Leuconostoc citrophilum and application thereof in preparation of nano-selenium

Abstract

The invention discloses leuconostoc citrophilum and application of the leuconostoc citrophilum in preparation of nano-selenium, and relates to the technical field of screening and cultivation of selenium-rich strains. The leuconostoc citrophilum provided by the invention is preserved in the China Center for Type Culture Collection on September 29, 2024, the preservation number is CCTCC NO: M 20242123, and the preservation address is Luojia mountain on eight road, Wuchang District, Wuhan City, Hubei Province. The Leuconostoc citrophilum has the advantages that the Leuconostoc citrophilum is good in selenite resistance and selenite reduction capacity, and the nano elemental selenium can be generated by means of reduction; the bacterial strain also has good bacteriostatic and antibacterial effects, especially gram-negative bacteria; the acid-resistant and cholate-resistant performance is good, and the survival rate in gastrointestinal tracts is relatively high.

Description

Technical Field

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

[0002] In nature, selenium exists in two forms: organic selenium and inorganic selenium. Among them, inorganic selenium has low utilization rate, narrow safe dosage range, and potential threat to the environment; while organic selenium has high bioavailability, wide relative safe dosage, and good palatability. Compared with inorganic and organic selenium, red nano-selenium has low toxicity, high biological activity, and the characteristics of photoelectronic properties of nanomaterials and multiple anti-biological activities. It is widely used in animal production, medicine and health products, biosensor preparation, solar cells, rectifiers, electrostatic copying and other technical fields.

[0003] Microorganisms play an important role in the natural cycle of selenium. It has been found that many bacteria, such as Escherichia coli, Bacillus subtilis, Rhodospirillum Molisch, Bacillus licheniformis, Pseudomonas fluorescens, etc., can reduce oxidized selenium to non-toxic and highly efficient red nano-selenium, and the synthesized biological nano-selenium is stable and highly active. Therefore, using bacteria to synthesize biological nano-selenium is an efficient way of selenium biotransformation. Summary of the invention

[0004] The present invention provides a Leuconostoc citracis strain and its application in preparing nano-selenium, and obtains a Leuconostoc citracis strain with high selenium resistance by extracting and screening from kimchi water. The specific implementation is through the following technologies.

[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 a deposit number of CCTCCNO: M 20242123, and a deposit address of 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, has good antibacterial and antimicrobial ability, and is acid-resistant and bile-resistant, 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 citricola, characterized in that it is used to prepare nano-selenium, or to prepare animal feed additives, or to prepare selenium-enriched fermented products, or to prepare bacterial agents for microbial transformation 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 citracis to reduce selenite to generate nano-selenium.

[0009] Furthermore, the activated Leuconostoc citricola 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 centrifuged to obtain a precipitate, resuspended, cell broken, and centrifuged to obtain a precipitate, namely nano-selenium.

[0011] Furthermore, according to the method for preparing nano-selenium according to claim 4, it 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 less than 2.0.

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

[0016] Compared with the prior art, the advantages of the present invention are as follows: the Leuconostoc citreum provided by the present invention has good resistance to selenite and the ability to reduce selenite, and reduces to generate nano-elemental selenium; the strain also has good antibacterial and antimicrobial effects, especially for Gram-negative bacteria; it has good acid and bile resistance, and its survival rate in the gastrointestinal tract is also relatively high. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The growth of strain PC-7 in 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 hours.

[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 technology (B).

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

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

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

[0026] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1: Isolation of kimchi-derived bacteria, determination of selenium resistance and molecular biological identification

[0028] 1. Isolation of kimchi source bacteria

[0029] Use a disposable syringe to take the kimchi water stock solution of homemade radish kimchi in the laboratory, dilute it in gradients, and apply 100 μL of each gradient on MRS solid medium. After 24 hours, observe the morphological structure of the colonies, select single colonies for numbering, and streak the isolated and purified bacteria onto a new plate. After repeating the plate streaking purification twice, pick out a single colony for subsequent experiments.

[0030] 2. Determination of selenium tolerance of strains

[0031] Several isolated strains were inoculated into MRS liquid culture medium and cultured at 30°C and 180 r / min until the logarithmic phase. Then 200 μL of the bacterial solution was added to a screw-mouth glass sample bottle (1% inoculation amount), which contained 20 mL of MRS liquid culture medium containing different concentrations (0, 5, 25, 50, 100 mmol / L) of sodium selenite; cultured at 30°C and 180 r / min for 3 days, and the color change of the liquid was observed.

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

[0033] from Figure 1 It can be seen that as the concentration of sodium selenite increases, the fermentation liquid of strain PC-7 becomes darker red, indicating that the elemental nano-selenium gradually increases; when the concentration of sodium selenite reaches 100mmol / L, the fermentation liquid is still red, indicating that the strain can grow in the presence of a higher concentration of sodium selenite and reduce sodium selenite to red nano-selenium. It can be seen that the selenium tolerance of strain PC-7 is 100mmol / L, and it is a highly selenium-resistant lactic acid bacteria strain. Therefore, strain PC-7 was selected for subsequent 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, 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 spliced ​​and compared in the NBCI gene library, it was found that the sequence similarity was the highest with that of Leuconostoc citriodora.

[0041] MEGA-X software was used for multiple comparisons and a phylogenetic tree based on 16S rDNA sequences was constructed using the maximum natural method. Figure 2 As shown, the results showed that the strain PC-7 was most similar to Leuconostoc citreum, and the strain PC-7 could be preliminarily identified as Leuconostoc citreum. The strain was deposited in China Center for Type Culture Collection on September 29, 2024, with the deposit number: CCTCC NO: M 20242123, and the address was: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The name of the deposited strain is Leuconostoc citreum PC-7.

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

[0043] The strain PC-7 was inoculated in MRS solid medium and activated overnight; a single colony was picked up the next day and cultured in MRS liquid medium until the stable period; the bacterial solution was then inoculated into 100 mL of MRS liquid medium containing different concentrations (5, 10, 25 mmol / L) of sodium selenite at an inoculum volume fraction of 1% for culture, and MRS medium without selenium was used as a blank control; the optical density value (OD) of the bacterial solution at 600 nm was measured every 3 hours. 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 it took for strain PC-7 to reach the logarithmic growth phase. The higher the concentration, the longer it took strain PC-7 to reach the logarithmic growth phase, but the maximum OD 600 Higher than 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 ascorbic acid reduction method, and a Na2SeO3 standard curve was prepared. The strain was inoculated into 100mL MRS liquid culture medium containing 5, 10, and 25mmol / L sodium selenite, and cultured in a shaker at 30℃ and 180r / min for 3d; samples were taken every 12h, centrifuged at 12000r / min for 15min, and 0.8mL of supernatant was taken; 0.4mL 4mol / L hydrochloric acid and 0.8mL 1mol / L ascorbic acid were added to the supernatant, shaken and mixed, and allowed to stand at room temperature for 10min, and the optical density value (OD) of the reaction solution at 500nm 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% in 12 hours, 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℃180r / min to the logarithmic phase. Then 200μL of the above bacterial solution was added to 20mL (i.e., 1% inoculation amount) of MRS liquid culture medium containing different concentrations (20, 40, 80, 160, 320μg / mL and 5mmol / L) of sodium selenite in a screw-mouthed glass sample bottle, cultured at 30℃180r / min for 3d, and the color change of the liquid was observed. The appearance of red in the system was observed with the naked eye to explore the concentration conditions at which the bacteria began to synthesize nano-selenium.

[0051] The results are shown in Table 1 below. When the concentration of sodium selenite was 320 μg / mL (about 1.85 mmol / L), the fermentation broth of strain PC-7 turned reddish. Under low concentration inorganic selenium treatment conditions, the bacteria converted inorganic selenium into non-nano selenium (the literature reports that it is mainly organic selenium). Under high concentration inorganic selenium conditions, the bacteria began to convert inorganic selenium into nano selenium (5 mmol / L, about 863 μg / mL) or more.

[0052] This is also the selenium conversion characteristic of PC-7. Under low concentration (20-160μg / mL) sodium selenite culture conditions, strain PC-7 mainly produces organic selenium, and under high concentration conditions (5mmol / L), it produces nano-selenium. Different concentrations of added sodium selenite will produce different products of bioconversion.

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

[0054]

[0055] Example 4: Nano-selenium particle size and zeta potential determination 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 out 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 containing 10 mmol / L sodium selenite at a concentration of 1% at an inoculation rate, and cultured in a shaking incubator 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 min, the supernatant was discarded, the precipitate was washed with sterile water and resuspended, and the cells were disrupted 4 times by ultra-high pressure and low temperature; the disrupted liquid was centrifuged at 12000 r / min for 10 min, 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 1.33, and the viscosity was 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 were recorded.

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

[0059] 2. Scanning electron microscope image

[0060] Inoculate the seed liquid into three bottles of MRS liquid culture medium containing 100mL 10mmol / L sodium selenite at a 1% inoculation rate, and culture in a shaker at 30℃ and 180r / min for 48h; centrifuge the bacterial liquid at 12000r / min for 10min, discard the supernatant, wash the precipitate with sterile water and resuspend; ultra-high pressure low temperature cell disruption 4 times, centrifuge the disrupted liquid at 12000r / min for 10min; resuspend the precipitate and centrifuge it at 12000r / min for 10min. Resuspend the precipitate and measure the particle size and potential. Centrifuge the sample and then freeze-dry it. Use a scanning electron microscope to observe its morphological characteristics. The scanning electron microscope results are as follows: Figure 6, 7 As shown in Table 2 below, it can be seen that strain PC-7 produced nano-selenium with distinct particle sizes, with an average particle size of 150nm. According to the Se characteristic peak in the EDX spectrum and the elemental composition analysis, it was found that the main component element was selenium, and it also contained a certain amount of C, N, O and S. This also shows that the surface of the nano-selenium particles is likely to be 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] According to the inoculation amount of 1%, the bacterial liquid was inoculated into the MRS acid-resistant liquid culture medium with low pH = 2.0, 3.0, and 4.0, and the MRS bile salt (0.1, 0.2, 0.3%) liquid culture medium, and cultured at 30℃ and 90r / min for 4h, and then the viable bacteria were counted after dilution and coating. 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 simulated gastric fluid. Trypsin was dissolved in PBS at pH 8.0 to prepare 1 g / L simulated intestinal fluid, and then sterilized by filtration with a 0.22 μm filter membrane for later use.

[0070] The experimental strain PC-7 was cultured for 18 hours. 1 mL of bacterial suspension was mixed with 9.0 mL of artificial gastric juice, cultured at 30℃ and 90 r / min for 3 hours, and then diluted and coated for viable bacteria count. 1 mL of bacterial suspension was mixed with 9.0 mL of artificial intestinal juice, cultured at 30℃ and 90 r / min for 4 hours, and then diluted and coated for viable bacteria count. 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 Fig. 9 As shown, the survival rates of strain PC-7 in artificial gastric juice without sodium selenite and artificial gastric juice containing 10mmol / L sodium selenite after 3 hours of culture were 47.42% and 72.59%, respectively; the survival rates in artificial intestinal juice without sodium selenite and artificial intestinal juice containing 10mmol / L sodium selenite after 3 hours of culture were 65.11% and 87.78%, respectively. This shows that after adding selenium, the survival rate of strain PC-7 in simulated artificial gastric juice and intestinal juice was significantly enhanced.

[0074] Example 7: Antibacterial effect test of strains

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

[0076] Incubate the pathogen 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 ability of the strain. Take 15mL of LB medium and pour it into the culture dish. After solidification, use it as the lower medium. Put in 4 Oxford cups. Take 5mL of medium containing 1% suspension of different indicator bacteria and add it to the solidified lower medium as the upper layer. After the upper medium solidifies, take out the Oxford cup and set it aside.

[0078] Take one of the Oxford cup wells and add 200 μL of PBS solution, and add 200 μL of conventional PC-7 fermentation liquid or selenium-enriched PC-7 fermentation liquid to the other Oxford cup wells, culture 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 on 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 in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

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

2. An application of the Leuconostoc citrachondriasis according to claim 1, characterized in that: It is used for preparing nano-selenium, or for preparing animal feed additives, or for preparing selenium-enriched fermented products, or for preparing bacterial agents for synthesizing nano-selenium through microbial transformation, or for preparing antibacterial agents.

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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: The pH value of the fermentation culture system is not less than 2.

0.

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

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