A probiotic composition and its application in traditional Chinese medicine fermentation

Abstract

This invention relates to the field of bio-fermentation, and discloses a probiotic composition and its application in the fermentation of traditional Chinese medicine. The probiotic composition of this invention includes *Saccharomyces cerevisiae* and *Lactobacillus*. This probiotic composition exhibits excellent fermentation performance, possesses a rapid fermentation rate, and can significantly shorten the production cycle. It is suitable for the fermentation of traditional Chinese medicine, especially in the synthesis of flavonoids and polyphenolic compounds. These strains can not only achieve efficient conversion but also effectively increase the yield of the final product. Furthermore, the flavonoids and polyphenols produced by these strains have extremely high antioxidant activity, and the efficacy of the product can be guaranteed even over a long shelf life.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation, and more particularly to a probiotic composition and its application in the fermentation of traditional Chinese medicine. Background Technology

[0002] Traditional Chinese medicine (TCM) fermentation technology has gradually transitioned from traditional fermentation methods to modern fermentation methods. Modern TCM fermentation technology has expanded upon traditional fermentation methods by drawing on modern technologies such as pharmaceutical engineering and bioengineering, and has incorporated probiotics into the fermentation process to achieve the biotransformation of TCM by microorganisms.

[0003] Currently, fermentation methods are mainly classified into three categories based on the fermentation form and technology of traditional Chinese medicine: solid-state fermentation, liquid fermentation, and two-way fermentation of medicinal fungi. Liquid fermentation technology uses nutrient-rich liquid culture media to cultivate microorganisms, controlling conditions such as temperature, time, and initial pH to obtain the desired fermentation product. Liquid fermentation technology not only enables large-scale production and allows for easy control of fermentation conditions, but also effectively improves production efficiency and yield, making it suitable for industrialized and automated production.

[0004] Traditional Chinese medicine preparations fermented with probiotics typically contain abundant polyphenols, flavonoids, saponins, and other antioxidants, which can scavenge harmful free radicals in the body, slow down the aging process, and prevent the occurrence and development of various chronic diseases. The choice of probiotics has a significant impact on the composition and antioxidant activity of the products obtained after fermentation of traditional Chinese medicine. Therefore, continuously discovering new probiotic combinations suitable for traditional Chinese medicine fermentation is of positive significance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a probiotic composition and its application in the fermentation of traditional Chinese medicine. The probiotic composition of this invention exhibits excellent fermentation performance, possesses a rapid fermentation rate, and can significantly shorten the production cycle. It is suitable for the fermentation of traditional Chinese medicine, especially in the synthesis of flavonoids and polyphenolic compounds. These strains not only achieve efficient conversion but also effectively increase the yield of the final product. Furthermore, the combination of flavonoids and polyphenols metabolized by these strains with specific combinations of traditional Chinese medicinal materials possesses extremely high antioxidant activity.

[0006] The specific technical solution of this invention is as follows:

[0007] In a first aspect, the present invention provides a probiotic composition comprising Saccharomyces cerevisiae and Lactobacillus; wherein Lactobacillus includes at least one of the following:

[0008] a) Lacticaseibacillus paracasei ML-001 was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with accession number CGMCC No. 33434 and microbiological classification name Lacticaseibacillus paracasei.

[0009] b) Lactobacillus johnsonii 021, deposited on July 24, 2002 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.3221, and classified as Lactobacillus johnsonii; c) Lactobacillus casei ML-003, deposited on January 16, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33436, and classified as Lactobacillus casei.

[0010] In our previous studies, we independently isolated several probiotics, including the strains mentioned above (excluding *Lactobacillus johnsonii* O21, which was commercially available), and confirmed through research that they possess excellent activity in fermenting traditional Chinese medicine (TCM). Subsequent studies revealed that several of these strains exhibited outstanding free radical scavenging activity after fermenting TCM. Compared to other reported TCM fermentation products, their most notable characteristic is the high antioxidant activity of the fermentation broth obtained at relatively low initial inoculum levels. Furthermore, we found that the fermentation activity of these strains is highly stable. TCM fermentation products obtained through multi-stage fermentation in combination with *Saccharomyces cerevisiae* maintained high levels of antioxidant activity even after storage at different temperatures and low pH conditions, indicating that they can retain their efficacy in the human gut environment. Moreover, TCM products obtained through multi-stage fermentation of these strains still exhibit high antioxidant activity over a long shelf life at room temperature, a level of antioxidant activity not yet observed in studies on multi-stage fermentation products of this type of strain.

[0011] Preferably, the brewing yeast includes at least one of the following:

[0012] d) Saccharomyces cerevisiae ML-002 was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with accession number CGMCC No. 33435 and microbiological classification name Saccharomyces cerevisiae.

[0013] e) Saccharomyces cerevisiae XL3-1 was deposited on June 5, 2008, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 2.3857 and microbiological classification name Saccharomyces cerevisiae.

[0014] Through extensive experiments, this invention has found that in multi-stage fermentation processes, using the two aforementioned brewing yeast strains in combination with the aforementioned lactobacillus strains of this invention results in better multi-stage fermentation effects.

[0015] Preferably, the probiotic composition is selected from the following combinations:

[0016] Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii O21; or

[0017] Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus casei ML-003.

[0018] The present invention has found that the combination of the above two groups has the best effect.

[0019] Secondly, the present invention provides the application of the above-mentioned probiotic composition in the fermentation of traditional Chinese medicine.

[0020] The probiotic composition of this invention exhibits excellent fermentation performance and a rapid fermentation rate, significantly shortening the production cycle. It is highly suitable for the fermentation of traditional Chinese medicine, particularly in the synthesis of flavonoids and polyphenols. These strains not only achieve efficient conversion but also effectively increase the yield of the final product. Furthermore, the flavonoids and polyphenols produced by these strains possess extremely high antioxidant activity.

[0021] Preferably, the fermentation of the traditional Chinese medicine is a two-stage or three-stage fermentation.

[0022] Preferably, the probiotic composition contains two types of lactobacilli. More preferably, the probiotic composition is selected from the following combination: Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii 021; or Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus casei ML-003.

[0023] Preferably, in the fermentation process of the traditional Chinese medicine, *Saccharomyces cerevisiae* is inoculated first, followed by inoculation with one type of *Lactobacillus* after fermentation, and then inoculation with another type of *Lactobacillus* after further fermentation. More preferably, in the fermentation process of the traditional Chinese medicine, *Saccharomyces cerevisiae* is inoculated first for primary fermentation, followed by inoculation with *Lactobacillus paracasei* for secondary fermentation, and finally inoculation with *Lactobacillus johnsonii* or *Lactobacillus casei* for tertiary fermentation.

[0024] The probiotic composition of this invention is suitable for multi-stage fermentation processes, especially three-stage fermentation processes. Specifically, the inoculation sequence is to first inoculate *Saccharomyces cerevisiae*, followed by *Lactobacillus paracasei* and *Lactobacillus johnsonii* or *Lactobacillus casei* in sequence. The traditional Chinese medicine fermentation products prepared using the probiotic composition of this invention under multi-stage fermentation exhibit high antioxidant activity and maintain their efficacy over a long shelf life. Compared with existing technologies, the beneficial effects of this invention are:

[0025] (1) The strains in the probiotic composition of the present invention exhibit excellent free radical scavenging activity after fermenting traditional Chinese medicine. In particular, the fermentation broth obtained under low initial inoculum conditions has high antioxidant activity. Furthermore, the fermentation activity of these strains is very stable. The fermented traditional Chinese medicine products obtained through multi-stage fermentation maintain a high level of antioxidant activity even after storage at different temperatures and low pH conditions, and can maintain their efficacy in the human gastrointestinal environment.

[0026] (2) The probiotic composition of the present invention has excellent fermentation performance and a rapid fermentation rate, which can significantly shorten the production cycle. It is very suitable for the fermentation of traditional Chinese medicine, especially in the process of synthesizing flavonoids and polyphenols. These strains can not only achieve efficient conversion, but also effectively increase the yield of the final product. In addition, the flavonoids and polyphenols produced by these strains have extremely high antioxidant activity.

[0027] (3) The probiotic composition of the present invention is particularly suitable for multi-stage fermentation processes, and the synergistic effect of the various strains in the probiotic composition during multi-stage fermentation is good. The traditional Chinese medicine fermentation products prepared by fermenting the probiotic composition of the present invention under multi-stage fermentation have high antioxidant activity and can maintain the efficacy of the products within a long shelf life. Attached Figure Description

[0028] Figure 1 The changes in flavonoids, polyphenols, and polysaccharides in the fermentation broth of different strains are shown.

[0029] Figure 2 The changes in antioxidant activity of strain ML-001 under different initial inoculum amounts and fermentation times are shown.

[0030] Figure 3 The changes in antioxidant activity after 14 days of fermentation with different transfer days in the secondary fermentation stage.

[0031] Figure 4 The changes in antioxidant activity after 16 days of fermentation with different transfer days in the three-stage fermentation process. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments.

[0033] First, a probiotic composition comprising Saccharomyces cerevisiae and Lactobacillus; wherein the Lactobacillus includes at least one of the following: a) Lactobacillus paracasei ML-001, which was deposited on January 16, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 33434, and microbiologically classified as Lactobacillus paracasei;

[0034] b) Lactobacillus johnsonii 021, deposited on July 24, 2002 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.3221, and classified as Lactobacillus johnsonii; c) Lactobacillus casei ML-003, deposited on January 16, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33436, and classified as Lactobacillus casei.

[0035] In some preferred embodiments, the brewing yeast includes at least one of the following:

[0036] d) Saccharomyces cerevisiae ML-002 was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with accession number CGMCC No. 33435 and microbiological classification name Saccharomyces cerevisiae.

[0037] e) Saccharomyces cerevisiae XL3-1 was deposited on June 5, 2008, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 2.3857 and microbiological classification name Saccharomyces cerevisiae.

[0038] In some preferred embodiments, the probiotic composition is selected from the following combinations:

[0039] Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii O21; or

[0040] Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus casei ML-003.

[0041] Secondly, a method for applying the above-mentioned probiotic composition in the fermentation of traditional Chinese medicine.

[0042] In some preferred embodiments, the fermentation of traditional Chinese medicine is a two-stage or three-stage fermentation.

[0043] In some preferred embodiments, the probiotic composition contains two types of lactobacilli.

[0044] In some preferred embodiments, during the fermentation process of the traditional Chinese medicine, Saccharomyces cerevisiae is first inoculated, followed by inoculation with one type of Lactobacillus after fermentation, and then inoculation with another type of Lactobacillus after fermentation.

[0045] In some preferred embodiments, the probiotic composition is selected from the following combinations:

[0046] Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii O21; or

[0047] Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus casei ML-003.

[0048] In some preferred embodiments, the fermentation process of the traditional Chinese medicine involves first inoculating with *Saccharomyces cerevisiae* for primary fermentation, then inoculating with *Lactobacillus paracasei* for secondary fermentation, and finally inoculating with *Lactobacillus johnsonii* or *Lactobacillus casei* for tertiary fermentation. In some preferred embodiments, the primary fermentation is carried out at a temperature of 28-37℃ for 4-7 days, with an inoculation amount of 2.5 × 10⁻⁶. 5 -1×10 6 CFU / m³; Secondary fermentation: Temperature 28-45℃, fermentation time 1-6 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL; Three-stage fermentation: temperature 28-45℃, fermentation time 5-10 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL.

[0049] In the three-stage fermentation process, the timing of inoculation for each strain has a significant impact on the fermentation effect. The inoculation timing for multi-stage fermentation needs to consider pH changes and the metabolic characteristics of the microbial community during fermentation. Primary fermentation (4-7 days) ensures that the brewer's yeast fully degrades polysaccharides and other macromolecules, and slowly lowers the pH to 4.0-4.5, avoiding premature acidification that inhibits the yeast. Secondary fermentation (1-6 days) is inoculated with *Lactobacillus paracasei* after the pH has dropped to the suitable range, rapidly producing acid (pH = 3.5-4.0). Tertiary fermentation (5-10 days) is inoculated with *Lactobacillus johnsonii* under strongly acidic conditions, further metabolizing complex carbon sources, deeply acidifying (pH = 3.0-3.5), and generating antimicrobial substances, ensuring thorough fermentation and product stability. Timing is designed to match the metabolic rate and pH adaptability of each strain, achieving efficient and stable multi-stage fermentation.

[0050] In some preferred embodiments, in step 1), the Chinese medicine raw material is a Chinese medicine raw material powder or a Chinese medicine raw material extract.

[0051] In some preferred embodiments, in step 2), the number of viable bacteria in the traditional Chinese medicine base is ≥1×10⁻⁶. 9 CFU / mL. Specific Implementation

[0053] Example 1: Optimization of Microbial Strains Fermentation of traditional Chinese medicine is a process that transforms medicinal herbs into a new form through microbial action. This process not only significantly affects the effective components of the herbs but also enhances their biological activity. Fermentation can increase the content of flavonoids, polyphenols, saponins, and other components in traditional Chinese medicine, ultimately contributing to enhanced antioxidant activity. This invention conducted a systematic fermentation screening study based on existing resources of various Lactobacillus and Saccharomyces cerevisiae. Through a series of experiments and evaluations, laboratory-developed strains and commercially available strains were used to ultimately screen out six strains with significant antioxidant activity, as shown in the table below. These strains exhibited excellent antioxidant capacity during fermentation, demonstrating their potential application in food or health products. The specific process is as follows.

[0054] 1.1 Fermentation of Traditional Chinese Medicine:

[0055] Six fermentation strains, as shown in the table below, were isolated from a mixed bacterial culture stored in the laboratory. These six strains were inoculated into 10 mL liquid tubes using 200 μL of glycerol and cultured at 30°C for 24 h. 200 μL of the first-generation seed culture was inoculated into 10 mL liquid tubes and cultured overnight at 30°C. 1.5 mL of the second-generation seed culture was centrifuged, the supernatant was discarded, and the culture was washed once with PBS buffer and resuspended in 1 mL of PBS buffer. 200 μL of the resuspended culture (approximately 10 mL) was then added. 7 The inoculum (CFU / mL) was inoculated into 30mL of traditional Chinese medicine fermentation liquid (20% lily, 20% sea buckthorn, 20% wolfberry, 20% raspberry, 20% angelica) and fermented at 30℃ for 10 days.

[0056] 1.2 Determination of Antioxidant Activity of Fermentation Broth Traditional Chinese medicine produces many antioxidants after fermentation, which can enhance the antioxidant activity of Fe... 3+ Reduced to Fe 2+ The latter can form stable complexes with phenanthrene-like substances, and its antioxidant capacity can be measured by colorimetry.

[0057] The strength of the body's antioxidant capacity is closely related to its overall health. This defense system comprises both enzymatic and non-enzymatic components. Many enzymes use trace elements as their active sites, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), and glutathione S-transferase (GST). The non-enzymatic reaction system mainly consists of vitamins, amino acids, and metalloproteins. After fermentation, traditional Chinese medicine (TCM) exhibits an increase in its antioxidant components, such as polyphenols, flavonoids, amino acids, and other microbial metabolites. Consuming these fermented TCM products can effectively enhance the body's antioxidant capacity, thus combating oxidative stress and delaying aging.

[0058] Take the supernatant and add Fe 3+ The control group was not given any sample. After incubating in a 37℃ water bath for 10 min, 1 mL of phenanthroline solution was added, mixed well, and incubated in a 37℃ water bath for 30 min. Then, 100 μL of 1 mol / L hydrochloric acid solution was added to terminate the reaction. At this time, an equal amount of sample was added to the control group. After incubating in a 37℃ water bath for 15 min, the absorbance was detected by a UV spectrophotometer at 510 nm. The experiment was repeated in triplicate, and the average value of the results was taken.

[0059] R = (AB) / A * 100%

[0060] R: Antioxidant capacity of fermentation broth sample

[0061] A: Absorbance of the reference standard

[0062] B: Absorbance in the sample group

[0063] After centrifuging an appropriate amount of fermentation broth at high speed and low temperature, collect the supernatant. Determine the antioxidant activity using the method described above. The results of the antioxidant activity determination are shown in the table below.

[0064]

[0065] The data in the table above show that during fermentation, the various flavonoids and polyphenols produced by the microbial decomposition and metabolism have an inhibitory effect on oxidative stress. Different strains produce different types and activities of metabolites, and even different strains of the same genus show significant differences. The antioxidant capacity of the fermentation broth is closely related to the type and strain of bacteria used. This invention measured the antioxidant activity of fermentation broths from various existing laboratory microbial strains. The antioxidant activities varied considerably, with the fermentation broth of *Lactobacillus paracasei* ML-001 exhibiting the highest antioxidant activity, reaching 91.59%.

[0066] 1.3 Changes in polyphenols, flavonoids, and polysaccharides in fermentation broths of traditional Chinese medicine obtained from fermentation with different strains: The above-mentioned single-strain fermentation broths were sterilized at 95℃ for 10 min. Samples were taken during the fermentation process to detect changes in flavonoids, polyphenols, and polysaccharides. The results are shown in […]. Figure 1 Two *Saccharomyces cerevisiae* strains, ML-002 and 2.3857, exhibited high polysaccharide utilization, and the low polysaccharide content avoids the metabolic burden of sugar intake on the body. The changes in flavonoids revealed the fermentation characteristics of different strains. Flavonoids from strains 6038 and ML-001 initially increased and then decreased, while strains 021 (1.3321) and ML-002 showed little change in flavonoid activity. Flavonoids from strain ML-003 increased in the later stages of fermentation. Although the polyphenol content of strain XL3-1 (2.3857) initially decreased, it reached its peak on day 8 of fermentation, and ML-002 showed a similar characteristic, initially decreasing and then increasing. The polyphenol content in the fermentation substrate increased after fermentation by all six strains. In summary, both *Saccharomyces cerevisiae* strains possess good characteristics for fermenting traditional Chinese medicine, and the fermentation components of strain ML-001 are superior to those of commercially available strains 6038, ML-003, and 021 (1.3321).

[0067] Example 2: Content of various components in multi-stage fermentation

[0068] This invention investigates and compares the various chemical components and antioxidant activities under different fermentation methods. It mainly focuses on two fermentation methods: "single-strain fermentation" and "multi-stage fermentation," analyzing their performance in terms of pH value, polysaccharides, flavonoids, polyphenols, saponins, and antioxidant activity.

[0069] In a medicinal powder substrate (lily: angelica: sea buckthorn: raspberry: wolfberry = 1:1:2:2:2), single-strain fermentation and multi-stage fermentation were carried out respectively. Under a fermentation temperature of 37℃, the fermentation was carried out at a rate of 5 × 10⁻⁶. 5 Inoculation with single bacteria was performed at a CFU / mL inoculum. Special attention was paid to the fact that products from secondary and tertiary fermentations required sterilization at 115℃ for 15 minutes before re-inoculation. Secondary fermentation was inoculated on day 10, and tertiary fermentation on days 5 and 10, respectively. Components were analyzed after 20 days of fermentation; the results are shown in the table below. Within a certain culture period, the inhibitory effect of the fermentation broth on antioxidant activity increased with prolonged fermentation time, indicating that antioxidants in the fermentation broth were produced during the fermentation process. The results showed that single-strain fermentation had a higher pH and weaker acidity, while multi-stage fermentation had a pH at a moderate level. This indicates that different fermentation methods have a significant impact on the acidity or alkalinity of the environment. Furthermore, lactic acid bacteria fermentation showed low polysaccharide utilization, while yeast fermentation performed better in terms of flavonoids and antioxidant capacity. Multi-stage fermentation showed component diversity and overall performance superior to single-strain fermentation. Yeast fermentation showed the best antioxidant capacity, suggesting it may be more effective in enhancing the efficacy of traditional Chinese medicine health products.

[0070]

[0071] Example 3: Preparation of Fermented Traditional Chinese Medicine Products for Ovary Repair

[0072] A method for preparing a fermented traditional Chinese medicine product for ovarian repair includes the following steps:

[0073] Fermentation broth preparation: Accurately weigh the required herbal powders and mix them according to the mass ratio (lily bulb: angelica: sea buckthorn: raspberry: wolfberry = 1:1:2:2:2). Gradually add an appropriate amount of water until the total moisture content of the mixture reaches 65 wt%. Ensure thorough stirring during this process to allow the powder to absorb water evenly, thus achieving the ideal moist state and preparing for subsequent processing and fermentation.

[0074] Sterilization: The fermentation broth obtained in step 1) is subjected to high-pressure sterilization, then sterilized at 115°C for 30 minutes, and cooled to 42°C to obtain sterile cooled broth.

[0075] Inoculation and fermentation: Add 5×10 to the sterilized and cooled liquid obtained in step 2). 5 CFU / mL Saccharomyces cerevisiae ML-002 was mixed thoroughly and placed in a constant temperature incubator at 30℃ and 220 rpm for 5 days. The viable count should be ≥1×10⁻⁶. 9 CFU / mL, added on day 5 at 5×10 5 CFU / mL Lactobacillus paracasei ML-001 was placed in a constant temperature incubator and fermented at 37℃ and 220 rpm for 4 days. Finally, on the tenth day of fermentation, 5 × 10⁻⁶ CFU / mL Lactobacillus paracasei was added. 5 Lactobacillus casei ML-003 was inoculated at a CFU / mL level and cultured at 37℃ for 220 rpm for 11 days to obtain the traditional Chinese medicine base material. This base material exhibited the highest antioxidant activity of 96.30%, with saponin content reaching 14.33 mg / mL, polysaccharide content 10.47 mg / mL, polyphenol content 25.63 mg / mL, and flavonoid content 22.08 mg / mL.

[0076] The herbal residue and impurities are removed. The herbal base is centrifuged. During centrifugation, the solid portion of the herbal base will settle to the bottom of the centrifuge tube due to centrifugal force, while the liquid portion will form the upper crude extract. Subsequently, the supernatant is carefully poured off or separated, and the crude extract is collected for subsequent analysis and application, ensuring the concentration and purity of the active ingredients in the extract.

[0077] 5) Filling and sterilization: Fill the centrifuged liquid from step 4) into HDPE packaging containers and seal them. Sterilize at 87℃ for 18 minutes and then cool to obtain a room-temperature, long-shelf-life oral liquid for ovarian repair.

[0078] Example 4: Antioxidant and free radical scavenging rates of traditional Chinese medicine fermentation broth at different inoculation amounts

[0079] Inoculation rate is a crucial factor in the fermentation process of traditional Chinese medicine (TCM). Optimizing the inoculation rate can significantly improve the fermentation effect and the quality of the final product. Therefore, in practice, it is necessary to determine the appropriate inoculation rate based on the specific TCM material and the target product to obtain the best fermentation results. (A ratio of 0-1×10⁻⁶ is recommended.) 6 Inoculation with CFU / mL of multi-stage fermentation strains was performed, and the fermentation process parameters were the same as in Example 4. The changes in antioxidant activity over time were then detected. Figure 2 As shown, antioxidant activity initially increased and then decreased over time, while the increase was not significant in the uninoculated herbal substrate, indicating that the fermentation process played a decisive role in the metabolic transformation of antioxidant components. The inoculum size was 1×10⁻⁶. 6 Although the antioxidant activity of the fermentation broth at CFU / mL initially increased rapidly, it fell below the level of the inoculum size of 5×10⁻⁶ on day 6 of fermentation. 5 CFU / mL and 7.5×10 5 CFU / mL, and the inoculum size is 5×10 5 The CFU / mL value reached its highest level on day 9, significantly higher than other values. Although the antioxidant capacity decreased somewhat during the later stages of fermentation, it remained high, reaching a level comparable to an inoculum of 7.5 × 10⁻⁶ on day 20. 5 The antioxidant capacity was the same at CFU / mL.

[0080] Example 5: Changes in Components During Three-Stage Fermentation with Different Transfer Days

[0081] Multistage fermentation, by dividing the fermentation process into multiple stages, can significantly improve yield and fermentation efficiency. Optimizing parameters such as temperature, pH, and oxygen supply at different stages can specifically meet the needs of microorganisms while reducing the inhibitory effects of metabolites on microbial growth. Furthermore, multistage fermentation allows for the selection of different microorganisms or strains, promoting the production of diversified products and improving economic efficiency and resource utilization. This flexible control and optimization makes multistage fermentation of significant application value in modern biotechnology. This example tested the fermentation process at 5 × 10⁻⁶... 5 The antioxidant activity of secondary and tertiary fermentation broths under CFU / mL inoculation conditions was investigated. Primary fermentation was inoculated with strain ML-002 at 220 rpm and 30°C. Secondary fermentation was carried out on days 4, 5, and 6 using strain ML-001, with subsequent cultures conducted at 220 rpm and 37°C. Changes in antioxidant activity were observed. Figure 3We found that transferring the secondary fermentation strain on day 5 of the primary fermentation yielded the best results, with its antioxidant activity continuously increasing and peaking on day 8. However, the antioxidant activity then declined on day 10. Therefore, we inoculated ML-003 on days 8, 9, and 10 for tertiary fermentation, using the same culture conditions as the secondary fermentation. The changes in antioxidant activity over time were as follows: Figure 4 As shown.

[0082] After the fermentation process, we conducted detailed component analysis on the obtained fermentation products, focusing on the content of bioactive components such as polyphenols, flavonoids, saponins, and polysaccharides (see table below). We also measured the pH of the fermentation broth to assess its acid-base characteristics. In the secondary fermentation stage: On day 4, the pH was 3.28, at which point the contents of polysaccharides, flavonoids, polyphenols, and saponins were relatively high. With increasing transfer days, especially on days 5 and 6, the pH decreased after fermentation, and the contents of polysaccharides and flavonoids also decreased significantly, indicating that these components may have been consumed or transformed during fermentation. In the tertiary fermentation stage: From day 8 to day 10, the pH gradually increased to 4.08, showing the change in the fermentation environment over time. The contents of polysaccharides and flavonoids reached 16.00 mg / mL and 15.00 mg / mL on day 8, respectively, and then gradually decreased. However, when the tertiary strain was transferred on day 10, the flavonoid content rebounded to 22.08 mg / mL after fermentation, which may be related to the different metabolic pathways of the microorganisms.

[0083] Based on the quantitative analysis of various components and parameters above, secondary strain transfer was performed on day 5 and tertiary strain transfer was performed on day 10 to achieve the best results for various components of fermentation. The results are shown in the table below.

[0084]

Claims

1. A probiotic composition, characterized in that: Choose from the following combinations: Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii O21; or Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001 and Lactobacillus casei ML-003; in: Lactobacillus paracasei ML-001 was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33434, and its microbiological classification is Lactobacillus paracasei (…). Lacticaseibacillus paracasei ); Lactobacillus johnsonii 021 was deposited on July 24, 2002, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.3221, and is classified as Lactobacillus johnsonii (CGMCC 021). Lactobacillus johnsonii ); Lactobacillus casei ML-003 was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33436, and its microbiological classification is Lactobacillus casei (…). Lactobacillus casei ); Saccharomyces cerevisiae ML-002 was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33435, and its microbiological classification is Saccharomyces cerevisiae (…). Saccharomyces cerevisiae ).

2. The application of the probiotic composition according to claim 1 in the fermentation of traditional Chinese medicine, characterized in that: In the fermentation process of traditional Chinese medicine, the first fermentation is carried out by inoculating Saccharomyces cerevisiae, followed by secondary fermentation by inoculating Lactobacillus paracasei, and finally tertiary fermentation by inoculating Lactobacillus johnsonii or Lactobacillus casei. The Chinese medicine used in the fermentation process is a medicinal powder base composed of lily, angelica, sea buckthorn, raspberry and wolfberry in a mass ratio of 1:1:2:2:2.

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