CN111349588B - Bacillus belgii and application thereof - Google Patents
Bacillus belgii and application thereof Download PDFInfo
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- CN111349588B CN111349588B CN202010353675.XA CN202010353675A CN111349588B CN 111349588 B CN111349588 B CN 111349588B CN 202010353675 A CN202010353675 A CN 202010353675A CN 111349588 B CN111349588 B CN 111349588B
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
Abstract
The invention discloses a Bacillus belgii strain and application thereof, wherein an Fb strain is obtained through enrichment culture, primary screening, secondary screening and the like, and belongs to Bacillus belgii, and gram-positive bacteria are obtained. The strain is characterized by comprising the following morphological characteristics: the thallus is rod-shaped, 0.4-0.6 μm × 0.9-4.0 μm, arranged singly or in pairs, with the spore being nearly round, growing proximally and the cyst being enlarged. Through detection, the Bacillus belgii Fb can tolerate the high temperature of 55 ℃, the optimal growth temperature is 45 ℃, and the optimal growth pH value is 3.0-7.6. The strain can efficiently degrade tea leaves, improve protein, amino acid and the like of the tea leaves, reduce water, cellulose and the like of the tea leaves, and the tea leaves after fermentation treatment can be used as feed raw materials; can realize smokeless, odorless and sewage discharge, and can realize the recycling of organic waste, thereby reducing the pollution of the tea leaves to the environment, and being a technology which accords with green environmental protection.
Description
Technical Field
The invention relates to the field of industrial microorganisms, in particular to a bacillus beiLeisi and application thereof.
Background
Bacillus belgii (B.), (Bacillus velezensis) Is a new species of bacillus, which can be used as biological control regulator in agricultural production due to its inhibition on pathogenic bacteria, and can produce secondary metabolite with broad-spectrum antibacterial activity for increasing crop yield and maintaining ecological ringThe first choice of biological agents for environmental and agroecological systems. Application research reports of the Bacillus belgii in degrading industrial and agricultural byproducts are not seen at home and abroad. The tea residue is a byproduct of tea used for producing instant tea or extracting tea polyphenol. As one of the main tea production countries in the world, China has the tea residue yield of tens of thousands of tons every year. Analysis shows that the tea leaves contain 17-23% of Crude Protein (CP), 1.5-4.5% of crude fat (EE) and 3.3-5.3% of Ash (Ash), and the tea leaves are good protein feed resources and have a certain feeding value. The tea residue fed by the feed can reduce fat accumulation in the abdomen of the chicken, enhance the oxidation resistance of milk cow plasma, and improve the growth performance and meat quality of the fattening pig. The water-insoluble tea protein in the tea residue is a protein which has the functions of nutrition, radiation protection and blood fat reduction. In addition, the tea protein can also improve the quality and flavor of meat products.
At present, most of tea leaves are discarded, so that not only is resource waste caused, but also the ecological environment is polluted. Therefore, the screening of the microorganisms capable of efficiently degrading the tea residues has great significance.
Disclosure of Invention
The invention aims to provide a bacillus belgii strain and application thereof.
In order to realize the purpose, the following technical scheme is adopted:
the strain is Bacillus belgii (B)Bacillus velezensis) Fb is preserved in China general microbiological culture Collection center at 28.2.2020, with the preservation number of CGMCC No.19438, and the address of No. 3, institute of microbiology, China academy of sciences, North Chen Xilu No.1, of the Chaoyang district, Beijing.
The application of the strain in tea residue degradation.
The specific method comprises the following steps:
culture medium:
(1) LB broth (L): 10g of yeast extract powder, 10g of peptone, 5g of beef extract, 10g of NaCl, 10g, pH6.0, adding distilled water to a constant volume of 1L, and sterilizing at 121 ℃ for 30 min.
(2) Purifying the solid culture medium: adding 20g/L agar powder into LB culture solution, and sterilizing at 121 deg.C for 30 min.
(3) Separating a solid culture medium: adding distilled water 500ml into fresh tea residue 50g, steaming for 30min, adding agar powder 20g into the filtered tea residue liquid, and sterilizing at 121 deg.C for 30 min.
(3) Liquid fermentation medium: 10g of yeast extract powder, 10g of peptone, 5g of beef extract, 10g of NaCl, 10g, pH6.0, adding distilled water to a constant volume of 1L, and sterilizing at 121 ℃ for 30 min.
The separation and screening method comprises the following steps:
(1) enrichment culture: 10g of a sample is taken from tea leaves piled up in a year, the sample is added into a 250mL conical flask containing 50mLLB culture medium, enrichment culture is carried out for 24h under the conditions of 45 ℃ and 120r/min, and 1mL of culture after enrichment culture is inoculated into fresh LB culture medium and culture is carried out under the same conditions. This was repeated 3 times.
(2) Primary screening: the enrichment culture was streaked on isolated solid medium and incubated at 45 ℃ for 24 h. Colonies with obvious growth were picked and rescreened.
(3) Re-screening: and streaking and separating the primary screened colony on a purified solid culture medium for 3 times to obtain a pure culture, and selecting a strain which grows vigorously according to the growth condition of the strain in the separated solid culture medium.
(4) Colony morphology and strain morphology observations
Screening the obtained strains, streaking the strains on a flat plate, culturing for 16h at 45 ℃, and observing the colony morphology under natural light; after gram staining, the strain morphology was observed.
(5) Determination of growth conditions of the strains: 50mL of LB medium was added to a 250mL Erlenmeyer flask and 0.5mL of about 8X 10 was inoculated6The optimal growth conditions were determined by culturing bacterial suspensions at cfu/ml (OD =1) at different initial pH and temperature, respectively, at 150 r/min.
The Fb strain obtained by separation and screening of the invention is cultured for 16h at 45 ℃ in an LB culture medium, and the colony is light yellow and round, has a dry surface, is opaque and has irregular edges.
The Fb strain obtained by separation and screening belongs to Bacillus belgii, is gram-positive, and has the morphological characteristics that: the thallus is rod-shaped, 0.4-0.6 μm × 0.9-4.0 μm, arranged singly or in pairs, with the spore being nearly round, growing proximally and the cyst being enlarged. Through detection, the Bacillus belgii can tolerate the high temperature of 55 ℃, the optimal growth temperature is 45 ℃, and the optimal growth pH value is 3.0-7.6.
The invention has the advantages that: because the Bacillus belgii can tolerate the high temperature of 55 ℃, the oxygen concentration can be conveniently controlled by controlling the temperature, and the requirements of production process and equipment are reduced; can grow in a very simple fermentation device (such as a device without preventing the pollution of foreign bacteria) by taking the tea dregs as a carbon-nitrogen source; the equipment investment and the cost of fermentation substrates are reduced; and other non-high temperature-resistant bacteria can be killed by increasing the temperature, so that the unicity of the target thalli is ensured. The strain can efficiently degrade tea leaves, improve protein, amino acid and the like of the tea leaves, reduce water, cellulose and the like of the tea leaves, and the tea leaves after fermentation treatment can be used as feed raw materials; can realize smokeless, odorless and sewage discharge, and can realize the recycling of organic waste, thereby reducing the pollution of the tea leaves to the environment, and being a technology which accords with green environmental protection.
Drawings
FIG. 1 is a natural-light morphological diagram of a colony.
FIG. 2 is a strain morphology diagram under an electron microscope.
FIG. 3 phylogenetic trees of the 16S rDNA sequences of the Fb strain and related species.
FIG. 4 of Fb Strain and related speciesgyrBThe gene sequence phylogenetically develops the tree.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1
The process flow comprises the following steps:
collecting samples → pretreating samples → enriching culture → selecting high temperature resistant strains → measuring growth performance → measuring optimum growth pH value → measuring optimum growth temperature → observing morphology of bacteria → obtaining Bacillus belgii: (Bacillus velezensis）Fb。
Culture medium:
(1) LB broth (L): 10g of yeast extract powder, 10g of peptone, 5g of beef extract, 10g of NaCl, 10g, pH6.0, adding distilled water to a constant volume of 1L, and sterilizing at 121 ℃ for 30 min.
(2) Purifying the solid culture medium: adding 20g/L agar powder into LB culture solution, and sterilizing at 121 deg.C for 30 min.
(3) Separating a solid culture medium: adding distilled water 500ml into fresh tea residue 50g, steaming for 30min, adding agar powder 20g into the filtered tea residue liquid, and sterilizing at 121 deg.C for 30 min.
(3) Liquid fermentation medium: 10g of yeast extract powder, 10g of peptone, 5g of beef extract, 10g of NaCl, 10g, pH6.0, adding distilled water to a constant volume of 1L, and sterilizing at 121 ℃ for 30 min.
The present invention relates to Bacillus belgiiBacillus velezensis) The Fb separation and screening method is obtained by adopting a high-temperature special habitat separation method, and comprises the following steps:
collecting a sample: collecting tea residues accumulated in the long-term;
(1) enrichment culture: 10g of a sample is taken from tea leaves piled up in a long year, and the sample is added into a 250mL conical flask containing 50mLLB culture medium and is subjected to enrichment culture for 24h at 45 ℃ at 120 r/min. 1mL of the enriched culture was inoculated into a fresh LB medium and cultured under the same conditions. This was repeated 3 times.
(2) Primary screening: the enrichment culture was streaked on isolated solid medium and incubated at 45 ℃ for 24 h. Colonies with obvious growth were picked and rescreened.
(3) Re-screening: streaking and separating the primary screened colony on a purified solid culture medium for 3 times to obtain a pure culture, and selecting a strain which grows vigorously according to the growth condition of the strain in the separated solid culture medium; selecting single colonies with different morphological characteristics, respectively propagating on an LB culture medium at 45 ℃, adding glycerol with the final volume fraction of 20%, uniformly mixing, and storing at-80 ℃;
(4) colony morphology and strain morphology observations
Screening the obtained strains, streaking the strains on a flat plate, culturing for 16h at 45 ℃, and observing the colony morphology under natural light; after gram staining, the strain morphology was observed.
(5) Determination of growth conditions of the strains:adjusting the activated cultured strain to about 8 x 106Bacterial suspension concentration cfu/mL (OD =1), 0.5mL of the suspension was aspirated and added to 250mL Erlenmeyer flask containing 50mLLB medium, and cultivation was performed at different initial pH and temperature under 150r/min, respectively, to determine the optimal growth conditions.
Determination of optimal growth pH: preparing LB culture solution with different pH values, respectively taking 50mL, subpackaging into 6 conical flasks with the volume of 250mL, inoculating 0.5mL of bacterial suspension, culturing at 45 ℃ for 16h at 120r/min, taking the LB culture solution as a blank, and measuring the optical density of the bacterial suspension at the wavelength of 630 nm.
Determination of optimum growth temperature: placing 50mL of LB culture solution with pH of 6.0 into a 250mL conical flask, inoculating 0.5mL of bacterial suspension, and culturing at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ for 16h respectively, wherein each temperature gradient is repeated for 6 times; the optical density value was measured at a wavelength of 630 nm.
The shaking frequency of the culture shaking is 120 r/min.
Example 2
A strain of Bacillus belgii was obtained by the isolation screening method described in example 1 (Bacillus velezensis) Fb. The results of the identification are as follows:
(1) observing colony morphology and strain morphology of Fb, culturing Fb strain in LB culture medium at 45 deg.C for 16h, wherein the colony morphology is characterized in that thallus is rod-shaped, 0.4-0.6 μm × 0.9-4.0 μm, arranged singly or in pairs, spore is nearly round, is near-end, cyst is enlarged, belongs to Bacillus belgii, is gram-positive, and is shown in figure 1-2.
Through detection, the Bacillus belgii can tolerate the high temperature of 55 ℃, the optimal growth temperature is 45 ℃, and the optimal growth pH value is 3.0-7.6.
(2) Physiological and biochemical characteristics of the strain:
(3) molecular characterization of strains
The sequencing result shows that the length of the 16S rDNA sequence of the strain Fb is 1440 bp. The results of Blast program analysis in NCBI database showed that the strain has the highest homology with Bacillus siamensis KCTC 13613T (AJVF01000043) and Bacillus amyloliquefaciens DSM 7T (FN597644), and the similarity reaches 99.93%. The 16S rDNA sequence phylogenetic tree of the Fb strain and related species was shown by the orthotopic ligation using MEGA5.0 software (FIG. 3). The gyrB sequencing analysis of the strain Fb shows that the sequence is 1176 bp long. The result of the analysis of MEGA5.0 software shows that the protein is combined with the proteinBacillus velezensis BCRC 17467T(DQ903176) has higher homology and the similarity reaches 100%. The gyrB gene of this strain was used to construct a phylogenetic tree with the gyrB genes of 10 strains of the other defined species (FIG. 4).
The strain Fb is identified as the Bacillus belgii by combining the strain morphology, physiological and biochemical characteristics and the sequence comparison analysis of conserved genesBacillus velezensis）。
Example 3 Bacillus belgii ((II))Bacillus velezensis) Preparation method of Fb bacteria powder
(1) Activating strains:
bacillus belgii preserved at 4 ℃ slant (Bacillus velezensis) Fb activation, respectively streaking on corresponding solid culture media, and culturing for 16h at 45 ℃;
the strain source is as follows: bacillus belgii (B.), (Bacillus velezensis) Fb is obtained by self-screening and is preserved in China general microbiological culture Collection center with the serial number of CGMCC No. 19438;
(2) seed culture:
picking up the activated colony, and inoculating Bacillus belgii (Bacillus belgii) (II)Bacillus velezensis) Fb is inoculated in a seed culture medium for seed culture, and shaking culture is carried out for 16h at 45 ℃ and 150 r/min;
the seed culture medium comprises the following components in mass concentration:
10g/L yeast extract powder, 10g/L peptone, 5g/L beef extract powder, 10g/L NaCl and 6.0 PH;
(3) and (3) tank loading fermentation culture:
the Bacillus belgii obtained in the previous step (B), (B)Bacillus velezensis) Inoculating Fb seed liquid into a fermentation tank according to the inoculation amount of 10%, wherein the fermentation medium is the same as the seed culture medium, the ventilation ratio is 1:1.05, the temperature is 45 ℃, the speed is 180r/min, and collecting bacterial liquid after culturing for 16 h;
(4) spray drying:
spray drying the bacterial liquid, and drying by using 5% of skimmed milk powder as a protective agent to obtain powder, wherein the spray drying conditions are as follows: the air inlet temperature is 150 ℃, the air outlet temperature is 90 ℃, and the feeding speed is 15 ml/min.
The bacterial liquid is packaged by an aseptic sealing bag after being sprayed and dried into bacterial powder so as to prevent moisture absorption.
Example 4: bacillus belgii (B.), (Bacillus velezensis) Application of Fb bacterial powder
The method comprises the following steps:
(1) pretreating fresh tea leaves, and cooling the tea leaves which are just discharged to 50 ℃.
(2) Tea residue compost fermentation: adding Fb bacteria powder according to 1% of the weight of the tea residues, uniformly mixing, piling on a hardened floor with rain shelter at the thickness of 80cm, turning the pile once every 24h at 0-5d, naturally evaporating water through compost for 2-3d without turning the pile, and taking the obtained product as a feed raw material when the water content is within 30%. The period is about 7-8 days.
According to the conventional nutrient components of the tea leaves, the crude protein of the fermented tea leaves is improved by 14.88 percent, the crude fiber is reduced by 9.69 percent, and the obvious difference exists; see table 1.
Table 1 conventional nutritional ingredients of tea leaves (dry basis, n =9,%)
Note: in the same column data, the same letters are annotated to indicate insignificant difference (P > 0.05), and adjacent or spaced letters are annotated to indicate significant difference (P < 0.05) or very significant difference (P < 0.01), respectively. The same as in Table 2.
The comparison of the tea residue fiber components shows that the neutral detergent fiber, the acidic detergent fiber and the lignin are respectively reduced by 10.72%, 4.47% and 11.37%, and the differences are obvious; see table 2.
Table 2 comparison of fiber content of tea leaves (dry basis, n = 9%)
As can be seen from the components and contents of the amino acids in the tea leaves, the two groups have the highest glutamic acid content and the second aspartic acid content except that the cystine, the methionine and the histidine are not different before and after fermentation; the content of glutamic acid and aspartic acid after fermentation is respectively improved by 6.88 percent and 7.01 percent; other variations in the amino acid content are also possible. The change trend of the total amount of amino acid is consistent with that of crude protein, the content of the amino acid is improved by 5.98 percent, and the differences are obvious, which is shown in a table 3.
Table 3 comparison of nutritional ingredients before and after tea leaf treatment (dry basis, n = 9%)
Note: for peer data, the same letters are annotated to indicate insignificant difference (P > 0.05), and adjacent or spaced letters are annotated to indicate significant difference (P < 0.05) or very significant difference (P < 0.01), respectively.
The results show that the Fb strain can efficiently degrade the tea leaves, improve the protein, amino acid and the like of the tea leaves, reduce the water content, cellulose and the like of the tea leaves, and the tea leaves after fermentation treatment can be used as feed raw materials. Can realize smokeless, odorless and sewage discharge, and can realize the recycling of organic waste, thereby reducing the pollution of the tea leaves to the environment, and being a technology which accords with green environmental protection. Has wide application prospect.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (2)
1. A strain of Bacillus belgii is characterized in that: the Bacillus belgii is Bacillus belgii (Bacillus belgii)Bacillus velezensis) Fb ofThe strain is preserved in China general microbiological culture collection center at 28.2.2020, with the preservation number of CGMCC No. 19438.
2. Use of bacillus beilesiensis according to claim 1 for degrading tea leaves.
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| CN112043002A (en) * | 2020-09-07 | 2020-12-08 | 湖北中烟工业有限责任公司 | Application of Bacillus belgii in degrading cellulose in tobacco flavor raw material |
| CN112094775B (en) * | 2020-09-22 | 2022-07-26 | 中国石油天然气集团有限公司 | Bacillus belgii and screening culture method and application thereof |
| CN114369542B (en) * | 2020-10-15 | 2023-06-09 | 南京理工大学 | Alkane degrading bacteria and application thereof |
| CN112662599B (en) * | 2021-01-27 | 2022-06-14 | 吉林省农业科学院 | Poultry source Bacillus belgii CL-4 and application thereof |
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