Process for preparing vitamin B12 through anaerobic fermentation of apple residues

Abstract

The invention discloses a process for preparing vitamin B12 through anaerobic fermentation of apple residues, and belongs to the technical field of biochemistry and fermentation. According to the process for preparing the vitamin B12 through anaerobic fermentation of the apple residues, a production strain of the vitamin B12 is cultured under the optimal fermentation condition through strain growth optimized through a large number of experiments and a culture medium used in the fermentation process; the fermentation process is carried out for 120 hours under a static condition of 30 DEG C, and L-cysteine hydrochloride is added into the culture medium to create an anaerobic condition, so that after the fermentation is finished, the highest concentration of the vitamin B12 obtained in the wet biomass of the propionibacterium can reach 290 mu g / 100g. According to the method disclosed by the invention, the vitamin B12 is prepared from the apple residues and the industrial wastes under anaerobic fermentation, and relatively high production efficiency of the vitamin B12 can be realized only by using the industrial residues without any enzyme or chemical pretreatment and enrichment.

Description

Technical Field

[0001] The present invention relates to the field of fermentation technology, and particularly relates to a process for preparing vitamin B12 by anaerobic fermentation using apple residues. Background Art

[0002] Vitamin B12, also known as cobalamin, is an important metabolite synthesized only by microorganisms and is widely present in animal foods. As a key nutrient, vitamin B12 plays an irreplaceable role in multiple important physiological processes in the human body, including erythropoiesis, DNA and RNA synthesis, and the normal operation of the nervous system. B12 helps maintain genomic stability by participating in the metabolism of folic acid and the conversion of homocysteine, and plays a central role in the formation of myelin and neurotransmitters. Its deficiency may lead to serious health problems such as cognitive impairment, nerve damage, and blood diseases. In recent years, due to changes in the global diet structure, especially the rapid popularity of vegetarianism and plant-based diets, the demand for vitamin B12 in the population has increased significantly. However, since plant-based foods naturally do not contain vitamin B12, this poses a huge nutritional fortification challenge for food producers, and there is an urgent need to find cost-effective sources of vitamin B12 to meet market demand.

[0003] Microbial fermentation is one of the main ways to produce vitamin B12. Propionibacterium has received extensive attention due to its generally recognized as safe status and stable metabolic characteristics. However, the cost of producing vitamin B12 by the microbial method remains high, and the cost of the culture medium accounts for a large proportion of the total cost. To reduce production costs, researchers have begun to explore new ways to use industrial by-products as microbial culture media.

[0004] Industrial by-products (such as fruit residues, potato wastewater, and waste glycerol) are usually rich in organic components such as sugars, amino acids, and trace elements, making them potential low-cost raw materials. However, the traditional treatment methods for these by-products (such as composting, landfilling, or incineration) are not only costly but may also cause environmental pollution. For example, millions of tons of fruit residues are generated each year during juice production, and only a small portion of them is recycled for animal feed or other uses, while most are directly discarded or landfilled, wasting valuable biological resources. Similarly, the large amount of wastewater generated during potato processing, due to its high chemical and biological oxygen demand, will damage the water and soil ecosystems if directly used for irrigation or discharged without proper treatment. And waste glycerol, as a by-product of biodiesel production, contains impurities and pollutants, and the cost of its further purification process is high, resulting in a large amount of waste glycerol being idle or wasted.

[0005] Using industrial by-products as a culture medium for microbial fermentation can not only provide a high-value application route for waste materials, but also significantly reduce production costs, promote resource recycling and environmental protection. For example, by using fruit pomace as a microbial culture medium, Propionibacterium can efficiently produce vitamin B12 under relatively inexpensive conditions. However, due to the complex composition of these by-products, the utilization efficiency of microorganisms is limited to a certain extent, resulting in low production efficiency and becoming the main bottleneck for industrial applications. Summary of the Invention

[0006] The present invention provides a process for preparing vitamin B12 by anaerobic fermentation of apple residues, aiming to develop an economical and efficient microbial culture system based on industrial by-products, which can not only alleviate the problem of tight supply of vitamin B12, but also realize the high-value utilization of waste resources, provide new ideas for the green production of vitamin B12, and at the same time reduce the environmental impact of industrial waste. To achieve the above object, the technical solution adopted by the present invention is as follows: A process for preparing vitamin B12 by anaerobic fermentation of apple residues, comprising the following steps: 1) Under suitable culture conditions, a production strain of vitamin B12 is cultured in a culture medium for propagating Propionibacterium, and the production strain of vitamin B12 is Propionibacterium freudenreichii.

[0007] 2) After the strain grows stably, it is inoculated onto the culture medium and inoculated for 24 hours under static conditions, then the inoculated culture medium is collected, centrifuged, and the obtained biomass is suspended in the experimental culture medium and fermented to produce vitamin B12.

[0008] 3) Vitamin B12 is isolated and purified from the experimental culture medium.

[0009] Further, in step 1, the culture medium for propagating Propionibacterium comprises the following components in mass percentage: 0.1-0.3% glucose, 0.2-0.4% meat extract, 0.8-1.2% peptone, 0.4-0.6% sodium chloride, 0.4-0.6% yeast extract, and 97-98% distilled water.

[0010] Further, before inoculation, the culture medium for propagating Propionibacterium is sterilized in an autoclave at a temperature of 117 °C for 30 minutes.

[0011] Further, the culture medium for propagating Propionibacterium is adjusted to a pH range of 6.5-7.0 with 10% NaOH solution before inoculation.

[0012] Further, the preparation of the experimental culture medium comprises the following steps: 1) Add an appropriate amount of apple residue to distilled water, heat and stir for extraction.

[0013] 2) Remove the residual solids by centrifugation, then sterilize at 121 °C for 30 minutes, and add 0.1 - 0.5 mg / L of plant tricin to obtain the experimental culture medium.

[0014] Further, the experimental culture medium contains the following components by mass percentage: 16.7 - 50% of apple residue and 50 - 83.3% of distilled water.

[0015] Further, the experimental culture medium is stirred and heated at 75 °C for 30 minutes, then the extract is centrifuged to remove the residual solids, and the supernatant is used to prepare the experimental culture medium, and then 0.1 - 0.3 mg / L of plant tricin is added.

[0016] Further, the pH value of the experimental culture medium is adjusted to 7.0 with NaOH before use.

[0017] Further, the preparation process of the plant tricin includes: Obtain a microbial flora, screen to obtain strains, and expand the culture in a specific culture medium to obtain a seed solution; Inoculate the seed solution into a fermentation tank containing a plant extract substrate, and perform triple - MAX triple glycolysis to decompose the substrate in stages, generate intermediate products, and form the basic framework of plant tricin; Extract the fermentation broth, add a specific enzyme preparation for "Multi - Point" enzymatic oligopeptide reaction to modify oligopeptides and polysaccharides, and improve the activity and stability; Transfer the treated product to a biosynthetic system, add coenzyme substances, and perform "Bio - Synthesis biosynthesis" to synthesize mesolignin, high - quality lignin, and glutin; Use methods such as centrifugation, filtration, and chromatography to separate and purify the fermentation product, and obtain a high - purity plant tricin product through crystallization and freeze - drying.

[0018] Further, L - cysteine hydrochloride (0.40 g / L) is added to the experimental culture medium to create an anaerobic condition.

[0019] Further, the fermentation culture is carried out under static conditions in a conical flask containing 250 mL of the experimental culture medium, the culture temperature is 30 °C, the time is 120 hours, and the experimental culture medium is neutralized with 10% NaOH solution every 24 hours to optimize the pH value range of propionic acid bacteria to 6.5 - 7.0.

[0020] The beneficial effects obtained by the above - mentioned present invention are as follows: Practical application and cost - benefit improvement 1. The present invention can provide a suitable culture environment for the production of propionic acid and vitamin B12 by using industrial waste such as apple pomace without additional pretreatment or supplementation of expensive laboratory reagents. This method significantly reduces the production cost. For example, apple pomace contains rich nitrogen and carbon sources, which can replace expensive peptone or yeast extract, enabling microorganisms to utilize lignocellulosic resources for effective metabolism and growth. Due to the low cost and easy availability of industrial waste, this will greatly reduce the raw material cost, thereby improving the overall economic efficiency. Most traditional industrial vitamin B12 production relies on pure carbon and nitrogen sources, such as glucose and yeast extract, which have high costs and are not conducive to large-scale industrial applications. However, using industrial waste as a culture medium can significantly reduce the production cost, making the biological production of vitamin B12 more economically feasible; 2. In the present invention, the industrial by-products of fruit processing are used, which not only reduces environmental pollution but also realizes the recycling of resources. By biotechnologically treating these wastes and converting them into useful metabolites such as vitamin B12, it helps to alleviate the problem of industrial waste accumulation, which is in line with the concept of current and future sustainable development. Especially in the context of the global advocacy of environmental protection and sustainable development, this biological production method using industrial waste has important ecological and economic significance. By converting waste into valuable products, it not only reduces the cost of waste treatment but also lessens the burden on the environment, embodying the concept of circular economy; 3. In the present invention, the industrial waste does not require complex pretreatment, and only its naturally occurring components can provide the nutrients required for bacterial growth, such as carbon sources (sugars and glycerol), nitrogen sources, vitamins, and trace elements. This greatly simplifies the production process, reduces the cost and time investment, and enhances the simplicity and practicality of the process. In addition, through experimental verification, the microbial culture medium prepared from apple pomace can effectively promote the production of vitamin B12. Not only is the process simple, but it can also make full use of the organic matter in the waste to directly provide the necessary nutrients for microbial metabolism. Such a process simplifies the preparation steps of the culture medium, making the entire production process more economical and efficient, with high potential for industrial application and worthy of further development and promotion; 4. The DSM 20271 strain used in the present invention can grow in a culture medium provided with industrial waste and effectively synthesize vitamin B12. Since this strain has a safe status and is widely used in food and animal feed, it can ensure the safety and acceptability of products. This strain is very suitable for microbial fortification of food, especially for the development of new nutritional products for vegetarian food and animal feed. The propionic acid and vitamin B12 produced by this strain can be directly applied in the food industry. In addition, the propionic acid produced by this strain has preservative properties. As a natural preservative, propionic acid is widely used in bread and dairy products, while vitamin B12 is an important additive in animal feed and functional foods. The products produced by using this strain are not only safe and reliable but also meet the needs of modern consumers for natural, safe, and green foods; 5. Although the current chemical production method of propionic acid has a relatively low cost, the production of both propionic acid and vitamin B12 by using biotechnology with industrial waste still shows significant market advantages and economic potential. By using waste to reduce raw material costs, it can gain an advantageous position in market competition and promote the wide application of biotechnology in the production of propionic acid and vitamin B12. This not only helps to reduce production costs but also improves the economic benefits and market competitiveness of enterprises. With the increasingly strict environmental protection regulations and the enhanced environmental awareness of consumers, the green production method will become the trend of industrial development, and this new production method will be more and more favored by enterprises and the market; 6. The method for producing propionic acid and vitamin B12 by using industrial waste apple pomace proposed in the present invention realizes the goal of converting waste into valuable products, and has significant economic benefits and environmental protection advantages. By effectively using industrial waste, it reduces production costs, simplifies the production process, and promotes the recycling of resources. In addition, the propionic acid and vitamin B12 produced in the production process have various application values and can be widely applied in the food industry and animal husbandry, further enhancing the practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present application will become obvious and easy to understand from the following description of the embodiments in conjunction with the drawings, where: Figure 1 It is a comparison of the total sugar content in the culture media with different components in the initial stage (0 h) and the end stage (120 h) of the fermentation process in the embodiments and comparative examples of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The technical solution of the present invention will be described below through specific specific examples. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or other method steps can be inserted between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of the method steps are only convenient tools for identifying the method steps, rather than limiting the arrangement order of the method steps or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope in which the present invention can be implemented.

[0023] To better understand the above technical solution, the exemplary embodiments of the present invention will be described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention can be understood more thoroughly and the scope of the present invention can be fully communicated to those skilled in the art.

[0024] The present invention will be further described below in conjunction with the following embodiments.

[0025] Example 1 The present invention provides a process for preparing vitamin B12 by anaerobic fermentation of apple residues, comprising the following steps: 1) Under suitable culture conditions, a production strain of vitamin B12 is cultured in a medium for propagating propionic acid bacteria, and the production strain of vitamin B12 is propionic acid bacteria.

[0026] 2) After the strain grows stably, it is inoculated onto the medium and inoculated for 24 hours under static conditions, then the inoculated medium is collected, centrifuged, and the obtained biomass is suspended in the experimental medium and fermented to produce vitamin B12.

[0027] 3) Vitamin B12 is separated and purified from the experimental culture medium.

[0028] In this example, in step 1, the medium for propagating propionic acid bacteria contains the following components in mass percentage: 0.2% glucose, 0.2% meat extract, 0.8% peptone, 0.4% sodium chloride, 0.4% yeast extract, 98% distilled water.

[0029] In this embodiment, before inoculation, the medium for culturing Propionibacterium is sterilized in an autoclave at a temperature of 117 °C for 30 minutes.

[0030] In this embodiment, the medium for culturing Propionibacterium is adjusted to a pH range of 6.5 - 7.0 with 10% NaOH solution before inoculation.

[0031] In this embodiment, the preparation of the experimental medium includes the following steps: 1) Add an appropriate amount of apple residue to distilled water, heat and stir for extraction.

[0032] 2) Remove the residual solids by centrifugation, then sterilize at 121 °C for 30 minutes, add 0.1 mg / L of phytohormones to obtain the experimental medium, and then add 0.2 mg / L of phytohormones.

[0033] In this embodiment, the experimental medium contains the following components by mass percentage: 50% apple residue, 50% distilled water.

[0034] In this embodiment, the experimental medium is stirred and heated at 75 °C for 30 minutes, then the extract is centrifuged to remove the residual solids, and the supernatant is used to prepare the experimental medium.

[0035] In this embodiment, the pH value of the experimental medium is adjusted to 7.0 with NaOH before use.

[0036] In this embodiment, L-cysteine hydrochloride (0.40 g / L) is added to the experimental medium to create anaerobic conditions.

[0037] In this embodiment, the fermentation culture is carried out under static conditions in a conical flask containing 250 mL of the experimental medium at a culture temperature of 30 °C for 120 hours, and the experimental medium is neutralized with 10% NaOH solution every 24 hours to optimize the pH range of Propionibacterium to 6.5 - 7.0.

[0038] Example 2 The present invention provides a process for anaerobic fermentation of apple residue to prepare vitamin B12, comprising the following steps: 1) Under suitable culture conditions, culture the production strain of vitamin B12, which is Propionibacterium, in the medium for culturing Propionibacterium.

[0039] 2) After the strain grows stably, inoculate it onto the medium and inoculate for 24 hours under static conditions, then collect the inoculated medium, centrifuge and separate, suspend the obtained biomass in the experimental medium, and ferment to produce vitamin B12.

[0040] 3) Purify vitamin B12 from the experimental culture medium.

[0041] In this embodiment, in step 1, the culture medium for propagating propionic acid bacteria comprises the following components by mass percentage: 0.4% glucose, 0.2% meat extract, 1.2% peptone, 0.6% sodium chloride, 0.6% yeast extract, 97% distilled water.

[0042] In this embodiment, before inoculation, the culture medium for propagating propionic acid bacteria is sterilized in an autoclave at a temperature of 117 °C for 30 minutes.

[0043] In this embodiment, the culture medium for propagating propionic acid bacteria is adjusted to a pH range of 6.5 - 7.0 with 10% NaOH solution before inoculation.

[0044] In this embodiment, the preparation of the experimental culture medium comprises the following steps: 1) Add an appropriate amount of apple residue to distilled water, heat and stir for extraction.

[0045] 2) Remove the residual solids by centrifugation, then sterilize at 121 °C for 30 minutes, and add 0.2 mg / L of phytotriol to obtain the experimental culture medium.

[0046] In this embodiment, the experimental culture medium comprises the following components by mass percentage: 50% apple residue, 50% distilled water.

[0047] In this embodiment, the experimental culture medium is stirred and heated at 75 °C for 30 minutes, then the extract is centrifuged to remove the residual solids, and the supernatant is used to prepare the experimental culture medium, and then 0.2 mg / L of phytotriol is added.

[0048] In this embodiment, the pH value of the experimental culture medium is adjusted to 7.0 with NaOH before use.

[0049] In this embodiment, L-cysteine hydrochloride (0.40 g / L) is added to the experimental culture medium to create anaerobic conditions.

[0050] In this example, the fermentation culture is carried out in a conical flask containing 250 mL of experimental medium under static conditions. The culture temperature is 30 °C and the time is 120 hours. And every 24 hours, the experimental medium is neutralized with 10% NaOH solution to optimize the pH value range of propionic acid bacteria to 6.5 - 7.0.

[0051] Example 3 The present invention provides a process for preparing vitamin B12 by anaerobic fermentation of apple residues, comprising the following steps: 1) Under suitable culture conditions, a production strain of vitamin B12 is cultured in a medium for propagating propionic acid bacteria, and the production strain of vitamin B12 is propionic acid bacteria.

[0052] 2) After the strain grows stably, it is inoculated onto the medium and inoculated for 24 hours under static conditions. Then the inoculated medium is collected, centrifuged, and the obtained biomass is suspended in the experimental medium and fermented to produce vitamin B12.

[0053] 3) Vitamin B12 is isolated and purified from the experimental medium solution.

[0054] In this example, in step 1, the medium for propagating propionic acid bacteria contains the following components by mass percentage: 0.2% glucose, 0.2% meat extract, 0.8% peptone, 0.4% sodium chloride, 0.4% yeast extract, 98% distilled water.

[0055] In this example, before inoculation, the medium for propagating propionic acid bacteria is sterilized in an autoclave at a temperature of 117 °C for 30 minutes.

[0056] In this example, the medium for propagating propionic acid bacteria is adjusted to a pH range of 6.5 - 7.0 with 10% NaOH solution before inoculation.

[0057] In this example, the preparation of the experimental medium comprises the following steps: 1) An appropriate amount of apple residues is added to distilled water, and heated and stirred for extraction.

[0058] 2) Residual solids are removed by centrifugation, and then sterilized at 121 °C for 30 minutes, and 0.3 mg / L of phytohormones is added to obtain the experimental medium.

[0059] In this example, the experimental medium contains the following components by mass percentage: 33.3% apple residue, 66.7% distilled water.

[0060] In this example, the experimental medium is stirred and heated at 75 °C for 30 minutes, then the extract is centrifuged to remove residual solids, and the supernatant is used to prepare the experimental medium, and 0.2 mg / L phytotriol is added.

[0061] In this example, the pH value of the experimental medium is adjusted to 7.0 with NaOH before use.

[0062] In this example, L-cysteine hydrochloride (0.40 g / L) is added to the experimental medium to create anaerobic conditions.

[0063] In this example, the fermentation culture is carried out under static conditions in a conical flask containing 250 mL of the experimental medium at a culture temperature of 30 °C for 120 hours, and the experimental medium is neutralized with 10% NaOH solution every 24 hours to optimize the pH value range of propionic acid bacteria to 6.5 - 7.0.

[0064] Example 4 The present invention provides a process for preparing vitamin B12 by anaerobic fermentation of apple residue, comprising the following steps: 1) Under suitable culture conditions, a production strain of vitamin B12 is cultured in a medium for propagating propionic acid bacteria, and the production strain of vitamin B12 is propionic acid bacteria.

[0065] 2) After the strain grows stably, it is inoculated onto the medium and inoculated for 24 hours under static conditions, then the inoculated medium is collected, centrifuged, and the obtained biomass is suspended in the experimental medium and fermented to produce vitamin B12.

[0066] 3) Vitamin B12 is isolated and purified from the experimental culture medium.

[0067] In this example, in step 1, the medium for propagating propionic acid bacteria contains the following components by mass percentage: 0.2% glucose, 0.2% meat extract, 0.8% peptone, 0.4% sodium chloride, 0.4% yeast extract, 98% distilled water.

[0068] In this example, before inoculation, the medium for propagating propionic acid bacteria is sterilized in an autoclave at a temperature of 117 °C for 30 minutes.

[0069] In this embodiment, the medium for culturing Propionibacterium is adjusted to a pH range of 6.5 - 7.0 with 10% NaOH solution before inoculation.

[0070] In this embodiment, the preparation of the experimental medium includes the following steps: 1) Add an appropriate amount of apple residue to distilled water, and heat and stir for extraction.

[0071] 2) Remove the residual solids by centrifugation, then sterilize at 121 °C for 30 minutes, and add 0.4 mg / L of phytohormones to obtain the experimental medium.

[0072] In this embodiment, the experimental medium contains the following components by mass percentage: 16.7% apple residue, 83.3% distilled water.

[0073] In this embodiment, the experimental medium is stirred and heated at 75 °C for 30 minutes, then the extract is centrifuged to remove the residual solids, and the supernatant is used to prepare the experimental medium, and then 0.2 mg / L of phytohormones is added.

[0074] In this embodiment, the pH value of the experimental medium is adjusted to 7.0 with NaOH before use.

[0075] In this embodiment, L-cysteine hydrochloride (0.40 g / L) is added to the experimental medium to create anaerobic conditions.

[0076] In this embodiment, the fermentation culture is carried out under static conditions in a conical flask containing 250 mL of the experimental medium, the culture temperature is 30 °C, the time is 120 hours, and the experimental medium is neutralized with 10% NaOH solution every 24 hours to optimize the pH value range of Propionibacterium to 6.5 - 7.0.

[0077] Comparative Example 1 A process for anaerobic fermentation of apple residue to prepare vitamin B12, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components by mass percentage: 50% potato wastewater, 0% distilled water.

[0078] The remaining components and their contents are the same as those in Example 1.

[0079] Comparative Example 2 A process for anaerobic fermentation of apple residue to prepare vitamin B12, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components by mass percentage: 2.5% glycerol, 97.5% distilled water.

[0080] The remaining components and their contents are the same as those in Example 1.

[0081] Comparative Example 3 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components by mass percentage: 50% apple pomace, 50% potato wastewater, 0% distilled water.

[0082] The remaining components and their contents are the same as those in Example 1.

[0083] Comparative Example 4 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components by mass percentage: 50% apple pomace, 1.25% glycerol, 48.75% distilled water.

[0084] The remaining components and their contents are the same as those in Example 1.

[0085] Comparative Example 5 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components by mass percentage: 50% potato wastewater, 1.25% glycerol, 48.75% distilled water.

[0086] The remaining components and their contents are the same as those in Example 1.

[0087] Comparative Example 6 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components by mass percentage: 33.3% apple pomace, 33.3% potato wastewater, 1.25% glycerol, 32.15% distilled water.

[0088] The remaining components and their contents are the same as those in Example 1.

[0089] Comparative Example 7 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components in mass percentages: 66.7% apple pomace, 16.7% potato wastewater, 1.25% glycerol, 15.35% distilled water.

[0090] The remaining components and their contents are the same as those in Example 1.

[0091] Comparative Example 8 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components in mass percentages: 16.7% apple pomace, 66.7% potato wastewater, 1.25% glycerol, 15.35% distilled water.

[0092] The remaining components and their contents are the same as those in Example 1.

[0093] Comparative Example 9 A process for preparing vitamin B12 by anaerobic fermentation of apple residues, which is different from Example 1 only in that in this comparative example, the experimental medium contains the following components in mass percentages: 16.7% apple pomace, 16.7% potato wastewater, 1.25% glycerol, 65.35% distilled water.

[0094] The remaining components and their contents are the same as those in Example 1.

[0095] Performance Test Table 1 Sugar, glycerol and nitrogen contents, and carbon-nitrogen ratio of the media in different examples and comparative examples

[0096] Table 2 Propionic acid, acetic acid and propionic acid / acetic acid ratio, and vitamin B12 yield in different examples and comparative examples

[0097] The present invention provides a process for preparing vitamin B12 by anaerobic fermentation of apple residues, which has significant economic benefits and environmental protection advantages. The waste apple pomace provides a rich carbon source and nitrogen source, creating ideal conditions for the growth and metabolism of DSM 20271 strain.

[0098] As shown in Table 1 and Figure 1 as indicated, in apple pomace, the total sugar content is 23.15 g / L and the nitrogen content is 0.32 g / L, while in potato wastewater, the total sugar content is 5.06 g / L and the nitrogen content is 2.91 g / L. These industrial wastes can be directly used as a culture medium without complex pretreatment, simplifying the process flow and reducing production costs. Compared with traditional methods, this biological production method using industrial wastes is more economical. For example, potato wastewater can replace expensive peptone or yeast extract, and the sugars in apple pomace can provide sufficient carbon sources for microorganisms.

[0099] As shown in Table 2, in the optimal culture medium (containing potato wastewater, apple pomace, and waste glycerol), the propionic acid yield reached 8.15 g / L, the yield efficiency was 0.48 g / g, the productivity was 0.068 g / L / h, while the yield efficiency of acetic acid was 0.13 g / g, the P / A ratio was 3.68:1, and the highest concentration of vitamin B12 obtained in the wet biomass of propionic acid bacteria could reach 290 μg / 100 g.

[0100] In addition, this process realizes the efficient utilization of resources by using by-products of the agricultural and food processing industries, reducing the environmental burden of waste. Compared with traditional chemical production methods, this biotechnological method not only reduces production costs but also reduces environmental pollution, meeting the concept of sustainable development. The product can be widely applied in food and animal feed, meeting the market demand for natural, safe, and green products. Therefore, the present invention provides a green, efficient, and economical biological production route for vitamin B12 and has broad market application prospects, promising to make important contributions to industrial production and environmental protection.

[0101] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A process for preparing vitamin B12 by anaerobic fermentation of apple residues, characterized in that, Comprising the following steps: 1) Under suitable culture conditions, cultivate the production strain of vitamin B12, which is Propionibacterium, in a culture medium for propagating Propionibacterium; 2) After the strain grows stably, inoculate the Propionibacterium onto the culture medium and inoculate for 24 hours under static conditions, then collect the inoculated culture medium, centrifuge, suspend the obtained biomass in the experimental culture medium, and ferment to produce vitamin B12; 3) Isolate and purify vitamin B12 from the experimental culture medium solution.

2. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 1, characterized in that, In step 1, the culture medium for propagating Propionibacterium contains the following components by mass percentage: 0.1 - 0.3% glucose, 0.2 - 0.4% meat extract, 0.8 - 1.2% peptone, 0.4 - 0.6% sodium chloride, 0.4 - 0.6% yeast extract, and 97 - 98% distilled water.

3. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 1, characterized in that, Before inoculating the Propionibacterium onto the culture medium, sterilize the culture medium for propagating Propionibacterium in an autoclave at a temperature of 117 °C for 30 minutes.

4. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 1, characterized in that, The culture medium for propagating Propionibacterium is adjusted to a pH range of 6.5 - 7.0 with 10% NaOH solution before inoculation.

5. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 1, characterized in that, The preparation of the experimental culture medium includes the following steps: 1) Add an appropriate amount of apple residue to distilled water, heat and stir for extraction; 2) Remove the residual solids by centrifugation, then sterilize at 121 °C for 30 minutes, and add 0.1 - 0.5 mg / L of phytotriol to obtain the experimental culture medium.

6. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 5, characterized in that, The experimental culture medium contains the following components by mass percentage: 16.7 - 50% apple residue and 50 - 83.3% distilled water.

7. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 5, characterized in that, The experimental culture medium is stirred and heated at 75 °C for 30 minutes, then the extract is centrifuged to remove the residual solids, and the supernatant is used to prepare the experimental culture medium, and then 0.1 - 0.3 mg / L of phytotriol is added.

8. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 5, characterized in that, The pH value of the experimental culture medium is adjusted to 7.0 with NaOH before use.

9. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 5, characterized in that, L-cysteine hydrochloride (0.40 g / L) is added to the experimental culture medium to create an anaerobic condition.

10. A process for preparing vitamin B12 by anaerobic fermentation of apple residues according to claim 1, characterized in that, The fermentation culture is carried out under static conditions in a conical flask containing 250 mL of the experimental culture medium at a culture temperature of 30 °C for 120 hours, and the experimental culture medium is neutralized with 10% NaOH solution every 24 hours to optimize the pH value range of Propionibacterium to 6.5 - 7.0.

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