Compound culture medium for double-bacterial compound cocultivation fermentation and application thereof
By using a modularly designed composite culture medium and a strategy of post-sterilization or segmented addition of herbal extracts, the problems of unstable growth and degradation of herbal extracts in the co-culture of Bifidobacterium longum and Lactobacillus casei were solved, achieving stable fermentation and product accumulation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHENJIANG SENXING E-COMMERCE TECHNOLOGY CO LTD
- Filing Date
- 2026-04-17
- Publication Date
- 2026-06-09
AI Technical Summary
In existing technologies, the co-culture of Bifidobacterium longum and Lactobacillus casei results in unstable growth and fluctuating acidification processes. The addition of herbal extracts leads to degradation of active ingredients and initial growth inhibition. There is a lack of systematic modular culture medium design and addition strategies.
The modularly designed composite culture medium includes modules for carbon source, nitrogen source, trace elements and cofactors, and herbal extract modules that are added after sterilization or in stages to avoid co-sterilization degradation and reduce the risk of initial inhibition.
Stable growth and synergistic acidification of Bifidobacterium longum and Lactobacillus casei were achieved under herbaceous conditions, which improved the accumulation of fermentation products and enhanced the overall fermentation performance of the dual-culture system.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a composite culture medium for co-culture and fermentation of two strains and its application. Background Technology
[0002] Bifidobacterium longum and Lactobacillus casei are two commonly used core strains in probiotic preparations and functional fermented foods. Co-culturing these two strains is expected to yield a richer combination of fermentation products through synergistic metabolism and reduce the process complexity associated with stepwise single-strain cultivation. However, Bifidobacterium longum has a relatively slow growth rate and high nutritional requirements, while Lactobacillus casei responds quickly to environmental changes. Improperly designed culture medium components can easily lead to overall growth instability, fluctuations in acidification processes, and decreased fermentation repeatability in the co-culture system. Therefore, it is necessary to develop a culture medium system that can accommodate the overall nutritional needs of both strains while maintaining the stability of the fermentation process.
[0003] On the other hand, introducing herbal extracts into microbial fermentation systems has become a hot topic in the development of functional raw materials in recent years. However, herbal extracts are complex in composition and vary greatly from batch to batch, and there is a lack of systematic research on the effects of their addition methods, timing, and proportions on compound fermentation systems. In existing technologies, herbal components are usually sterilized together with the culture medium and then added all at once, but this method can easily cause degradation of heat-sensitive active ingredients, and the one-time addition of high-concentration herbal components may also inhibit the initial growth of the compound system.
[0004] Therefore, there is an urgent need to provide a modular culture medium design and herb addition strategy to achieve effective utilization of herb components and improve the overall growth performance of the fermentation system while ensuring the feasibility of co-culture. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a composite culture medium for co-culture fermentation of two strains and its application. By decoupling the basic nutrient module from the herbal extract module and employing a post-sterilization or segmented addition strategy for the herbal extract, the overall growth and acidification process of the composite system can still be maintained under herbal conditions.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A composite culture medium for co-culture fermentation of two strains, the composite culture medium comprising:
[0008] Carbon source module, nitrogen source module, herbal extract module, and trace elements and cofactors module;
[0009] The herbal extract module includes one or more of the following: dandelion extract, salvia miltiorrhiza extract, wolfberry extract, or schisandra chinensis extract;
[0010] The herbal extract module is added as follows: after the carbon source module, nitrogen source module, trace elements and cofactor module are sterilized and cooled to the inoculation temperature, it is added to the compound culture medium by post-sterilization or segmented addition.
[0011] Preferably, the aforementioned carbon source module is one or more of glucose, oligosaccharides, and dietary fiber hydrolysate, and the amount added is 10-20 g / L based on the culture medium.
[0012] Preferably, the aforementioned nitrogen source module includes yeast extract and peptone, with addition amounts of 3-10 g / L and 1-5 g / L respectively, based on the culture medium.
[0013] Preferably, the total amount of the aforementioned herbal extract module added based on the culture medium is 2-5 g / L, the herbal extract is a water extract, an alcohol extract, or a hydroalcohol extract, its solvent residue is less than 50 ppm, and the total polyphenol content is 50-200 mg / L.
[0014] Preferably, the aforementioned trace element and cofactor module includes B vitamins and ZnSO4, with the addition amounts of each component being 0.05-0.2 g / L and 0.01-0.05 g / L, respectively, based on the culture medium.
[0015] Preferably, the initial pH of the aforementioned composite culture medium is adjusted to 6.0-6.5 before sterilization.
[0016] Preferably, the aforementioned herbal extract module is a combination of dandelion extract and salvia miltiorrhiza extract in a mass ratio of 1:1 to 1:2.
[0017] Application of compound culture medium in the co-culture of Bifidobacterium longum and Lactobacillus casei.
[0018] Application of compound culture medium in the preparation of dual-strain fermentation products containing herbal extracts.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0020] (1) This invention constructs a composite culture medium as a combination of a carbon source module, a nitrogen source module, a trace element module and a herbal extract module, and introduces the herbal module independently by post-sterilization or segmented addition, thereby achieving a decoupled design between basic nutrient supply and herbal functional components, effectively reducing the risk of degradation of active ingredients caused by co-sterilization of herbs and culture medium;
[0021] (2) The herbal addition strategy adopted in this invention reduces the risk of inhibition of the initial growth of the compound system by the one-time addition of high concentration herbal components, so that Bifidobacterium longum and Lactobacillus casei can still maintain the overall growth and acidification process under herbal conditions;
[0022] (3) The preferred combination of dandelion and salvia miltiorrhiza in this invention maintains normal fermentation acidification capacity while exhibiting the technical effect of synergistically promoting cell proliferation, which can improve the overall fermentation performance of the dual-strain co-culture system. Attached Figure Description
[0023] Figure 1 This is a comparison graph of pH during the fermentation process of Comparative Example 1 and Example 1;
[0024] Figure 2 This is a comparison graph of absorbance during the fermentation process of Comparative Example 1 and Example 1;
[0025] Figure 3 This is a comparison chart of the number of viable bacteria that can be recovered after 48 hours of fermentation between Comparative Example 1 and Example 1. Detailed Implementation
[0026] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.
[0027] A composite culture medium for co-culture fermentation of two strains, the composite culture medium comprising:
[0028] Carbon source module, which is one or more of glucose, oligosaccharides, and dietary fiber hydrolysate, is added at a rate of 10-20 g / L based on the culture medium.
[0029] The nitrogen source module includes yeast extract and peptone, with addition amounts of 3-10 g / L and 1-5 g / L respectively based on the culture medium.
[0030] The herbal extract module contains one or more of the following: dandelion extract, salvia miltiorrhiza extract, wolfberry extract, or schisandra chinensis extract. The total amount of the herbal extract module added based on the culture medium is 2-5 g / L. The herbal extract is a water extract, an alcohol extract, or a water-alcohol extract, with a solvent residue of less than 50 ppm and a total polyphenol content of 50-200 mg / L.
[0031] The micronutrient and cofactor module includes a vitamin B complex and a ZnSO4 buffer system, with the addition amounts of each component based on the culture medium being 0.05-0.2 g / L and 0.01-0.05 g / L, respectively.
[0032] The initial pH of the compound culture medium should be adjusted to 6.0-6.5 before sterilization.
[0033] The herbal extract module is added as follows: after the carbon source module, nitrogen source module, trace elements and cofactor module are sterilized and cooled to the inoculation temperature, it is added to the compound culture medium by post-sterilization or segmented addition.
[0034] Example 1
[0035] The culture medium consisted of: carbon source module MC (15 g / L glucose), nitrogen source module MN (5 g / L yeast extract, 3 g / L peptone), trace element and cofactor module MT (0.1 g / L vitamin B complex, 0.02 g / L ZnSO4), and herbal extract module MH. The initial pH of the culture medium was 6.2. MH was a combination of dandelion extract and salvia miltiorrhiza extract, added at 3 g / L and 2 g / L, respectively. MH was added by post-sterilization: the basal culture medium modules (MC, MN, MT) were mixed and sterilized, cooled to the inoculation temperature, and then the aseptically treated MH module was added.
[0036] Example 2
[0037] The culture medium formulation is the same as in Example 1, except that the MH is added in stages, with the total MH divided into two parts (50% each). The first part is added after sterilization at the time of inoculation, and the second part is added 24 hours after fermentation.
[0038] Example 3
[0039] The culture medium formulation and MH addition method are the same as in Example 1, except that the addition amounts of dandelion and salvia miltiorrhiza are adjusted to 4.5 g / L and 1.5 g / L respectively, while the total addition amount remains unchanged.
[0040] Comparative Example 1
[0041] The basal medium used was free of herbal extract modules and contained the following: carbon source module MC (15 g / L glucose), nitrogen source module MN (5 g / L yeast extract, 3 g / L peptone), and trace element and cofactor module MT (0.1 g / L B vitamins and 0.02 g / L ZnSO4). The initial pH was adjusted to 6.2 before sterilization.
[0042] Comparative Example 2
[0043] The culture medium formulation was the same as that of Comparative Example 1, except that 3 g / L of dandelion extract was added in addition, and the herbal addition method was the same as that of Example 1 (added after sterilization).
[0044] Comparative Example 3
[0045] The culture medium formulation is the same as in Example 1, except that the herbal extract module MH and the basic culture medium module are mixed and then sterilized together.
[0046] Comparative Example 4
[0047] The culture medium formulation and MH addition method are the same as in Example 1, except that ZnSO4 in the trace element module MT is removed.
[0048] Performance testing
[0049] (1) The specific detection methods used in each embodiment of this application are as follows:
[0050] pH measurement: The pH of the fermentation broth was directly measured at 0h, 12h, 24h, 36h and 48h using a calibrated pH meter.
[0051] Absorbance measurement: Fermentation broth at different time points was taken, appropriately diluted, and the absorbance value was measured at a wavelength of 600 nm using a UV spectrophotometer to characterize the relative growth of the cells.
[0052] Viable cell count (CFU): Fermentation broth from 48 hours prior was serially diluted 10-fold with sterile physiological saline. Appropriate dilutions were then plated onto MRS solid medium and anaerobically incubated at 37°C for 48 hours. Colony formation units (CFU) were then counted per milliliter of fermentation broth. This index is used for relative comparisons between different groups.
[0053] (2) Co-cultivation of basic module and herbal compound module
[0054] Both Example 1 and Comparative Example 1 were fermented separately. The specific steps were as follows: Seed cultures of *Bifidobacterium longum* and *Lactobacillus casei* were simultaneously inoculated at a 1:1 volume ratio into 500 mL shake flasks containing the aforementioned culture medium, with a total volume of 300 mL. The flasks were placed in a 37°C incubator and incubated statically for 48 h. Samples were taken at 0 h, 12 h, 24 h, 36 h, and 48 h to measure the pH, absorbance, and viable cell count of the fermentation broth. Three replicates were set up for each group, and the average value was taken. Specific results are shown in Table 1 and... Figure 1-3 .
[0055] Table 1. Indicators of Example 1 and Comparative Example 1
[0056]
[0057] As shown in Table 1, the pH value of both fermentation broths decreased continuously from approximately 6.2 initially to 3.8-3.9 after 48 hours, which was basically consistent with that of Comparative Example 1, indicating that the herbal compound module and its addition method used in this invention did not affect the normal metabolic acid production process of the bacteria. During fermentation from 0 to 24 hours, the OD values of the two groups were not significantly different, but from 36 hours onwards, the OD value of Example 1 increased significantly faster. By 48 hours, the average OD value of Example 1 reached 4.27, significantly higher than the 2.02 of Comparative Example 1. This indicates that the dandelion and salvia miltiorrhiza compound module significantly promoted bacterial growth or the accumulation of metabolites in the later stages of fermentation. At the fermentation endpoint of 48 hours, the average viable cell count of Example 1 was approximately 2.4 times that of Comparative Example 1, indicating that the herbal compound module of this invention can improve the relative viable cell level of the dual-bacterial co-culture system.
[0058] (3) Effects of herb combination and addition method on co-cultivation effect
[0059] Examples 1, 2, 3 and Comparative Examples 2-4 were fermented for 48 h according to the fermentation method in Experiment (2). The absorbance and viable cell count of the fermentation broth were measured. Three parallel samples were set up for each group and the average value was taken. The specific results are shown in Table 2.
[0060] Table 2. Effects of different herbal combinations and addition methods on co-cultivation effects.
[0061]
[0062] As shown in Table 2, although the OD value and CFU of Comparative Example 2 were higher than those of Comparative Example 1, they were significantly lower than those of Example 1, indicating that the combination of dandelion and salvia miltiorrhiza had a better growth-promoting effect than the single dandelion herb group. The OD value and CFU of Comparative Example 3 were significantly lower than those of Example 1, indicating that co-sterilization of the herbal extract and the basal culture medium weakened its growth-promoting effect, further proving the necessity of the post-sterilization supplementation strategy. The OD value and CFU of Example 2 were slightly lower than those of Example 1, but also much higher than those of Comparative Examples 1, 3, and 4, indicating that the segmented supplementation method could also achieve good co-culture results. After removing ZnSO4, the OD value and CFU of Comparative Example 4 were lower than those of Example 1, indicating that ZnSO4 in the trace element and cofactor module was beneficial to improving the overall fermentation performance of the system.
[0063] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
Claims
1. A composite culture medium for dual bacterial co-culture fermentation, characterized in that, The composite culture medium includes: a carbon source module, a nitrogen source module, a herbal extract module, and a trace element and cofactor module; The herbal extract module is one or more of dandelion extract, salvia miltiorrhiza extract, wolfberry extract, or schisandra chinensis extract; The herbal extract module is added as follows: after the carbon source module, nitrogen source module, trace elements and cofactor module are sterilized and cooled to the inoculation temperature, it is added to the composite culture medium by post-sterilization or segmented addition.
2. The composite culture medium according to claim 1, characterized in that, The carbon source module is one or more of glucose, oligosaccharides, and dietary fiber hydrolysate, and the amount added is 10-20 g / L based on the culture medium.
3. The composite culture medium according to claim 1, characterized in that, The nitrogen source module includes yeast extract and peptone, with addition amounts of 3-10 g / L and 1-5 g / L respectively, based on the culture medium.
4. The composite culture medium according to claim 1, characterized in that, The total amount of herbal extract module added based on the culture medium is 2-5 g / L. The herbal extract is a water extract, an alcohol extract, or a hydroalcohol extract, with a solvent residue of less than 50 ppm and a total polyphenol content of 50-200 mg / L.
5. The composite culture medium according to claim 1, characterized in that, The trace element and cofactor module includes B vitamins and ZnSO4, with the addition amounts of each component being 0.05-0.2 g / L and 0.01-0.05 g / L, respectively, based on the culture medium.
6. The composite culture medium according to claim 1, characterized in that, The initial pH of the composite culture medium was adjusted to 6.0-6.5 before sterilization.
7. The composite culture medium according to claim 1, characterized in that, The herbal extract module is a combination of dandelion extract and salvia miltiorrhiza extract in a mass ratio of 1:1 to 1:
2.
8. The application of the composite culture medium according to any one of claims 1-7 in the co-culture of Bifidobacterium longum and Lactobacillus casei.
9. The use of the composite culture medium according to any one of claims 1-7 in the preparation of a dual-strain fermentation product containing herbal extracts.