Composite bacterial agent with antibacterial activity and its application
Through co-culture and optimization of culture conditions of Lactobacillus casei and Lactobacillus rhamnosus, the antibacterial activity of lactic acid bacteria was improved, the problem of low antibacterial activity of lactic acid bacteria was solved, and efficient inhibition of E. coli and significant improvement of lactic acid production were achieved.
Patent Information
- Application Number
- CN202211654185.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-22
- Publication Date
- 2025-08-22
- Estimated Expiration
- 2042-12-22
AI Technical Summary
In the prior art, the antibacterial activity of lactic acid bacteria is low and it is difficult to effectively inhibit the growth of pathogenic bacteria.
The culture conditions were optimized to improve the antibacterial activity of lactic acid bacteria by co-culturing the complex bacterial agent composed of Lactobacillus casei and Lactobacillus rhamnosus.
The antibacterial activity of lactic acid bacteria was significantly improved, especially the inhibitory effect on E. coli. The lactic acid production increased by 32.1%, and the antibacterial circle diameter increased by 20.7%. The optimized compound bacterial agent performed more significantly in antibacterial activity and lactic acid production.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a composite bacterial agent with antibacterial activity and application thereof. Background Art
[0002] Lactic acid bacteria are a class of generally recognized as safe (GRAS) food-grade microorganisms beneficial to human health. They secrete organic acids, hydrogen peroxide, and bacteriocins, which inhibit the growth of spoilage and pathogenic bacteria. This antibacterial activity is the most important characteristic of lactic acid bacteria, and its application in food storage, regulating intestinal microbial composition, and treating human pathogenic infections has yielded promising results.
[0003] Compared to single microorganisms, diverse microorganisms in natural communities can grow synergistically, activating gene expression in certain metabolic pathways, reducing metabolite accumulation and metabolic burden, and achieving relatively high material conversion and metabolic efficiency. Simulating natural communities through the co-cultivation of artificial microorganisms to acquire functions not possessed by a single microorganism has become a hot topic in recent research and application. The addition of other microorganisms to pure microbial cultures creates interspecies competitive pressure, often increasing the production of certain metabolites. This effect is related to signaling molecules and interspecies quorum sensing. Slattery et al. found that 12 marine bacterial species can induce increased production of istamycin, an antibacterial substance secreted by Streptomyces tenjimariensis. When lactic acid bacteria are co-cultured with other bacteria, their production of bacteriocins and lactic acid is significantly increased. Therefore, a key strategy for enhancing the antibacterial activity of lactic acid bacteria is to select suitable inducing bacteria for co-cultivation with lactic acid bacteria to induce and enhance the synthesis of antibacterial substances. Summary of the Invention
[0004] The main problem to be solved by the present invention is how to improve the antibacterial activity of lactic acid bacteria.
[0005] In order to solve the above problems, the present invention provides a composite bacterial agent.
[0006] The active ingredients of the composite bacterial agent provided by the invention consist of Lactobacillus casei and Lactobacillus rhamnosus.
[0007] The composite bacterial agent is a composite bacterial agent for preparing antibacterial products, the Lactobacillus casei is Lactobacillus casei NA-2, and the Lactobacillus rhamnosus is Lactobacillus rhamnosus LGG, CGMCC No.12920.
[0008] The composite bacterial agent has antibacterial activity against Escherichia coli.
[0009] In the above bacterial agent, the CFU ratio of Lactobacillus casei and Lactobacillus rhamnosus is: 1.03×10 9 :5.4×10 8 .
[0010] The present invention also provides a probiotic preparation, which contains the composite bacterial agent described above.
[0011] The microecological preparation can be a culture of the composite bacterial agent.
[0012] The culture is a fermentation product obtained by culturing the composite bacterial agent in a microbial culture medium.
[0013] In the above culture, the substances include the composite bacterial agent and metabolites of the composite bacterial agent.
[0014] In the above culture, the microbial culture medium may be a solid culture medium or a liquid culture medium.
[0015] The term "culture" refers to any liquid or solid product (i.e., all materials within a culture vessel, i.e., fermentation products) containing a microbial population after artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, and / or other components produced during the culture process. The term "culture" also includes subcultures obtained by subculturing microorganisms, which can be cultures of a single generation or a mixture of several generations.
[0016] Herein, the metabolite can be obtained from the fermentation broth of the composite bacterial agent. The metabolite can be a sterile metabolite of the composite bacterial agent. The sterile metabolite of the composite bacterial agent (sterile fermentation filtrate) can be prepared according to the following method: culturing the composite bacterial agent in a liquid culture medium, filtering and removing the composite bacterial agent from the liquid culture (fermentation broth) to obtain the sterile metabolite of the composite bacterial agent.
[0017] The present invention also provides the use of the aforementioned composite bacterial agent in the preparation of a microecological preparation.
[0018] The microecological preparation has antibacterial activity against Escherichia coli.
[0019] The present invention also provides a method for preparing a probiotic preparation, comprising the following steps: inoculating the aforementioned composite bacterial agent into a liquid fermentation medium for culturing, collecting the fermentation liquid, and removing the bacterial cells to obtain the probiotic preparation.
[0020] In the above method, the nitrogen source of the liquid fermentation medium is beef extract powder.
[0021] In the above method, the carbon source of the liquid fermentation medium is lactose.
[0022] In the above method, the inoculation ratio of the composite bacterial agent and the liquid fermentation medium satisfies (based on the Lactobacillus casei): 2×10 9 CFU of the Lactobacillus casei: 1000 g of the fermentation medium.
[0023] The application of the above-mentioned composite bacterial agents and microecological preparations in the preparation of products for inhibiting pathogens also falls within the scope of protection of the present invention.
[0024] The pathogen may be Escherichia coli.
[0025] The present invention also provides a method for preparing lactic acid, which comprises inoculating the composite bacterial agent described above into a fermentation medium and fermenting to obtain lactic acid.
[0026] The present invention introduces another lactic acid bacterium, Lactobacillus rhamnosus LGG, into the culture system of Lactobacillus casei NA-2. The co-culture conditions, including inoculation ratio, culture temperature, culture time, ventilation volume, carbon and nitrogen sources, and pH, are optimized through single-factor experiments. The resulting microecological preparation has an antibacterial activity that is 20.7% higher than that of Lactobacillus casei NA-2 culture alone. The lactic acid production of Lactobacillus casei NA-2 increases from an initial 18.37±0.26 g / L to 24.26±0.07 g / L, a 32.1% increase. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is the effect of different inoculation ratios of Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG on the inhibition of Escherichia coli K88.
[0028] Figure 2 The effects of different culture conditions on the inhibition of Escherichia coli K88 in co-culture medium include: a. the effect of different culture temperatures; b. the effect of different ventilation volumes; c. the effect of different culture times.
[0029] Figure 3 The effects of different culture medium compositions on the inhibition of E. coli K88 in co-culture medium include: a. the effect of different carbon sources; b. the effect of different nitrogen sources; c. the effect of culture medium pH. DETAILED DESCRIPTION
[0030] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.
[0031] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0032] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.
[0033] The Lactobacillus casei NA-2 in the present invention has been recorded in: Xiaoqing Xu, Qing Peng, Yuwei Zhang, et al., Antibacterial potential of a novel Lactobacillus casei strain isolatedfrom Chinese northeast sauerkraut and the antibiofilm activity of its exopolysaccharides. Food Funct, 2020, 11(5): 4697-4706). The public can obtain the biological material from the Feed Research Institute of the Chinese Academy of Agricultural Sciences. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.
[0034] The Escherichia coli K88 in the present invention has been recorded in: Yang Jiajun, Huang Kehe, Qin Shunyi, et al. Study on the inhibition of Escherichia coli K88 by four probiotics in vitro. Journal of Jiangsu Agricultural Sciences, 2008, 24(3): 302-306). The public can obtain the biological material from the Feed Research Institute of the Chinese Academy of Agricultural Sciences. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.
[0035] The Lactobacillus rhamnosus LGG in the present invention has been recorded in: Gao Sheng, Qiao Yu, Zhang Yuwei, et al. Screening, identification and preliminary study of beneficial activities of lactic acid bacteria from human breast milk. Food Industry Science and Technology, 2017, 38(10): 205-210). The public can obtain the biological material from the Feed Research Institute of the Chinese Academy of Agricultural Sciences. The biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.
[0036] The MRS culture medium in the present invention was purchased from Beijing Solebow Technology Co., Ltd., item number M8540.
[0037] Preparation of LB medium: Prepare a medium containing 1% tryptone, 0.5% yeast extract, and 1% sodium chloride, adjust the pH to 7.2 with 5 mol / L NaOH, and sterilize at 121°C for 30 min.
[0038] Example 1: Determination of antibacterial activity of composite bacterial agents and probiotics
[0039] 1. Preparation of Lactobacillus casei NA-2 agent and NA-2 single culture microecological preparation
[0040] Take Lactobacillus casei NA-2 stored at -80℃ and streak it on MRS solid medium. After anaerobic culture at 37℃ for 36h, pick a single colony and inoculate it into 5mL liquid MRS medium (Beijing Solebaugh Technology Co., Ltd., product number M8540). After anaerobic culture at 37℃ for 36h, the Lactobacillus casei NA-2 seed liquid is obtained. The Lactobacillus casei NA-2 seed liquid is adjusted to OD 600 The content of Lactobacillus casei NA-2 in the Lactobacillus casei NA-2 agent was 1.2×10 9 CFU / mL.
[0041] The Lactobacillus casei NA-2 bacterial agent was centrifuged at 12000 rpm / min for 10 min, and the supernatant was taken. The supernatant was sterilized by filtration through a 0.22 μm sterile filter membrane to obtain a fermentation supernatant that does not contain Lactobacillus casei NA-2. The supernatant is the NA-2 single culture microecological preparation.
[0042] 2. Preparation of Lactobacillus rhamnosus LGG bacterial agent and LGG single culture microecological preparation
[0043] Take Lactobacillus rhamnosus LGG stored at -80℃ and streak it on MRS solid medium. After anaerobically incubating at 37℃ for 36h, pick a single colony and inoculate it into 5mL liquid MRS medium (Beijing Solebaugh Technology Co., Ltd., product number M8540). Then, incubate it anaerobically at 37℃ for 36h to obtain Lactobacillus rhamnosus LGG seed liquid. Adjust the Lactobacillus rhamnosus LGG seed liquid to OD 600 The content of Lactobacillus rhamnosus LGG in the Lactobacillus rhamnosus LGG agent was 2.8×10 9 CFU / mL.
[0044] The Lactobacillus rhamnosus LGG bacterial agent was centrifuged at 12000 rpm / min for 10 min, and the supernatant was taken. The supernatant was sterilized by filtering through a 0.22 μm sterile filter membrane to obtain a fermentation supernatant that does not contain Lactobacillus rhamnosus LGG. The supernatant is the Lactobacillus rhamnosus LGG single culture microecological preparation.
[0045] 3. Preparation of composite bacterial agent
[0046] The Lactobacillus casei NA-2 agent of step 1 and the Lactobacillus rhamnosus LGG agent of step 2 were mixed at a CFU ratio of 20:1 to obtain a composite agent.
[0047] 4. Preparation of Probiotics Before Co-cultivation-Optimization
[0048] The composite bacterial agent of step 3 was inoculated into liquid MRS culture medium (Beijing Suolebao Technology Co., Ltd., article number M8540) at an inoculum size of 2% (V / V), and cultured at 37°C for 30 h. The fermentation broth (Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG co-culture broth) was collected. The content of Lactobacillus casei NA-2 in the co-culture broth of Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG was 1.9 × 10 8 CFU / mL, the content of Lactobacillus rhamnosus LGG co-culture solution was 2.55×10 9 CFU / mL, the total bacterial content was 2.73×10 9 CFU / mL.
[0049] The co-culture liquid of Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG obtained under the above different culture medium conditions was centrifuged at 12000 rpm / min for 10 min, and the supernatant was taken and sterilized by filtration with a 0.22 μm filter membrane to finally obtain the fermentation supernatant without Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG. This supernatant was the co-culture-pre-optimization microecological preparation.
[0050] 5. Co-culture-Preparation of Optimized Probiotics
[0051] The composite bacterial agent of step 3 was inoculated into the optimized liquid MRS culture medium at an inoculum size of 2% (V / V), and cultured at 37°C for 30h, and the fermentation liquid (Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG co-culture liquid) was collected. The content of Lactobacillus casei NA-2 in the co-culture liquid of Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG was 1.03×10 9 CFU / mL, the content of Lactobacillus rhamnosus LGG co-culture solution was 5.4×10 8 CFU / mL, the total bacterial content was 1.57×10 9 CFU / mL.
[0052] The co-culture liquid of Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG obtained under the above different culture medium conditions was centrifuged at 12000 rpm / min for 10 min, and the supernatant was taken and sterilized by filtration with a 0.22 μm filter membrane to finally obtain a fermentation supernatant that did not contain Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG. This supernatant was the co-culture-optimized microecological preparation.
[0053] The optimized liquid MRS medium consists of: 1.9% beef extract powder, 2% lactose, 0.2% potassium phosphate dibasic, 0.02% magnesium sulfate, 0.2% triammonium citrate, 0.005% manganese sulfate, 0.5% sodium acetate, 0.1% Tween 80, and the remainder water, with a pH of 5.5. The medium is prepared by dissolving the various components in water according to the aforementioned proportions, adjusting the pH, and sterilizing under high pressure at 115°C for 30 minutes.
[0054] 6. Determination of the antibacterial activity of probiotics against Escherichia coli by the inhibition zone method
[0055] The experiment was repeated three times, with four treatments in each replicate: NA-2 monoculture probiotic preparation group, LGG monoculture probiotic preparation group, coculture-pre-optimized probiotic preparation group, and coculture-post-optimized probiotic preparation group. All treatments were identical except for the different probiotic preparations used. The details are as follows:
[0056] Pour 15 mL of 2% LB agar (the agar content is 2%) on a square petri dish, and after solidification, pour 15 mL of liquid containing E. coli K88 (the E. coli content obtained by adding E. coli K88 to 1.5% LB agar (the agar content is 1.5%) at 50°C is 1×10 6 After the plate solidifies, place the Oxford cup on the plate. Add 100 μL of the probiotic preparation to each Oxford cup. Incubate at 37°C for 16 hours and measure the diameter of the inhibition zone. Three culture dishes are used for each treatment.
[0057] The results showed that the diameter of the inhibition zone of the NA-2 single culture proecological preparation group was 12.17±0.18 mm, the diameter of the inhibition zone of the LGG single culture proecological preparation group was 11.78±0.28 mm, the diameter of the inhibition zone of the co-culture-pre-optimization proecological preparation group was 13.23±0.11 mm, and the diameter of the inhibition zone of the co-culture-post-optimization proecological preparation group was 14.69±0.17. The diameters of the inhibition zones of the co-culture-pre-optimization proecological preparation group and the co-culture-post-optimization proecological preparation group were significantly higher than those of the NA-2 single culture proecological preparation group and the LGG single culture proecological preparation group, and the diameter of the inhibition zone of the co-culture-post-optimization proecological preparation group was significantly higher than that of the co-culture-pre-optimization proecological preparation group (Table 1, treatments with different lowercase letters have significant differences). This shows that in the composite bacterial agent, Lactobacillus casei NA-2 and Lactobacillus rhamnosus have a synergistic effect in inhibiting Escherichia coli; compared with the liquid MRS culture medium, the optimized liquid MRS culture medium has a significant increase in promoting the composite bacterial agent to produce substances that inhibit Escherichia coli.
[0058] 7. Determination of lactic acid content
[0059] The lactic acid content in the above-mentioned NA-2 single-culture proecological preparation, LGG single-culture proecological preparation, co-culture-pre-optimization proecological preparation and co-culture-optimization proecological preparation was determined by high performance liquid chromatography.
[0060] Chromatographic system: Waters 2695 high performance liquid chromatograph, equipped with UV detector (215 nm), chromatographic column: Bio-Rad Aminex HPX-87H ion exclusion column (300 mm × 7.8 mm, 9 μm), mobile phase: 0.005 mol / L sulfuric acid solution, flow rate: 0.6 mL / min, column temperature: 40°C, injection volume: 20 μl, run time: 15 min.
[0061] Prepare lactic acid standards of different concentrations (Dr. Ehrenstorfer, Germany, catalog number 1058591), use the above detection conditions, and use the external standard method to integrate the chromatographic peaks to draw a lactic acid standard curve. The lactic acid standard curve is y = 326417x + 136539 (R 2 =0.9993).
[0062] The results showed that the lactic acid contents in NA-2 single culture proecological preparation, LGG single culture proecological preparation, co-culture-pre-optimization proecological preparation and co-culture-post-optimization proecological preparation were 18.37±0.26 g / L, 21.05±0.43 g / L, 22.62±0.11 g / L and 24.26±0.07 g / L, respectively (Table 1).
[0063] Table 1 Bacterial growth and antibacterial activity under different culture conditions
[0064]
[0065] The antibacterial substances secreted by lactic acid bacteria are mainly organic acids, bacteriocins and hydrogen peroxide. The antibacterial substances of Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG were analyzed by adjusting the pH of the culture medium and treating them with catalase and multiple proteases. Lactobacillus casei NA-2 and Lactobacillus rhamnosus LGG do not secrete bacteriocins and hydrogen peroxide, and their main antibacterial substance is lactic acid.
[0066] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.
Claims
1. A method for preparing a probiotic preparation, comprising the steps of: inoculating a composite bacterial agent into a liquid fermentation medium for culturing, collecting the fermentation broth, and removing the bacterial cells to obtain the probiotic preparation; The active ingredient of the composite bacterial agent is Lactobacillus casei ( Lactobacillus casei ) and Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), wherein, The Lactobacillus casei ( Lactobacillus casei ) is Lactobacillus casei ( Lactobacillus casei ) NA-2; the Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) is Lactobacillus rhamnosus LGG, CGMCC No.12920; The liquid fermentation medium is composed of: 1.9% beef extract powder, 2% lactose, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.2% triammonium citrate, 0.005% manganese sulfate, 0.5% sodium acetate, 0.1% Tween 80, and the rest is water, with a pH of 5.
5.
2. The microecological preparation prepared by the method according to claim 1.
3. Use of the probiotic preparation according to claim 2 in the preparation of a product for inhibiting Escherichia coli.
4. A method for preparing lactic acid, characterized in that: The method comprises inoculating a composite bacterial agent into a fermentation medium and fermenting to obtain lactic acid; The active ingredient of the composite bacterial agent is Lactobacillus casei ( Lactobacillus casei ) and Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) composition, wherein the Lactobacillus casei ( Lactobacillus casei ) is Lactobacillus casei ( Lactobacillus casei ) NA-2; the Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) is Lactobacillus rhamnosus LGG, CGMCC No.12920; The fermentation medium is composed of: 1.9% beef extract powder, 2% lactose, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.2% triammonium citrate, 0.005% manganese sulfate, 0.5% sodium acetate, 0.1% Tween 80, and the rest is water, with a pH of 5.5.