A preparation method for enhancing the stability of Lactobacillus johnsonii across the entire chain.

Abstract

This invention provides a whole-chain enhancement method for Lactobacillus johnsonii ( Lactobacillus johnsonii This invention discloses a method for improving the stability of *Lactobacillus johnsonii*. This method utilizes a non-animal-derived fermentation medium and a multifunctional synergistic pH control system, employing a segmented pH control strategy of "constant pH followed by relaxed pH," and is combined with a six-component compound freeze-drying protectant. This achieves a comprehensive improvement in the processing stability and product quality of *Lactobacillus johnsonii*. The preparation method provided by this invention significantly improves the viable count and stability of *Lactobacillus johnsonii* during fermentation, freeze-drying, and storage, and enhances its tolerance to digestive solutions. It addresses industry pain points such as the difficulty in culturing important probiotic varieties, easy inactivation during processing, rapid degradation during storage and transportation, and weak resistance to digestive solutions. Furthermore, this preparation method is simple, uses widely available raw materials, has controllable costs, and is easy to implement for large-scale, standardized production of *Lactobacillus johnsonii*, demonstrating significant economic benefits and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and probiotic preparation technology, specifically relating to a method for enhancing the entire chain of Lactobacillus johnsonii (…). Lactobacillus johnsonii The preparation method for improving stability, including its processing, storage and application stability, has been comprehensively improved. Background Technology

[0002] Lactobacillus johnsonii ( Lactobacillus johnsonii Lactobacillus johnsonii is an important probiotic, naturally occurring in the human digestive tract, possessing various beneficial effects such as regulating intestinal microecological balance, inhibiting pathogen colonization, and enhancing the body's immune function. In recent years, with the continuous expansion of the global probiotic market, the industrial demand for Lactobacillus johnsonii has been increasing. However, compared with other common lactobacilli (such as Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus), Lactobacillus johnsonii has significantly different physiological and metabolic characteristics, directly affecting its performance in industrial production. In terms of fermentation performance, Lactobacillus johnsonii has a relatively conservative metabolic pattern, with a limited range of efficiently utilized carbon sources, and its growth rate is usually slow, requiring the assistance of other nutrients to improve fermentation efficiency. Simultaneously, this strain generally has weak acid production and acid tolerance, resulting in a low survival rate in strongly acidic environments. Regarding freeze-drying performance, the freeze-drying survival rate of Lactobacillus johnsonii is typically between 40% and 70%, generally lower than that of the more tolerant Lactobacillus plantarum (which can achieve a freeze-drying survival rate of around 80%). The fundamental reason for this difference lies in the different cell membrane composition and structure. Due to the physiological and metabolic characteristics of Lactobacillus johnsonii, the following technical bottlenecks exist in its industrial production and application: 1. Low viable cell count and high dependence of fermentation medium on animal-derived raw materials. Although the commonly used MRS medium can meet the basic growth requirements of *Lactobacillus johnsonii*, the viable cell count is usually low, making high-density culture difficult. While existing research has optimized the culture medium, technical shortcomings still exist. For example, CN102417892A discloses a culture medium formulation for *Lactobacillus johnsonii* using glucose as the carbon source and tryptone, beef extract / beef meal, and yeast extract as a compound nitrogen source, which can increase the viable cell count to 4.9 × 10⁻⁶. 10 While the culture medium achieves a high viable count (CFU / mL), it is highly dependent on animal-derived raw materials (such as tryptone and beef extract). These raw materials pose a potential risk of animal diseases, severely limiting the product's application range. Furthermore, the complex composition of animal-derived components and large batch-to-batch quality fluctuations affect the stability of the fermentation process and hinder consistent product quality control.

[0003] 2. Short fermentation stationary period, posing significant challenges for industrial-scale production. Due to the metabolic characteristics of *Lactobacillus johnsonii*, the viable cell count in the fermentation broth often declines rapidly after reaching its peak during high-density cultivation, resulting in an extremely narrow operating window for tank collection and significantly increasing the difficulty of control in industrial production. Existing fermentation optimization research mainly focuses on how to increase the maximum viable cell count in the fermentation broth, while there are few reports on extending the fermentation stationary period and delaying the decline in viable cell count, lacking effective process strategies for maintaining cell viability in the later stages of fermentation.

[0004] 3. The freeze-drying protectant's effectiveness is unsatisfactory, and stability studies are insufficient. On the one hand, there is a lack of dedicated freeze-drying protectants designed based on the physiological and metabolic characteristics of *Lactobacillus johnsonii*. Industrially, general-purpose formulations (such as those composed of skim milk, sucrose, and soluble starch) are commonly used. These protectants only achieve basic cell encapsulation and vitrification protection, lacking a systematic design for synergistic effects among components. This results in low freeze-drying survival rates and rapid decline in viable cell counts during long-term storage. For example, CN102453681A discloses a freeze-drying protectant composed of skim milk, trehalose, soluble starch, monosodium glutamate, and glycerol in a specific ratio. Its protective effect on *Lactobacillus johnsonii* is poor, with the viable cell count in the freeze-dried powder remaining at only 10. 9 CFU / g level. On the other hand, existing technologies for evaluating the performance of freeze-drying protectants are mostly limited to freeze-drying survival rates, with few studies on long-term storage stability, and even fewer studies on whether protectants can improve the tolerance of bacterial powder after it enters the digestive tract. However, the development of freeze-drying protectant formulations should not only focus on the protective effect on bacterial cells during the freeze-drying process, but also take into account the stability of the bacterial powder during storage and application.

[0005] Therefore, existing technologies fail to provide a comprehensive, systematic solution for the entire process from fermentation to freeze-drying and product application. More importantly, the technical deficiencies at each stage are interconnected and mutually influential: the reliance on animal-derived raw materials and the short stability period in the fermentation stage directly reduce the initial cell quality and yield; insufficient protection in the freeze-drying stage further exacerbates cell damage and storage degradation; ultimately, the overall stability of the product during processing, storage, and the digestive tract cannot be guaranteed. Therefore, developing a novel preparation method that can significantly improve the overall stability of *Lactobacillus johnsonii* in fermentation, freeze-drying, long-term storage, and application has become a core technological need urgently needing to be addressed in this field. This invention addresses these systemic deficiencies by providing a *Lactobacillus johnsonii* preparation technology that balances processing and application stability, possessing significant industrial application value and market potential. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a preparation method that simultaneously improves the processing and storage stability of *Lactobacillus johnsonii* and its tolerance to digestive tracts. Fermentation is carried out using a non-animal-derived, highly stable fermentation medium formulation. This formulation employs a multifunctional synergistic pH regulation system integrating pH buffering, acidity neutralization, and nutrient utilization. A segmented pH control strategy is used during fermentation to effectively increase the viable cell count of the *Lactobacillus johnsonii* fermentation broth, enhance the stability of the fermentation stage, strengthen the cell's resistance to stress, and prolong the fermentation stability period. After fermentation, a six-component composite freeze-drying protectant formulation with synergistic effects is used in the preparation of the bacterial powder, effectively improving the freeze-drying survival rate and storage stability of the bacterial powder. Through the above fermentation and freeze-drying processes, the obtained *Lactobacillus johnsonii* active bacterial powder possesses excellent digestive tract tolerance.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A whole-chain enhancement of Lactobacillus johnsonii ( Lactobacillus johnsonii The method for preparing stable microbial culture medium includes: fermentation culture using a non-animal-derived fermentation medium combined with a multifunctional synergistic pH control system; a segmented pH control strategy of "constant pH first, then loose pH" is adopted during the culture process, that is, the pH is kept constant in the early stage of fermentation, and the pH control is released in the later stage of fermentation to allow the fermentation broth to naturally acidify; after the fermentation is completed, the microbial sludge is collected, and the microbial sludge is made into microbial powder using a six-element composite freeze-drying protectant; the stability of the microbial powder is improved throughout the entire chain of processing, storage and application.

[0008] In the above technical solution, the nitrogen source in the culture medium is a combination of yeast peptone, wheat peptone, and yeast extract.

[0009] Furthermore, the multifunctional synergistic pH regulation system in the culture medium consists of a buffer salt and an acidity regulator, wherein the buffer salt is a combination of citric acid, potassium citrate, and ammonium acetate, and the acidity regulator is ammonia.

[0010] Furthermore, based on a mass-volume ratio of g / 100 mL, the culture medium consists of: maltose 2-10, yeast peptone 2-8, wheat peptone 2-8, yeast extract 1-5, citric acid 0.1-0.8, potassium citrate 0.2-0.8, ammonium acetate 0.2-0.8, trace elements 0.01-0.02, with the remainder being deionized water; the pH is adjusted to 6.5±0.1.

[0011] Furthermore, the segmented pH control strategy involves maintaining the pH of the fermentation broth at 5.5±0.1 by adding ammonia water during the early stage of fermentation (usually 9-10 hours after fermentation begins, during which the number of viable bacteria can approach its peak). Then, in the later stage of fermentation, the fermentation broth is allowed to naturally acidify, and the pH value gradually decreases from 5.5 to 4.0~4.2 (the total fermentation time is usually 12-13 hours).

[0012] Furthermore, the hexa-component lyophilization protectant is composed of sucrose, trehalose, pea protein, PVP-K30, polydextrose, and sodium isovitamin C.

[0013] Furthermore, based on a mass-volume ratio of g / 100mL, the composition of the hexavalent composite freeze-drying protectant is as follows: 2-8g sucrose, 2-8g trehalose, 4-10g pea protein, 5-15g PVP-K30, 1-8g polydextrose, 0.5-2.0g sodium isovitamin C, with the remainder being deionized water.

[0014] Furthermore, the sterilized hexa-component lyophilization protectant is thoroughly mixed with the collected bacterial sludge at a mass ratio of 1:1 to 4, stirred in an ice bath for 10 to 15 minutes, and then freeze-dried and pulverized to obtain bacterial powder.

[0015] Furthermore, the stability of the entire chain is improved, specifically including: stable viable cell count at the end of fermentation, improved batch-to-batch stability, extended fermentation stability period, improved freeze-drying survival rate, improved storage stability, and improved digestion fluid tolerance.

[0016] The present invention also provides Lactobacillus johnsonii powder prepared by the above preparation method, which has relatively better application stability and good digestive tract tolerance.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. Fermentation efficiency and safety are synergistically improved, with controllable batch-to-batch stability. Addressing the shortcomings of existing Lactobacillus johnsonii culture media, such as low viable cell counts, reliance on animal-derived raw materials, and poor batch-to-batch stability, this invention constructs a non-animal-derived fermentation medium using maltose, yeast peptone, wheat peptone, and yeast extract, eliminating the risk of exogenous factors from animal-derived raw materials at the source. Using this invention's non-animal-derived culture medium, the viable Lactobacillus johnsonii count can reach 4.79 × 10⁻⁶ after 10 hours of fermentation. 10 CFU / mL, compared to 9.0 × 10⁻⁶ in MRS medium. 8 The CFU / mL increased by approximately 53 times. Meanwhile, three independent fermentation experiments verified that using non-animal-derived culture media resulted in more stable terminal viable cell counts with a coefficient of variation ≤10%, significantly superior to culture media containing animal nitrogen sources, demonstrating a substantial improvement in batch-to-batch stability.

[0018] 2. Significantly enhanced fermentation stability, effectively delaying cell death. Existing industrial fermentation systems using buffer salts only possess a single pH buffering function, failing to provide nutritional support for cell metabolism and lacking synergistic effects with neutralizing agents, leading to easy cell death and a rapid decline in viable cell count in the later stages of fermentation. Simultaneously, conventional constant-pH fermentation keeps cells in a constant environment for extended periods, resulting in weak resistance and a very narrow operating window for tank closing. While some studies have explored segmented pH control, they haven't been adapted to the metabolic characteristics of *Lactobacillus johnsonii*. To address these shortcomings, this invention provides an integrated solution combining a multifunctional synergistic pH control system with a "constant then relaxed" segmented pH control strategy. First, the citric acid-potassium citrate-ammonium acetate buffer system used in this invention has a synergistic effect with the neutralizing agent ammonia, combining buffering and nutritional functions: citric acid, as a key intermediate in the glycolysis pathway, can directly provide energy for *Lactobacillus johnsonii* metabolism; NH4... + It can serve as a nitrogen source to further promote cell proliferation and maintain cell activity. Based on this, the present invention further implements a "constant then relaxed" segmented pH control strategy—maintaining a constant pH in the early stage of fermentation to ensure high-density and rapid cell proliferation; and appropriately relaxing pH control in the later stage of fermentation to induce acid stress adaptation in the cells. Under the above synergistic effect, the peak viable cell count of 6.84 × 10⁻⁶ was reached after 10 hours of fermentation. 10 CFU / mL, the viable bacterial count remained at 6.52 × 10⁻⁶ after 12 h. 10 Compared to the strategy of maintaining a constant pH of 5.5 throughout the entire fermentation process, the segmented pH control strategy of this invention extends the fermentation stabilization period by nearly 3 hours, providing ample time for tank collection in large-scale industrial production. This effectively solves the industry pain point of rapid death of Lactobacillus johnsonii and difficulty in tank collection during the later stages of fermentation.

[0019] 3. Significantly improved freeze-drying survival rate and storage stability. Addressing the shortcomings of existing freeze-drying protectants, which are mostly simple compound formulations lacking synergistic effects, this invention provides a six-component composite freeze-drying protectant. The components work synergistically through multiple mechanisms: disaccharides (sucrose + trehalose) replace water molecules to maintain cell membrane and protein structure; pea protein forms a protective layer to mitigate ice crystal damage; PVP-K30 increases the glass transition temperature and enhances physical support; polydextrose constructs a three-dimensional framework and isolates oxygen; sodium isovitamin C scavenges free radicals and prevents oxidative damage. The six components synergistically enhance each other from multiple dimensions, including structural maintenance, physical barrier function, and oxidative protection. Using this protectant, the viable bacterial count in the freeze-dried bacterial powder reaches as high as 1.12 × 10⁻⁶. 12 The CFU / g count was maintained at 94.87% after freeze-drying. Furthermore, after storage at 4℃ and 25℃ for 360 days, the viable bacterial count remained at 6.74 × 10⁻⁶. 11 CFU / g and 5.59×10 11 After storage at 37℃ for 90 days, the viable bacterial count (CFU / g) remained at 4.61 × 10⁻⁶. 11CFU / g, all of the above indicators are significantly better than the levels of existing protective agents.

[0020] 4. Significantly improved digestive fluid tolerance, ensuring effective practical application. Existing technologies often focus on the processing and storage indicators of *Lactobacillus johnsonii*, neglecting its digestive tract tolerance during in vivo application. This leads to the bacterial powder being easily damaged by digestive fluids after oral administration, hindering effective colonization and efficacy. This invention significantly improves the digestive tract tolerance of the bacteria through synergistic optimization of the fermentation process and freeze-drying protection system. The *Lactobacillus johnsonii* freeze-dried bacterial powder prepared using the method provided by this invention achieves a survival rate of 90.50% after 2 hours of treatment with simulated gastric juice at pH 2.0, and more than half still survive after 4 hours, ensuring that the bacteria can effectively pass through gastric juices after oral administration and exert the expected efficacy.

[0021] 5. The process is simple and easy to industrialize. The raw materials used in this invention are widely available, the process steps are clear, no special equipment is required, the cost is controllable, and it is easy to achieve large-scale and standardized production. Attached Figure Description

[0022] Figure 1 This is a graph showing the trend of Lactobacillus johnsonii viable count over time in each experimental group under different buffer salt systems and neutralizing agent combinations in Example 3. Figure 2 This is a graph showing the trend of the number of viable Lactobacillus johnsonii bacteria over time in each experimental group under different pH control strategies in Example 4. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0024] Lactobacillus johnsonii HL-RH01 of this invention was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34776 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0025] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0026] According to some embodiments of the present invention, a preparation method capable of enhancing the stability of Lactobacillus johnsonii across the entire chain is provided, specifically comprising the following steps: Step 1: Preparation of non-animal-derived fermentation medium The non-animal-derived high-density culture medium, by mass-volume ratio (g / 100 mL), comprises: maltose 2-10, yeast peptone 2-8, wheat peptone 2-8, yeast extract 1-5, citric acid 0.1-0.8, potassium citrate 0.2-0.8, ammonium acetate 0.2-0.8, trace elements 0.01-0.02, with the remainder being deionized water; adjust the pH to 6.5±0.1, sterilize at 115℃ for 20 min, and cool for later use.

[0027] Step 2: Strain activation and high-density culture Lactobacillus johnsonii HL-RH01 glycerol culture medium was inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured statically at 37 ℃ for 24 h to obtain the first-generation seed culture. The first-generation seed culture was then passaged again under the same conditions to obtain the second-generation seed culture. The second-generation seed culture was then inoculated into the medium prepared in step one at a 4% (v / v) inoculum and cultured at 37 ℃ and 100 rpm for 12 h. Sterile nitrogen gas was continuously introduced during the culture process at a rate of 0.5~1.0 L / min.

[0028] Step 3: Implementation of the segmented pH control strategy A segmented pH control strategy of "constant first, then relaxed" is adopted, specifically including: (1) Initial constant pH control stage: 20%~30% ammonia water is added from 0 to 9 h to maintain the pH value of the fermentation broth at 5.5±0.1, ensuring rapid proliferation of cells.

[0029] (2) Later stage of natural acidification: Stop adding neutralizing agent after 9-12 hours, turn off the pH control system, and let the fermentation broth acidify naturally. The pH value gradually decreases from 5.5 to 4.0~4.2.

[0030] Step 4: Bacterial cell collection After fermentation, centrifuge at 4 ℃ and 4000~6000 rpm for 10~15 min to collect the bacterial sludge.

[0031] Step 5: Preparation of the hexa-component composite freeze-drying protectant The hexavalent composite freeze-drying protectant, by mass-volume ratio (g / 100mL), comprises: 2-8g sucrose, 2-8g trehalose, 4-10g pea protein, 5-15g PVP-K30, 1-8g polydextrose, 0.5-2.0g sodium isovitamin C, with the balance being deionized water. All components are mixed thoroughly, sterilized at 105℃ for 10 min, and cooled for later use.

[0032] Step Six: Preparation of the Mycotic Mushroom-Protective Agent Mixture After sterilization, the protective agent solution and the bacterial sludge obtained in step four are thoroughly mixed at a mass ratio of 1:1~4 (m / m), and stirred in an ice bath for 10~15 min.

[0033] Step 7: Freeze-drying The mixture obtained in step six is ​​filled into the tray of a freeze dryer and freeze-dried according to the set program. After freeze-drying, the appearance of the freeze-dried product is checked, and after crushing, sieving, and packaging, active freeze-dried Lactobacillus johnsonii powder is obtained.

[0034] Using the preparation method of this invention, the viable count of Lactobacillus johnsonii fermentation broth reached as high as 6.84 × 10⁻⁶. 10 CFU / mL, the viable count of the lyophilized bacterial powder reached 1.12 × 10⁻⁶ CFU / mL. 12 The CFU / g count was 94.87% after freeze-drying. After storage at 4℃ and 25℃ for 360 days, the viable bacterial counts were 6.74 × 10⁻⁶ CFU / g and 94.87% respectively. 11 CFU / g and 5.59×10 11 After storage at 37℃ for 90 days, the viable bacterial count (CFU / g) remained at 4.61 × 10⁻⁶. 11 CFU / g. After treatment with simulated gastric juice at pH 2.0 for 2 h, the survival rate of the strain was 90.50%, and more than half remained viable after 4 h. This method significantly improves the viable count and stability of *Lactobacillus johnsonii* during fermentation, freeze-drying, and storage, and enhances its tolerance to digestive juices. It achieves a comprehensive improvement in the processing stability and product quality of *Lactobacillus johnsonii*, solving industry pain points such as difficulty in culturing important probiotic varieties, easy inactivation during processing, rapid degradation during storage and transportation, and weak resistance to digestive juices. Furthermore, this preparation method is simple, uses widely available raw materials, has controllable costs, and is easy to implement for large-scale, standardized production of *Lactobacillus johnsonii*, demonstrating significant economic benefits and application prospects.

[0035] Example 1: Construction of non-animal-derived culture medium and comparison of fermentation performance Using MRS medium (commercially available MRS broth produced by Qingdao Haibo Biotechnology Co., Ltd.) as a control, seven different carbon and nitrogen source combinations were designed, as shown in Table 1. Except for the components listed in Table 1, all other components were identical. The components, by mass-volume ratio (g / mL), were: triammonium citrate 0.2%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.01%, manganese sulfate 0.005%, Tween-80 0.1%, and deionized water to a final volume of 100 mL. The pH was adjusted to 6.5 ± 0.1.

[0036] Fermentation experiments were conducted in a 5 L fermenter with a liquid volume of 3 L and an inoculum size of 4% (v / v). The culture temperature was 37 ℃, and the stirring speed was 100 rpm. During fermentation, NaOH was added to maintain the pH at 5.5 ± 0.1. Samples were taken at 8, 9, 10, 11, and 12 h of fermentation. Plate counting was performed using an appropriate dilution gradient, with three plates for each dilution. Results are expressed as the mean ± standard deviation of the three plate counts. The effects of different culture media on the proliferation ability of Lactobacillus johnsonii were investigated.

[0037] Table 1. Carbon and nitrogen source composition of different culture media

[0038] Table 2. Changes in viable Lactobacillus johnsonii count over time in different culture media (×10⁻¹⁰) 10 CFU / mL

[0039] Table 2 shows the changes in viable counts of *Lactobacillus johnsonii* in different culture media over time. As can be seen from Table 2, the type, combination, and amount of carbon and nitrogen sources in the culture medium all significantly affected the viable counts of *Lactobacillus johnsonii* in the fermentation broth. The control group reached its highest viable count of 9.0 × 10⁻¹⁰ cells / hour at 9–10 h. 8 CFU / mL. Compared with the control group, Experiment 1 increased the amount of carbon and nitrogen sources proportionally. The results showed a significant increase in viable cell counts at all detection time points, indicating that appropriately increasing the concentration of nutrients can stimulate cell proliferation. Experiment 2, after changing the type of carbon source, further increased the viable cell count, indicating that maltose is more conducive to cell proliferation. Experiment 3, replacing tryptone with yeast peptone, significantly increased the viable cell count, indicating that yeast peptone provides nutrients more suitable for the growth and reproduction of *Lactobacillus johnsonii*. However, in Experiment 4, after replacing beef extract with yeast peptone and yeast extract, the viable cell count dropped sharply to the level of MRS medium, indicating that beef extract provides the necessary nutrients, and simple removal cannot meet the needs of high-density culture of *Lactobacillus johnsonii*. Experiments 5-7 used different plant-derived peptones to replace beef extract, and simultaneously increased the amount of yeast extract added. The results showed that plant-derived peptone could not only effectively replace beef extract powder, but also significantly increase the viable cell count in the fermentation broth at each detection time point. Experiment 7 showed the best results, with a peak viable cell count as high as 4.79 × 10⁻⁶. 10 CFU / mL. The differences in peptone from different plant sources may be related to the higher glutamic acid and proline content and suitable peptide molecular weight distribution in wheat peptone.

[0040] Compared to existing research reports, the viable count of *Lactobacillus johnsonii* in conventional MRS medium or animal-derived modified medium is typically 5 × 10⁻⁶. 8 ~1×10 10The CFU / mL count was lower than that in Experiment 7 of this invention, where the peak viable count reached 4.79 × 10⁻⁶ under non-animal-derived culture conditions. 10 The CFU / mL level is significantly higher than existing fermentation levels. These results indicate that the culture medium provided by this invention, using maltose as a carbon source and a combination of yeast peptone, yeast extract, and wheat peptone as a non-animal source nitrogen source, can synergistically support high-density cultivation of *Lactobacillus johnsonii*, meeting the requirements for industrial production and safety.

[0041] Example 2: Effect of non-animal-derived culture media on batch-to-batch stability of fermentation To investigate the batch-to-batch stability differences between animal-derived and non-animal-derived culture media, the following culture media (Table 3) were subjected to three consecutive independent fermentations under the same fermentation conditions as in Example 1. Based on previous experience, samples were taken when the viable cell count in the fermentation broth reached its peak. Plate counting was performed using an appropriate dilution gradient, with three plates spread for each dilution. The results from each batch were expressed as the average value. The coefficient of variation was then calculated based on the average value and standard deviation of the three batches to assess the fermentation stability of each culture medium.

[0042] Table 3. Composition of culture media for fermentation stability studies

[0043] Table 4. Peak viable cell counts and their variations in three consecutive batches of independent fermentation on different culture media.

[0044] Table 4 shows that the coefficients of variation for Experiments 1 and 2 both exceeded 15%, indicating that simply adjusting the carbon source had no effect on fermentation stability. In Experiment 3, after replacing tryptone with yeast peptone in the nitrogen source, the coefficient of variation decreased to 12.34%, indicating that removing the undefined composition of tryptone improved the stability of the fermentation process to some extent. In Experiments 4 and 7, after completely removing the animal-derived nitrogen source, the results showed that the coefficient of variation decreased to below 10%, with small batch-to-batch differences and good stability. These results demonstrate that the non-animal-derived culture medium constructed in this invention not only enables high-density cultivation of *Lactobacillus johnsonii* but also possesses excellent batch consistency, effectively avoiding product quality fluctuations caused by batch differences.

[0045] Example 3: The effect of synergistic pH regulation by buffer salts and neutralizers on fermentation stability Analysis of the data in Examples 1 and 2 revealed that although non-animal-derived components significantly improved batch-to-batch stability during fermentation, the viable cell count still decreased significantly in the later stages of a single fermentation process. This may be due to the inherent sensitivity of *Lactobacillus johnsonii* to acidic environments. Therefore, in this example, four different combinations of buffer salt systems and neutralizing agents were set up based on the non-animal-derived culture medium. The buffer salt and NaOH neutralizing agent in MRS medium were used as a comparative example (see Table 5). The pH value was controlled at 5.5 ± 0.1 during fermentation, and the other culture medium components and fermentation conditions were the same as in Experiment 7 of Example 1. Samples were taken at 8, 9, 10, 11, and 12 h of fermentation, and plate counting was performed using a suitable dilution gradient. Three plates were spread for each dilution, and the results are expressed as the mean ± standard deviation of the three plate counts.

[0046] Table 5. Combinations of different buffer salt systems with neutralizing agents

[0047] Table 6. Changes in viable Lactobacillus johnsonii count over time under different combinations of buffer salt systems and neutralizing agents (×10) 10 CFU / mL

[0048] Table 6 shows the changes in viable cell count over time in fermentation broths with different buffer salt systems and neutralizing agent combinations. The comparative example used a sodium acetate-dipotassium hydrogen phosphate-triammonium citrate buffer system with NaOH as the neutralizing agent, a commonly used combination in traditional culture. In Experiment 8, sodium acetate was replaced with ammonium acetate in the buffer salt, while the remaining components remained the same as in the comparative example, aiming to initially reduce the introduction of sodium ions. Experiment 9 further replaced the neutralizing agent from NaOH to ammonia water to evaluate the effect of ammonia water on fermentation stability. The results showed that the viable cell count in Experiments 8 and 9 still showed a significant downward trend after 10 h of fermentation, and the peak viable cell count was not significantly higher than that in the comparative example, indicating that simply replacing sodium acetate or using ammonia water did not effectively delay cell death. Experiments 10 and 11 adjusted the buffer salt system to an ammonium acetate-citric acid-potassium citrate combination, with NaOH used as the neutralizing agent in Experiment 10 and ammonia water used in Experiment 11. The results showed that the viable cell count in Experiment 10 reached 5.50 × 10⁻⁶ after 10 h of fermentation. 10 CFU / mL, compared to 4.79 × 10⁻⁶ in the control group. 10 The CFU / mL increased, but the viable bacterial count began to decline after 10 h, dropping to 3.05 × 10⁻⁶ after 12 h. 10 CFU / mL. In Experiment 11, the viable cell count reached 5.58 × 10⁻⁶ after 10 h of fermentation. 10 The viable count remained stable at 4.50 × 10⁻⁶ CFU / mL from 10 to 12 hours. 10The optimal fermentation stability was observed at approximately CFU / mL. The trends in viable cell counts for each group are shown below. Figure 1 As shown.

[0049] Experiment 11 demonstrated good fermentation stability, which may be related to the participation of citric acid as an intermediate product of glycolysis in cell metabolism. Simultaneously, under this buffer salt system, the NH4+ produced by the reaction of organic acids with ammonia water can provide a small amount of nitrogen source for the strain's growth without introducing excessive sodium ions, thus reducing the inhibitory effect of excessively high ion concentrations on strain proliferation. In summary, the synergistic effect of the ammonium acetate-citric acid-potassium citrate buffer salt system and ammonia water can effectively increase the viable cell count and fermentation stability of *Lactobacillus johnsonii*, further enhancing its industrial fermentation performance.

[0050] Example 4: The effect of segmented pH control strategy on the fermentation stationary phase Example 3 revealed that pH changes during fermentation significantly affect the fermentation stability of *Lactobacillus johnsonii*, and good pH control can effectively reduce cell death. This example uses the non-animal-derived culture medium, buffer salt system, and neutralizing agent from Experiment 11 of Example 3, and sets up six different pH control strategies (Table 7) to monitor changes in viable cell counts during fermentation. Plate counting was used, with appropriate dilution gradients selected for plating, and three plates were spread for each dilution. Results are expressed as the mean ± standard deviation of the three plate counts.

[0051] Table 7. Settings for different pH control strategies

[0052] Table 8. Changes in viable Lactobacillus johnsonii count over time under different pH control strategies (×10) 10 CFU / mL

[0053] Table 8 shows that the viable cell counts in all active pH control groups (Experiments 13-17) were significantly higher than those in the naturally acidified group (Experiment 12), indicating that active addition of neutralizing agent can prevent excessive acidification of the fermentation broth and is beneficial to cell growth. Among the constant pH control groups, Experiment 14 showed the highest viable cell counts at all test time points, indicating that the optimal pH for *Lactobacillus johnsonii* growth is 5.5. Experiment 17 employed a segmented pH control strategy, which aimed to provide the optimal pH environment in the early stage to promote rapid cell proliferation, and then induce cell stress adaptation through moderate acid stress in the later stage, thereby delaying cell death. The results showed that the viable cell count in Experiment 17 reached a peak of 6.84 × 10⁻⁶ after 10 h of fermentation. 10 The CFU / mL count was approximately 1.2 times that of the optimal constant pH group (Experiment 14); although the viable bacterial count decreased slightly after 10 h, it remained at 6.52 × 10⁻⁶ at 12 h. 10The CFU / mL count decreased by 4.7%, which was significantly better than Experiment 14 (4.56 × 10⁻⁶ viable bacteria count after 12 h). 10 CFU / mL, a decrease of 21.6%. The trends of change in each experimental group are as follows: Figure 2 As shown.

[0054] As can be seen from the above two embodiments, the multifunctional synergistic pH regulation system and the segmented pH control strategy described in this invention can synergistically increase the number of viable fermentation bacteria of Lactobacillus johnsonii, prolong the stationary period, and slow down the rate of decline, thus providing a stable and efficient fermentation process for its industrial production.

[0055] Example 5: Effect of a hexavalent composite preservative on the survival and storage stability of Lactobacillus johnsonii freeze-dried bacteria. Fermentation was carried out using the process described in Experiment 17 of Example 4 of this invention. The mixture was centrifuged at 4 ℃ and 4000 rpm for 10-15 min, and the bacterial sludge was collected. The obtained bacterial sludge was thoroughly mixed with different protectants listed in Table 9 at a mass ratio of 1:2. After thorough mixing, samples were immediately taken from each mixture, and the viable cell count was determined using the plate count method. This viable cell count was used to calculate the freeze-drying survival rate. Comparative Examples 1-4 were selected from existing publicly available technologies (screened based on the characteristics of the microbial species and the availability of raw materials). Experiment 18 used a general-purpose protectant commonly used in the laboratory of this invention, while Experiments 19-22 used specialized protectants designed specifically for the growth and metabolic characteristics of *Lactobacillus johnsonii*.

[0056] Table 9. Composition of different freeze-drying protectants

[0057] The bacterial powder was prepared by freeze-drying according to the set procedure. Immediately after freeze-drying, samples were taken to determine the viable count of the bacterial powder, which was taken as the viable count for day 0. According to the formula, freeze-drying survival rate (%) = viable count of bacterial powder after freeze-drying / viable count before freeze-drying * 100%, the freeze-drying survival rate of each group was calculated, and the results are shown in Table 10.

[0058] The bacterial powder was stored at 4 ℃, 25 ℃, and 37 ℃ respectively, for 360 days at 4 ℃ and 25 ℃, and for 90 days at 37 ℃. During storage, samples were taken periodically to determine the viable bacterial count, and the results are shown in Tables 11-13.

[0059] The viable cell count was determined using the plate count method. 3.00 g of bacterial powder sample was accurately weighed and reconstituted in physiological saline. After serial dilution, an appropriate dilution gradient was selected for plate counting. Each dilution was plated on 3 plates. The results are expressed as the mean of the three plate counts ± standard deviation.

[0060] Table 10. Lyophilized survival rate of Lactobacillus johnsonii under different protectants

[0061] Table 10 shows that the freeze-drying survival rates of the four specific protectant formulations (Experiments 19-22) designed for Lactobacillus johnsonii were all superior to those of the general-purpose protectant (Experiment 18). Among them, the protectant in Experiment 19 was the most effective, with a freeze-drying survival rate as high as 94.87% and a viable count of 1.12 × 10⁻⁶ after freeze-drying. 12 CFU / g. Experiments 20-22 removed pea protein, PVP-K30, and polydextrose from the formulation of Experiment 19, respectively, resulting in lyophilization survival rates of 65.08%, 44.07%, and 72.60%, respectively. The varying degrees of decrease in lyophilization survival rates indicate that all six components in the formulation of Experiment 19 are necessary for the lyophilization protection of *Lactobacillus johnsonii*.

[0062] The freeze-drying survival rates of the protectants provided in Comparative Examples 1 and 2 for *Lactobacillus johnsonii* were 58.33% and 49.58%, respectively, which are similar to existing reports but significantly lower than those in Experiment 19 of this invention. The freeze-drying survival rates of Comparative Examples 3 and 4 were 81.5% and 72.85%, respectively, lower than the reported data (92.90% and 93.22%) and also lower than Experiment 2 (94.87%). The reason for this is that Comparative Examples 3 and 4 achieved better freeze-drying protection (reported freeze-drying survival rates exceeding 90%) for naturally resistant strains, such as *Lactobacillus plantarum*. However, the same protectant formulation significantly weakened the protective effect when applied to *Lactobacillus johnsonii*. This indicates that the protective effect of the protectant has significant species selectivity. For strains with poor natural tolerance, a specialized protectant formulation needs to be designed based on their growth and metabolic characteristics to achieve better freeze-drying protection.

[0063] To further investigate the effect of the six-component composite preservative formulation on the storage stability of Lactobacillus johnsonii, the changes in viable cell count were monitored under the possible temperature conditions during the storage of the bacterial powder.

[0064] Table 11. Viable bacterial count in powder at 4℃ (×10⁻¹⁰) 10 Changes in CFU / g

[0065] 4 ℃ was the standard refrigeration condition for the mycelium powder. As shown in Table 11, the viable cell counts in all groups decreased with prolonged storage. After 30 days of storage, the relative survival rate of Experiment 19 was basically the same as that of Comparative Example 1, Comparative Example 3, and Experiment 22; however, from 60 days onwards, the protective effects of each group showed differences, and the attenuation rate of Experiment 19 slowed significantly. Even after 360 days of storage, the viable cell count of Experiment 19 remained at 6.74 × 10⁻⁶. 11At the CFU / g level, the relative survival rate was 60.02%, significantly better than Comparative Example 1 (34.78%), Comparative Example 2 (26.08%), Comparative Example 3 (45.97%), and Comparative Example 4 (40.52%), and also significantly higher than Experiment 18 (43.18%), Experiment 20 (45.83%), Experiment 21 (44.39%), and Experiment 22 (52.26%). These results indicate that the hexa-component composite protectant of this invention can provide a more sustained and stable protective effect under long-term low-temperature storage conditions.

[0066] Table 12. Viable bacterial count in powder at 25℃ (×10⁻¹⁰) 10 Changes in CFU / g

[0067] 25 ℃ represents room temperature storage conditions. At this temperature, bacterial metabolic activity is enhanced, and the rate of viable cell count decline is significantly faster than at 4 ℃. Table 12 shows that Experiment 19 exhibited optimal storage stability at all time points, with its stability advantage becoming increasingly apparent as storage time increased. After 360 days of storage, the viable cell count in Experiment 19 remained as high as 5.59 × 10⁻⁶. 11 The relative survival rate was 49.81% (CFU / g), while the viable counts in all comparative examples and other experiments decreased significantly. Specifically, the survival rates of Comparative Example 4 and Experiment 18 dropped to <0.01% by day 360; Comparative Example 3 showed good performance in the early stages of storage, but its survival rate was only 3.87% by day 360. Experiments 20-22, lacking pea protein, PVP-K30, and polydextrose respectively, had survival rates of only 5.80%, 2.10%, and 10.74% by day 360, respectively, showing an order-of-magnitude difference compared to Experiment 19, further verifying the necessity of the synergistic effect of the six-component combination for room temperature storage protection. These results indicate that the six-component composite protectant provided by this invention can significantly improve the room temperature storage stability of probiotic powder, effectively overcoming the industry bottleneck of easy inactivation of probiotic powder at room temperature.

[0068] Table 13. Viable bacterial count in powder under storage conditions at 37℃ (×10⁻¹⁰) 10 Changes in CFU / g

[0069] Based on the 25 ℃ condition, the stability of the mycelium powder was further investigated at 37 ℃ to assess the impact of potential exposure to high temperatures during storage and distribution on its viable cell count. Table 13 shows that the viable cell count decline was more severe at 37 ℃ than at 4 ℃ and 25 ℃, exhibiting a rapid decline, with significant differences in the decline rate among the groups. Comparative Examples 1-3 showed relatively gradual decline in the early stages, while Comparative Example 4 showed the most rapid decline. In contrast, except for Experiment 18, the survival rate of the other experimental groups remained above 50% on day 15, with Experiment 19 having the highest survival rate (70.54%). After 90 days of storage, the differences between the groups widened further, with Experiment 19 maintaining a viable cell count of 4.61 × 10⁻⁶. 11 The relative survival rate was 41.07% (CFU / g), while the survival rates of Comparative Example 4 and Experiment 18 were only 0.11% and 3.37%, respectively. These results indicate that the hexa-component composite freeze-drying protectant provided by this invention can significantly improve the high-temperature resistance of the bacterial powder and effectively reduce the decline in the number of viable bacteria in the powder under high-temperature conditions.

[0070] Based on data on storage stability under low temperature, room temperature, and high temperature conditions, the six-element composite freeze-drying protectant formulation provided by this invention can not only improve the survival of Lactobacillus johnsonii during the freeze-drying process, but also improve its storage stability under different temperature conditions, especially at 25℃ and 37℃.

[0071] Example 6: Tolerance assessment of bacterial powder in simulated gastric fluid Accurately weigh 0.50 g of the bacterial powder from day 0 in Example 5 and dissolve it in 4.5 mL of artificial gastric fluid (pH=2.0). After thorough shaking and mixing, incubate at 37 ℃ for 1, 2, and 4 h, vortexing for 20 s every 10 min during the incubation period. Measure the viable count of the bacterial suspension, using the viable count of the freeze-dried bacterial powder measured in Example 5 as the initial viable count.

[0072] Viable bacterial counts were determined using the plate count method. Appropriate dilution gradients were selected for plate counting, with three plates for each dilution. Results were expressed as the average of the three plate counts ± standard deviation. The survival rate of each bacterial powder in gastric juice was calculated using the formula: Gastric juice viability (%) = (Number of viable bacteria at each test time / Initial number of viable bacteria) * 100% (Table 14).

[0073] Table 14 Viable bacteria count of bacterial powder in artificial gastric fluid (pH 2.0) (×10⁻¹⁰) 10 CFU / g change over time

[0074] The tolerance of probiotics to digestive juices significantly affects their efficacy. Human gastric juice is highly acidic, with a pH value generally fluctuating around 2.0. Most probiotics are easily inactivated after entering the gastric juice environment. As shown in Table 14, the bacterial powders in Experiment 19 exhibited the best gastric acid resistance. After incubation in artificial gastric juice for 1 hour, the survival rate reached 95.33%, and after incubation for 2 hours, it still maintained a high survival rate of 90.50%. After 4 hours of treatment, the survival rate of the strains remained at 50.85%, significantly better than the comparative groups and the other experimental groups. These results indicate that the six-component composite lyophilization protectant can effectively resist the damage to bacterial cells caused by a highly acidic environment and significantly improve the gastric acid tolerance of Lactobacillus johnsonii.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A whole-chain enhancement method for Lactobacillus johnsonii ( Lactobacillus johnsonii The method for preparing the stability is characterized by, include: Fermentation was carried out using a non-animal-derived fermentation medium with a multifunctional synergistic pH control system. During the cultivation process, a segmented pH control strategy of "constant pH first, then loose pH" was adopted, that is, the pH was kept constant in the early stage of fermentation and the pH control was released in the later stage of fermentation. After the fermentation was completed, the mycelium sludge was collected and mycelium powder was made into mycelium powder using a six-element composite freeze-drying protectant. The stability of the mycelium powder was improved throughout the entire chain of processing, storage and application.

2. The preparation method according to claim 1, characterized in that, The nitrogen source in the culture medium is a combination of yeast peptone, wheat peptone, and yeast extract.

3. The preparation method according to claim 1, characterized in that, The multifunctional synergistic pH regulation system in the culture medium consists of a buffer salt and an acidity regulator, wherein the buffer salt is a combination of citric acid, potassium citrate and ammonium acetate, and the acidity regulator is ammonia.

4. The preparation method according to claim 1, characterized in that, The culture medium, based on a mass-volume ratio of g / 100 mL, consists of: maltose 2-10, yeast peptone 2-8, wheat peptone 2-8, yeast extract 1-5, citric acid 0.1-0.8, potassium citrate 0.2-0.8, ammonium acetate 0.2-0.8, trace elements 0.01-0.02, with the remainder being deionized water; the pH is adjusted to 6.5±0.

1.

5. The preparation method according to claim 1, characterized in that, In the segmented pH control strategy, the pH is kept constant during the early stage of fermentation, from the start of fermentation until 9-10 hours of fermentation. During this process, the pH of the fermentation broth is maintained at 5.5±0.1 by adding ammonia water. After that, in the later stage of fermentation, the fermentation broth is allowed to naturally acidify, and the pH value gradually decreases from 5.5 to 4.0~4.2, at which point the fermentation ends.

6. The preparation method according to claim 1, characterized in that, The hexavalent composite freeze-drying protectant is composed of sucrose, trehalose, pea protein, PVP-K30, polydextrose, and sodium isovitamin C.

7. The preparation method according to claim 6, characterized in that, The composition of the hexavalent composite freeze-drying protectant, calculated by mass-volume ratio (g / 100 mL), is as follows: 2-8 g of sucrose, 2-8 g of trehalose, 4-10 g of pea protein, 5-15 g of PVP-K30, 1-8 g of polydextrose, 0.5-2.0 g of sodium isovitamin C, with the remainder being deionized water.

8. The preparation method according to claim 1, characterized in that, The sterilized hexa-component lyophilization protectant was thoroughly mixed with the collected mycelial mud at a mass ratio of 1:1 to 4, stirred in an ice bath for 10 to 15 minutes, and then freeze-dried and pulverized to obtain mycelial powder.

9. The preparation method according to claim 1, characterized in that, The stability of the entire chain has been improved, specifically including: stable viable cell count at the end of fermentation, improved batch-to-batch stability, extended fermentation stability period, improved freeze-drying survival rate, improved storage stability, and improved digestion fluid tolerance.

10. A Lactobacillus johnsonii powder, characterized in that, It is prepared by the method described in any one of claims 1-9.

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