Efficient slow-release hydrogen microbial fermentation additive as well as preparation method and application thereof

Abstract

The invention provides an efficient slow-release hydrogen microbial fermentation additive as well as a preparation method and application thereof, and belongs to the technical field of microbial fermention.The efficient slow-release hydrogen microbial fermentation additive comprises the following components in parts by weight: 1-10 parts of capsules; 0.001 to 6 parts of a slow-release hydrogen fermentation composition contained in the capsule; the slow-release hydrogen fermentation composition comprises a fermentable carbohydrate, the fermentable carbohydrate is configured to adjust the spatial distribution gradient of the active hydrogen concentration in the material, and the fermentable carbohydrate is 1-30 parts; the slow-release hydrogen fermentation composition further comprises a slow-release hydrogen material, and the slow-release hydrogen material comprises one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium phosphate and monopotassium phosphate. The additive is efficient, slow-released, stable and convenient to use, realizes uniform and directional release of hydrogen in bacterial liquid, and promotes growth and development of microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture and fermentation, and particularly relates to a highly efficient slow-release hydrogen microbial fermentation additive, its preparation method, and its application. Background Technology

[0002] Microorganisms are among the oldest life forms on Earth, forming the cornerstone of the biosphere. They not only reveal the mysteries of the origin of life but also provide crucial technological support for medicine, agriculture, and energy development. Hydrogen (H2), as a novel bioactive gas molecule, exhibits multiple biological effects in the fields of soil microbiology and plant physiological regulation. Hydrogen treatment can significantly activate the bio-driven efficiency of soil organic matter transformation and nutrient cycling. Simultaneously, exogenous hydrogen treatment can significantly promote seed germination index. Faisal Li et al. (2021) in *PLANT SCIENCES*, Zhiying et al. (2018) in *MICROBIOLOGY*, and Yang Haiyan et al. (2024) in *Science & Technology Review* reported that hydrogen treatment can significantly affect soil rhizosphere microorganisms, increasing soil microbial diversity. Furthermore, hydrogen can regulate soil carbon, nitrogen, and phosphorus cycles, and significantly increase seed emergence rate and growth potential. Clearly, soil microbial diversity after hydrogen treatment is of great significance to ecosystems and plant growth; however, its application in microbial fermentation has not yet been reported.

[0003] Although hydrogen (H2), as an emerging green functional molecule, has shown multi-dimensional application potential in agricultural efficiency enhancement, environmental remediation, and clean energy, its high volatility and difficulty in storage present numerous challenges in practical applications. Furthermore, the application of hydrogen in microbial fermentation still requires further research. Summary of the Invention

[0004] In view of this, the present invention provides a highly efficient slow-release hydrogen microbial fermentation additive, its preparation method and application, to solve the problem of hydrogen being difficult to store, and can be applied in the field of microbial fermentation to improve hydrogen utilization efficiency and promote microbial growth and development.

[0005] A first aspect of the present invention provides a highly efficient slow-release hydrogen microbial fermentation additive, comprising, by weight: 1-10 capsules; 0.001-6 parts of the sustained-release hydrogen fermentation composition contained in the capsule; The slow-release hydrogen fermentation composition comprises fermentable carbohydrates configured to regulate the spatial distribution gradient of active hydrogen concentration in the material, wherein the fermentable carbohydrates are 1-30 parts. The slow-release hydrogen fermentation composition further includes a slow-release hydrogen material, which includes one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0006] In one embodiment of the present invention, the fermentable carbohydrate includes one or more of maltose, lactose, sucrose, trehalose, maltodextrin, starch, soluble starch, mannose, glucose, xylose, galactose, raffinose, and fructose.

[0007] In one embodiment of the present invention, the mass ratio of the slow-release hydrogen material to the fermentable carbohydrate in the slow-release hydrogen fermentation composition is 1:(10-100).

[0008] In one embodiment of the present invention, the capsule includes one or more of gelatin capsules, hydroxypropyl methylcellulose capsules, pullulan capsules, and enteric-coated capsules.

[0009] In one embodiment of the present invention, the capsule structure includes at least one of a hard capsule, a soft capsule, a sustained-release capsule, and a targeted capsule.

[0010] In one embodiment of the present invention, the highly efficient slow-release hydrogen microbial fermentation additive is one or more of the following shapes: strip, sphere, and pellet.

[0011] Secondly, the present invention also provides a method for preparing a highly efficient slow-release hydrogen microbial fermentation additive, comprising the following steps: The raw materials provided include 1-10 parts of capsules, 0.001-6 parts of a slow-release hydrogen fermentation composition, wherein the slow-release hydrogen fermentation composition contains fermentable carbohydrates configured to adjust the spatial distribution gradient of active hydrogen concentration in the material, wherein the fermentable carbohydrates are 1-30 parts, and the slow-release hydrogen fermentation composition further includes a slow-release hydrogen material, wherein the slow-release hydrogen material includes one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; The sustained-release hydrogen material is mixed with the fermentable carbohydrate to obtain a sustained-release hydrogen mixture, and the sustained-release hydrogen mixture is filled into the capsule; The capsule containing the slow-release hydrogen mixture is subjected to a molding process to obtain the high-efficiency slow-release hydrogen microbial fermentation additive.

[0012] In one embodiment of the present invention, the slow-release hydrogen material is mixed with the fermentable carbohydrate to obtain the slow-release hydrogen mixture, comprising: The slow-release hydrogen material is mixed with the fermentable carbohydrate at a mass ratio of 1:(10-100).

[0013] The mixture of the slow-release hydrogen material and fermentable carbohydrates was mixed using a high-shear mixer at a speed of 15,000 rpm, with a three-stage stator, and a processing time of 40 min.

[0014] In one embodiment of the present invention, the preparation method further includes sterilizing the formed capsules by using γ-irradiation with a minimum sterilization dose of 28 (kGy), a maximum tolerated dose of 50 (kGy), and a dose uniformity requirement of DUR≤1.8.

[0015] Thirdly, the present invention also discloses the application of a high-efficiency slow-release hydrogen microbial fermentation additive in microbial fermentation, food fermentation, trace magnesium elements in fermentation culture media or agricultural planting, and agricultural remediation, wherein the high-efficiency slow-release hydrogen microbial fermentation additive is the high-efficiency slow-release hydrogen microbial fermentation additive of the first aspect.

[0016] The present invention has the following advantages and beneficial effects: The highly efficient slow-release hydrogen microbial fermentation additive provided by this invention achieves precise and targeted hydrogen release by uniformly compounding slow-release hydrogen materials with fermentable carbohydrates and using microencapsulation technology to achieve spatial isolation of functional components. This facilitates hydrogen release, prolongs the hydrogen release time, and ensures the uniform and directional release of the slow-release hydrogen materials in the bacterial culture. Simultaneously, the fermentable carbohydrates are released in a gradient to provide a continuous energy supply for microbial growth, thereby effectively improving microbial growth and development as well as the accumulation of metabolites. Attached Figure Description

[0017] Figure 1 The following are charts showing the OD600 and pH measurement results of Bacillus subtilis fermentation broth in Examples 1-5 and Comparative Example 1 of this invention; Figure 2 The charts show the colony count results corresponding to Examples 1-5 and Comparative Example 1 of this invention; Figure 3 This is a bar graph showing the changes in Bacillus subtilis concentration in Examples 1-5 and Comparative Example 1 of the present invention. Figure 4 The following are charts showing the OD600 and pH measurement results of Bacillus subtilis fermentation broth in Examples 1, 6-8 and Comparative Example 1 of this invention; Figure 5 The graphs show the colony count results corresponding to Examples 1, 6-8 and Comparative Example 1 of this invention. Figure 6This is a bar graph showing the changes in Bacillus subtilis concentration in Examples 1, 6-8, and Comparative Example 1 of the present invention. Figure 7 The growth curves of Bacillus subtilis fermentation broth after 16 hours are shown for Example 6 and Comparative Example 1 of this invention. Detailed Implementation

[0018] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0019] To facilitate understanding of the technical solutions of the embodiments of this application, the technical problems to be solved by the embodiments of this application will be explained first.

[0020] As mentioned above, hydrogen is highly volatile and difficult to store, presenting numerous challenges in practical applications. For instance, slow-release hydrogen materials can achieve significant bioactivity under low-dose conditions, but how to uniformly mix them into the culture medium remains a challenge. Previous studies have commonly used magnesium sulfate as a microbial fermentation additive. For example, patent application CN 115466765 A describes a dual innovation of "magnesium sulfate additive + feeding process" that significantly improves the efficiency of Bacillus subtilis D by optimizing the fermentation medium (adding magnesium sulfate) and significantly improving the feeding process, providing a feasible solution to the aflatoxin contamination problem. However, the application of hydrogen in the field of microbial fermentation is still in its infancy, and how to utilize hydrogen, which is difficult to store, in microbial fermentation is an urgent problem to be solved.

[0021] To address the aforementioned technical problems, this invention provides a highly efficient slow-release hydrogen microbial fermentation additive. This additive involves uniformly compounding a slow-release hydrogen material with fermentable carbohydrates and employing microencapsulation technology to achieve spatial isolation of the functional components. This enables precise, targeted hydrogen release, facilitating hydrogen release and extending the release time. It also ensures the uniform and directional release of the slow-release hydrogen material within the bacterial culture, while the simultaneous gradient release of fermentable carbohydrates provides a continuous energy supply for microbial growth, effectively enhancing microbial growth, development, and the accumulation of metabolites.

[0022] The technical solution of the high-efficiency slow-release hydrogen microbial fermentation additive of the present invention will be described below with reference to specific embodiments.

[0023] The highly efficient slow-release hydrogen microbial fermentation additive provided in this invention comprises, by weight: 1-10 parts of capsules; and 0.001-6 parts of a slow-release hydrogen fermentation composition contained within the capsules. The slow-release hydrogen fermentation composition contains fermentable carbohydrates configured to regulate the spatial distribution gradient of active hydrogen concentration in the material, and the fermentable carbohydrates comprise 1-30 parts. The slow-release hydrogen fermentation composition also includes a slow-release hydrogen material, which comprises one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0024] The high-efficiency slow-release hydrogen microbial fermentation additive of this invention achieves spatial isolation of functional components by uniformly compounding slow-release hydrogen materials with fermentable carbohydrates and employing microencapsulation technology. This enables precise targeted hydrogen release, facilitating hydrogen release and extending the hydrogen release time. It also ensures uniform and directional release of the slow-release hydrogen materials in the bacterial culture, while the simultaneous gradient release of fermentable carbohydrates provides a continuous energy supply for microbial growth, effectively enhancing microbial growth, development, and metabolic accumulation. Furthermore, the controllable component ratios and better integration of the slow-release hydrogen materials and fermentable carbohydrates result in more stable slow-release capsules, effectively enhancing the slow-release performance of hydrogen.

[0025] In one embodiment of the present invention, the fermentable carbohydrate may include one or more of maltose, lactose, sucrose, trehalose, maltodextrin, starch, soluble starch, mannose, glucose, xylose, galactose, raffinose, and fructose.

[0026] In one embodiment of the present invention, the mass ratio of the slow-release hydrogen material to the fermentable carbohydrate in the slow-release hydrogen fermentation composition is 1:(10-100).

[0027] In one embodiment of the present invention, the capsule includes one or more of gelatin capsules, hydroxypropyl methylcellulose capsules, pullulan capsules, and enteric-coated capsules.

[0028] In one embodiment of the present invention, the capsule structure includes at least one of hard capsules, soft capsules, sustained-release capsules, and targeted capsules. The structure is diverse, and a suitable capsule structure can be selected according to requirements.

[0029] In one embodiment of the present invention, the highly efficient slow-release hydrogen microbial fermentation additive is one or more of the following shapes: strip, sphere, and pellet.

[0030] The high-efficiency slow-release hydrogen microbial fermentation additive of this application embodiment possesses excellent pH regulation capabilities. Its components can be adjusted according to the fermentation time and acid production time of different microorganisms to ensure effective pH regulation during microbial fermentation. This is achieved through the reaction of magnesium hydride with water to produce alkalinity, or by adding acid-base regulators. The slow-release capsules can effectively regulate the pH of the fermentation broth, making it more suitable for microbial growth. Especially in ultra-high-density microbial fermentation environments, it can effectively improve microbial growth and development, significantly increasing microbial cell count and metabolic products. Furthermore, by combining it with food deep processing and food microbial fermentation, its application scope is further expanded, making the slow-release capsules not only suitable for traditional microbial fermentation culture but also widely applicable in food deep processing and ecological environment management.

[0031] Secondly, the present invention also provides a method for preparing a highly efficient slow-release hydrogen microbial fermentation additive, comprising the following steps: The raw materials provided include 1-10 parts of capsules, 0.001-6 parts of a slow-release hydrogen fermentation composition, wherein the slow-release hydrogen fermentation composition contains fermentable carbohydrates configured to adjust the spatial distribution gradient of active hydrogen concentration in the material, wherein the fermentable carbohydrates are 1-30 parts, and the slow-release hydrogen fermentation composition further includes a slow-release hydrogen material, wherein the slow-release hydrogen material includes one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; The sustained-release hydrogen material is mixed with the fermentable carbohydrate to obtain a sustained-release hydrogen mixture, and the sustained-release hydrogen mixture is filled into the capsule; The capsule containing the slow-release hydrogen mixture is subjected to a molding process to obtain the high-efficiency slow-release hydrogen microbial fermentation additive.

[0032] In one embodiment of the present invention, the slow-release hydrogen material is mixed with the fermentable carbohydrate to obtain the slow-release hydrogen mixture, comprising: The slow-release hydrogen material is mixed with the fermentable carbohydrate at a mass ratio of 1:20.

[0033] The mixture of the slow-release hydrogen material and fermentable carbohydrates was mixed using a high-shear mixer at a speed of 15,000 rpm, with a three-stage stator, and a processing time of 40 min.

[0034] In one embodiment of the present invention, the preparation method further includes sterilizing the formed capsules by using γ-irradiation with a minimum sterilization dose of 28 (kGy), a maximum tolerated dose of 50 (kGy), and a dose uniformity requirement of DUR≤1.8.

[0035] The method for preparing a highly efficient slow-release hydrogen microbial fermentation additive according to embodiments of the present invention is simple. Through mixing, filling, coating, and other molding processes, it can generate slow-release capsules that are easy to store, transport, and use, with controllable component ratios, diverse structures, and stability, while effectively enhancing the slow-release performance of hydrogen. Through these processes, the slow-release hydrogen material is combined with fermentable carbohydrates to achieve targeted and time-controlled hydrogen release. This invention improves hydrogen utilization. The sustained-release capsules of this invention are simple and convenient to manufacture, store, transport, and use, and can achieve uniform distribution and sustained release of hydrogen, thereby improving the efficiency of hydrogen absorption by microorganisms, optimizing the microbial fermentation environment, and promoting microbial growth.

[0036] Thirdly, the present invention also discloses the application of a high-efficiency slow-release hydrogen microbial fermentation additive in microbial fermentation, food fermentation, trace magnesium elements in fermentation culture media or agricultural planting, and agricultural remediation, wherein the high-efficiency slow-release hydrogen microbial fermentation additive is the high-efficiency slow-release hydrogen microbial fermentation additive of the first aspect.

[0037] The following specific embodiments further describe the highly efficient slow-release hydrogen microbial fermentation additive and its preparation method according to the present invention.

[0038] In this invention, Bacillus subtilis ( Bacillus subtilis The tested strain was *Bacillus subtilis*, a Gram-positive bacterium widely found in soil and plant roots and stems. Due to its strong environmental adaptability and metabolic diversity, it is widely used in food fermentation, environmental remediation, probiotic preparations, agriculture and biological control, and biopesticides. Adding sustained-release capsules to the culture medium significantly increased the bacterial concentration compared to the conventional medium without these capsules.

[0039] Based on the LB basal medium formula, the following optimized formula was used: 5-30g peptone, 1-10g yeast powder, 1-10g glucose, 1-15g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.2g of sustained-release hydrogen material and 0.16g of soluble starch).

[0040] The culture medium formulation of this invention includes the following components: 10g peptone, 3g yeast powder, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.05g sustained-release hydrogen material and 1g soluble starch). It should be noted that the sustained-release capsules are not sterilized together with the culture medium.

[0041] Preferably, the pH of the culture medium is 5.2 to 6.2, the sterilization temperature of the culture medium is 121°C, the sterilization time is 20 min, and the sustained-release capsule is added 2 h after inoculation.

[0042] Preparation of Bacillus subtilis seed culture: Bacillus subtilis seeds were spread on LB solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing LB liquid medium to obtain the seed culture.

[0043] Example 1 The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.02g magnesium hydride and 0.4g soluble starch).

[0044] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. After inoculation, a slow-release capsule was added, with the capsule placed in the lower layer of the fermentation broth. The flask was placed in a shaker and cultured at 37°C and 180 rpm for 12 h to obtain the Bacillus subtilis fermentation broth.

[0045] The difference between Example 2 and Example 1 lies in the different raw materials used in the samples. The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.05g magnesium hydride and 1g soluble starch).

[0046] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. After inoculation, a slow-release capsule was added, with the capsule placed in the lower layer of the fermentation broth. The flask was placed in a shaker and cultured at 37°C and 180 rpm for 12 h to obtain the Bacillus subtilis fermentation broth.

[0047] The difference between Example 3 and Example 2 lies in the different raw materials used in the samples. The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.06g magnesium hydride and 0.6g soluble starch).

[0048] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. After inoculation, a slow-release capsule was added, with the capsule placed in the lower layer of the fermentation broth. The flask was placed in a shaker and cultured at 37°C and 180 rpm for 12 h to obtain the Bacillus subtilis fermentation broth.

[0049] The difference between Example 4 and Example 3 lies in the different raw materials used in the samples. The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.1g magnesium hydride and 10g soluble starch).

[0050] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. After inoculation, a slow-release capsule was added, with the capsule placed in the lower layer of the fermentation broth. The flask was placed in a shaker and cultured at 37°C and 180 rpm for 12 h to obtain the Bacillus subtilis fermentation broth.

[0051] The difference between Example 5 and Example 4 lies in the different raw materials of the sample. The culture medium components include: 10g peptone, 3g yeast powder, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.2g magnesium hydride and 3g soluble starch).

[0052] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. After inoculation, a slow-release capsule was added, with the capsule placed in the lower layer of the fermentation broth. The flask was placed in a shaker and cultured at 37°C and 180 rpm for 12 h to obtain the Bacillus subtilis fermentation broth.

[0053] Comparative Example 1 The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, and 1000ml distilled water.

[0054] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. The flask was placed in a shaker and cultured at 37°C and 180 rpm for 12 h to obtain Bacillus subtilis fermentation broth.

[0055] Example 6 The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.05g magnesium hydroxide and 1g soluble starch).

[0056] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial suspension. After inoculation, a slow-release capsule was added 2 hours later, with the capsule at the bottom of the fermentation broth. The flask was then placed in a shaker and cultured at 37°C and 180 rpm for 12 hours to obtain the Bacillus subtilis fermentation broth.

[0057] The difference between Example 7 and Example 6 lies in the different raw materials used in the samples. The culture medium consists of: 10g peptone, 3g yeast extract, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.03g magnesium hydroxide and 1g soluble starch).

[0058] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial suspension. After inoculation, a slow-release capsule was added 4 hours later, with the capsule at the bottom of the fermentation broth. The flask was then placed in a shaker and cultured at 37°C and 180 rpm for 12 hours to obtain the Bacillus subtilis fermentation broth.

[0059] The difference between Example 8 and Example 7 lies in the different raw materials of the sample. The culture medium components include: 10g peptone, 3g yeast powder, 3g glucose, 5g sodium chloride, 1000ml distilled water, and sustained-release capsules (0.05g magnesium hydroxide and 1g soluble starch).

[0060] 400 ml of the culture medium was placed in a 1000 ml Erlenmeyer flask, the pH was adjusted to 5.2, and the flask was sterilized at high temperature. Bacillus subtilis was inoculated with 20 ml of bacterial solution. After inoculation, a slow-release capsule was added 6 hours later, with the capsule at the bottom of the fermentation broth. The flask was then placed in a shaker and cultured at 37°C and 180 rpm for 12 hours to obtain the Bacillus subtilis fermentation broth.

[0061] Evaluation Experiment The results of OD600, colony count, and pH measurements of Bacillus subtilis fermentation broth using Examples 1 to 5 and Comparative Example 1 are as follows: Figures 1-3 As shown, when Bacillus subtilis was cultured in Example 2 and Comparative Example 1, the bacterial count in Example 2 was significantly higher than that in Comparative Example 1.

[0062] The results of OD600, colony count, and pH measurements of Bacillus subtilis fermentation broth using Examples 1, 6-8, and Comparative Example 1 are as follows: Figures 4-6 As shown, Bacillus subtilis was cultured in Example 6 and Comparative Example 1. The bacterial count in Example 6 was significantly higher than that in Comparative Example 1. After inoculation, a slow-release capsule was added. By adjusting the ratio of slow-release hydrogen material to fermentable carbohydrates, the amount of each component was adjusted based on the target hydrogen demand, thereby achieving synergistic control of the total amount and rate of hydrogen release.

[0063] like Figure 7 As shown, the growth curve of Bacillus subtilis fermentation broth was determined after 16 hours using Example 6 and Comparative Example 1. Samples were taken every two hours after inoculation, and colony counting results were performed. Figure 7 As shown, in Example 6 and Comparative Example 1, colony counting was performed by continuous sampling for 16 hours. Example 6 showed a significant improvement in both growth rate and bacterial count compared to Comparative Example 1.

[0064] In summary, the highly efficient slow-release hydrogen microbial fermentation additive developed in this invention exhibits significant advantages in the field of microbial culture. Comparative experiments revealed that Bacillus subtilis cultured using this additive… Bacillus subtilisThe bacterial cell concentration is increased by 3-5 times compared to traditional LB medium. Example 6, which optimizes the hydrogen release rate of the capsule, shows the most outstanding effect. This breakthrough stems from the capsule's unique double-layer encapsulation structure: the core magnesium-based hydrogen storage material continuously releases hydrogen through a controlled hydrolysis reaction, while the outer polyelectrolyte membrane precisely regulates the release rate through a pH-responsive mechanism. This capsule system improves hydrogen energy conversion efficiency and avoids the industry pain point of excessive hydrogen inhibiting microbial growth. The high-efficiency slow-release hydrogen microbial fermentation additive provided by this invention has a high efficiency and stable hydrogen release effect, high hydrogen utilization efficiency, and can promote microbial growth and development. Furthermore, the capsule is convenient to use, store, and transport, making it suitable for large-scale application.

[0065] The above are some specific embodiments of the present invention. It should be noted that for those skilled in the art, several changes, improvements and modifications can be made without departing from the principle of the present invention. These changes, improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A highly efficient slow-release hydrogen microbial fermentation additive, characterized in that, By weight, it includes: 1-10 capsules; 0.001-6 parts of the sustained-release hydrogen fermentation composition contained in the capsule; The slow-release hydrogen fermentation composition comprises fermentable carbohydrates, which are configured to adjust the spatial distribution gradient of active hydrogen concentration in the material, wherein the fermentable carbohydrates are 1-30 parts. The slow-release hydrogen fermentation composition further includes a slow-release hydrogen material, which includes one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

2. The high-efficiency slow-release hydrogen microbial fermentation additive as described in claim 1, characterized in that, The fermentable carbohydrates include one or more of maltose, lactose, sucrose, trehalose, maltodextrin, starch, soluble starch, mannose, glucose, xylose, galactose, raffinose, and fructose.

3. The high-efficiency slow-release hydrogen microbial fermentation additive as described in claim 1 or 2, characterized in that, The mass ratio of the slow-release hydrogen material to the fermentable carbohydrate in the slow-release hydrogen fermentation composition is 1:(10-100).

4. The high-efficiency slow-release hydrogen microbial fermentation additive as described in claim 1 or 2, characterized in that, The capsules include one or more of gelatin capsules, hydroxypropyl methylcellulose capsules, pullulan capsules, and enteric-coated capsules.

5. The high-efficiency slow-release hydrogen microbial fermentation additive as described in claim 1 or 2, characterized in that, The capsule structure includes at least one of hard capsules, soft capsules, sustained-release capsules, and targeted capsules.

6. The high-efficiency slow-release hydrogen microbial fermentation additive as described in claim 1 or 2, characterized in that, The highly efficient slow-release hydrogen microbial fermentation additive is one or more of the following shapes: strip, sphere, and pellet.

7. A method for preparing a highly efficient slow-release hydrogen microbial fermentation additive, characterized in that, Includes the following steps: The raw materials provided include 1-10 parts of capsules, 0.001-6 parts of a slow-release hydrogen fermentation composition, wherein the slow-release hydrogen fermentation composition contains fermentable carbohydrates configured to adjust the spatial distribution gradient of active hydrogen concentration in the material, wherein the fermentable carbohydrates are 1-30 parts, and the slow-release hydrogen fermentation composition further includes a slow-release hydrogen material, wherein the slow-release hydrogen material includes one or more of diammonium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium hydroxide, magnesium hydride, diammonium hydrogen citrate, disodium hydrogen sulfate, sodium hydroxide, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; The sustained-release hydrogen material is mixed with the fermentable carbohydrate to obtain a sustained-release hydrogen mixture, and the sustained-release hydrogen mixture is filled into the capsule; The capsule containing the slow-release hydrogen mixture is subjected to a molding process to obtain the high-efficiency slow-release hydrogen microbial fermentation additive.

8. The preparation method according to claim 7, characterized in that, The slow-release hydrogen material is mixed with the fermentable carbohydrate to obtain the slow-release hydrogen mixture, comprising: The slow-release hydrogen material is mixed with the fermentable carbohydrate at a mass ratio of 1:(10-100); The mixture of the slow-release hydrogen material and fermentable carbohydrates was mixed using a high-shear mixer at a speed of 15,000 rpm, with a three-stage stator, and a processing time of 40 min.

9. The preparation method according to claim 7, characterized in that, It also includes sterilizing the formed capsules using a minimum sterilization dose of 28 kGy, a maximum tolerated dose of 50 kGy, and a dose uniformity requirement of DUR≤1.

8.

10. The application of a highly efficient slow-release hydrogen microbial fermentation additive in microbial fermentation, food fermentation, trace magnesium elements in fermentation culture media, or agricultural planting and agricultural remediation, wherein, The high-efficiency slow-release hydrogen microbial fermentation additive is the high-efficiency slow-release hydrogen microbial fermentation additive according to claims 1-6.

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