Segmented fermentation method for optimizing formula of probiotic compound preparation with homology of medicine and food

Abstract

The invention discloses a medicinal and edible probiotic composite preparation formula optimization segmented fermentation method, and relates to the technical field of fermentation engineering, and the method comprises the following steps: S1, raw material pretreatment: selecting red ginseng, donkey-hide gelatin and red rose according to a weight ratio of 3: 2: 1 to form a composite raw material, crushing the red ginseng and the red rose, sieving with a 80-mesh sieve, dissolving the donkey-hide gelatin with warm water, mixing with the crushed red ginseng and red rose, and carrying out uniform mixing to obtain a mixed material; adding sterile water until the ratio of the total mass of the raw materials to the weight of the sterile water is 1: 5, adjusting the initial pH value to 6.8-7.0, putting the raw materials into a fermentation tank, sterilizing at 121 DEG C for 20 minutes, and cooling to room temperature; by adopting a multi-temperature-zone dynamic segmented fermentation design, four dynamic temperature zones and matched stirring and inoculating parameters are accurately set by combining a probiotic growth curve and a conversion rule of active ingredients in red ginseng, donkey-hide gelatin and red roses, so that the core contradiction between probiotic proliferation and active ingredient conversion in existing fixed temperature zone fermentation is effectively solved; good growth and reproduction of probiotics are guaranteed, and full conversion of active ingredients such as saponin and flavone in the raw materials is promoted.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, specifically to a segmented fermentation method for optimizing the formulation of a compound preparation of medicinal and edible probiotics. Background Technology

[0002] Probiotics are a class of live microorganisms that have beneficial effects on the health of the host. Common examples include Lactobacillus, Bifidobacterium, and Bacillus. They can regulate the balance of the host's intestinal flora, enhance immune function, and are widely present in the natural environment and the human gut. The medicinal and edible probiotic complex system is a compound preparation formed by organically combining medicinal and edible raw materials such as red ginseng, donkey-hide gelatin, and red rose—which possess both food and medicinal value—with probiotics. Its core significance lies in eliminating the potential risks of chemical health care ingredients. Through the synergistic effect of the natural nutrients of the raw materials and the physiological activity of probiotics, it meets people's demand for safe and natural health products, achieving the dual goals of nutritional supplementation and health conditioning.

[0003] Fermentation technology plays an irreplaceable role in the preparation of probiotic compound formulations that are both food and medicine: the fermentation process provides a suitable growth environment for probiotics, promotes their proliferation, ensures the number of live bacteria in the preparation, and the enzymes produced by probiotics during metabolism can decompose macromolecular polysaccharides, proteins, saponins and other substances in the raw materials that are both food and medicine into small molecule active ingredients. This not only improves the absorption efficiency of the ingredients by the human body, but also enhances the health benefits of the preparation, while improving the flavor and stability of the preparation.

[0004] However, existing probiotic fermentation technologies still have certain shortcomings in use. Currently, the segmented fermentation of medicinal and edible probiotic preparations mostly adopts a fixed dual-temperature zone mode, without dynamic adjustment based on the probiotic growth curve and the transformation law of medicinal and edible components. A single or fixed temperature zone cannot adapt to the growth characteristics of different strains, nor can it meet the staged requirements of active ingredient transformation. This leads to a prominent contradiction between probiotic proliferation and active ingredient transformation, either resulting in insufficient live bacteria or low active ingredient transformation rate, and poor structural stability of the final metabolites, which seriously affects the efficacy and quality consistency of the preparation. Therefore, it is of great significance to develop a segmented fermentation method for optimizing the formulation of medicinal and edible probiotic compound preparations. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an optimized segmented fermentation method for the formulation of probiotic compound preparations that are both food and medicine. This method can precisely set four dynamic temperature zones and matching stirring and inoculation parameters by adopting a multi-temperature zone dynamic segmented fermentation design, combined with the growth curve of probiotics and the conversion law of active ingredients in red ginseng, donkey-hide gelatin, and red rose. This not only ensures the good growth and reproduction of probiotics, but also promotes the full conversion of active ingredients such as saponins and flavonoids in the raw materials. Through the precise adaptation of strains and process parameters at each stage and the design of the final stable phase, the structural stability of metabolites is significantly improved, the bioavailability of the preparation is enhanced, and the quality consistency between batches of products is guaranteed.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a segmented fermentation method for optimizing the formulation of a compound preparation of medicinal and edible probiotics, the fermentation method comprising the following steps: S1. Raw material pretreatment: Select red ginseng, donkey-hide gelatin, and red rose in a weight ratio of 3:2:1 to form a compound raw material. Crush the red ginseng and red rose and pass them through an 80-mesh sieve. Dissolve the donkey-hide gelatin in warm water and mix it with the crushed red ginseng and red rose. Add sterile water to make the total mass of raw materials to the weight ratio of sterile water 1:5. Adjust the initial pH to 6.8-7.0. Place the mixture in a fermentation tank and sterilize it at 121℃ for 20 minutes, then cool it to room temperature. S2. Strains preparation: Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium were selected as fermentation strains and activated and cultured using specialized culture media. The concentration of each strain was adjusted to 1×10⁻⁶. 9 CFU / mL; S3. First stage fermentation: Adjust the temperature of the fermenter to 28-30℃, set the stirring speed to 100r / min, inoculate with Bacillus subtilis bacterial solution at 3% of the volume of the raw material mixture, and ferment for 8-10 hours. S4. Second stage fermentation: Raise the temperature of the fermenter to 35-37℃, adjust the stirring speed to 80r / min, inoculate with Lactobacillus acidophilus culture at an inoculation amount of 5% of the volume of the raw material mixture, and ferment for 12-14 hours. S5. Third stage fermentation: raise the temperature of the fermenter to 38-40℃, adjust the stirring speed to 60r / min, inoculate with Bifidobacterium bacterial solution at 4% of the volume of the raw material mixture, and ferment for 6-8 hours. S6. The fourth stage of phase stabilization treatment involves lowering the fermenter temperature to 32°C and maintaining this temperature for 2 hours to promote the stability of the metabolite structure. S7. Centrifugation and mixing: After fermentation, the material is transferred to a centrifuge for centrifugation. The supernatant after centrifugation is taken and mixed with maltodextrin at a weight ratio of 4:1. S8. Drying treatment: The mixed materials are treated using a spray drying process to obtain a composite preparation powder.

[0007] Furthermore, in step S1, the temperature of the warm water used to dissolve the donkey-hide gelatin is 40℃. During dissolution, a magnetic stirrer is used with a stirring power of 50W for 20-30 minutes until the donkey-hide gelatin is completely dissolved and there are no visible particles. The dissolved donkey-hide gelatin solution is first mixed with red ginseng powder in a mixing tank at a stirring rate of 120r / min for 10 minutes. Then, red rose powder is added and stirring is continued for 5-8 minutes. Throughout the mixing process, the material temperature is controlled to be maintained at 35-40℃ by a constant temperature water bath.

[0008] Furthermore, in step S2, the special culture medium used for the activation culture of the strain is MRS medium. The activation environment for Bacillus subtilis is an aerobic environment, with a culture temperature of 37°C and a culture time of 24 hours. The activation environment for Lactobacillus acidophilus is a facultative anaerobic environment, achieved using anaerobic bags or gas replacement, with a culture temperature of 37°C and a culture time of 24 hours. The activation environment for Bifidobacterium is an anaerobic environment, with a culture temperature of 37°C and a culture time of 36 hours. During the activation process, the culture medium is shaken once every 8 hours at a shaking rate of 120 r / min, with each shaking lasting for 10 minutes. After shaking, the medium is allowed to stand for culture.

[0009] Furthermore, before the first stage of fermentation in step S3, sterile air is introduced into the fermenter for 30 minutes at a rate of 50 L / min. During the fermentation process, the dissolved oxygen level of the substrate is monitored every 2 hours using a dissolved oxygen meter. When the dissolved oxygen level is lower than 2 mg / L, the aeration rate is temporarily increased to 60 L / min for 5 minutes and then restored to the initial aeration rate of 50 L / min. The stirring device is turned off 1 hour before the end of fermentation, and the fermentation is allowed to proceed statically.

[0010] Furthermore, before inoculating the Lactobacillus acidophilus culture in the second stage of fermentation in step S4, the pH of the fermentation system is adjusted to 6.0-6.2 using sterile citric acid solution or sterile sodium bicarbonate solution. For the first 6 hours after inoculation, the stirring rate is maintained at 80 r / min, and then reduced to 60 r / min for the next 6-14 hours. Samples are taken every 4 hours during the fermentation process, and the pH value of the system is detected using a pH meter. When the pH is lower than 5.5, sterile sodium bicarbonate solution with a concentration of 0.1 mol / L is slowly added to adjust the pH to 5.8-6.0.

[0011] Furthermore, before the third stage of fermentation in step S5, a sterile glucose solution with a concentration of 50 g / L is added to the fermentation system. The amount added is calculated based on the volume of the fermentation system to maintain the glucose concentration in the system at 10-15 g / L. The stirring rate is maintained at 60 r / min for the first 3 hours of fermentation, and then reduced to 40 r / min for the next 3-8 hours. The vent valve of the fermenter is closed throughout the fermentation process to keep the tank sealed. Before fermentation, the air in the tank is replaced with nitrogen three times, each replacement lasting 10 minutes, and the nitrogen introduction rate is 30 L / min.

[0012] Furthermore, during the fourth stage of the phase stabilization process in step S6, the fermenter is kept in a sealed state, and the air inside the tank is replaced by nitrogen at a rate of 20 L / min for 15 min. During the phase stabilization process, a temperature sensor is used to monitor the material temperature in real time, and the temperature is adjusted by the fermenter temperature control system to keep the temperature fluctuation within ±0.5℃. No stirring is performed during the phase stabilization process, and the material is kept in a static state.

[0013] Furthermore, in step S7, the centrifugation and mixing process, the centrifugation parameters are 8000 r / min and 15 min. After centrifugation, the supernatant is taken and filtered at normal pressure using a 200-mesh filter cloth made of nylon or cotton to remove residual solid particles. The filtered supernatant is then transferred to a refrigeration unit and allowed to stand at 4°C for 2 hours. During the mixing process, the supernatant is taken out and placed in a mixing tank. It is stirred at a stirring rate of 150 r / min while maltodextrin is added. The stirring time is 20 min until the maltodextrin is completely dissolved.

[0014] Furthermore, in step S8 of the spray drying process, the inlet air temperature is controlled at 175-185℃, the outlet air temperature is controlled at 75-85℃, the feed rate is 50mL / min, the atomization pressure is 0.3MPa, and the maltodextrin used is selected from products with a DE value of 10-15. The DE value refers to the glucose equivalent value. When mixing, the equal incremental method is adopted. First, the maltodextrin is divided into three equal parts. The first part is added to the supernatant and stirred for 5 minutes until uniform. Then the second part is added and stirred for another 5 minutes. Finally, the third part is added and stirred for 10 minutes. After mixing evenly, spray drying is carried out.

[0015] Compared with existing technologies, the optimized segmented fermentation method of this medicinal and edible probiotic compound preparation has the following beneficial effects: This invention employs a multi-temperature zone dynamic segmented fermentation design, precisely setting four dynamic temperature zones and corresponding stirring and inoculation parameters based on the probiotic growth curve and the conversion patterns of active ingredients in red ginseng, donkey-hide gelatin, and red rose. This effectively solves the core contradiction between probiotic proliferation and active ingredient conversion in existing fixed-temperature zone fermentation. It ensures good growth and reproduction of probiotics while promoting the full conversion of active ingredients such as saponins and flavonoids in the raw materials. Through precise adaptation of strains and process parameters at each stage and the design of the final stable phase, the structural stability of metabolites is significantly improved, enhancing the bioavailability of the formulation. At the same time, it ensures the consistency of product quality between batches, fully leveraging the synergistic health benefits of medicinal and edible raw materials and probiotics, and meeting the safety and efficacy requirements of natural functional foods.

[0016] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0018] Figure 1 Flowchart for optimizing the segmented fermentation method for compound preparations of medicinal and edible probiotics; Figure 2 A flowchart of the segmented fermentation method for optimizing the formulation of a compound preparation of probiotics that are both food and medicine. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] The present invention provides an optimized segmented fermentation method for a compound preparation of medicinal and edible probiotics, constructing a complete technical solution. (See attached document.) Figure 1 and Figure 2 The core content is as follows: This technical solution uses red ginseng, donkey-hide gelatin, and red rose as compound raw materials, formulated in a weight ratio of 3:2:1. After pretreatment, the raw materials are combined with specific probiotic strains and prepared through a four-stage dynamic fermentation and subsequent processing. The raw material pretreatment process is clearly defined: red ginseng and red rose are pulverized and passed through an 80-mesh sieve; donkey-hide gelatin is dissolved in 40℃ warm water with magnetic stirring, and then mixed sequentially with red ginseng and red rose powders. The temperature is controlled at 35-40℃ throughout the process. The total mass of raw materials is mixed with sterile water at a ratio of 1:5, the pH is adjusted to 6.8-7.0, and the mixture is sterilized at 121℃ for 20 minutes and then cooled for later use.

[0021] Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium were selected as bacterial strains and activated using MRS medium. Suitable aerobic, facultative anaerobic, and anaerobic environments were provided, respectively. The culture temperature was 37℃, and the culture time was 24-36 hours. The culture was shaken periodically during activation. The final bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0022] The fermentation process employs a multi-temperature zone dynamic segmented design: In the first stage, at 28-30℃, 3% Bacillus subtilis is inoculated at a stirring rate of 100 rpm and fermented for 8-10 hours. Sterile air is introduced before fermentation, and dissolved oxygen levels are monitored and adjusted throughout the process. In the second stage, the temperature is raised to 35-37℃, the stirring rate is adjusted to 80 rpm (later reduced to 60 rpm), and 5% Lactobacillus acidophilus is inoculated. Fermentation lasts 12-14 hours, with the pH maintained between 5.8 and 6.2 throughout. In the third stage, the temperature is raised to 38-40℃, the stirring rate is 60 rpm (later reduced to 40 rpm), and 4% Bifidobacterium is inoculated. Fermentation lasts 6-8 hours, with glucose added to maintain concentration and ensure an anaerobic environment within the tank. In the fourth stage, the temperature is lowered to 32℃ for 2 hours for phase stabilization treatment, followed by a sealed, static environment.

[0023] The subsequent processing steps stipulate that: the fermented material is centrifuged at 8000 r / min for 15 min, the supernatant is filtered and refrigerated, and then mixed with maltodextrin at a weight ratio of 4:1 using an equal incremental method; during spray drying, the inlet air temperature is controlled at 175-185℃, the outlet air temperature at 75-85℃, the feed rate at 50 mL / min, and the atomization pressure at 0.3 MPa. Maltodextrin with a DE value of 10-15 is selected to finally obtain a compound preparation powder.

[0024] The entire technical solution forms a complete closed loop by precisely setting the raw material ratio, strain activation conditions, temperature, stirring rate, inoculation amount, environmental parameters and post-processing, which not only ensures the proliferation of probiotics, but also promotes the conversion of active ingredients in raw materials, and improves the stability and bioavailability of the formulation. Example 1

[0025] This embodiment strictly follows the core technical solution of the invention patent, using red ginseng, donkey-hide gelatin, and red rose in a 3:2:1 weight ratio as composite raw materials. Through standardized raw material pretreatment, strain activation, four-stage dynamic segmented fermentation, and post-processing, parameters at each stage are precisely controlled to verify the stability and effectiveness of the basic process. This provides a reliable benchmark for large-scale formulation production, ensuring the basic consistency of product quality and the repeatability of the process. See [link to relevant documentation]. Figure 1 and Figure 2 The complete technical solution is as follows: Raw material pretreatment: A compound raw material consisting of 30g red ginseng, 20g donkey-hide gelatin, and 10g red rose is selected. The red ginseng and red rose are pulverized and passed through an 80-mesh sieve to ensure fine powder. The donkey-hide gelatin is dissolved in 40℃ warm water using a 50W magnetic stirrer for 25 minutes until completely dissolved and free of visible particles. The dissolved donkey-hide gelatin solution is then placed in a mixing tank with the red ginseng powder and stirred at 120 rpm for 10 minutes. The red rose powder is then added and stirred for another 6 minutes. The entire mixing process is carried out using a constant temperature water bath to maintain the material temperature at 38℃. 300g of sterile water is added to the total raw material mass at a 1:5 ratio to adjust the initial pH to 6.9. The mixture is then placed in a fermentation tank, sterilized at 121℃ for 20 minutes, and then cooled to room temperature for later use.

[0026] Strains Preparation: Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium were selected as fermentation strains, all of which were activated and cultured on MRS medium. Bacillus subtilis was cultured at 37°C for 24 hours under aerobic conditions, Lactobacillus acidophilus for 24 hours under facultative anaerobic conditions, and Bifidobacterium for 36 hours under anaerobic conditions. During activation, the medium was shaken every 8 hours at a rate of 120 rpm for 10 minutes each time. After shaking, the medium was allowed to stand for further incubation. Finally, the concentration of each strain was uniformly adjusted to 1×10⁻⁶. 9 CFU per milliliter.

[0027] First stage fermentation: Adjust the fermenter temperature to 29℃ and set the stirring speed to 100 rpm. Before fermentation, introduce sterile air into the tank for 30 minutes at a flow rate of 50 liters per minute. Inoculate with Bacillus subtilis culture at 3% of the volume of the raw material mixture, and ferment for 9 hours. During fermentation, monitor the dissolved oxygen level of the substrate every 2 hours using a dissolved oxygen meter. When the dissolved oxygen level is below 2 mg / L, briefly increase the aeration rate to 60 liters per minute for 5 minutes, then return to the initial aeration rate. Turn off the stirring device 1 hour before the end of fermentation and allow it to stand for fermentation.

[0028] Second stage fermentation: The fermenter temperature was raised to 36℃, and the pH of the fermentation system was adjusted to 6.1 using sterile sodium bicarbonate solution. The stirring speed was adjusted to 80 rpm, and Lactobacillus acidophilus culture was inoculated at 5% of the volume of the raw material mixture. For the first 6 hours after inoculation, the stirring speed was maintained at 80 rpm, and then reduced to 60 rpm for the next 8 hours. Samples were taken every 4 hours during fermentation, and the pH was monitored using a pH meter. When the pH fell below 5.5, sterile sodium bicarbonate solution with a concentration of 0.1 mol / L was slowly added to adjust the pH to 5.9. The entire fermentation stage lasted 13 hours.

[0029] The third stage of fermentation: The temperature of the fermenter was raised to 39°C, and a sterile glucose solution with a concentration of 50 g / L was added to the fermentation system to maintain a glucose concentration of 12 g / L. The stirring speed was set to 60 rpm, maintained for the first 3 hours, and then reduced to 40 rpm for the next 5 hours. Bifidobacterium culture was inoculated at 4% of the volume of the raw material mixture. The vent valve of the fermenter was closed throughout the fermentation process to maintain a sealed environment. Before fermentation, the air inside the tank was replaced three times with nitrogen, each replacement lasting 10 minutes, at a nitrogen inlet rate of 30 liters per minute. This stage of fermentation lasted 7 hours.

[0030] The fourth stage of phase stabilization: The temperature of the fermenter is lowered to 32℃, and the fermenter is kept sealed. Nitrogen gas is introduced at a rate of 20 liters per minute for 15 minutes to replace the air inside the tank. During the phase stabilization process, the temperature of the material is monitored in real time using a temperature sensor, and the temperature is adjusted through the fermenter temperature control system to keep the temperature fluctuation within ±0.5℃. The mixture is then left to stand at this temperature for 2 hours.

[0031] Centrifugation and Mixing: After fermentation, the material is transferred to a centrifuge and centrifuged at 8000 rpm for 15 minutes. The supernatant is collected and filtered under normal pressure using a 200-mesh filter cloth to remove residual solid particles. The filtered supernatant is then transferred to a refrigeration unit and allowed to stand at 4°C for 2 hours. Maltodextrin is added to the supernatant at a weight ratio of 4:1 and stirred at 150 rpm for 20 minutes until the maltodextrin is completely dissolved.

[0032] Drying process: Maltodextrin with a DE value of 12 was selected. During mixing, an equal-incremental method was used, dividing the maltodextrin into three equal portions. The first portion was added to the supernatant and stirred for 5 minutes until homogeneous. The second portion was added and stirred for another 5 minutes, and finally the third portion was added and stirred for 10 minutes. Spray drying parameters were set as follows: inlet air temperature 180℃, outlet air temperature 80℃, feed rate 50 ml / min, and atomization pressure 0.3 MPa. The mixed material was then spray-dried to obtain a compound formulation powder.

[0033] In summary, this embodiment strictly adhered to all requirements of the patented technical solution. All process parameters were precisely controllable, raw material pretreatment was thorough, and strain activation conditions were tailored to the growth needs of each strain. The parameter settings for the four-stage fermentation and phase stabilization treatment were highly matched to the growth characteristics and active ingredient conversion patterns of different strains. The resulting compound preparation powder had a uniform texture, sufficient live probiotic count, high conversion rates of active ingredients such as red ginsenosides and rose flavonoids, stable metabolite structures, and good batch-to-batch quality consistency. This fully validated the feasibility, stability, and superiority of the basic process, laying a solid foundation for subsequent process optimization and large-scale production. Example 2

[0034] This embodiment, within the patented basic process framework, focuses on improving fermentation efficiency and active ingredient conversion, optimizing and adjusting key process parameters. The fermentation temperature in the third stage is increased to 40°C, the glucose concentration is maintained at 15 g / L, and the fermentation time in the second stage is extended to 14 hours. This explores the synergistic effect of probiotic proliferation and active ingredient conversion under higher temperature and higher carbon source conditions, further investigating the potential for process optimization to obtain a higher-quality compound formulation. (See [link to relevant documentation]). Figure 1 and Figure 2 The complete technical solution is as follows: Raw material pretreatment: Select 60g of red ginseng, 40g of donkey-hide gelatin, and 20g of red rose. The raw material treatment method is consistent with that in Example 1. Grind the red ginseng and red rose into powder and pass it through an 80-mesh sieve. Dissolve the donkey-hide gelatin in 40℃ warm water using magnetic stirring, then mix it sequentially with the red ginseng and red rose powders, maintaining a temperature of 38℃ during the mixing process. Add 600g of sterile water, adjust the pH to 7.0, sterilize at 121℃ for 20 minutes, and then cool for later use.

[0035] Strains were prepared in the same manner as in Example 1, with the selection and activation culture conditions being exactly the same. The final concentration of each strain was adjusted to 1×10⁻⁶. 9 CFU per milliliter.

[0036] First stage fermentation: The fermenter temperature was adjusted to 28℃, the stirring rate was set to 100 rpm, and the aeration rate was controlled in the same way as in Example 1. A 3% inoculum of Bacillus subtilis was added, and the fermentation time was 8 hours.

[0037] Second stage fermentation: The fermenter temperature was raised to 37°C, and the pH was adjusted to 6.2 using sterile sodium bicarbonate solution. A 5% inoculum of *Lactobacillus acidophilus* was added, and the stirring rate was maintained at 80 rpm for the first 6 hours, then reduced to 60 rpm for the next 8 hours. The pH was continuously monitored during fermentation and maintained between 5.8 and 6.0. This stage of fermentation lasted for 14 hours.

[0038] Third stage fermentation: The temperature of the fermenter was raised to 40°C, and sterile glucose solution was added to the fermentation system to maintain a glucose concentration of 15 g / L. The stirring rate was set to 60 rpm, maintained for the first 3 hours, and then reduced to 40 rpm for the next 5 hours. A 4% inoculum of Bifidobacterium was added, and fermentation was carried out in a sealed environment for 8 hours. The nitrogen purging procedure was the same as in Example 1.

[0039] The fourth stage of stabilization is completely consistent with the operation process and parameter settings of Example 1 to ensure the stability of the metabolite structure.

[0040] Centrifugation and mixing: The centrifugation parameters, filtration operation, refrigeration and settling and mixing methods are consistent with those in Example 1 to ensure the purity of the supernatant and the uniformity of mixing.

[0041] Drying treatment: Maltodextrin with a DE value of 15 was selected. The spray drying parameters were set to an inlet air temperature of 185°C and an outlet air temperature of 85°C. The other parameters were the same as in Example 1. The mixed materials were spray dried to obtain a compound preparation powder.

[0042] In summary, this embodiment, through targeted optimization of the second-stage fermentation time and the third-stage temperature and carbon source concentration, provided a more suitable growth and metabolic environment for *Lactobacillus acidophilus* and *Bifidobacterium*. Higher temperatures and ample glucose supply enhanced the metabolic activity of *Lactobacillus acidophilus* and the proliferation efficiency of *Bifidobacterium*, providing more sufficient energy and reaction conditions for the conversion of active ingredients. In the final formulation, the number of viable *Bifidobacterium* bacteria was significantly increased compared to Example 1, and the total conversion rate of active ingredients such as red ginsenosides and rose flavonoids also slightly increased, further optimizing product quality. This optimization scheme verifies that adjusting key process parameters within a reasonable range can effectively improve the core quality indicators of the formulation, providing a new process reference for the production of high-quality compound formulations. Example 3

[0043] This embodiment focuses on the efficiency requirements of industrial production, making targeted fine-tuning of process parameters based on the patented technology solution. The stirring rate during raw material mixing is increased to 130 rpm to improve the uniformity of raw material dispersion; the initial aeration rate for the first stage fermentation is adjusted to 55 liters per minute to ensure the aerobic metabolic needs of Bacillus subtilis; the third stage fermentation time is shortened to 6 hours, improving production efficiency while ensuring core product quality. This verifies the comprehensive impact of process parameter fine-tuning on production efficiency and product quality, adapting to the actual needs of industrial-scale mass production. See [link to relevant documentation]. Figure 1 and Figure 2 The complete technical solution is as follows: Raw material pretreatment: Select 45g of red ginseng, 30g of donkey-hide gelatin, and 15g of red rose. Grind the red ginseng and red rose into powder and pass it through an 80-mesh sieve. Dissolve the donkey-hide gelatin in 40℃ warm water using magnetic stirring, then mix it with the red ginseng powder at a speed of 130 rpm for 10 minutes. Add the red rose powder and continue stirring for 7 minutes. During the mixing process, maintain the material temperature at 35℃ using a constant temperature water bath. Add 450g of sterile water, adjust the pH to 6.8, sterilize at 121℃ for 20 minutes, and then cool for later use.

[0044] Strain preparation: The activation and culture conditions of the strain are exactly the same as those in Example 1 to ensure that the strain activity meets the standard.

[0045] First stage fermentation: Adjust the fermenter temperature to 30℃, and the initial aeration rate to 55 liters per minute. Aerate for 30 minutes before fermentation. The dissolved oxygen monitoring and adjustment method is consistent with Example 1. Inoculate with 3% Bacillus subtilis culture, and the fermentation time is 10 hours.

[0046] Second stage fermentation: The temperature of the fermenter was raised to 35°C, and the pH was adjusted to 6.0 using sterile citric acid solution or sterile sodium bicarbonate solution. A 5% inoculum of Lactobacillus acidophilus was added, and the stirring rate and pH control method were the same as in Example 1. The fermentation time for this stage was 12 hours.

[0047] Third stage fermentation: The fermenter temperature was raised to 38℃, and sterile glucose solution was added to the fermentation system to maintain a glucose concentration of 10 g / L. The stirring rate was set to 60 rpm, maintained for the first 3 hours, and then reduced to 40 rpm for the next 3 hours. A 4% inoculum of Bifidobacterium was added, and fermentation was carried out in a sealed environment for 6 hours, with nitrogen purging performed as required.

[0048] The fourth stage of stabilization is completely consistent with the operation process and parameters of Example 1, ensuring the stability of metabolites.

[0049] Centrifugation and mixing: Centrifugation parameters, filtration, refrigeration and mixing operations are consistent with those in Example 1 to ensure the treatment effect.

[0050] Drying process: Maltodextrin with a DE value of 10 was selected. The spray drying parameters were set to an inlet air temperature of 175℃ and an outlet air temperature of 75℃, with other parameters remaining unchanged. The mixed materials were spray dried to obtain a compound preparation powder.

[0051] In summary, this embodiment achieves a balance between production efficiency and product quality through multi-dimensional parameter fine-tuning. Increasing the raw material mixing and stirring rate effectively improves the uniformity of raw material dispersion, creating favorable conditions for sufficient contact between the strain and raw materials during subsequent fermentation. Optimizing the aeration rate in the first stage more fully meets the aerobic metabolic needs of Bacillus subtilis, ensuring the proliferation efficiency of the strain. Shortening the fermentation time in the third stage significantly improves overall production efficiency without significantly affecting the core quality of the product. Although the number of viable probiotics and the conversion rate of active ingredients in the final formulation are slightly lower than in Example 2, they are still at a high level. Furthermore, the production cycle is shorter, the production cost is lower, and it is more suitable for large-scale industrial production, possessing significant industrial application value. Comparative Example

[0052] This comparative example uses a common fixed dual-temperature zone fermentation mode in existing technologies, and does not employ the multi-temperature zone dynamic segmented fermentation and phase-stabilized treatment process described in the patent. It only maintains consistency with Example 1 in terms of raw material ratio, strain selection, and post-treatment process. Through comparative experiments, the significant advantages of the patented technology over traditional fermentation processes in terms of probiotic proliferation, active ingredient conversion, and product stability are visually verified. The complete technical solution is as follows: Raw material pretreatment: exactly the same as in Example 1, 30g of red ginseng, 20g of donkey-hide gelatin and 10g of red rose were selected and operated according to the same ratio, treatment method and sterilization and cooling parameters to ensure the consistency of the raw material pretreatment process and eliminate the interference of this process on the experimental results.

[0053] Strains preparation: Consistent with Example 1, the same Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium were selected, and the same activation culture conditions were used. Finally, the strain concentration was adjusted to 1×10⁻⁶. 9 CFU per milliliter ensures consistent initial state of the strain.

[0054] Fermentation process: A fixed dual-temperature zone fermentation mode was adopted, without dynamic temperature zone adjustment or phase stabilization treatment steps. In the first stage, the temperature was fixed at 37℃, the stirring rate was set to 80 rpm, and 3% Bacillus subtilis inoculum and 5% Lactobacillus acidophilus inoculum were added simultaneously for aerobic fermentation for 15 hours. In the second stage, the temperature remained unchanged at 37℃, the stirring rate was adjusted to 60 rpm, and 4% Bifidobacterium inoculum was added for anaerobic fermentation for 10 hours. There was no phase stabilization treatment step in the entire fermentation process.

[0055] Centrifugation and mixing: The operation process and parameters are completely consistent with those in Example 1 to ensure that the post-processing steps have the same impact on the experimental results.

[0056] Drying treatment: The spray drying parameters were kept consistent with those in Example 1. The mixed materials were dried to obtain the composite formulation powder.

[0057] In summary, the traditional fixed dual-temperature zone fermentation process used in this comparative example has significant limitations. The fixed temperature and stirring rate cannot adapt to the growth characteristics of different bacterial strains. The optimal growth conditions for Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium differ considerably. A uniform fermentation environment prevents each strain from reaching its optimal growth state, especially resulting in a low survival rate for Bifidobacterium. Furthermore, the lack of dynamic parameter adjustment and phase stabilization treatment leads to insufficient conversion of active ingredients in the raw materials and poor structural stability of metabolites. The final formulation has a significantly insufficient number of live probiotics, low conversion rates of active ingredients such as red ginsenosides and flavonoids, poor structural stability of metabolites, large batch-to-batch quality fluctuations, and a low product qualification rate. Compared with the three examples, the patented multi-temperature zone dynamic segmented fermentation technology fully demonstrates its advanced and superior advantages in adapting to strain growth, promoting active ingredient conversion, and improving product stability.

[0058]

[0059] A comprehensive comparison of the three examples and comparative examples in the table above clearly demonstrates that the multi-temperature zone dynamic segmented fermentation and phase-stabilized treatment in the patented technology are the core technological highlights for improving the quality of compound preparations. Example 1, as the basic process scheme, achieves the dual goals of efficient probiotic proliferation and full conversion of active ingredients by precisely matching parameters such as temperature, stirring rate, and aeration rate for each strain, providing a stable and reliable benchmark for subsequent process optimization. Example 2 further enhances the metabolic activity of the strains and the conversion efficiency of active ingredients by optimizing fermentation temperature, carbon source concentration, and fermentation time, resulting in the best performance in core product quality indicators. Example 3 focuses on the needs of industrial production, significantly improving production efficiency while ensuring high product quality through parameter fine-tuning, demonstrating strong industrial application value.

[0060] The traditional fixed-temperature-zone fermentation process used in the comparative examples, due to its inability to adapt to the growth characteristics of different bacterial strains and the lack of a crucial phase stabilization treatment step, resulted in significantly inferior performance compared to the three examples in key indicators such as probiotic viable count, active ingredient conversion rate, and product stability. In summary, this patented technical solution, through scientific process design, effectively resolves the core contradiction between probiotic proliferation and active ingredient conversion in traditional fermentation processes. It not only ensures high product quality and stability but also allows for flexible parameter optimization to adapt to different production needs, demonstrating significant technical advantages and broad application prospects compared to traditional processes.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An optimized segmented fermentation method for a compound preparation of medicinal and edible probiotics, characterized in that... The fermentation method includes the following steps: S1. Raw material pretreatment: Select red ginseng, donkey-hide gelatin, and red rose in a weight ratio of 3:2:1 to form a compound raw material. Crush the red ginseng and red rose and pass them through an 80-mesh sieve. Dissolve the donkey-hide gelatin in warm water and mix it with the crushed red ginseng and red rose. Add sterile water to make the total mass of raw materials to the weight ratio of sterile water 1:

5. Adjust the initial pH to 6.8-7.

0. Place the mixture in a fermentation tank and sterilize it at 121℃ for 20 minutes, then cool it to room temperature. S2. Strains preparation: Bacillus subtilis, Lactobacillus acidophilus, and Bifidobacterium were selected as fermentation strains and activated and cultured using specialized culture media. The concentration of each strain was adjusted to 1×10⁻⁶. 9 CFU / mL; S3. First stage fermentation: Adjust the temperature of the fermenter to 28-30℃, set the stirring speed to 100r / min, inoculate with Bacillus subtilis bacterial solution at 3% of the volume of the raw material mixture, and ferment for 8-10 hours. S4. Second stage fermentation: Raise the temperature of the fermenter to 35-37℃, adjust the stirring speed to 80r / min, inoculate with Lactobacillus acidophilus culture at an inoculation amount of 5% of the volume of the raw material mixture, and ferment for 12-14 hours. S5. Third stage fermentation: raise the temperature of the fermenter to 38-40℃, adjust the stirring speed to 60r / min, inoculate with Bifidobacterium bacterial solution at 4% of the volume of the raw material mixture, and ferment for 6-8 hours. S6. Fourth stage of phase stabilization treatment: reduce the fermenter temperature to 32°C and maintain this temperature for 2 hours. S7. Centrifugation and mixing: After fermentation, the material is transferred to a centrifuge for centrifugation. The supernatant after centrifugation is taken and mixed with maltodextrin at a weight ratio of 4:

1. S8. Drying treatment: The mixed materials are treated using a spray drying process to obtain a composite preparation powder.

2. The optimized segmented fermentation method for the formulation of the medicinal and edible probiotic compound preparation according to claim 1, characterized in that, In step S1, the temperature of the warm water used to dissolve the donkey-hide gelatin is 40℃. During dissolution, a magnetic stirrer is used with a stirring power of 50W for 20-30 minutes until the donkey-hide gelatin is completely dissolved and there are no visible particles. The dissolved donkey-hide gelatin solution is first mixed with red ginseng powder in a mixing tank at a stirring rate of 120r / min for 10 minutes. Then, red rose powder is added and stirring is continued for 5-8 minutes. Throughout the mixing process, the material temperature is controlled to be maintained at 35-40℃ by a constant temperature water bath.

3. The optimized segmented fermentation method for the formulation of the medicinal and edible probiotic compound preparation according to claim 1, characterized in that, In step S2, the special culture medium used for the activation culture of the strains is MRS medium. The activation environment for Bacillus subtilis is an aerobic environment, with a culture temperature of 37°C and a culture time of 24 hours. The activation environment for Lactobacillus acidophilus is a facultative anaerobic environment, with a culture temperature of 37°C and a culture time of 24 hours. The activation environment for Bifidobacterium is an anaerobic environment, with a culture temperature of 37°C and a culture time of 36 hours. During the activation process, the culture medium is shaken once every 8 hours at a shaking rate of 120 r / min for 10 minutes each time. After shaking, the medium is allowed to stand for culture.

4. The optimized segmented fermentation method for the formulation of the medicinal and edible probiotic compound preparation according to claim 1, characterized in that, Before the first stage of fermentation in step S3, sterile air is introduced into the fermenter for 30 minutes at a rate of 50 L / min. During the fermentation process, the dissolved oxygen level of the substrate is monitored every 2 hours using a dissolved oxygen meter. When the dissolved oxygen level is lower than 2 mg / L, the aeration rate is temporarily increased to 60 L / min for 5 minutes and then restored to the initial aeration rate of 50 L / min. The stirring device is turned off 1 hour before the end of fermentation, and the fermentation is allowed to proceed in a static state.

5. The optimized segmented fermentation method for the formulation of the medicinal and edible probiotic compound preparation according to claim 1, characterized in that, Before inoculating the Lactobacillus acidophilus culture in the second stage of fermentation in step S4, the pH of the fermentation system is adjusted to 6.0-6.2 using sterile citric acid solution or sterile sodium bicarbonate solution. For the first 6 hours after inoculation, the stirring rate is maintained at 80 r / min, and then reduced to 60 r / min for the next 6-14 hours. During the fermentation process, samples are taken every 4 hours, and the pH value of the system is detected using a pH meter. When the pH is lower than 5.5, sterile sodium bicarbonate solution with a concentration of 0.1 mol / L is slowly added to adjust the pH to 5.8-6.

0.

6. The optimized segmented fermentation method for the formulation of the medicinal and edible probiotic compound preparation according to claim 1, characterized in that, Before the third stage of fermentation in step S5, a sterile glucose solution with a concentration of 50 g / L is added to the fermentation system. The amount added is calculated based on the volume of the fermentation system to maintain the glucose concentration in the system at 10-15 g / L. The stirring rate is maintained at 60 r / min for the first 3 hours of fermentation, and then reduced to 40 r / min for the next 3-8 hours. The vent valve of the fermenter is closed throughout the fermentation process to keep the tank sealed. Before fermentation, the air in the tank is replaced with nitrogen three times, each replacement lasting 10 minutes, and the nitrogen introduction rate is 30 L / min.

7. The optimized segmented fermentation method for the formulation of a medicinal and edible probiotic compound preparation according to claim 1, characterized in that, During the fourth stage of the phase stabilization process in step S6, the fermenter is kept in a sealed state. The air inside the tank is replaced by nitrogen at a rate of 20 L / min for 15 min. During the phase stabilization process, a temperature sensor is used to monitor the material temperature in real time, and the temperature is adjusted by the fermenter temperature control system to keep the temperature fluctuation within ±0.5℃. No stirring is performed during the phase stabilization process, and the material is kept in a static state.

8. The optimized segmented fermentation method for the formulation of a medicinal and edible probiotic compound preparation according to claim 1, characterized in that, In step S7, centrifugation and mixing, the centrifugation parameters are 8000 r / min and 15 min. After centrifugation, the supernatant is taken and filtered under normal pressure using a 200-mesh filter cloth to remove residual solid particles. The filtered supernatant is then transferred to a refrigeration unit and allowed to stand at 4°C for 2 hours. During the mixing process, the supernatant is taken out and placed in a mixing tank. It is stirred at a stirring rate of 150 r / min while maltodextrin is added. The stirring time is 20 min until the maltodextrin is completely dissolved.

9. The optimized segmented fermentation method for the formulation of a medicinal and edible probiotic compound preparation according to claim 1, characterized in that, In step S8 of the spray drying process, the inlet air temperature is controlled at 175-185℃, the outlet air temperature is controlled at 75-85℃, the feed rate is 50mL / min, the atomization pressure is 0.3MPa, and the maltodextrin used is selected from products with a DE value of 10-15. When mixing, the equal incremental method is adopted. First, the maltodextrin is divided into three equal parts. The first part is added to the supernatant and stirred for 5 minutes until uniform. Then the second part is added and stirred for another 5 minutes. Finally, the third part is added and stirred for 10 minutes. After mixing evenly, spray drying is carried out.

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