CN117165480B - Bacillus verdans capable of producing DDP-IV inhibitor and siderophore and application thereof - Google Patents

Abstract

The invention discloses a bacillus verdans capable of producing a DDP-IV inhibitor and a siderophore and application thereof, and relates to a strain and application thereof. The invention provides a DDP-IV inhibitor and siderobacillus ferrocarrier, which is the siderobacillus verdans (Bacillus wiedmannii) LSQ6 and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 28085. The bacillus verdans (Bacillus wiedmannii) LSQ6 of the invention is a strain which has both hypoglycemic and siderophore production. When the bacillus verdans (Bacillus wiedmannii) LSQ6 disclosed by the invention is added into a rice field in a rice-crab comprehensive planting and breeding mode, the feed digestion utilization rate of procambarus clarkia can be improved, and the antibacterial effect is generated.

Description

A strain of Bacillus Wiedemannii producing DDP-IV inhibitor and siderophore and its application

Technical field

The present invention relates to a bacterial strain and its use.

Background technique

Procambarus clarkii, commonly known as crayfish, belongs to the phylum Arthropoda, the class Crustacea, the order Decapoda, the family Procambarus, and the genus Procambarus. It has omnivorous habits, such as burrowing and molting. The rice-shrimp co-cropping model is a composite ecosystem based on latent rice fields, centered on rice cultivation, and characterized by the return of rice straw to the fields for culture of Procambarus clarkii. This model makes full use of the shallow water environment and winter slack period of rice fields, organically combines planting and breeding, and achieves the goal of "dual uses of water and double harvests in one field" and has good economic and social benefits.

As people's living standards continue to improve, food safety requirements are becoming more stringent. In the feed link, which is the source of safety in aquatic product breeding, microbial fermented feed, as a new type of ecologically healthy feed, has very broad prospects. The output value of Procambarus clarkii will exceed 400 billion yuan in 2021, and its new and healthy breeding is still in its infancy. The use of beneficial bacteria to ferment feed, improve feed quality and increase feed utilization is an important direction of current research. Wang Tianshen’s research shows that adding 25% to 35% probiotics to feed can improve the growth performance of Procambarus clarkii; probiotics in biofermented feed can optimize the microbial flora in the intestine, increase digestive enzyme activity, and improve feed utilization. , promote the absorption of nutrients and enhance the specific immunity and disease resistance of Procambarus clarkii. Studies by Xu Zenghong and others found that biological feed has a significant impact on the growth and gonad maturation and development of Procambarus clarkii (P<0.05), indicating that biological feed has a good feeding effect on aquatic animal breeding such as Procambarus clarkii. It also has the ability to accelerate The growth rate of shrimps and crabs and the effect of improving the quality of commercial shrimps and crabs.

The main physiological function of sugar is to provide energy and is the cheapest feed energy. It is generally believed that aquatic animals have a low ability to utilize sugar, and aquatic animals are considered to have a congenital "diabetic constitution." Therefore, an important topic in the field of aquatic animal nutrition research is how to improve the utilization of feed sugars.

Contents of the invention

The invention provides a strain of Bacillus Wiedemannii that produces DDP-IV inhibitors and siderophores and its application.

The Bacillus wiedmannii that produces DDP-IV inhibitors and siderophores of the present invention is Bacillus wiedmannii (Bacillus wiedmannii) LSQ6, which is deposited in the General Microorganism Center of the China Microbial Culture Collection Committee, and the deposit number is CGMCC No.28085.

The use of the above-mentioned DDP-IV inhibitor and siderophore-producing Bacillus Wiedemannii in the rice-shrimp co-culture model of the present invention.

In the present invention, Bacillus wiedmannii LSQ6 bacterial liquid that produces DDP-IV inhibitors and siderophores is added to rice fields in a rice-shrimp co-cropping model to promote the growth of Procambarus clarkii.

The colonies of Bacillus wiedmannii LSQ6 of the present invention on the LB solid culture medium are milky white, the bacterial cells are larger in size, slightly convex in the center, round, translucent, and the surface is moist, smooth, shiny, and sticky. , Gram-positive bacteria after Gram staining.

Bacillus wiedmannii LSQ6 can produce siderophores with high iron chelation ability, and the Su value of siderophores produced in a 37°C environment is 47%. Under iron-deficient conditions, Bacilluswiedmannii LSQ6 can produce higher-affinity siderophores, which absorb and utilize iron in the environment, thereby acting on themselves and animals and plants. When interacting with pathogenic bacteria When competing for limited iron nutrition, it will inhibit the growth and reproduction of pathogenic bacteria, thereby achieving the biological control effect on Procambarus clarkii and rice, improving the immune defense ability of Procambarus clarkii, and reducing the presence of pathogenic bacteria in the intestinal flora of Procambarus clarkii. proportion.

In a 37°C culture environment, the hydrolysis circle diameter D of milk protein of Bacillus wiedmannii LSQ6 on SKM solid medium is 42.56mm, the colony diameter d is 13.29mm, and D/d is 3.20; at 37°C The yield of DDP-IV inhibitor in the environment is as high as 52.83%. Bacillus wiedmannii LSQ6 can produce DDP-IV inhibitors, which can control the blood sugar of Chinese mitten crabs and better absorb sugars in the feed, especially some non-starch polysaccharides; thus it can improve Improve feed digestion and utilization and enhance growth performance.

Bacillus wiedmannii LSQ6 of the present invention is a strain that has both the ability to lower blood sugar and produce siderophores. Adding Bacillus wiedmannii LSQ6 of the present invention to the rice fields of the comprehensive rice crab cultivation model can improve the digestion and utilization rate of Procambarus clarkii feed and produce antibacterial effects.

The ability to secrete IAA is considered to be a characteristic of rice growth-promoting bacteria with high colonization efficiency, and is closely related to whether the growth-promoting strain can stably exert its growth-promoting effect in the plant and its colonization efficiency. The Bacillus wiedmannii LSQ6 of the present invention contains 35.78 μg IAA per milliliter of fermentation liquid and has high IAA secretion, which will help to improve the effect of comprehensive cultivation of rice crabs and has the ability to produce auxin. Plants have growth-promoting effects.

Bacillus wiedmannii LSQ6 has a certain tolerance to acidity and extremely high tolerance to bile salts at 37°C; it can survive and play its role in the intestine of Procambarus clarkii. The Bacillus wiedmannii LSQ6 strain is safe and reliable and does not cause the death of Procambarus clarkii. Compared with the control, the feed coefficient of the Bacillus wiedmannii LSQ6 group was significantly lower than that of the control group. The weight gain rate and specific growth rate of the treatment group were significantly higher than those of the control group, and Bacillus wiedmannii LSQ6 could significantly improve feed utilization.

Bacillus wiedmannii LSQ6 is Bacillus wiedmannii, belonging to the genus Bacillus; it is deposited at the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures, and the deposit address is Chaoyang District, Beijing No. 3, No. 1, Beichen West Road, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No. 28085, and the deposit date is August 2, 2023.

Description of the drawings

Figure 1 is a graph showing the results of the siderophore screening test of Bacillus wiedmannii LSQ6 on CAS solid detection medium;

Figure 2 is a diagram showing the results of the hydrolysis test of milk protein by Bacillus wiedmannii LSQ6 on SKM culture medium;

Figure 3 is a graph showing the results of measuring the IAA-producing ability of Bacillus wiedmannii LSQ6 using the Salkowski colorimetric method;

Figure 4 is a phylogenetic tree constructed from Bacillus wiedmannii LSQ6.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without any creative work fall within the scope of protection of the present invention.

It should be noted that, as long as there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

Specific Embodiment 1: This embodiment produces DDP-IV inhibitor and siderophore Bacillus wiedmannii, which is Bacillus wiedmannii (Bacillus wiedmannii) LSQ6 and is deposited in the General Microorganisms Committee of China Microorganism Culture Collection and Management Committee. Center, the collection number is CGMCC No.28085.

In May 2023, a population of Procambarus clarkii was selected from the Hulan Fisheries Experimental Field of the Heilongjiang Fisheries Research Institute of the Chinese Academy of Fishery Sciences, and 300 healthy Procambarus clarkii (25.0±1.87) g were randomly selected, filled with water and packed with air, and then transported to Northeast Agricultural University College of Animal Science and Technology, selected 20 Procambarus clarkii with normal body color and active reaction, and anesthetized them with MS-222 anesthetic (250mg/L). Use absolute ethanol to wipe the surface of Procambarus clarkii, dissect it with sterilized scissors and tweezers on a clean workbench, take out the entire intestine of Procambarus clarkii, gently squeeze out the contents and place it in a glass bead. and 50 mL of sterile water in a conical flask, shake at room temperature for 30 minutes at a speed of 180 r/min, and then perform gradient dilution. Take 100 μL of the gradient of 10 ^-3 , 10 ^-4 , and 10 ^-5 and spread it on the LB solid culture medium plate. , each gradient was repeated three times, and cultured at 28°C for 24 to 48 hours. After culturing for 48 hours, strains of different shapes were selected for isolation and purification, and strain LSQ6 was obtained.

The colony isolate of strain LSQ6 was inoculated into R ₂ A liquid medium containing L-tryptophan and placed in a constant temperature shaker at 28°C for 4 days with shaking at 180 r/min. Pipette 500 μL of bacterial suspension into a 2 mL glass bottle, and add 500 μL of Salkowski colorimetric solution. At the same time, 500 mg/L IAA was added to Salkowski colorimetric solution as a positive control. Store the 2 mL glass bottle at room temperature in the dark for 30 minutes and observe the color change. If the color turns red, it means that the strain has the function of producing IAA. After adding Salkowski colorimetric solution dropwise to the bacterial suspension of strain LSQ6, the color in the 1mL glass bottle turned red at room temperature in the dark (as shown in Figure 3); this shows that strain LSQ6 has the ability to produce auxins and has certain effects on plants. Growth-promoting effect.

Accurately weigh 10 mg of IAA, first dissolve it with a small amount of absolute ethanol, then add distilled water to adjust the volume to 100 mL, prepare an IAA solution with a concentration of 100 μg/mL as a stock solution, and then dilute the stock solution to a concentration of 0 (blank ), 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0μg/mL series of standard solutions as working solutions. Take 2 mL of the above working solution and add it to 8 test tubes in sequence, add 2 times the volume of Salkowski colorimetric solution, incubate it at 40°C in the dark for 30 minutes, and then measure the absorbance value at a wavelength of 530 nm. With OD ₅₃₀ as the abscissa and IAA concentration as the ordinate, draw the IAA standard curve. The colony isolate of strain LSQ6 was inoculated into the R ₂ A liquid medium containing L-tryptophan, placed in a constant temperature shaker at 28°C and cultured at 180r/min for 4 days, and then the bacterial suspension at a wavelength of 600nm was measured spectrophotometrically. Then take the bacterial suspension and centrifuge it at 10000r/min for 10 minutes. Add the supernatant to an equal volume of Salkowski colorimetric solution, place it in the dark at 40°C for 30 minutes to develop color, and measure the OD value at a wavelength of 530nm. . Calculate the concentration of IAA in unit volume of fermentation broth when the OD ₆₀₀ value is 1 (if the concentration of the bacterial broth is too high, dilution is required). The measurement results showed that strain LSQ6 contained 35.78 μg IAA per milliliter of fermentation broth, and strain LSQ6 had strong IAA secretion ability.

Then, the purified strain LSQ6 was reactivated and transferred to an LB plate and cultured for 24 hours, and then a single colony was picked with a sterilized toothpick and transferred to Chromeazurol S (CAS) solid detection medium, and cultured upside down at 37°C for 2 ~3d, it was observed that an obvious discoloration circle formed around the colony of strain LSQ6 (as shown in Figure 1). The colony diameter d and the discoloration circle diameter D were measured using the cross method. The discoloration circle diameter D of strain LSQ6 was 6.90mm, and the colony diameter d is 2.16mm, and D/d is 3.19, indicating that strain LSQ6 has extremely strong production of siderophores with high iron chelation ability.

Further testing on strain LSQ6:

(1) Inoculate the activated bacterial strain LSQ6 lawn into iron-limited SA liquid medium and culture it on a shaking table at 37°C for 48 hours;

(2) Transfer the bacterial suspension to be tested that has grown for 48 hours into a sterilized 10mL centrifuge tube, and centrifuge at 13,000 rpm for 15 minutes;

(3) Transfer the supernatant to a test tube treated with concentrated hydrochloric acid, add a certain amount of freshly prepared CAS detection solution to make the volume ratio of supernatant to detection solution 1:1, mix thoroughly and let stand at room temperature for 1 hour. ;

(4) Measure the absorbance value (A) at a wavelength of 630 nm, use double-distilled water as a control for zero adjustment, and use the absorbance value at a wavelength of 630 nm after mixing the uninoculated SA iron-limited culture medium and the test solution measured in the same method as the reference value. (Ar), and the siderophore activity unit is represented by the following formula:

Su≈(Ar-As)/Ar×100;

In the formula: Su is the siderophore content; Ar is the OD value of the tested uninoculated SA iron-limited culture medium and the supernatant of the test solution; As is the OD value of the tested culture medium supernatant. (When the siderophore activity unit is less than 10, it is generally considered negative, and the mixture of siderophore and detection solution has no color change.)

It was measured through experiments that the Su value of siderophore production by strain LSQ6 at 37°C was 47%, indicating that this strain has strong siderophore production ability.

Strain LSQ6 is coated on the SKM (2% skimmed milk powder medium) medium plate to produce a transparent circle, and an obvious hydrolysis circle is formed around the colony of strain LSQ6 (as shown in Figure 2). The diameter D of the hydrolysis circle of strain LSQ6 is 42.56mm. , the colony diameter d is 13.29mm, and D/d is 3.20. Centrifuge the cultured 2nd generation strain LSQ6 bacterial liquid at 8000r/min for 15 minutes to remove the bacterial sludge remaining in the supernatant. Wash three times with 0.1 mol/L sterile phosphate buffer solution (PBS, pH 6.8), resuspend the bacteria in PBS, and adjust the absorbance value of the bacterial solution to 1.0. Incubate at 37°C for 24 hours, and then incubate at 4°C. Centrifuge at 8000r/min for 15min, filter the supernatant through a 0.22pm water-based membrane filter to obtain cell metabolites (CFS), and store at -80°C. Further test: In a 96-well plate, accurately drop 25 μL of 1.6 mmol/L glycyl-prolyl-p-nitroanilide and 25 μL of CFS. React at 37°C for 15 minutes, then add 50 μL, 0.01 U/mL DDP-IV, continue the reaction at 37°C for 1 hour, finally add 100 μL, 1 mol/L sodium acetate buffer solution (pH=4.0) to terminate the reaction, and use a microplate reader to read at 405 nm Detect the absorbance value of the reaction solution. DDP-IV inhibition rate calculation formula:

A _{sample to be tested} : 25 μL sample + 25 μL Gly-pro-phy + 50 μL DDP-IV + 100 μL sodium acetate;

A _{sample blank} : 25 μL sample + 50 μL Tris-HCl + 25 μL Gly-pro-phy + 100 μL sodium acetate;

A _{negative control} : 25μL Tris-HCl+25μL Gly-pro-phy+50μL DDP-IV+100μL sodium acetate;

A _{negative blank} : 75μL Tris-HCl+25μL Gly-pro-phy+100μL sodium acetate.

The DDP-IV inhibitor production rate of strain LSQ6 was measured to be as high as 52.83% at 37°C, indicating that strain LSQ6 has a strong ability to produce DDP-IV inhibitors.

Stress resistance test of strain LSQ6:

Acid resistance performance test: Inoculate strain LSQ6 into LB liquid culture medium with pH of 2.0, 2.5, and 3.0 at an inoculation amount of 2% (v/v), and draw the bacterial liquid and apply it on the plate at 0, 1, 2, and 3 hours respectively. 37 After incubation at ℃ for 24 hours, the number of viable bacteria was measured.

Bile salt resistance test: Dilute the activated strain LSQ6 with sterile saline, select an appropriate dilution gradient, and place 1 mL of the dilution into a sterilized plate. Then pour the plate with LB solid medium containing 0.3% and 1.0% sodium taurocholate, and use MRS solid medium without sodium taurocholate as the control group. Incubate at 37°C for 48 hours, count the colonies, and calculate the number of strains. survival rate.

The test results are shown in Table 1. When the pH is 4.0, it has little effect on the number of spores of strain LSQ6. When the pH is 2.0, the number of spores of strain LSQ6 decreases significantly. When the bile salt concentration increased, the number of spores of strain LSQ6 changed slightly. This shows that strain LSQ6 has a certain tolerance to acidity and extremely high tolerance to bile salts.

Table 1

Physiological and biochemical identification of strain LSQ6: The preserved strain LSQ6 was streaked in three areas on a solid LB medium plate, a single colony was isolated and its morphology was described, and the strain was Gram stained according to the "Manual for the Identification of Commonly Used Bacterial Systems" and physiological and biochemical identification.

The colonies of strain LSQ6 are milky white, with larger bacterial cells, slightly convex center, round shape, translucent, moist and smooth surface, shiny and sticky, and are Gram-positive bacteria after Gram staining. Some physiological and biochemical indicators of strain LSQ6 are shown in Table 2. According to the description of the physiological characteristics of Enterobacteriaceae in the Bergey's Bacterial Identification Manual, strain LSQ6 has the same physiological and biochemical characteristics as the model species Bacillus wiedmannii. It is inferred from various physiological and biochemical characteristics that strain LSQ6 may be Bacillus wiedmannii.

Table 2

Identification of 16S rRNA of strain LSQ6: Use the bacterial genomic DNA extraction kit from Beijing Solebao Biotechnology Company to extract the isolated and purified strain DNA. Use bacterial universal primer 27F/1492R for PCR amplification. The PCR amplification system is a 25 μL system: 2.5 μL of 10× buffer, 0.5 μL of Taq enzyme, 0.5 μL of primer 27F, 0.5 μL of primer 1492R, 1 μL of DNA template, and 20 μL of ddH ₂ O . The reaction program was set as pre-denaturation at 95°C for 5 min; denaturation at 94°C for 50 s, annealing at 56°C for 30 s, extension at 72°C for 1.5 min, 30 cycles, extension at 72°C for 10 min, and storage at 4°C. The PCR amplification products were sent to RuiBiotech for sequencing. The 16S rRNA sequencing results of the strains were compared through the NCBI database, and a phylogenetic tree was constructed (as shown in Figure 4). A BLAST comparison in NCBI found that the 16S rRNA gene sequence of strain LSQ6 was 99% similar to Bacillus wiedmannii. From the phylogenetic tree of strain LSQ6, it can be seen that strain LSQ6 and Bacillus wiedmannii (NR 152692.1) are in the smallest branch and have a close evolutionary distance. Based on the comprehensive physiological and biochemical indicators, the LSQ6 strain was identified as Wiedmannii. Bacillus wiedmannii.

Example 1

Effect of Bacillus wiedmannii LSQ6 on the growth performance of Procambarus clarkii.

Procambarus clarkii breeding experiments were conducted in the circulating pool of the laboratory of the Heilongjiang Fisheries Research Institute of the Chinese Academy of Fisheries Sciences. The Procambarus clarkii used in the experiments were all sourced from the Hulan Fisheries Experimental Site of the Heilongjiang Fisheries Research Institute of the Chinese Academy of Fisheries Sciences. Select 200 Procambarus clarkii with an initial weight of about 25.0±1.87g that year and raise them in a 4.3m×1.5m×1.0m cement tank (water depth 10cm) with a large number of aquatic plants as shelter for one week. In order to adapt to the experimental environment, the control group feed ( Special expanded feed for shrimp). After temporary breeding, 120 Procambarus clarkii with similar weight, good vitality and no body surface injuries were selected and randomly divided into 2 groups (control group and treatment group), with 3 parallel groups in each group, and 20 tails in each parallel group were placed in 6 In a 70cm × 80cm × 40cm circulating water breeding tank, the water body height is 10cm and equipped with a large number of aquatic plants as shelters. Tap water aerated for more than 24 hours is used as the test water body. During the test period, an aeration pump is used to aerate 24 hours a day at 08:00 every day. 00 and 16:00 feeding.

The treatment group was fed with Bacillus wiedmannii LSQ6 bacterial liquid mixed with 1.5×10 ⁶ CFU/mL. Special expanded feed for shrimp, the bacterial liquid and feed are fully mixed before feeding (the material-to-liquid ratio is 1:0.1), and all the bacterial liquid is adsorbed on the feed. The control group was fed the control group feed (/> Special extruded feed for shrimp); feeding twice a day, apparently full, sucking the bottom once a day, changing the water once a day, the water change amount is about 1/2, the water temperature during the test was (23±1)℃, the dissolved The oxygen is (6±0.5) mg/L, the pH is 7.1±0.5, and the breeding cycle is 7 days. Observe and record whether the appendages are complete at 09:00 every day, and calculate the mortality rate and residual limb rate; after the breeding, weigh the Procambarus clarkii in each treatment and calculate the average weight, weight growth rate, and feed coefficient. The specific calculation formula is as follows :

Weight growth rate=[(W ₁ -W ₀ )/W ₀ ]×100%

Mortality rate=D/Z×100%

Residual limb rate=C/Z×100%

Feed coefficient=W ₂ /(W ₁ -W ₀ )×100%

Among them, W ₀ is the initial weight, W ₁ is the final weight, W ₂ is the feed consumption, D is the number of deaths in each treatment within 24 hours, C is the number of shrimp tails with missing appendages in each treatment within 24 hours, and Z is the number of shrimp tails with missing appendages in each treatment within 24 hours. The number of tails of Procambarus clarkii released in each treatment.

The experimental results are shown in Table 3. From Table 3, it can be seen that there is no significant difference in the mortality rate and limb loss rate of Procambarus clarkii among the groups, indicating that Bacillus wiedmannii LSQ6 does not affect the survival rate of Procambarus clarkii. The mortality rate and limb loss rate of Procambarus crayfish are safe and reliable. The feed coefficient of the treatment group was significantly lower than that of the control group, and the weight growth rate of the treatment group was significantly higher than that of the control group, indicating that Bacillus wiedmannii LSQ6 can significantly improve feed utilization.

table 3

In this example, 1000ml of Bacillus wiedmannii LSQ6 culture medium consists of 40g of sucrose, 12g of yeast extract, 7.8g of casein peptone, 18g of sodium nitrate, 2.7g of zinc sulfate and the remainder. of distilled water.

Inoculate the Bacillus wiedmannii (Bacillus wiedmannii) LSQ6 seed liquid into the Bacillus wiedmannii (Bacillus wiedmannii) LSQ6 bacterial liquid culture medium at an inoculation amount of 0.5%, under the conditions of 37°C, 180r/min, and pH 7. After 24 hours of culture, the number of viable bacteria in the Bacillus wiedmannii LSQ6 bacterial solution reached 1.73×10 ⁸ CFU/mL. The number of viable bacteria before optimization was 5.49×10 ⁷ CFU/mL (the unoptimized medium was bean sprout juice medium, bean sprout juice medium: 100 mL bean sprout juice, 10 g sucrose, (NH ₄ ) ₂ SO ₄ 2 g, NaCl 0.4 g, ZnSO ₄ 0.08 g, distilled water to 1000mL, pH 7, autoclave at 115°C for 30 minutes).

Claims (2)

1. A strain of Bacillus wiedmannii that produces DDP-IV inhibitors and siderophores. It is Bacillus wiedmannii ( Bacillus wiedmannii) LSQ6. It is deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. The deposit number is It is CGMCC No.28085. 2. The use of Bacillus Wiedemannii producing DDP-IV inhibitors and siderophores according to claim 1 in a rice-shrimp co-cropping model.