadrant Q2 represents mechanically damaged cells, quadrant Q3 represents live cells, and quadrant Q4 represents unstained cell fragments or debris. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described therein; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and all reagents used can be purchased through conventional commercial channels.

[0017] The Lactobacillus rhamnosus used in this invention ( Lacticaseibacillus rhamnosus The strain number is ATCC 53103.

[0018] The culture medium used in this invention and its preparation method: MRS broth medium: comprising 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate (CAS: 7758-11-4), 2 g / L diammonium hydrogen citrate (CAS: 3012-65-5), 5 g / L sodium acetate (CAS: 127-09-3), 0.2 g / L magnesium sulfate (CAS: 7487-88-9), 0.04 g / L manganese sulfate (CAS: 1034-96-5), and 1 g / L Tween 80. The medium is brought to a final volume of 1 L with deionized water, and the pH is adjusted to 5.5-5.9 (25℃). The medium is then dispensed into test tubes (10 mL / tube) and sterilized at 121℃ for 20 min.

[0019] MRS agar medium: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 14 g / L agar, 1 g / L Tween 80. Adjust the volume to 1 L with deionized water, adjust the pH to 6.5 ± 0.2 (25℃), sterilize at 121℃ for 20 min, and pour into glass petri dishes, 15 mL per dish.

[0020] Osmotic stress medium: Add 0.6M sodium lactate (CAS: 72-17-3) to 1L of MRS broth medium, dispense into test tubes, 10mL / tube, and sterilize at 121℃ for 20min.

[0021] Proline-substituted medium: Add 1.6 g / L proline (CAS: 147-85-3) to 1 L of osmotic stress medium, dispense into test tubes, 10 mL / tube, and sterilize at 121 °C for 20 min.

[0022] Example 1 Proline on Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The effect of osmotic pressure stress resistance is investigated through the following steps: (1) Take out Lactobacillus rhamnosus, which was stored in glycerol stock solution at -80℃. Lacticaseibacillus rhamnosus After thawing, it was inoculated into MRS broth medium for activation, and Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The inoculum size was 2%, and the cells were incubated at 37°C for 12 hours until the mid-log phase to obtain the F0 generation. The F0 generation was then inoculated into new MRS broth medium for one subculture, with the volume percentage of the F0 generation being 2%, and incubated at 37°C for 12 hours to obtain the F1 generation. The F1 generation was then inoculated into new MRS broth medium for one subculture, with the volume percentage of the F1 generation being 2%, and incubated at 37°C for 12 hours to obtain the seed culture solution.

[0023] (2) Seed culture solutions were collected and inoculated into MRS broth medium (MRS broth culture), osmotic stress medium (sodium lactate stress culture), and proline replacement medium (proline supplementation culture under sodium lactate stress), respectively. The inoculation amount was 2%, and the cultures were incubated statically at 37°C for 24 hours. During the culture period, the biomass (OD) in each culture medium was measured every 2 hours. 600 ), until the lactic acid bacteria grow to the stationary phase after 24 hours, and the results are as follows [[ID= 32]]Figure 1 As shown, under sodium lactate stress culture, the levels of *Lactobacillus rhamnosus* (Lactobacillus casei) decreased. Lacticaseibacillus rhamnosus The biomass of *Lactobacillus rhamnosus* was reduced by adding proline to culture under sodium lactate stress; and sodium lactate stress reduced the effect of sodium lactate stress on *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosusThe effect on biomass indicates that exogenous proline addition helps improve the growth performance of lactic acid bacteria under osmotic stress.

[0024] (3) Take the seed culture from step (1) and inoculate it into MRS broth medium at an inoculation rate of 2%. Incubate at 37°C until the end of the logarithmic growth phase. Prepare two MRS agar media, one of which is sterilized and cooled to room temperature, and then 1.6 g / L proline is added. Determine the viable count using plate culture. Take Lactobacillus rhamnosus at the end of the logarithmic growth phase ( Lacticaseibacillus rhamnosus (), serially diluted in physiological saline, and take 10% of the diluted solution. 6 The bacterial suspension was plated on MRS agar medium and MRS agar medium supplemented with proline, and incubated statically at 37°C for 48 hours before colony counting; the results are as follows. As shown, *Lactobacillus rhamnosus* cultured with 1.6 g / L proline (…) Figure 2 The biomass is large, and the number of viable bacteria reaches 13.5 × 10⁻⁶. 8 CFU / mL, significantly higher than the MRS agar medium group ( p <0.05, indicating that the addition of proline helps to increase the levels of Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus This enhances the osmotic stress resistance of the strain and improves its growth characteristics.

[0025] Example 2 Take the seed culture from step (1) of Example 1 and inoculate it into MRS broth medium at an inoculation rate of 2%. Incubate at 37°C until the end of the logarithmic growth phase. Prepare two MRS agar media, one of which is sterilized and cooled to room temperature, and then has 1.2 g / L proline added. Determine the viable count using plate culture. Take Lactobacillus rhamnosus at the end of the logarithmic growth phase ( Lacticaseibacillus rhamnosus (), serially diluted in physiological saline, and take 10% of the diluted solution. 6 The bacterial suspension was plated on MRS agar medium and MRS agar medium supplemented with proline, and incubated statically at 37°C for 48 hours before colony counting; the results are as follows. Lacticaseibacillus rhamnosus As shown, the viable count of the strain under these conditions was low, lower than that of the group treated with 1.6 g / L proline.

[0026] Example 3 Proline's effect on Lactobacillus rhamnosus under osmotic stress ( Figure 1 The effects on cell integrity are explained in the following steps: The seed culture from step (1) of Example 1 was inoculated into osmotic stress medium (sodium lactate stress) and proline replacement medium (proline added under sodium lactate stress) at an inoculation rate of 2%. The culture was incubated at 37°C until the end of the logarithmic growth phase. The precipitate was collected by centrifugation, washed with PBS, and then fixed with glutaraldehyde (CAS: 111-30-8), ensuring the glutaraldehyde level covered the cells. After the precipitate dried, it was sputter-coated with gold and imaged using a scanning electron microscope. The results are as follows: Lacticaseibacillus rhamnosus As shown in (a), after culturing in osmotic stress medium, *Lactobacillus rhamnosus* (…) Figure 3 The morphology of the bacteria undergoes significant changes, manifesting as marked bending, folding, and severe deformation of the cells, along with cell rupture and leakage of contents; while the bacteria caused by… Lacticaseibacillus rhamnosus (b) It is evident that the addition of proline alleviated the cell deformation and rupture caused by osmotic stress, indicating that proline can maintain *Lactobacillus rhamnosus* under osmotic pressure treatment. Figure 3 Cell integrity.

[0027] Example 4 Proline's effect on Lactobacillus rhamnosus under osmotic stress ( Lacticaseibacillus rhamnosus The effects of membrane integrity, fluidity, and membrane damage on cell membranes are detailed in the following steps: (1) Cell membrane integrity test: Bacterial cultures in the late logarithmic growth phase of the osmotic stress medium (sodium lactate stress) and proline-substituted medium (proline added under sodium lactate stress) from Example 3 were collected after centrifugation. LDH activity was measured according to the instructions of the lactate dehydrogenase (LDH) test kit (Nanjing Jiancheng Bioengineering Institute, A020-1). Under normal circumstances, due to the integrity of the cell membrane, LDH cannot permeate the cell membrane into the extracellular fluid (such as blood or culture medium). When cell membrane permeability increases, LDH is released from the cytoplasm into the extracellular space. The results are as follows: Lacticaseibacillus rhamnosus As shown, the LDH activity in the sodium lactate stress treatment group was 84.41±3.62 U / gprot. Notably, the addition of proline significantly alleviated the osmotic pressure damage effect, reducing LDH activity to 58.32±2.27 U / gprot (a decrease of 30.9%). This means that exogenous addition of proline can effectively maintain cell membrane integrity and control LDH leakage.

[0028] (2) Membrane fluidity test: Bacterial suspensions at the end of the logarithmic growth phase were collected from the osmotic stress medium (sodium lactate stress treatment group) and proline replacement medium (proline treatment group under sodium lactate stress) of Example 3. After centrifugation, the cells were collected, washed twice with PBS, and the precipitate was collected. 1,6-diphenyl-1,3,5-hextriene (DPH, CAS: 1720-32-7) was added. DPH was prepared as a stock solution using tetrahydrofuran (CAS: 109-99-9) before use and added to the precipitate to make the final concentration of the mixed system (bacterial suspension + DPH) 2 μmol / L. The mixture was incubated at 37°C in the dark for 20 min. After centrifugation, the precipitate was collected, washed with PBS, resuspended, and subjected to fluorescence measurement. The smaller the P value (polarization), r value (anisotropy), and η value (microviscosity), the better the cell membrane fluidity. The measurement results are as follows: Figure 4 As shown, *Lactobacillus rhamnosus* (Lactobacillus) under sodium lactate stress... Figure 5 The cell membrane has low fluidity, indicating that osmotic stress has a significant impact on *Lactobacillus rhamnosus* (Lactobacillus). Lacticaseibacillus rhamnosus The addition of proline under sodium lactate stress negatively impacts cell membrane fluidity. Low fluidity reflects damage to cell membrane structure, leading to host cell dysfunction, homeostasis disruption, and ultimately cell death. Lacticaseibacillus rhamnosus The P-value, r-value, and η-value of *Lactobacillus rhamnosus* under sodium lactate stress were all lower than those of *Lactobacillus rhamnosus* (Lactobacillus casei) under sodium lactate stress. Lacticaseibacillus rhamnosus ), indicating that Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus Lacticaseibacillus rhamnosus The cell membrane fluidity was significantly improved. p <0.05, demonstrating that proline enhances cell membrane fluidity and improves the activity of Lactobacillus rhamnosus (Lactobacillus). Lacticaseibacillus rhamnosus Potential for growth performance.

[0029] (3) Cell membrane damage detection: Bacterial suspensions in the late logarithmic growth phase of osmotic stress medium (sodium lactate stress treatment group) and proline replacement medium (proline treatment group under sodium lactate stress) from Example 3 were collected by centrifugation. The bacterial cells were stained with the fluorescent dyes propidium iodide (PI, CAS: 25535-16-4) and SYTO 9 (CAS: 854647-88-4). The amount of fluorescent dye added was equal to the volume of the bacterial suspension. SYTO 9 can penetrate the cell membrane, while PI cannot. The change in the permeability of the lactic acid bacteria cell membrane is negatively correlated with the cell viability; increased permeability leads to a significant decrease in cell viability. The Luminex Guava EasyCyte flow cytometer was used to detect and analyze the changes in the ratio of apoptotic cells to normal cells under different culture conditions. The results are as follows: Figure 6 As shown, compared with the cells under sodium lactate stress, the number of cells in Q1 and Q2 was significantly reduced after the addition of proline. p<0.05%, the percentage of viable cells increased to 17.27±1.36%; these results indicate that under osmotic stress, the addition of proline can effectively reduce the osmotic pressure on *Lactobacillus rhamnosus* (Lactobacillus). Lacticaseibacillus rhamnosus It can reduce cell membrane damage and decrease the activity of Lactobacillus rhamnosus (Lactobacillus rhamnosus). Lacticaseibacillus rhamnosus Loss of cell viability.

[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of proline in improving the osmotic stress resistance of Lactobacillus rhamnosus.

2. The application of proline according to claim 1 in improving the osmotic stress resistance of Lactobacillus rhamnosus, characterized in that, The specific application method is as follows: In culturing Lactobacillus rhamnosus... Lacticaseibacillus rhamnosus Add proline to the culture medium.

3. The application of proline according to claim 2 in improving the osmotic stress resistance of Lactobacillus rhamnosus, characterized in that, The concentration of proline in the culture medium is 1.2-1.6 g / L.

4. The application of proline according to claim 2 in improving the osmotic stress resistance of Lactobacillus rhamnosus, characterized in that, The inoculation amount of *Lactobacillus rhamnosus* was 2%.

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