Pichia kudriavzevii, application thereof and rotifer culture method

Abstract

The invention relates to the technical field of microorganisms, and discloses pichia kudriavzevii, application of pichia kudriavzevii and a rotifer culture method. The preservation number of the pichia kudriavzevii provided by the invention is GDMCC No: 66038, and the name of the pichia kudriavzevii in the Latin literature is Pichia kudriavzevii. According to the application, a strain of pichia kudriavzevii is screened from a seawater environment, the pichia kudriavzevii is natural and has the capability of adapting to high salinity of seawater, and the problems that bacillus subtilis and other conventional saccharomycetes in the current freshwater environment cannot be efficiently used and cause water pollution in rotifer culture are solved; meanwhile, when the bacillus subtilis is used as bait for feeding rotifers, the culture efficiency is extremely remarkable, and compared with conventional bacillus subtilis and other saccharomycetes, the yield of the rotifers can be remarkably increased within a shorter time, so that rotifer culture can be carried out in a low-cost, high-efficiency and environment-friendly manner.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a Pichia kudriavzevii yeast and an application thereof, as well as a method for cultivating rotifers. Background Art

[0002] Rotifers are micro filter-feeding organisms with sizes ranging from 40 to 2000 μm. Depending on the species, they are widely found in all water layers of natural salty and fresh water bodies. They feed on algae, fungi and various organic debris of appropriate sizes, and are excellent biological baits for fish fry during the nursery period. When rotifers are artificially cultured, microalgae (Chlorella vulgaris, Microchlorophylla, Chrysophyte, etc.) or fungi (Breadcrumbsaccharomyces cerevisiae, Bacillus subtilis, etc.) are often used as bait. When algae are used to feed rotifers, there is a defect of high cost, so fungi are often used as rotifer bait in actual production.

[0003] When using fungi to feed marine rotifers, the water quality will be seriously deteriorated and the yield will be low. The main reason for this phenomenon is the lack of suitable marine bacteria. In the past, bacteria such as yeast and Bacillus subtilis survived in fresh water. When the bacteria that survive in fresh water enter the seawater, the high osmotic pressure caused by the high salinity of the seawater often causes the bacteria to dehydrate and shrink, resulting in a decrease in the vitality of the bacteria or even death, and then quickly sink to the bottom of the water and cannot be used by rotifers. At the same time, the toxic and harmful substances produced by the corruption of the dead and sunk bacteria will destroy the rotifer breeding water, further hindering the production of rotifers. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide a Pichia kudriavzewi yeast, so that when the Pichia kudriavzewi yeast is used for rotifer cultivation, the growth rate and yield of rotifers can be significantly improved compared with conventional fungus baits; Another object of the present application is to provide related applications of the above-mentioned Pichia kudriavzewi in preparing bait for culturing rotifers and in culturing rotifers, as well as a method for culturing rotifers using the above-mentioned Pichia kudriavzewi and a bait product for culturing rotifers.

[0005] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, a yeast Pichia kudriavzevii is provided, with a preservation number of GDMCC No: 66038 and a Latin name of Pichia kudriavzevii .

[0006] As a second aspect of the present application, provided is the use of the Pichia kudriavzevii described in the present application in preparing bait for culturing rotifers or in culturing rotifers.

[0007] As the third aspect of the present application, a bait for culturing rotifers is provided, which comprises the Pichia kudriavzevii described in the present application.

[0008] Optionally, the bait is a bacterial liquid obtained by culturing the Pichia kudriavzevii described in the present application in an expanded culture medium; the expanded culture medium comprises a carbon source, a nitrogen source, trace elements, a growth-promoting substance and seawater.

[0009] Further optionally, the expanded culture medium comprises brown sugar, corn steep liquor dry powder, fish meal, yeast extract powder, copper sulfate, zinc sulfate, ferric chloride, natural seawater, beet molasses yeast fermentation concentrate, ammonium sulfate and fish oil. More specifically, the expanded culture medium comprises 200 - 300 g of brown sugar, 100 - 200 g of corn steep liquor dry powder, 100 - 200 g of fish meal, 10 - 12 g of yeast extract powder, 4 - 6 mg of copper sulfate, 4 - 6 mg of zinc sulfate, 2 - 3 mg of ferric chloride, 10 liters of natural seawater with a salinity of 20 - 30‰, 30 - 50 ml of beet molasses yeast fermentation concentrate, 50 - 60 g of ammonium sulfate and 20 - 50 ml of fish oil.

[0010] As the fourth aspect of the present application, a method for culturing rotifers is provided, comprising: Adding the bacterial liquid obtained by culturing the Pichia kudriavzevii described in the present application into the seawater culture solution of rotifers to culture the rotifers.

[0011] Optionally, the seawater culture solution of rotifers comprises rotifers and natural seawater, and the salinity of the natural seawater is adjusted to the suitable range for the Pichia kudriavzevii described in the present application.

[0012] Further optionally, the density of rotifers in the culture solution of rotifers is 5 - 10 individuals / mL.

[0013] Optionally, the cell density of the bacterial liquid is above 2×10 8 cells / mL.

[0014] In the present application, a strain of Pichia kudriavzevii is screened from the seawater environment, which naturally has the ability to adapt to the high salinity of seawater, avoiding the problems that the current Bacillus subtilis in the freshwater environment and other conventional yeasts cannot be efficiently utilized in rotifer culture and cause water pollution; at the same time, when used as bait to feed rotifers, it has extremely remarkable culture efficiency, and can significantly improve the yield of rotifers in a shorter time compared with conventional Bacillus subtilis and other yeasts, enabling rotifer farming to be carried out in a low-cost, high-efficiency and green and environmentally friendly manner.

[0015] Biological deposit description Pichia kudriavzevii ELJ2024HNSYBGJ02, taxonomic name:Pichia kudriavzevii It was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 21, 2025. The address is the 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou. It is the Institute of Microbiology, Guangdong Academy of Sciences, and the deposit number is GDMCC No: 66038. Description of the Drawings

[0016] Figure 1 Shown is the Brachionus plicatilis fed with the application of the present application Pichia kudriavzevii in seawater; Figure 2 Shown is the Brachionus plicatilis fed with baker's yeast in seawater; Figure 3 Shown is the Brachionus plicatilis fed with Bacillus subtilis in seawater. Detailed Embodiments

[0017] The present application discloses a Pichia kudriavzevii, its application and a method for culturing rotifers. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present application. The products, processes and applications described in the present application have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods described in this article without departing from the content, spirit and scope of the present application to implement and apply the technology of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0018] It should be noted that in this article, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of another identical element in the process, method, article or device including the said element. At the same time, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0019] In the first aspect of the present application, through multiple screenings and isolations, a strain of Pichia kudriavzevii ( Pichia kudriavzevii ELJ2024HNSYBGJ02) was isolated and purified from a seawater grouper culture pond with a large number of rotifers. The cell length of this strain ranges from 4 to 12 μm, the suitable growth temperature range is 15 to 40 °C, the optimal growth temperature is 25 to 30 °C, the suitable salinity range is 0 to 50‰, and the most suitable salinity range is 20 to 30‰. It is preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC No: 66038, and the Latin taxonomic name is Pichia kudriavzevii .

[0020] In the second aspect of the present application, the Pichia kudriavzevii preserved in the present application was used to feed seawater rotifers. Compared with the conventional feeding of yeast and Bacillus subtilis, the final yield (the maximum density that can be achieved in a single culture) increased by about 3.3 to 5 times. Among them, the highest yield of rotifers fed with the strain of the present application reached 200,000 individuals / L, the highest yield of rotifers fed with conventional yeast reached 60,000 individuals / L, and the highest yield of rotifers fed with Bacillus subtilis reached 39,000 individuals / L. The growth rate of rotifers increased by about 2 to 3 times. At the same inoculation density, the feeding group with the strain of the present application reached a density of 39,000 individuals / L in 2 days, while the conventional yeast feeding group reached a density of 39,000 individuals / L in 4 days, and the Bacillus subtilis feeding group reached a density of 39,000 individuals / L in 5 days. The above two aspects of the aquaculture experiment results fully prove that the Pichia kudriavzevii provided by the present application can significantly improve the growth rate and yield of rotifers, and the strain itself can tolerate the high salinity of seawater and will not damage the aquaculture water body.

[0021] Based on this, the present application proposes the application of the above-mentioned Pichia kudriavzevii in the preparation of bait for culturing rotifers or in culturing rotifers; wherein, the rotifers are seawater rotifers, including but not limited to seawater Brachionus plicatilis, seawater Brachionus rotundiformis, etc.

[0022] In the third aspect of the present application, according to the application scope, a bait for culturing rotifers is proposed, which includes the Pichia kudriavzevii described in the present application.

[0023] In some embodiments of the present application, the bait is a bacterial liquid obtained by expanding and culturing the Pichia kudriavzevii described in the present application in an expansion culture medium; the expansion culture medium includes a carbon source, a nitrogen source, trace elements, growth-promoting substances, and seawater.

[0024] In some other embodiments of the present application, the amplification culture medium comprises a carbon source: brown sugar, fish oil; a nitrogen source: ammonium sulfate, corn steep liquor powder, fish meal; a growth-promoting substance: beet molasses yeast fermentation concentrate, yeast extract powder; trace elements: copper sulfate, zinc sulfate, ferric chloride; and the rest is natural seawater. Compared with the culture medium for conventional Pichia pastoris, this culture medium is optimized for rotifer culture. For example, adding fish oil can increase the DHA and EPA contents in the bacterial cells, and adding fish meal makes the protein composition of the bacterial cells more comprehensive. This optimization makes the cultured bacterial cells more nutritious and beneficial for rotifer culture. At the same time, nutrient sources containing growth-promoting factors such as beet molasses yeast fermentation concentrate and yeast extract powder are added to promote the growth of Pichia pastoris and increase the yield of bacterial cells. More specifically, the culture medium comprises 200 - 300 g of brown sugar, 100 - 200 g of corn steep liquor powder, 100 - 200 g of fish meal, 10 - 12 g of yeast extract powder, 4 - 6 mg of copper sulfate, 4 - 6 mg of zinc sulfate, 2 - 3 mg of ferric chloride, 10 liters of natural seawater with a salinity of 20 - 30‰, 30 - 50 ml of beet molasses yeast fermentation concentrate, 50 - 60 g of ammonium sulfate, and 20 - 50 ml of fish oil. The amplification culture medium of other specifications can be adjusted proportionally according to the above concentrations, which also belongs to the protection scope of the amplification culture medium of the present application.

[0025] In the fourth aspect of the present application, a method for culturing rotifers is provided, comprising: Adding the bacterial liquid after amplification culture of the Pichia kudriavzevii described in the present application into the seawater culture solution of rotifers to culture the rotifers.

[0026] In some embodiments of the present application, the amplification culture of the Pichia kudriavzevii comprises: Preparing an amplification culture medium; Inoculating the Pichia kudriavzevii into the amplification culture medium, and culturing it at a temperature in the range of 25 - 30°C with intermittent aeration.

[0027] In some embodiments of the present application, the preparation of the amplification culture medium comprises: Taking brown sugar, corn steep liquor powder, fish meal, yeast extract powder, copper sulfate, zinc sulfate, ferric chloride, mixing and crushing them, adding natural seawater with a salinity within the suitable range for the Pichia kudriavzevii described in the present application, and beet molasses yeast fermentation concentrate, mixing evenly, boiling, cooling, and then adding ammonium sulfate and fish oil in seawater, and mixing evenly.

[0028] In some other embodiments of the present application, the enrichment culture medium comprises 200 - 300 g of brown sugar, 100 - 200 g of dried corn steep liquor, 100 - 200 g of fish meal, 10 - 12 g of yeast extract powder, 4 - 6 mg of copper sulfate, 4 - 6 mg of zinc sulfate, 2 - 3 mg of ferric chloride, 10 liters of natural seawater with a salinity of 0 - 50‰, 30 - 50 ml of beet molasses yeast fermentation concentrate, 50 - 60 g of ammonium sulfate, and 20 - 50 ml of fish oil.

[0029] In some embodiments of the present application, intermittent aeration during the enrichment process is to introduce mixed air containing 40 - 50% oxygen for 5 minutes every 30 - 40 minutes; the enrichment time is determined according to the required cell density, usually not less than 2×10 8 cells / mL.

[0030] In some embodiments of the present application, the seawater culture solution of the rotifers comprises rotifers and natural seawater, and the salinity of the natural seawater is adjusted to the suitable range for Pichia kudriavzevii described in the present application, that is, 0 - 50‰, more preferably 20 - 30‰; the water temperature of the natural seawater is maintained at 20 - 30°C, and air is introduced to adjust it to a slightly boiling state.

[0031] In some other embodiments of the present application, the density of rotifers in the seawater culture solution of the rotifers is 5 - 10 individuals / mL.

[0032] In some embodiments of the present application, based on the culture volume of the rotifers, take the enriched bacterial solution, and add 0.8 - 1.2 mL of the bacterial solution to every 10 liters of the seawater culture solution of the rotifers. The cell density of the bacterial solution is above 2×10 8 cells / mL. Feed 2 - 5 times a day. After about 8 - 12 days, when the rotifer density reaches 200 individuals / mL (200,000 individuals / L), the rotifers can be harvested. Among them, the feeding days are adjusted according to the daily feeding amount and frequency. The highest yield of a single culture of rotifers in the present application can reach 200,000 - 250,000 individuals / L.

[0033] In each group of comparative experiments provided in the present application, unless otherwise specified, except for the differences pointed out in each group, other experimental conditions, materials, etc. are kept consistent for comparability. In addition, the materials used in the present application can all be obtained through commercial channels.

[0034] The following further describes a Pichia kudriavzevii and its application and a method for culturing rotifers provided by the present application.

[0035] Example 1: Cultivation of Brachionus plicatilis in seawater (1) Prepare the culture medium Take 220 g of brown sugar, 180 g of corn syrup dry powder, 120 g of fish meal, 12 g of yeast extract powder, 4 mg of copper sulfate, 4 mg of zinc sulfate, and 2 mg of ferric chloride. Mix them, crush through a 100-mesh sieve, put them into a 30-L electric heating boiling bucket, add 10 liters of natural seawater with a salinity of 20‰ and 40 ml of beet molasses yeast fermentation concentrate, mix well, boil for 30 min, cool to 38 °C, and add 60 g of ammonium sulfate and 40 ml of seawater fish oil, and mix well.

[0036] (2) Expand and culture the strain Take Pichia kudriavzevii One bottle of ELJ2024HNSYBGJ02 strain, add it to the above medium, put in a heating rod and adjust to 28 °C, introduce mixed air containing 45% oxygen for 5 min every 30 min, culture for 5 days, shake well once in the morning and evening every day, and the cell density of the finally cultured bacterial liquid is about 2×10 8 cells / mL.

[0037] (3) Use of the strain Take a 300-L fiberglass culture bucket, fill it with natural seawater with a salinity of 20‰, put in a heating rod, maintain the water temperature at 26 °C, charge air to adjust to a slightly boiling state, and inoculate L-shaped Brachionus plicatilis with an average size of 320 μm in good growth and development to a density of 8 individuals / mL. Immediately after the rotifers are inoculated, feed 30 mL of the bacterial liquid, and then feed 3 times a day, 30 mL of the bacterial liquid each time. After 9 days, the average density of the rotifers reaches 223 individuals / mL, and then the rotifers are harvested.

[0038] Example 2: Culture of marine Brachionus rotundiformis (1) Prepare the medium Take 200 g of brown sugar, 150 g of corn syrup dry powder, 100 g of fish meal, 12 g of yeast extract powder, 6 mg of copper sulfate, 4 mg of zinc sulfate, and 2 mg of ferric chloride. Mix them, crush through a 100-mesh sieve, put them into a 30-L electric heating boiling bucket, add 10 liters of natural seawater with a salinity of 20‰ and 35 ml of beet molasses yeast fermentation concentrate, mix well, boil for 30 min, cool to 33 °C, and add 55 g of ammonium sulfate and 45 ml of seawater fish oil, and mix well.

[0039] (2) Expand and culture the strain Take Pichia kudriavzevii One bottle of ELJ2024HNSYBGJ02 strain, add it to the above medium, put in a heating rod and adjust to 27 °C, introduce mixed air containing 43% oxygen for 5 min every 35 min, culture for 6 days, shake well once in the morning and evening every day, and the cell density of the finally cultured bacterial liquid is about 2.2×10 8 cells / mL.

[0040] (3) Use of the strain Take a 300L fiberglass culture barrel, fill it with natural seawater with a salinity of 20‰, place a heating rod, maintain the water temperature at 26°C, charge air to adjust it to a slightly boiling state, and introduce well-developed S-shaped Brachionus rotundiformis with an average size of 160μm to a density of 10 individuals / mL. Immediately after the rotifers are introduced, feed 30 mL of the bacterial solution, and then feed 3 times a day, 30 mL of the bacterial solution each time. After 9 days, the average density of the rotifers reaches 213 individuals / mL, and then the rotifers are harvested.

[0041] Experimental example: Refer to the method of Example 1 and use the strains Pichia kudriavzevii ELJ2024HNSYBGJ02, baker's yeast (Angel), and Bacillus subtilis (self-isolated in the laboratory) to culture L-type Brachionus rotundiformis. Except for the different feeding strains, other culture steps are kept the same for each group (the expansion culture medium and culture conditions of baker's yeast and Bacillus subtilis are the same as Pichia kudriavzevii those of the strain ELJ2024HNSYBGJ02), and the culture results are shown in Table 1 below and Figures 1-3 ; Table 1

[0042] It can be seen from Table 1 above that in the experiment of the maximum density reached in a single culture, the highest yield of rotifers fed with the strain of the present application reaches 200,000 individuals / L, the highest yield of rotifers fed with conventional baker's yeast reaches 60,000 individuals / L, and the highest yield of rotifers fed with Bacillus subtilis reaches 39,000 individuals / L.

[0043] In the experiment of the density reached under the condition of the same culture days, the yield of rotifers fed with the strain of the present application reaches 96,000 individuals / L, the yield of rotifers fed with conventional baker's yeast reaches 44,000 individuals / L, and the yield of rotifers fed with Bacillus subtilis reaches 39,000 individuals / L.

[0044] In the experiment of the culture days required to reach the same density, the density of 39,000 individuals / L is reached in 2 days for the group fed with the strain of the present application, while it takes 4 days for the group fed with conventional baker's yeast to reach a density of 39,000 individuals / L, and it takes 5 days for the group fed with Bacillus subtilis to reach a density of 39,000 individuals / L.

[0045] The above three aspects of the aquaculture experiment results fully prove that the Pichia kudriavzevii provided by the present application can significantly improve the growth rate and yield of rotifers, and the strain itself can tolerate the high salinity of seawater and will not damage the aquaculture water body.

[0046] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A Pichia kudriavzevii yeast, characterized in that The deposit number is GDMCC No: 66038, and its Latin name is Pichia kudriavzevii .

2. Use of the Pichia kudriavzevii according to claim 1 in preparing bait for culturing rotifers or in culturing rotifers.

3. A bait for cultivating rotifers, characterized in that: The invention comprises the Pichia kudriavzevii described in claim 1.

4. The bait according to claim 3, characterized in that The bait is the bacterial liquid of the Pichia kudriavzevii described in claim 1 after expansion with an expansion medium; the expansion medium comprises a carbon source, a nitrogen source, trace elements, growth-promoting substances and seawater.

5. The bait according to claim 4, characterized in that The culture medium comprises brown sugar, fish oil, ammonium sulfate, corn steep liquor powder, fish meal, beet molasses yeast fermentation concentrate, yeast extract powder, copper sulfate, zinc sulfate, ferric chloride, and the rest is natural seawater.

6. The bait according to claim 5, characterized in that The expansion culture medium comprises 200-300 g of brown sugar, 100-200 g of corn steep liquor powder, 100-200 g of fish meal, 10-12 g of yeast extract powder, 4-6 mg of copper sulfate, 4-6 mg of zinc sulfate, 2-3 mg of ferric chloride, 10 liters of natural seawater with a salinity of 20-30‰, 30-50 ml of beet molasses yeast fermentation concentrate, 50-60 g of ammonium sulfate, and 20-50 ml of fish oil.

7. A method for cultivating rotifers, characterized in that: include: The bacterial solution of Pichia kudriavzevii according to claim 1 is added to the seawater culture solution of rotifers to culture the rotifers.

8. The culture method according to claim 7, characterized in that: The seawater culture solution of rotifers comprises rotifers and natural seawater, and the salinity of the natural seawater is adjusted to a suitable range for the Pichia kudriavzevii described in claim 1.

9. The culture method according to claim 7 or 8, characterized in that: The density of rotifers in the seawater culture solution of rotifers is 5 to 10 rotifers / mL.

10. The culture method according to claim 7, characterized in that: The bacterial cell density of the bacterial solution is 2×10 8 cells / mL or above.

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