Phytobacterium plantarum WJ28 as well as culture method and application thereof

Abstract

The invention relates to the technical field of microbial engineering, in particular to lactobacillus plantarum WJ28 as well as a culture method and application of the lactobacillus plantarum WJ28. The lactobacillus plantarum WJ28 is preserved in the Guangdong Province Microbial Culture Collection Center, the preservation number is GDMCC No: 65644, the preservation date is December 16, 2024, and the preservation address is the fifth floor, No. 59 floor, No. 100, Xianlie Middle Road, Xianlie District, Guangzhou City. The lactobacillus plantarum WJ28 provided by the invention has acid resistance and acid production capacity and also has the capacity of producing high-yield ketone compounds, and when the lactobacillus plantarum WJ28 is applied to preparation of fermented dairy products, a large amount of 2-heptanone, 2-nonanone and 2-pentanone can be produced, so that the fermented dairy products are endowed with fruity flavor; meanwhile, the lactobacillus plantarum WJ28 can be used for adsorbing the micro-plastic, and can be discharged out of the body along with the eating of the fermented dairy product.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial engineering, and in particular to a Lactobacillus plantarum WJ28 and a culture method and application thereof. Background Art

[0002] In recent years, plastic pollution has sparked global concern for the environment, biodiversity, and public health. In particular, non-recyclable and non-degradable plastic micro- and nanoparticles suspended in water have been found in plants, animals, and even human blood, posing an urgent need for pollution control. Among the pollution caused by traditional plastics, environmental microplastics (plastic particles <5μm) have received widespread attention in recent years due to their high concentrations and significant impact on ecosystems. Recent studies have demonstrated the presence of microplastics in human blood, immune cells, and even the placenta, revealing their direct impact on human health. Currently, microplastics can be detected in the air, soil, rivers, and oceans.

[0003] Animal milk and dairy products are rich in dietary fat, energy, protein, and other nutrients, playing a key role in healthy nutrition and development throughout human life. They are also part of a growing globalized commodity with fixed incomes; world dairy exports have expanded to 75 million tons (measured in milk equivalent), and global milk production was estimated at 843 million tons in 2018. Microplastic contamination can occur at different stages of the dairy supply chain. They can enter the milk during milking on the farm, during downstream processing, or through final packaging. Fluorescence microscopy, SEM-EDS, and Raman spectroscopy have revealed 3 to 11 microplastics / liter (MPs) in the form of fibers and fragments >11 μm in size in whole, skim, light, and lactose-free milk packaged in Mexican polysulfone packaging. FTIR imaging has revealed 10 to 110 MPs / kg in the form of fibers and fragments in milk powder packaged in cartons with inner plastic. Turkish yogurt has been found to contain 20 to 580 MPs / liter in the form of fibers and fragments in Turkish yogurt by SEM and ATR-FTIR. Using Raman imaging, optical microscopy, and SEM-EDX, researchers report that Swiss liquid milk and milk powder contain 2,040 to 10,040 MPs / L, with a size of >5 μm and a fragmented shape. Developing an efficient and safe way to adsorb microplastics smaller than 5 μm from dairy products is an urgent need for food safety.

[0004] During the fermentation of dairy products, lactic acid bacteria produce ketones. Due to their low odor threshold, these compounds contribute significantly to the overall flavor of fermented products. These compounds primarily originate from the Strecker degradation pathway of amino acids. Generally, ketone compounds impart pleasant fruity and mushroom flavors. However, lactic acid bacteria are excellent biosorbents, and some possess exceptional adhesion capabilities, enabling them to adsorb certain harmful substances, such as heavy metal ions, mycotoxins, and plasticizers.

[0005] Therefore, it is crucial to screen a lactic acid bacteria strain that has both efficient fermentation performance and microplastic adsorption ability. Summary of the Invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a plant lactobacillus (Lactiplantibacillus plantarum) WJ28 and its culture method and application. The plant lactobacillus (Lactiplantibacillus plantarum) WJ28 provided by the present invention has acid resistance and acid production ability, and also has the ability to produce high ketone compounds. When it is applied to the preparation of fermented dairy products, it can produce a large amount of 2-heptanone, 2-nonanone and 2-pentanone, thereby giving the fermented milk a fruity flavor; at the same time, the plant lactobacillus (Lactiplantibacillus plantarum) WJ28 can absorb microplastics and can be excreted from the body with the consumption of fermented dairy products.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The first object of the present invention is to provide a plant lactobacillus (Lactiplantibacillus plantarum) WJ28, wherein the plant lactobacillus (Lactiplantibacillus plantarum) WJ28 is preserved in the Guangdong Provincial Microbial Culture Collection Center, with a preservation number of GDMCC No: 65644, a preservation date of December 16, 2024, and a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City.

[0009] In one embodiment of the present invention, the 16S rRNA gene sequence of Lactiplantibacillus plantarum WJ28 is shown as SEQ ID NO.1.

[0010] In the present invention, by solid culture medium plate culture, observing the bacterium colony of plant lactobacillus (Lactiplantibacillus plantarum) WJ28 is circular, raised, neatly edged, milky white in color, opaque, and smooth and moist surface, and picking can be drawn into threads. Gram staining is adopted to carry out Gram staining observation on plant lactobacillus (Lactiplantibacillus plantarum) WJ28, and the result shows that the thalline is non-motile rod-shaped, mostly arranged in chains of varying lengths, and also has a single dispersed arrangement, does not produce spores, and is Gram-positive.

[0011] A second object of the present invention is to provide a culture method of Lactobacillus plantarum WJ28, comprising the following steps:

[0012] (S1) Lactiplantibacillus plantarum WJ28 was inoculated into MRS liquid medium for activation culture;

[0013] (S2) The activated cultured Lactobacillus plantarum WJ28 is inoculated into MRS medium and cultured continuously.

[0014] The third object of the present invention is to provide an application of Lactiplantibacillus plantarum WJ28 in microplastic adsorption.

[0015] In one embodiment of the present invention, Lactiplantibacillus plantarum WJ28 adsorbs microplastics under acidic conditions.

[0016] In one embodiment of the present invention, during the microplastic adsorption process, the pH is 2.5 to 6.5;

[0017] Preferably, the microplastic is 5 μm PET microplastic.

[0018] The fourth object of the present invention is to provide an application of Lactobacillus plantarum WJ28 in the field of microbial acid production.

[0019] In one embodiment of the present invention, the field of microbial acid production includes fermented food production (preferably, fermented dairy product production), sour agent production, organic acid production or microbial preparation production.

[0020] A fifth object of the present invention is to provide an application of Lactiplantibacillus plantarum WJ28 in the field of microbial ketone production.

[0021] In one embodiment of the present invention, the field of microbial ketone production includes ketone flavor production, ketone food additive production, wastewater treatment or pharmaceutical intermediate production.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The content of ketone compounds in the milk fermented by Lactiplantibacillus plantarum WJ28 provided by the present invention is relatively high, and is higher than that of milk fermented by commercial starter cultures. It has a good adsorption effect on microplastics PET. Compared with traditional methods, it not only has the advantages of green and efficient biological adsorption, but also, due to its own characteristics, it is a beneficial bacteria, has certain advantages in the fields of food and so on, and has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the adsorption rates of different Lactobacillus plantarum strains on microplastics PET in Example 2;

[0025] Figure 2 This is a graph showing the acid production capacity of Lactiplantibacillus plantarum WJ28 in Example 3;

[0026] Figure 3 This is a graph showing the measurement results of the adsorption rates of different plant lactobacilli on microplastics PET under acidic conditions in Example 6. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0029] Example 1

[0030] This embodiment provides the isolation and purification, morphological identification, molecular identification and culture method of Lactobacillus plantarum, which are as follows:

[0031] (1) Isolation and purification of strains:

[0032] (A1) Lettuce, kimchi, sauerkraut and other samples were diluted with sterile saline to 10 -6Each dilution gradient was applied to an MRS plate in sequence and incubated at 37°C for 72 h. After the incubation, colonies with different morphologies were selected using an inoculation needle and streaked onto the MRS plate until single colonies of uniform size and morphology appeared.

[0033] (A2) After step (A1), the selected single colonies were Gram-stained, and strains that were Gram-purple, catalase-negative, and non-spore-forming were selected and tentatively identified as lactic acid bacteria (the isolated strains were labeled A1, A2, A3, A4, and WJ28, respectively);

[0034] (A3) After step (A2), the isolated strain tentatively identified as lactic acid bacteria is activated for three generations in MRS liquid culture medium and then subjected to physiological and biochemical identification and molecular biological identification.

[0035] (2) Morphological identification of strains

[0036] The strain isolated in step (1) was streaked on an MRS plate and cultured anaerobically at 37°C for 48 hours. The strain grew well and had round, raised colonies with neat edges, milky white, opaque color, and a moist and smooth surface that could be drawn into threads.

[0037] Gram staining of strain WJ28 revealed that the bacteria were non-motile rods, mostly arranged in chains of varying lengths, and also individually dispersed. They did not produce spores and were Gram-positive.

[0038] (3) Molecular identification

[0039] The 16S rRNA gene of the isolated strain WJ28 was cloned and sequenced, and the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID No. 1. The 16S rRNA gene sequence of the strain was compared with the sequence of Lactobacillus plantarum from NCBI, and it was found that the 16S rRNA gene sequence of the strain was 99% similar to that of Lactiplantibacillus plantarum.

[0040] Furthermore, the strain was deposited in the Guangdong Provincial Microbial Culture Collection Center, and its taxonomic name was Lactiplantibacillus plantarum, specifically Lactiplantibacillus plantarum WJ28; its preservation number was GDMCC No: 65644, the preservation date was December 16, 2024, and the preservation address was 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou.

[0041] Among them, SEQ ID No.1 is as follows:

[0042]

[0043] (4) Culture method of strains

[0044] (S1) Lactiplantibacillus plantarum WJ28 was inoculated into MRS liquid medium and cultured at 37°C for 12 hours;

[0045] (S2) The activated Lactobacillus plantarum WJ28 was inoculated into MRS medium and cultured at 37° C. for 12 hours.

[0046] Example 2

[0047] This example provides a measurement of the adsorption capacity of Lactiplantibacillus plantarum WJ28 on 5 μm microplastic PET, as follows:

[0048] The five strains isolated in Example 1 (A1, A2, A3, A4, and WJ28) were activated in a centrifuge tube containing 2 mL of MRS medium (37°C, 12 h) for three generations. 1 mL of the cultured bacterial solution was centrifuged, the supernatant was removed, and the resulting bacterial cells were washed once with physiological saline and resuspended in physiological saline. The cell concentration was adjusted to 5 × 10 8 CFU / mL, and place at 4°C for later use. Take 1mL of bacterial suspension and centrifuge for 10 minutes (4°C, 4000rpm), remove the supernatant, add 1mL of PET microplastic solution with a particle size of 5μm (30μg / mL) and mix well, and adsorb at 37°C for 4h. After the adsorption is completed, centrifuge again for 10 minutes (4°C, 6000rpm), collect the supernatant, and calculate its adsorption rate using the direct counting method under a microscope. The blank control is a microplastic solution without the addition of strains, and each group is repeated 3 times. The adsorption rate of the strain on microplastics is calculated according to the following formula:

[0049] Adsorption rate (%) = (number of microplastics in blank - number of microplastics in sample) / number of microplastics in blank × 100%

[0050] The results are as follows Figure 1 As shown, through Figure 1 It can be found that all five strains have the ability to adsorb microplastics PET, among which Lactiplantibacillus plantarum WJ28 has the best adsorption effect of 88.76%.

[0051] Example 3

[0052] This example provides a determination of the acid production capacity of Lactiplantibacillus plantarum WJ28, as follows:

[0053] After activation, commercial starter cultures and Lactiplantibacillus plantarum WJ28 were inoculated into sealed containers containing MRS liquid culture medium and cultured at 37°C. 15 mL of culture medium was taken out every 2 hours and the pH value was measured with a precision acidometer for a total of 24 hours. With time as the horizontal axis and the pH value of the fermentation culture medium as the vertical axis, a dynamic change curve of the pH value of the acid production of the strain was obtained (see Figure 2). Figure 2 shown).

[0054] pass Figure 2 It can be found that the pH of Lactiplantibacillus plantarum WJ28 can reach 3.99 after 24 hours of fermentation, and its acid production capacity is higher than that of commercial starter.

[0055] Example 4

[0056] This example provides an acid resistance test of Lactiplantibacillus plantarum WJ28 (using MRS medium with a pH of 2.5 to verify the acid resistance of Lactiplantibacillus plantarum WJ28), as follows:

[0057] Take 1 mL of bacterial suspension and centrifuge at 4000 rpm for 10 minutes. Discard the supernatant and wash once with 1 mL of PBS. After centrifugation at 4000 rpm for 10 minutes, resuspend the pellet to 1 mL in MRS medium (pH 2.5). Incubate at 37°C for 3 hours. Samples are taken at 0 and 3 hours. After centrifugation, the samples are resuspended in PBS and serially diluted. The diluted samples are spread on MRS agar plates and incubated anaerobically at 37°C for 16 hours before colony count. Survival rate is calculated as follows: Acid-resistant survival rate (%) = Number of colonies at 3 hours / Number of colonies at 0 hours × 100%.

[0058] The results are shown in Table 1. It can be found from Table 1 that the survival rate of Lactiplantibacillus plantarum WJ28 after being cultured in MRS medium with a pH of 2.5 for 3 hours was 89.78±2.53%, indicating that it has strong acid resistance.

[0059] Table 1 Determination of acid resistance of Lactobacillus plantarum WJ28

[0060]

[0061] Example 5

[0062] This example provides a determination of the ability of Lactiplantibacillus plantarum WJ28 to produce ketone compounds, as follows:

[0063] Lactiplantibacillus plantarum WJ28 was cultured in MRS liquid medium at 37°C for 12 hours and then subjected to secondary activation at a 2% (v / v) inoculum size. The activated bacterial solution was centrifuged at 6000 rpm for 10 minutes at 4°C to obtain bacterial cells. The bacterial cells were rinsed three times with physiological saline, and then 1 mL of physiological saline was added to mix the cells for later use.

[0064] A 12% by mass reconstituted milk was prepared using skim milk powder, which was heat-pasteurized at 85°C for 15 minutes or 95°C for 5 minutes. The bacterial suspension was added at a 4% (v / v) rate, and a commercial starter culture, JOINTEC VB540, was added to the sterilized reconstituted milk at a 4% (v / v) rate. The mixture was mixed and fermented at 37°C until the pH reached 4.6. The mixture was then cooled to room temperature and stored under a refrigeration at 4°C.

[0065] 5.00 g of the fermented milk after refrigeration for 12 hours was weighed and placed in a 20 mL headspace solid phase microextraction vial. The headspace vial was placed in a constant temperature water bath at 55°C, and an aged extraction head (50 / 30 μm DVB / CAR / PDMS) was inserted. The headspace adsorption was completed for 40 minutes. After the extraction was completed, the extraction head was placed in GC-MS analysis for 5 minutes to complete the injection.

[0066] GC-MS program conditions were as follows: HP-INNOWAX column (60 m × 0.25 mm × 0.25 mm); inlet temperature: 230°C; temperature program: 40°C for 3 min, then 120°C at 4°C / min, and finally 230°C at 5°C / min, held for 10 min; carrier gas: He; flow rate: 1 mL / min; injection mode: splitless. Mass spectrometry conditions: ion source temperature: 230°C; ion source: EI; electron energy: 70 eV; scan range: 30–300 amu; full ion scan mode. Quantitative analysis was performed using an external standard method. The experimental results are shown in Table 2. Table 2 shows that the ketone compound content in milk fermented with Lactobacillus plantarum WJ28 is relatively high, and higher than that in milk fermented with a commercial starter culture.

[0067] Table 2 Determination of aroma production ability of Lactobacillus plantarum WJ28

[0068]

[0069] Example 6

[0070] This example provides a determination of the microplastic adsorption capacity of Lactiplantibacillus plantarum WJ28 under acidic conditions, as follows:

[0071] Take 1 mL of bacterial solution (respectively, the five strains isolated in Example 1 (A1, A2, A3, A4, and WJ28)), centrifuge at 4000 rpm for 10 minutes, discard the supernatant, and then add 1 mL of PBS to wash once. After centrifugation at 4000 rpm for 10 minutes, the precipitate is resuspended to 1 mL in MRS medium with a pH of 2.5. Add 5 μm PET microplastics (final concentration of 30 μg / mL) and incubate at 37°C for 3 hours. After that, the adsorption rate is measured according to Example 2.

[0072] The results are as follows Figure 3 As shown, the five strains all have the ability to adsorb microplastics PET under acidic conditions. Among them, Lactobacillus plantarum WJ28 has the best adsorption effect, with an adsorption rate of 80.87%, indicating that Lactobacillus plantarum WJ28 can also have good adsorption capacity in an acidic environment.

[0073] Comparative Example 1

[0074] This comparative example provides the microplastic adsorption capacity of Lactobacillus plantarum DT55 of patent CN117089486A. Specifically, the adsorption rate of Lactobacillus plantarum DT55 on 0.1 μm PS microplastics is 83.90%.

[0075] Compared with Example 2, it is demonstrated that the Lactiplantibacillus plantarum WJ28 of the present invention exhibits a better adsorption rate for 5 μm PET microplastics.

[0076] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A Lactobacillus plantarum WJ28, characterized in that The Lactobacillus plantarum WJ28 is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No: 65644, a deposit date of December 16, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou.

2. A plant lactobacillus (Lactiplantibacillus plantarum) WJ28 according to claim 1, characterized in that, The 16S rRNA gene sequence of the Lactiplantibacillus plantarum WJ28 is shown in SEQ ID NO.

1.

3. A method for culturing Lactobacillus plantarum WJ28 according to any one of claims 1 to 2, characterized in that: The following steps are involved: (S1) Lactiplantibacillus plantarum WJ28 was inoculated into MRS liquid medium for activation culture; (S2) The activated cultured Lactobacillus plantarum WJ28 is inoculated into MRS medium and cultured continuously.

4. A use of Lactiplantibacillus plantarum WJ28 as described in any one of claims 1 to 2 in microplastic adsorption.

5. The use according to claim 4, characterized in that Lactiplantibacillus plantarum WJ28 adsorbs microplastics under acidic conditions.

6. The use according to claim 4, characterized in that During the microplastic adsorption process, the pH is 2.5 to 6.

5.

7. Use of Lactiplantibacillus plantarum WJ28 according to any one of claims 1 to 2 in the field of microbial acid production.

8. The use according to claim 7, characterized in that The field of microbial acid production includes fermented food production, acidulant production, organic acid production or microbial preparation production.

9. Use of Lactiplantibacillus plantarum WJ28 according to any one of claims 1 to 2 in the field of microbial ketone production.

10. The use according to claim 9, characterized in that The field of microbial ketone production includes the production of ketone flavoring agents, the production of ketone food additives, wastewater treatment or the production of pharmaceutical intermediates.

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