Probiotic composition and application thereof in traditional Chinese medicine fermentation

Abstract

The invention relates to the field of biological fermentation, and discloses a probiotic composition and application thereof in traditional Chinese medicine fermentation. The probiotic composition comprises saccharomyces cerevisiae and lactobacillus, has excellent fermentation performance and high fermentation speed, can significantly shorten the production cycle, is suitable for fermentation of traditional Chinese medicines, and especially in the process of synthesizing flavones and polyphenol compounds, the strains not only can realize efficient conversion, but also can realize fermentation of the flavones and polyphenol compounds. And the yield of the final product can be effectively improved. In addition, flavones and polyphenols generated by the strains have extremely high antioxidant activity, and the efficacy of the product can also be ensured within a long shelf life.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation, and in particular to a probiotic composition and its application in traditional Chinese medicine fermentation. Background Art

[0002] The technology of traditional Chinese medicine fermentation has gradually transitioned from traditional fermentation methods to modern fermentation methods. Modern traditional Chinese medicine fermentation technology has expanded the traditional fermentation method by drawing on modern technologies such as pharmaceutical engineering technology and biological engineering technology, and applied probiotics to the fermentation of traditional Chinese medicine to achieve the biological transformation of traditional Chinese medicine by microorganisms.

[0003] Currently, fermentation methods are mainly divided into three categories: solid fermentation, liquid fermentation, and two-way fermentation of medicinal fungi, according to the different fermentation forms and techniques of traditional Chinese medicine. Among them, liquid fermentation technology uses a nutrient-rich liquid medium to culture strains, and through controlling conditions such as temperature, time, and initial pH, the fermentation products with established goals are obtained. Liquid fermentation technology can not only achieve large-scale production, but also the fermentation conditions are easy to control, and it can effectively improve production efficiency and yield. Therefore, it is suitable for industrialized and automated production.

[0004] The traditional Chinese medicine preparations fermented by probiotics usually contain a large amount of antioxidant substances such as polyphenols, flavonoids, and saponins, which can scavenge harmful free radicals in the body, delay the aging process, and prevent the occurrence and development of various chronic diseases. The selection of probiotics has a crucial impact on the composition and antioxidant activity of the products obtained after traditional Chinese medicine fermentation. Therefore, it is of positive significance to continuously discover new probiotic combinations suitable for traditional Chinese medicine fermentation. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a probiotic composition and its application in traditional Chinese medicine fermentation. The probiotic composition of the present invention has excellent fermentation performance, with a rapid fermentation rate, which can significantly shorten the production cycle, and is suitable for traditional Chinese medicine fermentation. Especially in the process of synthesizing flavonoids and polyphenolic compounds, these strains can not only achieve efficient transformation, but also effectively increase the yield of the final product. In addition, the combination of the effective components such as flavonoids and polyphenols metabolized after the combination of these strains and specific traditional Chinese medicinal materials has extremely high antioxidant activity.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a probiotic composition, comprising Saccharomyces cerevisiae and Lactobacillus; wherein the Lactobacillus comprises at least one of the following: a) Lactobacillus paracasei ML-001, deposited with the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33434, and the microbial taxonomic name is Lactobacillus paracasei; b) Lactobacillus johnsonii 021, deposited with the China General Microbiological Culture Collection Center on July 24, 2002, with the deposit number CGMCC No. 1.3221, and the microbial taxonomic name is Lactobacillus johnsonii; c) Lactobacillus casei ML-003, deposited with the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33436, and the microbial taxonomic name is Lactobacillus casei.

[0007] The applicant has previously isolated several probiotics including the above strains (except Lactobacillus johnsonii 021, which was purchased commercially) independently, and through research, it has been confirmed that they have good activity in fermenting traditional Chinese medicine. In subsequent research, we also found that several of the above strains have excellent free radical scavenging activity after fermenting traditional Chinese medicine. Compared with other reported traditional Chinese medicine fermentation products, their characteristics are particularly that the fermented liquid obtained under the condition of a lower initial inoculation amount has a very high antioxidant activity. Furthermore, we also found that the fermentation activity of these strains is very stable. The traditional Chinese medicine fermentation products prepared by multi-stage fermentation in combination with Saccharomyces cerevisiae still maintain a high level of antioxidant activity after being stored under different temperatures and a lower pH condition, indicating that they can maintain their efficacy in the human gastrointestinal environment. In addition, the traditional Chinese medicine products obtained by multi-stage fermentation of this type of strain still have a very high antioxidant activity during the long shelf life at room temperature, and currently, there is no research on the antioxidant level of the multi-stage fermentation products of this type of strain.

[0008] Preferably, the Saccharomyces cerevisiae includes at least one of the following: d) Saccharomyces cerevisiae ML-002, deposited with the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33435, and the microbial taxonomic name is Saccharomyces cerevisiae; e) Saccharomyces cerevisiae XL3-1, deposited with the China General Microbiological Culture Collection Center on June 5, 2008, with the deposit number CGMCC No. 2.3857, and the microbial taxonomic name is Saccharomyces cerevisiae.

[0009] Through a large number of experiments, the present invention discovers that in the multi-stage fermentation process, when the above two strains of Saccharomyces cerevisiae are combined with the several strains of Lactobacillus mentioned above in the present invention, the multi-stage fermentation effect is better.

[0010] Preferably, the probiotic composition is selected from the following combinations: 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.

[0011] The present invention discovers that the combination effect of the above two groups of combinations is the best.

[0012] In a second aspect, the present invention provides the application of the above probiotic composition in traditional Chinese medicine fermentation.

[0013] The probiotic composition of the present invention has excellent fermentation performance, with a fast fermentation speed, can significantly shorten the production cycle, and is very suitable for traditional Chinese medicine fermentation. Especially in the process of synthesizing flavonoids and polyphenolic compounds, 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.

[0014] Preferably, the traditional Chinese medicine fermentation is secondary fermentation or tertiary fermentation.

[0015] Preferably, the probiotic composition contains 2 kinds of Lactobacillus. Further preferably, the probiotic composition is selected from the following combinations: 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.

[0016] Preferably, during the traditional Chinese medicine fermentation process, Saccharomyces cerevisiae is inoculated first, and then one of the Lactobacillus is inoculated after fermentation, and then the other Lactobacillus is inoculated after fermentation. Further preferably, during the traditional Chinese medicine fermentation process, Saccharomyces cerevisiae is inoculated first for primary fermentation, then Lactobacillus paracasei is inoculated for secondary fermentation, and finally Lactobacillus johnsonii or Lactobacillus casei is inoculated for tertiary fermentation.

[0017] The probiotic composition of the present invention is applicable to the multi-stage fermentation process, especially the tertiary fermentation process. The specific inoculation sequence is to inoculate Saccharomyces cerevisiae first, and then sequentially inoculate Lactobacillus paracasei and Lactobacillus johnsonii or Lactobacillus casei. The traditional Chinese medicine fermentation product prepared by fermenting with the probiotic composition of the present invention under multi-stage fermentation has relatively high antioxidant activity, and the efficacy of the product can also be guaranteed within a long shelf life. Compared with the prior art, the beneficial effects of the present invention are: (1) The strains in the probiotic composition of the present invention have excellent free radical scavenging activity after fermenting traditional Chinese medicine. In particular, the fermented liquid obtained under the condition of a relatively low initial inoculation amount has high antioxidant activity. Moreover, the fermentation activity of these strains is very stable. The traditional Chinese medicine fermentation products prepared by multi-stage fermentation still maintain a relatively high level of antioxidant activity after being stored under different temperatures and relatively low pH conditions, and can maintain their efficacy in the human gastrointestinal environment.

[0018] (2) The probiotic composition of the present invention has excellent fermentation performance, with a rapid fermentation rate, which can significantly shorten the production cycle and is very suitable for the fermentation of traditional Chinese medicine. Especially in the process of synthesizing flavonoids and polyphenolic compounds, 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.

[0019] (3) The probiotic composition of the present invention is particularly suitable for multi-stage fermentation processes, and several types of strains in the probiotic composition have good cooperation effects in multi-stage fermentation. The traditional Chinese medicine fermentation products prepared by fermenting with the probiotic composition of the present invention under multi-stage fermentation have relatively high antioxidant activity and can also ensure the efficacy of the products during a long shelf life. Description of the Drawings

[0020] Figure 1 Shows the changes in flavonoids, polyphenols and polysaccharides in the fermented liquids of different strains.

[0021] Figure 2 Shows the changes in the antioxidant activity of strain ML-001 at different initial inoculation amounts and different fermentation times.

[0022] Figure 3 Shows the changes in the antioxidant activity during fermentation for 14 days at different transfer days in secondary fermentation.

[0023] Figure 4 Shows the changes in the antioxidant activity during fermentation for 16 days at different transfer days in tertiary fermentation. Detailed Embodiments

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

[0025] First, a probiotic composition includes 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 the deposit number CGMCC No. 33434, and the microbial taxonomic name is Lactobacillus paracasei (Lacticaseibacillus paracasei); b) Lactobacillus johnsonii 021, deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 24, 2002, with the deposit number CGMCC No. 1.3221, and the microbial taxonomic name is Lactobacillus johnsonii; c) Lactobacillus casei ML-003, deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33436, and the microbial taxonomic name is Lactobacillus casei.

[0026] In some preferred embodiments, the Saccharomyces cerevisiae includes at least one of the following: d) Saccharomyces cerevisiae ML-002, deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33435, and the microbial taxonomic name is Saccharomyces cerevisiae; e) Saccharomyces cerevisiae XL3-1, deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 5, 2008, with the deposit number CGMCC No. 2.3857, and the microbial taxonomic name is Saccharomyces cerevisiae.

[0027] In some preferred embodiments, the probiotic composition is selected from the following combinations: 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.

[0028] Secondly, a method for applying the above probiotic composition in traditional Chinese medicine fermentation.

[0029] In some preferred embodiments, the traditional Chinese medicine fermentation is secondary fermentation or tertiary fermentation.

[0030] In some preferred embodiments, the probiotic composition contains 2 kinds of Lactobacillus.

[0031] In some more preferred embodiments, during the traditional Chinese medicine fermentation process, Saccharomyces cerevisiae is inoculated first, and then one kind of Lactobacillus is inoculated after fermentation, and another kind of Lactobacillus is inoculated after further fermentation.

[0032] In some preferred embodiments, the probiotic composition is selected from the following combinations: 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.

[0033] In some more preferred embodiments, during the traditional Chinese medicine fermentation process, Saccharomyces cerevisiae is first inoculated for primary fermentation, then Lactobacillus paracasei is inoculated for secondary fermentation, and finally Lactobacillus johnsonii or Lactobacillus casei is inoculated for tertiary fermentation. In some preferred embodiments, for primary fermentation: the temperature is 28 - 37°C, the fermentation lasts for 4 - 7 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / m; for secondary fermentation: the temperature is 28 - 45°C, the fermentation lasts for 1 - 6 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL; for tertiary fermentation: the temperature is 28 - 45°C, the fermentation lasts for 5 - 10 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL.

[0034] During the tertiary fermentation process, the inoculation timing of each strain has a significant impact on the fermentation effect. The inoculation timing of multi-stage fermentation needs to consider the pH change and the metabolic characteristics of the bacterial community during the fermentation process. The 4 - 7 days of primary fermentation ensures that Saccharomyces cerevisiae fully degrades macromolecules such as polysaccharides and slowly reduces the pH to 4.0 - 4.5, avoiding premature acidification that inhibits yeast. The 1 - 6 days of secondary fermentation inoculates Lactobacillus paracasei after the pH drops to the appropriate range, quickly producing acid (pH = 3.5 - 4.0). The 5 - 10 days of tertiary fermentation inoculates Lactobacillus johnsonii under strong acidic conditions, further metabolizing complex carbon sources, deeply acidifying (pH = 3.0 - 3.5) and generating antibacterial substances to ensure complete fermentation and product stability. The time design matches the metabolic rates and pH adaptabilities of each strain to achieve efficient and stable multi-stage fermentation.

[0035] In some preferred embodiments, in step 1), the traditional Chinese medicine raw material is traditional Chinese medicine raw material powder or traditional Chinese medicine raw material extract.

[0036] In some preferred embodiments, in step 2), the viable bacteria count in the traditional Chinese medicine base material ≥ 1×10 9 CFU / mL. Specific embodiments Example 1: Optimization of Strains for Traditional Chinese Medicine Fermentation Traditional Chinese medicine fermentation is a process in which traditional Chinese medicine is transformed into a new form through the action of microorganisms. This process not only has an important impact on the active ingredients of the medicine but also enhances its biological activity. Fermentation can increase the content of components such as flavonoids, polyphenols, and saponins in traditional Chinese medicine, ultimately contributing to enhancing its antioxidant activity. Based on the existing resources of various Lactobacillus and Saccharomyces cerevisiae strains, the present invention carried out a systematic fermentation screening study. Through a series of experiments and evaluations on self-owned strains in the laboratory and commercially available strains, six strains with significant antioxidant activity were finally screened out, as shown in the following table. During the fermentation process, these strains demonstrated excellent antioxidant capabilities, indicating their potential applications in food or health products. The specific process is as follows.

[0038] 1.1 Traditional Chinese Medicine Fermentation: The following 6 fermentation strains were isolated from the mixed strains stored in the laboratory. 200 μL of these 6 strains were inoculated into 10 mL liquid test tubes through glycerol tubes and cultured at 30 °C for 24 h. 200 μL of the first-generation seed liquid was inoculated into 10 mL liquid test tubes and cultured overnight at 30 °C. 1.5 mL of the second-generation seed liquid was centrifuged to remove the supernatant, washed once with PBS buffer, and resuspended in 1 mL of PBS buffer. 200 μL of the resuspended liquid (inoculation amount of approximately 10 7 CFU / mL) was inoculated into 30 mL of traditional Chinese medicine fermentation broth (20% lily, 20% seabuckthorn, 20% wolfberry, 20% raspberry, 20% angelica) and fermented at 30 °C for 10 d.

[0039] 1.2 Determination of Antioxidant Activity of Fermentation Broth After fermentation, traditional Chinese medicine produces many antioxidant substances. These substances can reduce Fe 3+ to Fe 2+ , and the latter can form a stable complex with phenanthroline substances. The antioxidant ability can be measured by colorimetry.

[0040] There is a close relationship between the antioxidant ability of the body's defense system and health. This defense system has two systems: enzymatic and non-enzymatic. Many enzymes have trace elements as their active centers. For example, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), glutathione S-transferase (GST), etc. In the non-enzymatic reaction system, it is mainly vitamins, amino acids, and metal proteins. After fermentation of traditional Chinese medicine, the components with antioxidant ability increase, such as polyphenols, flavonoids, amino acids, and other microbial metabolites. By ingesting this traditional Chinese medicine fermentation product, the antioxidant ability of the body can be effectively enhanced to cope with oxidative stress and delay aging.

[0041] Take the supernatant and add Fe 3+, in the control group, no sample was added. After incubating in a 37°C water bath for 10 min, 1 mL of phenanthroline solution was added. After mixing evenly, it was incubated in a 37°C 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°C water bath for 15 min, it was detected by an ultraviolet spectrophotometer, and the change in absorbance was detected at 510 nm. The parallel experiment was carried out 3 times, and the results were averaged.

[0042] R = (A - B) / A * 100% R: Antioxidant capacity of the fermentation broth sample A: Absorbance of the reference substance B: Absorbance in the sample group After taking an appropriate amount of the fermentation broth and centrifuging it at high speed and low temperature, the supernatant was taken. The antioxidant activity was determined according to the above method. The results of the antioxidant activity determination are shown in the following table.

[0043] It can be seen from the data in the above table that during the fermentation process, various flavonoid substances and polyphenol substances produced by the catabolism of the strains have an inhibitory effect on oxidative stress. Different strains have different types and activities of their metabolites, and there are obvious differences even among different strains of the same genus. The antioxidant property of the fermentation broth is closely related to the types and strains of the bacteria used. In this invention, the antioxidant activities of fermentation broths of various types of strains existing in the laboratory were determined, and there were significant differences in antioxidant activities. Among them, the fermentation broth of Lactobacillus paracasei ML-001 had the highest antioxidant activity, reaching 91.59%.

[0044] 1.3 Changes in polyphenols, flavonoids, and polysaccharides in traditional Chinese medicine fermentation broths obtained by fermenting different strains. The above-mentioned several single-strain fermentation broths were sterilized at 95°C for 10 min, and samples were taken during the fermentation process to detect the changes in flavonoids, polyphenols, and polysaccharides. The results are shown in Figure 1 . Two strains of Saccharomyces cerevisiae ML-002 and 2.3857 showed a relatively high polysaccharide utilization rate. A low polysaccharide content can avoid the metabolic burden on the body caused by sugar intake. The change in flavonoids showed the fermentation characteristics of different strains. The flavonoids fermented by strains 6038 and ML-001 first increased and then decreased. The flavonoid change trends of strain 021 (1.3321) and ML-002 were not significant. The flavonoids of strain ML-003 increased in the later stage of fermentation. Although the polyphenols of strain XL3-1 (2.3857) first decreased, they reached the highest level on the 8th day of fermentation, and ML-002 also showed similar characteristics, first decreasing and then rising in the initial stage of fermentation. The polyphenols in the fermentation substrate increased after being fermented by the six strains. In summary, the two strains of Saccharomyces cerevisiae have good characteristics for fermenting traditional Chinese medicine, and the fermentation components of strain ML-001 are also superior to the commercially available strains 6038, ML-003, and 021 (1.3321).

[0045] Example 2: Contents of Various Components in Multi - stage Fermentation The present invention explores the comparison of various chemical components and antioxidant activities under different fermentation methods. It is mainly divided into two fermentation methods: "single - strain fermentation" and "multi - stage fermentation", and their performances in terms of pH value, polysaccharides, flavonoids, polyphenols, saponins and antioxidant activities are analyzed.

[0046] In the medicinal powder base material (Lilium brownii: Angelica sinensis: Hippophae rhamnoides: Rubus idaeus: Lycium barbarum = 1:1:2:2:2), single - strain fermentation and multi - stage fermentation are carried out respectively. Under the condition of a fermentation temperature of 37°C, a single strain is inoculated at an inoculum size of 5×10 5 CFU / mL. Special attention should be paid that the products of secondary fermentation and tertiary fermentation need to be sterilized at 115°C for 15 min and then inoculated again; the secondary fermentation is inoculated on the 10th day of fermentation, and the tertiary fermentation is inoculated on the 5th day and 10th day of fermentation respectively. After 20 days of fermentation, the relevant components are detected, and the detection results are shown in the following table. Within a certain culture time, as the fermentation time prolongs, the antioxidant activity inhibition effect of the fermentation broth also increases, which indicates that the antioxidant substances in the fermentation broth are produced during the culture fermentation process. The results show that the pH value of single - strain fermentation is higher, the fermentation acidity is weaker, while the pH value of multi - stage fermentation is at an intermediate level. This indicates that different fermentation methods have a significant impact on the acidity and alkalinity of the environment. In addition, lactic acid bacteria fermentation shows a low utilization rate of polysaccharides, while yeast fermentation performs better in terms of flavonoids and antioxidant capacity. Multi - stage fermentation shows the diversity of components and the overall performance is better than that of single - strain fermentation. Yeast fermentation shows the best antioxidant capacity, indicating that it may be more effective in enhancing the efficacy of traditional Chinese medicine health products.

[0047] Example 3: Preparation of Traditional Chinese Medicine Fermentation Product for Ovarian Repair A preparation method of a traditional Chinese medicine fermentation product for ovarian repair includes the following steps: Fermentation broth preparation: After accurately weighing the traditional Chinese medicine powder of the required formula, it is mixed according to the mass ratio (Lilium brownii: Angelica sinensis: Hippophae rhamnoides: Rubus idaeus: Lycium barbarum = 1:1:2:2:2), and an appropriate amount of water is gradually added until the water content of the whole mixture reaches 65 wt%. Ensure that during this process, it is fully stirred so that the powder can uniformly absorb water, thus obtaining an ideal wet state to prepare for subsequent processing and fermentation.

[0048] Sterilization: The fermentation broth obtained in step 1) is sterilized under high pressure, and then sterilized at 115°C for 30 minutes and cooled to 42°C to obtain a sterile cooled feed liquid.

[0049] Inoculation and fermentation: Add 5×10 5CFU / mL of Saccharomyces cerevisiae ML-002, stir and mix evenly, place in a constant temperature incubator and ferment at 30 °C at a rotation speed of 220 rpm for 5 days, the viable count requirement is ≥1×10 9 CFU / mL, add 5×10 5 CFU / mL of Lactobacillus paracasei ML-001 on the 5th day, place in a constant temperature incubator and ferment at 37 °C at a rotation speed of 220 rpm for 4 days, and finally inoculate Lactobacillus casei ML-003 at an inoculation amount of 5×10 5 CFU / mL and continue to culture at 37 °C at a rotation speed of 220 rpm for 11 days to obtain a traditional Chinese medicine base material. The highest antioxidant activity of this traditional Chinese medicine base material reaches 96.30%, the saponin content reaches 14.33 mg / mL, the polysaccharide content is 10.47 mg / mL, the polyphenol is 25.63 mg / mL, and the flavonoid content is 22.08 mg / mL.

[0050] The medicinal residues and impurities therein. Centrifuge the traditional Chinese medicine base material. During centrifugation, the solid part in the traditional Chinese medicine base material will precipitate to the bottom of the centrifuge tube due to the action of centrifugal force, while the liquid part will form the crude extraction liquid medicine on the upper layer. Subsequently, carefully pour or separate the supernatant, and collect the obtained crude extraction liquid medicine for subsequent analysis and application to ensure the concentration and purity of the active ingredients in the liquid medicine.

[0051] 5) Filling and sterilization: Fill the centrifuged liquid material in step 4) into a packaging container made of HDPE material and seal it, and sterilize it by pasteurization at 87 °C for 18 min and then cool it to obtain a traditional Chinese medicine oral liquid with a long shelf life at room temperature for ovarian repair.

[0052] Example 4: Antioxidant and free radical scavenging rate of traditional Chinese medicine fermentation broth under different inoculation amounts The inoculation amount is a key factor in the traditional Chinese medicine fermentation process. Reasonably optimizing the inoculation amount can significantly improve the fermentation effect of medicinal materials and the quality of the final product. Therefore, in actual operation, it is necessary to determine the appropriate inoculation amount according to specific traditional Chinese medicinal materials and target products to obtain the best fermentation effect. Inoculate the multi-stage fermentation strain at an inoculation amount of 0 - 1×10 6 CFU / mL, and the fermentation process parameters are the same as in Example 4. Detect the change of its antioxidant activity over time as Figure 2 shown. The antioxidant activity first increases and then decreases with the increase of time. The increase of the traditional Chinese medicine base material without inoculating bacteria is not obvious, indicating that the fermentation process plays a decisive role in the metabolic transformation of antioxidant components. The antioxidant activity of the fermentation broth with an inoculation amount of 1×10 6 CFU / mL increases rapidly initially, but is lower than that of the fermentation broth with an inoculation amounts of 5×10 5 CFU / mL and 7.5×10 5 CFU / mL on the 6th day of fermentation, and the fermentation broth with an inoculation amount of 5×10 5The CFU / mL reached the highest value on the 9th day, far higher than other values, and although the antioxidant activity decreased during the later fermentation, it remained at a high level. On the 20th day, it was comparable to the antioxidant activity of the inoculum density of 7.5×10 5 CFU / mL.

[0053] Example 5: Changes in the components of the third-stage fermentation at different transfer days Multistage fermentation divides the fermentation process into multiple stages, which can significantly improve the yield and fermentation efficiency. At different stages, optimizing various parameters such as temperature, pH, and oxygen supply can specifically meet the needs of microorganisms, while reducing the inhibitory effect of metabolites on the growth of microorganisms. In addition, multistage fermentation also allows the selection of different microorganisms or strains, promoting the production of diverse products and enhancing economic benefits and resource utilization efficiency. This flexible control and optimization make multistage fermentation of great application value in modern biotechnology. In this example, the antioxidant activities of the broth in the secondary fermentation and the third-stage fermentation were respectively detected under the inoculation condition of 5×10 5 CFU / mL. The strain ML-002 was inoculated in the first-stage fermentation and cultured at a rotation speed of 220 rpm and a temperature of 30°C. The secondary fermentation was inoculated on the 4th, 5th, and 6th days of fermentation respectively, and the strain ML-001 was selected. The subsequent culture was carried out at a rotation speed of 220 rpm and a temperature of 37°C. The changes in its antioxidant activity are shown in Figure 3 . We found that transferring the secondary fermentation strain on the 5th day of the first-stage fermentation had the best effect, and its antioxidant activity continued to increase and reached a peak on the 8th day. However, the antioxidant activity decreased on the 10th day. Therefore, we inoculated ML-003 for the third-stage fermentation on the 8th, 9th, and 10th days, and the culture conditions were the same as those of the secondary fermentation. The changes in the antioxidant activity over time are as shown in Figure 4 .

[0054] After the fermentation process ended, we conducted a detailed component analysis of the obtained fermentation products, focusing on the contents of bioactive components such as polyphenols, flavonoids, saponins, and polysaccharides as shown in the following table. At the same time, we also measured the pH value of the fermentation broth to evaluate its acid-base properties. In the secondary fermentation stage: on the 4th day, the pH was 3.28, and at this time, the contents of polysaccharides, flavonoids, polyphenols, and saponins were relatively high. As the transfer days increased, especially on the 5th and 6th days, the pH decreased after fermentation ended, and the contents of polysaccharides and flavonoids also decreased significantly, indicating that these components may be consumed or transformed during the fermentation process. In the third-stage fermentation stage: from the 8th day to the 10th day, the pH gradually increased to 4.08, indicating the change of the fermentation environment over time. The contents of polysaccharides and flavonoids reached 16.00 mg / mL and 15.00 mg / mL on the 8th day, and then gradually decreased. When the third-stage strain was transferred on the 10th day, the flavonoid content recovered to 22.08 mg / mL after fermentation ended, which may be related to the metabolic modes of different microorganisms.

[0055] Based on the above quantitative analysis of various components and parameters, the second-stage strain transfer was carried out on the 5th day, and the third-stage strain transfer was carried out on the 10th day to make the various components of the fermentation reach the best effect. The results are shown in the following table.

Claims

1. A probiotic composition, characterized in that: It includes Saccharomyces cerevisiae and Lactobacillus; among which, the Lactobacillus includes at least one of the following: a) Lactobacillus paracasei ML-001, deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33434, and the microbial taxonomic name is Lactobacillus paracasei ( Lacticaseibacillus paracasei ) b) Lactobacillus johnsonii 021, deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 24, 2002, with the deposit number CGMCC No. 1.3221, and the microbial taxonomic name is Lactobacillus johnsonii ( Lactobacillus johnsonii ) c) Lactobacillus casei ML-003, which was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33436, and the microbial taxonomic name is Lactobacillus casei ( Lactobacillus casei ) 2. The probiotic composition according to claim 1, wherein: The Saccharomyces cerevisiae includes at least one of the following: d) Saccharomyces cerevisiae ML-002, deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33435, and the microbial taxonomic name is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) e) Saccharomyces cerevisiae XL3-1, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 5, 2008, with the deposit number CGMCC No. 2.3857, and the microbial taxonomic name is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) 3. The probiotic composition according to claim 1 or 2, characterized in that: Selected from the following combinations: Saccharomyces cerevisiae ML-002, Lactiplantibacillus paraplantarum ML-001 and Lactobacillus johnsonii 021; or Saccharomyces cerevisiae ML-002, Lactiplantibacillus paraplantarum ML-001 and Lactobacillus casei ML-003.

4. Use of the probiotic composition according to any one of claims 1-3 in traditional Chinese medicine fermentation.

5. The application according to claim 4, characterized in that: The traditional Chinese medicine fermentation is secondary fermentation or tertiary fermentation.

6. The application according to claim 4, wherein: The probiotic composition contains 2 kinds of Lactobacillus.

7. The application according to claim 6, wherein: During the traditional Chinese medicine fermentation process, Saccharomyces cerevisiae is inoculated first, and after fermentation, one of the Lactobacillus is continuously inoculated, and then the other Lactobacillus is inoculated after fermentation.

8. The application according to claim 6, wherein: The probiotic composition is selected from the following combinations: Saccharomyces cerevisiae ML-002, Lactiplantibacillus paraplantarum ML-001 and Lactobacillus johnsonii 021; or Saccharomyces cerevisiae ML-002, Lactiplantibacillus paraplantarum ML-001 and Lactobacillus casei ML-003.

9. The application according to claim 8, wherein: During the traditional Chinese medicine fermentation process, Saccharomyces cerevisiae is inoculated first for primary fermentation, then Lactiplantibacillus paraplantarum is inoculated for secondary fermentation, and finally Lactobacillus johnsonii or Lactobacillus casei is inoculated for tertiary fermentation.

10. The application according to claim 4, wherein: The medicinal raw materials in the traditional Chinese medicine fermentation are traditional Chinese medicine raw material powders or traditional Chinese medicine raw material extracts.

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