Methods to reduce mercury accumulation in fish using fish-derived nano-selenium-enriched lactic acid bacteria

Abstract

This method utilizes fish-derived, selenium-enriched nano-sized lactic acid bacteria to reduce mercury accumulation in fish, belonging to the field of aquaculture technology. Method: Bio-sized selenium powder is prepared using selenium-enriched nano-sized lactic acid bacteria; a selenium-enriched fish feed is prepared, containing 0.5 mg / kg selenium and 10 mg / kg of selenium-enriched nano-sized lactic acid bacteria. 5 CFU / kg; From the fry stage to the marketable fish stage, the fish are fed selenium-enriched feed. Using the method of reducing mercury accumulation in fish as described in this invention, the mercury content in the muscle and viscera of farmed fish is relatively low. However, in fish farmed in mercury-polluted water, the mercury content in the muscle reaches 0.91 μg / g, and the mercury content in the viscera reaches 21.34 μg / g. Adding selenium-enriched lactic acid bacteria to the feed can reduce the mercury content in the muscle of fish from mercury-polluted water by 75.82% and the mercury content in the viscera by 81.87%. The use of selenium-enriched lactic acid bacteria provides protection for the health of the fish and the safety of the fish meat for consumption.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology; specifically, it relates to a method for reducing mercury accumulation in fish by using fish-derived nano-selenium-enriched lactic acid bacteria. Background Technology

[0002] Selenium (Se) is an essential trace element required by many organisms, including humans. It is a component of more than 30 different selenoproteins, including glutathione peroxidase, some of which are antioxidant enzymes that help protect cells from free radical damage. Selenium is typically obtained through consuming selenium-rich foods (such as black fungus, seafood, meat, and vegetables) or inorganic selenium salts. However, the selenium content in food varies from region to region and may not be sufficient to meet daily needs. A recent trend in the food industry is to address this issue by producing biomass with increased organically bound selenium content through the cultivation of yeast or algae in selenium-enriched media, which is more suitable for human consumption than potentially toxic inorganic selenium salts.

[0003] Lactic acid bacteria are widely used as probiotics. When administered in appropriate doses, they can survive, proliferate, and colonize the animal gut to promote a healthy balance of the gut microbiota. They also facilitate the transformation of gut microbiota into beneficial outcomes, such as eliminating invading microbial pathogens, improving digestion, and enhancing growth performance. More importantly, some members of these bacteria possess certain antibiotic resistance mechanisms.

[0004] Currently, human dietary supplementation relies on proper food selection and a balanced diet. Organic selenium (selenium-enriched eggs, selenium-enriched meat products) and inorganic selenium (selenite tablets, bread, beverages, etc.) products have been developed. However, the narrow range of nutritional and toxic dosages, low safety profile, and difficulty in controlling dosage have limited the development of selenium supplements. Research has found that nano-selenium has the lowest toxicity, less than organic selenium (selenoproteins) and less than inorganic selenium (sodium selenite).

[0005] Mercury is a global pollutant affecting human and ecosystem health. It is a widespread natural and anthropogenic pollutant, with both organic and inorganic mercury potentially harming humans and aquatic life. Mercury accumulates relatively high levels in animals, and its buildup in the body from consuming high levels of aquatic organisms or plants can lead to oxidative damage over time, resulting in the production of reactive oxygen species (ROS). This damages biomolecules and can cause illness. Summary of the Invention

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

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

[0008] I. Preparation of Bio-nano Selenium Powder:

[0009] The nano-selenium-enriched lactic acid bacteria were inoculated into MRS medium for activation culture to obtain nano-selenium-enriched lactic acid bacteria liquid. Then, the nano-selenium-enriched lactic acid bacteria liquid and Na2SeO3 solution were added to MRS medium for fermentation culture. After the fermentation culture, the product was centrifuged, washed and dried to obtain biological nano-selenium dry powder.

[0010] II. Preparation of selenium-enriched feed for fish:

[0011] The above-mentioned bio-nano selenium dry powder is suspended in distilled water to obtain selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred evenly. After drying, the fish selenium-enriched feed is obtained.

[0012] Third, from the fry stage to the market fish stage, farmed fish should be fed selenium-enriched feed, with a daily feeding amount of 2-4% of the fish's body weight. This will reduce mercury accumulation in the fish by utilizing fish-derived nano-selenium-enriched lactic acid bacteria.

[0013] The selenium-enriched fish feed obtained in step two contains 0.5 mg / kg of selenium and 10 mg / kg of nano-selenium-enriched lactic acid bacteria. 5 cfu / kg.

[0014] Furthermore, the nano-selenium-enriched lactic acid bacteria mentioned in step one is Lactobacillus plantarum L1, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20242780, deposit date December 10, 2024, and deposit address Wuhan University, Wuhan, China. Its name is Lactobacillus plantarum HRFRI-L1.

[0015] Furthermore, the activation culture described in step one involves shaking the culture at 37°C for 16 hours at a shaking speed of 150 rpm.

[0016] Furthermore, the bacterial count in the nano-selenium-enriched lactic acid bacteria solution described in step one is 1×10⁻⁶. 4 CFU / ml.

[0017] Furthermore, the concentration of the Na2SeO3 solution mentioned in step one is 500–2000 μg / ml.

[0018] Furthermore, in step one, the amount of the nano-selenium-enriched lactic acid bacteria solution used is 50 μl, the amount of Na2SeO3 solution used is 50 μl, and the amount of MRS culture medium used is 10 mL.

[0019] Furthermore, the fermentation culture described in step one: the culture was carried out at 37°C on a shaker for 24 hours at a shaking speed of 150 rpm.

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

[0021] Furthermore, the nutritional requirements for the commercial fish feed described in step two are as follows: 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 two is 37°C.

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

[0024] Advantages of this invention:

[0025] This invention utilizes fish-derived, nano-selenium-enriched lactic acid bacteria to reduce mercury accumulation in fish. Firstly, the bio-nanoselenium itself is low in toxicity and has high biological activity. Secondly, it employs *Lactobacillus plantarum* L1, isolated from the intestines of healthy fish. Besides promoting a healthy balance of the fish's intestinal microbiota, it can also adsorb some heavy metals. This invention uses two methods to simultaneously reduce mercury accumulation in the body. Consuming nano-selenium-enriched lactic acid bacteria can significantly reduce mercury accumulation in fish. Moreover, consuming nano-selenium-enriched lactic acid bacteria beforehand is more effective than consuming nano-selenium-enriched lactic acid bacteria and mercury simultaneously, or consuming mercury first and then consuming nano-selenium-enriched lactic acid bacteria.

[0026] The method of reducing mercury accumulation in fish by using fish-derived selenium-enriched nano-selenium lactic acid bacteria, as described in this invention, results in lower mercury content in the muscle and viscera of farmed fish. However, in fish farmed in mercury-polluted water, the mercury content in the muscle reaches 0.91 μg / g, and the mercury content in the viscera reaches 21.34 μg / g. By adding selenium-enriched lactic acid bacteria to the feed, the mercury content in the muscle of fish in mercury-polluted water can be reduced by 75.82%, and the mercury content in the viscera can be reduced by 81.87%. The use of selenium-enriched lactic acid bacteria provides protection for the health of the fish and the safety of fish meat for consumption.

[0027] This invention is applicable to reducing the accumulation of mercury in fish during aquaculture. Attached Figure Description

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

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

[0030] Figure 3The image shown is a transmission electron microscope image of Lactobacillus plantarum L1 after fermentation culture in the example. Detailed Implementation

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

[0032] Specific Implementation Method 1: This implementation method utilizes fish-derived, nano-selenium-enriched lactic acid bacteria to reduce mercury accumulation in fish. It is achieved through the following steps:

[0033] I. Preparation of Bio-nano Selenium Powder:

[0034] The nano-selenium-enriched lactic acid bacteria were inoculated into MRS medium for activation culture to obtain nano-selenium-enriched lactic acid bacteria liquid. Then, the nano-selenium-enriched lactic acid bacteria liquid and Na2SeO3 solution were added to MRS medium for fermentation culture. After the fermentation culture, the product was centrifuged, washed and dried to obtain biological nano-selenium dry powder.

[0035] II. Preparation of selenium-enriched feed for fish:

[0036] The above-mentioned bio-nano selenium dry powder is suspended in distilled water to obtain selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred evenly. After drying, the fish selenium-enriched feed is obtained.

[0037] Third, from the fry stage to the market fish stage, farmed fish should be fed selenium-enriched feed, with a daily feeding amount of 2-4% of the fish's body weight. This will reduce mercury accumulation in the fish by utilizing fish-derived nano-selenium-enriched lactic acid bacteria.

[0038] The selenium-enriched fish feed obtained in step two contains 0.5 mg / kg of selenium and 10 mg / kg of nano-selenium-enriched lactic acid bacteria. 5 cfu / kg.

[0039] In step three of this implementation method, the daily feeding amount should be adjusted reasonably according to the actual situation such as weather, water temperature, fish size, and feeding intensity.

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

[0041] The selenium-enriched fish feed obtained in step two of this embodiment is sampled and tested for selenium and bacterial content. Qualified products are then stored at 4°C.

[0042] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the nano-selenium-enriched lactic acid bacteria mentioned in step one is *Lactobacillus plantarum* L1, which has been deposited at the China Center for Type Culture Collection (CCTCCNO: M 20242780) on December 10, 2024, at Wuhan University, Wuhan, China. Its classification is *Lactobacillus plantarum* HRFRI-L1. Other steps and parameters are the same as in Specific Implementation Method One.

[0043] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One in that the activation culture in step one is: the culture is carried out at 37°C on a shaker for 16 hours at a shaker speed of 150 rpm. Other steps and parameters are the same as in Specific Implementation Method One.

[0044] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method One in that the bacterial count in the nano-selenium-enriched lactic acid bacteria solution in step one is 1×10⁻⁶. 4 CFU / ml. Other steps and parameters are the same as in Specific Implementation Method 1.

[0045] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method One in that the concentration of the Na₂SeO₃ solution in step one is 500–2000 μg / ml. Other steps and parameters are the same as in Specific Implementation Method One.

[0046] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method One in that the amount of nano-selenium-enriched lactic acid bacteria solution used in step one 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 in Specific Implementation Method One.

[0047] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method One in that the fermentation culture in step one is carried out at 37°C with shaking for 24 hours at a shaking speed of 150 rpm. Other steps and parameters are the same as in Specific Implementation Method One.

[0048] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method One in that, in step one, the centrifugation, washing, and drying are performed as follows: After the fermentation culture is completed, the product is centrifuged at 3000 rpm for 10 min, the supernatant is discarded, and then 10 ml of distilled water is added. The mixture is then centrifuged three times repeatedly to remove selenium from the liquid. The precipitate is then dried in a 37°C oven. Other steps and parameters are the same as in Specific Implementation Method One.

[0049] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method One in that the nutritional requirements for the commercial fish feed described in step two 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 in Specific Implementation Method One.

[0050] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method One in that the drying temperature in step two is 37°C. Other steps and parameters are the same as in Specific Implementation Method One.

[0051] Specific Implementation Method Eleven: This implementation method differs from Specific Implementation Method One in that the daily feeding amount in step three is 3% of the fish's body weight. Other steps and parameters are the same as in Specific Implementation Method One.

[0052] The beneficial effects of the present invention are verified through the following embodiments:

[0053] Example:

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

[0055] I. Preparation of Bio-nano Selenium Powder:

[0056] The nano-selenium-enriched lactic acid bacteria were inoculated into MRS medium for activation culture to obtain nano-selenium-enriched lactic acid bacteria liquid. Then, the nano-selenium-enriched lactic acid bacteria liquid and Na2SeO3 solution were added to MRS medium for fermentation culture. After the fermentation culture, the product was centrifuged, washed and dried to obtain biological nano-selenium dry powder.

[0057] II. Preparation of selenium-enriched feed for fish:

[0058] The above-mentioned bio-nano selenium dry powder is suspended in distilled water to obtain selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred evenly. After drying, the fish selenium-enriched feed is obtained.

[0059] Third, from the fry stage to the marketable fish stage, the above-mentioned fish should be fed selenium-enriched feed at a daily rate of 3% of the fish's body weight, which will reduce the accumulation of mercury in the fish by utilizing fish-derived nano-selenium-enriched lactic acid bacteria.

[0060] The selenium-enriched fish feed obtained in step two contains 0.5 mg / kg of selenium and 10 mg / kg of nano-selenium-enriched lactic acid bacteria. 5 cfu / kg;

[0061] The nano-selenium-enriched lactic acid bacteria mentioned in step one is Lactobacillus plantarum L1 isolated from the intestines of healthy fish and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20242780, deposit date December 10, 2024, and deposit address Wuhan University, Wuhan, China. Its name is Lactobacillus plantarum HRFRI-L1.

[0062] The activation culture described in step one: cultured in a shaker at 37°C for 16 hours at a speed of 150 rpm;

[0063] The bacterial count in the nano-selenium-enriched lactic acid bacteria solution mentioned in step one is 1×10⁻⁶. 4 CFU / ml;

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

[0065] In step one, the amount of nano-selenium-enriched lactic acid bacteria solution used is 50 μl, the amount of Na2SeO3 solution used is 50 μl, and the amount of MRS culture medium used is 10 mL.

[0066] The fermentation culture described in step one: cultured on a shaker at 37°C for 24 hours with a shaking speed of 150 rpm;

[0067] Centrifugation, washing and drying as described in step one: After the fermentation culture is completed, the product obtained is centrifuged at 3000 rpm for 10 min, the supernatant is discarded, then 10 ml of distilled water is added, and the mixture is centrifuged three times to remove selenium from the liquid. The precipitate is then dried in an oven at 37℃.

[0068] The nutritional requirements for commercial fish feed described in step two 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 two is 37°C.

[0070] After the fermentation culture in step one of this embodiment is completed, the color change of Lactobacillus plantarum L1 is observed, such as... Figure 1 As shown, a large amount of red selenium nanoparticles precipitate out at this time.

[0071] The bio-nano selenium powder obtained in step one of this embodiment, analyzed by atomic fluorescence spectrometry, contains approximately 862.236 μg / L (diluted 10000) selenium on *Lactobacillus plantarum* L1. Figure 2 and Figure 3 As can be seen, there are nanoparticles on the surface of Lactobacillus plantarum L1, with a size of approximately 42.9 nm.

[0072] In this embodiment, the selenium-enriched fish feed A prepared under laboratory conditions was used to feed carp for 30 days to observe their growth and mercury levels. Mercury was added to the water to achieve a final concentration of 0.03 mg / L, with half the water replaced daily and the corresponding amount of mercury added. After 30 days, the fish were dissected, and the mercury content in the muscle was measured. The control group (water free of mercury and fed commercial fish feed) had a mercury content of 0.053 mg / kg in muscle, the mercury group (water containing mercury and fed commercial fish feed) had 0.58 mg / kg, and the selenium-enriched lactic acid bacteria plus mercury group (water containing mercury and fed selenium-enriched fish feed A) had 0.22 mg / kg. During the one-month feeding period, feeding with feed supplemented with selenium-enriched lactic acid bacteria significantly reduced the mercury content in the muscle. During this period, we also fed carp separately with feed supplemented with selenium-enriched lactic acid bacteria for 30 days, then added the same mercury concentration daily for 30 days, and measured the mercury concentration in the muscle afterward. Adding selenium-enriched lactic acid bacteria to the feed reduced the mercury content in the muscle of fish in mercury-polluted water by 75.82% and reduced the mercury content in the visceral mass of fish by 81.87%. Feeding fish with selenium-enriched lactic acid bacteria for one month, followed by the addition of mercury to the water, resulted in a mercury concentration of 0.057 mg / kg in the fish muscle and 1.62 mg / kg in the visceral mass. This feeding method reduced mercury levels in fish muscle by 93.74% and in the visceral mass by 92.40% (Table 1). The results indicate that feeding selenium-enriched lactic acid bacteria significantly reduces mercury accumulation in fish. Furthermore, feeding fish with selenium-enriched lactic acid bacteria first is more effective than feeding them with both selenium-enriched lactic acid bacteria and mercury simultaneously, significantly reducing mercury accumulation in fish and protecting aquatic organisms from mercury harm, thereby promoting the development of aquaculture.

[0073] Table 1. Mercury accumulation in fish

[0074]

[0075] Note: Different letters indicate significant differences between different groups within the same organization, P<0.05.

[0076] The above description is merely 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for reducing mercury accumulation in fish using fish-derived nano-selenium-enriched lactic acid bacteria, characterized in that... It is implemented in the following steps: I. Preparation of Bio-nano Selenium Powder: The nano-selenium-enriched lactic acid bacteria were inoculated into MRS medium for activation culture to obtain nano-selenium-enriched lactic acid bacteria liquid. Then, the nano-selenium-enriched lactic acid bacteria liquid and Na2SeO3 solution were added to MRS medium for fermentation culture. After the fermentation culture, the product was centrifuged, washed and dried to obtain biological nano-selenium dry powder. II. Preparation of selenium-enriched feed for fish: The above-mentioned bio-nano selenium dry powder is suspended in distilled water to obtain selenium-enriched bacterial solution, which is then evenly sprayed into commercial fish feed and stirred evenly. After drying, the fish selenium-enriched feed is obtained. Third, from the fry stage to the market fish stage, the above-mentioned fish should be fed selenium-enriched feed, with a daily feeding amount of 2-4% of the fish's body weight, which will reduce the accumulation of mercury in the fish by utilizing fish-derived nano-selenium-enriched lactic acid bacteria. The selenium-enriched fish feed obtained in step two contains 0.5 mg / kg of selenium and 10 mg / kg of nano-selenium-enriched lactic acid bacteria. 5 cfu / kg; The nano-selenium-enriched lactic acid bacteria mentioned in step one is *Lactobacillus plantarum* L1, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242780, on December 10, 2024, at Wuhan University, Wuhan, China. Lactobacillus plantarum HRFRI-L1; The activation culture described in step one: cultured in a shaker at 37°C for 16 hours at a speed of 150 rpm; The bacterial count in the nano-selenium-enriched lactic acid bacteria solution mentioned in step one is 1×10⁻⁶. 4 CFU / ml; The concentration of the Na2SeO3 solution mentioned in step one is 500~2000 μg / ml; In step one, the amount of nano-selenium-enriched lactic acid bacteria solution used is 50 μl, the amount of Na2SeO3 solution used is 50 μl, and the amount of MRS culture medium used is 10 mL. The fermentation culture described in step one: cultured on a shaker at 37°C for 24 hours at a shaking speed of 150 rpm; Centrifugation, washing and drying as described in step one: After the fermentation culture is completed, the product obtained is centrifuged at 3000 rpm for 10 min, the supernatant is discarded, then 10 ml of distilled water is added, and the mixture is centrifuged three times to remove selenium from the liquid. The precipitate is then dried in an oven at 37℃. The nutritional requirements for the commercial fish feed described in step two are: crude protein 40%, crude fat 4.0%, crude fiber 8.0%, crude ash 16%, total phosphorus 0.5%, and lysine 1.8%. The drying temperature in step two is 37°C.

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