Lactobacillus kefir and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses a lactobacillus kefir strain and an application of the lactobacillus kefir strain. The preservation number of the lactobacillus kefir is CGMCC (China General Microbiological Culture Collection Center) No.33431. The lactobacillus kefir provided by the invention can effectively remove mycotoxin, especially zearalenone, and the lactobacillus kefir is used for detoxification of mycotoxin and has important significance for guaranteeing safe production of animal husbandry and agriculture.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus kefir and applications thereof. Background Art

[0002] Zearalenone (ZEN), a common mycotoxin that contaminates corn and animal feed, is produced by Fusarium spp. ZEN is found at higher levels in animal feed than other mycotoxins. ZEN is lipophilic and accumulates in animal-derived foods. Its removal methods primarily include physical, chemical, and biological methods. Physical methods may destroy the nutritional content of feed, while chemical methods are prone to secondary contamination and have limited scope of application. Therefore, neither physical nor chemical methods are optimal for ZEN detoxification.

[0003] Biological methods involve the degradation of toxins by microorganisms, which metabolize them into low-toxic or non-toxic products, and the adsorption of toxins by microorganisms. Biological detoxification is safe, environmentally friendly, highly efficient, highly specific, and boasts high detoxification rates. Currently, the most studied microorganisms capable of detoxifying ZEN include Bacillus, Pseudomonas, and yeast. However, further research is needed to identify efficient ZEN-degrading strains and refine the ZEN biological detoxification system. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and provide a kefir lactobacillus strain and its application, wherein the strain can effectively remove mycotoxins, especially zearalenone.

[0005] In order to achieve the above object, the present invention provides a first aspect of a Lactobacillus kefir strain ( Lactobacillus kefiri ), the preservation number of the kefir lactobacillus is CGMCC No.33431.

[0006] The second aspect of the present invention provides a microbial product, comprising the aforementioned Lactobacillus kefir; and / or, the microbial preparation comprises a cell lysate of Lactobacillus kefir as described above; And / or, the microbial preparation comprises the cell metabolites of Lactobacillus kefir as described above.

[0007] The third aspect of the present invention provides the use of the aforementioned Lactobacillus kefir or the aforementioned microbial product in removing mycotoxins.

[0008] A fourth aspect of the present invention provides the use of the aforementioned Lactobacillus kefir, or the aforementioned microbial product, or the aforementioned application in mycotoxin detoxification.

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The Lactobacillus kefir provided by the present invention can effectively remove mycotoxins in a substrate, especially zearalenone.

[0010] Biological Deposits Lactobacillus kefir of the present invention Lactobacillus kefiri , numbered X34, and was deposited on January 15, 2025 at the General Microbiology Center of China Culture Collection Committee (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) (the abbreviation of the depository is CGMCC), and the deposit number is CGMCC No.33431. DETAILED DESCRIPTION

[0011] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0012] The first aspect of the present invention provides a strain of Lactobacillus kefir ( Lactobacillus kefiri ), the preservation number of the kefir lactobacillus is CGMCC No.33431.

[0013] The inventors of the present invention accidentally isolated a strain of Lactobacillus kefir from fermented dairy products. The strain can effectively remove mycotoxins, especially zearalenone.

[0014] According to the present invention, the 16S rDNA sequence of the Lactobacillus kefir is shown as SEQ ID NO: 1.

[0015] SEQ ID NO: 1:

[0016] A second aspect of the present invention provides a microbial product, comprising the Lactobacillus kefir as described above.

[0017] According to the present invention, the microbial preparation comprises the cell lysate of Lactobacillus kefir as described above.

[0018] According to the present invention, the microbial preparation comprises the cell metabolites of Lactobacillus kefir as described above.

[0019] In the present invention, the cell metabolites refer to the components remaining in the clear liquid after the cultured Lactobacillus kefir is separated from the culture medium by centrifugation.

[0020] In the present invention, the microbial preparation contains at least one of the above components, so that the microbial preparation has the ability to remove mycotoxins.

[0021] According to the present invention, preferably, the microbial preparation is a liquid preparation.

[0022] The third aspect of the present invention provides the use of the aforementioned Lactobacillus kefir or the aforementioned microbial product in removing mycotoxins.

[0023] In the present invention, the mycotoxins are metabolites produced by fungi and are widely present in polluted environments. Common types include aflatoxins, ochratoxins and zearalenone.

[0024] According to the present invention, preferably, the mycotoxin is zearalenone.

[0025] The Lactobacillus kefir and / or microbial products thereof provided by the present invention have excellent removal effects on zearalenone (ZEN).

[0026] According to the present invention, preferably, the use comprises: contacting the aforementioned Lactobacillus kefir or the aforementioned microbial preparation with a substrate containing mycotoxins; The substrate is water, feed or soil.

[0027] In the present invention, the Lactobacillus kefir or microbial product can be used in various environments contaminated by mycotoxins.

[0028] In the present invention, the amount of the Lactobacillus kefir or microbial product can be determined according to the degree of mycotoxin contamination of the substrate.

[0029] According to the present invention, preferably, the amount of Lactobacillus kefir is 10 3 -10 9 CFU. 10 3-10 9 CFU means that the content of Kefir Lactobacillus in the system reaches 10 3 -10 9 of the order of magnitude, that is, 1×10 3 CFU, 5×10 3 CFU, 9.9×10 3 CFU etc. are all 10 3 The order of magnitude of CFU.

[0030] Further preferably, the amount of Lactobacillus kefir is 10 5 -10 8 CFU.

[0031] According to the present invention, preferably, the contact conditions include: temperature of 36-38° C., time of 12-50 h, and pH of 5-5.5.

[0032] A fourth aspect of the present invention provides the use of the aforementioned Lactobacillus kefir, or the aforementioned microbial product, or the aforementioned application in mycotoxin detoxification.

[0033] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were purchased from regular chemical reagent suppliers and were of analytical grade.

[0035] In the following examples, zearalenone is represented by the abbreviation ZEN.

[0036] In the following examples, the ZEN content was detected by centrifuging the tested sample at 10,000 rpm for 8 minutes, adding 2,400 μL of dichloromethane to 800 μL of the supernatant, and mixing in a glass test tube. 1,600 μL of the organic phase of the mixed sample was placed in a new glass test tube, heated at 60° C. for 20 minutes to dryness, and then 1 mL of mobile phase was added to the evaporated sample for reconstitution. After vortexing for 1 minute, the sample solution was filtered through a 0.22 μm organic phase needle filter to obtain a sample solution, which was then detected by HPLC.

[0037] The detection conditions of HPLC are: Column: Pursuit XRs C18 250 × 4.6 mm; Mobile phase: acetonitrile: water (glacial acetic acid: water = 2:100) = 5:5 Flow rate: 1 mL / min; column temperature: 30°C; Injection volume: 15 μL; detection wavelength: excitation wavelength 274 nm, emission wavelength 440 nm.

[0038] ZEN removal rate (%) = (the amount of ZEN initially added - the content of ZEN remaining in the sample) / the amount of ZEN initially added × 100%.

[0039] The culture medium formula used in the following examples is as follows: MRS liquid medium (1 L): peptone 10 g, beef powder 8 g, yeast powder 4 g, glucose 20 g, sodium acetate trihydrate 5 g, magnesium sulfate heptahydrate 0.2 g, potassium hydrogen phosphate trihydrate 2 g, ammonium citrate 2 g, manganese sulfate heptahydrate 0.05 g, Tween 80 1 mL, distilled water 1 L, agar 15 g.

[0040] Preparation Example 1 The glycerol-preserved Lactobacillus kefir X34 was inoculated on MRS medium and cultured at 37°C, 180 rpm, and pH 5.7 for 12 h. The initial bacterial agent was obtained after three generations of culture. The concentration of Lactobacillus kefir X34 in the initial bacterial agent reached 10 4 CFU / mL.

[0041] Take 10 mL of the above initial bacterial agent, centrifuge at 10,000 rpm at 4°C for 5 min, remove the supernatant, add an equal volume of PBS buffer to the supernatant, and mix evenly to obtain bacterial agent 1 (live bacteria).

[0042] Take 10 mL of the above initial bacterial agent, centrifuge at 10,000 rpm for 5 min, and filter the supernatant through a 0.22 μm filter to obtain bacterial agent 2 (cellular metabolite).

[0043] Take 10 mL of the above initial bacterial agent and sterilize it at 121°C for 15 min to obtain bacterial agent 3 (inactivated bacteria).

[0044] Take 10 mL of the above initial bacterial agent and centrifuge at 10,000 rpm for 5 min. After removing the supernatant, add an equal volume of PBS buffer to the supernatant and mix evenly. Use an ultrasonic disruptor to treat at 4°C for 30 min. Take all the supernatant and pass it through a 0.22 μm filter membrane to obtain bacterial agent 4 (cell lysate).

[0045] Example 1 990 μL of the initial bacterial agent prepared in Preparation Example 1 was taken, and 10 μL of a ZEN solution with a concentration of 1000 μg / mL was added thereto. A total of four groups were prepared. After culturing at 37°C for 12 h, 24 h, 36 h, and 48 h, the ZEN content (removal rate was calculated) and the number of viable bacteria were tested. The results are shown in Table 1.

[0046] Table 1

[0047] Example 2 990 μL of the bacterial agents 1-4 prepared in Preparation Example 1 were taken respectively, and 10 μL of a 1000 μg / mL ZEN solution was added thereto. After incubation at 37°C for 48 hours, the ZEN content was tested and the removal rate was calculated. The results are shown in Table 2.

[0048] Table 2

[0049] As shown in Table 2, the bacteria (agent 1) removed 57.64% of ZEN, while the inactivated bacterial solution (agent 3) removed only 3.44%. Therefore, the bacteria's ability to remove ZEN is primarily due to degradation.

[0050] The cell lysate (agent 4) achieved a ZEN removal rate of 94.76%, while the free supernatant (agent 2) achieved a ZEN removal rate of 18.03%. This result indicates that some of the degradation substances are present outside the cell body, and that the bacterial cell's ability to remove ZEN is significantly weaker than that of the cell lysate, further demonstrating that the ZEN removal process primarily relies on the bacterial strain's ability to degrade ZEN.

[0051] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus kefir ( Lactobacillus kefiri ), characterized in that, The deposit number of the Lactobacillus kefir is CGMCC No.33431.

2. The Lactobacillus kefir according to claim 1, wherein The sequence of 16S rDNA of the Lactobacillus kefir is shown in SEQ ID NO:

1.

3. A microbial product, characterized in that The microbial product comprises the Lactobacillus kefir according to claim 1 or 2; and / or, the microbial preparation comprises the cell lysate of Lactobacillus kefir according to claim 1 or 2; And / or, the microbial product comprises the cell metabolites of Lactobacillus kefir according to claim 1 or 2.

4. The microbial preparation according to claim 3, wherein The microbial preparation is a liquid preparation.

5. Use of the Lactobacillus kefir according to claim 1 or 2 or the microbial preparation according to claim 3 or 4 in removing mycotoxins.

6. The use according to claim 5, wherein: The mycotoxin is zearalenone.

7. The use according to claim 5 or 6, wherein: The application comprises: contacting the Lactobacillus kefir described in claim 1 or 2 or the microbial preparation described in claim 3 or 4 with a substrate containing mycotoxins; The substrate is water, feed or soil.

8. The use according to claim 7, wherein: The amount of Lactobacillus kefir is 10 3 -10 9 CFU, preferably 10 5 -10 8 CFU.

9. The use according to claim 7, wherein: The contact conditions include: temperature of 36-38° C., time of 12-50 h, and pH of 5-5.

5.

10. Use of the Lactobacillus kefir according to claim 1 or 2, or the microbial preparation according to claim 3 or 4, or the use according to any one of claims 5 to 9 in mycotoxin detoxification.

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