Bacillus with effects of promoting abalone growth and enhancing immunity

Abstract

The invention provides bacillus capable of promoting abalone growth and enhancing immunity, which is bacillus velezensis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.27210. The bacillus velezensis capable of producing alginate lyase is screened from intestinal tissues of abalones and is compatible with abalone feed, so that cell walls of brown algae can be efficiently cracked, and the digestion and absorption of organisms on effective components in the brown algae are promoted, thereby promoting the growth of the abalones and enhancing the immunity of the abalones.

Description

Technical Field

[0001] The invention belongs to the technical field of marine aquaculture probiotics, and particularly relates to a bacillus strain capable of promoting abalone growth and enhancing immunity. Background Art

[0002] Abalone is an algae-eating marine primitive gastropod. Young abalone mainly eat benthic diatoms, while abalone from 1 cm to adult abalone mainly eats large seaweed, including brown algae such as kelp, wakame, and sargassum. The outer layer of the brown algae cell wall is a large amount of alginic substances, the most common of which is alginate. Hydrolysis will produce brown algae oligosaccharides, which have a variety of biological activities, including immunomodulation, prebiotics, anti-inflammatory, anti-apoptosis, antioxidant and even anti-tumor. After the brown algae wall is broken, the bioactive substances such as phycoxanthin and brown algae polyphenols in the brown algae are more easily absorbed. The Korean Marine Biological Resources Museum and Jeju University found that the loliolide component in copper algae can effectively inhibit the excess of reactive oxygen that causes cell aging damage in the experimental group. By treating abalone feed with special probiotics, alginate is degraded, brown algae oligosaccharides are produced, and more active substances are released at the same time, which is more conducive to abalone digestion and absorption, promotes abalone growth, and enhances abalone immunity. Compared with drugs, probiotics are green and healthy. However, there are no relevant reports on probiotics suitable for abalone at this stage. Summary of the invention

[0003] The invention provides a bacillus with the functions of promoting the growth of abalone and enhancing immunity. The bacillus has the ability to produce alginate lyase and can significantly promote the growth of abalone and enhance immunity.

[0004] The present invention first provides a strain of Bacillus velezensis. The Bacillus velezensis (Bacillus velezensis) HKY-B-20210201 strain was deposited on April 27, 2023 in the General Microbiological Center of China National Microbiological Culture Collection Committee located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 27210.

[0005] The Bacillus Velez subtilis provided by the present invention has a 16SrDNA gene sequence of SEQ ID NO: 1.

[0006] The present invention also provides a use of the screened Bacillus Velezii in the preparation of alginate lyase;

[0007] The present invention also provides another use of the screened Bacillus Velez subtilis, which is use in preparing a product for improving the growth of abalone;

[0008] The product can be prepared into feed additive.

[0009] The invention also provides application of the screened Bacillus Velez in preparing a product for improving the disease resistance of abalone.

[0010] Another aspect of the present invention provides a feed additive for abalone, wherein the feed additive contains live bacteria of screened Bacillus Velezii.

[0011] The invention also provides an abalone feed, wherein the feed additive is added into the feed.

[0012] The present invention also provides a product for improving the immunity of abalone, wherein the product contains live bacteria of screened Bacillus Velezii.

[0013] The product is a probiotic agent.

[0014] The invention obtains a Bacillus Velezii strain producing alginate lyase from the intestinal tissue of abalone, and the Bacillus Velezii strain is matched with abalone feed to efficiently lyse the cell wall of brown algae, promote the digestion and absorption of effective components in the brown algae by organisms, thereby promoting the growth of abalone and enhancing the immunity of abalone. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :Photographs of strain screening;

[0016] Figure 2 : Phylogenetic tree of strain B12. DETAILED DESCRIPTION

[0017] Bacillus is a type of Gram-positive bacteria that can produce spores and have rod-shaped cells. The endospores produced by Bacillus cells enable the bacteria to resist many adverse growth conditions (such as high temperature, low temperature, high salt, etc.).

[0018] The present invention is described in detail below in conjunction with embodiments and drawings.

[0019] Example 1: Screening of target strains

[0020] After cleaning the intestines of fresh abalone, grind them with a sterilized mortar, place them in a conical flask filled with liquid culture medium, and ferment them at 28℃ and 160r / min for 7 days. The liquid culture medium formula is: 10g kelp, 5g peptone, 1g yeast powder, 5g sodium alginate, 24.7g sodium chloride, 6.3g magnesium sulfate, 0.7g potassium chloride, 8.13g magnesium chloride hexahydrate, 1000mL filtered old sea water, pH adjusted to 7.0, and sterilized at 121℃ for 20min.

[0021] Take 1 mL of culture medium with a pipette and transfer it into a test tube containing 9 mL of sterile 9‰ saline to obtain 10 -1 The same method was used to dilute the bacterial solution to obtain 10-2 , 10 -3 , 10 -4 Dilution multiples of bacterial solution.

[0022] After sterilizing the selection medium at 121°C for 20 minutes, cool it to 40-60°C and pour it into a plate. The formula of the selection medium is as follows: 5g peptone, 1g yeast powder, 5g sodium alginate, 24.7g sodium chloride, 6.3g magnesium sulfate, 0.7g potassium chloride, 8.13g magnesium chloride hexahydrate, 1000mL filtered old sea water, 15g nutrient agar, pH adjusted to 7.0, sterilized at 121°C for 20 minutes, cooled to 40°C and poured into a plate.

[0023] After the plate of the selection medium solidifies, take 200 μL of the diluted bacterial solution and spread it on the plate, culture it at 28°C for 24 hours, check the hydrolysis circle formed by the colony, and select the bacteria with larger hydrolysis circle for subsequent experiments. The strain was separated and purified by the three-step streaking method to obtain a single strain. The strain formed white or milky white colonies on the plate medium, with a dense texture and wrinkles on the surface of the colony. The strain was named B12.

[0024] Example 2: Taxonomic identification and physicochemical property analysis of target strains

[0025] The V3-V4 region of the screening strain B12 was amplified using 16SrDNA universal primers, forward primer: 5′-AGAGTTTGATCCTGGCTC AG-3′ (E.coli 27F), reverse primer: 5′-TACGGCTACCTTGTTACGACTT-3′ (E.coli 1492R). The PCR product (about 1.5 kb) was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with CLUSTAL X using the Blast program, and the results showed that strain B12 was closely related to Bacillus velezensis.

[0026] The 16SrDNA gene of B12 (SEQ ID NO: 1) was searched for homology in NCBI. Among the 100 most similar sequences, 20 gene sequences were selected to construct a phylogenetic tree with B12. B12 and Bacillus velez were clustered into one branch with a genetic distance of 0.01, indicating that strain B12 was most closely related to Bacillus velez ( Figure 2 ). Through 16rsDNA analysis combined with physical and chemical properties analysis, it was preliminarily identified as Bacillus velez.

[0027] The B12 strain was named Bacillus velezensis HKY-B-20210201, and was deposited at the General Microbiology Center of China National Microbiological Culture Collection Committee at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on April 27, 2023, with a deposit number of CGMCC No. 27210. In this specification and the accompanying drawings, B12 or strain B12 is used as the code for Bacillus velezensis HKY-B-20210201 strain with a deposit number of CGMCC No. 27210.

[0028] The physiological and biochemical characteristics of strain B12 are shown in Table 1. It can be seen that in addition to producing protease, the strain can also metabolize glucose, D-xylose, L-arabinose, D-mannitol, and hydrolyze starch.

[0029] Table 1: Physicochemical properties of strain B12

[0030]

[0031] Example 2: Determination of alginate lyase activity of strain B12

[0032] The strain was placed in a liquid culture medium (5g peptone, 1g yeast powder, 5g sodium alginate, 24.7g sodium chloride, 6.3g magnesium sulfate, 0.7g potassium chloride, 8.13g magnesium chloride hexahydrate, 1000mL filtered old seawater, pH adjusted to 7.0, sterilized at 121℃ for 20min), and placed at 28℃, 180r / min shaking fermentation for 24d. After the bacterial solution was centrifuged at 10000rpm for 10min at 4℃, the supernatant was taken as the crude enzyme solution. The enzyme activity was determined by ultraviolet spectrophotometry. 400μL of 5g / L sodium alginate solution (solvent was 10mmol / LTris-HCl buffer) was taken, 100μL of crude enzyme solution was added, and after reacting in a 40℃ water bath for 20min, 500μL DNS was added to terminate the enzymatic reaction, boiling water bath for 10min, cold water cooled to room temperature, and the light absorption value was detected at 540nm to determine the amount of reducing sugar generated. The inactivated enzyme solution was used as the blank control group. The enzyme activity unit (U) is defined as the amount of enzyme required to degrade the substrate into 1 μmol reducing sugar per unit time at a certain temperature. The final determination of the alginate lyase activity in the fermentation broth was 41±3.86U / mL.

[0033] Example 3: Effects of strain B12 on growth and immunity of abalone

[0034] The abalone was placed in the water tank for 5 days before the experiment began. There were two treatments in the experiment, namely the control group and the experimental group. The control group was fed with ordinary abalone seedlings compound feed (containing brown algae), and the experimental group was fed with mixed feed (ordinary abalone seedlings compound feed mixed with 10% bacterial liquid). Each group had 5 replicates, with a total of 10 tanks. About 10 abalones of the same size were stocked in each well. After 30 days of cultivation, 5 abalone individuals were randomly selected from each replicate to measure the corresponding growth and immune indicators. The experiment can be adjusted according to the results of the previous experiment.

[0035] Hemolymph collection: Transfer 5 abalones from each replicate to a sterile culture dish. Blood was collected by cutting the ventral foot. After absorbing the water on the surface of the abalone ventral foot with absorbent paper, gently cut the abalone ventral foot with a scalpel, and then use a 0.2mL sterile pipette to absorb the hemolymph overflowing from the wound, and quickly place the hemolymph in a pre-cooled 1.5mL centrifuge tube. The abalone hemolymph in each replicate was 1 sample, and then an equal volume of PBS solution (pH 7.4) was added, mixed, and centrifuged at 3000r / min and 40℃ for 10min. The supernatant was stored at -80℃ for the detection of abalone immune and antioxidant indicators. The results are shown in Table 2.

[0036] The results showed that the daily increase in the body weight of abalone in the test group with bacterial liquid added was greater than that in the control group, and the activities of immune enzymes such as alkaline phosphatase, acid phosphatase, lysozyme, catalase in blood lymphocytes, and superoxide dismutase in the test group were higher than those in the control group. It can be seen that strain B12 can promote the growth of abalone and enhance its immunity to a certain extent.

[0037] Table 2: Effects of strain B12 on abalone growth and immunity

[0038]

[0039] Example 4: Disease resistance analysis of strain B12

[0040] Cultivation test: Abalones were placed in a water tank for 5 days before the test was started. The test was divided into three groups: an experimental group, a control group, and a blank group. The control group and the blank group were fed with ordinary abalone seedling compound feed, and the experimental group was fed with a mixture (ordinary abalone seedling compound feed with 10% bacterial solution added). Each group was repeated 3 times, for a total of 9 tanks. About 10 abalones of the same size were stocked in each tank, and the cultivation period was 20 days.

[0041] Virus infection test: The activated pathogenic Vibrio was cultured in nutrient broth medium at 28°C for 24 h, and the concentration was adjusted to 10 8cfu / mL. In the challenge test, each head of the test group and the control group was injected with a syringe into the middle and lower part of the ventral foot muscle twice with a dose of 0.1 mL of pathogenic Vibrio dilution, and the blank group was injected with the same amount of sterile saline. The daily mortality was recorded, and the cumulative mortality was calculated after 20 days. The calculation formula is as follows: cumulative mortality / % = cumulative number of deaths / initial number × 100.

[0042] Table 3: Data table of disease resistance test results of strain B12

[0043]

[0044] The results show that the strain B12 screened by the present invention has obvious disease resistance.

Claims

1. A Bacillus Velez, characterized in that The deposit number of the Bacillus Velez is CGMCC No.27210.

2. The Bacillus Velezii according to claim 1, characterized in that The sequence of the 16SrDNA gene of the Bacillus Velez is SEQ ID NO:

1.

3. Use of the Bacillus Velezii described in claim 1 in the preparation of alginate lyase.

4. Use of the Bacillus Velezii according to claim 1 in preparing a product for improving the growth of abalone.

5. The use according to claim 4, characterized in that The product is a feed additive.

6. Use of the Bacillus Velezii described in claim 1 in preparing a product for improving the disease resistance of abalone.

7. A feed additive for abalone, characterized in that: The feed additive contains the live bacteria of Bacillus Velezii described in claim 1.

8. An abalone feed, characterized in that: The abalone feed contains the feed additive according to claim 7.

9. A product for improving the immunity of abalone, characterized in that: The product contains the live bacteria of Bacillus Velezii according to claim 1.

10. The article according to claim 9, characterized in that The product is a probiotic agent.

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