CN113652379B - Bacillus cell wall depolymerization GIEC and application thereof - Google Patents
Bacillus cell wall depolymerization GIEC and application thereof Download PDFInfo
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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
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- 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
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Abstract
The invention discloses bacillus cell wall depolymerization GIEC and application thereof, belonging to the field of biotechnology, wherein the cell wall depolymerization GIEC is stored in the Guangdong province microorganism strain collection center at 2021, 2 and 5 days, and the storage number is GDMCC No. 61501. According to the invention, a plurality of experiments prove that cell wall depolymerization GIEC can effectively degrade cell walls, for example, spirulina is degraded by using the strain, and the recovery rate of carbohydrate in the obtained depolymerization liquid is more than or equal to 40%; the strain is used for degrading hybrid pennisetum, pennisetum and corn stalks, the carbohydrate in the obtained degradation liquid is more than or equal to 2g/L, the protein is more than or equal to 0.7g/L, the cellulose degradation rate is more than or equal to 17%, the hemicellulose degradation rate is more than or equal to 19%, and the lignin degradation rate is more than or equal to 16%.
Description
Technical Field
The invention relates to the technical field of biology, in particular to bacillus cell wall depolymerization GIEC and application thereof.
Background
Spirulina is a cyanobacteria rich in proteins, polysaccharides, lipids, vitamins and minerals, and also high value substances such as chlorophyll, carotenoids and unsaturated fatty acids. Spirulina has a typical resistant cell wall consisting of multiple layers of dextran and peptidoglycan polymers, covered externally with a sheath of acidic polysaccharide. The cell wall of lignocellulosic biomass is composed of cellulose, hemicellulose, pectin, lignin, cell wall proteins, and other components, between which there are very complex covalent and non-covalent bonds.
Due to the complex structure of the cell wall, the limitation of the cell wall directly leads to poor high-value utilization effect of biomass. In order to realize the recycling of spirulina and lignocellulosic biomass, various pretreatment means are indispensable steps for cell wall disruption.
The pretreatment method mainly comprises a physical method, a chemical method, a physicochemical method and a biological method. At present, most manufacturers at home and abroad pretreat microalgae and lignocellulose biomass by a chemical method. The biggest disadvantage of using chemical pretreatment is that the high temperature of strong alkali can destroy the activity of the endogenous substances of microalgae and generate black liquor in the pretreatment process of the lignocellulose biomass. The principle of the biological fermentation method is that microorganisms are utilized to generate various enzymes in the metabolic process taking spirulina, lignocellulose biomass and the like as nutrients, so that macromolecular substances forming the cell wall are degraded into micromolecules and water-soluble substances. The microbial fermentation does not need energy consumption and high pressure of a physical method, and does not need strong alkali and high temperature of a chemical method, so that the microbial fermentation is a mild pretreatment mode. Accordingly, the present invention is directed to screening for microorganisms capable of efficiently depolymerizing cell walls.
Disclosure of Invention
The first object of the present invention is to provide a bacillusBacillussp.), designated as cell wall depolymerized GIEC, stored in the microorganism culture collection in guangdong, 2 months, 5 days of 2021, at floor 5 of the national institute of advanced middle road 100, university, 5 th floor, in the viewye district, guangdong, and post code: 510070 with deposit number GDMCC No. 61501.
The cell wall depolymerization GIEC has the advantages of 10-18mm colony size, no color, transparency, round shape, neat edge, smooth and moist surface, rod-shaped bacteria observed under a microscope, mesogenic spores and positive gram reaction. And combining with whole genome analysis to identify it as bacillusBacillus). Experiments prove that the cell wall depolymerization GIEC has stronger cell wall depolymerization capability, and particularly can efficiently degrade the complex cell wall structure of the spirulina.
The second object of the present invention is to provide a microbial preparation comprising the culture of cell wall depolymerized GIEC or a fermentation broth thereof as an active ingredient.
A third object of the present invention is to provide the use of the cell wall depolymerizing GIEC or microbial preparation thereof of the present invention for depolymerizing a biomass cell wall;
preferably, the biomass is any one of spirulina, hybrid pennisetum, pennisetum or corn stalks;
further, the cell wall depolymerizing GIEC or the microbial preparation thereof of the present invention is used for depolymerizing biomass cell walls to prepare foods or feeds or additives.
A fourth object of the present invention is to provide a method for depolymerizing a biomass cell wall comprising the steps of:
a. preparing a fermentation medium: adding deionized water into biomass to obtain a required fermentation medium;
b. inoculating and fermenting: the cell wall depolymerized GIEC of the present invention was inoculated into a fermentation medium for fermentation.
Preferably, the ingredients of the fermentation medium are 100-150 parts by weight of biomass, and deionized water is added to 1000 parts by weight;
preferably, the inoculation is to transfer the cell wall depolymerized GIEC from the preserved liquid medium to a fresh liquid medium for activation, provided that: shaking table 37 ℃,150r/min, shake culture for 48h;
preferably, the fermentation conditions are a temperature of 37 ℃, an oscillating rotation speed of 140-150rpm, and a ventilation N 2 :CO 2 =4:1。
Compared with the prior art, the invention has the following beneficial effects:
(1) The bacillus has stronger cell wall depolymerization capability, and the strain is used for degrading spirulina, so that the recovery rate of carbohydrate in the depolymerization liquid is more than or equal to 40%;
(2) The bacillus provided by the invention has stronger cell wall depolymerization capability, and the strain is used for degrading hybrid pennisetum, pennisetum and corn stalks, so that the carbohydrate in the degradation liquid is more than or equal to 2g/L, the protein is more than or equal to 0.7g/L, the cellulose degradation rate is more than or equal to 17%, the hemicellulose degradation rate is more than or equal to 19%, and the lignin degradation rate is more than or equal to 16%.
The invention is characterized in thatBacillus sp, cell wall depolymerization GIEC, stored in Guangdong at 2021, 2 and 5 daysThe provincial microorganism strain collection center has an address of building 5 of No. 59 of Mitsui No. 100 of Xiuzhou district of Guangzhou, guangdong province, and the post code is as follows: 510070 with deposit number GDMCC No. 61501.
Drawings
FIG. 1 is a microscopic view of cell wall depolymerizing GIEC cell morphology (A) and spirulina state (B).
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The present invention will be further described with reference to examples, in which the test methods are conventional test methods unless otherwise specified, and the test reagents and consumables described in the examples are from conventional Biochemical reagent company unless otherwise specified.
Example 1 screening and identification of strains
1.1 Screening of bacterial Strain cell wall depolymerizing GIEC
And diluting the probiotic capsules obtained from the market with a proper amount of sterile water, then coating the diluted probiotic capsules on a dilution plate, and selecting the grown bacterial colonies into a liquid culture medium to obtain target bacterial colonies. The liquid culture medium is obtained by diluting spirulina in logarithmic culture period to proper concentration, and sterilizing at 115deg.C for 20min under high pressure; the solid medium was obtained by adding 1% agar to the above liquid medium.
1.2 Identification of bacterial Strain cell wall depolymerizing GIEC
Morphological characteristics of the strain: the colony is 10-18mm in size, colorless, transparent, round, neat in edge, smooth and moist in surface, and the bacteria are observed to be rod-shaped under a microscope, and the spores are mesogenic, so that gram reaction is positive. And combining with whole genome analysis to identify it as bacillusBacillus）。
Thus, the strain is named by combining morphological, molecular biological and physiological biochemical characteristicsBacillus sp, cell wall depolymerization GIEC, stored in the microorganism strain collection center of guangdong, 2 months 5 days 2021, at the address of building 5 of first institute No. 100, the xiu district of guangzhou city, guangdong, post code: 510070 with deposit number GDMCC No. 61501.
EXAMPLE 2 cell wall depolymerization GIEC depolymerization of Spirulina cell wall
(1) Cell wall disaggregation GIEC seed activation
The cell wall depolymerized GIEC obtained by the screening in example 1 was transferred from the preserved liquid medium to a fresh liquid medium, and cultured by shaking at 37℃in a shaker at 150r/min for 48 hours. Seed quality standard: OD value reaches 0.5 when measured at the wavelength of 600nm, and the microscopic bacteria are full. The inoculation amount is 10%. The preserved liquid culture medium is obtained by diluting spirulina in logarithmic culture stage to proper concentration, and sterilizing at 115deg.C for 20min under high pressure.
(2) Preparing fermentation medium
Collecting spirulina (dry weight of about 1.7 mg/ml) in logarithmic growth period to obtain the desired fermentation medium.
(3) Inoculating and fermenting
And (3) inoculating 10% of cell wall depolymerized GIEC into a fermentation medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotational speed: 150rpm, aeration rate; n (N) 2 :CO 2 =4:1 (v/v), samples were taken at 0, 3, 6, 24 hours after inoculation, then taken every 24 hours, and the cell morphology and spirulina state were microscopic, and the results are shown in fig. 1. The samples were then centrifuged at 8000rpm for 5min, and the supernatant and the slag were separated and stored separately.
EXAMPLE 3 cell wall depolymerization of GIEC depolymerization of hybrid pennisetum cell walls
(1) Cell wall disaggregation GIEC seed activation
The cell wall depolymerized GIEC obtained by the screening in example 1 was transferred from the preserved liquid medium to a fresh liquid medium, and cultured by shaking at 37℃in a shaker at 150r/min for 48 hours. Seed quality standard: OD (optical density) 600 The value reaches 0.5, and the microscopic bacteria are full. The inoculation amount is 10%.
(2) Crushing hybrid pennisetum
Pulverizing hybrid pennisetum with pulverizer for 10min.
(3) Preparation of fermentation Medium
Taking 100g of crushed hybrid pennisetum, and adding deionized water to 1000ml to obtain the required fermentation medium.
(4) Inoculating and fermenting
Inoculating the cell wall depolymerized GIEC into a fermentation medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotational speed: 150rpm, aeration rate; n (N) 2 :CO 2 =4:1 (v/v), samples were taken at 0, 3, 6, 24 hours after inoculation, respectively, and then taken every 24 hours. The sample was then centrifuged for 5min, and the supernatant and the residue were separated and stored separately.
EXAMPLE 4 cell wall depolymerization of GIEC depolymerization of Japanese butterflybush
(1) Cell wall disaggregation GIEC seed activation
The cell wall depolymerized GIEC obtained by the screening in example 1 was transferred from the preserved liquid medium to a fresh liquid medium, and cultured by shaking at 37℃in a shaker at 150r/min for 48 hours. Seed quality standard: OD (optical density) 600 The value reaches 0.5, and the microscopic bacteria are full. The inoculation amount is 10%.
(2) Pulverizing herba Ardisiae Japonicae
Pulverizing herba Ardisiae Japonicae with pulverizer for 10min.
(3) Preparation of fermentation Medium
150g of crushed Japanese butterflybush herb is taken, deionized water is added to 1000ml, and the required fermentation medium is obtained.
(4) Inoculating and fermenting
Inoculating the cell wall depolymerized GIEC into a fermentation medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotational speed: 140rpm, aeration rate; n (N) 2 :CO 2 =4:1 (v/v), samples were taken at 0, 3, 6, 24 hours after inoculation, respectively, and then taken every 24 hours. The sample was then centrifuged for 5min, and the supernatant and the residue were separated and stored separately.
Example 5 cell wall depolymerization of GIEC depolymerized corn stover
(1) Cell wall disaggregation GIEC seed activation
The cell wall depolymerized GIEC obtained by the screening in example 1 was transferred from the preserved liquid medium to a fresh liquid medium, and cultured by shaking at 37℃in a shaker at 140r/min for 48 hours. Seed quality standard: OD (optical density) 600 The value reaches 0.5, and the microscopic bacteria are full. Inoculum size 15%。
(2) Corn stalk crushing
The corn stalks are crushed for 10min by a crusher.
(3) Preparation of fermentation Medium
100g of crushed corn straw is taken, deionized water is added to 1000ml, and the required fermentation medium is obtained.
(4) Inoculating and fermenting
Inoculating the cell wall depolymerized GIEC into a fermentation medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotational speed: 140rpm, aeration rate; n (N) 2 :CO 2 =4:1 (v/v), samples were taken at 0, 6, 24 hours after inoculation, respectively, and then taken every 24 hours. The sample was then centrifuged for 5min, and the supernatant and the residue were separated and stored separately.
The method is respectively applied to the implementation of the degradation of the spirulina by the strain, and the recovery rate of the carbohydrate in the obtained depolymerization liquid is more than or equal to 40 percent; the strain is used for degrading hybrid pennisetum, pennisetum and corn stalks, and the carbohydrate in the degradation liquid is more than or equal to 2g/L, the protein is more than or equal to 0.7g/L, so that the degradation rate of cellulose in the cell wall is more than 17%, the degradation rate of hemicellulose is more than 19% and the degradation rate of lignin is more than 16%. The degradation rate is calculated as follows:
degradation rate =
Wherein C (r) and C (F) respectively represent the percentage content of cellulose, hemicellulose and lignin before and after bacterial treatment; m (r) and m (F) represent the mass of the solid before and after the treatment, respectively.
Claims (8)
1. Bacillus cellBacillus sp.) cell wall depolymerizing GIEC, characterized in that it has deposit number GDMCC No. 61501.
2. A microbial preparation comprising the fermentation broth of the bacillus cell wall depolymerization GIEC according to claim 1 as an active ingredient.
3. Use of the bacillus cell wall depolymerizing GIEC of claim 1 or the microbial preparation of claim 2 for depolymerizing a biomass cell wall.
4. The use according to claim 3, wherein the biomass is any one of spirulina, hybrid pennisetum, pennisetum or maize straw.
5. A method of depolymerizing a biomass cell wall, comprising the steps of:
a. preparing a fermentation medium: adding deionized water into biomass to obtain a required fermentation medium;
b. inoculating and fermenting: inoculating the bacillus cell wall depolymerized GIEC of claim 1 into a fermentation medium for fermentation.
6. The method according to claim 5, wherein the fermentation medium comprises 100-150 parts by weight biomass and deionized water to 1000 parts by weight.
7. The method of claim 5, wherein the inoculating is performed by transferring the bacillus cell wall depolymerized GIEC of claim 1 from the stored liquid medium to a fresh liquid medium for activation, provided that: shaking table 37 ℃,150r/min, shake culture 48h.
8. The method according to claim 5, wherein the fermentation conditions are a temperature of 37℃and a shaking speed of 140-150rpm, and a ventilation N 2 :CO 2 =4:1。
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| CN105385629A (en) * | 2015-12-01 | 2016-03-09 | 北京德瑞丰农业科技有限责任公司 | Lignocellulos substance high-efficiency degrading bacterium M2 and application thereof |
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