CN116333906B - Bacillus and method for preparing ectoin by using same - Google Patents

Bacillus and method for preparing ectoin by using same Download PDF

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CN116333906B
CN116333906B CN202210828214.2A CN202210828214A CN116333906B CN 116333906 B CN116333906 B CN 116333906B CN 202210828214 A CN202210828214 A CN 202210828214A CN 116333906 B CN116333906 B CN 116333906B
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ectoin
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qyj
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CN116333906A (en
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谢小保
杨平
彭如群
李彩玲
袁英姿
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Institute of Microbiology of Guangdong Academy of Sciences
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Abstract

The invention discloses bacillus and a method for preparing ectoin by using the same. The invention firstly separates a strain with the secretion characteristics of the ectoin from the root of the polygonum hydropiper, and then identifies the strain as bacillus Bacillus atrophaeus QYJ, and the preservation number of the strain is GDMCC No. 62486. The strain QYJ is cultivated by non-salinity and aerobic fermentation at 37 ℃ to produce the ectoin, and the fermentation yield is 7.348g/L. The method for preparing the ectoin by using the strain has the advantages of simple process, low cost of raw materials, environmental protection and potential industrial application prospect.

Description

Bacillus and method for preparing ectoin by using same
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus and a method for preparing ectoin by using the same.
Background
Ekedorine, chemical name is (S) -2-methyl-3, 4,5, 6-tetrahydropyrimidine-4-carboxylic acid, which is called tetrahydropyrimidine for short, CAS number is 96702-03-3, molecular formula is C 6 H 10 N 2 O 2 The molecular weight is 142.1, and is a cyclic amino acid. The ectoin can be used as a humectant, and can prevent skin dryness and aging, and reduce skin injury caused by ultraviolet rays. Therefore, the ectoin is widely applied to anti-wrinkle, tightening, repairing and anti-aging skin care products.
Currently, escitalopram is obtained by bacterial milking. Bacterial milking is a process in which the production bacteria are stimulated by repeated hypertonicity and hypotonicity, causing the accumulation of exendin in the cells and eventually excreted into the medium. Since the synthesis of ectoine is regulated by osmotic pressure, high yields of tetrahydropyrimidine are currently generally dependent on high salt concentrations. However, the use of high salinity media in fermentation processes not only places significant limitations on the equipment cost, design and durability of the fermenter system, but also places significant pressure on the treatment of high salinity wastewater. In addition, the process involves complex process operating strategies, thereby increasing production costs. In addition, very high extracellular salt concentrations can also have a significant negative impact on cell homeostasis and structural function of the protein, adversely affecting cell growth and function.
In order to solve the problem of high salinity fermentation, a few fermentation strains with reduced salinity or no salinity requirements are searched for producing the ectoin, so that the production cost can be reduced, the process is simplified, the wastewater treatment difficulty can be reduced, the environment is protected, and the method has important practical significance.
Disclosure of Invention
The first object of the present invention is to provide a bacillus Bacillus atrophaeus QYJ strain. The strain was deposited at the Cantonese microbiological strain collection center (GDMCC) at 5.19, 2022, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62486. the strain can produce the ectoine under the conditions of no salinity and aerobic fermentation, and has the advantages of simple preparation process and low raw material cost.
The invention separates a strain Bacillus atrophaeus QYJ37 with quick growth speed and the characteristic of secondaries from the root of the polygonum hydropiper. The colony characteristics of the strain are shown in figure 1, the colony is nearly round but has uneven edges, is opaque, has raised lawn, has wrinkled surface and is not sticky and jelly-like on the surface, and the diameter of the colony reaches 10-15mm after 24 hours of culture. The 16S rRNA sequence is shown as SEQ ID NO. 1. As shown in FIG. 2, the microscopic photograph of Bacillus Bacillus atrophaeus QYJ shows gram-positive staining, short rod-like, spores in the center of the cell, and aerobic growth.
A second object of the invention is to provide the use of said Bacillus Bacillus atrophaeus QYJ37 for the preparation of ectoin.
The third object of the present invention is to provide a method for preparing ectoin, which comprises the steps of:
(1) Seed liquid culture: inoculating the bacillus Bacillus atrophaeus QYJ strain into a seed culture medium for shake cultivation for 16-24 hours to obtain seed liquid;
(2) Aerobic fermentation: inoculating the seed solution into a fermentation medium for culturing for 24-48 hours to obtain a fermentation liquor containing the ectoin.
Preferably, the seed culture medium or the fermentation culture medium comprises the following components in percentage by weight: 2g/L of beef extract powder, 1.5g/L of soluble starch and 17.5g/L of casein acid hydrolysate, wherein the solvent is water.
Preferably, the inoculation amount of the inoculation is 1-5% by volume.
Preferably, the culture temperature of the culture is 37 ℃.
A fourth object of the present invention is to provide the use of said preparation method for the production of escitalopram.
A fifth object of the present invention is to provide a method for detecting ectoin, which comprises the following steps: and after fermentation is finished, adding three volumes of ethanol into a certain volume of fermentation liquid, standing for 2 hours, filtering with a 0.45 micrometer filter membrane, removing a solvent by rotary evaporation under reduced pressure, adding water with the same volume as the obtained fermentation liquid for dissolution, diluting to a proper concentration, and detecting by using a liquid chromatography-mass spectrometer.
Wherein, the chromatographic conditions are as follows: c18 column 2.1X100 mm,3.5 μm mobile phase water: methanol volume ratio=90:10, flow rate 0.3mL/min, column temperature 40 ℃; the mass spectrum conditions are as follows: the ion source is ESI positive ion mode, the ionization voltage is 5500V, and the declustering voltage (DP) is 120V. The qualitative ion pair was 143.0 to 97.0 (collision energy (CE) was 24V), 143.0 to 68.1 (collision energy (CE) was 33V). The quantitative ion pair was 143.0.fwdarw.97.0.
The invention has the following beneficial effects:
the bacillus Bacillus atrophaeus QYJ37 can prepare the ectoine by a fermentation method, and is a novel ectoine preparation method, and is environment-friendly. The bacillus Bacillus atrophaeus QYJ is rapid in growth under the conditions of no salinity and no oxygen, the required fermentation culture medium is simple and easy to obtain, the culture is simple, the industrial development is facilitated, and the bacillus Bacillus atrophaeus QYJ has important significance and value in the biotechnology field and the cosmetic field.
Bacillus Bacillus atrophaeus QYJ, deposited at the cantonese institute of microbiological bacterial strain (GDMCC) at 2022, 5, 19, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62486.
drawings
FIG. 1 is a photograph of colony morphology of Bacillus Bacillus atrophaeus QYJ37.
FIG. 2 is a photomicrograph of Bacillus Bacillus atrophaeus QYJ37.
FIG. 3 shows the results of quantitative determination of ectoin in Bacillus Bacillus atrophaeus QYJ37 fermentation broth.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1: screening of Epideodone-producing strains
Cleaning the surface of a sample of the root of the polygonum hydropiper which is collected in Hunan by using sterile water, collecting cleaning liquid, coating the cleaning liquid on a nutrient agar culture medium (the formula is 10g/L of peptone, 3g/L of beef extract powder, 5g/L of sodium chloride and 15g/L of agar, and the solvent is water; the preparation steps are that each component is dissolved in water, sterilized and cooled to obtain) plate, culturing at 37 ℃, selecting single colony after colony grows out, continuing to separate and purify until pure bacteria are obtained, determining whether the ectoin exists and the yield by using a liquid phase mass spectrometry, and screening to obtain the strain with the highest ectoin which is named as QYJ strain.
Example 2: morphological observations of strain QYJ37
The strain QYJ grows faster on a nutrient agar medium, and is cultivated aerobically for 24 hours at 37 ℃, the colony diameter reaches 10-15mm, the colony is nearly round but the edge is uneven, the colony is opaque, the lawn is raised, the surface is wrinkled, the surface is not sticky and is like a jelly, and the morphological characteristics of the colony are shown in figure 1. The strain was gram positive with short rods and spores located in the center of the cells, see figure 2.
Example 3: molecular biological characteristics of Strain QYJ37
Amplification, sequencing and sequence alignment of 16S rRNA of strain QYJ:
collecting fresh thalli, extracting a total DNA template by using an Ezup column type bacterial genome DNA extraction kit, amplifying a 16S rRNA gene by using a universal primer 27F/1492R, detecting and purifying a PCR product, and directly carrying out sequence determination by the Guangzhou qing department biotechnology Co-Ltd.
The primer sequences were as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；
1492R：5'-TACGGCTACCTTGTTACGACTT-3'；
the 50. Mu.L PCR reaction system was as follows: template 1. Mu.L; 1. Mu.L each of the upstream primer and the downstream primer; dream Taq Green PCR Master Mix 25 μL; ddH 2 O 22μL。
PCR conditions: 95℃for 5min,94℃for 30s,52℃for 2min and 72℃for 1min; cycling was performed 30 times.
The full sequence results were measured as follows: the 16S rRNA sequence of the strain is shown as SEQ ID NO.1, and the obtained complete sequence is uploaded to NCBI website for sequence alignment, the similarity of the strain QYJ37 and bacillus Bacillus atrophaeus is found to be 99.53%, and the strain is identified as bacillus Bacillus atrophaeus QYJ by combining colony characteristics and physiological and biochemical characteristics. The strain has been deposited at the Guangdong province microorganism strain collection (GDMCC) at 2022, 5 and 19, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070, accession number is: GDMCC No:62486.
example 4: preparation of Exclerodine Using Bacillus QYJ37
A method for preparing ectoin by fermenting bacillus Bacillus atrophaeus QYJ37, comprising the following steps:
(1) Seed liquid culture: inoculating bacillus Bacillus atrophaeus QYJ strain to a seed culture medium, and performing shake culture at 37 ℃ for 24 hours to obtain seed liquid; the formula of the seed culture medium is as follows: 2g/L of beef extract powder, 1.5g/L of soluble starch and 17.5g/L of casein acid hydrolysate, wherein the solvent is water. The preparation method comprises the following steps: dissolving the above materials in water, stirring for dissolving, and sterilizing.
(2) Aerobic fermentation: inoculating the seed solution into a fermentation culture medium in an inoculum size of 5% by volume, and culturing at 37 ℃ for 48 hours to obtain a fermentation broth containing the ectoin. The formula of the fermentation medium is as follows: 2g/L of beef extract powder, 1.5g/L of soluble starch and 17.5g/L of casein acid hydrolysate, wherein the solvent is water. The preparation method comprises the following steps: dissolving the above materials in water, stirring for dissolving, and sterilizing.
Example 5: detection of escidodine
The bacillus Bacillus atrophaeus QYJ37 fermentation broth in example 4 was used as a test sample, which was diluted with water by a hundred thousand times and subjected to the content determination of ectoin by a liquid-phase mass spectrometry method. Liquid phase conditions: the chromatographic column is a C18 column, the inner diameter and the length are 2.1X100 mm, and the particle size of the filling is 3.5 μm; column temperature is 40 ℃; the sample injection amount is 1 mu L; the mobile phase is water: methanol volume ratio = 90:10; the flow rate was 0.3mL/min. Mass spectrometry conditions: the ion source is ESI positive ion mode, the ionization voltage is 5500V, and the declustering voltage (DP) is 120V. The qualitative ion pair was 143.0 to 97.0 (collision energy (CE) was 24V), 143.0 to 68.1 (collision energy (CE) was 33V). The quantitative ion pair was 143.0.fwdarw.97.0. The results are shown in FIG. 3.
The yield of ectoin in the Bacillus Bacillus atrophaeus QYJ37 fermentation broth was 7.348g/L.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (7)

1. Bacillus Bacillus atrophaeus QYJ, accession No.: GDMCC No. 62486.
2. The use of bacillus Bacillus atrophaeus QYJ37 of claim 1 for the preparation of ectoin.
3. A process for preparing ectoin, which comprises fermenting with Bacillus Bacillusatrophaeus QYJ37 as claimed in claim 1.
4. A method according to claim 3, comprising the steps of:
(1) Seed liquid culture: inoculating the bacillus Bacillus atrophaeus QYJ strain according to claim 1 into a seed culture medium for shake cultivation for 16-24 hours to obtain seed liquid;
(2) Aerobic fermentation: inoculating the seed solution into a fermentation medium for culturing for 24-48 hours to obtain a fermentation liquor containing the ectoin.
5. The method according to claim 4, wherein the seed culture medium or the fermentation medium comprises the following components in percentage by weight: 2g/L of beef extract powder, 1.5g/L of soluble starch and 17.5g/L of casein acid hydrolysate, wherein the solvent is water.
6. The method according to claim 4, wherein the inoculation amount is 1-5% by volume.
7. The method according to claim 4, wherein the culturing temperature of the culturing is 37 ℃.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2743350A1 (en) * 2012-12-13 2014-06-18 Artes Biotechnologie GmbH A method for producing ectoine or a derivative thereof and a yeast cell for use as a host cell in such a method
CN104195074A (en) * 2014-08-13 2014-12-10 甘肃农业大学 Endophytic bacteria bacillus atrophaeus bacterial strain of forage in alpine grassland, microbial agent as well as preparation method and application of microbial agent
CN111304106A (en) * 2018-12-11 2020-06-19 北京百奥茵诺生物科技有限公司 Bacillus clausii and method for producing tetrahydropyrimidine by using same
CN112300957A (en) * 2019-12-12 2021-02-02 山东福瑞达生物科技有限公司 Bacillus halophilus and method for industrially producing ectoin by using same
KR20220003924A (en) * 2020-07-02 2022-01-11 서강대학교산학협력단 Methanogenic mutant strain for improved ectoine production and Method for producing ectoin using the same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2743350A1 (en) * 2012-12-13 2014-06-18 Artes Biotechnologie GmbH A method for producing ectoine or a derivative thereof and a yeast cell for use as a host cell in such a method
CN104195074A (en) * 2014-08-13 2014-12-10 甘肃农业大学 Endophytic bacteria bacillus atrophaeus bacterial strain of forage in alpine grassland, microbial agent as well as preparation method and application of microbial agent
CN111304106A (en) * 2018-12-11 2020-06-19 北京百奥茵诺生物科技有限公司 Bacillus clausii and method for producing tetrahydropyrimidine by using same
CN112300957A (en) * 2019-12-12 2021-02-02 山东福瑞达生物科技有限公司 Bacillus halophilus and method for industrially producing ectoin by using same
KR20220003924A (en) * 2020-07-02 2022-01-11 서강대학교산학협력단 Methanogenic mutant strain for improved ectoine production and Method for producing ectoin using the same

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烟台海域一株中度嗜盐芽孢杆菌YTM-5的鉴定及其耐盐机制研究;王磊;冯二梅;宿红艳;;新乡学院学报(自然科学版)(03);全文 *

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