Lactobacillus paracasei
By using the antibacterial extract prepared by fermentation of CGMCC No. 28782 Lactobacillus paracasei strain, the problem of difficulty in inhibiting drug-resistant pathogenic bacteria in the existing technology is solved, and effective inhibition of multiple pathogenic bacteria is achieved. Avoid adverse effects on probiotics.
Patent Information
- Application Number
- CN202411482372.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art is difficult to effectively inhibit drug-resistant pathogenic bacteria, such as vancomycin-resistant Enterococcus and cavitary nocardia, and the use of traditional chemical preservatives and antibiotics has problems with residual and drug resistance.
Provides a strain of Lacticaseibacillus paracasei, with the preservation number CGMCC No. 28782, which is used to prepare an antibacterial extract through fermentation. The extract is processed through ammonium sulfate precipitation and ultrafiltration, has high molecular weight and thermal stability, and can Effectively inhibits a variety of pathogenic bacteria.
The antibacterial extract has a significant inhibitory effect on intestinal pathogenic bacteria, oral pathogenic bacteria, environmental pathogenic bacteria and antibiotic-resistant microorganisms, is not sensitive to catalase and protease, does not inhibit probiotics, and avoids the Adverse effects of probiotics.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to a Lactobacillus paracasei and its application. Background Art
[0002] The rampant proliferation of environmental pathogens and spoilage bacteria is one of the main reasons for the frequent occurrence of food safety problems in recent years. These microorganisms directly or indirectly cause various harms to the human body and cause a series of diseases, such as diarrhea caused by Escherichia coli. In addition to the risk of pathogenicity from exogenous ingestion, the inherent enterococci in the human body are also an uncontrollable factor. This conditional pathogen causes bacteremia, respiratory tract and endocarditis in hosts with low immunity. In recent years, with the abuse of antibiotics, the drug resistance of enterococci has gradually increased. In some clinical infection cases, Enterococcus faecium and Enterococcus faecalis are the most common infectious bacteria. Among them, vancomycin-resistant Enterococci (VRE) are particularly common. The widespread spread of this strain around the world has had a great impact on public health and social security. In addition, a large number of pathogenic bacteria are also parasitic in the human oral cavity. Take Nocardia cavitaria as an example. It germinates and grows in a specific oral environment, eventually leading to periodontitis and odontogenic infection.
[0003] At present, the preferred option to inhibit these pathogenic microorganisms is to use chemical preservatives. However, excessive abuse of such preservative technology will lead to a large amount of residues in feed and even food, which will become one of the potential factors threatening human health. Although Nisin, a bacteriocin from Streptococcus lactis, is safe and non-toxic and avoids the residual hazards caused by chemical preservatives, the "lanthionine ring" in its molecule is also harmful to probiotics. Antibiotics are another means of inhibiting bacteria, but long-term use will cause the problem of "antibiotic resistance". Some microorganisms can even survive by withstanding strong antibiotics at the level of vancomycin, such as vancomycin-resistant Enterococcus faecium.
[0004] Lactobacillus paracasei is one of important members of lactobacillus family, it is widely present in fermented food, human oral cavity and intestinal tract, has good acid and bile salt tolerance ability, can tolerate the defense mechanism of human gastrointestinal tract, colonizes in intestinal tract.It has multiple physiological effects, such as enhancing human immunity, preventing diseases etc., but the research or report of this type of microorganism antibacterial is relatively rare.Therefore, it is a problem for those skilled in the art to find a strain to pathogenic microorganisms, especially that the microorganism of tolerance antibiotic has the Lactobacillus paracasei of inhibition function. Summary of the invention
[0005] In order to solve the above technical problems, the present application provides a Lactobacillus paracasei, whose preservation number is CGMCC No.28782.
[0006] The deposit information of the strain in this application is as follows:
[0007] Strain name: Lacticaseibacillus paracasei
[0008] The accession number is: CGMCC No.28782;
[0009] Deposit date: October 26, 2023;
[0010] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;
[0011] Abbreviation of depository unit: CGMCC;
[0012] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0013] The present application also provides a fermentation broth extract, which is obtained by fermenting the above-mentioned Lactobacillus paracasei.
[0014] The present application also provides a method for preparing the fermentation broth extract, comprising the following steps: 1) inoculating the seed liquid of the above-mentioned Lactobacillus paracasei into a liquid culture medium, culturing, and obtaining a fermentation broth; 2) centrifuging the fermentation broth to obtain a supernatant; 3) adding ammonium sulfate to the supernatant, centrifuging, and collecting a precipitate; 4) re-dissolving the precipitate with distilled water, ultrafiltering with an ultrafiltration tube, and freeze-drying the obtained macromolecules to obtain a fermentation broth extract.
[0015] The present application also provides a food, medicine or health product, wherein the food, medicine or health product contains the above-mentioned Lactobacillus paracasei or the above-mentioned fermentation broth extract.
[0016] The beneficial effects brought by the present application include but are not limited to: (1) The present application discovered a strain of Lactobacillus paracasei, with the deposit number of CGMCC No. 28782. The antibacterial extract prepared by using the Lactobacillus paracasei of the present application has the effect of inhibiting intestinal pathogens, oral pathogens, environmental pathogens and antibiotic-resistant microorganisms. (2) The antibacterial extract prepared by the present application is insensitive to catalase and protease; (3) The antibacterial extract prepared by the present application has no inhibitory effect on conventional probiotics. DETAILED DESCRIPTION
[0017] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.
[0018] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.
[0019] The present application provides a Lactobacillus paracasei, whose preservation number is CGMCC No.28782.
[0020] In some embodiments, the 16S rRNA coding sequence of Lactobacillus paracasei may include the nucleotide sequence shown as SEQ ID NO.1.
[0021] The present application provides a fermentation broth extract, which is prepared from the fermentation broth of the above-mentioned Lactobacillus paracasei.
[0022] In some embodiments, the molecular weight of the antibacterial substance in the fermentation broth extract may be greater than 3000 kDa.
[0023] In some embodiments, the fermentation broth extract is thermostable.
[0024] In some embodiments, the fermentation broth extract is not sensitive to catalase or protease.
[0025] The present application also provides a method for preparing the fermentation broth extract, comprising the following steps: 1) inoculating the seed liquid of the above-mentioned Lactobacillus paracasei into a liquid culture medium, culturing, and obtaining a fermentation broth; 2) centrifuging the fermentation broth to obtain a supernatant; 3) adding ammonium sulfate to the supernatant, centrifuging, and collecting a precipitate; 4) re-dissolving the precipitate with distilled water, ultrafiltering with an ultrafiltration tube, and freeze-drying the obtained macromolecules to obtain a fermentation broth extract.
[0026] In some embodiments, in step 1), the volume ratio of the seed solution to the liquid culture medium may be 1-5%. For example, the volume ratio of the seed solution to the liquid culture medium may be 1, 2, 3, 4 or 5%. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0027] In some embodiments, in step 1), the effective viable count in the seed solution may be 0.5 to 1.5 x 109 CFU / mL. For example, in step 1), the effective viable count in the seed solution can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 x 10 9 CFU / mL. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 1), the effective viable count in the seed solution can be 1.0x10 9 CFU / mL.
[0028] In some embodiments, in step 1), the culture may be anaerobic culture.
[0029] In some embodiments, in step 1), the liquid culture medium may be TPY liquid culture medium.
[0030] In some embodiments, in step 1), the culture temperature may be 25-45° C. For example, in step 1), the culture temperature may be 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5 or 45° C. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0031] In some embodiments, in step 1), the culture time may be 24 to 48 hours. For example, in step 1), the culture time may be 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 hours. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0032] In some embodiments, in step 2), the centrifugal speed may be 8000-12000 g. For example, in step 2), the centrifugal speed may be 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500 or 12000 g. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0033] In some embodiments, in step 2), the centrifugation time may be 10 to 20 minutes. For example, in step 2), the centrifugation time may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minutes. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0034] In some embodiments, in step 3), the saturation of ammonium sulfate in the supernatant can be 30-70%. For example, in step 3), the saturation of ammonium sulfate in the supernatant can be 30, 40, 50, 60 or 70%. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0035] In some embodiments, in step 3), the centrifugal speed may be 8000-12000 g. For example, in step 3), the centrifugal speed may be 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500 or 12000 g. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0036] In some embodiments, in step 3), the centrifugation time may be 10 to 20 min. For example, in step 3), the centrifugation time may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 min. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0037] In some embodiments, in step 4), the ultrafiltration tube molecular weight cutoff can be 3 kDa.
[0038] The present application also provides a food, medicine or health product, wherein the food, medicine or health product contains the above-mentioned Lactobacillus paracasei or the above-mentioned fermentation broth extract.
[0039] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.
[0040] Example 1 - Screening, Identification and Preservation of Lactobacillus paracasei RF7
[0041] The Lactobacillus paracasei RF7 in the present invention is obtained by the following method: 1 g of a Xinjiang Kuitun kefir sample is aseptically weighed, serially diluted with sterile physiological saline, and the dilution is evenly spread on a sterile MRS agar medium (purchased from Merck, Germany) by coating, anaerobically cultured at 37°C for 48 hours, a single colony with a smooth colony edge and white color is selected and streaked again on the MRS agar medium, and after anaerobically cultured at 37°C for 24 hours, a single colony is selected and entrusted to the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration for 16S rRNA gene sequencing and physiological and biochemical detection (as shown in Table 1).
[0042] The 16S rRNA gene sequence of this strain is as follows:
[0043]
[0044] Table 1 Results of physical and chemical tests on strains
[0045]
[0046]
[0047] The strain was identified as Lactobacillus paracasei by 16S rRNA gene sequence comparison and physiological and biochemical analysis at the China General Microbiology Center (CGMCC). The strain was deposited at the China General Microbiology Center (CGMCC) on October 26, 2023, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The strain's deposit number is CGMCC No. 28782.
[0048] Example 2 - Application of Lactobacillus paracasei RF7 in the preparation of antibacterial extracts
[0049] 1. Materials and methods
[0050] Preparation of fermentation seed liquid of Lactobacillus paracasei RF7: the lyophilized powder of Lactobacillus paracasei RF7 was dissolved with a small amount of sterile distilled water, a ring was streaked on MRS solid medium (purchased from Merck, Germany) with an inoculating loop, 37 ℃ of anaerobic culture was taken out for 48h, single colony was picked up with an inoculating loop and put into 10mL MRS liquid medium (purchased from Merck, Germany), vortex oscillator was used to evenly disperse the colony in the liquid medium, 37 ℃ of cultivation for 24h was taken out, and the inoculum was inoculated into MRS liquid medium with 2% (v / v) inoculum size, and 37 ℃ was cultivated again for 24h, and the culture was centrifuged at 15,000rpm for 10 minutes, and the supernatant was discarded. After the thalline was washed twice with sterile distilled water, the sterile distilled water of the original culture volume was suspended to obtain the fermentation seed liquid of Lactobacillus paracasei RF7. After measuring, the viable count of this seed liquid was 1x10 9 CFU / mL.
[0051] 2. Preparation of antibacterial extract of Lactobacillus paracasei RF7
[0052] The seed liquid of Lactobacillus paracasei RF7 is aseptically inoculated in a TPY liquid medium at an inoculum amount of 3% (v / v), and the fermentation liquid is obtained by anaerobically culturing at 35°C for 36 hours, and the fermentation liquid is centrifuged at 10,000g for 15 minutes to obtain the supernatant. Ammonium sulfate is slowly added to the supernatant, and the precipitate is collected by centrifugation at 10,000g for 15 minutes when the ammonium sulfate saturation is 30% to 70%, and the precipitate is re-dissolved with a small amount of distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the antibacterial extract A of Lactobacillus paracasei RF7.
[0053] Example 3 - Application of Lactobacillus paracasei RF7 in the preparation of antibacterial extracts
[0054] 1. Materials and methods
[0055] Preparation of fermentation seed liquid of Lactobacillus paracasei RF7: same as in Example 2.
[0056] 2. Preparation of antibacterial extract of Lactobacillus paracasei RF7
[0057] The seed liquid of Lactobacillus paracasei RF7 is aseptically inoculated in a TPY liquid medium at an inoculum amount of 1% (v / v), anaerobically cultured at 45°C for 48 hours to obtain a fermentation liquid, centrifuged at 12,000g for 10 minutes, and the supernatant is taken. Ammonium sulfate is slowly added to the supernatant, and the precipitate precipitated when the ammonium sulfate saturation is 60% to 70% is collected by centrifugation at 12,000g for 10 minutes, and the precipitate is re-dissolved with a small amount of distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the antibacterial extract B of Lactobacillus paracasei RF7.
[0058] Example 4 - Application of Lactobacillus paracasei RF7 in the preparation of antibacterial extracts
[0059] 1. Materials and methods
[0060] Preparation of fermentation seed liquid of Lactobacillus paracasei RF7: same as in Example 2.
[0061] 2. Preparation of antibacterial extract of Lactobacillus paracasei RF7
[0062] The seed liquid of Lactobacillus paracasei RF7 is aseptically inoculated in a TPY liquid medium at an inoculum amount of 5% (v / v), and the fermentation liquid is obtained by anaerobically culturing at 25°C for 30 hours, and the fermentation liquid is centrifuged at 8,000g for 20 minutes to obtain the supernatant. Ammonium sulfate is slowly added to the supernatant, and the precipitate is collected at an ammonium sulfate saturation of 40% to an ammonium sulfate saturation of 60% by centrifugation at 8,000g for 20 minutes, and the precipitate is reconstituted with a small amount of distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the antibacterial extract C of Lactobacillus paracasei RF7.
[0063] Example 5 - Application of Lactobacillus paracasei RF7 in the preparation of antibacterial extracts
[0064] 1. Materials and methods
[0065] Preparation of fermentation seed liquid of Lactobacillus paracasei RF7: same as in Example 2.
[0066] 2. Preparation of antibacterial extract of Lactobacillus paracasei RF7
[0067] The seed liquid of Lactobacillus paracasei RF7 was aseptically inoculated into a TPY liquid medium at an inoculum amount of 2% (v / v), and the fermentation liquid was obtained by anaerobically culturing at 40°C for 24 hours, and the fermentation liquid was centrifuged at 11,000g for 13 minutes to obtain the supernatant. Ammonium sulfate was slowly added to the supernatant, and the precipitate was collected at an ammonium sulfate saturation of 50% to an ammonium sulfate saturation of 70% by centrifugation at 11,000g for 13 minutes, and the precipitate was reconstituted with a small amount of distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part was freeze-dried to obtain the antibacterial extract D of Lactobacillus paracasei RF7.
[0068] Example 6 - Application of Lactobacillus paracasei RF7 in the preparation of antibacterial extracts
[0069] 1. Materials and methods
[0070] Preparation of fermentation seed liquid of Lactobacillus paracasei RF7: same as in Example 2.
[0071] 2. Preparation of antibacterial extract of Lactobacillus paracasei RF7
[0072] The seed liquid of Lactobacillus paracasei RF7 is aseptically inoculated in a TPY liquid medium at an inoculum amount of 4% (v / v), and the fermentation liquid is obtained by anaerobically culturing at 30°C for 42 hours, and the fermentation liquid is centrifuged at 9,000g for 17 minutes to obtain the supernatant. Ammonium sulfate is slowly added to the supernatant, and the precipitate is collected at an ammonium sulfate saturation of 50% to an ammonium sulfate saturation of 60% by centrifugation at 9,000g for 17 minutes, and the precipitate is reconstituted with a small amount of distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the antibacterial extract E of Lactobacillus paracasei RF7.
[0073] Effect Example 1 - Antibacterial Effect of Antibacterial Extract of Lactobacillus paracasei RF7
[0074] (a) Preparation of indicator bacterial suspension: Lyophilized powders of Listeria monocytogenes ATCC 19115 (purchased from CGMCC, China), Nocardia coeliaca ATCC 17041 (purchased from ATCC, USA), Enterococcus faecium CICC24261 (purchased from CICC, China), Bacillus cereus ATCC 11778 (purchased from ATCC, USA) and Pseudomonas fluorescens CICC23250 (purchased from CICC, China) were dissolved in a small amount of sterile distilled water, and a loop of the suspension was streaked onto TPY solid medium (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., China) using an inoculation loop, and cultured at 37°C for 48 h. A single colony was picked up using an inoculation loop and placed in 10 mL of 4% paraformaldehyde solution. TPY liquid culture medium (purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., China) was used. The colonies were evenly dispersed in the liquid culture medium using a vortex oscillator, cultured at 37°C for 24 h, taken out, and inoculated into TPY liquid culture medium with a 2% (v / v) inoculation amount. After culturing again at 37°C for 24 h, the culture was centrifuged at 15,000 rpm for 10 min, the supernatant was discarded, the bacteria were washed twice with sterile distilled water, and then suspended with sterile distilled water of the original culture volume to obtain a bacterial suspension of each indicator bacteria.
[0075] (b) Preparation of indicator bacteria plate: dilute the indicator bacteria suspension prepared by the above method to 10 6 CFU / mL, take an appropriate amount of bacterial solution and evenly spread it on the TPY plate to obtain the indicator bacteria plate containing different pathogenic bacteria.
[0076] (c) Antibacterial effect of Lactobacillus paracasei RF7 antibacterial extract
[0077] The prepared Lactobacillus paracasei RF7 antibacterial extracts A, B, C, D and E were dissolved in sterile water to obtain the corresponding antibacterial extract solutions (15 mg / mL). The antibacterial extract solutions were placed on the indicator bacteria plates previously inoculated with pathogenic bacteria using the Oxford cup method (100 uL / cup), and cultured at 37° C. for 2 days. The diameters of the inhibition zones were measured. The results are shown in Table 2 below.
[0078] Table 2 The results of inhibition of pathogenic bacteria by the antibacterial extract of Lactobacillus paracasei RF7
[0079]
[0080] As can be seen from Table 2 above, the diameter of the inhibition zone of the antibacterial extract of Lactobacillus paracasei RF7 against intestinal pathogens is ≥11mm, the diameter of the inhibition zone against oral pathogens is ≥14mm, the diameter of the inhibition zone against environmental pathogens is ≥8mm, and the diameter of the inhibition zone against antibiotic-resistant microorganisms is ≥18mm.
[0081] Effect Example 2 - Molecular Weight of the Active Ingredient in the Antibacterial Extract of Lactobacillus paracasei RF7
[0082] With reference to the method described in Example 2, the macromolecular part of the ammonium sulfate precipitation of the supernatant of Lactobacillus paracasei RF7 fermentation liquid and the small molecule part freeze-dried powder of ultrafiltration interception (molecular weight cut-off is 3kDa) and the permeation are prepared. The above two parts of freeze-dried powder are detected by the method described in Effect Example 1 to inhibit Nocardia cavitas, and the results are shown in Table 3 below.
[0083] Table 3 Inhibitory effects of different fractions obtained by ultrafiltration of ammonium sulfate precipitation from the supernatant of Lactobacillus paracasei RF7 fermentation broth on Nocardia cavitas
[0084] Ultrafiltration components Molecular weight (Da) Diameter of inhibition zone against Nocardia cavitas (mm) Macromolecules ＞3000 14.5 Small molecules ＜3000 -
[0085] It can be seen from Table 3 above that the molecular weight of the component in the antibacterial extract of Lactobacillus paracasei RF7 that effectively inhibits pathogenic bacteria is greater than 3000 Da.
[0086] Effect Example 3 - Sensitivity test of the antibacterial extract of Lactobacillus paracasei RF7 to catalase and protease
[0087] Catalase, pepsin, trypsin, papain and proteinase K were added to the antibacterial extract solution A (described in Effect Example 1) adjusted to the optimal pH of each enzyme to a final concentration of 1 mg / mL. After 2 h in a 37°C water bath, the pH of each sample was adjusted back to the initial pH of the antibacterial extract solution A. At the same time, the antibacterial extract solution A without enzyme treatment was used as a control. The antibacterial activity of the sample against Nocardia cavitas was detected by the method described in Effect Example 1. The results are shown in Table 4 below.
[0088] Table 4 Results of sensitivity test of the antibacterial extract of Lactobacillus paracasei RF7 to catalase and protease
[0089] Diameter of inhibition zone against Nocardia cavitas (mm) Catalase 13.9 Pepsin 10.9 Trypsin 10.3 Papain 12.4 Proteinase K 9.9 Control group 14.4
[0090] As shown in Table 4 above, the antibacterial extract of Lactobacillus paracasei RF7 still has a stable inhibitory effect on Nocardia cavitas after being treated with different enzymes, indicating that the antibacterial extract is insensitive to catalase and protease.
[0091] Effect Example 4 - Inhibitory Effect of Antibacterial Extract of Lactobacillus paracasei RF7 on Probiotics
[0092] 1. Materials and methods
[0093] (a) Preparation of probiotic suspension: Referring to the preparation method of the indicator bacteria suspension described in Example 1, bacterial suspensions of Lactobacillus delbruechii subsp. Bulgaricus (purchased from CGMCC, China), Bifidobacterium animalis subsp. Lactis (purchased from CGMCC, China), Lactobacillus salivarius (Lactobacillus salivarius) CGMCC No. 23518 and Limosilactobacillus fermentum (Limosilactobacillus fermentum) CGMCC 17321 (provided by Bright Dairy Co., Ltd.) were prepared respectively.
[0094] (b) Preparation of indicator bacteria plates
[0095] Referring to the method described in Effect Example 1, the above-mentioned probiotic suspension was used to prepare indicator bacteria plates containing different probiotics.
[0096] 2. Inhibitory effect of Lactobacillus paracasei RF7 antibacterial extract on probiotics
[0097] An Oxford cup was placed on the above-mentioned probiotic indicator bacteria plate, and 100 μL of the antibacterial extract solution A prepared in the above-mentioned Effective Example 1 was added to each Oxford cup. The mixture was incubated at 37° C. for 2 days, and the diameter of the antibacterial zone was measured. The results are shown in Table 5 below.
[0098] Table 5 The inhibition results of the antibacterial extract of Lactobacillus paracasei RF7 on probiotics
[0099] Diameter of inhibition zone (mm) Lactobacillus paracasei RF7 antibacterial extract solution A Lactobacillus bulgaricus - Bifidobacterium - Lactobacillus salivarius - Lactobacillus mucilaginosus fermentation -
[0100] It can be seen from Table 5 above that the antibacterial extract solution of Lactobacillus paracasei RF7 has no inhibitory effect on probiotics including Lactobacillus bulgaricus, Bifidobacterium, Lactobacillus salivarius and Lactobacillus fermentum.
[0101] Comparative Example 1 - Inhibitory effects of fermentation broth extracts of different strains on pathogenic bacteria
[0102] With reference to the method described in Example 2, the inhibitory effects of TPY fermentation broth extracts prepared by Lactobacillus paracasei RF7, Lactobacillus paracasei ATCC334 (Lacticaseibacillus paracasei, purchased from ATCC, the U.S.), Lactobacillus plantarum CGMCC 0847 (Lactiplantibacillus plantarum, provided by Bright Dairy Co., Ltd.), and Streptococcus thermophilus CGMCC1.8748 (Streptococcus thermophilus, purchased from CGMCC, China) on different pathogenic bacteria were compared, and the specific operations were as follows:
[0103] 1. Materials and methods
[0104] (a) Preparation of seed solution or bacterial suspension: Seed solution or bacterial suspension of Lactobacillus paracasei RF7, Lactobacillus paracasei ATCC334, Lactobacillus plantarum CGMCC 0847 (provided by Bright Dairy Co., Ltd.), Streptococcus thermophilus CGMCC 1.8748 and pathogenic bacteria (Listeria monocytogenes ATCC 19115, Nocardia cavitas ATCC17041, vancomycin-resistant Enterococcus faecium CICC 24261, Bacillus cereus ATCC 11778 and Pseudomonas fluorescens CICC23250) were prepared respectively according to the method described in Example 1.
[0105] (b) Preparation of indicator bacteria plates
[0106] Referring to the method described in Effect Example 1, the above pathogenic bacteria suspension was used to prepare indicator bacteria plates containing different pathogenic bacteria.
[0107] (c) Preparation of fermentation broth extracts of different strains
[0108] The seed liquid of Lactobacillus paracasei RF7, Lactobacillus paracasei ATCC334, Lactobacillus plantarum CGMCC 0847 and Streptococcus thermophilus CGMCC 1.8748 is aseptically inoculated in a TPY liquid culture medium at an inoculum amount of 3% (v / v), and the fermentation liquid is obtained by anaerobic culture at 35°C for 36 hours, and the fermentation liquid is centrifuged at 10,000g for 15 minutes to obtain the supernatant. Ammonium sulfate is slowly added to the supernatant, and the precipitate is collected when the ammonium sulfate saturation is 30-70% by centrifugation at 10,000g for 15 minutes, and the precipitate is re-dissolved with a small amount of distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the fermentation extract of the corresponding strain.
[0109] 2. Inhibitory effects of fermentation extracts of different strains on pathogenic bacteria
[0110] An Oxford cup was placed on the above pathogenic bacteria indicator bacteria plate, and 100 μL of the fermentation extract solution (15 mg / mL) of the different strains prepared above was added to each Oxford cup. The culture was incubated at 37° C. for 2 days, and the diameter of the inhibition zone was measured. The results are shown in Table 6 below.
[0111] Table 6 Inhibition results of fermentation extracts of different strains on pathogenic bacteria
[0112]
[0113] It can be seen from Table 6 above that TPY fermentation strains, including the same strain (Lactobacillus paracasei), do not have the ability to ferment TPY to produce antibacterial substances, while Lactobacillus paracasei RF7 can use TPY fermentation to synthesize specific products that inhibit various pathogenic bacteria, which indicates that Lactobacillus paracasei RF7 has unique fermentation properties different from other strains.
[0114] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.
[0115] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.
[0116] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values is as accurate as possible within the feasible range.
[0117] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.
Claims
1. A Lactobacillus paracasei, whose preserving number is CGMCC No.28782.
2. Lactobacillus paracasei as claimed in claim 1, characterized in that The 16S rRNA coding sequence of Lactobacillus paracasei includes the nucleotide sequence shown in SEQ ID NO.
1.
3. A fermentation broth extract, prepared from the fermentation broth of Lactobacillus paracasei according to claim 1 or 2.
4. The fermentation broth extract according to claim 3, characterized in that The molecular weight of the antibacterial substance in the fermentation broth extract is greater than 3000Da; and / or, the fermentation broth extract is thermostable; And / or, the fermentation broth extract is insensitive to catalase or protease.
5. The method for preparing the fermentation broth extract according to claim 3 or 4, comprising the following steps: 1) inoculating the seed liquid of Lactobacillus paracasei according to claim 1 or 2 into a liquid culture medium, culturing, and obtaining a fermentation liquid; 2) centrifuging the fermentation broth to obtain a supernatant; 3) adding ammonium sulfate to the supernatant, centrifuging, and collecting the precipitate; 4) re-dissolving the precipitate with distilled water, ultrafiltration with an ultrafiltration tube, and freeze-drying the obtained macromolecules to obtain a fermentation broth extract.
6. The preparation method according to claim 5, characterized in that: The step 1) includes the following features: The volume ratio of the seed solution to the liquid culture medium is 1 to 5%; And / or, the effective viable bacteria count in the seed solution is 0.5 to 1.5 x 10 9 CFU / mL; And / or, the culture is anaerobic culture; And / or, the liquid culture medium is TPY liquid culture medium; And / or, the culture temperature is 25-45°C; And / or, the culture time is 24 to 48 hours.
7. The preparation method according to claim 5, characterized in that: The step 2) includes the following features: The centrifugal speed is 8000-12000g; And / or, the centrifugation time is 10 to 20 minutes.
8. The preparation method according to claim 5, characterized in that: The step 3) includes the following features: The saturation of ammonium sulfate in the supernatant is 30-70%; And / or, the centrifugal speed is 8000-12000g; And / or, the centrifugation time is 10 to 20 minutes.
9. The preparation method according to claim 5, characterized in that: In the step 4), the molecular weight cut-off of the ultrafiltration tube is 3 kDa.
10. A food, medicine or health product, characterized in that: The food, medicine or health product contains the Lactobacillus paracasei according to claim 1 or 2 or the fermentation broth extract according to claim 3 or 4.