Zygosaccharomyces parabailii Y4 and application thereof

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a strain Zygosaccharomyces parabailii Y4 and application thereof. A strain Y4, the taxonomic name of the selenium-resistant strain Y4 is Zygosaccharomyces parabailii; the strain Y4 was preserved in the Guangdong Provincial Center for Disease Prevention and Control on July 17, 2023, and the preservation number is GDMCC No. 63656. The present application screens the selenium-resistant strain in molasses, identifies the selenium-resistant strain through morphological observation and ITS rRNA sequencing, and determines the organic selenium conversion rate of the selenium-resistant strain. The screened strain Y4 is identified as Zygosaccharomyces parabailii; in the study of the organic selenium conversion rate, the organic selenium conversion rate of the strain Y4 reaches 75.89% under the selenium concentration of 50 μg / mL, which can provide strain resources for the bioremediation of selenium-polluted areas and provide a reference for the development and utilization of selenium resources.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, specifically relating to a strain Zygosaccharomyces parabailii Y4 and its applications. Background Technology

[0002] Selenium exists in nature primarily in four forms, namely selenides (Se... 2- ), elemental selenium (Se) 0 ), selenite (SeO3) 2- ) and selenate (SeO4) 2- Selenium, in particular, is highly susceptible to oxidation states, and the transformation of valence states between its various forms is closely related to microorganisms. The safe range for selenium in soil is 0.1-3.0 μg / g; levels below or above this range can lead to significant selenium deficiency or poisoning in animals. In recent years, selenium pollution has become a recurring problem globally. Cases of selenium poisoning have occurred in Enshi, Hubei Province, and Ziyang, Shaanxi Province, my country, the most famous being the selenium poisoning incident in the Yutangba area of ​​Enshi. Environmental remediation in selenium-polluted areas has become a hot research topic in recent years.

[0003] High-selenium environments are a potential source of selenium-tolerant microbial strains. Studies have shown that microorganisms under high-selenium stress produce stress responses, converting highly toxic inorganic selenium into less toxic elemental selenium. Peng Zuoquan et al. and Wang Mingyi et al. previously screened selenium-tolerant strains from selenium-rich soil in Enshi, Hubei Province, and were able to reduce inorganic selenium to red elemental selenium in selenium-containing culture media, which is of great significance in the remediation of environmental selenium pollution.

[0004] Currently, domestic research on selenium mainly focuses on the behavior of selenium in soil, selenium enrichment in plants, and nano-selenium materials. However, there is relatively little research on microorganisms in selenium-rich soils. To address this, the inventors collected soil samples from selenium-rich areas in Guangxi, identified selenium-resistant strains, and determined the organic selenium conversion rate of these strains, aiming to provide microbial resources for the development and utilization of selenium resources from a microbiological perspective.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a strain Zygosaccharomyces parabailii Y4 and its applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The strain Y4 provided by this invention was isolated from molasses produced in Nanning City, Guangxi Province. It was identified as Zygosaccharomyces parabailii by morphology and molecular biology. It was deposited on July 17, 2023 at the Guangdong Provincial Institute of Microbiology and Microbial Culture Conservation Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China) with accession number GDMCC No. 63656.

[0009] The present invention also provides the use of the strain Y4 in a transformation environment for selenium.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] This invention screens selenium-tolerant strains from molasses, identifies these strains through morphological observation and ITS rRNA sequencing, and determines their organic selenium conversion rate. The screened strain Y4 was identified as *Zygosaccharomyces parabailii*. In the organic selenium conversion rate study, strain Y4 achieved an organic selenium conversion rate of 75.89% at a selenium concentration of 50 μg / mL, providing a strain resource for bioremediation in selenium-polluted areas and a reference for the development and utilization of selenium resources.

[0012] Preservation Information

[0013] Zygosaccharomycesparabailii Y4, accession number GDMCC No.63656, accession date July 17, 2023, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0014] Figure 1 This describes the colony morphology of the selenium-resistant strain Y4 of this invention.

[0015] Figure 2 This describes the liquid culture characteristics of the selenium-resistant strain Y4 of the present invention;

[0016] Figure 3 Microscopic examination of the selenium-resistant strain Y4 of this invention;

[0017] Figure 4 This is a gel electrophoresis image of the PCR products of the colonies isolated in this invention;

[0018] Figure 5 This is a phylogenetic tree of the ITS rRNA gene sequence of the selenium-resistant strain Y4 of this invention;

[0019] Figure 6 This is a growth curve of the selenium-resistant strain Y4 of the present invention.

[0020] Explanation of key figure labels:

[0021] Figure 3 In the table, Marker: Marker DL2000; 1-4: PCR products of some strains, of which 1 is the PCR product of strain Y4. Detailed Implementation

[0022] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all experimental methods used in the following examples are conventional methods.

[0024] Example

[0025] 1. Materials and Methods

[0026] 1.1 Materials

[0027] 1.1.1 Screening molasses

[0028] The strains were screened from sugarcane molasses samples produced in Nanning City, Guangxi Province (provided by Guangxi Tingni Biological Products Co., Ltd.). After being shaken well, the molasses was poured into sterile blue-capped bottles and stored in an ice box. The samples were then brought back to the laboratory on the same day and stored at 4°C for later use. Some components of the molasses are shown in Table 1.

[0029] Table 1. Some components of molasses

[0030]

[0031] 1.1.2 Culture medium

[0032] Yeast extract peptone glucose medium: 20.0g glucose, 20.0g peptone, 10.0g yeast extract, pH 6.0±0.2, bring to a final volume of 1L, stir and heat to boiling until completely dissolved, then autoclave at 121℃ for 20min.

[0033] Yeast extract powder peptone glucose agar medium (YPD): purchased from High-Tech Microbial Technology 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 heated to boiling until completely dissolved, autoclaved at 121℃ for 20min.

[0034] Glucose peptone medium: 5.0g glucose, 5.0g peptone, 2.0g dipotassium hydrogen phosphate, pH 7.0±0.2, 1L distilled water. Stir and heat to boiling until completely dissolved, then steam sterilize at 115℃ for 30min.

[0035] McFarland medium: 2.5g yeast extract, 1.8g potassium chloride, 1.0g glucose, 8.2g sodium acetate, 15g agar, pH 6.4±0.2, 1L distilled water. Stir and heat to boiling until completely dissolved, then autoclave at 115℃ for 30min.

[0036] 1.1.3 Selenium solution

[0037] Weigh out sodium selenite, then dilute to a final volume with deionized water to prepare a solution with a selenium concentration of 100 mg / mL. Filter the solution using a 0.22 μm sterile filter and set aside.

[0038] 1.2 Isolation and purification of selenium-resistant strains

[0039] Measure 10 mL of molasses into an Erlenmeyer flask containing 10 glass beads and 90 mL of sterile water. Incubate at 30°C, 200 rpm, and shake for 30 min. Then, prepare 10 mL of molasses using a serial dilution method. -2 -10 -4 The diluted solution was plated at 100 μL onto YPD medium and incubated at 28 °C for 2 days. Single colonies with different colors, shapes, sizes, textures, and edges were picked and purified three times using the streak plate method to obtain pure cultures of the strain, which were then preserved on YPD slant medium.

[0040] 1.3 Screening of selenium-resistant strains

[0041] The isolated and purified microbial strains were inoculated into liquid culture medium and cultured for 3 days. Then, 5 μL of the culture medium was inoculated into YPD culture medium with different selenium concentrations. The selenium concentration in the selenium-containing YPD culture medium was increased in a gradient of 500 μg / mL until strains with high selenium tolerance were selected.

[0042] 1.4 Morphological observation of selenium-resistant strains

[0043] The colony morphology and liquid culture characteristics of selenium-resistant strains were observed, and the cell morphology and reproduction methods of the bacteria were described.

[0044] 1.5 Molecular biological identification of selenium-resistant strains

[0045] The pure cultures of the selected selenium-resistant strains were subjected to ITS rRNA sequence amplification: single colonies were picked and placed in 1.5 mL centrifuge tubes for colony PCR, using universal fungal primers ITS1 and ITS4. The primer sequences are shown in Table 2, and the PCR reaction system is shown in Table 3.

[0046] Table 2 ITS rRNA primers

[0047]

[0048] Table 3 PCR reaction system

[0049]

[0050] The PCR program was as follows: 95℃ for 3 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 30 s, for 35 cycles; 72℃ for 5 min.

[0051] After detecting the PCR products by agarose gel electrophoresis, the PCR amplification products were sequenced, and the sequencing sequences were compared with BLAST in the NCBI database to determine the species information.

[0052] 1.6 Constructing a phylogenetic tree

[0053] The ITS rRNA sequences of the selected selenium-tolerant strains were entered into the NCBI database for BLAST comparison. Twelve sequences with high consistency were selected from each strain to construct a phylogenetic tree.

[0054] 1.7 Growth status of selenium-tolerant strains

[0055] To plot the growth curves of selenium-tolerant strains: Prepare yeast extract peptone glucose medium with three replicates for each treatment. Inoculate with a 5% inoculation of pre-shaken yeast suspension and culture at 28℃ and 200 r / min with shaking. Take samples at 0h, 4h, 8h, 12h, 16h, 20h, 24h and 28h to measure the OD value (600nm wavelength) and plot the growth curves of selenium-tolerant strains.

[0056] 1.8 Determination of the selenium conversion ability of selenium-resistant strains

[0057] The study on the organic selenium conversion rate of selenium-tolerant strains was conducted in accordance with the National Standard of the People's Republic of China, "National Food Safety Standard for Food Fortification Agent: Selenium-Enriched Yeast" (GB 1903.21-2016). A filtered and sterilized sodium selenite solution was added to the yeast extract peptone glucose medium to achieve a selenium concentration of 50 μg / mL in the liquid medium. Three replicates were established, with 5% inoculation into the liquid medium. The cultures were incubated at 28℃ and 200 rpm with shaking for 24 h. After incubation, the cultures were centrifuged at 4000 rpm for 10 min, washed twice with sterile physiological saline, centrifuged again to collect the cells, pre-frozen at -80℃ for 12 h, and then freeze-dried. After digestion, the total selenium content and inorganic selenium content in the freeze-dried yeast samples were determined using an atomic fluorescence spectrophotometer. The organic selenium conversion rate was calculated according to Equation 1-1.

[0058] Organic selenium conversion rate (%) = (C i -C f ) / C i ×100% (1-1)

[0059] In the above formula, C i C represents the total selenium content of yeast. f This refers to the inorganic selenium content of yeast.

[0060] 1.9 Acid resistance test of strains

[0061] The pH of YPD medium was adjusted with HCl and NaOH to create seven pH gradient treatments: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. After sterilization at 120°C for 20 min, the medium was poured into 9 cm diameter petri dishes, 20 mL per dish. 10 μL of Y4 bacterial culture in the logarithmic growth phase was pipetted onto a pH gradient D YPD plate and incubated at 28°C for 48 h. The growth of strain Y4 was then observed.

[0062] 1.10 Data Processing

[0063] Phylogenetic trees were constructed using the Neighbor-Joining method in MEGA 5.22, and plotted using Excel 2007.

[0064] 2. Results and Analysis

[0065] 2.1 Isolation and purification of selenium-resistant strains

[0066] Sugarcane molasses samples were added to yeast extract peptone glucose liquid medium for initial screening. Single colonies with different colors, shapes, sizes, textures and edges were picked and purified by streak plating. A total of 4 yeast strains were screened out. The single colonies screened in the initial screening were then further screened for selenium tolerance.

[0067] 2.2 Screening of selenium-resistant strains

[0068] The growth of the isolated and purified strains in YPD medium with different selenium concentrations is shown in Table 4 below.

[0069] Table 4. Growth status of strains under different selenium concentrations.

[0070]

[0071] Note: + indicates growth; - indicates no growth.

[0072] As shown in Table 4, the highest selenium tolerance levels for strains Y4, Y7, Y9, and Y12 were 3500 μg / mL, 2000 μg / mL, 0 μg / mL, and 3000 μg / mL, respectively. Among them, strain Y4 showed the strongest tolerance to selenium.

[0073] 2.3 Identification of selenium-resistant strain Y4

[0074] 2.3.1 Morphological observation

[0075] The morphology of the pure culture of selenium-tolerant strain Y4, after being cultured at 28℃ for 24 hours, is as follows. Figure 1 As shown.

[0076] Depend on Figure 1 It can be seen that the colonies of the selenium-resistant strain Y4 are milky white, round, raised, 1-3 mm in size, with neat edges, and the colonies are viscous and easy to pick up.

[0077] After culturing selenium-tolerant strain Y4 in glucose peptone medium for 3 days, the characteristics of the culture medium are as follows: Figure 2 As shown.

[0078] Depend on Figure 2 It can be seen that the culture medium of strain Y4 became turbid, formed obvious rings, had a small amount of granules, no islands, and produced a lot of sediment when left to stand.

[0079] After culturing selenium-resistant strain Y4 in McFarland solid medium for one week, the bacterial cell morphology under a microscope is as follows: Figure 3 As shown.

[0080] Depend on Figure 3 It can be seen that when selenium-resistant strain Y4 is stained with malachite green, the bacterial cells are elliptical, and a small number of strains have pale green (or no red) ascospores.

[0081] 2.4 Molecular biological identification of selenium-resistant strain Y4

[0082] The isolated and purified selenium-resistant strain Y4 was subjected to colony PCR amplification. The PCR products were detected by agarose gel electrophoresis. The electrophoretic detection of the PCR products is as follows: Figure 4 As shown.

[0083] Depend on Figure 4The presence of the target band in the PCR product, and the clear band, indicates that the PCR product was successfully amplified.

[0084] The PCR amplification products were sequenced, and the ITS rRNA sequence of strain Y4 is shown in SEQ ID No. 3. The sequencing sequences were compared with BLAST data from the NCBI database, and the comparison results are shown in Table 5.

[0085] Table 5. BLAST comparison results of strains

[0086]

[0087] As shown in Table 5, strain Y4 and Zygosaccharomyces parabailii MC-5K3 have a similarity of 99.86%.

[0088] 2.5 Phylogenetic tree of selenium-tolerant strain Y4

[0089] The ITS rRNA sequence of the selenium-tolerant strain Y4 was compared with the NCBI database. Twelve sequences with high homology were selected from each strain, and a phylogenetic tree was constructed using MEGA 5.22. The constructed phylogenetic tree is shown below. Figure 5 As shown.

[0090] Depend on Figure 5 It can be seen that strain Y4 has a high similarity to the genus Zygosaccharomyces, and strain Y4 is identified as Bayer Zygosaccharomyces, named Zygosaccharomyces parabailii Y4.

[0091] 2.6 Growth curve of selenium-tolerant strain Y4

[0092] The selected selenium-tolerant strain Y4 was inoculated into yeast extract peptone dextrose agar medium, and the OD of the bacterial culture was measured every 4 hours. 600 Values ​​were used to plot growth curves, and the results are as follows: Figure 6 As shown.

[0093] Depend on Figure 6 It can be seen that the selenium-tolerant strain Y4 has a rapid growth period from 0 to 12 hours; growth slows down from 12 to 20 hours; and growth stabilizes from 20 to 28 hours.

[0094] 2.7 Selenium conversion ability of selenium-tolerant strain Y4

[0095] Yeast extract peptone glucose medium with a selenium concentration of 50 μg / mL was cultured with shaking for 24 h, and the selenium content of the yeast was measured. The results are shown in Table 6.

[0096] As shown in Table 6, strain Y4 achieved a 75.89% conversion rate of organic selenium at a selenium concentration of 50 μg / mL.

[0097] Table 6. Selenium conversion rate (%) of strains

[0098]

[0099] In summary, the selenium-resistant strains screened in this invention have a high efficiency in organic selenium conversion.

[0100] 2.8 Acid tolerance of strains

[0101] The results are shown in Table 7.

[0102] Table 7 Growth of the strain at different pH values

[0103]

[0104] Note: + indicates growth; - indicates no growth.

[0105] As shown in Table 7, strain Y4 can grow on YPD plates at pH 4.5-7.0.

[0106] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain Y4, characterized in that, The strain Y4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 17, 2023, with accession number GDMCC No. 63656, and its taxonomic name is Bayer conjugative yeast (GDMCC No. 63656). Zygosaccharomyces parabailii ).

2. The application of strain Y4 according to claim 1 in activating selenium activity in an acidic environment and converting inorganic selenium into organic selenium.

Images