Lactobacillus reuteri YG 01 and application thereof
By developing Lactobacillus reuteri YG 01, this strain grew in a high-concentration selenium environment and had an efficient selenium conversion rate, solving the problem of low selenium conversion ability of existing selenium-rich lactic acid bacteria, and achieving efficient selenium-rich animal-derived lactic acid bacteria Additives reduce feed costs.
Patent Information
- Application Number
- CN202411777433.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-02-11
AI Technical Summary
The existing selenium-rich lactic acid bacteria have low conversion ability to selenium, which affects the cost in the feed production process.
A Lactobacillus reuteri YG 01 strain was developed, which is able to grow in high selenium concentrations and has an efficient selenium conversion rate. Sodium selenite was significantly improved by placing Lactobacillus reuteri YG 01 in MRS liquid medium with high selenium concentration and combining appropriate medium ingredients adjustments, such as the addition of CaCO3, glutathione and ammonium citrate. Conversion rate.
Lactobacillus reuteri YG 01 significantly improves the conversion rate of inorganic selenium, can grow in an environment with a selenium concentration of ≥1mg/L, reduces the feed cost of the breeding industry, and provides efficient selenium-rich animal-derived lactic acid bacteria additives.
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Figure CN119410550A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus reuteri YG 01 and an application thereof. Background Art
[0002] Selenium-enriched lactic acid bacteria have outstanding antioxidant, anti-inflammatory and anti-cancer activities. They can use their conversion ability to convert inorganic selenium into organic selenium and extract organic selenium, which makes the development of selenium-enriched lactic acid bacteria attract much attention. Many lactic acid bacteria have the function of enriching organic selenium, and their selenium-enriching characteristics are different.
[0003] The problem with the prior art is that selenium-enriched lactic acid bacteria have a low ability to convert selenium, which greatly affects the cost of feed production. In order to improve the selenium conversion ability, new strains need to be developed. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a strain of Lactobacillus reuteri YG 01 and its application. Lactobacillus reuteri YG 01 is used for efficiently converting selenium in sodium selenite.
[0005] The technical solution of the present invention is as follows:
[0006] A Lactobacillus reuteri YG 01 was deposited in the General Microbiology Center of China National Culture Collection Administration on September 14, 2024, with a deposit number of CGMCCNO.31975 and a classification name of Lactobacillus reuteri.
[0007] The application of Lactobacillus reuteri YG 01 in improving the selenium conversion ability is to culture Lactobacillus reuteri YG 01 in a high-concentration selenium environment, wherein the high concentration refers to a selenium concentration of ≥1 mg / L in the selenium environment. Lactobacillus reuteri YG 01 can grow in an environment with a selenium concentration of ≥1 mg / L and has a good selenium conversion rate, while the selenium-rich lactic acid bacteria in the prior art are difficult to grow in a high-concentration sodium selenite solution of more than 1 mg / L and have poor selenium conversion ability.
[0008] Preferably, the selenium environment refers to an MRS liquid culture medium containing sodium selenite, and the concentration of sodium selenite in the MRS liquid culture medium is ≥1 mg / L.
[0009] Preferably, the concentration of sodium selenite in the MRS liquid culture medium is 3 mg / mL to 8 mg / mL.
[0010] Preferably, the inoculation amount of Lactobacillus reuteri YG 01 in the MRS liquid culture medium is 1 g / L to 100 g / L.
[0011] Preferably, Lactobacillus reuteri YG 01 is inoculated first, and then sodium selenite is supplemented in the MRS liquid culture medium, and the supplementation time is 0 h to 16 h after inoculation.
[0012] Preferably, the MRS liquid culture medium is further supplemented with CaCO3, and the concentration of CaCO3 in the MRS liquid culture medium is ≤30 g / L.
[0013] Preferably, the MRS liquid culture medium is further supplemented with glutathione, and the concentration of glutathione in the MRS liquid culture medium is ≤30 g / L.
[0014] Preferably, the MRS liquid culture medium is further added with ammonium citrate, and the concentration of ammonium citrate in the MRS liquid culture medium is ≤5 g / L.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The invention provides a Lactobacillus reuteri YG 01, which has a significant selenium-enriching effect and a high inorganic selenium conversion rate, and can be used to prepare a selenium-enriched animal-derived lactic acid bacteria additive for animal feed, thereby reducing the feed cost of the breeding industry. Lactobacillus reuteri YG 01 can also grow in an environment with a selenium concentration of ≥1 mg / L.
[0017] Information on the deposit of biological materials
[0018] YG 01, referred to as Lactobacillus reuteri YG 01 in the present invention, was deposited in the General Microbiology Center of China National Culture Collection Administration on September 14, 2024, with a deposit number of CGMCCNO.31975. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the classification name is Lactobacillus reuteri. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is the Gram staining result of Lactobacillus reuteri 01, 1000×.
[0020] Figure 2 16S rRNA PCR amplification results of Lactobacillus reuteri 01, M: DNAMarkerDL 2000; 1: 16S RRNA PCR amplification results of Lactobacillus reuteri 01; 2: negative control.
[0021] Figure 3 This is the growth curve of Lactobacillus reuteri 01.
[0022] Figure 4 This is the selenium resistance acclimation process of Lactobacillus reuteri 01.
[0023] Figure 5 This is the growth curve of Lactobacillus reuteri YG 01 in MRS medium with a sodium selenite concentration of 0 mg / mL to 8 mg / mL.
[0024] Figure 6 This is the standard curve of sodium selenite content.
[0025] Figure 7 The conversion rate of sodium selenite in MRS liquid culture medium with different concentrations of sodium selenite added.
[0026] Figure 8 is the conversion rate of sodium selenite when sodium selenite was added at different culture periods.
[0027] Fig. 9 is the conversion rate of sodium selenite under different inoculation amounts of Lactobacillus reuteri YG 01.
[0028] Fig.10 The conversion rate of sodium selenite with different CaCO3 addition amounts.
[0029] Fig.11 The conversion rate of sodium selenite with different glutathione addition amounts.
[0030] Fig.12 The sodium selenite conversion rate of Lactobacillus reuteri YG 01 with different ammonium citrate addition amounts. DETAILED DESCRIPTION
[0031] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.
[0032] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.
[0033] Example 1: Isolation and identification of Lactobacillus reuteri.
[0034] 1. Isolation of bacterial strains
[0035] A strain of Lactobacillus reuteri 01 was isolated from pig feces. The Lactobacillus reuteri was streaked onto an MRS agar plate and cultured at 37°C for 24 hours. Milky white colonies with protrusions or flattened in the center were observed to grow, with regular edges, smooth, shiny, opaque, and soft texture. Gram staining results showed that it was Gram-positive and the bacteria were rod-shaped, see Figure 1 .
[0036] 2. Strain Sequencing and Homology Comparison
[0037] The 16S rRNA gene was amplified by PCR using the extracted genomic DNA of Lactobacillus reuteri 01 as a template. The results are shown in Figure 2The size of the PCR product was about 1600 bp. After sequencing, Blast alignment was performed and the homology with the ribosomal RNA gene of Lactobacillus reuteri strain 45516S was as high as 100%, indicating that the strain was Lactobacillus reuteri.
[0038] The spliced sequence of Lactobacillus reuteri 01 is shown in SEQ ID NO.1, and is as follows:
[0039]
[0040] 3. Biochemical Identification of Bacterial Species
[0041] Biochemical identification was performed with reference to the "Classification, Identification and Experimental Methods of Lactic Acid Bacteria". The results of biochemical identification are shown in Table 1. Lactobacillus reuteri 01 can metabolize maltose, sucrose, raffinose and lactose, and can metabolize tryptophan to produce indole.
[0042] Table 1 Biochemical identification results of Lactobacillus reuteri 01
[0043]
[0044]
[0045] Note: + is positive, – is negative
[0046] 4. Growth curve determination of bacterial strains
[0047] The absorbance of the cultured bacteria at a wavelength of 600 nm was measured every 1 hour. Figure 3 As shown, from the growth curve of Lactobacillus reuteri 01, it can be seen that 0h~2h is the lag phase, 2h~14h is the logarithmic growth phase, 14h~24h is the plateau phase, and it begins to decline after 24h.
[0048] Example 2: Preparation method of Lactobacillus reuteri YG 01.
[0049] 1. Domestication of Lactobacillus reuteri 01
[0050] Prepare an MRS liquid medium with a selenium concentration of 10 μg / mL using sodium selenite: Pick a single colony of Lactobacillus reuteri 01 and inoculate it in an MRS liquid medium containing the above-mentioned selenium concentration and culture it at 37°C for 24 hours. Pipette 0.1mL of the culture solution and streak it on a plate with a 10μg / mL selenium concentration MRS medium, purify it twice and store it. Continue to inoculate the preserved strain in an MRS liquid medium containing 100μg / mL sodium selenite, culture it at 37°C for 24 hours, aspirate the bacterial solution and streak it on a plate with a 100μg / mL selenium concentration MRS medium, purify it and store it. Continue to inoculate the preserved strain in an MRS liquid medium containing 1mg / mL sodium selenite, culture it at 37°C for 24 hours, aspirate the bacterial solution and streak it on a plate with a 1mg / mL selenium concentration MRS medium, purify it and store it. Then, the strain was taken from the MRS liquid medium containing 1 mg / mL sodium selenite and inoculated into the MRS liquid medium containing 5 mg / ml sodium selenite. Then, the strain was taken from the liquid of MRS containing 5 mg / ml sodium selenite and inoculated into the MRS liquid medium containing 6 mg / ml sodium selenite. In this way, the concentration of sodium selenite in the MRS liquid medium was increased by 1 mg / ml each time until the concentration of sodium selenite was increased by 5 mg / ml until the concentration of sodium selenite was increased to 8 mg / ml, thereby gradually improving the ability of Lactobacillus reuteri 01 to tolerate sodium selenite. Figure 4 This is the selenium-resistant acclimation process of Lactobacillus reuteri 01, and finally Lactobacillus reuteri YG 01 was screened out.
[0051] In addition, the absorbance of the Lactobacillus reuteri YG 01 bacterial solution at each selenium concentration at a wavelength of 600 nm was measured every 4 hours to draw a growth curve. Figure 5 Figure 2 is the growth curve of Lactobacillus reuteri YG 01 in MRS medium with a sodium selenite concentration of 0 mg / mL to 8 mg / mL. It can be seen that the domesticated Lactobacillus reuteri YG 01 can grow normally in a medium with a high sodium selenite concentration. Figure 5 .
[0052] 2. Determination of Selenium Conversion Conditions of Lactobacillus reuteri YG 01
[0053] 1. Draw the standard curve of sodium selenite
[0054] Accurately pipette 0mL, 0.5mL, 1mL, 1.5mL, 2mL, and 2.5mL of 1mg / mL sodium selenite standard solution into a 100mL beaker, add water to 35mL, add 1mL of 5g / 100mL EDTA-2Na (disodium ethylenediaminetetraacetic acid) solution respectively, shake well, adjust the pH of the solution to 2.5 with 50% hydrochloric acid solution, add 4mL of 0.5g / 100mL DAB (diaminobenzidine) solution respectively, shake well, place in a dark place for 30min, and then adjust the pH of the solution to 7.0 with 5g / 100mL NaOH, transfer to a separatory funnel, add 10mL of xylene and shake for 2min, let stand to separate, and discard the aqueous layer. The sodium selenite in the solution was extracted with xylene, and the absorbance was measured at a wavelength of 420 nm using a spectrophotometer. A standard curve was drawn, y = 2.0595x + 0.1645R2 = 0.9979, see Figure 6 .
[0055] 2. Determination of inorganic selenium
[0056] Take 1 mL and culture until OD 600 Lactobacillus reuteri YG 01 bacterial liquid with a pH value of 1.4, centrifuged at 4000rpm for 10min, all supernatants were taken in a 50mL beaker, water was added to 35mL, 1mL 5g / 100mL EDTA-2Na solution was added, shaken, pH value of the solution was adjusted to 2.5 with a volume fraction 50% hydrochloric acid solution, 4mL 0.5g / 100mL DAB solution was added, shaken, placed in a dark place for 30min, pH value of the solution was adjusted to 7.0 with 5g / 100mL NaOH, transferred to a separating funnel, 10mL xylene was added to shake for 2min, stratified, and the water layer was discarded. Xylene was used to extract selenium in the solution, absorbance was measured at 420nm wavelength with an ultraviolet spectrophotometer, and the corresponding selenium content was found from the standard curve, which was the inorganic selenium content. Organic selenium content = total selenium content - residual inorganic selenium content. The value of the total organic selenium divided by the total selenium content is the sodium selenite conversion rate. The higher the organic selenium content, the higher the selenium-enriched rate of Lactobacillus reuteri YG 01, and the higher the conversion rate of Lactobacillus reuteri YG 01 to inorganic selenium (sodium selenite).
[0057] 3. Effect of sodium selenite concentration on the conversion rate of sodium selenite in Lactobacillus reuteri YG 01
[0058] Lactobacillus reuteri YG 01 was inoculated into MRS liquid culture medium containing 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, and 7 mg / mL sodium selenite, respectively, and cultured at 37°C for 24 h. The inorganic selenium content in the culture medium was determined, and the organic selenium content was converted to obtain the sodium selenite conversion rate of Lactobacillus reuteri YG 01, that is, the sodium selenite conversion rate. It can be concluded that when the sodium selenite concentration is 6 mg / mL, the sodium selenite conversion rate of Lactobacillus reuteri YG 01 is the highest, see Figure 7 Therefore, MRS medium containing 6 mg / mL sodium selenite was selected for subsequent experiments.
[0059] 4. Determination of the best time to supplement sodium selenite
[0060] Add 6 mg / mL sodium selenite to MRS liquid medium as the basic selenium-enriched medium, and add sodium selenite at the beginning, early stage, early logarithmic stage, mid-logarithmic stage, and late logarithmic stage of Lactobacillus reuteri YG 01 culture, that is, add sodium selenite at 0h, 4h, 8h, 12h, and 16h of culture, so that the final concentration of sodium selenite in the culture medium is 6 mg / mL. After sodium selenite is added, it is cultured at a constant temperature of 37°C for 24h, and the residual inorganic selenium content in the culture supernatant of Lactobacillus reuteri YG 01 at different growth stages is analyzed. It is found that the conversion of sodium selenite can be shown from 0h to 16h after inoculation, and the optimal time for sodium selenite supplementation is 8h after inoculation when the conversion rate of sodium selenite is the highest. The results are shown in Figure 8 .
[0061] 5. Effect of inoculation amount on sodium selenite conversion rate of Lactobacillus reuteri YG 01
[0062] The activated Lactobacillus reuteri YG 01 was inoculated into 5 MRS liquid culture media at inoculation doses of 0.1 g / 100 mL, 0.5 g / 100 mL, 1 g / 100 mL, 5 g / 100 mL, and 10 g / 100 mL, respectively. Sodium selenite was added at 8 h to make the concentration of sodium selenite reach 6 mg / mL. The culture was carried out at 37° C. for 24 h. The conversion rate of sodium selenite was determined. It was found that when the inoculation amount was 5 g / 100 mL, the conversion rate of sodium selenite could reach 83.0%. The results are shown in Fig. 9 .
[0063] 6. Effect of CaCO3 addition in culture medium on sodium selenite conversion rate of Lactobacillus reuteri YG 01
[0064] Since lactic acid bacteria produce acid by fermentation, considering that pH value will affect the conversion rate of sodium selenite, 0mg / mL, 5mg / mL, 15mg / mL, 20mg / mL, 25mg / mL, and 30mg / mL CaCO3 were added to 6 MRS liquid culture media respectively, so that the inoculation amount of Lactobacillus reuteri YG 01 reached 5g / 00mL, sodium selenite was added at the 6th hour to make the sodium selenite concentration reach 6mg / mL, and the culture was carried out at 37℃ for 24h. The conversion rate of sodium selenite was measured. It can be concluded that when the amount of CaCO3 added was 20mg / mL, the conversion rate of sodium selenite reached 88.5%. The results are shown in Fig.10 .
[0065] 7. Effect of glutathione addition in culture medium on sodium selenite conversion rate of Lactobacillus reuteri YG 01
[0066] 0 mg / mL, 5 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, and 30 mg / mL glutathione were added to 6 MRS liquid culture media, respectively. The inoculation amount of Lactobacillus reuteri YG 01 reached 5 g / 00 mL. Sodium selenite was added at the 6th hour to make the concentration of sodium selenite reach 6 mg / mL. The culture was carried out at 37°C for 24 hours. The conversion rate of sodium selenite was determined. It was found that when the glutathione concentration was 15 mg / mL, the conversion rate of sodium selenite could reach 92.5%. The results are shown in Fig.11 .
[0067] 8. Effect of ammonium citrate addition in culture medium on sodium selenite conversion rate of Lactobacillus reuteri YG 01
[0068] 0 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL ammonium citrate were added to 6 MRS liquid culture media, respectively, to make the inoculum amount of Lactobacillus reuteri YG 01 reach 5 g / 00 mL, sodium selenite was added at the 6th hour to make the sodium selenite concentration reach 6 mg / mL, and the culture was carried out at 37°C for 24 hours. The conversion rate of sodium selenite was determined, and it was found that when the concentration of ammonium citrate was 2 mg / mL, the conversion rate of sodium selenite could reach 94.3%. The results are shown in Fig.12 .
[0069] In summary, the present invention discovered and utilized the enrichment effect of Lactobacillus reuteri YG 01 on sodium selenite in the culture system, and found that Lactobacillus reuteri YG 01 has a good selenium conversion effect. The present invention also achieves a significant increase in the sodium selenite conversion rate of Lactobacillus reuteri YG 01 by controlling the components of the culture medium, and achieves efficient completion of the bioconversion and accumulation of inorganic selenium to organic selenium in the process of vigorous growth of animal-derived lactobacilli, creating conditions for further improving the conversion efficiency and conversion amount of selenium.
[0070] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic creative concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.
[0071] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.
Claims
1. A strain of Lactobacillus reuteri YG 01, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 14, 2024, with the deposit number CGMCC NO.31975.
2. Application of Lactobacillus reuteri YG 01 in improving selenium conversion capacity.
3. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 2, characterized in that: Lactobacillus reuteri YG 01 is placed in a high-concentration selenium environment for cultivation, wherein the high concentration refers to a selenium concentration in the selenium environment of ≥1 mg / L.
4. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 3, characterized in that: The selenium environment refers to an MRS liquid culture medium containing sodium selenite, and the concentration of sodium selenite in the MRS liquid culture medium is ≥1 mg / L.
5. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 4, characterized in that: The concentration of sodium selenite in MRS liquid culture medium is 3 mg / mL to 8 mg / mL.
6. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 4, characterized in that: The inoculation amount of Lactobacillus reuteri YG 01 in the MRS liquid culture medium is 1 g / L to 100 g / L.
7. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 6, characterized in that: First, Lactobacillus reuteri YG 01 is inoculated into the MRS liquid culture medium, and then sodium selenite is supplemented into the MRS liquid culture medium, and the supplementation time is 0h to 16h after the inoculation.
8. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 3, characterized in that: The MRS liquid culture medium is also added with CaCO3, and the concentration of CaCO3 in the MRS liquid culture medium is ≤30g / L.
9. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 3, characterized in that: The MRS liquid culture medium is also supplemented with glutathione, and the concentration of glutathione in the MRS liquid culture medium is ≤30 g / L.
10. The use of Lactobacillus reuteri YG 01 in improving selenium conversion ability according to claim 3, characterized in that: The MRS liquid culture medium is also added with ammonium citrate, and the concentration of ammonium citrate in the MRS liquid culture medium is ≤5g / L.