Selenium-enriched culture method and application of a strain of animal Bifidobacterium

Abstract

The present invention relates to a selenium-enriched culture method and application of animal bifidobacterium. The selenium-enriched culture method comprises the following steps: reviving the animal bifidobacterium; activating the animal bifidobacterium through a CM0231 solid culture medium; inoculating the animal bifidobacterium into a CM0231 liquid culture medium with a pH of 7.5 after activation, and culturing the same through shaking to obtain a seed solution; inoculating the seed solution into a CM0231 liquid culture medium with a pH of 9, and culturing the same through shaking to obtain a logarithmic phase bacterial solution; and adding sodium selenite to the bacterial solution to obtain a selenium content of 100 mg·L ‑1 The animal bifidobacterium of the present invention can be used to prepare selenium-supplemented food, medicine or feed to meet the selenium needs of humans and animals.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an efficient selenium-enriched culture method and application of animal bifidobacteria capable of converting sodium selenite into red nano-selenium with high bioavailability. Background Art

[0002] Selenium has been recognized by the United Nations FAO / IAEA / WHO as an essential trace element for the human body. It is crucial for the normal development of animals and humans and has physiological functions such as antioxidant and immune-enhancing properties. Selenium (Se) was discovered by Swedish chemist Jakob Berzelius and was initially considered a toxic element. However, continued research into the various physiological reactions involved in selenium has led to its recognition as an essential dietary supplement. The World Health Organization recommends a daily intake of 40 to 400 μg of selenium. Studies have shown that within safe limits, selenium not only exhibits antioxidant, anti-aging, cell protection, and repair activities, but also enhances the immune system and detoxification functions. However, outside these safe limits, selenium deficiency can lead to Keshan disease and Kashin-Beck disease, while excessive selenium intake can lead to selenium poisoning. Currently, most selenium supplementation methods use an inorganic selenium compound, sodium selenite, in the form of an oral formulation. However, sodium selenite has significant toxic side effects. Research has shown that inorganic selenium undergoes biotransformation to form organic selenides. Organic and nano-selenium are less toxic, more readily absorbed and safely utilized by plants and animals, and significantly more effectively stimulate immune responses than inorganic selenium. These results demonstrate significant potential for promoting and addressing selenium deficiency in my country. While organic selenium and nano-selenium can be synthesized through physical and chemical methods, their high production costs and low yields limit their application. Consequently, considerable research has focused on their biological absorption and conversion. The use of microorganisms to convert inorganic selenium into highly nutritious organic and nano-selenium forms has become a growing area of research.

[0003] Using probiotics and other microorganisms as carriers of selenium supplementation simultaneously provides both health benefits and selenium supplementation. Most selenium supplements use inorganic selenium (such as sodium selenite), which inhibits microbial growth. Excessive consumption of unused inorganic salts can also be harmful. Probiotic fermentation converts inorganic selenium into organic selenium, which is then absorbed and utilized by the body, ensuring safe selenium supplementation.

[0004] Probiotics are the main research objects of selenium-enriched microorganisms, including selenium-enriched yeast, selenium-enriched lactic acid bacteria and selenium-enriched bifidobacteria. Selenium-enriched probiotics have multiple benefits, such as antioxidant, anti-pathogenic, anti-mutagenic, anti-cancer and anti-inflammatory activities. Selenium-enriched probiotics used in food can not only provide a gastrointestinal protective barrier, but also change the pH of the intestinal environment to provide the proliferation of non-pathogenic bacteria, thereby enhancing the host's immune response ability, improving antioxidant capacity, producing antibacterial substances to compete with them in the recipient's intestine, and directly providing a rich source of organic selenium (mainly selenoproteins). First, the biotransformation ability of probiotics for selenium provides a cheap source of organic selenium required for human and animal nutrition. Secondly, after long-term survival in the intestine, probiotics can accumulate selenium from the diet, thereby affecting the host's selenium intake and then changing the expression of several selenoproteins in the host. Therefore, the human body's need for selenium can be met from the perspectives of health preservation and nutritional element supplementation. However, the existing selenium-enriched yeast, selenium-enriched lactic acid bacteria and selenium-enriched bifidobacteria have low selenium conversion rates. Summary of the Invention

[0005] Technical issues to be solved:

[0006] The present invention proposes a method for efficiently culturing animal bifidobacteria with selenium enrichment and its application, aiming to provide a method for culturing animal bifidobacteria with selenium enrichment and high selenium enrichment conversion rate. The cultured animal bifidobacteria can efficiently convert sodium selenite into nano-selenium. The bacteria can convert inorganic sodium selenite into red nano-selenium with low toxicity and high bioavailability.

[0007] Technical solution:

[0008] The technical solutions of the present invention are as follows:

[0009] The first aspect of the present invention provides a method for cultivating animal Bifidobacterium with selenium-enriched properties, the specific steps of the method are:

[0010] a. The animal Bifidobacterium was revived and then transferred to CM0231 solid medium and cultured at 37°C for 48h;

[0011] b. The bacterial solution obtained in step a was transferred to 2-3 generations of activation through CM0231 solid medium;

[0012] c. The animal Bifidobacterium obtained in step b was inoculated into CM0231 liquid medium at pH 7.5 and cultured with shaking at 37 ° C and 160 rpm for 12 h to obtain a seed solution;

[0013] d. The seed solution obtained in step c was inoculated into CM0231 liquid medium at pH 9 at a 3% (V / V) inoculum size and cultured in a shaker at 35°C and 160 rpm for 9 h to obtain a logarithmic phase bacterial solution;

[0014] e. Sodium selenite was added to the bacterial solution obtained in step d to make the selenium content of the bacterial solution 100 mg·L -1 , culture under shaking conditions of 35°C and 160rpm for 24h, and observe that the bacterial solution turns red, thus obtaining selenium-enriched animal Bifidobacterium.

[0015] Optionally, the components of CM0231 solid culture medium per liter are: peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 5.0 g, K2HPO4 0.45 g, KH2PO4 0.33 g, NH4Cl 1.0 g, MgSO4 7H2O 0.1 g, L-cysteine 0.5 g, resazurin 0.001 g, Na2S 9H2O 0.5 g, and agar 15 g.

[0016] Optionally, the components of CM0231 liquid culture medium per liter are: peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 5.0 g, K2HPO4 0.45 g, KH2PO4 0.33 g, NH4Cl 1.0 g, MgSO4 7H2O 0.1 g, L-cysteine 0.5 g, resazurin 0.001 g, and Na2S 9H2O 0.5 g.

[0017] The second aspect of the present invention provides the use of animal Bifidobacterium cultured by the culture method in the preparation of selenium-supplemented food, medicine or feed.

[0018] Optionally, the selenium-supplemented food, medicine or feed comprises a live strain or a dried strain of Bifidobacterium animalis.

[0019] Optionally, the selenium-supplementing food or medicine includes a pharmaceutically acceptable carrier, and the carrier is a tablet, capsule, oral solution or lyophilized powder.

[0020] Beneficial effects:

[0021] The present invention develops a method for selenium enrichment of animal Bifidobacterium. The method utilizes animal Bifidobacterium as a selenium supplementation carrier to enhance its selenium enrichment capacity, ultimately yielding a healthy, highly nutritious, selenium-enriched animal Bifidobacterium. After culturing in an incubator at 35°C and shaking at 160 rpm for 9 hours, sodium selenite is added and cultured under these conditions for 24 hours, resulting in a sodium selenite conversion rate of 95.59% for the animal Bifidobacterium. The animal Bifidobacterium of the present invention can be used in foods and medicines for selenium supplementation in humans, or in animal feed, thereby ensuring the selenium needs of humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a process flow for cultivating selenium-enriched animal Bifidobacterium;

[0023] Figure 2 is the selenium standard curve;

[0024] Figure 3 The concentration and conversion efficiency of unconverted selenium in the supernatant after 24 h of conversion of Na2SeO3 by animal Bifidobacterium in CM0231 medium at different initial pH;

[0025] Figure 4 It is the concentration and conversion efficiency of unconverted selenium in the supernatant after animal Bifidobacterium converts Na2SeO3 for 24 hours under different temperature culture conditions. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0027] Example 1: Figure 1 As shown, a strain of Bifidobacterium animalis was identified as the dominant selenium-rich probiotic.

[0028] A strain of Bifidobacterium animalis capable of converting sodium selenite was purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC 6165 and a platform resource number of 1511C0005000004655. This strain is a predominantly selenium-rich probiotic. The strain can be further acclimated and cultured to produce selenium-enriched Bifidobacterium animalis.

[0029] The specific steps of the selenium-enriched culture method of animal Bifidobacterium are:

[0030] (1) Revitalization of bacterial strains: Take a vial of animal Bifidobacterium stored at -20°C, wipe the surface of the vial with 75% alcohol cotton for disinfection, use a sterile pipette to draw about 0.5 ml of CM0231 liquid culture medium into the vial to dissolve all the lyophilized powder, transfer the dissolved bacterial suspension to a test tube containing 4-5 ml of CM0231 liquid culture medium and mix well, transfer the 1-2 drops of bacterial suspension remaining in the pipette to CM0231 solid culture medium, and incubate at 37°C for 48 h.

[0031] (2) Activation of bacterial strains: The obtained bacterial suspension and plates were transferred to the 2nd-3rd generation to restore the activity. Then, the animal Bifidobacterium on the solid culture medium plate was inoculated into CM0231 liquid culture medium with pH 7.5, and cultured in a shaking incubator at 37°C and 160 rpm for 12 hours to obtain seed solution.

[0032] (3) Preparation of sodium selenite stock solution: First, weigh 2.19 g of sodium selenite and dissolve it in 100 mL of deionized water to prepare 10 g·L -1Selenium stock solution.

[0033] (4) Conversion Verification: The seed solution was inoculated at a 3% (V / V) inoculum into an anaerobic bottle containing CM0231 liquid culture medium at pH 9, and cultured in a shaker at 37°C and 160 rpm for 9 h. After that, a sterile sodium selenite stock solution (prepared with a sterile 0.22 μm nylon membrane using a sterile syringe) was added, and cultured in a shaker at 37°C and 160 rpm for 24 h. Since the sodium selenite solution is colorless and the nano-selenium after bioconversion is red, the color change from colorless to red can be observed before and after the bacterial suspension is cultured.

[0034] Example 2: Selenium-enriched culture of animal Bifidobacterium converting sodium selenite into nano-selenium

[0035] (1) Revitalization of bacterial strains: Take a vial of animal Bifidobacterium stored at -20°C, wipe the surface of the vial with 75% alcohol cotton for disinfection, use a sterile pipette to draw about 0.5 ml of CM0231 liquid culture medium into the vial to dissolve all the lyophilized powder, transfer the dissolved bacterial suspension to a test tube containing 4-5 ml of CM0231 liquid culture medium and mix well, transfer the 1-2 drops of bacterial suspension remaining in the pipette to CM0231 solid culture medium, and incubate at 37°C for 48 h.

[0036] (2) Activation of bacterial strains: The obtained bacterial suspension and plates were transferred to the 2nd-3rd generation to restore the activity. Then, the animal Bifidobacterium on the solid culture medium plate was inoculated into CM0231 liquid culture medium with pH 7.5, and cultured in a shaking incubator at 37°C and 160 rpm for 12 hours to obtain seed solution.

[0037] (3) Preparation of sodium selenite stock solution: First, weigh 2.19 g of sodium selenite and dissolve it in 100 mL of deionized water to prepare a 10 g·L-1 selenium-containing stock solution.

[0038] (4) Preparation of Selenium-Enriched Animal Bifidobacterium: The seed liquid was inoculated into an anaerobic bottle containing CM0231 liquid culture medium at pH 9 at a rate of 3% (V / V), and cultured in an anaerobic flask at 35°C with shaking at 160 rpm for 9 h. After that, a sterile sodium selenite stock solution (prepared with a sterile 0.22 μm nylon membrane using a sterile syringe) was added, and cultured in an anaerobic flask at 35°C with shaking at 160 rpm for 24 h. Since the sodium selenite solution is colorless and the nano-selenium after bioconversion is red, the color of the bacterial suspension can be observed to change from colorless to red before and after culture.

[0039] (5) Conversion rate of animal Bifidobacterium to sodium selenite

[0040] Draw a selenium standard curve: Take 1000 mg·L selenium -11 mL of the standard solution was diluted to 100 mL with 10% dilute hydrochloric acid to obtain 10 mg·L -1 Selenium standard intermediate solution was further diluted with 10% dilute hydrochloric acid to 1, 2, 5, 10, and 20 μg·L -1 Different concentration gradients. Detection was performed using a hydrogenation atomic fluorescence spectrometer, with the instrument's reducing solution being a 15% potassium borohydride aqueous solution. A standard curve was drawn with the concentration gradient as the horizontal axis and the fluorescence intensity as the vertical axis, as shown in the following example: Figure 2 shown.

[0041] The red bacterial suspension after biotransformation was centrifuged, and the supernatant was diluted to a certain multiple and the concentration of untransformed selenium was detected by atomic fluorescence spectrometry. t The reducing liquid of the instrument is 15% potassium borohydride aqueous solution.

[0042]

[0043] In the above formula:

[0044] C0 – initial selenium concentration of sodium selenite converted by Bifidobacterium animalis;

[0045] C t ——The concentration of unconverted selenium in the supernatant after time t.

[0046] Example 3: Biotransformation of Sodium Selenite by Animal Bifidobacterium in CM0231 Liquid Medium at Different Initial pH

[0047] Animal Bifidobacterium was inoculated into CM0231 liquid culture medium and cultured for 12 hours as seed liquid. The biotransformation of sodium selenite by animal Bifidobacterium was investigated under different initial pH conditions of CM0231 liquid culture medium. CM0231 culture medium was divided into 7 groups, with 3 replicates in each group. Before use, 0.1 mol·L -1 HCl and 0.1 mol·L -1 The initial pH value of the CM0231 culture medium was corrected with NaOH solution, and the initial pH values of the CM0231 culture medium in the seven groups were 3, 4, 5, 6, 7, 8, and 9, respectively. 3% of the seed solution was inoculated into CM0231 liquid culture medium with different initial pH values and placed in an incubator at 37°C and 160 rpm for continuous fermentation for 9 h. Subsequently, sterile sodium selenite stock solution was added to make the selenium concentration in the bacterial suspension 100 mg·L -1 , and continued to ferment for 24 hours to obtain the terminal animal Bifidobacterium bacterial solution. The terminal bacterial solution was centrifuged to separate the supernatant, and the concentration of unconverted selenium in the supernatant was detected by atomic fluorescence analyzer, thereby determining the animal Bifidobacterium's ability to convert selenium to 100 mg·L at different pH conditions. -1 Biotransformation of sodium selenite.

[0048] Table 1 Conversion efficiency of Na2SeO3 by animal Bifidobacterium under CM0231 culture medium with different initial pH

[0049]

[0050]

[0051] like Figure 3 As shown, when the initial pH of the CM0231 culture medium was between 3 and 6, the concentration of unconverted selenium in the supernatant after 24 hours of sodium selenite conversion by Bifidobacterium animalis decreased with increasing pH, indicating a better bioconversion of sodium selenite. When the pH was between 6 and 9, the concentration of unconverted selenium in the supernatant increased with increasing pH. Therefore, the conversion rate of sodium selenite by Bifidobacterium animalis was highest at 9, reaching 94.84%.

[0052] Example 4: Biotransformation of Sodium Selenite by Animal Bifidobacterium at Different Temperatures

[0053] Animal Bifidobacterium was inoculated into CM0231 liquid culture medium and cultured for 12 hours. This culture medium served as a seed solution to investigate the conversion of sodium selenite by animal Bifidobacterium at different temperatures. The CM0231 culture medium was divided into five groups, each with three replicates. The seed solution was added to each group of CM0231 liquid culture medium at a 3% inoculum rate. Each group was cultured in an incubator at 20, 25, 30, 35, and 40°C, with shaking at 160 rpm for 9 hours. Sterile sodium selenite stock solution was then added to the suspension to achieve a selenium concentration of 100 mg·L. -1 The culture was allowed to grow for 24 hours to obtain the endpoint bacterial solution of animal bifidobacterium. The endpoint bacterial solution was centrifuged to separate the supernatant, and the concentration of unconverted selenium in the supernatant was measured by atomic fluorescence analyzer to determine the biotransformation of sodium selenite by animal bifidobacterium under different culture temperature conditions.

[0054] Table 2 Conversion efficiency of Na2SeO3 by animal bifidobacteria under different culture temperature conditions

[0055]

[0056] like Figure 4 As shown in the results, animal Bifidobacterium converted Na2SeO3 for 24 h under different culture conditions. When the temperature was 20-35°C, the concentration of unconverted selenium in the supernatant after sodium selenite was converted by animal Bifidobacterium for 24 h decreased with increasing temperature. When the temperature was 35-40°C, the concentration of unconverted selenium in the supernatant increased with increasing temperature. The concentration of unconverted selenium in the supernatant at 35°C was the lowest, which was 4.41 mg·L -1Therefore, the conversion rate of animal Bifidobacterium to sodium selenite was the highest at 35°C, which was 95.59%.

[0057] In summary, sodium selenite was added after shaking culture at 160 rpm at 35° C. for 9 h in an incubator, and culture was continued for 24 h under this condition so that the conversion rate of sodium selenite by animal Bifidobacterium was 95.59%.

[0058] Comparative Example 1

[0059] Xu Zhou et al. (2017) determined the optimal culture conditions for Phaffia rhodozyma by studying the amount of selenium added, incubation time, and incubation volume. They found that Phaffia rhodozyma grows vigorously in PDA medium, and adding selenium solution twice in a single incubation period facilitates the conversion of organic selenium. The optimal culture conditions were: 80 mL / 500 mL incubation volume, 20 mg / L selenium concentration, and 30 h incubation time. Under these conditions, the organic selenium conversion rate reached 63.2%. The conversion rate of sodium selenite by animal Bifidobacterium in Example 4 of the present invention reached 95.59%, exceeding the 51.25% in the comparative example.

[0060] The animal Bifidobacterium strain of the present invention has a clear source, is safe and healthy, and can be used to prepare selenium-supplemented foods, medicines, or feed after selenium-enriched cultivation, thereby ensuring the selenium needs of humans and animals. Specifically, it can be applied to industries such as dairy fermentation, food and beverage production, health product production, and animal feed.

[0061] The above is a detailed description of the embodiments of the present invention, but the contents are not considered to limit the scope of implementation of the present invention. Any modifications and improvements made by those skilled in the art based on the present invention and relying on the spirit of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for cultivating animal bifidobacteria with selenium-enriched properties, characterized in that: The specific steps of this method are: a. Bifidobacterium animalis Bifidobacterium animalis The animal Bifidobacterium was purchased from the China Industrial Microorganism Culture Collection with the collection number CICC 6165. The animal Bifidobacterium was revived and then transferred to CM0231 solid culture medium and cultured at 37°C for 48 hours. b. The bacterial solution obtained in step a was transferred to 2-3 generations of activation through CM0231 solid medium; c. The animal Bifidobacterium obtained in step b was inoculated into a CM0231 liquid medium at pH 7.5 and cultured with shaking at 37 ° C and 160 rpm for 12 h to obtain a seed solution; d. The seed solution obtained in step c was inoculated into CM0231 liquid medium at pH 9 at a 3% (V / V) inoculum size and cultured at 35°C with shaking at 160 rpm for 9 h to obtain a logarithmic phase bacterial solution; e. Add sodium selenite to the bacterial solution obtained in step d to make the selenium content of the bacterial solution 100 mg∙L -1 , cultured under shaking conditions of 35℃ and 160 rpm for 24 h, and the color of the bacterial solution turned red, indicating that selenium-enriched animal Bifidobacterium was obtained.

2. The method for cultivating animal bifidobacteria by selenium enrichment according to claim 1, wherein: The components of CM0231 solid culture medium per liter are: peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 5.0 g, K2HPO4 0.45 g, KH2PO4 0.33 g, NH4Cl 1.0 g, MgSO4 7H2O 0.1 g, L-cysteine 0.5 g, resazurin 0.001 g, Na2S 9H2O 0.5 g, and agar 15 g.

3. The method for cultivating animal bifidobacteria with selenium-enriched properties according to claim 1, wherein: The composition of CM0231 liquid culture medium per liter is: peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 5.0 g, K2HPO4 0.45 g, KH2PO4 0.33 g, NH4Cl 1.0 g, MgSO4 7H2O 0.1 g, L-cysteine 0.5 g, resazurin 0.001 g, and Na2S 9H2O 0.5 g.

4. Use of animal bifidobacteria cultured by the culture method according to claim 1 in preparing selenium-supplemented food, medicine or feed.

5. The use according to claim 4, characterized in that: The selenium-supplemented food, medicine or feed comprises a live strain or a dried strain of animal Bifidobacterium.

6. The use according to claim 4, characterized in that: The selenium-supplementing food and medicine include a pharmaceutically acceptable carrier, and the carrier is a tablet, a capsule, an oral solution or a freeze-dried powder.

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