Lipomyces lipoferus and application thereof in preparation of nano-selenium

Abstract

The application discloses a Leuconostoc citreum and application of the Leuconostoc citreum in preparation of nano selenium, and relates to the technical field of selenium-rich bacterial strain screening and cultivation. The Leuconostoc citreum provided by the application is preserved in the China Center for Type Culture Collection on September 29, 2024, and the preservation number is CCTCC NO: M 20242123, and the preservation address is Luojia Mountain, Baying Road, Wuchang District, Wuhan City, Hubei Province. The Leuconostoc citreum has good tolerance to selenite and the ability to reduce selenite to generate nano elemental selenium; the strain also has good antibacterial effect, especially on gram-negative bacteria; and the strain has good acid and bile salt tolerance, and also has a high survival rate in the gastrointestinal tract.

Description

Technical Field

[0001] The present invention relates to the technical field of screening and cultivating selenium-rich bacteria, and in particular to Leuconostoc citriodora and application thereof in preparing nano-selenium. Background Art

[0002] In nature, selenium exists primarily in two forms: organic and inorganic. Inorganic selenium has a low bioavailability, a narrow safe dose range, and potential environmental risks. Organic selenium, on the other hand, has high bioavailability, a wide relative safe dose range, and good palatability. Compared to inorganic and organic selenium, red nano-selenium exhibits low toxicity, high bioactivity, and possesses the optoelectronic properties of nanomaterials, as well as multiple antimicrobial activities. It has widespread applications in animal production, pharmaceuticals and healthcare products, biosensor production, solar cells, rectifiers, and electrostatic copying.

[0003] Microorganisms play a vital role in the natural cycle of selenium. Many bacteria, such as Escherichia coli, Bacillus subtilis, Rhodospirillum Molisch, Bacillus licheniformis, and Pseudomonas fluorescens, have been found to reduce oxidized selenium into non-toxic, highly effective red nanoselenium. The synthesized bio-nanoselenium is stable and highly active. Therefore, using bacteria to synthesize bio-nanoselenium is a highly efficient method for selenium biotransformation. Summary of the Invention

[0004] The present invention provides a strain of Leuconostoc citrovida and its use in the preparation of nano-selenium. By extracting and screening the strain from kimchi water, a strain of Leuconostoc citrovida with high selenium tolerance was obtained. This is achieved specifically through the following techniques.

[0005] The first aspect of the present invention provides a Leuconostoc citreum, which was deposited in the China Center for Type Culture Collection on September 29, 2024, with the deposit number: CCTCCNO: M 20242123, and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The Leuconostoc citreum provided by the present invention has a high selenite reducing ability, produces red nano-elemental selenium, and has good antibacterial and antimicrobial abilities, as well as acid and bile salt resistance, and can survive and grow normally in the gastrointestinal tract.

[0007] The second aspect of the present invention provides an application of the above-mentioned Leuconostoc citriodora, which is characterized in that it is used to prepare nano-selenium, or to prepare animal feed additives, or to prepare selenium-rich fermented products, or to prepare bacterial agents for microbial conversion and synthesis of nano-selenium, or to prepare antibacterial agents.

[0008] The third aspect of the present invention provides a method for preparing nano-selenium, which utilizes the above-mentioned Leuconostoc citracophilus to reduce selenite to generate nano-selenium.

[0009] Furthermore, the activated Leuconostoc citriodora is inoculated into a fermentation medium containing selenite, and fermented and cultured to synthesize nano-selenium.

[0010] Furthermore, the composite system obtained after fermentation and culture is sequentially subjected to centrifugation to obtain precipitation, resuspended, cell broken, and centrifuged to obtain the precipitation, namely nano-selenium.

[0011] Furthermore, the method for preparing nano-selenium according to claim 4 is characterized in that the inoculation amount of the Leuconostoc citricola is 1-2%.

[0012] Furthermore, the fermentation culture temperature is 20-30°C.

[0013] Preferably, the fermentation temperature can be selected to be around 30°C.

[0014] Furthermore, the pH value of the fermentation culture system is not lower than 2.0.

[0015] Preferably, the pH value of the fermentation culture system can be selected to be around 6.0-7.0. Furthermore, the concentration of sodium selenite in the fermentation culture medium is 5-100 mmol / L.

[0016] Compared with the existing technology, the advantages of the present invention are: the Leuconostoc citreum provided by the present invention has good selenite resistance and the ability to reduce selenite to produce nano-elemental selenium; the strain also has good antibacterial and antimicrobial effects, especially against Gram-negative bacteria; it has good acid and bile salt resistance and a high survival rate in the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The growth of strain PC-7 in culture medium containing different concentrations of sodium selenite.

[0018] Figure 2 This is the phylogenetic tree of strain PC-7 based on the 16S rDNA gene sequence.

[0019] Figure 3This is the growth curve of strain PC-7 at different sodium selenite concentrations.

[0020] Figure 4 is the sodium selenite reduction rate of strain PC-7 within 72 h.

[0021] Figure 5 The particle size and zeta potential of strain PC-7 at different times under 10 mmol / L (A) and 25 mmol / L (B) sodium selenite concentrations.

[0022] Figure 6 SEM image of nano-selenium extracted by adding 10 mmol / L sodium selenite to strain PC-7 for 48 hours (A) and characteristic peaks of SeNPs analyzed by EDX spectroscopy (B).

[0023] Figure 7 EDX element distribution diagram of nano-selenium extracted by adding 10 mmol / L sodium selenite to strain PC-7 for 48 hours.

[0024] Figure 8 The results show the acid resistance (A) and bile resistance (B) of strain PC-7.

[0025] Figure 9 The results of the tolerance test of strain PC-7 in simulated gastrointestinal fluid are shown. DETAILED DESCRIPTION

[0026] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Isolation, selenium tolerance determination and molecular biological identification of kimchi-derived bacterial strains

[0028] 1. Isolation of kimchi source strains

[0029] Using a disposable syringe, dilute the original kimchi solution of homemade radish kimchi in a gradient dilution process. Apply 100 μL of each gradient dilution onto MRS solid medium. After 24 hours, observe the colony morphology and structure, select individual colonies, number them, and streak the isolated and purified bacteria onto new plates. Repeat the streak purification process twice, and select individual colonies for subsequent experiments.

[0030] 2. Determination of selenium tolerance of strains

[0031] Several isolated strains were inoculated into MRS liquid medium and cultured at 30°C, 180 rpm, until the logarithmic phase. Then, 200 μL of the bacterial solution was added (1% inoculum size) to a screw-capped glass vial containing 20 mL of MRS liquid medium containing varying concentrations of sodium selenite (0, 5, 25, 50, and 100 mmol / L). The vial was cultured at 30°C, 180 rpm, for 3 days, and the color of the liquid was observed.

[0032] In this example, six strains with selenium tolerance exceeding 5 mmol / L were screened from kimchi water. The six strains were numbered PC-2, PC-3, PC-5, PC-6, PC-7, and PC-9.

[0033] from Figure 1 As the sodium selenite concentration increased, the fermentation broth of strain PC-7 became increasingly red, indicating a gradual increase in elemental nano-selenium. Even at a sodium selenite concentration of 100 mmol / L, the fermentation broth remained red, demonstrating that the strain was capable of growing in the presence of higher concentrations of sodium selenite and reducing it to red nano-selenium. This indicates that strain PC-7 has a selenium tolerance of 100 mmol / L, making it a highly selenium-tolerant lactic acid bacteria strain. Therefore, strain PC-7 was selected for further research.

[0034] 3. Molecular biological identification of strains

[0035] The single colony PCR method was used: the strain PC-7 was inoculated into MRS solid culture medium and cultured in a 30°C constant temperature incubator for 36 hours; a single colony was picked and placed in 10 μL sterile water, and 1 μL was taken from it as a DNA template for PCR amplification.

[0036] Upstream primer 27F: 5′-agagtttgatcctggctcag-3′, as shown in SEQ ID NO.1.

[0037] Downstream primer 1492R: 5′-tacgacttaaccccaatcgc-3′, as shown in SEQ ID NO.2.

[0038] 16S rDNA amplification system: 0.5 μL of upstream and downstream primers, 1 μL of DNA template, 10.0 μL of M5 Taq HiFi PCR Mix (2×), and 8.0 μL of sterile water.

[0039] The PCR reaction conditions were as follows: 95°C for 3 min; 94°C for 25 s, 55°C for 25 s, and 72°C for 2 min, for a total of 30 cycles; 72°C for 5 min; and storage at 4°C.

[0040] The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. After the DNA sequencing results were assembled and compared with the NBCI gene library, it was found that the sequence had the highest similarity with that of Leuconostoc citriodora.

[0041] MEGA-X software was used for multiple comparisons and the phylogenetic tree based on 16S rDNA sequences was constructed using the maximum natural method. Figure 2 As shown, the results showed that the closest homology to strain PC-7 was Leuconostoc citreum, allowing for preliminary identification of strain PC-7 as Leuconostoc citreum. This strain was deposited with the China Center for Type Culture Collection on September 29, 2024, under the deposit number CCTCC NO: M 20242123. The address is Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposited strain is named Leuconostoc citreum PC-7.

[0042] 4. Growth curve determination of strain PC-7

[0043] The strain PC-7 was inoculated into MRS solid medium and activated overnight. The next day, a single colony was picked and cultured in MRS liquid medium until the stationary phase. The bacterial solution was then inoculated into 100 mL of MRS liquid medium containing different concentrations (5, 10, and 25 mmol / L) of sodium selenite at an inoculum volume fraction of 1%, and MRS medium without selenium was used as a blank control. The optical density (OD) of the bacterial solution at 600 nm was measured every 3 h. 600 ), 3 parallels per group, with OD 600 The value is the vertical axis and the culture time is the horizontal axis (h), and the growth curve is drawn.

[0044] like Figure 3 As shown in the figure, it can be seen that the concentrations of 5, 10, and 25 mmol / L sodium selenite prolonged the time for strain PC-7 to reach the logarithmic growth phase. The higher the concentration, the longer it took for strain PC-7 to reach the logarithmic growth phase, but the maximum OD 600 Higher than that of the blank control (without sodium selenite).

[0045] Example 2: Determination of Sodium Selenite Reduction Rate of Strain PC-7

[0046] The concentration of sodium selenite was determined by the ascorbic acid reduction method, and a Na2SeO3 standard curve was prepared. The strain was inoculated into 100 mL of MRS liquid culture medium containing 5, 10, and 25 mmol / L sodium selenite and cultured in a shaker at 30°C and 180 r / min for 3 days; samples were taken every 12 hours, centrifuged at 12000 r / min for 15 minutes, and 0.8 mL of the supernatant was taken; 0.4 mL of 4 mol / L hydrochloric acid and 0.8 mL of 1 mol / L ascorbic acid were added to the supernatant, shaken to mix, and allowed to stand at room temperature for 10 minutes. The optical density (OD) of the reaction solution at 500 nm was measured. 500 ). The OD measured above 500 Substitute the value into the standard curve formula to calculate the concentration of sodium selenite, and then substitute it into the following formula to obtain the sodium selenite reduction rate.

[0047]

[0048] like Figure 4 As shown, under the conditions of 5, 10, and 25 mmol / L sodium selenite, the conversion rates of strain PC-7 were 52.86%, 56.27%, and 75.88% within 12 h, respectively.

[0049] Example 3: Growth of strain PC-7 in culture medium containing different concentrations of sodium selenite

[0050] The isolated strain PC-7 was inoculated into MRS liquid culture medium and cultured at 30°C, 180 rpm, until the logarithmic phase. Then, 200 μL of the bacterial solution was added to 20 mL (i.e., 1% inoculum) of MRS liquid culture medium containing different concentrations of sodium selenite (20, 40, 80, 160, 320 μg / mL, and 5 mmol / L). The culture was then incubated at 30°C, 180 rpm, for 3 days, and the color change of the liquid was observed. The appearance of red in the system was observed visually to explore the concentration conditions at which the bacteria began to synthesize nano-selenium.

[0051] The results are shown in Table 1 below. At a sodium selenite concentration of 320 μg / mL (approximately 1.85 mmol / L), the fermentation broth of strain PC-7 turned reddish. Under low concentrations of inorganic selenium, the bacteria converted inorganic selenium into non-nanoselenium forms (literature reports primarily used organic selenium). Only at high concentrations of inorganic selenium did the bacteria begin to convert inorganic selenium into nanoselenium (5 mmol / L, approximately 863 μg / mL) and above.

[0052] This is also the selenium conversion characteristic of PC-7. When cultured with low concentrations of sodium selenite (20-160 μg / mL), strain PC-7 primarily produces organic selenium, while at higher concentrations (5 mmol / L), it produces nano-selenium. Different concentrations of added sodium selenite result in different biotransformation products.

[0053] Table 1 Growth of PC-7 strain in culture medium with different concentrations of sodium selenite for 36 h

[0054]

[0055] Example 4: Nanoselenium Particle Size and Zeta Potential Measurement and Scanning Electron Microscopy

[0056] 1. Determination of nano-selenium particle size and zeta potential

[0057] The strain was inoculated into MRS solid culture medium for overnight activation, and a single colony was picked the next day and cultured in MRS liquid culture medium until the stable period; the seed liquid was inoculated into 240 mL of MRS liquid culture medium with a concentration of 10 mmol / L sodium selenite at a 1% inoculation rate, and cultured in a shaker at 30°C and 180 r / min; 60 mL of bacterial liquid sample was taken out every 12 hours, the bacterial liquid was centrifuged at 12000 r / min for 10 minutes, the supernatant was discarded, the precipitate was washed with sterile water and resuspended, and the cells were disrupted by ultra-high pressure and low temperature for 4 times; the disrupted liquid was centrifuged at 12000 r / min for 10 minutes, and the precipitate was resuspended; 800 μL of sample was taken and added to a quartz dish, the instrument parameters were adjusted, the measurement temperature was set to 25°C, the refractive index was set to 1.33, and the viscosity was set to 0.8936, the quartz dish containing the sample was placed in the instrument, the particle size and potential of the nano-selenium particles were measured, and the data was recorded.

[0058] like Figure 5 As shown in the figure, under 10 mmol / L sodium selenite conditions, the particle size and potential of strain PC-7 after 12 hours of fermentation were 182.86 nm and -42.81, respectively, and after 48 hours of fermentation, the nano-selenium particle size and potential were 336.21 nm and -32.67, respectively. Under 25 mmol / L sodium selenite conditions, the nano-selenium particle size and potential of strain PC-7 after 12 hours of fermentation were 222.58 nm and -29.23, respectively, and after 48 hours of fermentation, the nano-selenium particle size and potential were 203.36 nm and -45.53, respectively.

[0059] 2. Scanning electron microscope image

[0060] The seed liquid was inoculated into three bottles of MRS liquid culture medium containing 100mL 10mmol / L sodium selenite at a 1% inoculation rate, and cultured in a shaker at 30℃ and 180r / min for 48h; the bacterial liquid was centrifuged at 12000r / min for 10min, the supernatant was discarded, and the precipitate was washed with sterile water and resuspended; the cells were crushed by ultra-high pressure and low temperature for 4 times, and the crushed liquid was centrifuged at 12000r / min for 10min; the precipitate was resuspended and centrifuged at 12000r / min for 10min. The precipitate was resuspended and the particle size and potential were measured. The sample was centrifuged and then freeze-dried and its morphological characteristics were observed using a scanning electron microscope. The scanning electron microscope results are as follows: Figure 6、 7 As shown in Table 2 below, strain PC-7 produced nanoselenium particles with distinct particle sizes, with an average particle size of 150 nm. Based on the characteristic Se peaks in the EDX spectrum and elemental composition analysis, the primary component was selenium, along with certain amounts of C, N, O, and S. This suggests that the surface of the nanoselenium particles is likely coated with substances such as proteins and polysaccharides.

[0061] Table 2

[0062]

[0063] Example 5: Determination of acid and bile resistance of strain PC-7

[0064] At a 1% inoculum, the bacterial solution was inoculated into MRS acid-resistant liquid culture medium at low pH = 2.0, 3.0, and 4.0, and MRS bile salt (0.1, 0.2, and 0.3%) liquid culture medium, respectively. After incubation at 30°C and 90 rpm for 4 hours, the viable bacteria were counted after dilution and plating. The calculation formula is as follows:

[0065]

[0066] Among them, N T N represents the number of viable bacteria at 4 h of culture, and N0 represents the initial number of viable bacteria during culture.

[0067] like Figure 8 As shown, when the bile salt concentration was 0.1%, 0.2% and 0.3%, the bile salt tolerance of strain PC-7 was 91%, 87.74% and 81.59% respectively after culturing for 4 hours; when the pH value was 2, 3 and 4, the acid resistance was 79.13%, 85.24% and 88.04% respectively after culturing for 4 hours.

[0068] Example 6: Tolerance of strain PC-7 to simulated gastrointestinal fluid

[0069] Pepsin was dissolved in phosphate buffered saline (PBS) at pH 3.0 to prepare 3 g / L of simulated gastric fluid. Trypsin was dissolved in PBS at pH 8.0 to prepare 1 g / L of simulated intestinal fluid, and the mixture was sterilized by filtration using a 0.22 μm filter membrane.

[0070] Culture the experimental strain PC-7 for 18 hours. Mix 1 mL of the bacterial suspension with 9.0 mL of artificial gastric fluid, incubate at 30°C at 90 rpm for 3 hours, then dilute and apply the solution for viable counts. Mix 1 mL of the bacterial suspension with 9.0 mL of artificial intestinal fluid, incubate at 30°C at 90 rpm for 4 hours, then dilute and apply the solution for viable counts. The calculation formula is as follows:

[0071]

[0072] Among them, N T N represents the number of viable bacteria at 4 h of culture, and N0 represents the initial number of viable bacteria during culture.

[0073] like Figure 9 As shown, the survival rates of strain PC-7 after 3 hours of culture in artificial gastric fluid without sodium selenite and in artificial gastric fluid containing 10 mmol / L sodium selenite were 47.42% and 72.59%, respectively. The survival rates after 3 hours of culture in artificial intestinal fluid without sodium selenite and in artificial intestinal fluid containing 10 mmol / L sodium selenite were 65.11% and 87.78%, respectively. This indicates that the addition of selenium significantly enhances the survival rate of strain PC-7 in both simulated gastric and intestinal fluids.

[0074] Example 7: Antibacterial effect test of strains

[0075] Pick a single colony of PC-7 and culture it in MRS liquid medium, shake it at 30℃ and 180r / min overnight. Dilute the seed solution to OD 600 =0.7±0.02, and inoculated with 1% inoculum into conventional MRS liquid medium and MRS liquid medium containing 10 mmol / L sodium selenite, respectively, and cultured at 30°C and 180 rpm for 24 h to obtain conventional PC-7 fermentation broth and selenium-enriched PC-7 fermentation broth.

[0076] Incubate the pathogenic indicator bacteria (Escherichia coli, Staphylococcus aureus and Salmonella) on a plate at 37°C for 12-24 hours. Pick the indicator bacteria colonies and place them in PBS solution until the OD 600 =0.5±0.02.

[0077] Use the double-layer plate method to determine the antibacterial activity of the strain. Pour 15 mL of LB medium into a Petri dish and allow it to solidify as the lower layer. Place four Oxford cups in the dish. Add 5 mL of medium containing a 1% suspension of different indicator bacteria to the solidified lower layer as the upper layer. Once the upper layer solidifies, remove the Oxford cups and set aside.

[0078] Add 200 μL of PBS solution to one of the wells of the Oxford cup, and add 200 μL of regular PC-7 fermentation liquid or selenium-enriched PC-7 fermentation liquid to the other wells of the Oxford cup. Incubate in a 30°C incubator for 24 h and observe whether there is an inhibition zone.

[0079] The results are shown in Table 2. It can be seen that the PC-7 strain has a significant antibacterial effect on the three pathogens, and the antibacterial effect on Gram-negative bacteria is more obvious, and the antibacterial effect of PC-7-Se on the three pathogens is enhanced.

[0080] Table 2 Antibacterial performance of strain PC-7 against different foodborne pathogens

[0081]

[0082] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A citraconicum Leuconostoc ( Leuconostoc citreum ), characterized in that, The Leuconostoc citriodora was deposited in the China Center for Type Culture Collection on September 29, 2024, with the collection number: CCTCC NO: M20242123, and the collection address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A use of Leuconostoc citracophilus according to claim 1, characterized in that: It can be used to prepare nano-selenium, or to prepare selenium-enriched fermentation products, or to prepare bacterial agents for microbial conversion and synthesis of nano-selenium, or to prepare antibacterial agents for inhibiting Escherichia coli, Staphylococcus aureus and Salmonella.

3. A method for preparing nano-selenium, characterized in that: The Leuconostoc citricola described in claim 1 is used to reduce selenite to generate nano-selenium.

4. The method for preparing nano-selenium according to claim 3, wherein The activated Leuconostoc citricola is inoculated into a fermentation medium containing selenite, and the nano-selenium is synthesized by fermentation and culture.

5. The method for preparing nano-selenium according to claim 4, wherein: The composite system obtained after fermentation and culture is sequentially subjected to centrifugation to obtain precipitation, resuspended, cell broken, and centrifuged to obtain the precipitation, namely nano-selenium.

6. The method for preparing nano-selenium according to claim 4, wherein: The inoculation amount of the Leuconostoc citricola is 1-2%.

7. The method for preparing nano-selenium according to claim 4, characterized in that: The fermentation temperature is 20-30°C.

8. The method for preparing nano-selenium according to claim 4, wherein: The pH value of the fermentation culture system is not lower than 2.

0.

9. The method for preparing nano-selenium according to claim 4, wherein: In the fermentation medium, the concentration of sodium selenite is 5-100 mmol / L.

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