Method for reducing accumulation of mercury in fish body by using fish-derived nano-selenium-rich lactic acid bacteria

Abstract

The invention discloses a method for reducing accumulation of mercury in fish bodies by using fish-derived nano-selenium-rich lactic acid bacteria, and belongs to the technical field of aquaculture. The method comprises the following steps: preparing biological nano-selenium dry powder by utilizing nano-selenium-rich lactic acid bacteria; the selenium content of the selenium-rich fish feed is 0.5 mg / kg, and the content of the nano-selenium-rich lactic acid bacteria is 105 cfu / kg; from a fry stage to a commercial fish stage, feeding the fish with the selenium-rich feed. By adopting the method for reducing accumulation of mercury in the fish body, the mercury content in muscles and visceral groups of the cultured fish is relatively low, but the mercury content in the muscles of the fish cultured in the mercury-containing polluted water body reaches 0.91 mu g / g, and the mercury content in the visceral groups reaches 21.34 mu g / g; by adding the selenium-rich lactic acid bacteria into the feed, the mercury content of fish muscles in mercury polluted water can be reduced by 75.82%, the mercury content of fish viscera can be reduced by 81.87%, and the use of the selenium-rich lactic acid bacteria provides guarantee for the health of fish bodies and the edible safety of fish meat.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and specifically relates to a method for reducing the accumulation of mercury in fish bodies by utilizing fish-derived nano-selenium-rich lactic acid bacteria. Background Art

[0002] Selenium (Se) is an essential trace element required by a variety of organisms, including humans. It is a component of more than 30 different selenoproteins, including glutathione peroxidase, some antioxidant enzymes that help protect cells from free radical damage. Typically, selenium is obtained by eating selenium-rich foods (such as fungus, seafood, meat and vegetables) or inorganic selenium salts. However, the selenium content in food varies in different regions and may not be enough to meet daily needs. The latest trend in the food industry is to solve this problem by culturing yeast or algae in selenium-enriched media to produce biomass with increased organically bound selenium content, which is more suitable for human consumption than potentially toxic inorganic selenium salts.

[0003] Lactic acid bacteria can be widely used as probiotics. When administered at appropriate doses, they can survive, proliferate and colonize the intestines of animals to promote a healthy balance of intestinal microbial populations. They also transform microorganisms in the intestine into beneficial results, such as excluding invading microbial pathogens, improving digestion, and enhancing growth performance. More importantly, some members of this bacteria have certain antibiotic resistance mechanisms.

[0004] At present, human dietary supplementation depends on the correct choice of food and a balanced diet. Organic selenium (selenium-enriched eggs, selenium-enriched meat products) and inorganic selenium (selenite tablets, bread, beverages, etc.) selenium supplement products have been developed. However, the narrow range of nutritional doses and toxic doses, low safety, and difficult control of doses have limited the development of selenium supplement products. Studies have found that nano-selenium is less toxic than organic selenium (selenoprotein) and inorganic selenium (sodium selenite), and has the lowest toxicity.

[0005] Mercury is a global pollutant that affects the health of humans and ecosystems. It is a widespread natural and anthropogenic pollutant. Both organic and inorganic mercury can cause harm to humans and aquatic life. Mercury accumulates in animals at a relatively high level. Once high levels of mercury are consumed in aquatic organisms or plants, it will accumulate in the body. Over time, it will cause oxidative damage to the body, leading to the production of reactive oxygen species (ROS), which in turn damage biological macromolecules and cause disease. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems existing in the prior art and to propose a method for reducing the accumulation of mercury in fish bodies by using fish-derived nano-selenium-rich lactic acid bacteria.

[0007] The method of reducing the accumulation of mercury in fish by using fish-derived nano-selenium-rich lactic acid bacteria is achieved by the following steps:

[0008] 1. Preparation of biological nano-selenium dry powder:

[0009] The nano-selenium-rich lactic acid bacteria are inoculated into an MRS medium for activation culture to obtain a nano-selenium-rich lactic acid bacteria liquid, and then the nano-selenium-rich lactic acid bacteria liquid and a Na2SeO3 solution are added into the MRS medium for fermentation culture. After the fermentation, the obtained product is centrifuged, washed and dried to obtain a biological nano-selenium dry powder;

[0010] 2. Preparation of selenium-rich fish feed:

[0011] The biological nano-selenium dry powder is suspended in distilled water to obtain a selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred, and dried to obtain a selenium-enriched fish feed;

[0012] 3. From the seedling stage to the commercial fish stage, farmed fish are fed with the above-mentioned fish selenium-rich feed, with a daily feeding amount of 2-4% of the fish body weight, thus completing the use of fish-derived nano-selenium-rich lactic acid bacteria to reduce the accumulation of mercury in the fish body;

[0013] The selenium content in the fish selenium-enriched feed obtained in step 2 is 0.5 mg / kg, and the content of nano-selenium-enriched lactic acid bacteria is 10 5 cfu / kg.

[0014] Furthermore, the nano-selenium-rich lactic acid bacteria in step one is Lactobacillus plantarum L1, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242780, a deposit date of December 10, 2024, and a deposit address of Wuhan University, Wuhan, China. It is Lactobacillus plantarum HRFRI-L1.

[0015] Furthermore, the activation culture in step 1 is cultured in a shaking incubator at 37° C. for 16 h at a shaking speed of 150 rpm.

[0016] Furthermore, the amount of bacteria in the nano-selenium-rich lactic acid bacteria liquid in step 1 is 1×10 4 CFU / ml.

[0017] Furthermore, the concentration of the Na2SeO3 solution in step 1 is 500-2000 μg / ml.

[0018] Furthermore, in step 1, the dosage of the nano-selenium-rich lactic acid bacteria liquid is 50 μl, the dosage of the Na2SeO3 solution is 50 μl, and the dosage of the MRS culture medium is 10 mL.

[0019] Furthermore, the fermentation culture in step 1 is cultured in a shaking incubator at 37° C. for 24 h at a shaking speed of 150 rpm.

[0020] Furthermore, the centrifugation, washing and drying described in step 1: after the fermentation culture is completed, the product is centrifuged at 3000 rpm for 10 minutes, the supernatant is discarded, and then 10 ml of distilled water is added, and the centrifugation is repeated three times to remove selenium in the liquid, and the precipitate is dried in a 37°C oven.

[0021] Furthermore, the nutritional level requirements of the commercial fish feed in step 2 are: crude protein 40%, crude fat 4.0%, crude fiber 8.0%, crude ash 16%, total phosphorus 0.5%, and lysine 1.8%.

[0022] Furthermore, the drying temperature in step 2 is 37°C.

[0023] Furthermore, the daily feeding amount in step 3 is 3% of the fish body weight.

[0024] Advantages of the present invention:

[0025] The method of using fish-derived nano-selenium lactic acid bacteria to reduce the accumulation of mercury in fish bodies in the present invention has the following characteristics: first, biological nano-selenium itself has low toxicity and high biological activity. Secondly, plant lactobacillus L1 isolated from the intestinal tract of healthy fish is used, which can not only promote the healthy balance of microbial populations in the intestinal tract of fish, but also absorb some heavy metals; the present invention uses two means to simultaneously reduce the accumulation of mercury in organisms, and eating nano-selenium lactic acid bacteria can greatly reduce the accumulation of mercury in fish bodies, and eating nano-selenium lactic acid bacteria in advance is better than eating nano-selenium lactic acid bacteria and mercury at the same time, or eating mercury first and then taking nano-selenium lactic acid bacteria.

[0026] By adopting the method of reducing the accumulation of mercury in fish bodies by using fish-derived nano-selenium lactic acid bacteria in the present invention, the mercury content in the muscle and visceral mass of cultured fish is low. However, the mercury content in the muscle of fish cultured in mercury-contaminated water reaches 0.91 μg / g, and the mercury content in the visceral mass reaches 21.34 μg / g. By adding selenium-rich lactic acid bacteria to feed, the mercury content in the muscle of fish cultured in mercury-contaminated water can be reduced by 75.82%, and the mercury content in the visceral mass of fish can be reduced by 81.87%. The use of selenium-rich lactic acid bacteria provides protection for the health of the fish itself and the safety of fish meat.

[0027] The invention is suitable for reducing the accumulation of mercury in fish bodies in aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a color change diagram of Lactobacillus plantarum L1 after the fermentation culture in the embodiment is completed;

[0029] Figure 2 This is a scanning electron microscope image of Lactobacillus plantarum L1 after the fermentation culture in the embodiment;

[0030] Figure 3This is a transmission electron micrograph of Lactobacillus plantarum L1 after fermentation and cultivation in the example. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0032] Specific implementation method 1: This implementation method uses fish-derived nano-selenium-rich lactic acid bacteria to reduce the accumulation of mercury in fish bodies, which is achieved by the following steps:

[0033] 1. Preparation of biological nano-selenium dry powder:

[0034] The nano-selenium-rich lactic acid bacteria are inoculated into an MRS medium for activation culture to obtain a nano-selenium-rich lactic acid bacteria liquid, and then the nano-selenium-rich lactic acid bacteria liquid and a Na2SeO3 solution are added into the MRS medium for fermentation culture. After the fermentation, the obtained product is centrifuged, washed and dried to obtain a biological nano-selenium dry powder;

[0035] 2. Preparation of selenium-rich fish feed:

[0036] The biological nano-selenium dry powder is suspended in distilled water to obtain a selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred, and dried to obtain a selenium-enriched fish feed;

[0037] 3. From the seedling stage to the commercial fish stage, farmed fish are fed with the above-mentioned fish selenium-rich feed, with a daily feeding amount of 2-4% of the fish body weight, thus completing the use of fish-derived nano-selenium-rich lactic acid bacteria to reduce the accumulation of mercury in the fish body;

[0038] The selenium content in the fish selenium-enriched feed obtained in step 2 is 0.5 mg / kg, and the content of nano-selenium-enriched lactic acid bacteria is 10 5 cfu / kg.

[0039] In step three of this embodiment, the daily feeding amount is reasonably adjusted according to actual conditions such as weather, water temperature, fish size, feeding intensity, etc.

[0040] In this embodiment, MRS culture medium and commercial fish feed are both commercially available products.

[0041] The selenium-enriched fish feed obtained in step 2 of this embodiment is sampled and tested for selenium content and bacteria content, and the qualified products are stored at a low temperature of 4°C.

[0042] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the nano-selenium-rich lactic acid bacteria in step 1 is Lactobacillus plantarum L1, which has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: M 20242780, a deposit date of December 10, 2024, and a deposit address of Wuhan University, Wuhan, China, and is Lactobacillus plantarum HRFRI-L1. The other steps and parameters are the same as those in specific embodiment 1.

[0043] Specific implementation method three: This implementation method is different from specific implementation method one in that the activation culture in step one is cultured in a shaking incubator at 37° C. for 16 h, with a shaking speed of 150 rpm. Other steps and parameters are the same as those in specific implementation method one.

[0044] Specific embodiment 4: This embodiment is different from the specific embodiment 1 in that the amount of bacteria in the nano-selenium-rich lactic acid bacteria liquid in step 1 is 1×10 4 CFU / ml. Other steps and parameters are the same as those in the first embodiment.

[0045] Specific implementation method 5: This implementation method is different from specific implementation method 1 in that the concentration of the Na2SeO3 solution in step 1 is 500-2000 μg / ml. Other steps and parameters are the same as those in specific implementation method 1.

[0046] Specific embodiment 6: This embodiment is different from specific embodiment 1 in that the amount of nano-selenium-rich lactic acid bacteria liquid in step 1 is 50 μl, the amount of Na2SeO3 solution is 50 μl, and the amount of MRS culture medium is 10 mL. Other steps and parameters are the same as those in specific embodiment 1.

[0047] Specific embodiment 7: This embodiment differs from specific embodiment 1 in that the fermentation culture in step 1 is cultured in a shaking incubator at 37° C. for 24 h, with a shaking speed of 150 rpm. Other steps and parameters are the same as those in specific embodiment 1.

[0048] Specific embodiment eight: This embodiment differs from specific embodiment one in that the centrifugation, washing and drying in step one: after the fermentation culture is completed, the obtained product is centrifuged at 3000rpm for 10min, the supernatant is discarded, and then 10ml of distilled water is added, and the centrifugation is repeated three times to remove selenium in the liquid, and the precipitate is dried in an oven at 37°C. The other steps and parameters are the same as those in specific embodiment one.

[0049] Specific embodiment 9: This embodiment is different from specific embodiment 1 in that the nutritional level requirements of the commercial fish feed in step 2 are: crude protein 40%, crude fat 4.0%, crude fiber 8.0%, crude ash 16%, total phosphorus 0.5%, and lysine 1.8%. Other steps and parameters are the same as those in specific embodiment 1.

[0050] Specific embodiment 10: This embodiment is different from the specific embodiment 1 in that the drying temperature in step 2 is 37° C. The other steps and parameters are the same as those in the specific embodiment 1.

[0051] Specific implementation eleven: This implementation differs from specific implementation one in that the daily feeding amount in step three is 3% of the fish body weight. Other steps and parameters are the same as those in specific implementation one.

[0052] The beneficial effects of the present invention are verified by the following examples:

[0053] Example:

[0054] The method of reducing the accumulation of mercury in fish by using fish-derived nano-selenium-rich lactic acid bacteria is achieved by the following steps:

[0055] 1. Preparation of biological nano-selenium dry powder:

[0056] The nano-selenium-rich lactic acid bacteria are inoculated into an MRS medium for activation culture to obtain a nano-selenium-rich lactic acid bacteria liquid, and then the nano-selenium-rich lactic acid bacteria liquid and a Na2SeO3 solution are added into the MRS medium for fermentation culture. After the fermentation, the obtained product is centrifuged, washed and dried to obtain a biological nano-selenium dry powder;

[0057] 2. Preparation of selenium-rich fish feed:

[0058] The biological nano-selenium dry powder is suspended in distilled water to obtain a selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred, and dried to obtain a selenium-enriched fish feed;

[0059] 3. From the seedling stage to the commercial fish stage, farmed fish are fed with the above-mentioned fish selenium-rich feed, with a daily feeding amount of 3% of the fish body weight, thus completing the use of fish-derived nano-selenium-rich lactic acid bacteria to reduce the accumulation of mercury in the fish body;

[0060] The selenium content in the fish selenium-enriched feed obtained in step 2 is 0.5 mg / kg, and the content of nano-selenium-enriched lactic acid bacteria is 10 5 cfu / kg;

[0061] The nano-selenium-rich lactic acid bacteria described in step 1 is Lactobacillus plantarum L1 isolated from the intestine of healthy fish, and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242780, a deposit date of December 10, 2024, and a deposit address of Wuhan University, Wuhan, China. It is Lactobacillus plantarum HRFRI-L1;

[0062] The activation culture in step 1 was cultured in a shaking incubator at 37°C for 16 h at a shaking speed of 150 rpm;

[0063] The amount of bacteria in the nano-selenium-rich lactic acid bacteria solution in step 1 is 1×10 4 CFU / ml;

[0064] The concentration of the Na2SeO3 solution in step 1 is 2000 μg / ml;

[0065] In step 1, the amount of the nano-selenium-rich lactic acid bacteria solution is 50 μl, the amount of the Na2SeO3 solution is 50 μl, and the amount of the MRS culture medium is 10 mL;

[0066] The fermentation culture in step 1: culture in a shaking incubator at 37°C for 24 hours at a shaking speed of 150 rpm;

[0067] Centrifugation, washing and drying in step 1: After the fermentation culture is completed, the product is centrifuged at 3000 rpm for 10 minutes, the supernatant is discarded, and then 10 ml of distilled water is added, and centrifugation is repeated three times to remove selenium in the liquid, and the precipitate is dried in an oven at 37°C;

[0068] The nutritional level requirements of the commercial fish feed in step 2 are: crude protein 40%, crude fat 4.0%, crude fiber 8.0%, crude ash 16%, total phosphorus 0.5%, and lysine 1.8%;

[0069] The drying temperature in step 2 is 37°C.

[0070] After the fermentation culture in step 1 of this embodiment is completed, the color change of Lactobacillus plantarum L1 is observed. Figure 1 As shown, a large amount of red nano-selenium is precipitated at this time.

[0071] The biological nano-selenium dry powder obtained in step 1 of this embodiment has a selenium content of about 862.236 μg / L (diluted 10000) in Lactobacillus plantarum L1 by atomic fluorescence analysis. Figure 2 and Figure 3 As can be seen, the surface nanoparticles of Lactobacillus plantarum L1 have a size of about 42.9 nm.

[0072] In this embodiment, the obtained fish selenium-enriched feed A was prepared under laboratory conditions to feed carp for 30 days to observe the growth status and the situation of mercury in the body. Mercury was put into water to make the concentration of water finally 0.03mg / L, and half of the water was changed every day to add corresponding mercury. After 30 days, the fish body was dissected and the mercury content in the muscle was measured. The control group (no mercury in the water and feeding fish commercial feed) had a mercury content of 0.053mg / kg in the muscle, the mercury group (mercury in the water and feeding fish commercial feed) had a mercury content of 0.58mg / kg, and the selenium-enriched lactic acid bacteria plus mercury group (i.e., mercury in the water fed fish selenium-enriched feed A) had a mercury content of 0.22mg / kg. In the feeding process of one month, feeding the feed with selenium-enriched lactic acid bacteria can significantly reduce the mercury content in the muscle. During this period, we also fed carp with feed with selenium-enriched lactic acid bacteria alone for 30 days, and then added the same mercury concentration as before every day to measure the mercury concentration in the muscle after 30 days. By adding selenium-enriched lactic acid bacteria to the feed, the mercury content of the muscle of fish in mercury-contaminated water bodies can be reduced by 75.82%, and the mercury content of the fish viscera can be reduced by 81.87%. Feeding selenium-enriched lactic acid bacteria for one month and then adding mercury to the water body, the mercury concentration in the muscle was 0.057mg / kg, and the mercury accumulation in the visceral mass was 1.62mg / kg. This feeding method can reduce the mercury content in the muscle of fish in mercury-contaminated water by 93.74% and reduce the mercury content in the visceral mass of fish by 92.40% (Table 1). The results show that feeding selenium-enriched lactic acid bacteria feed can significantly reduce the accumulation of mercury in fish, and the effect of feeding selenium-enriched lactic acid bacteria feed before is better than feeding selenium-enriched lactic acid bacteria and mercury at the same time, which can greatly reduce the accumulation of mercury in fish, protect aquatic organisms from the harm of mercury, and thus promote the development of aquaculture.

[0073] Table 1. Accumulation of mercury in fish

[0074]

[0075] Note: Items marked with different letters indicate significant differences among different groups in the same tissue, P<0.05.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for reducing mercury accumulation in fish using fish-derived nano-selenium-rich lactic acid bacteria, characterized in that It is implemented as follows:

1. Preparation of biological nano-selenium dry powder: The nano-selenium-rich lactic acid bacteria are inoculated into an MRS medium for activation culture to obtain a nano-selenium-rich lactic acid bacteria liquid, and then the nano-selenium-rich lactic acid bacteria liquid and a Na2SeO3 solution are added into the MRS medium for fermentation culture. After the fermentation, the obtained product is centrifuged, washed and dried to obtain a biological nano-selenium dry powder; 2. Preparation of selenium-rich fish feed: The biological nano-selenium dry powder is suspended in distilled water to obtain a selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred, and dried to obtain a selenium-enriched fish feed; 3. From the seedling stage to the commercial fish stage, farmed fish are fed with the above-mentioned fish selenium-rich feed, with a daily feeding amount of 2-4% of the fish body weight, thus completing the use of fish-derived nano-selenium-rich lactic acid bacteria to reduce the accumulation of mercury in the fish body; The selenium content in the fish selenium-enriched feed obtained in step 2 is 0.5 mg / kg, and the content of nano-selenium-enriched lactic acid bacteria is 10 5 cfu / kg.

2. The method of reducing mercury accumulation in fish by using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The nano-selenium-rich lactic acid bacteria described in step one is Lactobacillus plantarum L1, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242780, a deposit date of December 10, 2024, and a deposit address of Wuhan University, Wuhan, China. It is Lactobacillus plantarum HRFRI-L1.

3. The method of reducing mercury accumulation in fish by using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The activation culture in step 1 was cultured in a shaking incubator at 37° C. for 16 h at a shaking speed of 150 rpm.

4. The method for reducing mercury accumulation in fish using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The amount of bacteria in the nano-selenium-rich lactic acid bacteria solution in step 1 is 1×10 4 CFU / ml.

5. The method of reducing mercury accumulation in fish by using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The concentration of the Na2SeO3 solution in step 1 is 500-2000 μg / ml.

6. The method of reducing mercury accumulation in fish using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that In step 1, the dosage of the nano-selenium-rich lactic acid bacteria liquid is 50 μl, the dosage of the Na2SeO3 solution is 50 μl, and the dosage of the MRS culture medium is 10 mL.

7. The method of reducing mercury accumulation in fish by using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The fermentation culture in step 1 was cultured in a shaking incubator at 37° C. for 24 h at a shaking speed of 150 rpm.

8. The method of reducing mercury accumulation in fish by using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that Centrifugation, washing and drying as described in step 1: After the fermentation culture is completed, the product is centrifuged at 3000 rpm for 10 minutes, the supernatant is discarded, and then 10 ml of distilled water is added, and centrifugation is repeated three times to remove selenium in the liquid, and the precipitate is dried in a 37°C oven.

9. The method of reducing mercury accumulation in fish using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The nutritional level requirements of the commercial fish feed in step 2 are: crude protein 40%, crude fat 4.0%, crude fiber 8.0%, crude ash 16%, total phosphorus 0.5%, and lysine 1.8%.

10. The method for reducing mercury accumulation in fish using fish-derived nano-selenium-rich lactic acid bacteria according to claim 1, characterized in that The drying temperature in step 2 is 37° C.; the daily feeding amount in step 3 is 3% of the fish body weight.

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