CN113652379A - Bacillus cell wall depolymerized GIEC and application thereof - Google Patents
Bacillus cell wall depolymerized GIEC and application thereof Download PDFInfo
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Images
Classifications
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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
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- 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
Abstract
The invention discloses a bacillus cell wall depolymerized GIEC and application thereof, belonging to the field of biotechnology, wherein the cell wall depolymerized GIEC is stored in Guangdong province microorganism strain preservation center at 2021, 2 and 5 days, and the preservation number is GDMCC No. 61501. A plurality of experiments prove that the cell wall depolymerized GIEC can effectively degrade the cell wall, for example, the strain is used for degrading spirulina, and the recovery rate of carbohydrate in the obtained depolymerized liquid is more than or equal to 40 percent; the strain is used for degrading hybrid pennisetum alopecuroides, pennisetum purpureum and corn straws, carbohydrate in the obtained degradation liquid is more than or equal to 2g/L, protein is more than or equal to 0.7g/L, cellulose degradation rate is more than or equal to 17%, hemicellulose degradation rate is more than or equal to 19%, and lignin degradation rate is more than or equal to 16%.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to bacillus cell wall depolymerized GIEC and application thereof.
Background
Spirulina is a kind of cyanobacteria, and is rich in proteins, polysaccharides, lipids, vitamins and minerals, and high-value substances such as chlorophyll, carotenoid and unsaturated fatty acid. Spirulina has a typical resistant cell wall, which is composed of multiple layers of glucan and peptidoglycan polymers, covered externally with a sheath of acidic polysaccharides. The cell wall of lignocellulosic biomass is composed of cellulose, hemicellulose, pectin, lignin, cell wall proteins and other components, between which very complex covalent and non-covalent interactions exist.
Due to the complex cell wall structure, the limitation of the cell wall directly results in poor high-value utilization of biomass. In order to realize resource utilization of spirulina and wood fiber biomass, various pretreatment means are indispensable steps for cell wall breaking.
The pretreatment methods include physical methods, chemical methods, physicochemical methods, and biological methods. At present, manufacturers at home and abroad mostly adopt a chemical method to pretreat microalgae and wood fiber biomass. The greatest disadvantage of chemical pretreatment is that the activity of microalgae endogenous substances is damaged by strong alkali and high temperature, and black liquor is generated in the pretreatment process of the lignocellulose biomass. The principle of the biological fermentation method is that microorganisms can generate various enzymes in the metabolic process taking spirulina, wood fiber biomass and the like as nutrients, and macromolecular substances forming cell walls are degraded into micromolecular 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 method is a mild pretreatment mode. Therefore, the present invention is directed to screening microorganisms capable of efficiently disaggregating cell walls.
Disclosure of Invention
The first purpose of the invention is to provide a Bacillus (Bacillus sp.), named as cell wall depolymerization GIEC, which is preserved in the Guangdong province microorganism strain preservation center at 2021, 2 and 5 days, and is addressed to No. 59 building, 5 floor of Michelia Tokyo No. 100 Hotel in overseas district of Guangzhou city, Guangdong province, and the postal code is as follows: 510070 with the deposit number GDMCC No. 61501.
The cell wall depolymerized GIEC has the colony size of 10-18mm, is colorless, transparent, round, neat in edge, smooth and moist in surface, and is rod-shaped, spore-born and gram-reaction-positive when observed under a microscope. And combining the analysis of a whole genome, and identifying the Bacillus as Bacillus (Bacillus). Experiments prove that the cell wall depolymerized GIEC has stronger cell wall depolymerizing capability, and particularly can efficiently degrade the complex cell wall structure of the spirulina.
It is a second object of the present invention to provide a microbial preparation comprising, as an active ingredient, a culture of the murally depolymerized GIEC of the present invention or a fermentation broth thereof.
It is a third object of the present invention to provide use of the cell wall depolymerized GIEC or microbial preparation thereof according to the present invention for depolymerizing biomass cell walls;
preferably, the biomass is any one of spirulina, hybrid pennisetum alopecuroides, elephantopus japonicus or corn stalks;
further, the use of the murally depolymerized GIEC or the microbial preparation thereof according to the present invention for depolymerizing biomass cell walls to prepare food or feed or additives.
A fourth object of the present invention is to provide a method for depolymerizing biomass cell walls, comprising the steps of:
a. preparation of a fermentation medium: adding deionized water into biomass to obtain a required fermentation culture medium;
b. inoculation and fermentation: the cell wall depolymerized GIEC of the present invention was inoculated into a fermentation medium for fermentation.
Preferably, the fermentation medium comprises the following components of 100 parts by weight of biomass and 150 parts by weight of deionized water to 1000 parts by weight;
preferably, said inoculating is activating said muramyl depolymerized GIEC from a preserved liquid medium to a fresh liquid medium, provided that: shaking and culturing for 48h at 37 ℃ and 150 r/min;
preferably, the fermentation conditions are 37 ℃ of temperature, 140-150rpm of oscillation speed and N of ventilation2:CO2＝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 recycling rate of carbohydrate in the obtained depolymerized liquid is more than or equal to 40 percent by degrading the spirulina by using the strain;
(2) the bacillus has strong cell wall depolymerization capability, the strain is used for degrading hybrid pennisetum alopecuroides, pennisetum purpureum and corn straws, carbohydrate in the obtained degradation liquid is more than or equal to 2g/L, protein is more than or equal to 0.7g/L, cellulose degradation rate is more than or equal to 17%, hemicellulose degradation rate is more than or equal to 19%, and lignin degradation rate is more than or equal to 16%.
The Bacillus sp cell wall depolymerization GIEC of the invention is preserved in the microbial strain preservation center of Guangdong province at 2021, 2 and 5 days, and is addressed to No. 59 floor 5 of the Mineli Zhongluo No. 100 Hospital in the oversea city of Guangzhou province, and the zip code is as follows: 510070 with the deposit number GDMCC No. 61501.
Drawings
FIG. 1 is a microscopic examination of the morphology (A) of cell wall depolymerized GIEC cells and the state (B) of Spirulina cells.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The present invention will be further described with reference to the following examples, which are all conventional test methods unless otherwise specified, and the test reagents and consumables described in the following examples, which are all available from conventional biochemical reagents companies, unless otherwise specified.
Example 1 screening and identification of strains
1.1 screening of Strain cell wall depolymerized GIEC
Diluting the probiotic capsule purchased from the market with a proper amount of sterile water, then carrying out dilution plate coating, and selecting the grown bacterial colony to a liquid culture medium to obtain the target bacterial colony. The liquid culture medium is prepared by diluting spirulina in logarithmic culture stage to appropriate concentration, and autoclaving at 115 deg.C for 20 min; the solid medium was obtained by adding 1% agar to the above liquid medium.
1.2 identification of Strain mural depolymerized GIEC
The strain morphological characteristics are as follows: the size of the bacterial colony is 10-18mm, the bacterial colony is colorless, transparent, round, neat in edge, smooth and moist in surface, and the bacteria observed under a microscope are rod-shaped, spore is middle-born, and gram reaction is positive. And combining the analysis of a whole genome, and identifying the Bacillus as Bacillus (Bacillus).
Therefore, the strain is named as Bacillus sp cell wall depolymerization GIEC by combining morphological, molecular biology and physiological and biochemical characteristics, is stored in the microbial strain preservation center of Guangdong province at 2021, 2 and 5 days, and is addressed to No. 59 building of first furious Zhonglu 100 college in overseas City of Guangzhou province, Guangdong province, and is coded by a postal code: 510070 with the deposit number GDMCC No. 61501.
Example 2 cell wall depolymerization of GIEC depolymerized Spirulina cell walls
(1) Mural depolymerization GIEC seed activation
The cell wall disaggregated GIECs screened in example 1 were transferred from the preserved liquid medium to a fresh liquid medium and shake-cultured at 150r/min for 48 hours at 37 ℃ on a shaker. Seed quality standard: the OD value measured under the wavelength of 600nm reaches 0.5, and the thalli are plump through microscopic examination. The inoculation amount is 10%. The preserved liquid culture medium is prepared by diluting spirulina in logarithmic culture stage to appropriate concentration, and autoclaving at 115 deg.C for 20 min.
(2) Preparing fermentation medium
Collecting spirulina (dry weight about 1.7mg/ml) in logarithmic growth stage to obtain the desired fermentation medium.
(3) Inoculating and fermenting
Inoculating 10% of cell wall depolymerized GIEC into a fermentation culture medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotation speed: 150 rpm; ventilation capacity; n is a radical of2:CO2Samples were taken at 0, 3, 6 and 24 hours after inoculation, and then at 24-hour intervals, and the cell morphology and spirulina status were examined under the microscope, as shown in fig. 1. The sample was then centrifuged at 8000rpm for 5min and the supernatant and pellet were separated and stored separately.
Example 3 cell wall depolymerization GIEC depolymerization of hybrid pennisetum cell wall
(1) Mural depolymerization GIEC seed activation
The cell wall disaggregated GIECs screened in example 1 were transferred from the preserved liquid medium to a fresh liquid medium and shake-cultured at 150r/min for 48 hours at 37 ℃ on a shaker. Seed quality standard: OD600The value reaches 0.5, and the thalli are plump by microscopic examination. The inoculation amount is 10%.
(2) Pulverizing hybrid pennisetum alopecuroides
Pulverizing the hybrid pennisetum for 10min by a pulverizer.
(3) Preparation of fermentation medium
Taking 100g of crushed pennisetum hybrid, 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 culture medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotation speed: 150 rpm; ventilation capacity; n is a radical of2:CO2Samples were taken at 0, 3, 6, 24 hours post inoculation and then every 24 hours (v/v) at 4: 1. The samples were then centrifuged for 5min and the supernatant and pellet separated and stored separately.
Example 4 cell wall depolymerization GIEC depolymerized Bidens pilosa
(1) Mural depolymerization GIEC seed activation
The cell wall disaggregated GIECs screened in example 1 were transferred from the preserved liquid medium to a fresh liquid medium and shake-cultured at 150r/min for 48 hours at 37 ℃ on a shaker. Seed quality standard: OD600The value reaches 0.5, and the thalli are plump by microscopic examination. The inoculation amount is 10%.
(2) Pulverizing herba Pteridis Multifidae
Pulverizing the hybrid pennisetum for 10min by a pulverizer.
(3) Preparation of fermentation medium
Taking 150g of crushed pennisetum hybrid, 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 culture medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotation speed: 140 rpm; ventilation capacity; n is a radical of2:CO2Samples were taken at 0, 3, 6, 24 hours post inoculation and then every 24 hours (v/v) at 4: 1. The samples were then centrifuged for 5min and the supernatant and pellet separated and stored separately.
Example 5 cell wall depolymerized GIEC depolymerized corn stover
(1) Mural depolymerization GIEC seed activation
The cell wall disaggregated GIECs screened in example 1 were transferred from the preserved liquid medium to a fresh liquid medium and cultured with shaking at 140r/min for 48 hours at 37 ℃ on a shaker. Seed quality standard: OD600The value reaches 0.5, and the thalli are plump by microscopic examination. The inoculation amount is 15%.
(2) Pulverizing herba Pteridis Multifidae
Pulverizing the hybrid pennisetum for 10min by a pulverizer.
(3) Preparation of fermentation medium
Taking 100g of crushed pennisetum hybrid, 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 culture medium for fermentation to obtain the biomass degradation liquid. Fermentation parameters were controlled as follows: fermentation temperature: 37 ℃; oscillating rotation speed: 140 rpm; ventilation capacity; n is a radical of2:CO2Samples were taken at 0, 6, and 24 hours post inoculation, and then every 24 hours (v/v). The samples were then centrifuged for 5min and the supernatant and pellet separated and stored separately.
Respectively degrading spirulina by using the strain, wherein the recovery rate of carbohydrate in the obtained depolymerized liquid is more than or equal to 40%; the strain is used for degrading hybrid pennisetum alopecuroides, pennisetum purpureum and corn straws, the carbohydrate in the degradation solution is more than or equal to 2g/L, the protein is more than or equal to 0.7g/L, and the degradation rate of cellulose in cell walls is more than 17%, the degradation rate of hemicellulose is more than 19% and the degradation rate of lignin is more than 16% can be realized. The calculation formula of the degradation rate is as follows:
wherein C (F) and C (r) respectively represent the percentage contents of cellulose, hemicellulose and lignin before and after bacterial treatment; m (F) and m (r) represent the mass of the solid before and after treatment, respectively.
Claims (8)
1. A Bacillus sp cell wall depolymerized GIEC characterized by the accession number GDMCC No. 61501.
2. A microbial preparation comprising, as an active ingredient, a culture of the mural depolymerized GIEC of claim 1 or a fermentation broth thereof.
3. Use of the mural depolymerized GIEC of claim 1 or the microbial preparation of claim 2 for depolymerizing biomass cell walls.
4. The use of claim 3, wherein the biomass is any one of spirulina, pennisetum hydridum, elephant grass or corn stover.
5. A method for depolymerizing biomass cell walls, comprising the steps of:
a. preparation of a fermentation medium: adding deionized water into biomass to obtain a required fermentation culture medium;
b. inoculation and fermentation: inoculating the muramyl depolymerized GIEC of claim 1 into a fermentation medium for fermentation.
6. The method as claimed in claim 5, wherein the fermentation medium comprises 100 parts by weight of biomass and 150 parts by weight of deionized water to 1000 parts by weight.
7. The method of claim 5, wherein said inoculating is by activating said mural depolymerized GIEC from a preserved liquid medium to a fresh liquid medium, provided that: shaking and culturing at 37 deg.C and 150r/min for 48 h.
8. Root of herbaceous plantThe method as claimed in claim 5, wherein the fermentation conditions are a temperature of 37 ℃, an oscillation speed of 140-150rpm, and a ventilation N2:CO2＝4:1。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115340964A (en) * | 2022-06-26 | 2022-11-15 | 上海龙殷生物科技有限公司 | Bacillus strain with swollenin activity and application thereof |
| CN115404181A (en) * | 2022-07-25 | 2022-11-29 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as lactobacillus plantarum fermentation medium |
| CN115340964B (en) * | 2022-06-26 | 2024-05-14 | 上海龙殷生物科技有限公司 | Bacillus strain with swollenin activity and application thereof |
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| CN115340964B (en) * | 2022-06-26 | 2024-05-14 | 上海龙殷生物科技有限公司 | Bacillus strain with swollenin activity and application thereof |
| CN115404181A (en) * | 2022-07-25 | 2022-11-29 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as lactobacillus plantarum fermentation medium |
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