CN117448199A - Shrimp-derived bacillus coagulans and application thereof - Google Patents

Shrimp-derived bacillus coagulans and application thereof Download PDF

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Publication number
CN117448199A
CN117448199A CN202311190679.0A CN202311190679A CN117448199A CN 117448199 A CN117448199 A CN 117448199A CN 202311190679 A CN202311190679 A CN 202311190679A CN 117448199 A CN117448199 A CN 117448199A
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bacillus coagulans
cultivation
aquatic animals
aquatic
application
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徐洋
程海华
彭菲
高强
李喜莲
慎佩晶
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Zhejiang Institute of Freshwater Fisheries
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Zhejiang Institute of Freshwater Fisheries
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/07—Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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Abstract

The invention discloses a shrimp-derived bacillus coagulans and application thereof, belonging to the technical field of microorganisms, wherein the preservation number of the bacillus coagulans is CGMCC No.26707. The bacillus coagulans mr-1B obtained by separation has sensitive antibacterial activity on main pathogenic bacteria in aquatic animal cultivation, can improve the immune function of aquatic animals, improve the health level of the animals, reduce the content of ammonia nitrogen and nitrite in a cultivation water body, improve the ecological environment of cultivation, enable the benign development of cultivation production, improve the effect of the aquatic cultivation, and obtain better economic benefit and ecological benefit; and the bacillus coagulans mr-1B has the advantages of strong stress resistance, high temperature resistance, easy preservation, high survival rate and the like, and widens the popularization and application value of the lactobacillus probiotics in the feed industry.

Description

Shrimp-derived bacillus coagulans and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to shrimp-derived bacillus coagulans and application thereof.
Background
The most common problems in intensive cultivation are diseases and water quality, and common solutions are injection of immune vaccines, addition of immune promoters and microecologics to feeds. However, the vaccine is hindered to develop due to the complicated operation; immune promoters are also limited to animal body immunopotentiation and have little effect on water body changes. The lactobacillus probiotics are a safe microorganism strain with better effect at present. It has the following characteristics: acid resistance, can grow at pH 3.0-4.5, and is suitable for gastrointestinal acid environment; lactic acid bacteria are normal flora of animal intestinal tracts, colonize in intestinal systems, antagonize gram negative pathogenic bacteria in vivo by biological antagonism, and maintain normal ecological balance in the intestinal tracts; can lower intestinal pH value, prevent and inhibit harmful substances, and enhance anti-infection ability; the metabolite in living bacteria contains higher superoxide dismutase (SOD), can increase humoral immunity and cellular immunity of animals, strengthen immunoregulatory activity of organisms and promote growth.
However, common lactic acid bacteria do not form spores in the growth process, have poor stress resistance, are easy to inactivate and difficult to preserve, so that the application in the feed industry is limited to a certain extent.
Disclosure of Invention
The invention aims to provide the shrimp-derived bacillus coagulans and the application thereof, so as to solve the problems in the prior art, and the bacillus coagulans mr-1B can improve the immune function of aquatic animals, improve the health level of the animals, reduce the content of ammonia nitrogen and nitrite in a culture water body, improve the ecological environment of culture, enable the benign development of culture production, improve the effect of aquaculture, and obtain better economic benefit and ecological benefit.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides bacillus coagulans (Bacillus coagulans) mr-1B which is preserved in 2023 and 02 and 27 days to China general microbiological culture Collection center (CGMCC) No.26707, and the preservation address is North Chen Xili No. 1 and 3 in the Korean region of Beijing city.
The invention also provides an application of the bacillus coagulans mr-1B, which comprises any one of the following applications:
s1, application in improving the water quality of cultivation;
s2, application in preparation of broad-spectrum antibacterial agents;
s3, application of the composition in preparation of products for enhancing immune functions of aquatic animals.
Further, the bacillus coagulans mr-1B improves the culture water quality by reducing the content of ammonia nitrogen and nitrite in the culture water body.
Further, the bacillus coagulans mr-1B inhibits vibrio harveyi, aeromonas veronii, enterobacter cloacae and aeromonas hydrophila and exerts broad-spectrum antibacterial effects.
Further, the bacillus coagulans mr-1B exerts an effect of enhancing immune function by enhancing AKP, POD, POX and amylase activities of aquatic animals.
The invention also provides a microecological preparation for promoting the intensive culture of aquatic animals, which comprises bacillus coagulans mr-1B.
Further, a bait is also included.
The invention also provides a method for promoting the intensive culture of aquatic animals, which comprises the step of adding the bacillus coagulans mr-1B or the microecological preparation into a culture water body.
Further, the bacillus coagulans mr-1B or the microecological preparation can promote the intensive culture of aquatic animals by inhibiting aquatic pathogenic bacteria, enhancing the immune function of the aquatic animals, improving the water quality of the aquaculture.
The invention discloses the following technical effects:
the bacillus coagulans mr-1B obtained by separation has sensitive antibacterial activity on main pathogenic bacteria in aquatic animal cultivation, can improve the immune function of aquatic animals, improve the health level of the animals, reduce the content of ammonia nitrogen and nitrite in a cultivation water body, improve the ecological environment of cultivation, enable the benign development of cultivation production, improve the effect of the aquatic animal cultivation, and obtain better economic benefit and ecological benefit.
In addition, the bacillus coagulans used by the invention is a kind of lactobacillus capable of producing spores, has the characteristics of the lactobacillus, has the advantages of strong stress resistance, high temperature resistance, easy preservation, high survival rate and the like, and widens the popularization and application value of the lactobacillus probiotics in the feed industry.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromosome circle diagram of the whole gene of Bacillus coagulans mr-1B.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1. Separation, identification and safety evaluation of bacillus coagulans
1.1 isolation and purification of Strain
Diluting the liver and pancreas tissue homogenate of macrobrachium rosenbergii with sterile normal saline by 10 times dilution method, and taking 10 -5 ～10 -8 The dilution of the dilution range was spread on LB medium plates. Culturing at 30deg.C for 48 hr, picking typical single colony, and repeatedly streaking to obtain pure strain mr-1B.
1.2 Whole genome sequencing analysis and safety assessment
Collecting bacteria by adopting a logarithmic centrifugation method, and fully grinding by using liquid nitrogen; adopting SDS lysate to carry out pyrolysis, adding a proper amount of proteinase K and mercaptoethanol, and slightly reversing the pyrolysis process combination; cooling to room temperature after cracking and centrifuging; extracting the supernatant with chloroform/isoamyl alcohol (24:1), and repeating the extraction twice; precipitating the DNA with isopropanol, mixing slightly up and down, for centrifugation; removing waste liquid, washing the precipitate with 75% ethanol, and washing twice; purifying by a purification column (OMEGA); after purification, purification was performed with Amure XP beads; DNA quality control tests were performed using Nanodrop, qbuit and gel electrophoresis.
The DNA sample detection instrument is a NanoDrop One spectrometer (NanoDrop Technologies, wilmington): qubit 3.0 fluorometer (Life Technologies, karlsbard).
The construction of the sequencing library used the same equipment as the DNA sample detection equipment described above. The main kits for library construction are SQK-LSK109 and EXP-NBD104/114. The sequencing platform is PromethION (oxford nanohole technologies, oxford, UK).
The library construction and sequencing steps were as follows: 2.5 μg of qualified DNA sample and 1 x magnetic beads are purified, and 1 μl of sample is subjected to qubit quantification; damage and terminal repair.
Culture conditions: preserving at 65 ℃ for 40 minutes, preserving at 20 ℃ for 20 minutes, and preserving at 4 ℃; magnetic bead purified DNA,25 μl EB eluted DNA,1 μl qubit quantitated sample; bar code label ligation (700 ng of repair DNA per sample).
Culture conditions: incubation at 25℃for 30 min; magnetic bead purification, 25 μl EB eluted DNA,1 μl qubit quantification; a common library. The labeled samples were mixed in the same ratio to form a total volume of 65. Mu.l and a total volume of 2.5. Mu.g of library. The sequencing connector is connected.
Culture conditions: incubation at 25℃for 30 min; the DNA was purified using 0.5 Xmagnetic beads, eluted with 25. Mu.l elution buffer (SQK-LSK 109) and Qubit eluted with 1. Mu.l sample. And configuring a sequence operation library and operating the sequence. The library was loaded into an R9.4 sequencing chip and sequenced for 48-72h using a PromethION sequencer (Oxford Nanopore Technologies, oxford, UK).
The information analysis mainly comprises the following steps of controlling the quality of original data, and filtering low-quality and too short reading; genome assembly-filtering reads from head assembly and assembling sketch genomes is error correction; genomic structural analysis including repeat sequences, coding genes, non-coding RNAs, gene islands, CRISPR, and the like; and (5) function annotation. The genetic structure and nucleic acid level of Bacillus coagulans mr-1B were predicted and analyzed for their whole genomic DNA library. Different genetic elements of the predicted assembled genome, including coding genes, tRNA, rRNA, miscRNA, and the like. And finally summarizing the predicted genetic elements and performing preliminary annotation.
The whole genome sequencing analysis result shows that the novel bacillus coagulans mr-1B is separated and identified from the macrobrachium rosenbergii in the embodiment. In the genome annotation of the assembled sequence, 6026 genes were co-predicted and assigned. Genomic analysis showed that this bacterium lacks an active virulence factor gene. The risk of transferring genes is not high due to the lack of movable elements near the antibiotic resistance genes. The lack of a functionally active prophage sequence suggests that it has advantages in promoting genomic stability. This indicates that mr-1B strain is not pathogenic to human/host Macrobrachium rosenbergii, and has high safety. FIG. 1 is a chromosome circle diagram of the whole gene of Bacillus coagulans mr-1B.
1.3 preservation of Bacillus coagulans mr-1B
Bacillus coagulans (Bacillus coagulans) mr-1B has been deposited in 2023, 02 and 27 days to China general microbiological culture Collection center (CGMCC No. 26707) with a deposit address of North Chen Xili No. 1, 3 in the Korea of Beijing.
Example 2
2.1 test of antibacterial Activity against aquatic pathogenic bacteria
In the embodiment, aquatic pathogenic bacteria of vibrio harveyi, aeromonas veronii, enterobacter cloacae and aeromonas hydrophila preserved in a laboratory are used as indicator bacteria, bacterial culture supernatant is prepared, and a perforation method is adopted for bacteriostasis test. The agar plate punching method is adopted: 10ml of a heated and thawed agar medium (suitable for the growth of the test bacteria) was poured into a sterile plate, and after it had cooled sufficiently to solidify, a plurality of sterilized oxford cups were placed. Heating 30ml of TSA culture medium to melt, cooling to about 50deg.C, inoculating 0.15 μl of overnight cultured test bacterial liquid, rapidly mixing, and pouring into a plate. After cooling, the oxford cup was removed from the sterile forceps. 200 μl of the supernatant to be tested was aspirated under aseptic conditions and added to oxford cup wells, incubated at 30deg.C for 24h, and the diameter (mm) of the zone of inhibition was determined.
As shown in Table 1, the bacillus coagulans mr-1B has a sensitive inhibition effect on pathogenic bacteria of macrobrachium rosenbergii, namely enterobacter cloacae, has a strong inhibition effect on common conditional pathogenic bacteria of aeromonas veronii and aeromonas hydrophila in aquaculture, and has a relatively weak antibacterial activity on main pathogenic bacteria of the crustacean vibrio harveyi.
TABLE 1 microbial bacteriostatic Effect
2.2 Effect on immune function of Macrobrachium rosenbergii
Fermentation broth of Bacillus coagulans mr-1B (10 8 CFU/mL) is added with 2% of bait (macrobrachium rosenbergii pellet compound feed) according to the mass fraction to prepare medicine bait, and three repeated macrobrachium rosenbergii with the average initial weight of (18.04+/-3.15) g is fed, and the macrobrachium rosenbergii of the control group is fed with the bait without fermentation liquor, wherein each group has 12 tails. The experiments were carried out in a circulating aquarium with a volume of 80L/L. The temperature of the experimental water is (25+/-1) DEG C. Feeding continuously for 4 weeks, and then detecting the activity of the macrobrachium rosenbergii blood AKP, POD, POX and amylase to study the influence of bacterial cell components and metabolites on the immune function of the macrobrachium rosenbergii. The immunoenzymatic activity was measured with reference to a kit purchased from Nanjing established technology Co.
As shown in table 2, macrobrachium rosenbergii blood AKP, POD, POX and amylase activity were measured 4 weeks after feeding, and the difference between the test group and the control group was extremely remarkable (p < 0.01). Therefore, bacillus coagulans mr-1B has a certain effect of improving the immune function of the macrobrachium rosenbergii.
TABLE 2 various immunological enzyme Activity indicators 4 weeks after the addition of Bacillus coagulans
* Indicating significant differences p <0.05 compared to the control group, indicating very significant differences p <0.01 compared to the control group.
2.3 action on Ammonia nitrogen and nitrite
Test group 0.2% of 10 is added into pond water for breeding macrobrachium rosenbergii 9 CFU/mL bacterial liquid, ammonia nitrogen and nitrite in the water body are respectively measured at 0d, 1d and 4d, and no bacterial liquid is added in the control group.
As shown in Table 3, the contents of ammonia nitrogen and nitrite in the culture water body can be effectively reduced by the bacillus coagulans mr-1B compared with the control group, and the contents of ammonia nitrogen and nitrite in the culture water body are extremely obviously reduced (p is less than 0.01) on the 4 th day.
TABLE 3 variation of ammonia nitrogen and nitrite in Water body with Bacillus coagulans addition at different time periods
* Indicating significant differences p <0.05 compared to the control group, indicating very significant differences p <0.01 compared to the control group.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (9)

1. The bacillus coagulans (Bacillus coagulans) mr-1B is characterized in that the bacillus coagulans is preserved in China general microbiological culture collection center (CGMCC) No.26707 in 2023 and 27 months, and the preservation address is North Chen West Lu No. 1 of the Korean region of Beijing city No. 3.
2. Use of bacillus coagulans mr-1B according to claim 1, characterized in that the use comprises any one of the following applications:
s1, application in improving the water quality of cultivation;
s2, application in preparation of broad-spectrum antibacterial agents;
s3, application of the composition in preparation of products for enhancing immune functions of aquatic animals.
3. The use according to claim 2, wherein the bacillus coagulans mr-1B improves the quality of aquaculture water by reducing the ammonia nitrogen and nitrite content in the aquaculture water.
4. The use according to claim 2, wherein the bacillus coagulans mr-1B inhibits vibrio harveyi, aeromonas veronii, enterobacter cloacae and aeromonas hydrophila, exerting broad-spectrum antibacterial effect.
5. The use according to claim 2, wherein the bacillus coagulans mr-1B exerts an immune function enhancing effect by increasing AKP, POD, POX and amylase activities of aquatic animals.
6. A microecological preparation for promoting the intensive cultivation of aquatic animals, which is characterized by comprising bacillus coagulans mr-1B according to claim 1.
7. The probiotic formulation of claim 6, further comprising a bait.
8. A method for promoting intensive aquaculture of aquatic animals, comprising the step of adding bacillus coagulans mr-1B according to claim 1 or a microecological preparation according to claim 6 to a body of aquaculture water.
9. The method of claim 8, wherein the bacillus coagulans mr-1B or the microecological formulation promotes intensive farming of aquatic animals by inhibiting aquatic pathogenic bacteria, enhancing immune function of aquatic animals, and improving aquaculture water quality.
CN202311190679.0A 2023-09-15 2023-09-15 Shrimp-derived bacillus coagulans and application thereof Pending CN117448199A (en)

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Application Number Priority Date Filing Date Title
CN202311190679.0A CN117448199A (en) 2023-09-15 2023-09-15 Shrimp-derived bacillus coagulans and application thereof

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Application Number Priority Date Filing Date Title
CN202311190679.0A CN117448199A (en) 2023-09-15 2023-09-15 Shrimp-derived bacillus coagulans and application thereof

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