Efficient preparation method of compound probiotic preparation capable of effectively improving intestinal flora
Through the complex probiotic preparations of Lactobacillus rhamnosus and Streptococcus thermophilus, the lack of efficacy of the combination of single strains and traditional twin strains was solved, and the efficient regulation of intestinal flora and the promotion of intestinal health in mice was achieved.
Patent Information
- Application Number
- CN202510380404.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-08-12
AI Technical Summary
In the prior art, the probiotic effect of a single strain is insufficient. The traditional double strain combination has insufficient biocompatibility due to the antagonism between strains, and the culture conditions are difficult to maintain stability, which affects the regulation effect of intestinal microbiota.
Complex probiotic preparations of Lactobacillus rhamnosus and Streptococcus thermophilus were prepared through the coordinated proliferation of specific proportions and culture conditions to regulate the intestinal microbial structure.
It significantly improves the richness and diversity of intestinal flora in mice, promotes intestinal health, and has the potential for industrialization and clinical application.
Smart Images
Figure CN120459149A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of probiotic preparations, and more particularly to a method for efficiently preparing a composite probiotic preparation capable of effectively improving intestinal flora. Background Art
[0002] Probiotics are widely considered a promising alternative to antibiotics, leaving no residue and preventing drug resistance. They are widely used in human healthcare and clinical medicine to maintain intestinal health and treat intestinal disorders such as diarrhea. However, single-strain interventions are limited in their effectiveness in improving the intestinal microbiota, manifested in a lack of targeted regulation of gut-associated bacterial genera within the host's intestinal microbiota.
[0003] The traditional dual-strain combination has insufficient biocompatibility due to antagonism between strains, interspecies competition inhibition causes a decrease in the total number of viable bacteria, metabolite synthesis pathways are mutually exclusive, and there is a lack of process adaptability. Conventional culture conditions make it difficult to maintain the stability of the dual-bacteria symbiotic system. Summary of the Invention
[0004] In order to solve the above technical problems, the present invention provides a composite probiotic preparation that can effectively improve intestinal flora.
[0005] The present invention provides a composite probiotic preparation that can effectively improve intestinal flora. The composite probiotics include but are not limited to Lactobacillus rhamnosus and Streptococcus thermophilus.
[0006] An efficient preparation method of a composite probiotic preparation that can effectively improve intestinal flora comprises the following steps: Step 1: Lactobacillus rhamnosus and Streptococcus thermophilus were isolated and purified from the probiotic beverage, and inoculated into MRS broth medium respectively. The culture was then anaerobically cultured at 37°C for 24 hours to activate the strains. Step 2: Activated Lactobacillus rhamnosus and Streptococcus thermophilus were inoculated into MRS liquid culture medium at a ratio of 1:1.5 at a total inoculum volume of 2%. The pH of the MRS liquid culture medium was 6.5 and cultured at 37° C. for 13 h.
[0007] Step 3: Prepare the compound probiotic preparation.
[0008] The beneficial effects of the present invention are: the present invention adopts a composite thermophilic Streptococcus and rhamnosus Bacillus to enhance the system through strain synergy, thereby achieving efficient bacterial proliferation. The preparation can effectively regulate the intestinal flora structure of mice, thereby promoting the intestinal health of mice.
[0009] Animal experiments have shown that the mixture enhances the richness and diversity of the intestinal flora in mice by regulating the structure of the intestinal flora. The present invention also provides efficient mixed culture conditions, which has both industrial potential and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 This is the growth curve of the pure culture and co-culture of the composite bacteria of the present invention; Figure 2 This is a diagram showing the effect of culture temperature on strains in the mixed culture conditions of the present invention; Figure 3 This is a diagram showing the effect of medium pH on strains in the mixed culture conditions of the present invention; Figure 4 This is a diagram showing the effect of the inoculation ratio of the two strains on the strains in the mixed culture conditions of the present invention; Figure 5 This is a diagram showing the effect of inoculum size on strains in the mixed culture conditions of the present invention; Figure 6 Alpha diversity analysis of mouse fecal samples according to the present invention; Figure 7 This is the analysis of the dominant flora of the mouse intestinal flora of the present invention; Figure 8 It is the LDA Effect Size community difference analysis among groups of the present invention; Figure 9 The present invention measures parameters based on species information (the left picture shows differences in microorganisms at the family level; the right picture shows differences in microorganisms at the genus level). DETAILED DESCRIPTION
[0011] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that these embodiments are discussed solely to enable those skilled in the art to better understand and implement the subject matter described herein, and that the functions and arrangements of the elements discussed may be varied without departing from the scope of this specification. Various examples may omit, substitute, or add various processes or components as needed. Furthermore, features described in some examples may be combined in other examples.
[0012] At least one embodiment of the present invention discloses a composite probiotic preparation that can effectively improve intestinal flora, including but not limited to Lactobacillus rhamnosus and Streptococcus thermophilus.
[0013] An efficient preparation method of a composite probiotic preparation that can effectively improve intestinal flora comprises the following steps: Step 1: Lactobacillus rhamnosus and Streptococcus thermophilus were isolated and purified from the probiotic beverage, and inoculated into MRS broth medium respectively. The culture was then anaerobically cultured at 37°C for 24 hours to activate the strains. Step 2: Activated Lactobacillus rhamnosus and Streptococcus thermophilus were inoculated into MRS liquid culture medium at a ratio of 1:1.5 at a total inoculum volume of 2%. The pH of the MRS liquid culture medium was 6.5 and cultured at 37° C. for 13 h.
[0014] Step 3: Prepare the compound probiotic preparation.
[0015] We first isolated and purified Lactobacillus rhamnosus and Streptococcus thermophilus from a probiotic drink, inoculated them into MRS broth culture medium, and cultured them anaerobically at 37°C for 24 hours to activate the strains. We then measured the growth curves of the two bacteria in pure culture and co-culture.
[0016] Pure culture: Activated Lactobacillus rhamnosus and Streptococcus thermophilus were inoculated into MRS liquid culture medium at a 2% inoculation rate, cultured at 37°C for 36 hours, and samples were taken every 4 hours to measure the OD600nm value of the culture medium.
[0017] Co-culture: Activated Lactobacillus rhamnosus and Streptococcus thermophilus were inoculated into MRS liquid culture medium at a ratio of 1:1 at 2% of the total inoculum volume, and cultured at 37°C for 36 hours. Samples were taken every 4 hours, and the OD600nm value of the culture medium was measured.
[0018] Then, the mixed culture conditions of the two bacteria were optimized: the growth curves of pure culture and co-culture were drawn with the culture time as the horizontal axis and the OD600nm value as the vertical axis (see Figure 1 The optimal method and time for culturing the two bacteria were determined by the growth curve characteristics.
[0019] Lactobacillus rhamnosus and Streptococcus thermophilus were mixed and cultured in MRS liquid medium for 24 h. The medium temperature was set separately by single factor experiment. Figure 2 (25℃, 29℃, 33℃, 37℃, 41℃, 45℃), see Figure 3 , pH value of culture medium (5.6, 5.9, 6.2, 6.5, 6.8, 7.1), see Figure 4 , the inoculation ratio of the two strains (2:1, 1.5:1, 1:1, 1:1.5, 2:1), see Figure 5 , inoculation amount (1%, 2%, 3%, 4%, 5%, 6%). Measure the corresponding viable bacterial counts and determine the conditions that are conducive to the mixed culture of the two bacteria.
[0020] Then, see Figure 6-Figure 9We conducted animal experiments to determine the effects of the two strains on the intestinal microbiota of mice. Fecal DNA was extracted from each group of mice using a fecal genomic DNA extraction kit and stored at -20°C for subsequent data analysis. A two-step PCR amplification method was used to amplify bacterial 16S rRNA. Sequencing libraries were constructed from the resulting PCR products and sequenced using the Illumina Miseq platform. We found that the two strains exhibited significant complementary synergistic effects in mixed cultures, significantly enhancing strain activity and improving intestinal microbiota structure, thereby promoting intestinal health in mice.
[0021] The above describes an embodiment of the present invention, but this embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Ordinary technicians in this field can also make more forms of equivalent embodiments based on the inspiration of this embodiment, all of which are protected by this embodiment.
Claims
1. A composite probiotic preparation that can effectively improve intestinal flora, characterized in that: These include Lactobacillus rhamnosus and Streptococcus thermophilus.
2. An efficient preparation method of a composite probiotic preparation that can effectively improve intestinal flora, characterized in that: The steps include: Step 1: Lactobacillus rhamnosus and Streptococcus thermophilus were isolated and purified from the probiotic beverage, and inoculated into MRS broth medium respectively. The culture was then anaerobically cultured at 37°C for 24 hours to activate the strains. Step 2: 2% of the total inoculum of activated Lactobacillus rhamnosus and Streptococcus thermophilus was inoculated into MRS liquid culture medium at a ratio of 1:1.
5. The pH of the MRS liquid culture medium was set to 6.5, and cultured at 37°C for 13 hours. Step 3: Prepare the compound probiotic preparation.