High-temperature-resistant lactobacillus rhamnosus obtained by artp mutagenesis and adaptive evolution and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a thermoresistant strain of *Lactobacillus rhamnosus* obtained through ARTP mutagenesis and adaptive evolution, and its applications. This invention, through mutagenesis and adaptive evolution, obtains a thermoresistant strain of *Lactobacillus rhamnosus*, which, compared to the wild-type strain, can grow and reproduce at 50°C without sacrificing its probiotic properties and exhibits strong antioxidant capacity. Using this strain in the preparation of probiotic products can effectively reduce the loss of viable bacteria during the preparation process, lower production costs, improve probiotic performance, and achieve the goal of cost reduction and efficiency improvement, showing broad application prospects in industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to thermostable Lactobacillus rhamnosus obtained through ARTP mutagenesis and adaptive evolution, and its applications. Background Technology

[0002] Lactobacillus rhamnosus is a probiotic that colonizes the human body and alters the composition of the host's gut microbiota, creating a beneficial effect. Its key characteristic is its ability to adhere to the host's intestinal epithelial cells, reducing the adhesion of pathogenic bacteria, and inhibiting their growth and reproduction. Studies have shown that sufficient probiotics can enhance gastrointestinal function and stability, strengthen the immune system, and lower blood cholesterol levels. Due to a lack of domestically developed strains with proprietary intellectual property rights and superior quality, my country's probiotic industry has long been reliant on foreign imports. Ensuring the safety and diversity of my country's bacterial strains and overcoming the limitations imposed by commercially available foreign probiotic strains are crucial issues that need to be addressed.

[0003] Lactobacillus rhamnosus is subjected to various adverse stimuli during manufacturing and subsequent application. For example, during the manufacturing process, the bacteria may be exposed to different temperatures and face changes in external conditions such as osmotic pressure, oxygen concentration, and moisture content. These factors can further lead to lethal damage to the strain. This means that the abundance / viable cell count of probiotics gradually decreases during manufacturing. The reduction in cell number leads to decreased fermentation capacity and a decline in probiotic performance. Therefore, identifying stable and high-performing Lactobacillus rhamnosus strains is of significant research importance in industrial production and other fields. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide thermostable Lactobacillus rhamnosus obtained through ARTP mutagenesis and adaptive evolution and its applications.

[0005] This invention provides Lactobacillus rhamnosus with accession number CGMCC No. 21815.

[0006] This invention obtains a heat-resistant Lactobacillus rhamnosus strain through mutagenesis. It can maintain good growth at 50℃, and its tolerance to the gastric environment and bile salts is not reduced. In addition, compared with wild-type strains and existing Lactobacillus rhamnosus strains, it has outstanding antioxidant capacity and strong DPPH and ABTS free radical scavenging ability. The experimental results show that the bacterial cells fermented for 24 hours have a DPPH and ABTS free radical scavenging ability of more than 90%.

[0007] Furthermore, the Lactobacillus rhamnosus described in this invention is a strain obtained through mutagenesis that possesses strong probiotic capabilities and can grow at 50°C, exhibiting significant advantages compared to other mutagenic strains. For instance, in some specific embodiments of this invention, both the 19th and 20th generations obtained through subculture and domestication can grow at 50°C, but the 20th generation exhibits stronger survival capabilities.

[0008] The present invention provides a microbial community, which includes Lactobacillus rhamnosus as described in the present invention.

[0009] The present invention provides a microbial agent comprising Lactobacillus rhamnosus or the microbial community described in the present invention.

[0010] Furthermore, the microbial agent of the present invention is available in a dosage form including at least one of granules, liquids, and / or dry powders.

[0011] The present invention provides the fermentation product of the Lactobacillus rhamnosus, the bacterial flora, or the bacterial agent.

[0012] Furthermore, the fermentation products described in this invention include lactic acid, esters, and / or aromatic compounds.

[0013] This invention provides the use of at least one of the following (I) to (IV) in the preparation of fermented products and / or probiotic products:

[0014] I) The Lactobacillus rhamnosus described in this invention;

[0015] II) The microbial community described in this invention;

[0016] III) The bacterial agent described in this invention;

[0017] IV) The fermentation products described in this invention.

[0018] Furthermore, in the applications described in this invention, the probiotic includes at least one of antioxidant, gut microbiota regulation, cell proliferation promotion, and / or enhanced cellular anti-inflammatory capacity; in specific embodiments of this invention, the probiotic includes antioxidant, which includes scavenging DPPH free radicals and / or ABTS free radicals.

[0019] In this invention, the fermented products include cheese, yogurt, fruit juice, fruit juice beverages, etc.

[0020] This invention provides a method for preparing fermented products and / or probiotic products, which involves fermentation using at least one of the Lactobacillus rhamnosus, the bacterial flora, or the bacterial agent described in this invention.

[0021] The present invention provides a fermentation product obtained by fermentation of at least one of the Lactobacillus rhamnosus described in the present invention, the bacterial flora described in the present invention, or the bacterial agent described in the present invention.

[0022] This invention provides probiotic products or fermented products, the raw materials of which include at least one of the following i) to iv):

[0023] i) The Lactobacillus rhamnosus described in this invention;

[0024] ii) The microbial community described in this invention;

[0025] iii) The microbial agent described in this invention;

[0026] iv) The fermentation product described in this invention.

[0027] This invention provides a method for enhancing immunity, which involves using the probiotic product described in this invention.

[0028] This invention provides a method for preparing *Lactobacillus rhamnosus* with accession number CGMCC No. 21815, comprising the following steps:

[0029] Step 1: Perform ARTP mutagenesis on wild-type Lactobacillus rhamnosus strain;

[0030] Step 2: Enrich and subculture the strain obtained by mutagenesis to obtain Lactobacillus rhamnosus with accession number CGMCCNo.21815;

[0031] The conditions for ARTP mutagenesis are: power 120W, distance 2mm, gas flow rate 10SLM, and mutagenesis time 45s.

[0032] The enrichment temperature was 37°C;

[0033] The culture medium used for subculture domestication was MRS medium, and the culture speed during subculture domestication was 160 rpm.

[0034] The initial temperature for the domestication process was 37°C, and the final temperature was 50°C, with the temperature increasing by 0.5 to 1°C for each generation.

[0035] The criteria for allowing the next generation of domestication to proceed are: the final OD value of each generation is ≥ 70% of the final OD value of the wild type at 37°C.

[0036] The final OD value of the wild type at 37°C was 1.2–1.6.

[0037] In a specific embodiment of the present invention, when the wild type is domesticated into the next generation, the final OD value at 37°C is 1.4.

[0038] This invention obtains a heat-resistant strain of *Lactobacillus rhamnosus* through mutagenesis and adaptive evolution. Compared with the wild-type strain, it can grow and reproduce at 50°C without losing its probiotic properties and has strong antioxidant capacity. Using it in the preparation of probiotic products can effectively reduce the loss of live bacteria during the preparation process, reduce production costs, improve probiotic performance, and achieve the goal of cost reduction and efficiency improvement. It has broad application prospects in industrial production. Attached Figure Description

[0039] Figure 1 Showing the growth of wild-type and mutant strains at 37°C;

[0040] Figure 2 Showing the growth of wild-type and mutant strains at 50℃;

[0041] Figure 3 The mutant strain exhibits stable and heritable growth characteristics at 50℃.

[0042] Figure 4 Showing the heat treatment effects of 50–75℃ on wild-type and mutant strains;

[0043] Figure 5 The mutant strain showed no loss of its beneficial characteristics.

[0044] Biological Preservation Instructions

[0045] Biological material AUH2101, classified and named Lactobacillus rhamnosus, was deposited on February 5, 2021, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 21815. Detailed Implementation

[0046] This invention provides thermostable *Lactobacillus rhamnosus* obtained through ARTP mutagenesis and adaptive evolution, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] The reagents and consumables used in this invention are all commercially available products that can be purchased on the market.

[0048] The present invention will be further illustrated below with reference to the embodiments:

[0049] Example 1: Mutant strain and its performance

[0050] I. A mutant strain Mut was obtained.

[0051] 1. Acquisition and performance testing of mutant strains

[0052] ARTP mutagenesis was performed on wild-type *Lactobacillus rhamnosus* strains. The resulting mutant populations were first enriched at 37°C and then repeatedly passaged for adaptive evolution. Passages were performed using MRS medium (components / L: peptone 10g, beef extract 10g, yeast extract 5g, K₂HPO₄ 2g, diammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, MgSO₄·7H₂O 0.58g, MnSO₄·4H₂O 0.25g) at 160 rpm. 600 The cell density is used as the metric, and the culture period is 24 hours. If the final OD value of each generation is close to (≥70%) the final OD of the wild type at 37°C, then the adaptive evolution of this generation is considered successful, and the next round can begin. The passage is shown in Table 1, with the temperature increasing by 0.5 to 1°C for each passage.

[0053] The original strain used for mutagenesis in this invention is *Lactobacillus rhamnosus*, whose optimal growth temperature is 37°C. The mutant strain obtained through mutagenesis is named Mut. Compared to the wild-type WT, the mutant's growth rate and cell density at 37°C were not affected (e.g., ...). Figure 1 As shown), the mutant strain Mut can tolerate 50℃ and maintain a relatively good cell density, while the wild type cannot grow at 50℃ (e.g., Figure 2 (As shown).

[0054] Both the 19th and 20th generation strains in Table 1 can survive at 50°C. However, the 20th generation strain clearly performs better than the 19th generation strain. Some heat-resistant strains (such as the 19th generation) can be obtained through ARTP and laboratory adaptive evolution, but the 20th generation strain, i.e., the preserved strain, performs the best.

[0055] The mutant was passaged for 20 generations, and its growth characteristics remained unchanged. This indicates that its heat tolerance is stably inherited, as shown in the 20 passages. Figure 3 .

[0056] Table 1. Mutagenesis and passage of mutant strains

[0057]

[0058] 2. Testing for tolerance to higher temperatures

[0059] First, strains WT and Mut were cultured at 37°C for approximately 24 hours to reach their maximum OD value. At this point, their viable cell counts were both calculated to be 10⁻⁶. 9 CFU / mL. The bacterial culture was then added to preheated MRS medium and heat-treated for 1 minute. The OD values ​​in the system were controlled to be within the range of 0.5–0.6. The culture was then rapidly cooled to room temperature, and the viable cell count was determined using the spread plating method. The heat treatment temperature was set to 50–75°C. The results are as follows: Figure 4 As shown.

[0060] The results showed that the mutant Mut retained more than 55% viable count after heat treatment at 50–75 °C for 1 min, while the wild-type WT almost lost its activity at temperatures above 60 °C.

[0061] II. Comparison of partial beneficial properties between wild-type and mutant strains

[0062] Gastric environment tolerance test: PBS (pH 7.4) buffer was adjusted to pH 2.0, 2.5, 3.0, and 3.5 with 0.1 mol / L HCl. After dissolving in 3 g / L pepsin, the solution was filtered through a 0.22 μm sterile filter membrane. Solutions were prepared fresh for each use. The original strain WT and the mutant strain Mut were activated three times and inoculated into 10 mL of MRS liquid at a 2% inoculum. After incubation at 37°C for 24 h, the bacterial cells were collected by centrifugation. The cells were washed twice with sterile PBS buffer and then resuspended. 1.0 mL of the bacterial suspension was added to 9.0 mL of simulated gastric fluid at pH 2.0, 2.5, 3.0, and 3.5, respectively, and incubated at 37°C for 3 h. The culture media at 0 h and 3 h were diluted with sterile physiological saline at a 1:10 gradient. The viable cell count was determined by plate counting, and the survival rate (%) was calculated. Each group was repeated three times.

[0063] Bile salt tolerance: 0.1%, 0.2%, and 0.3% bovine bile salts were added to the prepared MRS medium, respectively. The bacterial suspension was prepared in the same way as above. The culture solutions at 0h and 3h were diluted with sterile physiological saline in a 1:10 gradient and plated. The survival rate of the strain in different concentrations of bile salts was calculated.

[0064] Survival rate is calculated as follows:

[0065] Survival rate = (A1 / A0) × 100%

[0066] Note: A0 represents the total number of untreated colonies, and A1 represents the total number of treated colonies.

[0067] The results are as follows Figure 5As shown, there was no significant difference in the basic probiotic properties between the wild type and the mutant, indicating that the mutant did not sacrifice its probiotic characteristics. For industrial production, probiotic products need to withstand higher heat treatment temperatures during manufacturing to ensure the quantity and quality of live bacteria at the factory. Therefore, the strains of this invention, when used in probiotic products, can maintain higher probiotic viability, have broader application potential, and superior probiotic performance.

[0068] Example 2 Other Performance

[0069] I. Scavenging rate of DPPH free radicals by mutant Mut

[0070] DPPH (1-diphenyl-2-trinitrophenylhydrazine) is a very stable nitrogen-centered free radical. It is widely used to quantitatively determine the in vitro antioxidant capacity of biological samples, pure compounds, and extracts by binding with free radical scavengers.

[0071] Wild-type WT and mutant Mut strains were cultured, and samples were taken at different time points to collect bacterial cells. The culture medium was washed off with sterile physiological saline, and the cells were resuspended to evaluate their free radical scavenging effect.

[0072] Preparation of DPPH standard solution: Weigh 0.02g of DPPH, dissolve it in 60% ethanol, and make up to 250mL to prepare a 0.2mM DPPH standard solution.

[0073] Add 150 μL of DPPH solution to 150 μL of appropriately diluted sample, mix thoroughly, and incubate at room temperature in the dark for 30 min. Then, measure the absorbance at 517 nm. Record the absorbance values ​​A1, A2, and A3 respectively, and calculate the DPPH scavenging rate (%). The results are shown in Table 3.

[0074] DPPH clearance rate (%) = [A3 - (A1 - A2)] / A3 × 100%

[0075] A1: Absorbance of the system without 60% ethanol

[0076] A2: Absorbance of the system without DPPH

[0077] A3: Absorbance of the system without sample

[0078] Table 3. DPPH removal rate (%)

[0079] Fermentation time WT Mut 12h 52.45％ 68.41％ 24h 65.02％ 92.25％

[0080] II. Experimental study on the ABTS radical scavenging rate of mutant Mut

[0081] ABTS (2,2'-Aza-bis-3-ethylbenzothiazoline-6-sulfonic acid) can be used to evaluate the antioxidant capacity of substances.

[0082] Wild-type WT and mutant Mut strains were cultured, and samples were taken at different time points to collect bacterial cells. The culture medium was washed off with sterile physiological saline, and the cells were resuspended to evaluate their free radical scavenging effect.

[0083] Preparation of ABTS standard solution: Prepare a 7mM ABTS solution with deionized water, then add potassium persulfate to prepare a 2.45mM potassium persulfate solution, and let it stand overnight at room temperature in the dark.

[0084] Add 150 μL of ABTS solution to 150 μL of appropriately diluted sample, mix thoroughly, and incubate at room temperature in the dark for 6 min. Then, measure the absorbance at 405 nm. Record the absorbance values ​​A1, A2, and A3 respectively, and calculate the ABTS scavenging rate (%). The results are shown in Table 3.

[0085] ABTS clearance rate (%) = [A3 - (A1 - A2)] / A3 × 100%

[0086] A1: Absorbance of the system without potassium persulfate

[0087] A2: Absorbance of the system without ABTS

[0088] A3: Absorbance of the system without sample

[0089] Table 3. ABTS clearance rate (%)

[0090] Fermentation time WT Mut 12h 49.58％ 72.54％ 24h 52.97％ 91.36％

[0091] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Lactobacillus rhamnosus with the preservation number of CGMCC No. 21815.

2. A bacterial population characterized in that, The Lactobacillus rhamnosus of claim 1.

3. A bacterial agent, characterized in that, The Lactobacillus rhamnosus of claim 1 or the flora of claim 2.

4. The bacterial agent of claim 3, characterized in that, The dosage form comprises at least one of granules, liquid and / or dry powder.

5. Use of at least one of the following I)~III) in the preparation of a fermented product: I) the Lactobacillus rhamnosus of claim 1; II) the flora of claim 2; III) the microbial agent of claim 3; The fermented product is cheese, yogurt, juice and / or juice beverage.

6. A fermentation product, characterized in that, The raw material comprises at least one of the following i)~iii): i) the Lactobacillus rhamnosus of claim 1; ii) the flora of claim 2; iii) the microbial agent of claim 3; The fermented product is cheese, yogurt, juice and / or juice beverage.

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