A chromium-resistant Lactobacillus salivarius strain and its application

Abstract

The present application discloses a chromium-resistant Lactobacillus salivarius strain and its application. The Lactobacillus salivarius can survive in a high-concentration chromium environment and has a strong adsorption effect on chromium, and can be used to remove chromium from animal feed. The chromium-resistant Lactobacillus salivarius is derived from healthy chicken feces and is obtained by culturing and screening on a chromium (VI)-containing MRS solid medium. The chromium (VI)-resistant Lactobacillus salivarius is prepared into a chromium adsorbent that can be used on a large scale in animal feed, especially under conditions of a pH of 3-5, with good chromium adsorption rate.

Description

Technical Field

[0001] The invention relates to the technical field of microbial separation, and in particular to a chromium-resistant Lactobacillus salivarius strain and application thereof. Background Art

[0002] Lactobacillus salivarius is widely distributed in nature, exhibiting rapid growth and stability, is easy to isolate and culture, is harmless to humans and animals, and does not pollute the environment. Its rich metabolites possess broad-spectrum antimicrobial activity and strong stress resistance, making it an increasingly important bacterium in many fields, including agriculture, the environment, and the fermentation industry.

[0003] Lactobacillus salivarius is a Gram-positive bacillus that lacks catalase and oxidase, yet produces lactic acid and bacteriocins. Lactobacillus salivarius is a common intestinal flora of humans, livestock, and aquatic animals. By competing with harmful microorganisms or expressing beneficial substances, it plays an important role in the host's nutritional metabolism, physiological, and immune processes. When administered in an appropriate dosage, Lactobacillus salivarius can be used as a probiotic to improve the host's health, such as regulating the intestinal microbial flora, providing antibacterial effects, stimulating protective immune responses, and optimizing intestinal acidification. Lactobacillus salivarius can be used as a feed additive to improve the growth performance, immune function, and disease resistance of livestock and poultry.

[0004] Chromium exists primarily in the environment as trivalent and hexavalent forms. It is considered the second most serious environmental pollutant on Earth. Chromium and its compounds are widely used in industries such as electroplating, metallurgy, dye production, metalworking, leather making, fertilizers, and printing and dyeing. These processes generate significant amounts of chromium-containing wastewater and residue, polluting water, air, and soil. Chromium, known for its environmental stability and resistance to degradation, can also pose a threat to human health through its accumulation and amplification effects within the food chain.

[0005] Studies have shown that the absorption of chromium by the digestive system occurs mainly in the small intestine, followed by the large intestine. The absorbed chromium is then transported through the blood to different tissues and organs of the body (such as the liver, kidneys, spleen and lungs). However, due to its slow metabolism, it can accumulate in large quantities in tissues and organs. Early investigations have shown that long-term exposure to high concentrations of Cr(VI) can lead to obvious gastrointestinal symptoms, accompanied by weight loss. In addition, autopsies found bleeding and ulcers in the gastric mucosa, suggesting that the morphology and physiology of the gastrointestinal tract are also seriously affected. In addition, related studies have also confirmed that hexavalent chromium is associated with organ failure, asthma, rhinitis and even cancer. Chromium is a toxic and harmful ingredient in animal feed. If animals ingest excessive levels of chromium, chromium will accumulate in the body, damaging the health of livestock and poultry, and then affecting human health through the food chain.

[0006] Feed itself has a certain background chromium content. Environmental pollution is an important source of feed chromium contamination. Controlling the chromium (VI) content in feed is a necessary measure to protect the health of livestock and poultry. In order to prevent and reduce the harm and risks of chromium to animals and humans, it is necessary to find ways to reduce the harm of feed chromium (VI) to animals. However, there is currently no specific antidote for chromium (VI) poisoning. In order to reduce the harm of feed chromium (VI) pollution to poultry, we draw on the mechanism of action of microorganisms in environmental pollution remediation and screen probiotics with tolerance and adsorption capacity for chromium (VI) in the intestinal microorganisms of poultry. By adding chromium-resistant probiotics to feed, we can effectively reduce the toxic damage of chromium to poultry and its accumulation in poultry products. Summary of the Invention

[0007] Purpose of the invention: The first purpose of the present invention is to provide a hexavalent chromium-resistant Lactobacillus salivarius. The chromium-resistant Lactobacillus salivarius is deposited in the China Center for Type Culture Collection with the accession number CCTCC M 20221599. The Lactobacillus salivarius can survive in a high concentration of chromium (VI) environment and has a strong adsorption effect on hexavalent chromium, and can be used to remove hexavalent chromium from animal feed.

[0008] The second object of the present invention is to provide a method for isolating the chromium-resistant Lactobacillus salivarius as described above.

[0009] The third object of the present invention is to provide the use of the above-mentioned chromium-resistant Lactobacillus salivarius in adsorbing chromium-contaminated substrates.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned chromium-resistant Lactobacillus salivarius in the preparation of a chromium adsorbent.

[0011] The fifth object of the present invention is to provide a chromium adsorbent comprising the above-mentioned chromium-resistant Lactobacillus salivarius.

[0012] Technical solution: In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solution for chromium-resistant Lactobacillus salivarius and its isolation method and application:

[0013] A chromium-resistant Lactobacillus salivarius, classified and named Lactobacillus salivarius, was deposited in the China Center for Type Culture Collection on October 19, 2022, with the accession number CCTCC M20221599, and the deposit address is Wuhan University, Wuhan, China.

[0014] Preferably, the 16S rRNA of the chromium-resistant Lactobacillus salivarius is shown in the sequence listing as SEQ ID NO.1.

[0015] A method for isolating chromium-resistant Lactobacillus salivarius comprises the following steps: taking fresh chicken manure from free-range healthy chickens, adding a sterile PBS solution, shaking and mixing, centrifuging to obtain a supernatant, then diluting the manure stepwise with the sterile PBS solution, and spreading the stepwise dilutions on a chromium-containing MRS solid culture medium. After anaerobic culture at 37°C for 24 hours, picking a single colony, and continuously inoculating the manure on a chromium (VI)-containing MRS solid culture medium. After further anaerobic culture for 24 hours, picking a single colony, and repeatedly inoculating the manure on a chromium (VI)-containing MRS solid culture medium. The single colony obtained after culturing for 24 hours is chromium (VI)-resistant Lactobacillus salivarius.

[0016] Preferably, the chromium (VI) concentration in the MRS solid culture medium is 80 mg / L.

[0017] The application of the above-mentioned chromium-resistant Lactobacillus salivarius in adsorbing chromium-contaminated matrix.

[0018] Preferably, chromium-resistant Lactobacillus salivarius is added to a chromium-contaminated substrate, and the chromium-contaminated substrate is animal feed.

[0019] The application of the above-mentioned chromium-resistant Lactobacillus salivarius in the preparation of chromium adsorbent.

[0020] A chromium adsorbent comprises the above-mentioned chromium-resistant Lactobacillus salivarius.

[0021] Beneficial effects: The present invention provides a chromium-resistant Lactobacillus salivarius, which can survive in a high-concentration chromium environment and has a strong adsorption effect on chromium, and can be used to remove chromium from animal feed. The chromium-resistant Lactobacillus salivarius is derived from healthy chicken feces and is obtained by culturing and screening in an MRS solid culture medium containing chromium (VI); the chromium (VI)-resistant Lactobacillus salivarius is prepared into a chromium adsorbent, which can be used on a large scale in animal feed, especially under conditions of pH 3-5, and has a good chromium adsorption rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a colony morphology diagram of the chromium-resistant Lactobacillus salivarius cultured in the present invention;

[0023] Figure 2 The figure is a Gram staining result of the chromium-resistant Lactobacillus salivarius isolated in Example 1;

[0024] Figure 3 Growth curve of Lactobacillus salivarius. DETAILED DESCRIPTION

[0025] The following embodiments describe the present invention in detail, but the protection scope of the present invention is not limited to the embodiments.

[0026] Example 1

[0027] The method for isolating chromium-resistant Lactobacillus salivarius comprises the following steps:

[0028] (1) Take 1 g of fresh chicken manure from healthy laying hens and place it in a sterile 15 ml centrifuge tube. Then add 10 ml of sterile PBS solution and shake for 30 minutes.

[0029] (2) Centrifuge at 4000 rpm for 10 min, and collect 1 ml of the supernatant into a new 15 ml centrifuge tube;

[0030] (3) and diluted with sterile PBS to 10 -5 100 μL of each dilution was dropped onto MRS solid medium containing 80 mg / L Cr(VI), and cultured anaerobically at 37°C for 24 h. Then, a single colony was picked.

[0031] (4) The single colony 1 removed in step (3) was inoculated on MRS solid medium containing 80 mg / L Cr(VI), and after culturing for 24 hours, single colony 2 was picked;

[0032] (5) The single colony picked in step (4) was inoculated twice on MRS solid culture medium containing 80 mg / L of Cr(VI). The single colony obtained after culturing for 24 hours was Lactobacillus salivarius resistant to 80 mg / L of Cr(VI).

[0033] The Lactobacillus salivarius separated in step (5) is used in animal feed to adsorb chromium, and can also be used to prepare a chromium adsorbent.

[0034] Identification:

[0035] 1. Gram staining

[0036] A single colony of the chromium-resistant Lactobacillus salivarius isolated in Example 1 was mixed with 10 μL of sterile PBS solution, spread on a sterile glass slide, quickly fixed with a flame, and after cooling, Gram staining was performed according to the procedure. Observation under a microscope using an oil immersion lens revealed that the bacteria were rod-shaped, bluish-purple, with intense staining at both ends, either alone or in clusters. It was determined to be a Gram-positive bacillus, such as Figure 2 shown.

[0037] 2. 16S rRNA biological identification

[0038] The chromium-resistant Lactobacillus salivarius isolated in Example 1 was cultured in MRS liquid medium at 37°C for 24 hours, and 2 mL of culture medium was taken. The strain DNA was extracted according to the requirements of the Tiangen Bacterial DNA Extraction Kit (No: W9125); and the strain 16S rRNA gene was amplified by PCR using universal primers for the bacterial 16S rRNA gene. The upstream primer sequence was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; the downstream primer sequence was 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'; the product size was 1451; the PCR amplification program was: 95°C: 1 min, 95°C: 30 s, 52°C: 30 s, 72°C: 2 min, repeated 35 cycles; and stored at 4°C.

[0039] 5 μL of the PCR amplification product was subjected to 1% agarose gel electrophoresis, and the remaining PCR product was sent to a biotechnology company for sequencing. The resulting gene sequence is shown as SEQ ID NO. 1. The sequenced gene sequence was compared with the NCBI database (https: / / www.ncbi.nlm.nih.gov) to determine the species of the lactic acid bacteria.

[0040] The sequencing results are shown in the sequence listing (SEQ ID NO. 1): Based on Gram staining and 16S rRNA biological identification, the strain isolated in Example 1 of the present invention was determined to be Lactobacillus salivarius.

[0041] 3 Growth curve determination

[0042] After activating the strain for 24 hours, the strain was inoculated into MRS liquid medium at a 3% inoculum volume. Three replicates were set up and cultured at 37°C. Every 2 hours, the OD600 value was measured at 600 nm using a UV spectrophotometer. The growth curve of chromium-resistant Lactobacillus salivarius was drawn based on the average value of the measurement results. Figure 3 In the first 12 hours, the strain grew slowly, entered the logarithmic phase after 12 hours, and reached its maximum value at 18 hours. From 18 to 36 hours, the strain grew steadily, and after 36 hours, OD 600 The growth curve of Lactobacillus salivarius is shown in Figure 3 .

[0043] Example 2 (maximum chromium tolerance)

[0044] The chromium (VI) resistant Lactobacillus salivarius obtained in Example 1 was inoculated into 5 mL of MRS liquid culture medium at a 1% inoculum volume and cultured for 24 h. The absorbance was adjusted to 1 (OD600), and the mixture was diluted stepwise to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5100 μL of the solution was dripped onto MRS solid culture medium containing 160 mg / L, 320 mg / L, 640 mg / L, and 1280 mg / L of Cr, respectively. The culture was then incubated at 37°C for 24 hours to determine the maximum Cr concentration tolerated by Lactobacillus salivarius. The strain with the highest Cr tolerance was then inoculated onto Cr-free MRS solid culture medium and incubated at 37°C for 24 hours. The results showed that it could tolerate growth at a maximum Cr(VI) concentration of 1280 mg / L.

[0045] Example 3 (Adsorption of chromium ions in solution)

[0046] The Lactobacillus salivarius that embodiment 1 is obtained is inoculated into the MRS liquid nutrient medium shaking culture of 5mL by 1% inoculum size after culturing 24h, with 10000r / min rotating speed centrifugal 10 minutes, remove supernatant, after ultrapure water cleaning precipitation, adjusting absorbance is 1 (OD600), get 0.5mL solution still with 10000r / min rotating speed, centrifugal 10 minutes, remove supernatant, in sedimentation material, add 0.5mL pH respectively and be 3.0,4.0,5.0 and 6.0 50mg / L chromium solution suspension precipitation, 37 ℃, after medium speed concussion 2h, 10000r / min rotating speed, centrifugal 10 minutes, get supernatant, after suitable multiple dilution, with the chromium content in the graphite furnace method determination solution. Utilize formula (1) to calculate the Lactobacillus salivarius chromium adsorption rate.

[0047] Formula (1) is as follows:

[0048] Adsorption rate (%) = 100% - (C1 / C0) × 100%

[0049] Where C0 is the initial chromium solution concentration, and C1 is the final chromium solution concentration in the supernatant.

[0050] The results are shown in Table 1 below

[0051] Table 1 Adsorption rate of chromium solution by Lactobacillus salivarius isolated from Example 1 of the present invention

[0052] pH Adsorption rate (%) 2.0 25.2±0.6 4.0 27.5±0.4 5.0 34.7±2.6 6.0 18.8±0.5

[0053] The results in Table 1 above show that pH significantly affects the strain's adsorption of chromium (VI). At pH values ​​of 2 to 5, the adsorption rate of chromium by Lactobacillus salivarius on a 50 mg / L chromium solution gradually increases, reaching nearly 35% at pH 5.0. As the pH increases further, the adsorption rate decreases, reaching 18.8% at pH 6.0.

[0054] Example 4 (antibacterial properties)

[0055] The chromium-resistant Lactobacillus salivarius obtained in Example 1 was inoculated into 5 mL of MRS liquid culture medium at a 1% inoculum size and cultured for 24 h. The absorbance was adjusted to 1 (OD600). 1 mL of the MRS culture medium was placed in a sterile tube, and 6 mm sterile filter paper pieces were respectively immersed in the MRS culture medium. The absorbance of Staphylococcus aureus, Escherichia coli, and Salmonella was adjusted to 0.08-0.1 (OD600). 50 μL of each of Staphylococcus aureus, Escherichia coli, and Salmonella were respectively evenly spread over the MRS culture medium. The filter paper pieces were respectively spread on bacterial culture plates. Staphylococcus aureus, Escherichia coli, and Salmonella were cultured at 37° C. for 24 h and the results were observed to determine the antibacterial properties of Lactobacillus salivarius.

[0056] The results are shown in Table 2 below

[0057] Table 2 Antibacterial test results of Lactobacillus salivarius isolated from Example 1 of the present invention

[0058]

[0059] The results in Table 2 above show that the Lactobacillus salivarius can inhibit the growth of Staphylococcus aureus, Escherichia coli and Salmonella.

[0060] Example 5 (Bile Salt Tolerance)

[0061] The chromium-resistant Lactobacillus salivarius obtained in Example 1 was inoculated into 5 mL of MRS liquid culture medium at a 1% inoculum volume and cultured for 18 h. The absorbance was adjusted to 1 (OD600), and the mixture was diluted stepwise to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , take 100 μL and drop it onto MRS solid culture plates containing 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1.0% ox bile salts, respectively, and use MRS culture plates without bile salts as controls. Culture at 37°C for 48 hours and observe the results to determine the bile salt concentration that Lactobacillus salivarius tolerates.

[0062] The results showed that the Lactobacillus salivarius could tolerate 0.5% bile salts.

[0063] In summary, the Lactobacillus salivarius isolated in Example 1 of the present invention has a maximum tolerance to chromium (VI) concentration of 1280 mg / L, a chromium adsorption rate of greater than 30% for a 50 mg / L chromium solution (pH = 5), can tolerate 0.5% bile salts, and can inhibit the growth of Staphylococcus aureus, Escherichia coli, and Salmonella.

[0064] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application.

Claims

1. A strain of hexavalent chromium-resistant Lactobacillus salivarius, deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20221599.

2. The use of the hexavalent chromium-resistant Lactobacillus salivarius according to claim 1 in adsorbing chromium (VI) from chromium-contaminated substrates, characterized in that: The chromium-contaminated matrix is ​​animal feed.

3. Use of the hexavalent chromium-resistant Lactobacillus salivarius according to claim 1 in the preparation of a chromium (VI) adsorbent.

4. A chromium (VI) adsorbent, characterized in that: The chromium (VI) adsorbent includes the hexavalent chromium-resistant Lactobacillus salivarius according to claim 1.

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