CN117535162A

CN117535162A - Zygosaccharomyces bailii strain Y19 and application thereof

Info

Publication number: CN117535162A
Application number: CN202311543912.9A
Authority: CN
Inventors: 廖青; 刘永贤; 邢颖
Original assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Current assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Priority date: 2023-11-20
Filing date: 2023-11-20
Publication date: 2024-02-09

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a zygosaccharomyces bailii strain Y19 and application thereof. A strain Y19, said strain Y19 having a taxonomic name of zygosaccharomyces bailii (Zygosaccharomyces parabailii); the strain Y19 is deposited with the Guangdong province biological strain protection center on the 6 th month 20 of 2023 with the deposit number of GDMCC No.63575. The invention screens selenium-resistant strains in molasses, identifies the selenium-resistant strains through morphological observation and ITS rRNA sequencing, and determines the organic selenium conversion rate of the selenium-resistant strains. The strain Y19 selected was identified as Zygosaccharomyces bailii; in the research of the organic selenium conversion rate, the organic selenium conversion rate of the strain Y19 reaches up to 73.26% at the concentration of 25 mug/mL selenium, so that strain resources can be provided for bioremediation of selenium-polluted areas, and references are provided for development and utilization of the selenium resources.

Description

Zygosaccharomyces bailii strain Y19 and application thereof

Technical Field

The invention belongs to the technical field of microbiology, and particularly relates to a zygosaccharomyces bailii strain Y19 and application thereof.

Background

Selenium exists in nature mainly in four forms, namely selenide (Se ^2- ) Elemental selenium (Se) ⁰ ) Selenite (SeO) ₃ ^2- ) And selenate (SeO) ₄ ^2- ) The conversion of the valence state between the various forms is closely related to the microorganism. The safety value of selenium in the soil is in the range of 0.1-3.0 mug/g, and less than or greater than the safety value can lead to obvious selenium deficiency disease or selenium poisoning disease of animals. Environmental remediation in selenium contaminated areas has become a hot spot of research in recent years.

The high-selenium environment is a potential source of selenium-resistant microorganism strains, and researches show that microorganisms generate stress reaction under the stress of the high-selenium environment, so that inorganic selenium with higher toxicity is converted into selenium simple substance with lower toxicity. Peng and Wang Mingyi are used for screening selenium-resistant strains from Hubei Enshi selenium-rich soil, and can reduce inorganic selenium into red elemental selenium in a selenium-containing culture medium, thereby having important significance in the aspect of treating environmental selenium pollution.

At present, domestic research on selenium is mainly focused on the behavior of selenium in soil, plant selenium enrichment and nano-selenium materials, and little research on microorganisms in selenium-enriched soil is performed, so that the inventor collects soil samples from a Guangxi selenium-enriched region, identifies selenium-resistant strains, and performs organic selenium conversion rate measurement of the selenium-resistant strains so as to provide strain resources for development and utilization of selenium resources from the microorganism direction.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Disclosure of Invention

The invention aims to provide a Zygosaccharomyces bailii strain Y19 and application thereof.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the strain Y19 provided by the invention is separated from molasses produced in Guangxi nan Ning City, is identified as Bayer zygosaccharomyces (Zygosaccharomyces parabailii) by morphological and molecular biological identification, and is preserved in Guangdong province biological strain protection center (GDMCC for short, address: guangzhou Mitsui No. 100 institute of China No. 59 building 5, and Guangdong province microbiological institute, post code: 510070) at the date of 2023, 6 and 20, and the preservation number is GDMCC No.63575.

The invention also provides application of the strain Y19 in selenium in a transformation environment.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens selenium-resistant strains in molasses, identifies the selenium-resistant strains through morphological observation and ITS rRNA sequencing, and determines the organic selenium conversion rate of the selenium-resistant strains. The screened strain Y19 was identified as Zygosaccharomyces bailii (Zygosaccharomyces parabailii); in the research of the organic selenium conversion rate, the organic selenium conversion rate of the strain Y19 reaches up to 73.26% at the concentration of 25 mug/mL selenium, so that strain resources can be provided for bioremediation of selenium-polluted areas, and references are provided for development and utilization of the selenium resources.

Preservation information

Zygosaccharomyces parabailiiY19 the deposit number is GDMCC No.63575, the deposit date is 2023, 6 and 20, the deposit unit is Guangdong province microbiological bacterial culture Collection center (GDMCC), and the deposit address is Guangzhou Miao No. 100 institute 59 building 5.

Drawings

FIG. 1 shows colony morphology of selenium-tolerant strain Y19 of the present invention;

FIG. 2 is a liquid culture profile of selenium tolerant strain Y19 of the present invention;

FIG. 3 shows the microscopic examination of the cells of selenium-resistant strain Y19 of the present invention;

FIG. 4 is a gel electrophoresis chart of a colony PCR product isolated according to the present invention;

FIG. 5 is a phylogenetic tree of the ITS rRNA gene sequences of selenium-tolerant strain Y19 of the present invention;

FIG. 6 is a graph showing the growth of selenium tolerant strain Y19 of the present invention;

FIG. 7 shows the organic selenium conversion of selenium tolerant strains of the present invention at various selenium concentrations.

The main reference numerals illustrate:

in fig. 3, marker: marker DL2000;1-4: PCR products of a part of the strains, wherein 4 is the PCR product of strain Y19.

Detailed Description

The following description of the embodiments of the present invention will be apparent from the description of the embodiments of the present invention, which is provided in part, but not in whole. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

Materials and reagents used in the examples described below, unless otherwise indicated, were all commercially available. The experimental methods used in the following examples are conventional methods unless otherwise specified.

Examples

1. Materials and methods

1.1 materials

1.1.1 Screen bacteria molasses

Strains were screened for a sample of cane molasses produced from Guangxi nan Ning, inc. (supplied by Guangxi Di you Biotechnology Co., ltd.). After shaking up the test molasses, pouring the test molasses into a sterilizing blue cap bottle, placing the test molasses into an ice box for preservation, taking the test molasses back to a laboratory for preservation at 4 ℃ for later use, and the components of the test molasses are shown in table 1.

TABLE 1 molasses partial Components

1.1.2 Medium

Yeast extract peptone glucose medium: glucose 20.0g, peptone 20.0g, yeast extract 10.0g, pH 6.0+ -0.2, constant volume 1L, stirring, heating and boiling to dissolve completely, and sterilizing at 121deg.C for 20min.

Yeast extract peptone glucose agar medium (YPD): purchased from GaocycloKai microorganism technologies Co., ltd., peptone 10.0g, yeast extract powder 5.0g, glucose 20.0g, agar 14.0g, pH 6.0.+ -. 0.2, distilled water 1L, stirred and boiled to complete dissolution, and autoclaved at 121℃for 20min.

Glucose peptone medium: glucose 5.0g, peptone 5.0g, dipotassium hydrogen phosphate 2.0g, pH 7.0+ -0.2, distilled water 1L, stirring, heating and boiling to dissolve completely, and steam sterilizing at 115 deg.C for 30min.

Wheat culture medium: 2.5g of yeast extract, 1.8g of potassium chloride, 1.0g of glucose, 8.2g of sodium acetate, 15g of agar, pH of 6.4+/-0.2 and 1L of distilled water, stirring, heating, boiling until the mixture is completely dissolved, and sterilizing at 115 ℃ for 30min.

1.1.3 selenium solution

Weighing sodium selenite, and then, using deionized water to fix the volume to prepare a solution with the selenium concentration of 100mg/mL, and filtering the solution by using a sterile filter head with the selenium concentration of 0.22 mu m for later use.

1.2 isolation and purification of selenium-resistant Strain

10mL of molasses was measured and placed in a triangular flask containing 10 glass beads and 90mL of sterile water. Shaking at 30deg.C for 30min at 200r/min for 30min, and preparing into 10 by gradient dilution method ^-2 -10 ^-4 100. Mu.L of the dilution was applied to YPD medium and incubated at 28℃for 2d. Single colonies with different colors, shapes, sizes, textures and edges are picked, repeated separation and purification are carried out for 3 times by adopting a plate streaking method, and pure cultures of the strains are obtained and preserved by adopting YPD slant culture medium.

1.3 selenium-resistant Strain screening

Inoculating the microbial strains obtained by separation and purification into a liquid culture medium, culturing for 3 days, and inoculating 5 mu L of culture solution into YPD culture mediums with different selenium concentrations; wherein the concentration of selenium in the selenium-containing YPD culture medium is increased gradually in a gradient of 500 mug/mL until the strain with higher selenium resistance is screened out.

1.4 morphological observations of selenium-resistant strains

The colony morphology and liquid culture characteristics of the selenium-resistant strain are observed, and the cell morphology structure and propagation mode of the thallus are described.

1.5 molecular biological characterization of selenium-resistant strains

Amplifying ITS rRNA sequences of pure cultures of the selenium-resistant strains obtained by screening: single colonies were picked and colony PCR was performed in 1.5mL centrifuge tubes, and fungal universal primers ITS1 and ITS4 were selected. Wherein, the primer sequences are shown in Table 2, and the PCR reaction system is shown in Table 3.

TABLE 2 ITS rRNA primers

TABLE 3 PCR reaction System

The PCR procedure was: 95 ℃ for 3min; cycling for 35 times at 95 ℃ for 15s,56 ℃ for 15s and 72 ℃ for 30 s; 72℃for 5min.

After agarose gel electrophoresis detection of the PCR products, the PCR amplified products were sequenced, and the sequenced sequences were aligned by BLAST on the NCBI database to determine species information.

1.6 construction of phylogenetic Tree

And (3) logging ITS rRNA sequences of the screened selenium-resistant strains into an NCBI database for BLAST comparison, and selecting 12 sequences with higher consistency from one strain to construct a phylogenetic tree.

1.7 growth status of selenium-resistant Strain

Drawing a growth curve of the selenium-resistant strain: preparing a yeast extract peptone glucose culture medium, repeating each treatment, inoculating a yeast suspension which is shaken in advance according to an inoculum size of 5%, shake culturing at 28 ℃ and 200r/min, sampling at 0h, 4h, 8h, 12h, 16h, 20h, 24h and 28h respectively, measuring OD values (600 nm wavelength), and drawing a growth curve of selenium-resistant strains.

1.8 determination of selenium-tolerant Strain for conversion of selenium

Organic selenium conversion rate research of selenium-resistant strains referring to food safety national Standard food nutrition enhancer selenium-enriched Yeast (GB 1903.21-2016) of the national Standard of the people's republic of China, a filtered and sterilized sodium selenite solution is added into a Yeast extract peptone glucose culture medium, so that the selenium concentration in the liquid culture medium is 0 mug/mL, 10 mug/mL, 25 mug/mL, 50 mug/mL and 100 mug/mL. Three treatments were arranged in parallel, inoculated into corresponding liquid culture media at an inoculum size of 5%, and shake-cultured at 28℃for 24h at 200 r/min. After the culture is finished, the culture is centrifuged for 10min at 4000r/min, washed for 2 times by sterile physiological saline, and the thalli are collected by centrifugation, pre-frozen for 12h at-80 ℃, and freeze-dried. And (3) after digestion of the freeze-dried yeast sample, measuring the total selenium content and the inorganic selenium content in the yeast by using an atomic fluorescence photometer, and calculating the conversion rate of the organic selenium according to the formula 1-1.

Organic selenium conversion (%) = (C _i -C _f )/C _i ×100％ (1-1)

In the above formula, C _i C is the total selenium content of yeast _f Is the inorganic selenium content of yeast.

1.9 data processing

Phylogenetic tree was constructed using the MEGA 5.22 orthorhombic ligation (Neighbor-Joining), and mapped using Excel 2007.

2. Results and analysis

2.1 isolation and purification of selenium-resistant Strain

Adding a cane molasses sample into a yeast extract peptone glucose liquid medium for primary screening, picking single colonies with different colors, shapes, sizes, textures and edges, carrying out flat plate streak purification, screening 4 strains of yeast strains together, and carrying out next screening on the single colonies subjected to primary screening.

2.2 selenium-resistant Strain screening

The growth of the isolated and purified strains in YPD medium containing different selenium concentrations is shown in Table 4 below.

TABLE 4 growth of strains at different selenium levels

Note that: + represents growth; -indicating no growth

As can be seen from Table 4, the highest tolerated selenium content of strains Y7, Y9, Y12 and Y19 was 2000. Mu.g/mL, 0. Mu.g/mL, 3000. Mu.g/mL and 3500. Mu.g/mL, respectively. Among them, the strain Y19 is more resistant to selenium.

2.3 identification of selenium-resistant Strain Y19

2.3.1 morphological observations

The morphology of the selenium-resistant strain Y19 cultured in pure form after 24 hours at 28 ℃ is shown in figure 1.

As can be seen from FIG. 1, the colony of selenium-resistant strain Y19 is milky white, round, convex, 1-3mm, and has neat edge, sticky colony texture and easy picking.

After selenium-resistant strain Y19 was cultured in glucose peptone culture solution for 3d, the characteristics of the culture solution are shown in FIG. 2.

As can be seen from FIG. 2, the culture solution of strain Y19 became turbid, formed an obvious ring, a small amount of uncut, no islands, and more precipitate on standing.

The morphology of the selenium-resistant strain Y19 after 1 week of culture in the solid medium for McGeorge is shown in FIG. 3.

As shown in FIG. 3, the selenium-resistant strain Y19 cells were elliptic and propagated in a budding manner.

2.4 molecular biological identification of selenium-resistant Strain Y19

The separated and purified selenium-resistant strain Y19 is subjected to colony PCR amplification, and PCR products are detected through agarose gel electrophoresis, and the electrophoresis detection of the PCR products is shown in figure 4.

As can be seen from FIG. 4, the target bands appear in the PCR products, and the bands are clear, indicating successful amplification of the PCR products.

Sequencing the PCR amplified product, wherein the ITS rRNA sequence of the strain Y19 is shown as SEQ ID No. 3. The sequenced sequences were aligned by BLAST on the NCBI database and the alignment results are shown in Table 5.

TABLE 5 BLAST alignment of strains

As shown in Table 5, the similarity between the strains Y19 and Zygosaccharomyces parabailiiMC-5K3 was 99.86%.

2.5 selenium-resistant Strain Y19 phylogenetic Tree

Comparing the ITS rRNA sequence of the screened selenium-resistant strain Y19 with NCBI database, selecting 12 sequences with higher consistency for each strain, and constructing a phylogenetic tree by using MEGA 5.22, wherein the constructed phylogenetic tree is shown in figure 5.

As is clear from FIG. 5, the strain Y19 has a high similarity to Zygosaccharomyces, and the strain Y19 is identified as Zygosaccharomyces bayer and designated as Zygosaccharomyces parabailii Y.

2.6 growth curves of selenium-resistant Strain Y19

Inoculating the screened selenium-resistant strain Y19 into a yeast extract peptone glucose agar medium, and measuring the OD of the bacterial liquid every 4 hours ₆₀₀ Values, growth curves were plotted and the results are shown in fig. 6.

As can be seen from FIG. 6, selenium-resistant strain Y19 is in rapid growth phase at 0-12 h; growth is slowed down in 12-20 hours; and the growth stability period is 20-28 h.

2.7 selenium-resistant Strain Y19 ability to convert selenium

The selenium content of the yeast is measured after the yeast extract peptone glucose culture medium with different selenium concentrations is arranged and shake-cultured for 24 hours, and the result is shown in figure 7.

As can be seen from FIG. 7, the strain was able to grow at a selenium concentration of 0-100. Mu.g/mL, wherein the Y19 strain had a maximum organic selenium conversion of 73.26% at a selenium concentration of 25. Mu.g/mL.

In fig. 7, the organic selenium conversion rate of the strain shows a tendency to rise and then fall, which may be due to the higher adsorption capacity of the strain at a relatively low selenium concentration, and as the selenium concentration in the liquid medium rises, the adsorption of the cells reaches a saturated state.

In conclusion, the selenium-resistant strain screened by the method has an efficient organic selenium conversion effect.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. A strain Y19, wherein said strain Y19 was deposited at the cantonese province biological seed protection center under accession number GDMCC No.63575 on day 20 of 2023 under the taxonomic designation bayer zygosaccharomyces paramabailii (zygosaccharomyces paraba).

2. Use of strain Y19 according to claim 1 for selenium in a transformation environment.