Lactobacillus buchneri strain r16 and application thereof

Abstract

The present application relates to a lactobacillus buchneri R16 and its application, and belongs to the technical field of microorganisms. The lactobacillus buchneri R16 can produce gamma-aminobutyric acid, reduce the cadmium content in soil, increase the biomass (dry weight) of lettuce, reduce the cadmium content in edible tissues and roots of lettuce, and significantly improve the chlorophyll content, vitamin C content, soluble protein content and IAA content in edible tissues of lettuce under cadmium-free or cadmium-containing conditions. The antioxidant capacity of lettuce is significantly enhanced. In addition, the strain R16 can also increase the available potassium content in soil, and has a very broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus brunelli R16 and its applications. Background Technology

[0002] Cadmium (Cd), a non-oxidizing heavy metal, is one of the most toxic heavy metals, posing a significant threat to human health. Accumulation of cadmium in soil not only affects the physicochemical properties and quality of farmland but also exerts a strong toxic effect on plants. Excessive cadmium levels in soil will lead to excessive cadmium content in edible crop tissues, and cadmium can enter the human body through the food chain, seriously threatening human health. Currently, cadmium pollution has become one of the core issues contributing to the degradation of farmland soil quality and food safety risks globally.

[0003] In cadmium pollution remediation technologies, in-situ fixation is widely used. Its core mechanism lies in inhibiting the migration of cadmium from the soil to crops by adding fixatives. Fixatives can reduce the bioavailability of cadmium through adsorption, complexation, or precipitation, thereby controlling its accumulation in plants. However, long-term or excessive use of these fixatives can alter soil aggregate structure, disrupt microbial communities, and even cause degradation of soil physicochemical properties, thus affecting the long-term health of the farmland ecosystem.

[0004] Microbial remediation is an environmentally friendly green remediation technology with significant advantages such as low cost, no damage to soil structure, and no secondary pollution. It enables in-situ remediation, is relatively simple to operate, and can achieve long-term treatment and ecological restoration of polluted environments. In existing research, most cases utilizing microorganisms to degrade heavy metals and promote plant growth rely on the combined use of multiple microorganisms or the combination of microorganisms with other substances to achieve multiple functions. In contrast, reports of a single strain of microorganism simultaneously performing multiple functions such as cadmium removal, plant growth promotion, and soil improvement are very limited. Therefore, developing multifunctional microbial strains has become a key issue urgently needing breakthroughs in the field of agricultural microbiology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain of Lactobacillus brunelli R16 and its applications.

[0006] The technical solution of this invention is as follows:

[0007] A strain of Lactobacillus bruneri ( Lactobacillus buchneri R16, deposited on September 25, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M 20252111.

[0008] The 16S rDNA sequence of Lactobacillus brunelli R16 is shown in SEQ ID NO.1.

[0009] The method for culturing Lactobacillus bruneri R16 includes the following steps: inoculating Lactobacillus bruneri R16 onto MRS solid medium and activating it at 28-37°C to obtain an activated strain; inoculating the activated strain into MRS liquid medium and culturing it with shaking at 150-200 rpm and 30-37°C to obtain Lactobacillus bruneri R16 bacterial suspension.

[0010] The application of Lactobacillus brunelli R16 in the production of γ-aminobutyric acid.

[0011] The application of Lactobacillus brunelli R16 in cadmium removal.

[0012] Preferably, the cadmium removal is to reduce the cadmium content in the soil and / or lettuce.

[0013] The application of Lactobacillus brunelli R16 in promoting lettuce growth.

[0014] The application of Lactobacillus brunelli R16 in increasing the chlorophyll content, vitamin C content, soluble protein content, and IAA content in the edible tissues of lettuce.

[0015] The application of Lactobacillus brunelli R16 in enhancing the antioxidant capacity of lettuce.

[0016] The application of Lactobacillus brunelli R16 in increasing the available potassium content in soil.

[0017] A live bacterial preparation with Lactobacillus bruneri R16 as the active ingredient.

[0018] The beneficial effects of this invention are:

[0019] The *Lactobacillus brunelli* R16 provided by this invention can produce γ-aminobutyric acid (GABA), reduce cadmium content in soil, increase lettuce biomass (dry weight), and reduce cadmium content in edible tissues and roots of lettuce. Under both cadmium-free and cadmium-containing conditions, it can significantly increase the chlorophyll content, vitamin C content, soluble protein content, and IAA content in edible tissues of lettuce, and the antioxidant capacity of lettuce is significantly enhanced. At the same time, strain R16 can also increase the content of available potassium in soil, and its application prospects are very broad. Attached Figure Description

[0020] Figure 1 The results show the determination of the removal capacity of strain R16 for cadmium in soil;

[0021] Figure 2 The effect of strain R16 on increasing the dry weight of lettuce;

[0022] Figure 3 The effect of strain R16 on reducing cadmium content in edible tissues of lettuce;

[0023] Figure 4 The effect of strain R16 on reducing cadmium content in lettuce roots;

[0024] Figure 5 The effect of strain R16 on increasing chlorophyll content in edible tissues of lettuce;

[0025] Figure 6 The effect of strain R16 on increasing the vitamin C content in edible tissues of lettuce;

[0026] Figure 7 The effect of strain R16 on increasing the soluble protein content in the edible tissues of lettuce;

[0027] Figure 8 The effect of strain R16 on increasing the IAA content in edible tissues of lettuce;

[0028] Figure 9 The effect of strain R16 on increasing SOD content in edible tissues of lettuce;

[0029] Figure 10 The effect of strain R16 on the DPPH free radical scavenging rate in edible tissues of lettuce;

[0030] Figure 11 The effect of strain R16 on increasing the content of available potassium in soil. Detailed Implementation

[0031] The following description is based on specific embodiments:

[0032] Preparation of the culture medium used in the examples:

[0033] MRS solid medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, agar 20 g / L, balance water.

[0034] MRS liquid culture medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, balance water.

[0035] Example 1: Isolation, screening and identification of Lactobacillus bruneri R16

[0036] The sample was collected from sauerkraut fermentation broth produced in laboratories in Jinan, Shandong Province, China. The specific separation method was as follows: the fermentation broth was diluted using a gradient dilution method to a gradient of 10. -3 10 -4 10 -5 The suspension was spread onto MRS solid medium and incubated upside down at 37°C for 48 hours, resulting in single colonies. Single colonies were picked and inoculated into MRS liquid medium (without Cd) and MRS solid medium containing 5 mg / L Cd, respectively, and cultured at 37°C upside down for 48 hours and at 37°C with shaking at 180 rpm for 48 hours. Colonies that could grow normally on MRS solid medium containing 5 mg / L Cd were selected, and after centrifugation of the culture medium in MRS liquid medium, the supernatant was collected. Thin-layer chromatography was used to screen for strains that could produce γ-aminobutyric acid, and finally a strain was obtained, which was named "R16".

[0037] The 16S rDNA gene sequence of strain R16 was sequenced, and the sequencing results are shown in SEQ ID NO.1. The obtained 16S rDNA sequence was compared with the gene sequences already available in the NCBI database. The results showed that strain R16 had a 99% similarity to *Lactobacillus bruneri*. Based on the physiological characteristics of the strain, it was identified as *Lactobacillus bruneri*. Lactobacillus buchneri ).

[0038] Lactobacillus bruneri ( Lactobacillus buchneri R16, deposited on September 25, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M 20252111.

[0039] Example 2: Cultivation and preparation of Lactobacillus bruneri R16 bacterial suspension

[0040] (1) The culture method is as follows: Lactobacillus bruneri R16 was inoculated onto MRS solid medium and activated at 37°C for 24 h to obtain activated strain; the activated strain was inoculated into MRS liquid medium and cultured at 180 rpm and 37°C for 48 h to obtain Lactobacillus bruneri R16 bacterial solution.

[0041] (2) Preparation of bacterial agent: Transfer the R16 bacterial solution from step (1) to a sterile centrifuge bottle, centrifuge at 5000 rpm for 5 min to collect the bacterial cells, wash the bacterial cells with sterile deionized water and resuspend them to make the bacterial concentration in the suspension 5 × 10⁻⁶. 8 cfu / mL, to obtain R16 bacterial agent.

[0042] Example 3: Determination of cadmium removal capacity of Lactobacillus bruneri R16

[0043] Add 10 L of deionized water to 2.5 kg of soil, shake at 150 rpm for 48 h, centrifuge at 5000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm microporous membrane and sterilize, then mix thoroughly with sterile MRS liquid culture medium at a volume ratio of 4:1 to obtain a sterile mixture; add CdCl2·2.5H2O to the above-prepared sterile mixture to reduce the Cd concentration in the sterile mixture. 2+ The final concentration was 5 mg / L; 100 mL was added to an Erlenmeyer flask, and the R16 bacterial agent prepared in Example 2 was inoculated at an inoculum size of 1% (v / v). The flask was incubated at 37°C with shaking at 180 rpm to obtain the culture medium. 2+ The sterile mixture was used as the control group (CK), and three replicates were set up for each group.

[0044] The culture medium was sampled every 24 hours. After centrifugation at 10,000 rpm for 5 min, the supernatant was filtered through a 0.45 μm microporous membrane and Cd was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (Optima 2100DV, Perkin-Elmer). 2+ Content, the test results are as follows Figure 1 As shown.

[0045] Depend on Figure 1 It can be seen that, with the extension of culture time, compared with the control group, the Cd in the culture medium after adding R16 bacterial agent is higher. 2+ The content continued to decrease; specifically, on the 4th day of culture, the Cd content in the culture medium decreased. 2+ The concentration was approximately 3.47 mg / L, and the cadmium removal rate was approximately 30.6%; on day 8 of culture, the Cd concentration in the culture medium was... 2+ The concentration tended to stabilize at approximately 2.7 mg / L, with a cadmium removal rate of approximately 46%. These results indicate that strain R16 can reduce the cadmium content in the soil.

[0046] Example 4: Application of Lactobacillus bruneri R16 in lettuce cultivation

[0047] Follow these steps:

[0048] Prepare 27 cultivation pots containing 1.5 kg of soil each, and divide them into 3 groups of 9 pots each. Add CdCl2·2.5H2O to the pots to achieve cadmium concentrations of 0 mg / kg, 0.5 mg / kg, and 1 mg / kg in the soil of the 3 groups, respectively. After thorough mixing with the soil, allow to equilibrate for 45 days. Sow surface-sterilized lettuce seeds into each pot. After germination, thin the seedlings to 5 plants per pot. Water the soil regularly to keep it moist. When the lettuce reaches the third leaf stage, divide the 9 cultivation pots from each group into a control group (CK group). Groups L-R16 and D-R16 were prepared, with three replicates in each group. Deionized water was added to the CK group, R16 inoculant prepared in Example 2 was added to the L-R16 group, and inactivated R16 inoculant was added to the D-R16 group. The addition method was as follows: dig a trench 1-2 cm deep around the lettuce roots, add deionized water or inoculant to the trench, and add 20 mL of liquid per pot. The lettuce was then cultivated in a greenhouse with a temperature of 10-22℃, a relative humidity of 30-45%, and normal light. The entire cultivation cycle was 45 days.

[0049] After cultivation, edible tissues and roots from each lettuce plant were collected for further analysis: the edible tissues (leaves) and roots were washed sequentially with 0.01M EDTA and distilled water, and then divided into two equal portions. One portion was dried for determining dry weight and Cd content; the other portion was left undried, and the contents of chlorophyll, vitamin C (Vc), soluble protein, IAA, SOD, and DPPH free radical scavenging rate in the fresh edible tissue were directly measured. The specific operating steps are as follows:

[0050] (1) Determination of lettuce dry weight: Lettuce leaves were inactivated at 105℃ for 30 min, and then dried at 65℃ to constant weight. The total weight after drying was recorded. The results are as follows: Figure 2 As shown in the figure, different treatment groups at the same cadmium concentration are labeled with different letters to indicate significant differences (P < 0.05, Tukey test).

[0051] Depend on Figure 2 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group can significantly increase the dry weight of lettuce under both cadmium-free and cadmium-containing conditions after the addition of R16 inoculant.

[0052] (2) Determination of Cd content in edible tissues and roots: The edible tissues and roots dried in step (1) were ground and digested to determine the Cd content. The specific method was as follows: 0.2000 g of dried sample was accurately weighed and placed in a polytetrafluoroethylene digestion tube. 10 mL of concentrated nitric acid was added and left overnight. Then, it was placed in a microwave digester for microwave digestion. After digestion, the acid was removed to about 1 mL. The volume was adjusted to 25 mL with distilled water. After filtration through a 0.45 μm filter membrane, the Cd content in the digestion solution was detected by ICP-AES. The results of the Cd content detection in edible tissues are as follows: Figure 3As shown in the figure, the Cd content detection results in lettuce roots are as follows: Figure 4 As shown.

[0053] Depend on Figures 3-4 It was found that, compared with the CK group and the D-R16 group, the Cd content in the edible tissue and roots of lettuce in the L-R16 group was significantly reduced after the addition of R16 inoculant. Specifically, under the condition of Cd concentration of 0.5 mg / kg, the Cd content in the edible tissue of lettuce in the L-R16 group met the edible standard (cadmium limit of 0.2 mg / kg) specified in the national standard GB 2762-2022.

[0054] (3) Chlorophyll content determination: Accurately weigh 0.2000g of fresh leaves, add 95% ethanol solution for grinding, centrifuge, take the supernatant, and measure the absorbance at 665nm and 649nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the chlorophyll content. The results are as follows: Figure 5 As shown.

[0055] Depend on Figure 5 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group, after adding R16 inoculant, can significantly increase the chlorophyll content in the edible tissue of lettuce under both cadmium-free and cadmium-containing conditions.

[0056] (4) Determination of Vitamin C Content: Fresh leaves were taken and the vitamin C content was determined according to the method in national standard GB 5009.86-2016. The results are as follows: Figure 6 As shown.

[0057] Depend on Figure 6 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group, after adding R16 inoculant, can significantly increase the vitamin C content in the edible tissue of lettuce under both cadmium-free and cadmium-containing conditions.

[0058] (5) Determination of soluble protein content: Fresh leaves were taken, and the content of soluble protein was determined using a BCA protein concentration assay kit. The results are as follows: Figure 7 As shown.

[0059] Depend on Figure 7 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group, after adding R16 inoculant, can significantly increase the soluble protein content in the edible tissue of lettuce under both cadmium-free and cadmium-containing conditions.

[0060] (6) IAA content determination: Fresh edible tissue was taken, and the IAA content was determined using a plant growth regulator enzyme-linked immunosorbent assay kit. The results are as follows: Figure 8 As shown.

[0061] Depend on Figure 8It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group, after adding R16 inoculant, can significantly increase the IAA content in the edible tissue of lettuce under both cadmium-free and cadmium-containing conditions.

[0062] (7) SOD content determination: Fresh edible tissues were taken, and the SOD content was determined using a total superoxide dismutase (SOD) activity assay kit. The results are as follows: Figure 9 As shown.

[0063] Depend on Figure 9 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group, after adding R16 inoculant, can significantly increase the SOD content in the edible tissue of lettuce under both cadmium-free and cadmium-containing conditions.

[0064] (8) DPPH free radical scavenging rate determination: Fresh edible tissues were taken, and the DPPH free radical scavenging rate was determined using a total antioxidant capacity (DPPH method) kit. The results are as follows: Figure 10 As shown.

[0065] Depend on Figure 10 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group showed a significant increase in the DPPH free radical scavenging rate in the edible tissue of lettuce after the addition of R16 inoculant, under both cadmium-free and cadmium-containing conditions.

[0066] (9) Determination of available potassium (usable potassium) content in soil: Weigh 38.5g of ammonium acetate, add deionized water to a final volume of 500mL, adjust the pH to 7.0 with 1mol / L acetic acid solution to obtain ammonium acetate solution for later use; take soil from the above cultivation pots and pass it through a 10-mesh sieve, dry it, and then grind it through a 30-mesh sieve; take 1g of the sieved soil into a 50mL centrifuge tube, add the above ammonium acetate solution, shake at 20℃ and 150rpm for 25min to obtain an extract; take 2mL of the extract, centrifuge at 10000rpm for 2min, take the supernatant, filter it thoroughly, and use ICP-AES to determine the available potassium content in the supernatant. The results are as follows. Figure 11 As shown.

[0067] Depend on Figure 11 It can be seen that, compared with the CK group and the D-R16 group, the L-R16 group, after adding R16 inoculant, can significantly increase the content of available potassium in the soil under both cadmium-free and cadmium-containing conditions.

[0068] In summary, the *Lactobacillus brunelli* R16 provided by this invention can produce γ-aminobutyric acid (GABA), reduce cadmium content in soil, increase lettuce biomass (dry weight), and reduce cadmium content in edible tissues and roots of lettuce. Furthermore, it can significantly increase chlorophyll content, vitamin C content, soluble protein content, and IAA content in edible tissues of lettuce under both cadmium-free and cadmium-containing conditions, and significantly enhance the antioxidant capacity of lettuce. Additionally, strain R16 can increase the available potassium content in the soil.

Claims

1. A strain of Lactobacillus brunelli R16, characterized in that, The Lactobacillus bruneri ( Lactobacillus buchneri R16 was deposited at the China Center for Type Culture Collection on September 25, 2025, with accession number CCTCC NO: M 20252111.

2. The method for culturing Lactobacillus brunelli R16 according to claim 1, characterized in that, The procedure includes the following steps: inoculating Lactobacillus bruneri R16 onto MRS solid medium and activating it at 28-37°C to obtain an activated strain; inoculating the activated strain onto MRS liquid medium and culturing it with shaking at 150-200 rpm and 30-37°C to obtain Lactobacillus bruneri R16 bacterial suspension.

3. The application of Lactobacillus brunelli R16 as described in claim 1, characterized in that, It is used for cadmium removal, which means reducing the cadmium content in the soil.

4. The application of Lactobacillus brunelli R16 as described in claim 1, characterized in that, It is used to promote the growth of lettuce.

5. A live bacteria preparation, characterized in that, It includes Lactobacillus brunelli R16 as described in claim 1.

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