Selenium-rich pediococcus acidilactici and application thereof
By using lacticococcus lactica to convert inorganic selenium into organic selenium, the problem that inorganic selenium is difficult to be effectively absorbed by animals and plants in aquaculture is solved, and the effect of improving the absorption and utilization rate of animal selenium and disease resistance is achieved.
Patent Information
- Application Number
- CN202510254772.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively convert inorganic selenium into organic selenium, which is easier to be absorbed and utilized by animals and plants, especially in aquaculture to prevent diseases caused by intestinal pathogenic bacteria.
Through the microbial transformation method, inorganic selenium was converted into organic selenium using Pediococcus acidilactici as a selenium-rich carrier and enriched in bacterial cells and used as feed additives for animal breeding.
The efficient conversion of inorganic selenium into organic selenium is achieved, which improves the selenium absorption and utilization rate of animals, enhances its immune function and disease resistance, and especially significantly improves the survival rate of aquatic animals under heat stress stress.
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Figure CN120098839A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a high-selenium-enriched Pediococcus acidilactici bacterium and an application thereof. Background Art
[0002] Selenium is one of the essential trace elements for humans and animals and is an important component of many enzymes. Selenium can also enhance immunity and prevent diseases caused by pathogenic bacteria that often occur in the intensive breeding of aquatic animals, especially diseases caused by intestinal pathogens.
[0003] Studies have shown that inorganic selenium is transformed into organic selenide after biotransformation, which is less toxic, more conducive to animal and plant absorption and safe utilization. It is significantly better than inorganic selenide in activating animal immune response. The biotransformation of selenium has gradually become the focus of attention. Probiotics are the main object of research in the field of selenium-rich microbial transformation. Selenium-rich probiotics have been experimentally confirmed to have a variety of benefits, such as antioxidant, anti-pathogenic, anti-mutagenic, anti-cancer and anti-inflammatory activities.
[0004] Lactic acid bacteria are a very promising probiotic strain in aquaculture and have been widely used in aquaculture. Relevant studies have shown that lactic acid bacteria can improve the growth performance, disease resistance, immune function and antioxidant capacity of aquatic animals when used as feed additives. It also has the effects of improving intestinal flora, promoting digestion and absorption, enhancing stress resistance and improving water quality. Lactic acid bacteria have a high ability to enrich selenium and can convert inorganic selenium into organic selenium. Selenium-enriched lactic acid bacteria are generally obtained by adding inorganic selenium in the form of sodium selenite during the cultivation of lactic acid bacteria, converting it into an organic selenium form and enriching it in selenium-enriched lactic acid bacteria. It is a microbial fermentation product. Summary of the invention
[0005] The invention provides a high-selenium-enriched lactic acid bacteria and application thereof, which can be used as a selenium-enriched carrier to convert inorganic selenium into organic selenium that is easily absorbed and utilized by organisms through a microbial conversion method, and is enriched in the bacterial cells of the lactic acid bacteria. The lactic acid bacteria can be used as a feed additive for animal breeding.
[0006] In a first aspect, the present invention provides a Pediococcus acidilactici or its progeny, wherein the scientific name of the Pediococcus acidilactici is Pediococcus acidilactici, and the Pediococcus acidilactici was deposited in the General Microbiological Center of China National Microorganism Culture Collection on February 19, 2025, and the registration number of the Pediococcus acidilactici in the General Microbiological Center of China National Microorganism Culture Collection is CGMCC No.33566.
[0007] The progeny of Pediococcus acidilactici as described above refers to daughter cells produced by the growth of the microorganism (eg, growth in culture medium). It is to be understood that the progeny of Pediococcus acidilactici still retains the function of Pediococcus acidilactici.
[0008] In a second aspect, the present invention provides a culture comprising the above-mentioned Pediococcus acidilactici or its progeny.
[0009] The culture as described above is based on the ability of the Pediococcus acidilactici to enrich selenium in the environment. The culture is obtained by culturing the Pediococcus acidilactici in a culture system containing selenium. Furthermore, the selenium is added in the form of inorganic selenium, such as sodium selenite.
[0010] The culture as described above may only include the bacterial bodies obtained after culturing the Pediococcus acidilactici in an environment containing selenium, and may also include a solid or liquid culture medium for culturing the Pediococcus acidilactici, or components in the culture medium.
[0011] The culture as described above is powdered bacterial cells.
[0012] In a third aspect, the present invention provides a composition comprising the above-mentioned Pediococcus acidilactici or its progeny or a culture of the above-mentioned Pediococcus acidilactici.
[0013] The composition as described above, in addition to the lactic acid Pediococcus or its offspring or the culture of lactic acid Pediococcus, may also include other biological components or non-biological components. In a specific embodiment, the composition may be a feed. When the composition is a feed, the composition may also include other necessary nutrients or other probiotics for animal growth, which may be added according to the needs of animal growth. For example, other necessary nutrients include dietary fiber, prebiotics, proteins (such as enzymes), carbohydrates, lipids (such as fats), minerals, vitamins, etc. Other probiotics may include microorganisms that are beneficial to animal growth, such as microorganisms selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, etc. In a specific embodiment, the composition may be a plant inoculant, used to provide the necessary selenium element for plant growth. In a specific embodiment, the composition may be a food fermentation inoculant, such as for use in the preparation of fermented food.
[0014] Furthermore, the composition is feed for aquatic animals, and the aquatic animals can be fish, shrimp, crab, shellfish, etc.; further, the aquatic animals can be zebrafish, California bass, carp, grass carp and other fish whose suitable growth temperature is not higher than 30°C.
[0015] In a fourth aspect, the present invention provides a use of the above-mentioned Pediococcus acidilactici or its progeny or the above-mentioned culture or the above-mentioned composition, wherein the use is selected from at least one of A1) to A3):
[0016] A1) Application in the preparation of selenium-resistant bacterial agents;
[0017] A2) Application in the preparation of selenium-enriched bacterial agents;
[0018] A3) Application in preparing feed.
[0019] In a fifth aspect, the present invention provides a bacterial agent, comprising the above-mentioned Pediococcus acidilactici or its progeny or any of the above-mentioned cultures or any of the above-mentioned compositions.
[0020] The bacterial agent as described above is a live bacterial preparation prepared from the above-mentioned Pediococcus acidilactici. The dosage form of the bacterial agent can be various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.
[0021] The bacterial agent as described above is a selenium-rich bacterial agent, that is, the selenium content of the bacterial agent is higher than that of conventional microbial agents.
[0022] The bacterial agent as described above is a selenium-resistant bacterial agent, that is, it can grow normally in a culture environment containing selenium.
[0023] The bacterial agent as described above has the ability to transform inorganic selenium.
[0024] In a sixth aspect, the present invention provides a method for enriching selenium in an environment, comprising culturing the above-mentioned Pediococcus acidilactici or its progeny in an environment containing selenium, and enriching selenium in the environment through the growth of the Pediococcus acidilactici.
[0025] In the above method, the environment containing selenium can be an artificial environment or a natural environment, and the artificial environment can be, for example, a liquid culture medium or a solid culture medium, and the culture medium at least includes inorganic selenium. Further, the inorganic selenium can be added in the form of sodium selenite.
[0026] In a seventh aspect, the present invention provides a feed for animal breeding, comprising the above-mentioned Pediococcus acidilactici or its progeny or any of the above-mentioned cultures or any of the above-mentioned compositions.
[0027] In an eighth aspect, the present invention provides an animal breeding method, comprising adding the above-mentioned Pediococcus acidilactici or its progeny or any of the above-mentioned cultures or any of the above-mentioned compositions during the animal breeding process.
[0028] In a ninth aspect, the present invention provides a method for improving the survival rate of animals under heat stress, comprising adding the above-mentioned Pediococcus acidilactici or its progeny or any of the above-mentioned cultures or any of the above-mentioned compositions during the animal breeding process.
[0029] In the above method, the animal can be an aquatic animal, such as fish, shrimp, crab, shellfish, etc. Further, the aquatic animal can be zebrafish, California bass, carp, grass carp and other fishes suitable for growth at a temperature not higher than 30°C.
[0030] Furthermore, the total selenium content of the Pediococcus acidilactici or its culture or its composition should not affect the safe growth of animals, that is, the growth condition of the animal with the addition of the Pediococcus acidilactici or its culture or its composition should not be significantly different from the growth condition without the addition of the Pediococcus acidilactici or its culture or its composition.
[0031] The lactic acid bacteria provided by the present invention can be used as a selenium-enriched carrier, and can transform inorganic selenium into organic selenium that is easily absorbed and utilized by organisms through microbial transformation, and is enriched in the lactic acid bacteria cells, and can be used as a feed additive for animal breeding.
[0032] Collection Instructions
[0033] Bacteria name: Pediococcus acidilactici
[0034] Strain ID: SKR-2
[0035] Depository: China National Microbiological Culture Collection Administration General Microbiology Center
[0036] Abbreviation of depository institution: CGMCC
[0037] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing
[0038] Date of deposit: February 19, 2025
[0039] CGMCC Registration Number: CGMCC No.33566 BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 This is a photo of the bacterial powder of Pediococcus acidilactici SKR-2 obtained by screening in Example 1 of the present invention;
[0041] Figure 2 This is a photo of bacterial powder of Pediococcus acidilactici SKR-2 after being cultured in a solid medium containing sodium selenite;
[0042] Figure 3 Selected images for added concentrations of sodium selenite;
[0043] Figure 4 This is a comparison chart of selenium content in Pediococcus acidilactici SKR-2 and selenium-enriched Pediococcus acidilactici SKR-2;
[0044] Figure 5 This is a comparison chart of selenium production in the bacterial cells of Pediococcus acidilactici SKR-2 and Selenium-enriched Pediococcus acidilactici SKR-2;
[0045] Figure 6 The statistical results of the weight change and feed efficiency of zebrafish after feeding with a feed containing inorganic selenium and selenium-enriched lactic acid Pediococcus SKR-2; A is the statistical result of the initial weight of zebrafish before feeding; B is the statistical result of the final weight of zebrafish after feeding; C is the statistical result of the weight gain rate of zebrafish before and after feeding; D is the statistical result of feed efficiency;
[0046] Figure 7 These are the statistical results of the survival rate of zebrafish under heat stress after being fed a diet containing inorganic selenium and selenium-enriched Pediococcus acidilactici SKR-2.
[0047] Figure 6-7 In the table, ns means p>0.05; * means p≤0.05; ** means p≤0.01; *** means p≤0.001. DETAILED DESCRIPTION
[0048] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.
[0049] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0050] Example 1: Screening out selenium-rich microorganisms by testing the tolerance of microorganisms to selenium
[0051] First, prepare MRS solid culture medium (its composition is: 10g / L protein Chen, 5g / L beef powder, 4g / L yeast powder, 2g / L glucose, 1ml / L Tween 80, 2g / L potassium dihydrogen phosphate, 5g / L sodium acetate, 2g / L ammonium citrate, 0.2g / L magnesium sulfate, 0.05g / L manganese sulfate, 15g / L agar powder), and sterilize the prepared culture medium at 121°C for 20min; at the same time, prepare a sodium selenite solution with a concentration of 500mg / ml and filter sterilize it. Before the MRS solid culture medium solidifies, add sterile sodium selenite solution, and the initial working concentration of sodium selenite is 100μg / ml. The microorganisms isolated from the animal samples were inoculated in MRS solid medium containing sodium selenite, and the microorganisms that can grow were screened, and the concentration of sodium selenite in the MRS solid medium was gradually increased (including 1000μg / ml, 2500μg / ml and 5000μg / ml), and the screened microorganisms were screened again at a higher tolerance concentration. Finally, when the sodium selenite concentration was 5000μg / ml, the microorganism with the best tolerance was screened, and the 16s rDNA of the microorganism was shown in SEQ ID NO: 1. The microorganism was identified as Pediococcus acidilactici and named Pediococcus acidilactici SKR-2.
[0052] SEQ ID NO: 1 is as follows:
[0053]
[0054] The screened Pediococcus acidilactici SKR-2 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on February 19, 2025, and the registration number of the Pediococcus acidilactici SKR-2 in the General Microbiology Center of China National Microbiological Culture Collection is CGMCC No.33566.
[0055] Example 2: Selection of sodium selenite addition concentration
[0056] The lactic acid Pediococcus SKR-2 screened in Example 1 was activated in MRS solid culture medium, and a single colony was picked and inoculated into MRS liquid (the formula is solid culture medium minus 15g / L agar) culture medium, and cultured at a constant temperature of 37°C to the late logarithmic period, and the OD value was adjusted to about 0.8 to prepare a seed solution. The seed solution was inoculated into MRS culture medium at a 2% (v / v) inoculation amount and sterile sodium selenite storage solution was added (the final concentration of sodium selenite in MRS culture medium is 2.5g / L, 4.5g / L, 6.5g / L, 8.5g / L), the strain was cultured at 37°C, and the OD value of the bacterial solution was tested at 4h, 6h, 8h, 12h, 18h, 24h, 30h, 34h, and 36h. The test results are as follows: Figure 3 As shown, it can be seen that the OD value of Pediococcus acidilactici SKR-2 is the highest and the growth condition is the best when the sodium selenite concentration is 2.5 g / L. 2.5 g / L is selected as the addition concentration of sodium selenite for subsequent experiments.
[0057] Example 3: Selenium-enriching ability test of Pediococcus acidilactici SKR-2
[0058] After activating the frozen and preserved Pediococcus acidilactici SKR-2 in MRS solid culture medium, pick a single colony and inoculate it into MRS liquid culture medium, culture it at a constant temperature of 37°C until the late logarithmic period, and adjust the OD value to about 0.8 to prepare a seed solution. Inoculate the seed solution into the MRS culture medium at a 2% (v / v) inoculation amount and add sterile sodium selenite storage solution (the final concentration of sodium selenite in the MRS culture medium is 2.5g / L), and culture it at 37°C for 24h. After the strain culture is completed, centrifuge the culture product at 8000rpm for 10min, collect the precipitate, and wash it with deionized water (to wash away the unconverted sodium selenite) 2-3 times, collect the bacterial body, and name it as selenium-enriched Pediococcus acidilactici SKR-2, and freeze-dry it to obtain the following Figure 2 The bacterial powder shown.
[0059] At the same time, the seed solution was inoculated into MRS liquid culture medium (without sodium selenite) at a 2% (v / v) inoculation amount and cultured at 37°C for 24 hours. After the culture of the strain was completed, the culture product was centrifuged at 8000 rpm for 10 minutes, the precipitate was collected, and washed with deionized water 2-3 times (to wash away the unconverted sodium selenite), and the bacterial cells were collected and named Pediococcus acidilactici SKR-2. After freeze-drying, the obtained Figure 1 The bacterial powder shown.
[0060] The selenium content of the powder of Pediococcus acidilactici SKR-2 and the powder of selenium-enriched Pediococcus acidilactici SKR-2 was detected by inductively coupled plasma (ICP), and the selenium yield was calculated according to selenium yield (mg / L) = selenium content (mg / kg) × biomass (g / L) / 1000. The test results are as follows: Figure 4-5 As shown, it can be seen that the selenium content of selenium-enriched lactic acid Pediococcus SKR-2 is significantly higher than that of lactic acid Pediococcus SKR-2, specifically 315.73 g / kg, and the selenium yield is 309.97 mg / L.
[0061] Example 4: Selenium-enriched Pediococcus acidilactici SKR-2 was added as a feed additive to the basic diet of zebrafish for breeding evaluation
[0062] 1. One-month-old healthy zebrafish of the same size were selected as the test subjects and randomly divided into 4 test groups, each with 8 parallels, 22 fish in each parallel, for a total of 704 fish. The initial weight (IBW, g) of zebrafish in each group was weighed, and the average value was taken to obtain the initial average weight. The 4 test groups were named inorganic selenium group (Se), microbial selenium 50% group (effective amount of selenium is 50% of inorganic selenium), microbial selenium 100% group (effective amount of selenium is 100% of inorganic selenium), and microbial selenium 150% group (effective amount of selenium is 150% of inorganic selenium). The feed formula of each group is shown in Table 1.
[0063] Table 1 Feed formula for each group of zebrafish experiment
[0064]
[0065]
[0066] After the zebrafish were fed nearly full food according to the formula shown in Table 1 for 4 weeks, the final weight (FBW, g) of the zebrafish was weighed, and the weight gain rate and feed efficiency were calculated according to the following formula. The statistical results are shown in Figure 6 shown.
[0067] Weight gain rate (WG, %) = 100% × (final average weight - initial average weight) / initial average weight;
[0068] Feed efficiency (FCR) = (final total fish weight + total weight of dead fish - initial total weight) / food intake.
[0069] according to Figure 6 As shown, compared with inorganic selenium, the addition of 50% microbial selenium had no effect on the growth of zebrafish, and 50% was determined to be a safe addition concentration of microbial selenium.
[0070] 2. After 4 weeks of culture, zebrafish were fasted for 12 hours, and then subjected to a heat stress test in 40°C water. Each group (inorganic selenium group, microbial selenium 50% group, microbial selenium 100% group, microbial selenium 150% group) was set up with 4 replicates, and each replicate had 10 fish. The number of dead zebrafish was recorded every 2 hours, and the dead fish were removed in time. The test lasted for 6 hours, and the survival rate was calculated after the end. No feeding was carried out during the heat stress period to evaluate the heat stress survival rate of zebrafish.
[0071] Heat stress survival rate (%) = number of zebrafish surviving at the heat stress test recording time point / number of zebrafish at the beginning of the heat stress test × 100.
[0072] Test results such as Figure 7 As shown, it can be seen that the survival rate of the 50% microbial selenium group was the highest and was significantly different from that of the inorganic selenium group, indicating that compared with inorganic selenium, adding 50% microbial selenium can effectively improve the survival rate of zebrafish under heat stress.
[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Pediococcus acidilactici or its progeny, characterized in that The lactic acid Pediococcus is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and the registration number of the lactic acid Pediococcus is CGMCC No.33566.
2. A culture, characterized in that The method comprises the Pediococcus acidilactici or its progeny according to claim 1.
3. The culture according to claim 2, characterized in that The culture is obtained by culturing the Pediococcus acidilactici in a culture system containing selenium.
4. A composition, characterized in that The method comprises the Pediococcus acidilactici or its progeny according to claim 1 or the culture according to any one of claims 2-3.
5. Use of the Pediococcus acidilactici or its progeny according to claim 1, or the culture according to any one of claims 2 to 3, or the composition according to claim 4, characterized in that: The application is selected from at least one of A1) to A3): A1) application in the preparation of a selenium-resistant bacterial agent; A2) Application in the preparation of selenium-enriched bacterial agents; A3) Application in preparing feed.
6. A microbial agent, characterized in that: The method comprises the Pediococcus acidilactici or its progeny according to claim 1, the culture according to any one of claims 2 to 3, or the composition according to claim 4.
7. A method for enriching selenium in an environment, characterized in that: The method comprises culturing the Pediococcus acidilactici or its progeny according to claim 1 in an environment containing selenium, and enriching the selenium in the environment through the growth of the Pediococcus acidilactici.
8. Feed for animal breeding, characterized in that: The method comprises the Pediococcus acidilactici or its progeny according to claim 1, the culture according to any one of claims 2 to 3, or the composition according to claim 4.
9. An animal breeding method, characterized in that: The method comprises adding the Pediococcus acidilactici or its progeny according to claim 1 or the culture according to any one of claims 2 to 3 or the composition according to claim 4 during the animal breeding process.
10. A method for improving the survival rate of animals under heat stress, characterized in that: The method comprises adding the Pediococcus acidilactici or its progeny according to claim 1 or the culture according to any one of claims 2 to 3 or the composition according to claim 4 during the animal breeding process.