Paenibacillus volcano ZTUXWS1 derived from excrement of wild black neck crane and application of paenibacillus volcano ZTUXWS1

Abstract

The invention discloses a strain of paenibacillus volcano ZTUXWS1 derived from excrement of wild black neck crane and application of the strain, and belongs to the field of microorganisms, the strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M 20242603. According to the present invention, a culture medium is selected through cellulose, and the Paenibacillus cinereus ZTUXWS1 is obtained by carrying out strain separation on the part, not in contact with the environment, in the fresh faeces of wild birds by using the cellulose to provide the only carbon source, such that the Paenibacillus cinereus ZTUXWS1 can survive in the intestinal tract and can provide the related effect; meanwhile, a nematode toxicity sensitivity test proves that the compound has no obvious toxic effect on nematodes and can be used for degrading cellulose.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a Paenibacillus cineris strain derived from wild black-necked crane feces and having cellulose degradation potential. Background Art

[0002] The black-necked crane (Grus nigricollis) is a globally endangered species (IUCN, 2015). It is the only crane species in the world that breeds on the plateau and lives on the plateau all year round. It is an omnivorous species, but its food is mainly plant-based. According to our synchronous monitoring data of black-necked cranes in Zhaotong Dashanbao Wetland and Maolin Wetland from January to February 2023, it was found that there are about 1,900 black-necked cranes living around Dashanbao and Maolin Wetland, and the distribution range is gradually expanding. This shows that the black-necked crane has a strong adaptability to the environment. At the same time, the diversity of intestinal flora is the result of strong selection and co-evolution between the host and the parasitic intestinal flora in the body in the process of the host adapting to the living environment. The intestinal flora exists in the entire digestive system of the host and forms a symbiotic relationship with the host. The host provides a living environment and nutritional supplements for the intestinal flora, and the intestinal flora also affects the physiological metabolic process of the host through its own metabolism. The host's phylogenetic relationship, physical health, dietary structure, and habitat environment have a close impact on the composition and structure of the intestinal flora. Related studies have confirmed that the intestinal flora plays a vital role in the host's food digestion, nutritional homeostasis, immune regulation, signal transduction, energy metabolism, growth and development, and other physiological activities. Some intestinal flora can help the host improve food digestibility, improve food utilization, and help the host accumulate energy, deposit fat, and improve intestinal immunity. Feces are also widely used to reveal important sources of material for animal intestinal flora.

[0003] At present, genomic methods have been used to prove that the intestinal bacteria of black-necked cranes have complex polysaccharide chitin and cellulose degradation, acetate production, orotic acid production, and genes that fix gaseous ammonia to glutamate, which can help the host obtain more energy from the diet. Therefore, it shows that the intestinal flora plays an important role in the digestion of host diets. However, there is still a lack of effective screening of relevant functional bacteria in the intestines of black-necked cranes. However, this type of bacteria plays an important role in helping the host to increase the rate of food digestion in poultry farming, and can also increase the intestinal immune efficiency of poultry and reduce the use of antibiotics. In addition, this type of bacteria is excreted from the body with feces, which can also achieve the rapid degradation of cellulose in livestock and poultry feces and the efficient utilization of biomass resources, solving the core technical problems in aerobic composting production. Therefore, screening the flora related to cellulose digestion in the intestines of black-necked cranes and analyzing their cellulase activity are of great significance for poultry farming and aerobic composting in high-altitude areas.

[0004] In recent years, with the rapid development of intestinal microbial research, through genomic technology, we have recognized and understood the potential mechanism of intestinal microorganisms on the host, and even obtained the genomic data of related strains, but the isolation and cultivation of these functional bacteria is still very limited. From the perspective of functional bacterial isolation, it is more based on environmental (soil) samples for separation and purification. However, the addition of functional bacteria isolated from the environment to animal nutrition has the risks of customization success rate and toxicity to the intestine.

[0005] In addition, our understanding of the role of Paenibacillus cineris in the animal intestine is still very limited. Reports on related strains show that they all come from environmental samples, and there are no reports on their classification from the animal intestine. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a functional bacterium Paenibacillus cineris ZTUXWS1 which is derived from the excrement of wild birds with strong environmental adaptability and has cellulose degradation function and is non-toxic.

[0007] The technical solution of the present invention is: a strain of Paenibacillus cineris ZTUXWS1, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC M 20242603.

[0008] A bacterial agent containing the above-mentioned Paenibacillus cineris ZTUXWS1.

[0009] The use of the Paenibacillus cineris ZTUXWS1 or bacterial agent described above in degrading cellulose.

[0010] Application of the above-mentioned Paenibacillus cineris ZTUXWS1 or bacterial agent in poultry farming.

[0011] The invention uses fresh feces samples of wild black-necked cranes as materials, uses 37-39°C which is close to the body temperature of birds as a culture temperature, and screens strains separated from the intestines of the strains which have the potential to utilize cellulose as a carbon source (cellulose degradation potential) through a cellulose selection culture medium.

[0012] Add 1g of fecal sample into a 10ml centrifuge tube and add physiological saline to make 10ml bacterial suspension, then take 4ml of bacterial suspension and put it into a 250ml Erlenmeyer flask containing 40ml enrichment culture medium, and culture at 37-39℃ and 160rpm shaking for 2-3d.

[0013] For strain isolation, take 1 mL of enriched culture medium, dilute it to a gradient concentration of 10-2, then take 0.1 mL and spread it on a screening medium plate, culture it at 37-39°C, and spread it on 3 plates. After the colonies grow, select strains of different morphologies and streak them to obtain single strains and purify the strains. Take the purified plate colonies in liquid seed culture medium (50 mL of seed culture medium in a 250 mL conical flask) and shake it at 37-39°C and 160 rpm for 24 hours. After turbidity, inoculate the liquid seeds with a 4% inoculation amount into a 250 mL conical flask containing 50 mL of basic enzyme production culture medium and shake it at 37-39°C and 160 rpm for 48 hours.

[0014] The 16S rRNA gene of strain ZTUXWS1 was amplified by PCR using primers 27F (5`-AGAGTTTGATCCTGGCTCAG-3`) and 1492R (5`-TACGGYTACCTTGTTAYGACTT-3`). The amplified product was sent to Shanghai Biotech Co., Ltd. for bidirectional sequencing. The sequencing results were spliced, and the spliced ​​sequence is shown in SEQ ID No. 1. The sequencing data was compared with the existing database using the NCBI database Blast tool to determine the strain type.

[0015] At the same time, 1% Congo red staining revealed an obvious transparent color reaction around the colonies, indicating that the strain has the potential to produce cellulase.

[0016] Finally, the purified strain ZTUXWS1 was streaked onto screening medium and cultured at 37-39°C for 48 hours. After observing obvious growth of the colonies, Caenorhabditis elegans was inoculated and the survival of the nematodes was observed through a stereoscope 72 hours later. It was found that the Caenorhabditis elegans grew well, indicating that the strain was not toxic.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] At present, the understanding of the role of Paenibacillus cineris in the intestines of animals is still very limited, and the strains obtained in the relevant reports are all obtained from environmental samples. The present invention uses cellulose selection culture medium to separate the strain Paenibacillus cineris ZTUXWS1 from the part of the fresh feces of wild birds that is not in contact with the environment with cellulose as the only carbon source, which fully demonstrates that it can survive in the intestine and play a relevant role. At the same time, it is confirmed by the nematode toxicity sensitivity test that it has no obvious toxic effect on nematodes. It will be used in poultry farming in high-altitude areas in the future to help them increase the rate of food digestion.

[0019] Collection information:

[0020] Paenibacillus cineris ZTUXWS1 was deposited on November 20, 2024 in the China Type Culture Collection (CCTCC) located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the strain accession number: CCTCC M 20242603. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Phylogenetic tree constructed for the purpose;

[0022] Figure 2 To test the toxicity of strain ZTUXWS1 to nematodes;

[0023] Figure 3 Tested for Congo red staining. DETAILED DESCRIPTION

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0025] Culture medium

[0026] (1) Enrichment medium: 15 g sodium carboxymethyl cellulose (CMC-Na), 1 g peptone, 1 g yeast extract, 1 g magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 0.5 g, potassium dihydrogen phosphate (KH 2 PO 4 )1g, distilled water 1000mL.

[0027] (2) Screening medium: 20 g sodium carboxymethyl cellulose (CMCNa), 20 g potassium dihydrogen phosphate (KH 2 PO 4 )2g, magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 0.3 g, ammonium sulfate (NH 4 ) 2 SO 4 1.4 g, calcium chloride (CaCl 2 )0.3g, zinc chloride (ZnCl 2 )0.001 7g, manganese sulfate (MnSO 4 )0.001 6g, cobalt chloride (CoCl 2 )0.002g, ferrous sulfate (FeSO 4 )0.005g, distilled water 1000mL, pH7.0~7.2.

[0028] (3) Basic enzyme production medium: 5 g sodium carboxymethyl cellulose (CMC-Na), 10 g peptone, 0.2 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 1000 mL distilled water, pH 8.

[0029] (4) Liquid SDY (seed) medium: 10 g tryptone, 5 g yeast powder, 5 g sodium chloride, pH 7.4, and 1000 mL distilled water.

[0030] Example 1 Isolation, identification and preservation of strains

[0031] (1) Isolation of strain ZTUXWS1

[0032] Using fresh feces samples of wild black-necked cranes as materials, 1g of feces sample was added to a 10ml centrifuge tube and physiological saline was added to make 10ml bacterial suspension, then 4ml of bacterial suspension was placed in a 250ml Erlenmeyer flask containing 40ml of enrichment culture medium, and cultured at 37℃ and 160rpm for 2-3 days. 1mL of enrichment culture medium was taken and gradiently diluted to 10 -2 Gradient concentration, then take 0.1 mL, spread on the screening medium plate, culture at 37°C, and spread on 3 plates. After the colonies grow, select strains with different morphologies and streak them on the screening medium to obtain single strains, purify the strains, and obtain the strain ZTUXWS1 of the present invention.

[0033] (2) Identification of strain ZTUXWS1

[0034] The 16S rRNA gene of strain ZTUXWS1 was amplified by PCR using primers 27F (5`-AGAGTTTGATCCTGGCTCAG-3`) and 1492R (5`-TACGGYTACCTTGTTAYGACTT-3`). The amplified product was sent to Shanghai Biotech Co., Ltd. for bidirectional sequencing. The sequencing results were spliced, and the spliced ​​sequence was shown in SEQID No. 1. The sequencing data was compared with the existing database using the NCBI database Blast tool, and the strain ZTUXWS1 was determined to be Paenibacillus cineris. At the same time, the top 6-7 species with the highest similarity ratio were used using Mega software or online tools. Based on the comparison results, a phylogenetic tree was constructed to identify the strain species, reveal the position of the obtained species in the microbial kingdom, and explore its evolutionary history and kinship ( Figure 1 ).

[0035] (3) Preservation of strains

[0036] The Paenibacillus cineris ZTUXWS1 of the present invention was deposited in the China Type Culture Collection (CCTCC) located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province on November 20, 2024, with the strain collection number: CCTCC M 20242603.

[0037] Example 2 Toxicity test of strain ZTUXWS1 on nematodes

[0038] The purified strain ZTUXWS1 was streaked onto the screening medium and cultured at 37-39°C for 48 hours. After observing the obvious growth of the colony, Caenorhabditis elegans was inoculated. After 72 hours, the survival of the nematodes was observed under a stereoscope, and it was found that Caenorhabditis elegans grew well, indicating that the strain was not toxic ( Figure 2 ).

[0039] Example 3 Congo red staining test

[0040] The purified strain ZTUXWS1 was streaked onto the screening medium and cultured at 37-39°C for 72 hours. After the colonies grew significantly, 1% Congo red staining revealed a clear transparent color reaction around the colonies, indicating that the strain had the activity of producing cellulase ( Figure 3 ).

[0041] Example 4 Cellulase activity assay

[0042] Take 50ml of seed culture medium and add it to 250ml of basic enzyme production medium. After culturing at 37-39℃, 160rpm shaking in the dark for 5-7 days, take the supernatant and use the source leaf cellulase detection kit (DNS colorimetric method, RT27130-100T) to test the cellulase production of the strain according to the instructions. The cellulase activity unit of the strain per milliliter of bacterial liquid is 22-23U. (The definition of cellulase activity unit: at 60℃, 1ml of bacterial liquid extract, 1min catalyzes the hydrolysis of sodium carboxymethyl cellulose to produce 1 microgram of glucose, which is defined as 1 enzyme activity unit (U)). This shows that the strain has the potential to degrade cellulose in the animal intestine.

[0043] In summary, this strain can effectively help the host improve its ability to digest foods such as cellulose in poultry farming.

Claims

1. A strain of Paenibacillus cineris ZTUXWS1, deposited in China Center for Type Culture Collection, with the deposit number: CCTCC M 20242603.

2. A bacterial agent comprising the Paenibacillus cineris ZTUXWS1 according to claim 1.

3. Use of Paenibacillus cineris ZTUXWS1 according to claim 1 or the bacterial agent according to claim 2 in degrading cellulose.

4. Use of the Paenibacillus cineris ZTUXWS1 according to claim 1 or the bacterial agent according to claim 2 in poultry farming.

Images