CN117987332A - Lactobacillus paracasei and application thereof - Google Patents
Lactobacillus paracasei and application thereof Download PDFInfo
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- CN117987332A CN117987332A CN202410404216.8A CN202410404216A CN117987332A CN 117987332 A CN117987332 A CN 117987332A CN 202410404216 A CN202410404216 A CN 202410404216A CN 117987332 A CN117987332 A CN 117987332A
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Abstract
The invention belongs to the technical field of biological application, and particularly relates to lactobacillus paracasei and application thereof. The lactobacillus paracasei has a preservation number of GDMCC No:63407 and is preserved in the China center for type culture Collection of microorganisms, guangdong province, and the biological material of the lactobacillus paracasei is Lacticaseibacillus paracasei 85:26; the lactobacillus paracasei is in a long rod shape, is gram-positive bacteria and catalase-negative, and the nucleic acid sequence of the 16S rDNA sequencing is shown as SEQ ID NO. 1; the strain is obtained by separating lactobacillus from pickle samples and screening strains with good removal capacity for patulin STC and zearalenone ZEN from the lactobacillus, can degrade both toxins of STC and ZEN simultaneously, has degradation rate higher than 80%, and has the effect of effectively weakening toxicity of STC and ZEN in the field of foods.
Description
Technical Field
The invention belongs to the technical field of biological application, and particularly relates to lactobacillus paracasei and application thereof.
Background
Zearalenone (ZEN) is also known as F-2 toxin, is a non-steroid mycotoxin produced by various Fusarium fungi through polyketides, has a strong estrogen effect, is very common in cereal feeds such as corns, wheat and the like which have mildewed, and is one of main mycotoxins which cause pollution to grains and animal feeds. ZEN causes various toxicities to humans and animals, including reproductive, developmental, cellular and genetic aspects, which can cause chronic poisoning, loss of developmental function, severe impairment of reproductive function, and in severe cases death.
Patulin (Sterigmatocystin, STC) is a polypeptide mycotoxin produced by metabolism of certain fungi, including aspergillus flavus (a. Flavus), aspergillus parasiticus (a. Patulirus), aspergillus nidulans (a. Nidulans), and aspergillus versicolor (a. Versolor), wherein aspergillus versicolor is the predominant species that metabolizes STC. In the biological metabolic process, STC is the penultimate synthetic precursor of AFB 1, which share the same metabolic pathway. The grain crops which can be polluted by STC include barley, wheat, peanut, corn, soybean, coffee beans and the like, and the pollution to forage grass of the peanut, the corn, the wheat and the like is particularly serious; but also has been detected in foods such as cheese, nuts, beer and spices, and finished products thereof. STC can remain in food for long periods of time, causing food safety problems, and then indirectly enter the human body through the food chain, threatening the health of humans and animals, and in some special cases, excessive intake of rice can present health risks for STC contamination. Animal toxicology studies show that STC has genotoxicity and carcinogenicity, and can induce cancers such as liver cancer.
In the prior art, physical, chemical and biological detoxification methods are mainly adopted to reduce toxin toxicity, and three methods are used for degrading ZEN, wherein the research of degrading ZEN by using microorganisms is mostly mixed strains, additives, degrading agents, bacterial agents and the like or other strains; while there is less research on STC, there is less research on degrading STC, and there is currently no single strain that can degrade both ZEN and STC.
Disclosure of Invention
In view of the above problems, an object of the present invention is to provide lactobacillus paracasei and uses thereof.
The technical content of the invention is as follows:
The invention provides lactobacillus paracasei with a preservation number of GDMCC No:63407, a classification name of Lacticaseibacillus paracasei and a biological material name of Lacticaseibacillus paracasei, wherein the strain is preserved in the microorganism strain preservation center of Guangdong province (GDMCC for short, the address is building 5 of No. 59 of Dai 100 in Guangzhou city martyr, and the unit is the microbiological institute of the university of Guangdong province) on the 26 th day of 2023;
the lactobacillus paracasei is in a long rod shape, is gram-positive bacteria and catalase-negative, and the nucleic acid sequence of the 16S rDNA sequencing is shown as SEQ ID NO. 1;
The lactobacillus paracasei is a strain obtained by separating lactobacillus from a kimchi sample and screening for excellent removal ability for patulin (Sterigmatocystin, STC) and Zearalenone (ZEN).
The invention also provides an application of the lactobacillus paracasei for degrading patulin (Sterigmatocystin, STC) and Zearalenone (ZEN).
The invention also provides an application of the lactobacillus paracasei in preparing a mould degradation microbial inoculum, wherein the microbial inoculum contains the lactobacillus paracasei and is used for degrading patulin (Sterigmatocystin, STC) and Zearalenone (ZEN);
the use concentration of lactobacillus paracasei in the microbial inoculum is adjusted according to the concentration of toxin.
The beneficial effects of the invention are as follows:
The lactobacillus paracasei with the preservation number of GDMCC No:63407 is obtained by separating lactobacillus from pickle samples and screening strains with good removal capacity for patulin (Sterigmatocystin, STC) and Zearalenone (ZEN) from the lactobacillus paracasei, can degrade both toxins of STC and ZEN, has degradation rate higher than 80%, has the effect of effectively weakening toxicity of STC and ZEN, and can be applied to the food field.
Drawings
FIG. 1 is a colony morphology of the Lactobacillus paracasei;
FIG. 2 is a microscopic morphology of the Lactobacillus paracasei (400X);
FIG. 3 is a scanning electron microscope image of the Lactobacillus paracasei.
Preservation description
Strain name: lactobacillus paracasei;
latin name: lacticaseibacillus paracasei;
Preservation number: GDMCC No:63407;
Preservation date: 2023, 04, 26;
preservation unit: the Guangdong province microorganism strain collection center is called GDMCC for short, and the address is building 5 of No. 59 of Dai 100 in Guangzhou city martyr.
Detailed Description
The application is described in further detail below with reference to specific embodiments and the accompanying drawings, it being understood that these embodiments are only for the purpose of illustrating the application and not for the purpose of limiting the same, and that various modifications of the application, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present application.
All materials and reagents of the invention are materials and reagents of the conventional market unless specified otherwise.
Example 1
Screening of Lactobacillus paracasei
The method for coating the flat plate after gradient dilution is adopted to separate lactobacillus from a farmyard natural fermentation pickle water sample manufactured by the traditional process: uniformly mixing the pickle water samples, diluting with sterile PBS buffer solution according to a 10-time gradient, sequentially obtaining 10 -4、10-5、10-6 dilutions, respectively sucking 100 mu L of the dilutions, coating the dilutions on an MRS solid culture medium containing 10 g/L calcium carbonate, culturing 48 and h at 37 ℃, picking single colony with a calcium dissolving ring on a new flat plate, and continuously streaking and culturing for 3 generations.
Gram staining: firstly, the single bacterial colonies are picked up and coated on a glass slide to form a thin layer and fixed, bacteria are dyed by crystal violet, the bacteria are usually washed clean slowly by distilled water after lasting for 1 minute, then the bacteria are mordant-dyed by iodine solution, the bacteria are washed clean slowly by distilled water after lasting for 1 minute, the bacteria are decolorized by 95% alcohol, the bacteria are washed clean slowly by distilled water after decolorizing time is about 30 seconds, and finally the bacteria are counterstained by safranine dye (thin) for 1 minute, the bacteria are washed clean slowly by distilled water, dried and microscopic examination: gram-positive bacteria appear bluish purple and gram-negative bacteria appear red.
Catalase assay: picking a colony-inoculating loop on a solid culture medium, placing the colony-inoculating loop in a clean test tube, dropwise adding 2mL of 3% hydrogen peroxide solution, and observing the result: the cells were positive when they were in half a minute, and the cells were negative when they were not in half a minute.
According to the characteristics of lactic acid bacteria, gram-positive bacterial colonies and bacterial colonies which are negative in the catalase tests are selected through gram-positive and bacterial colonies which are round, white in color, smooth in surface, bulges and neat in edge and microscopic in shape (400×) in FIG. 1, and the obtained bacterial strains are in a long rod shape and are positive in gram-positive.
FIG. 3 is a scanning electron microscope image of the selected strains, with smooth cell surface and complete structure, of a short or long rod-shaped bacteria terminal bacteria, arranged in short or long chain.
PCR amplification was performed on the selected colonies, further 16S rDNA sequencing analysis and strain identification:
Single colonies were picked and added to a PCR reaction system (50. Mu.L) for amplification, which included: 2X TAQ PCR MASTER Mix 25. Mu.L, 1. Mu.L of each of the upstream and downstream primers, 23. Mu.L of double distilled water and single colonies dipped as DNA templates. PCR amplification conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 90 s, and cycle number of 35; the extension is 10min at 72 ℃. Selecting a strain with a PCR product having an obvious band through 1% agarose gel electrophoresis, and carrying out sequence determination on the strain by a stock company of biological engineering (Shanghai);
the nucleic acid sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3;
The nucleic acid sequence of 16S rDNA sequencing of the selected colony is shown in SEQ ID NO. 1.
The strain selected was deposited at 26/04/2023 with the collection of microorganisms and cell cultures under the accession number GDMCC No:63407, under the taxonomic designation Lacticaseibacillus paracasei and under the name Lactobacillus paracasei Lacticaseibacillus paracasei.
The strains screened were further tested as follows:
1) Screening for strains with clearance to patulin (Sterigmatocystin, STC)
The single colonies were picked up and inoculated onto MRS solid medium plates with a final concentration of 1. Mu.g/mL STC, and the plates were placed in an incubator and incubated at 37℃for 48 hours.
Starting from 12h, observing the growth condition of the strain on the plate, observing the strain once every 12 hours, marking the strain which does not grow as "-", marking the strain which slowly grows as "++", marking the strain which can rapidly grow as "++", and screening 12 strains marked as "++";
The 12 primary screened strains were inoculated onto MRS solid medium for activation, cultured for 1d at 37 ℃, single colonies were picked up, inoculated into a test tube containing 10mL MRS liquid medium, subjected to stationary culture at 37 ℃ for 18h, inoculated into a conical flask containing 50 mL MRS liquid medium to which 5 μg/mL STC had been added at 37 ℃ and subjected to stationary culture at 48 h. The culture medium without inoculation is set as a blank control.
After the culture medium was sterilized at 121℃for 30min, it was poured into a 50ml centrifuge tube and centrifuged at 10000rpm for 10min. 10ml of the supernatant was pipetted into a 50ml centrifuge tube, 20ml of pure chloroform was added, vortexed for 10min to mix well and centrifuged at 8000rpm for 5min. The lower solution (chloroform layer) was placed in a 50ml centrifuge tube and spin-evaporated to dryness at 40 ℃. Then, re-dissolving with 10ml of acetonitrile/water (95/5, v/v) solution, swirling for 3min to mix uniformly, filtering with a 0.22 mu m filter membrane, collecting in a sample bottle, and carrying out HPLC-MS/MS detection to obtain the concentration of 4.74 mg/L after culture of a control group, wherein the concentration of 0.76 mg/L, 0.81 mg/L and 0.80 mg/L after culture of a treatment group and the average concentration of 0.79 mg/L respectively, and calculating according to a formula to obtain the toxin clearance of 83.94%, 82.83% and 83.22% of STC respectively and the average clearance of 83.3%.
2) Screening for a Strain having a clearing ability against Zearalenone (ZEN)
The isolated and purified lactic acid bacteria were inoculated at a density of 10 9 CFU/mL in MRS broth medium containing 1 mg/L ZEN, respectively, and incubated at 37℃for 48 h. To determine residual ZEN levels, cell suspensions were centrifuged at 10000 rpm at 4 ℃ for 10min, the supernatant was mixed with an equal volume of ethyl acetate, then centrifuged at 8000 rpm for 2 min to collect the ethyl acetate phase, the collected organic solvents were evaporated using a water bath at 40 ℃, the residue was dissolved in a methanol/water (50/50, v/v) mixture, 0.22 μm filter was filtered and collected in a sample bottle for HPLC-MS/MS detection to give a control cultured concentration of 0.94 mg/L, a treatment cultured concentration of 0.14 mg/L, 0.15 mg/L, 0.14 mg/L, an average concentration of 0.14 mg/L, toxin clearance calculated according to the formula to give STC of 85.14%, 84.40%, 85.45% and an average clearance of 85.0% respectively.
When the isolated and purified lactic acid bacteria were inoculated into MRS broth medium containing 5 mg/L ZEN at a density of 10 9 CFU/mL, HPLC-MS/MS detection was performed according to the above procedure, resulting in a concentration of 4.77 mg/L after control culture, a concentration of 1.06 mg/L, 1.11 mg/L, 1.02 mg/L, and an average concentration of 1.06 mg/L, respectively, after treatment culture, and a toxin clearance of 77.74%, 76.69%, 78.67%, respectively, and an average clearance of 77.7% for STC, calculated according to the formula.
The toxin clearance rate calculation formula is as follows: dr= (1-Ct/Cc) ×100%;
Where Dr represents the toxin removal rate and Ct and Cc are the toxin concentrations after incubation in the treatment and control groups, respectively. From the above, lactobacillus paracasei screened by the method has the capability of removing and degrading STC and ZEN, and can achieve the effect of effectively weakening the toxicity of STC and ZEN.
Claims (5)
1. Lactobacillus paracasei, characterized in that it has a deposit number GDMCC No:63407, deposited at the cantonese province microorganism strain collection at 26, 04, 2023, under the biological material name Lacticaseibacillus paracasei.
2. The lactobacillus paracasei according to claim 1, wherein the lactobacillus paracasei is in a long rod shape, is gram positive and catalase negative, and has a 16S rDNA sequenced nucleic acid sequence shown in SEQ ID No. 1.
3. Lactobacillus paracasei according to claim 1, characterized in that the lactobacillus paracasei is a strain isolated from kimchi samples and screened therefrom for good clearance of patulin and zearalenone, respectively.
4. Use of lactobacillus paracasei according to any of claims 1 to 3 for degrading patulin and zearalenone.
5. Use of the lactobacillus paracasei according to any of claims 1 to 3 for the preparation of a mould degrading bacterial agent, wherein the bacterial agent comprises the lactobacillus paracasei according to any of claims 1 to 3 for degrading patulin and zearalenone.
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| CN202410404216.8A CN117987332B (en) | 2024-04-07 | 2024-04-07 | Lactobacillus paracasei and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410404216.8A CN117987332B (en) | 2024-04-07 | 2024-04-07 | Lactobacillus paracasei and application thereof |
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| CN117987332A true CN117987332A (en) | 2024-05-07 |
| CN117987332B CN117987332B (en) | 2024-06-14 |
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