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WO2023038282A1 - Souche de bacillus subtilis présentant une excellente capacité de production de surfactine et d'enzymes, et son utilisation - Google Patents

Souche de bacillus subtilis présentant une excellente capacité de production de surfactine et d'enzymes, et son utilisation Download PDF

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WO2023038282A1
WO2023038282A1 PCT/KR2022/010753 KR2022010753W WO2023038282A1 WO 2023038282 A1 WO2023038282 A1 WO 2023038282A1 KR 2022010753 W KR2022010753 W KR 2022010753W WO 2023038282 A1 WO2023038282 A1 WO 2023038282A1
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bacillus subtilis
surfactin
seq
enzyme
subtilis
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조양래
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Proxenrem Co ltd
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    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the present invention relates to a Bacillus subtilis strain excellent in surfactin and enzyme production ability, and more particularly to Bacillus subtilis ps4060 KACC81161BP excellent in surfactin, proteolytic enzyme and polysaccharide degrading enzyme production ability.
  • surfactin which has excellent antibiotic efficacy as a bio-surfactant, is a substance with high potential to be used as an animal growth promoter replacing existing antibiotics.
  • Surfactin is made up of 7 amino acids (Glu, Asp, Val, Leu) of 4 types by 3 non-ribosomal pepetide synthase (NRPS), which are connected in a ring form, and 12-17 carbon backbones are attached to the surfactin. It is a lipopolypeptide that is composed of lipid acids bound together.
  • NRPS non-ribosomal pepetide synthase
  • B. subtillis Bacillus subtilis
  • Bacillus pmilus B. pumilus
  • Bacillus amyloliquefaciens B.
  • amyloliquefaciens Bacillus licheniformis ( B. licheniformis ), Bacillus mozaben It is produced by B. mojavensis ) et al. (Hmidet et al., 2017). Since most of the surfactin produced by the Bacillus subtilis produces a very small amount of surfactin during cultivation even when the fermentation conditions of the Bacillus subtilis are good (Chen et al., 2015), surfactin or Bacillus subtilis producing surfactin There is a limit to its use as a livestock growth promoter.
  • enzymes such as starch degrading enzymes (amylases), proteases (proteases), and plant cell wall degrading enzymes (cellulases, hemicelluloses, pectinases, xylanases) are used in various industries. These enzymes are generally known to be added to laundry detergents to increase washing efficiency, and industrial enzyme proteins are also used as feed supplements in the livestock industry to help animals digest and increase the effectiveness of feed. Industrial enzyme proteins are generally produced in large quantities in fungi. However, since most of the enzymes produced by fungi are not suitable for human or animal consumption, a new method is needed to produce and manufacture them in a form that can be consumed by humans and animals.
  • Korean Patent Registration No. 1155158 relates to a new Bacillus subtilis with excellent survival rate. It can efficiently decompose fibrous and semi-fibrous components, which are difficult to decompose in the body, to increase the efficiency of feed use for livestock animals, suggesting the possibility of using it as a feed additive.
  • the strain in the prior art is not only different from the strain in the present invention, but also the most important proteolytic enzyme and starch degrading enzyme production function and surfactin production function as components of the animal growth promoter are not disclosed at all. There is a difference from the present invention.
  • Subtilis ps4060 KACC81161BP was found to have excellent surfactin and digestive enzyme production capabilities, confirmed that it could be used as an animal growth promoter or feed additive, and the present invention was completed.
  • the main object of the present invention is to provide a novel microorganism, Bacillus subtilis ps4060 KACC81161BP, which is excellent in producing surfactin and digestive enzymes.
  • Another object of the present invention is to provide a method for producing surfactin using the Bacillus subtilis ps4060 KACC81161BP.
  • Another object of the present invention is to provide a composition for promoting animal feed supplements capable of promoting animal growth using the Bacillus subtilis ps4060 KACC81161BP or its culture and reducing the amount of harmful gases.
  • Another object of the present invention is to provide a composition for adding feed using the Bacillus subtilis ps4060 KACC81161BP or its culture.
  • the present invention provides Bacillus subtilis ps4060 KACC81161BP having the whole genome represented by SEQ ID NO: 1 that produces surfactin and enzymes.
  • Bacillus subtilis have been developed as beneficial bacteria (hereinafter referred to as 'probiotics', probiotics) for use in animals and humans. Some are used as antibiotic substitutes instead of adding antibiotics to animal feed. However, since Bacillus subtilis generally produces very small amounts of surfactin, even if it is used as an antibiotic substitute, various animals (particularly chicks and pigs) can still prevent or treat diseases (diarrhea, lethality, etc.) caused by harmful microorganisms infection. There were limits.
  • feed for economic animals commonly used in the market is composed of plant tissue composed of starch, cellulose, and protein, including 30% corn, 40% wheat, and 25% soybean.
  • starch cellulose
  • protein including 30% corn, 40% wheat, and 25% soybean.
  • amylase or cellulase was used as a feed supplement to facilitate the decomposition of starch or cellulose, but they were easily destroyed in the stomach and difficult to reach the small intestine, so it was difficult to maximize the digestive effect.
  • the present inventors have made research efforts to isolate and identify strains with excellent surfactin and enzyme production ability in order to promote animal growth by replacing classical antibiotics and promoting animal digestion.
  • Bacillus subtilis was isolated from, and their emulsifying ability was compared to finally select strains with excellent plant-derived nutrient degrading enzyme production ability among strains with excellent emulsifying ability, and whole genome sequencing and genetic analysis were performed.
  • the present inventors registered the whole genome sequence of the selected strain in NCBI's "GenomeProject database" (accession ID: CP081458).
  • the selected strain was named "Bacillus subtilis ps4060" as a new microorganism, and the Rural Development Administration It was deposited at the Microorganism Bank (KACC) of the National Academy of Agricultural Sciences on June 22, 2021 and was given an accession number (KACC81161BP).
  • nucleotide sequence of the entire genome of Bacillus subtilis ps4060 KACC81161BP selected in one embodiment of the present invention was read 200 times by Pacific Bioscience long reads sequencing, and then these sequences were concatenated to determine the entire genome.
  • the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention is characterized by including a 16S rDNA gene having the nucleotide sequence shown in SEQ ID NO: 2.
  • the enzyme may be a digestive enzyme that helps digestion of animals, such as polysaccharide degrading enzyme, monosaccharide degrading enzyme, disaccharide degrading enzyme, oligosaccharide degrading enzyme, proteolytic enzyme and It may be a lipolytic enzyme, preferably a proteolytic enzyme or a polysaccharide degrading enzyme.
  • the polysaccharide in the polysaccharide degrading enzyme may be dextrin, dextran, starch, glycogen and cellulose, preferably starch or cellulose.
  • the yield of surfactin of the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention is about 1.5 to 2.0 mg/ml, which produces a sufficient amount of surfactin to be used as an animal growth promoter.
  • the amount of surfactin produced by culturing the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention was confirmed to be about 1.5 to 2.0 mg/ml.
  • the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention has 5-10 times more proteolytic enzyme production than B. subtilis ATCC21332, a subspecies producing surfactin, and 20-70 times more than B. subtilis pb2441. It is characterized by being twice as high.
  • the amount of protease secreted by culturing the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention is up to 27.4ug/ml, whereas the subspecies Bacillus subtilis strain ( B. subtilis ATCC21332 ), the amount of protease secreted by culturing was up to 3.57ug/ml, and the amount of proteolytic enzyme secreted by culturing B. subtilis pb2441 was only up to 3.93 ug/ml (Experimental Example 4, FIGS. 7B to 7D reference).
  • the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention is characterized in that the production of polysaccharide degrading enzyme is 2 to 5 times higher than that of the Bacillus subtilis strain ( B. subtilis ATCC21332), a subspecies producing surfactin.
  • the production of secreted starch degrading enzyme by culturing the novel microorganism Bacillus subtilis ps4060 of the present invention is Bacillus subtilis It was more than twice as high as the production of secreted starch degrading enzyme by culturing ATCC21332, and in the case of cellulolytic enzyme production, Bacillus subtilis It was confirmed that it was 5 times higher than ATCC21332 (see Experimental Examples 5 and 6 and Tables 7 and 8).
  • starch degrading enzyme of Bacillus subtilis ps4060 is not significantly high, the reason why the corn contained in the feed is not digested well is because of the fibrous shell, and the Bacillus subtilis ps4060 of the present invention has the fibrous Since the production of cellulolytic enzyme capable of decomposing the skin is remarkably high, it is possible to improve starch digestion efficiency through effective decomposition of cellulose, so it can be easily used as an animal feed composition.
  • the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention secretes polysaccharides during growth. These polysaccharides are known to enhance the immunity of animals and affect the production of surfactin. Specifically, according to the results of research on the relationship between the growth of Bacillus subtilis and surfactin production, when biomass increases, pores are created by quorum sensing, and surfactin production is stopped. That is, as the number of Bacillus subtilis increases by culturing, the content of surfactin they produce temporarily increases.
  • Bacillus subtilis of the present invention is separated into polysaccharides, so there is a possibility that quorum sensing occurs late.
  • the polysaccharide material of the novel microorganism Bacillus subtilis ps4060 of the present invention is an important feature that can improve the production of surfactin.
  • the novel microorganism Bacillus subtilis ps4060 of the present invention grows vigorously when nutrients including starch are abundant at a high temperature of 55 ° C., where microorganisms generally do not grow well.
  • ATCC21332 grows vigorously at 37 ° C, but the growth rate decreases at 55 ° C
  • Bacillus subtilis ps4060 of the present invention grew more vigorously at 55 ° C than at 37 ° C (see Experimental Example 2 and FIG. 4).
  • contamination of infectious bacteria and fungi can be easily prevented when preparing animal feed supplements using a solid medium, so the Bacillus subtilis ps4060 of the present invention can be easily used as an animal feed composition.
  • the present invention provides (a) culturing Bacillus subtilis ps4060 KACC81161BP having the whole genome represented by SEQ ID NO: 1 of claim 1 on a solid medium to form colonies; (b) inoculating and culturing the colony in a liquid medium; and (c) obtaining surfactin from the precipitate after centrifuging the culture medium.
  • the liquid medium in step (b) is glucose, yeast extract, peptone, dibasic potassium phosphate (K 2 HPO 4 ), sodium chloride (NaCl), sodium carbonate (Na 2 CO 3 ) and magnesium sulfate (MgSO 4 ) are included, sucrose can be used instead of glucose as a carbon source, and ammonium can be used instead of peptone as a nitrogen source. Not limited.
  • the present invention provides a composition for promoting animal growth comprising Bacillus subtilis ps4060 KACC81161BP having the whole genome represented by SEQ ID NO: 1 or a culture medium thereof as an active ingredient.
  • the present invention provides a composition for adding feed containing Bacillus subtilis ps4060 KACC81161BP having the whole genome represented by SEQ ID NO: 1 or a culture medium thereof as an active ingredient.
  • the novel microorganism Bacillus subtilis ps4060 KACC81161BP of the present invention has a remarkably excellent ability to produce surfactin and digestive enzymes
  • the novel strain or its culture medium according to the present invention is used for animal growth promotion or feed It is very suitable as an additive composition.
  • the composition for promoting animal growth or the composition for adding feed of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate, polyphenol, and catechin. , alpha-tocopherol, vitamin C, chitosan, tannic acid, may further include any one or more than one of natural antioxidants such as phytic acid.
  • composition for promoting animal growth or the composition for adding feed according to the present invention may be in the form of a dry or liquid formulation, and may include an excipient.
  • Excipients include, for example, zeolite, jade powder or rice bran, but are not limited thereto.
  • Animals that can be used for the composition for promoting animal growth or the composition for adding feed of the present invention are, for example, livestock such as edible cattle, dairy cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, and chicks.
  • poultry such as, but not limited to, egg-laying chickens, meat chickens, domestic chickens, roosters, ducks, geese, turkeys, quails, small birds, etc.
  • Bacillus subtilis ps4060 KACC81161BP of the present invention has excellent production ability of surfactin and various nutrient digestive enzymes, Bacillus subtilis ps4060 KACC81161BP or its culture promotes the growth of animals and inhibits intestinal microbes. It can be used for the purpose of reducing the generation of harmful gases generated by
  • Figure 1 shows the phylogenetic tree of Bacillus subtilis ps4060 KACC81161BP ( Figure 1a: phylogenetic tree created by the Maximumlikelyhood method using 16S rDNA, Figure 1b: phylogenetic tree using 720 orthologs amino acid sequences. Figure 1c: bootstrap value 50 or less nodes are removed phylogenetic tree).
  • FIG. 2 is a diagram showing vegetative cells and spores of Bacillus subtilis ps4060 KACC81161BP (FIG. 2a: vegetative cells grown in liquid medium, FIG. 2b: cells that started to form spores while growing in liquid medium, FIG. 2c: Spore cells grown in sporulation medium).
  • Figure 3 is a diagram comparing the structure of the whole genome of Bacillus subtilis ps4060 KACC81161BP and other Bacillus subtilis.
  • Figure 4 is a result of comparing the growth rate of Bacillus subtilis ps4060 KACC81161BP at 37 ° C and 55 ° C (x-axis: time, y-axis: shortcut indicating the size of colonies).
  • Figure 7 is a result of comparing the amount of proteolytic enzyme secretion during culture in liquid medium. Bars included in the diagram represent standard deviation (FIG. 7a: clear zone comparison image, FIGS. 7b to 7c: protease production comparison).
  • Figure 8 is a result of comparing the secretion of starch degrading enzyme (Figure 8a: the amount of enzyme secreted for 16 hours in 1x LB culture medium, 8b. the amount of enzyme secreted for 16 hours in culture medium containing 1x LB + 5% starch, 8c Amount of enzyme secreted in culture medium containing 5% starch, 8d. Amount of enzyme secreted in culture medium containing 1/4x LB + 1% starch, 8e. As a result of tracking the amount of enzyme, as a result of tracking cell growth in a culture medium containing 8f 1/4x LB + 5% starch, and as a result of confirming the degree of cell growth and starch degradation in an agar medium containing 8g starch).
  • Figure 9 is a result of comparing the amount of cellulolytic enzyme secretion (relative amount of cellulolytic enzyme secreted during growth in LB culture medium containing 2% of methylcellulose) (9a and 9b: reaction indirectly indicating cellulolytic enzyme activity) Difference in glucose amount before and after, Fig. 9c: cell growth curve).
  • Feces of third instar larvae were collected to investigate the intestinal bacteria of Protaetia brevitarsis taughtensis larvae.
  • the larvae were collected from Taean-eup, Taean-gun, Chungcheongnam-do, and were maintained by supplying fermented sawdust as food.
  • the feces produced during this breeding were diluted in distilled water, heat-treated at 80 ° C for 15 minutes, spread on a 15 mm diameter plate on which LB agar medium was hardened, and incubated at 30 ° C for 16 hours. More than 100 bacterial colonies appeared on the cultured plate, and they were individually inoculated on a new plate at intervals of 0.5 to 1 cm to separate pure bacteria. Initially, colonies were classified according to size, shape, and color, and 400 colonies belonging to each group were examined as a unit.
  • Example 1 20-40 of the well-separated bacterial colonies in Example 1 were individually transferred to LB agar medium plates and cultured at 30° C. for 16-48 hours. After culturing, soybean oil was sprayed on the plate on which the colonies grew, and strains secreting surfactant were selected. At this time, a round transparent part is formed around the colony secreting the surfactant, and the larger the amount of the surfactant, the wider the transparent part appears. Each of the separated strains was inoculated into an Erlenmeyer flask containing 50ml LB and cultured for 48 hours to 72 hours while maintaining a temperature of 30° C. while shaking at a speed of 200 rpm on a stirrer.
  • the culture solution was centrifuged to remove microorganisms, and the remaining culture solution was tested by the water drop-collapse method.
  • strains and strain residues were completely removed by passing the culture solution remaining in the upper layer through a 0.45 ⁇ m filter.
  • Water drop-collapse assay was performed as follows. A drop of microbial culture was added to each well of an oiled 96-well plate. The culture medium without surfactant is water-based and remains in the form of water droplets on the oil, but the culture medium containing surfactant is mixed with the oil and the water droplets spread widely. Through the above experiments, strains were selected that allow the culture solution droplets to mix well with the oil on the film formed of oil.
  • strains with the most excellent emulsification ability of the cultured liquid were selected through an assay experiment.
  • mineral oil was used in the initial discovery experiment, and mineral oil and soybean oil were used in the additional confirmation verification experiment.
  • the 96-well plate has a diameter of 8 mm and a depth of 30 mm. 7ul of oil was added to each well and stored at room temperature for 24 hours. Thereafter, 20ul of the culture medium was carefully dropped into each well through a needle or pipette.
  • distilled water was used instead of the culture medium. After 1 minute had elapsed after the culture solution was added, the degree of change of the culture solution droplet was visually assessed. All experiments were repeated at least three times.
  • Example 3 Identification through 16S rRNA sequence of Bacillus subtilis ps4060 strain
  • the strain selected in Example 2 was presumed to be Bacillus subtilis when observed under a microscope, and the nucleotide sequence of 1600 nucleic acids of 16S rRNA was determined to be identified by molecular biological methods.
  • the strain was cultured on an LB agar plate for 24 hours. The cultured colonies were placed in an aliquot, washed with distilled water, heat-treated at 95° C. for 15 minutes, centrifuged to remove cell debris, and the supernatant was used as template DNA for PCR. For PCR amplification, DNA polymerase was used as described in the manual.
  • the nucleic acid sequences were compared with bacteria registered in public databases by the BlastN method. As a result, as shown in Table 1, the 16S rRNA sequence (SEQ ID NO: 2) obtained through PCR was 100% identical to the NCBI-registered B. subtilis 16S rRNA sequence.
  • SEQ ID NO: 2 (16S rRNA nucleic acid base sequence of Bacillus subtilis ps4060) gtagtggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtctgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgtgagtcggag
  • ITS sequence Internal transcribed spacer (ITS) sequence with high mutation fixation rate is used to distinguish subspecies within bacterial species or differences between strains. Since the 16S rRNA sequence of the ps4060 strain was the same as that of B. subtilis, the ITS sequence was checked to confirm the difference from known strains. When ITS was amplified using PCR, an elongation reaction was performed for 40 seconds using 16S_1094F 5'-CAGCTCGTGTCGTGAGATGT-3' (SEQ ID NO: 6) and 23S_197R 5'-TCCTCCGGGTACTTAGATGTTT-3' (SEQ ID NO: 7), Other conditions were the same as for amplifying 16S rRNA. For sequencing, the primers used for PCR were used.
  • SEQ ID NO: 3 Bacillus subtilis 16S rRNA, internal transcribed spacer (ITS), 23S rRNA sequence TTTTTCGAGTGT 1 TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCCGGT
  • the nucleotide sequence of this region was also identical to the sequence of B. subtilis registered in GenBank, and was identical to 15 strains identified as subspecies subtilis among B. subtilis.
  • the sites where these subspecies are found are diverse, such as plant tissue, soil, coastal-silt soil, and fermented materials including soybean paste. They have been shown to produce secondary metabolites that exhibit antibiotic efficacy or antifungal efficacy against certain fungi that cause plant diseases.
  • the ps4060 strain derived from the insect digestive system was identified as a subspecies subtilis (hereinafter Bacillus subtilis subsp. subtilis), a subspecies that synthesizes and releases large amounts of surfactin among Bacillus subtilis.
  • Example 5 Full-length genome sequencing of Bacillus subtilis ps4060 strain
  • the extracted DNA was sequenced by creating a library according to the protocol of PacBio's bacterial genome sequencing kits at DNA Link. After reading the nucleotide sequence of the whole genome 200 times, a map of the whole genome was completed using a known algorithm (Koren et al., 2013). The map of the whole genome was registered in NCBI's "GenomeProject database" (SubmissionID: SUB10211484 BioProjectID: PRJNA755476, accession ID: CP081458, organism: Bacillus subtilis subsp. subtilis ps4060).
  • Example 6 Establishment of a phylogenetic tree of Bacillus subtilis ps4060 strain
  • 16S rRNA described above may not represent ps4060, all rRNA sequences in the entire genome were confirmed.
  • 16S rRNA with a length of 1547 nt was repeated 10 times, and their base sequences were distinguished into four types. The four sequences are very similar, and mutations at 1547 nt appeared only in 4 places, and the maximum difference was only a difference of 3 bases among 1547 nt.
  • Sequence information of rRNA contained in the genomes of ps4060 and other Bacillus strains was extracted from the NCBI database. Other strains also possessed a large number of rRNAs with different nucleotide sequences and included all sequences with different nucleotide sequences. Sequences of rRNA of Bacillus subtilis containing 9 base sequences in ps4060 were aligned using MEGA software and a phylogenetic tree was created (Kumar et al., 2018).
  • a phylogenetic tree was constructed using 728 homologous protein genes as shown in FIG. 1B. Specifically, 12 strains representing Bacillus subtilis were downloaded from the NCBI database and annotation was performed using PGAP (Tatusova et al., 2016). Homologous proteins were selected using CD-HIT when the following conditions were met (Fu et al., 2012). 1) the length of the amino acid sequence is at least 90% higher than that of the longest sequence, 2) the amino acids in this region are at least 70% identical, and 3) 728 present in all 12 strains and ps4060 strains for which the whole genome sequence is available Dog genes were selected. A phylogenetic tree was reconstructed with the MEGA program using all of these genes (Kumar et al., 2018).
  • ps4060 was most similar to BAB1 and showed a more distant relationship with ATCC21332, pb2441, and ps4010.
  • ps4060 was different from ATCC21332 and known bacterial species among known B. subtilis sub species subtilis and was found to be the most similar to BAB1 (Fig. 1b, Fig. 1c).
  • Example 7 Biochemical characteristics of Bacillus subtilis ps4060 strain
  • the genetic information contained in the whole genome of the 8 strains was analyzed to confirm the G+C content, number of proteins, number of ribosomal RNA operons, and t-RNA of Bacillus subtilis ps4060 strain.
  • the G+C content of the whole genome was calculated using the algorithm used when assembling the whole genome (Koren et al., 2013), and the genes containing protein information were generated using the GeneMark.HMM (Besemer et al., 2001) package. 3,960 genes were predicted using .
  • tRNAs scattered throughout the entire genome were discovered using a package called tRNA-Scan (Schattner et al., 2005), and a total of 86 were identified.
  • Ribosomal RNA was discovered with RNAmmer (Lagesen et al., 2007), and it was confirmed that a total of 30 and 10 rRNA operons were composed (Table 3). As a result, as can be seen in Table 3 below, the length of the whole genome, the G + C content, the number of proteins, the number of ribosomal RNA operons, and the number of t-RNAs all show that the ps4060 strain is more Bacillus subtilis than other Bacillus subtilis species. It was confirmed to be similar to B. subtilis.
  • subtilis ps4060 Accession NZ_CP011252.1 NC_014551.1 NC_009725.1 NC_006322.1 NC_009848.4 NC_004722.1 NC_000964.3 CP068982 CP076445 CP081458 Genome size (bp) 3,897,521 3,980,199 3,918,589 4,222,645 3,704,641 5,411,809 4,215,606 4,288,724 4,106,342 4,030,808 G+C content (%) 46.09 46.08 46.48 46.19 41.29 35.28 43.51 43.46 43.78 43.88 CDS 3,752 3,870 3,687 4,223 3,598 5,210 4,328 4,328 4,068 3,960 rRNA 24 30 29 24 21 42 30 30 30 30 30 t-RNAs 81 94 88 72 72 108 86 86 86 86 86 86
  • Example 8 Morphological characteristics of Bacillus subtilis ps4060 strain
  • Colonies of Bacillus subtilis ps4060 strain grown on LB agar plates form a helper in a round shape when viewed externally, and when incubated for more than 16 hours, polysaccharides are secreted to reflect light, and the colonies are easily combined and flow like thick runny nose and become sticky. When the number of colonies is large, it flows down thickly, evenly covering the entire plate. Observed under a microscope, vegetative cells that grow in solid medium or liquid medium or actively undergo growth and metabolism are rod-shaped (Fig. 2a: cells grown in liquid medium, Fig. 2b: cells grown in liquid medium) At the stage of sporulation, part of the cells are darkened (Fig. 2C: after almost all cells have changed into spores).
  • Spores are formed when the growth environment deteriorates due to conditions such as lack of nutrients, increased cell density, and increased concentrations of metabolites.
  • a normal medium less than 10% of the cells turn into spores even when the conditions deteriorate, and when cultured in a sporulation medium, ⁇ 100% of the cells form spores under special conditions.
  • ps4060 turns ⁇ 100% of vegetative cells into spores (Berikashvili et al., 2018; Monteiro et al., 2014), Fig. 2a. shows only vegetative cells in the stage before spore cells are formed, 2b shows cells in the process of changing from vegetative cells to spore cells, and 2c shows no vegetative cells, only spores.
  • Example 9 Novelty of Bacillus subtilis ps4060 strain
  • ps4060 is another B. subtilis sub. There is no difference from B. subtilis, but when looking at the length of the whole genome and the arrangement (macrosyntany) of genes in the whole genome, it is distinctly different from the previously known B. subtilis (Fig. 3).
  • ps4060 is a novel strain that has no identical genome length and gene arrangement among known strains. The macro synteny is similar, but the genome of ps4060 appears to be the shortest. Specifically, compared to B. subtilis strains pb24441 and ps4010, which have similar sequences and genome structures, the genome sizes are 257,916 nt and 75,534 nt shorter. The number of genes is also 386 and 108 fewer (Fig. 3).
  • the Bacillus subtilis ps4060 strain is inoculated on LB agar solid medium and cultured for 24 to 48 hours in an incubator at 25 to 37 ° C, inoculated directly into a liquid culture medium, or stored at 4 ° C for up to 1 month. Inoculate one or more colonies properly grown in the solid medium into the five liquid media in Table 4 below, and incubate for 48-72 hours while shaking at 180-240 rpm while maintaining 30 ° C. in a shaking-incubator.
  • a quantitative method can be used to inoculate a liquid culture medium with a relatively accurate amount of strain. For example, it is similar to inoculating 1ml of a 50ml medium by dissolving colonies in sterile water and then diluting them to 600nm absorbance (OD600nm) of 0.8 ⁇ 1.5 using a spectrophotometer (UV-vis Spectrophotometer, Mecasys, Korea). . Additional strains may be inoculated, and surfactin production is not significantly affected by the amount of inoculation.
  • the ps4060 strain was cultured for 48 hours while shaking at 200 rpm while maintaining 30 ° C. in a shaking incubator by putting 50 ml medium in a 250 ml Erlenmeyer flask. After incubation, the culture medium was transferred to a clean tube, centrifuged at 8,000 g for 10 minutes, and 30 ml of the supernatant was transferred to a clean tube. After correcting the pH to 2.0 by adding 6M hydrochloric acid (HCl) to the supernatant, the mixture was aged at 4° C. for 16 hours and centrifuged at 8,000 g for 15 minutes. The supernatant was discarded and the remaining concentrated precipitate was referred to as crude surfactin. The concentrated surfactin precipitate was lyophilized and 10mg was dissolved in 1ml 100% methanol. The amount of surfactin was analyzed using HPLC, and the results are shown in Table 4.
  • the growth rate of ps4060 was compared with B. subtilis ATCC21332, pb2441, and ps4100, which are subspecies widely used as experimental species worldwide, and B. subtilis ps4060, a subspecies discovered by the present inventors, while culturing on a solid medium.
  • the solid medium is Luria-Bertani (LB), 1% starch with 1/4 x LB, 1% skim milk, 1% methyl-cellulose with 1/4 x LB, 0.3% pectin with 1/4 x agar.
  • LB, 1% xylan with 1/4 x LB was added.
  • the ps4060 strain was cultured in a 250ml Erlenmeyer flask with 50ml No. 5 medium (Table 4) maintained at 30° C. while shaking at 200rpm for 60 hours in a shaking incubator. After the incubation, 35 ml of the culture medium was transferred to a high-speed spin bottle and centrifuged at 8,000 g for 10 minutes. After transferring the supernatant to a clean tube, the supernatant was calibrated to pH 2.0 by adding 33% HCl. The pH-corrected supernatant was aged at 4° C. for >16 hours and then centrifuged at 12,000 g for 30 minutes.
  • the amount of surfactin produced by ps4060 was greater than that produced by ATCC21332, and as shown in FIG. 6, surfactin produced by ps4060 was Sigma Aldrich. Similar to the standard surfactin purchased from , the molecular weight increased by 14 and -CH 2 increased by one. Surfactin produced by ps4060 of the present invention showed similar yield and profile to surfactin produced by ATCC21332 strain.
  • Milk which contains a large amount of protein, can supply carbon and nitrogen at the same time and contains sufficient nutrients necessary for growth.
  • proteolytic enzymes must be secreted and digested to absorb nutrients.
  • ps4060 was difficult to distinguish from the subspecies B. subtilis ATCC21233 due to the small size of the colonies.
  • colonies were larger than that of pb2441, which has a slow growth rate.
  • Fig. 7a when continuously cultured for more than 1 week, the speed at which colonies of ps4060 strains grow faster than other strains, and the colonies appear larger than other strains, and the transparent part showing the efficiency of protease production appears very wide (Fig. 7a). . Specifically, when 1% of skim milk powder is included in the agar plate, it has an opaque milky color, but when the milk powder is decomposed, it becomes transparent. The area of the clear zone in the agar plate indirectly indicates the secreted amount of enzymes that degrade milk powder, especially enzymes that degrade protein. The diameter of the transparent area was significantly larger than that of other bacterial species after culturing for 3.5 days (Fig. 7a).
  • Amylase is expressed in a small amount at the initial stage of inoculation, but when the cells grow exponentially, the expression level increases, and the highest expression is maintained during the stable period (FIG. 8e).
  • the expression level of amylase was confirmed in the order of ps4100 > ps4060 > ATCC21332 > pb2441. That is, it was confirmed that ps4060 secreted twice as much starch degrading enzyme as ATCC21332 (Table 7).
  • Enzyme activity amount (unit) Relative Quantity Comparison (Ratio) incubation time ATCC 21332 pb2441 ps4060 ps4010 ps4060/ ATCC21332 ps4060/pb2441 ps4060/ ps4010 ps4010/ ATCC21332 ps4010/pb2441 9 hours 0.1 0.0 0.1 0.0 0.9 6.2 1.5 0.6 4.1 10 hours 0.1 0.0 0.1 0.1 1.7 3.3 1.9 0.9 1.7 18 hours 0.1 0.0 0.3 0.3 2.0 89.3 0.9 2.2 99.9 21 hours 0.2 0.0 0.3 0.3 1.7 111.0 0.8 2.1 135.7 43 hours 0.2 0.0 0.3 0.4 1.3 8.3 0.8 1.6 10.5
  • Bacillus subtilis ps4060 KACC81161BP
  • the applicability of the Bacillus subtilis ps4060 (KACC81161BP) of the present invention as a composition for promoting animal growth/feed additive was confirmed through an in vivo test.
  • a total of 100 finishing pigs [Landrace ⁇ Hampshire] ⁇ Duroc) with an average weight (BW) of 54.15 ⁇ 1.70 kg were used in the 10-week experiment.
  • the four experimental diets were randomly assigned to pigs according to their initial BW, but sex ratios were applied. There were 10 replicate pens for each treatment, with 5 pigs per pen (each averaging 50% castrated males and 50% females).
  • ADG Average daily gain
  • ADFI average daily feed intake
  • G:F gain/feed ratio
  • a total of 20 stool samples per group were stored at -20 °C until analysis, then dried at 70 °C for 72 h, and then finely ground to less than 1 mm in diameter.
  • Dietary dry matter (DM), nitrogen and digestible energy (DE) were analyzed according to the procedures of the International Society of Official Analytical Chemists (USDA, 2006).
  • USDA International Society of Official Analytical Chemists
  • the boxes were fermented at room temperature for 7 days and methyl mercaptan, NH 3 , H 2 S, acetic acid and CO 2 were measured using MultiRAe Lite (model PGE-6208, RAE, USA).
  • Basal diet corn-soybean meal-based diet
  • TRT Basal diet + supplements (including bacillus subtilis ps4060).
  • ADG average daily gain (average daily weight gain)
  • ADFI average daily feed intake (average daily feed intake)
  • CON Basal diet
  • TRT Basal diet + Bacillus subtilis supplement
  • the P-value represents the Student-t test result with the same mean distribution.
  • SEQ ID NO: 1 Bacillus subtilis ps4060 KACC81161BP
  • SEQ ID NO:2 Bacillus subtilis ps4060 KACC81161BP 16S rDNA
  • SEQ ID NO: 3 Bacillus subtilis 16S rRNA, internal transcribed spacer (ITS), 23S rRNA sequence
  • SEQ ID NO: 4 Bac27F (forward primer)

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Abstract

La présente invention concerne un nouveau micro-organisme Bacillus subtilis ps4060 KACC81161BP présentant une excellente capacité à produire de la surfactine, une protéase et une lyase de polysaccharide, le nouveau micro-organisme Bacillus subtilis ps4060 KACC81161BP présentant une excellente capacité à produire de la surfactine et une enzyme digestive et pouvant ainsi être utilisé comme promoteur de croissance animale ou comme additif alimentaire, et étant capable de réduire la quantité de gaz nocifs contenus dans les fèces.
PCT/KR2022/010753 2021-09-09 2022-07-22 Souche de bacillus subtilis présentant une excellente capacité de production de surfactine et d'enzymes, et son utilisation Ceased WO2023038282A1 (fr)

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CN117925865B (zh) * 2023-11-27 2025-09-26 广东省科学院微生物研究所(广东省微生物分析检测中心) 检测枯草芽胞杆菌的特异性新分子靶标及其快速检测方法
CN119752746A (zh) * 2024-11-29 2025-04-04 南京大学 高产特定表面活性素的枯草芽孢杆菌及其制备方法与应用
CN119715469A (zh) * 2024-12-23 2025-03-28 中国医学科学院医药生物技术研究所 一种抗菌药物的筛选靶点、应用及筛选方法
CN119859602B (zh) * 2025-01-23 2025-10-31 浙江工业大学 一种稳定高产d-泛酸的工程菌及其构建方法与应用
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