Lactobacillus for promoting growth of leavening dough and application thereof
Through the adsorption and metabolism of Levilactobacillus zymae, the problem of incomplete mycotoxin removal in the existing technology is solved, and the effect of efficient removal of ochratoxin A is achieved.
Patent Information
- Application Number
- CN202510675895.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-23
- Publication Date
- 2025-09-26
AI Technical Summary
Existing biological methods for removing mycotoxins, especially ochratoxin A, have problems with nonspecific adsorption and toxin re-release after adsorbent saturation. There is an urgent need to develop strains with efficient degradation and adsorption functions.
Provided is a strain of Lactobacillus zymae (CGMCC No. 33432) capable of removing mycotoxins, particularly ochratoxin A, through adsorption and cell metabolism.
The system achieves efficient removal of ochratoxin A, with a removal rate of up to 99.5%, while avoiding the re-release of toxins after the adsorbent is saturated.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus serrata and an application thereof. Background Art
[0002] Mycotoxins are a group of highly toxic compounds produced by fungi or yeasts, typically formed in areas where water resources are insufficient and unsuitable for bacterial growth. When animals ingest contaminated food, mycotoxins are metabolized, biotransformed, and transferred into animal products, such as milk or meat, posing a threat to human health. Ochratoxin A (OTA) is a secondary metabolite produced by various Aspergillus and Penicillium species, primarily found in grains and their by-products. OTA can cause various toxicological effects on organisms, including teratogenicity, carcinogenicity, mutagenicity, hepatotoxicity, and especially nephrotoxicity. OTA is difficult to remove; its structure can only be destroyed by heating above 250°C. OTA removal methods primarily include physical, chemical, and biological methods.
[0003] Compared with physical and chemical methods, biological methods exhibit better safety, sensory properties, and cost-effectiveness, while also maintaining its nutritional value. Currently, many bacteria and fungi have been found to be capable of removing OTA. For example, yeast, lactic acid bacteria, and Bacillus can effectively remove OTA through cell adsorption. Some microorganisms have also been found to degrade OTA or convert it into other non-toxic (or less toxic) products through active enzymes (such as carboxypeptidases, amidases, ochratoxinases, amidohydrolases, and lipases). Zhang, X., Yang, H., Apaliya, MT et al., 2018. The mechanisms involved in ochratoxin A elimination by Yarrowia lipolytica Y-2. Ann Appl Biol 173, 164–174. It was disclosed that OTA can be converted to Otα by carboxypeptidase in Yarrowia lipolytica through amide bond hydrolysis. Cho, SM, Jeong, SE, Lee, KR, et al., 2016. Biodegradation of ochratoxinA by Aspergillus tubingensis isolated from Meju. J Microbiol Biotechnol 26(10), 1687–1695. Disclosed is the enzymatic degradation of OTA by Aspergillus tubingensis. The crude enzyme prepared from the Aspergillus tubingensis culture removed more than 90% of OTA at pH 5.0, but the active protein was not purified.
[0004] While biological methods can effectively remove toxins through adsorption, microbial adsorption and removal using their tissue structures is non-specific. Factors such as organic solvents, substrates, and pH significantly influence the formation of complexes, causing the adsorbent to release toxins again. Once the bacteria are saturated with adsorption, further separation and processing are required. Therefore, there is an urgent need to develop strains that possess both efficient degradation and adsorption capabilities to achieve complete mycotoxin removal. Summary of the Invention
[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide a strain of Lactobacillus for promoting the growth of old dough and its application. The Lactobacillus for promoting the growth of old dough can effectively remove mycotoxins, especially ochratoxin A.
[0006] In order to achieve the above object, the present invention provides a first aspect of a Lactobacillus sp. Levilactobacillus zymae ), the preservation number of the old dough growth-promoting lactobacillus is CGMCC No.33432.
[0007] The second aspect of the present invention provides a microbial product, which comprises the aforementioned Lactobacillus sp. and / or, the microbial preparation comprises the inactivated bacteria of the aforementioned Lactobacillus sp. And / or, the microbial preparation comprises the cell lysate of Lactobacillus serrata as described above.
[0008] The third aspect of the present invention provides the use of the aforementioned Lactobacillus sp. or the aforementioned microbial product in removing mycotoxins.
[0009] The fourth aspect of the present invention provides the use of the aforementioned Lactobacillus serrata, or the aforementioned microbial product, or the aforementioned application in mycotoxin detoxification.
[0010] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The old dough growth-promoting lactobacillus provided by the present invention can effectively remove mycotoxins in a substrate through adsorption and cell metabolism.
[0011] Biological Deposits The old dough growth-promoting lactobacillus of the present invention Levilactobacillus zymae , numbered 19-1, and was deposited on January 15, 2025 in the General Microbiology Center of China Culture Collection of Microorganisms (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.33432. DETAILED DESCRIPTION
[0012] 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.
[0013] The first aspect of the present invention provides a strain of Lactobacillus spp. Levilactobacillus zymae ), the preservation number of the old dough growth-promoting lactobacillus is CGMCC No.33432. In the present invention, it is numbered 19-1.
[0014] The inventors of the present invention accidentally isolated a strain of Lactobacillus spp. from fermented sour bamboo shoots in Liuzhou. The strain can effectively remove mycotoxins, especially ochratoxin A.
[0015] According to the present invention, the sequence of 16S rDNA of the Lactobacillus sp. is shown as SEQ ID NO: 1.
[0016] SEQ ID NO: 1:
[0017] The second aspect of the present invention provides a microbial product, which contains the aforementioned Lactobacillus serrata.
[0018] According to the present invention, the microbial preparation comprises the inactivated bacteria of the aforementioned Lactobacillus sp.
[0019] According to the present invention, the microbial preparation comprises the cell lysate of the aforementioned Lactobacillus sp.
[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 sp. 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 comprises ochratoxin and / or a metabolite of ochratoxin.
[0025] More preferably, the ochratoxin is ochratoxin A.
[0026] The old dough growth-promoting Lactobacillus and / or microbial products thereof provided by the present invention have excellent removal effects on ochratoxin A (OTA).
[0027] According to the present invention, preferably, the application comprises: contacting the aforementioned Lactobacillus serrata or the aforementioned microbial preparation with a substrate containing mycotoxins; The substrate is water, feed or soil.
[0028] In the present invention, the dough-growing-promoting Lactobacillus or microbial product can be used in various environments contaminated by mycotoxins.
[0029] In the present invention, the dosage of the dough-growing-promoting lactobacillus or microbial product can be determined according to the degree of mycotoxin contamination of the substrate.
[0030] According to the present invention, preferably, the amount of the old dough promoting lactobacillus is 10 5 -10 10 CFU. 10 5 -1010 CFU means that the content of old dough growth-promoting lactobacillus in the system reaches 10 5 -10 10 The order of magnitude of CFU, that is, 1×10 5 CFU, 5×10 5 CFU, 9.9×10 5 CFU etc. are all 10 5 The order of magnitude of CFU.
[0031] More preferably, the dosage of the old dough growth promoting lactobacillus is 10 6 -10 9 CFU.
[0032] According to the present invention, preferably, the contact conditions include: temperature of 30-38° C., time of 12-50 h, and pH of 5-5.5.
[0033] The fourth aspect of the present invention provides the use of the aforementioned Lactobacillus serrata, or the aforementioned microbial product, or the aforementioned application in mycotoxin detoxification.
[0034] 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.
[0035] Unless otherwise specified, the reagents and materials used in the following examples were purchased from regular chemical reagent suppliers and were of analytical grade.
[0036] In the following examples, ochratoxin A is represented by the abbreviation OTA.
[0037] In the following examples, the OTA content was determined 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 the mixture 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 and evaporated to dryness. 1 mL of mobile phase was then added to the evaporated sample for reconstitution, vortexed for 1 minute, and filtered through a 0.22 μm organic phase needle filter to obtain a sample solution, which was then detected by HPLC.
[0038] 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: 10 μL; detection wavelength: excitation wavelength 333 nm, emission wavelength 460 nm.
[0039] OTA removal rate (%) = (the amount of OTA initially added - the amount of OTA remaining in the sample) / the amount of OTA initially added × 100%.
[0040] The culture medium formula used in the following examples is as follows: MRS liquid medium: 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.
[0041] Preparation Example 1 The glycerol-preserved Lactobacillus 19-1 was inoculated into 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 19-1 in the initial bacterial agent reached 10 7 CFU / mL.
[0042] 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).
[0043] Take 10 mL of the above initial bacterial agent, centrifuge at 10,000 rpm for 5 minutes, and filter the supernatant through a 0.22 μm filter to obtain bacterial agent 2 (cellular metabolite).
[0044] 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).
[0045] 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).
[0046] Example 1 990 μL of the initial bacterial agent prepared in Preparation Example 1 was taken, and 10 μL of OTA solution with a concentration of 100 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 OTA content (removal rate was calculated) and the number of viable bacteria were tested. The results are shown in Table 1.
[0047] Table 1
[0048] Example 2 990 μL of the bacterial agents 1-4 prepared in Preparation Example 1 were taken respectively, and 10 μL of a 100 μg / mL OTA solution was added thereto. After incubation at 37°C for 48 hours, the OTA content was tested and the removal rate was calculated. The results are shown in Table 2.
[0049] Table 2
[0050] As shown in Table 2, the bacteria (agent 1) achieved a 99.5% OTA removal rate, while the inactivated bacterial solution (agent 3) only removed 25.7%. Therefore, the OTA removal efficiency is partially attributed to adsorption by the bacteria, but complete removal is primarily due to degradation.
[0051] The cell lysate (agent 4) achieved a 44.7% OTA removal rate, while the free supernatant (agent 2) achieved a 4.73% OTA removal rate. This result indicates that some of the degradation substances are present outside the cell body, and that the bacterial cells are significantly more capable of removing OTA than the cell lysate, further demonstrating that the OTA removal process involves not only cellular adsorption but also degradation.
[0052] 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 spp. Levilactobacillus zymae ), characterized in that, The preservation number of the old dough growth-promoting lactobacillus is CGMCC No.33432.
2. The old dough growth-promoting lactobacillus according to claim 1, wherein The sequence of 16S rDNA of the Lactobacillus serrata is shown in SEQ ID NO:
1.
3. A microbial product, characterized in that The microbial product comprises the old dough growth-promoting Lactobacillus according to claim 1 or 2; And / or, the microbial preparation comprises the inactivated bacteria of the old dough growth-promoting Lactobacillus according to claim 1 or 2; And / or, the microbial product comprises the cell lysate of the old dough growth-promoting Lactobacillus 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 dough-growing-promoting Lactobacillus according to claim 1 or 2 or the microbial product according to claim 3 or 4 in removing mycotoxins.
6. The use according to claim 5, wherein: The mycotoxins include ochratoxin and / or metabolites of ochratoxin.
7. The use according to claim 5 or 6, wherein: The application comprises: contacting the dough-growing Lactobacillus according to claim 1 or 2 or the microbial preparation according to 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 dosage of the old dough growth promoting lactobacillus is 10 5 -10 10 CFU, preferably 10 6 -10 9 CFU.
9. The use according to claim 7, wherein: The contact conditions include: temperature of 30-38° C., time of 12-50 h, and pH of 5-5.
5.
10. Use of the dough-growing Lactobacillus according to claim 1 or 2, or the microbial product according to claim 3 or 4, or the use according to any one of claims 5 to 9 in mycotoxin detoxification.