Pichia kudriavzevii and its application and rotifer cultivation method
By using Pichia Kudri Azwitz, the problem of declining bacterial vitality in rotifera culture was solved, and the efficient and environmentally friendly rotifera culture effect was achieved, which significantly improved the growth rate and yield of rotifera.
Patent Information
- Application Number
- CN202510541829.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2045-04-28
AI Technical Summary
Existing bacterial baits in the culture of rotifers due to high salinity lead to decreased bacterial vitality or death, resulting in water quality deterioration and low rotifer yields. Conventional yeasts and Bacillus subtilis cannot effectively improve rotifer growth rate and yield.
Using Pichia kudriavzevii, this strain has the ability to adapt to high salinity in seawater, significantly improving the growth rate and yield of rotifers by optimizing the medium formulation and culture conditions.
Significantly improve the growth rate and yield of rotifers, achieve low-cost and high-efficiency rotifer farming, avoid water pollution, and increase the density and yield of single cultures.
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Figure CN120082446B_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a Pichia kudriavzevii yeast and its application, as well as a rotifer cultivation method. Background Art
[0002] Rotifers are microscopic, filter-feeding organisms ranging in size from 40 to 2000 μm. Depending on the species, they are widely found in all layers of natural brackish and freshwater bodies. They feed on appropriately sized algae, fungi, and various organic detritus, making them excellent biological feeds for fish fry during their nursery period. In artificial aquaculture, rotifers are often fed with microalgae (such as Chlorella vulgaris, Nannochloropsis chlorophylla, and Chrysophyte) or fungi (such as Baker's yeast and Bacillus subtilis). However, the cost of feeding rotifers with algae is prohibitive, so fungi are often used as rotifer bait in practice.
[0003] However, using fungi to feed marine rotifers can lead to severe deterioration of water quality and low production. This phenomenon is primarily due to a lack of suitable marine bacterial species. Previously used bacterial species, such as yeast and Bacillus subtilis, survive in freshwater. When freshwater-surviving bacteria enter seawater, the high osmotic pressure caused by the high salinity of the seawater often causes them to dehydrate and shrink, resulting in decreased vitality or even death. They then quickly sink to the bottom of the water, making them unusable by the rotifers. Furthermore, the toxic and harmful substances produced by the decay of these dead and sinking bacteria damage the water for rotifer cultivation, further hindering rotifer production. Summary of the Invention
[0004] In view of this, the purpose of the present application is to provide a Pichia kudriavzevii yeast, so that when the Pichia kudriavzevii yeast is used for rotifer culture, it can significantly increase the growth rate and yield of rotifers compared with conventional fungal baits;
[0005] Another object of the present application is to provide related applications of the above-mentioned Pichia kudriavzevii in preparing bait for culturing rotifers and in culturing rotifers, as well as a method for culturing rotifers using the above-mentioned Pichia kudriavzevii and a bait product for culturing rotifers.
[0006] 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, there is provided a Pichia kudriavzevii yeast, with a preservation number of GDMCC No: 66038, and a Latin name of Pichia kudriavzevii .
[0007] As a second aspect of the present application, provided is the use of the Pichia kudriavzii described in the present application in preparing bait for culturing rotifers or in culturing rotifers.
[0008] As a third aspect of the present application, a bait for culturing rotifers is provided, which includes the Pichia kudriavzii described in the present application.
[0009] Optionally, the bait is the bacterial liquid of the Pichia kudriavzii described in the present application after expansion in an expansion medium; the expansion medium includes a carbon source, a nitrogen source, trace elements, growth-promoting substances and seawater.
[0010] Further optionally, the propagation culture medium comprises brown sugar, corn steep liquor powder, fish meal, yeast extract, copper sulfate, zinc sulfate, ferric chloride, natural seawater, beet molasses yeast fermentation concentrate, ammonium sulfate, and fish oil. More specifically, the propagation culture medium comprises 200-300 g brown sugar, 100-200 g corn steep liquor powder, 100-200 g fish meal, 10-12 g yeast extract, 4-6 mg copper sulfate, 4-6 mg zinc sulfate, 2-3 mg ferric chloride, 10 liters of natural seawater with a salinity of 20-30‰, 30-50 ml beet molasses yeast fermentation concentrate, 50-60 g ammonium sulfate, and 20-50 ml fish oil.
[0011] As a fourth aspect of the present application, a method for cultivating rotifers is provided, comprising:
[0012] The bacterial solution of the expanded Pichia kudriavzii described in the present application is added to the seawater culture solution of rotifers to culture the rotifers.
[0013] Optionally, the seawater culture medium of the rotifer comprises rotifers and natural seawater, and the salinity of the natural seawater is adjusted to a suitable range for the Pichia kudriavzii described in the present application.
[0014] Further optionally, the density of rotifers in the culture solution of rotifers is 5 to 10 rotifers / mL.
[0015] Optionally, the bacterial cell density of the bacterial solution is 2×10 8 cells / mL or above.
[0016] The present application screened out a strain of Pichia kudriavzi from a seawater environment, which naturally has the ability to adapt to the high salinity of seawater, avoiding the problem that Bacillus subtilis and other conventional yeasts in the current freshwater environment cannot be used efficiently in rotifer cultivation and cause water pollution; at the same time, it has extremely significant cultivation efficiency when used as bait for rotifers, and can significantly increase the yield of rotifers in a shorter time than conventional Bacillus subtilis and other yeasts, so that rotifer cultivation can be carried out in a low-cost, high-efficiency, and green and environmentally friendly manner.
[0017] Biological Deposit Description
[0018] Pichia kudriavzeviiELJ2024HNSYBGJ02, classification and naming: Pichia kudriavzevii , was deposited on March 21, 2025 in the Guangdong Provincial Microbial Culture Collection, located at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number GDMCC No: 66038. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Shown is the application Pichia kudriavzevii Brachionus plicatilis (Brachium plicatilis) after feeding;
[0020] Figure 2 Shown are marine Brachionus plicatilis rotifers fed with baker's yeast;
[0021] Figure 3 Shown are marine Brachionus plicatilis rotifers fed with Bacillus subtilis. DETAILED DESCRIPTION
[0022] The present application discloses a method for cultivating Pichia kudriavzevii and its application as well as a method for cultivating rotifers. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.
[0023] It should be noted that, in this document, 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 actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the existence of other identical elements in the process, method, article or device that includes the elements. At the same time, the embodiments and features in the embodiments of this application can be combined with each other in the absence of conflict.
[0024] In the first aspect of the present application, after multiple screening and separation, the present application isolated and purified a strain of Pichia kudriavzii ( Pichia kudriavzevii ELJ2024HNSYBGJ02), the length of its bacteria is 4~12μm, the suitable growth temperature range is 15~40℃, the optimum growth temperature is 25~30℃, the suitable salinity range is 0~50‰, and the most suitable salinity range is 20~30‰. It is deposited in Guangdong Provincial Microbiological Culture Collection with the deposit number GDMCC No: 66038, and the Latin classification is named Pichia kudriavzevii .
[0025] In the second aspect of the present application, the use of the Pichia kudriavzi yeast preserved in the present application to feed marine rotifers increased the final yield (the maximum density that can be achieved in a single culture) by about 3.3 to 5 times compared to conventional feeding with yeast and Bacillus subtilis. Among them, the maximum yield of rotifers fed with the present application strain reached 200,000 / L, the maximum yield of rotifers fed with conventional yeast reached 60,000 / L, and the maximum yield of rotifers fed with Bacillus subtilis reached 39,000 / L. The growth rate of rotifers increased by about 2 to 3 times. At the same inoculation density, the group fed with the present application strain took 2 days to reach a density of 39,000 / L, while the group fed with conventional yeast took 4 days to reach a density of 39,000 / L, and the group fed with Bacillus subtilis took 5 days to reach a density of 39,000 / L. The above two aquaculture experimental results fully prove that the Pichia kudriavzii provided in this application can significantly increase the growth rate and yield of rotifers, and the strain itself can tolerate the high salinity of seawater and will not cause damage to the aquaculture water body.
[0026] Based on this, the present application proposes the use of the described Pichia kudriavzii in preparing bait for culturing rotifers or in culturing rotifers; wherein, the rotifers are marine rotifers, including but not limited to marine plicatilis Brachionus rotifers, marine round Brachionus rotifers, etc.
[0027] In the third aspect of the present application, according to the scope of application, a bait for culturing rotifers is proposed, which includes the Pichia kudriavzii described in the present application.
[0028] In certain embodiments of the present application, the bait is the bacterial liquid of the Pichia kudriavzii described in the present application after expansion in an expansion medium; the expansion medium includes a carbon source, a nitrogen source, trace elements, growth-promoting substances and seawater.
[0029] In other embodiments of the present application, the expansion culture medium includes the following carbon sources: brown sugar and fish oil; the following nitrogen sources: ammonium sulfate, corn steep liquor powder, and fish meal; the following growth-promoting substances: beet molasses yeast fermentation concentrate and yeast extract powder; the following trace elements: copper sulfate, zinc sulfate, and ferric chloride; and the remainder is natural seawater. Compared to conventional Pichia culture media, this culture medium is optimized for rotifer cultivation. For example, the addition of fish oil increases the DHA and EPA content of the bacteria, while the addition of fish meal provides a more comprehensive protein profile. This optimization results in a more nutritious culture, which is beneficial for rotifer cultivation. Furthermore, the addition of nutrient sources containing growth-promoting factors, such as beet molasses yeast fermentation concentrate and yeast extract powder, promotes Pichia growth and increases bacterial yield. More specifically, the culture medium includes 200-300g of brown sugar, 100-200g of corn steep liquor powder, 100-200g of fish meal, 10-12g of yeast extract powder, 4-6mg of copper sulfate, 4-6mg of zinc sulfate, 2-3mg of ferric chloride, 10 liters of natural seawater with a salinity of 20-30‰, 30-50ml of beet molasses yeast fermentation concentrate, 50-60g of ammonium sulfate, and 20-50ml of fish oil; other specifications of expansion culture medium can be adjusted in proportion according to the above concentrations, which also falls within the protection scope of the expansion culture medium of this application.
[0030] In a fourth aspect of the present application, a method for cultivating rotifers is provided, comprising:
[0031] The bacterial solution of the expanded Pichia kudriavzii described in the present application is added to the seawater culture solution of rotifers to culture the rotifers.
[0032] In certain embodiments of the present application, the expansion of Pichia kudriavzii comprises:
[0033] Prepare expansion culture medium;
[0034] The Pichia kudriavzevii is inoculated into an expansion culture medium, placed in a temperature range of 25-30° C., and cultured with intermittent ventilation.
[0035] In certain embodiments of the present application, the preparation of the expansion culture medium comprises:
[0036] Brown sugar, corn steep liquor powder, fish meal, yeast extract powder, copper sulfate, zinc sulfate, and ferric chloride are mixed and then crushed. Natural seawater with a salinity within the suitable range for Pichia kudriavzevii as described in the present application and beet molasses yeast fermentation concentrate are added, mixed and boiled, and after cooling, ammonium sulfate and seawater fish oil are added and mixed.
[0037] In some other embodiments of the present application, the expansion culture medium includes 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 0-50‰, 30-50 ml of beet molasses yeast fermentation concentrate, 50-60 g of ammonium sulfate, and 20-50 ml of fish oil.
[0038] In certain embodiments of the present application, intermittent ventilation during the expansion process is to introduce mixed air containing 40-50% oxygen for 5 minutes every 30-40 minutes; the expansion time is determined by the required bacterial cell density, usually not less than 2×10 8 cells / mL.
[0039] In certain embodiments of the present application, the seawater culture medium of the rotifer comprises rotifers and natural seawater, and the salinity of the natural seawater is adjusted to a suitable range for the Pichia kudriavzii described in the present application, i.e., 0~50‰, more preferably 20~30‰; the water temperature of the natural seawater is maintained at 20~30°C, and air is injected into the seawater to adjust it to a slightly boiling state.
[0040] In some other embodiments of the present application, the density of the rotifers in the seawater culture medium is 5 to 10 rotifers / mL.
[0041] In certain embodiments of the present application, based on the volume of rotifer culture, 0.8-1.2 mL of the culture solution was added to every 10 liters of rotifer seawater culture solution, and the cell density of the culture solution was 2×10 8 cells / mL, feed 2-5 times daily for approximately 8-12 days, and harvest rotifers when the density reaches 200 cells / mL (200,000 cells / L). The number of feeding days will be adjusted based on the daily feeding amount and frequency. The maximum yield of a single culture of rotifers in this application can reach 200,000-250,000 cells / L.
[0042] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials, except for the differences noted in each group, were kept consistent to ensure comparability. In addition, all materials used in this application can be purchased from commercial sources.
[0043] The following further describes the Pichia kudriavzia yeast, its application, and the rotifer culture method provided in this application.
[0044] Example 1: Cultivation of Brachionus plicatilis in seawater
[0045] (1) Preparation of culture medium
[0046] Take 220g of brown sugar, 180g of corn steep liquor powder, 120g of fish meal, 12g of yeast extract powder, 4mg of copper sulfate, 4mg of zinc sulfate, and 2mg of ferric chloride, mix them, grind them through a 100-mesh sieve, put them into a 30L electric boiling bucket, add 10L of natural seawater with a salinity of 20‰ and 40ml of beet molasses yeast fermentation concentrate, mix well, boil for 30min, cool to 38°C, add 60g of ammonium sulfate and 40ml of seawater fish oil, and mix well.
[0047] (2) Expansion of bacterial strains
[0048] Pick Pichia kudriavzevii Add the above culture medium to a bottle of ELJ2024HNSYBGJ02 strain, place a heating rod and adjust to 28°C, aerate with mixed air containing 45% oxygen for 5 minutes every 30 minutes, and culture for 5 days. Shake once in the morning and evening every day. The final cell density of the cultured bacterial solution is about 2×10 8 cells / mL.
[0049] (3) Use of bacterial strains
[0050] A 300L fiberglass culture tank was filled with natural seawater at a salinity of 20‰. A heating rod was placed to maintain the water temperature at 26°C. Air was added to the tank to bring it to a slight boil. Well-developed L-shaped Brachionus plicatilis rotifers, averaging 320 μm in size, were seeded to a density of 8 per mL. Immediately after seeding, 30 mL of bacterial solution was added, followed by three feedings per day, 30 mL each time. After nine days, the average density of the rotifers reached 223 per mL, at which point the rotifers were harvested.
[0051] Example 2: Cultivation of Brachionus rotifer in seawater
[0052] (1) Preparation of culture medium
[0053] Take 200g of brown sugar, 150g of corn steep liquor powder, 100g of fish meal, 12g of yeast extract powder, 6mg of copper sulfate, 4mg of zinc sulfate, and 2mg of ferric chloride, mix them, grind them through a 100-mesh sieve, put them into a 30L electric boiling bucket, add 10L of natural seawater with a salinity of 20‰ and 35ml of beet molasses yeast fermentation concentrate, mix well, boil for 30min, cool to 33°C, add 55g of ammonium sulfate and 45ml of seawater fish oil, and mix well.
[0054] (2) Expansion of bacterial strains
[0055] Pick Pichia kudriavzevii Add the above culture medium to a bottle of ELJ2024HNSYBGJ02 strain, place a heating rod and adjust to 27°C, aerate with mixed air containing 43% oxygen for 5 minutes every 35 minutes, and culture for 6 days. Shake once in the morning and evening every day. The final cell density of the cultured bacterial solution is about 2.2×10 8 cells / mL.
[0056] (3) Use of bacterial strains
[0057] Take a 300L fiberglass culture tank, fill it with natural seawater with a salinity of 20‰, insert a heater to maintain the water temperature at 26°C, and aerate it until it is slightly boiling. Inoculate the tank with well-developed, S-shaped, round-shaped Brachionus rotifers, averaging 160 μm in size, to a density of 10 per mL. Immediately after inoculation, feed the rotifers with 30 mL of bacterial solution, then feed them three times daily, 30 mL each time. After nine days, when the average density reaches 213 per mL, the rotifers are harvested.
[0058] Experimental example:
[0059] Refer to the method of Example 1 and adopt the method of this application Pichia kudriavzevii ELJ2024HNSYBGJ02, baker's yeast (Angel), and Bacillus subtilis (isolated in the laboratory) were used to culture L-shaped Brachionus plicatilis. Except for the different feeding strains, other culture steps were consistent among the groups (the culture medium and culture conditions of baker's yeast and Bacillus subtilis were the same). Pichia kudriavzevii ELJ2024HNSYBGJ02 strain), the culture results are shown in Tables 1 and Figures 1 - 3 ;
[0060] Table 1
[0061]
[0062] According to Table 1 above, in the maximum density experiment achieved in a single culture, the maximum yield of rotifers fed by the strain of the present application reached 200,000 / L, the maximum yield of rotifers fed by conventional baker's yeast reached 60,000 / L, and the maximum yield of rotifers fed by Bacillus subtilis reached 39,000 / L.
[0063] In the density experiment achieved under the same culture days, the yield of rotifers fed by the strain of the present application reached 96,000 / L, the yield of rotifers fed by conventional baker's yeast reached 44,000 / L, and the yield of rotifers fed by Bacillus subtilis reached 39,000 / L.
[0064] In the experiment of the number of culture days required to reach the same density, the group fed with the strain of the present application reached a density of 39,000 / L in 2 days, while the group fed with conventional baker's yeast reached a density of 39,000 / L in 4 days, and the group fed with Bacillus subtilis reached a density of 39,000 / L in 5 days.
[0065] The above three aspects of the aquaculture experimental results fully prove that the Pichia kudriavzii provided by the present application can significantly increase the growth rate and yield of rotifers, and the strain itself can tolerate the high salinity of seawater and will not cause damage to the aquaculture water body.
[0066] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.
Claims
1. A Pichia kudriavzevii yeast ( Pichia kudriavzevii ), characterized in that, The deposit number is GDMCC No: 66038.
2. Use of the Pichia kudriavzii according to claim 1 in preparing bait for culturing rotifers or in culturing rotifers; the rotifers are Brachionus plicatilis or Brachionus roundus.
3. A bait for cultivating rotifers, characterized in that: The bait is the bacterial liquid of the Pichia kudriavzii described in claim 1 after expansion in an expansion medium; the expansion medium includes 200-300g of brown sugar, 100-200g of corn steep liquor powder, 100-200g of fish meal, 10-12g of yeast extract powder, 4-6mg of copper sulfate, 4-6mg of zinc sulfate, 2-3mg of ferric chloride, 10 liters of natural seawater with a salinity of 20-30‰, 30-50ml of beet molasses yeast fermentation concentrate, 50-60g of ammonium sulfate, and 20-50ml of fish oil; the rotifer is seawater Brachionus plicatilis rotifer or seawater Brachionus round rotifer.
4. A method for cultivating rotifers, characterized in that: include: The bacterial solution of the expanded Pichia kudriavzevii according to claim 1 is added to the seawater culture solution of rotifers to culture the rotifers; the rotifers are seawater Brachionus plicatilis or seawater Brachionus roundus; 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 of the Pichia kudriavzevii according to claim 1; the cell density of the bacterial solution is 2×10 8 cells / mL or above.
5. The culture method according to claim 4, characterized in that The density of the rotifers in the seawater culture solution of the rotifers is 5 to 10 rotifers / mL.