Lactobacillus rhamnosus fermentation composition, method of preparation and use thereof in improving levels of inflammation induced by tumor cells, prevention and inhibition of breast tumors

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to a *Lactobacillus rhamnosus* fermentation composition, its preparation method, and its application in improving the level of inflammation induced by tumor cells and preventing and inhibiting breast tumors. This invention provides a *Lactobacillus rhamnosus* fermentation composition, which is composed of raw materials and *Lactobacillus rhamnosus* NKU ML1-2; the raw materials include raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel. The *Lactobacillus rhamnosus* fermentation composition of this invention has the effects of nourishing yin and clearing heat, soothing the liver and regulating qi, and strengthening the spleen and stomach. The results of the examples show that the *Lactobacillus rhamnosus* fermentation composition can, to a certain extent, prevent the proliferation, invasion, and migration of breast cancer tumor cells and has an improving effect on the level of inflammation induced by tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to Lactobacillus rhamnosus fermentation composition, preparation method, and its application in improving the level of inflammation induced by tumor cells and preventing and inhibiting breast tumors. Background Technology

[0002] Breast cancer, the most common malignant tumor among women worldwide and the second leading cause of cancer-related deaths among women in China, has become a significant public health issue threatening women's lives and health. Currently, clinical treatment for breast cancer primarily involves surgical resection combined with radiotherapy and chemotherapy. However, commonly used chemotherapy drugs, while killing tumor cells, often cause severe adverse reactions such as bone marrow suppression and liver and kidney damage, significantly impacting patients' quality of life. Therefore, there is an urgent need for a safe adjuvant therapy or health intervention method with minimal side effects.

[0003] Food and medicine homology substances are not only rich in bioactive substances with medicinal value, but can also be used as natural edible materials in daily food, holding an important place in traditional Chinese medicine and dietary culture. These substances are used to prevent diseases, improve poor physical conditions, and meet nutritional needs, embodying the concept of "medicine and food sharing the same roots and origins." Research on probiotic fermentation of food and medicine homology substances has become a hot topic in food science, nutrition, and biotechnology in recent years. Combining traditional Chinese medicine theory with modern fermentation technology, this research aims to increase the content of active ingredients in the fermentation broth of food and medicine homology substances through probiotic fermentation, generating new functional substances and enhancing their bioactivity. This provides an innovative path for developing new functional foods that combine nutritional supply and health benefits. Summary of the Invention

[0004] The purpose of this invention is to provide a *Lactobacillus rhamnosus* fermentation composition, its preparation method, and its application in improving the level of inflammation induced by tumor cells and preventing and inhibiting breast tumors. The *Lactobacillus rhamnosus* fermentation composition provided by this invention can, to a certain extent, prevent the proliferation, invasion, and migration of breast cancer tumor cells and improve the level of inflammation induced by tumor cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a Lactobacillus rhamnosus fermentation composition, wherein the Lactobacillus rhamnosus fermentation composition is composed of raw materials and Lactobacillus rhamnosus NKU ML1-2.

[0006] Preferably, the raw materials are composed of the following components by weight: 15-20 parts raspberry, 5-15 parts poria cocos, 5-10 parts gardenia, 1-5 parts rehmannia glutinosa, 1-5 parts licorice, and 5 parts tangerine peel.

[0007] Preferably, the concentration of Lactobacillus rhamnosus NKU ML1-2 in the Lactobacillus rhamnosus composition is 1×10⁻⁶. 6 -5×10 8 CFU / mL, the preservation number of the Lactobacillus rhamnosus NKU ML1-2 is GDMCC No: 66837.

[0008] The present invention also provides a method for preparing the Lactobacillus rhamnosus fermentation composition as described above, comprising the following steps: mixing raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel extracts in proportion, fully dissolving and homogenizing them, filtering and concentrating them to obtain a concentrated aqueous extract; inoculating the obtained concentrated aqueous extract with Lactobacillus rhamnosus NKU ML1-2 for fermentation to obtain a fermentation product; centrifuging the obtained fermentation product, collecting the supernatant, and obtaining the Lactobacillus rhamnosus fermentation composition.

[0009] Preferably, the concentration of the Lactobacillus rhamnosus fermentation composition is 1 mg / mL; the ratio is 4:3:2:2:1:1.

[0010] Preferably, the inoculation amount of Lactobacillus rhamnosus NKU ML1-2 is 1%-10%.

[0011] Preferably, the fermentation time is 20-36 hours, the fermentation temperature is 35-37°C, the centrifugation time is 10-15 minutes, and the centrifugation speed is 7000-9000 rpm.

[0012] The present invention also provides the use of the Lactobacillus rhamnosus fermentation composition as described above or the Lactobacillus rhamnosus fermentation composition prepared by the preparation method described above in the preparation of a medicament for preventing and / or inhibiting the proliferation, invasion and migration of breast tumor cells.

[0013] The present invention also provides the application of the Lactobacillus rhamnosus fermentation composition as described above or the Lactobacillus rhamnosus fermentation composition prepared by the preparation method described above in the preparation of a drug for improving the level of inflammation induced by tumor cells.

[0014] The present invention also provides the use of a food-medicine homology composition in the preparation of products that help enhance immunity and / or regulate intestinal flora, the composition comprising the above-described Lactobacillus rhamnosus fermentation composition; the product comprising food or pharmaceutical.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The Lactobacillus rhamnosus fermentation composition provided by this invention has the effects of nourishing yin and clearing heat, soothing the liver and regulating qi, and strengthening the spleen and stomach.

[0016] The Lactobacillus rhamnosus fermentation composition provided by this invention can, to a certain extent, prevent the proliferation, invasion and migration of breast cancer tumor cells and improve the level of inflammation induced by tumor cells. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The graph shows the changes in pH value and viable cell count during fermentation. Group A represents the complete formula, Group B lacks orange peel, Group C lacks licorice, Group D lacks rehmannia, Group E lacks gardenia, Group F lacks poria, and Group G lacks raspberry.

[0019] Figure 2 This is a graph showing the changes in total sugar content during fermentation.

[0020] Figure 3 This is a schematic diagram showing the total sugar content of each formula after 18 hours of fermentation.

[0021] Figure 4 The differences in amino acids and related metabolites among the fermentation broths of each formula after 18 hours of fermentation were identified. Among them, 1 was the fermentation broth of the complete formula, 5 was the fermentation broth lacking Gardenia, 6 was the fermentation broth lacking Poria, and 7 was the fermentation broth lacking Raspberry.

[0022] Figure 5 This is a schematic diagram showing the analysis results of the total flavonoid content in the fermentation broth of each formula after 18 hours of fermentation. Among them, the T value refers to the sum of the index values ​​of all experiments containing that level of a certain factor at a certain level, and the T value refers to the range (R value), which is the difference between the maximum and minimum values ​​of the average index under different levels of the same factor.

[0023] Figure 6 This is a schematic diagram showing the analysis results of the total content of polypeptides in the fermentation broth of each formula after 18 hours of fermentation.

[0024] Figure 7 The study was conducted to determine the viability of CCK-8 cells. Group A consisted of the full formula, while A-1, 5, and 10% consisted of 1%, 5%, and 10% of the full formula inoculation amounts, respectively. Group E was deficient in Gardenia jasminoides, Group F was deficient in Poria cocos, and Group G was deficient in Rubus idaeus.

[0025] Figure 8 This is a schematic diagram of cell colony formation assay.

[0026] Figure 9 This is a schematic diagram of the results of the scratch test.

[0027] Figure 10 This is a schematic diagram illustrating the effect of each intervention group on the total apoptosis level of 4T1 cells.

[0028] Figure 11 This is a graph showing the impact of each intervention group on the total apoptosis level of 4T1 cells.

[0029] Figure 12 This is a graph showing the expression level analysis of the inflammation-related protein tumor necrosis factor-α.

[0030] Figure 13 This is a graph showing the expression level analysis of the inflammation-related protein nuclear factor κB. Detailed Implementation

[0031] Preservation certificate information Preservation name: Lacticaseibacillus rhamnosus NKU ML1-2; Taxonomical name: Lacticaseibacillus rhamnosus; Preservation institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC); Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Accession number: GDMCC No: 66837; Deposit date: August 13, 2025.

[0032] The present invention provides a Lactobacillus rhamnosus fermentation composition, wherein the Lactobacillus rhamnosus composition is composed of raw materials and Lactobacillus rhamnosus NKU ML1-2.

[0033] Preferably, the raw materials are composed of the following components by weight: 15-20 parts raspberry, 5-15 parts poria cocos, 5-10 parts gardenia, 1-5 parts rehmannia glutinosa, 1-5 parts licorice, and 5 parts tangerine peel.

[0034] Preferably, the concentration of Lactobacillus rhamnosus NKU ML1-2 in the Lactobacillus rhamnosus NKU ML1-2 composition is 1×10⁻⁶. 6 -5×10 8 CFU / mL, the preservation number of the Lactobacillus rhamnosus NKU ML1-2 is GDMCC No: 66837.

[0035] The preferred concentration of *Lactobacillus rhamnosus* NKU ML1-2 is 1 × 10⁻⁶. 6 CFU / mL The present invention also provides a method for preparing the Lactobacillus rhamnosus fermentation composition as described above, comprising the following steps: mixing raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel extracts in proportion, fully dissolving and homogenizing them, filtering and concentrating them to obtain a concentrated aqueous extract; inoculating the obtained concentrated aqueous extract with Lactobacillus rhamnosus NKU ML1-2 for fermentation to obtain a fermentation product; centrifuging the obtained fermentation product, collecting the supernatant, and obtaining the Lactobacillus rhamnosus fermentation composition.

[0036] Preferably, the concentration of the Lactobacillus rhamnosus fermentation product is 1 mg / mL; the ratio is 4:3:2:2:1:1.

[0037] Preferably, the inoculation amount of Lactobacillus rhamnosus NKU ML1-2 is 1%-10%.

[0038] The preferred inoculation amount is 1%, 5%, or 10%, and more preferably 5%.

[0039] Preferably, the fermentation time is 20-36 hours, the fermentation temperature is 35-37°C, the centrifugation time is 10-15 minutes, and the centrifugation speed is 7000-9000 rpm.

[0040] The fermentation time is preferably 0, 6, 12, 18, 24, 30, or 36 hours; more preferably 18 hours; the fermentation temperature is preferably 37°C; the centrifugation time is preferably 15 minutes, and the centrifugation speed is 8000 rpm.

[0041] The present invention also provides the use of the Lactobacillus rhamnosus fermentation composition as described above or the Lactobacillus rhamnosus fermentation composition prepared by the preparation method described above in the preparation of a medicament for preventing and / or inhibiting the proliferation, invasion and migration of breast tumor cells.

[0042] The present invention also provides the application of the Lactobacillus rhamnosus fermentation composition as described above or the Lactobacillus rhamnosus fermentation composition prepared by the preparation method described above in the preparation of a drug for improving the level of inflammation induced by tumor cells.

[0043] The present invention also provides the use of a food-medicine homology composition in the preparation of products that help enhance immunity and / or regulate intestinal flora, the composition comprising the above-described Lactobacillus rhamnosus fermentation composition.

[0044] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0045] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0046] Example 1: Preparation of raw materials for Lactobacillus rhamnosus fermentation composition This invention provides a food-medicine composition, the ingredients of which are: raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel are mixed in the following proportions: 20 parts raspberry, 15 parts poria cocos, 10 parts gardenia, 10 parts rehmannia glutinosa, 5 parts licorice, and 5 parts tangerine peel.

[0047] The preparation method steps are as follows: The above-mentioned raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel were mixed and dissolved in drinking water to prepare an extract of Lactobacillus rhamnosus fermentation composition at a concentration of 1 mg / mL.

[0048] Extracts of the following seven Lactobacillus rhamnosus fermentation compositions were prepared by mixing the remaining components in proportions to a total volume of 30 mL: A: Contains extracts of raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel; B: Extracts of raspberry, poria cocos, gardenia, rehmannia glutinosa, and licorice; C: Extracts of raspberry, poria cocos, gardenia, rehmannia glutinosa, and tangerine peel; D: Extracts of raspberry, poria cocos, gardenia, licorice, and tangerine peel; E: Extracts of raspberry, poria cocos, rehmannia glutinosa, licorice, and tangerine peel; F: Extracts of raspberry, gardenia, rehmannia, licorice, and tangerine peel; G: Extracts of Poria cocos, Gardenia jasminoides, Rehmannia glutinosa, Glycyrrhiza uralensis, and Citrus reticulata peel; Each formula has 3 parallels.

[0049] Example 2 Preparation of Lactobacillus rhamnosus fermentation composition This invention provides a fermentation method for a Lactobacillus rhamnosus fermentation composition.

[0050] In this invention, during fermentation, an extract of raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel is first prepared and fermentation broth is prepared in a ratio of 4:3:2:2:1:1. Then, fermentation is carried out using Lactobacillus rhamnosus NKU ML1-2.

[0051] To prepare the NKU ML1-2 bacterial suspension, Lactobacillus rhamnosus NKU ML1-2 was cultured in MRS liquid medium. The culture was centrifuged, and the precipitate was resuspended to obtain a 1×10⁻⁶ Lactobacillus rhamnosus NKU ML1-2 bacterial suspension. 6 CFU / mL.

[0052] The aqueous extracts of the seven Lactobacillus rhamnosus fermentation compositions prepared in Example 1 were inoculated at inoculation rates of 1%, 5%, and 10% and fermented at 37°C for 36 hours. The fermentation broth was collected at 0, 6, 12, 18, 24, 30, and 36 hours of fermentation, with 1 mL collected each time. The mixture was centrifuged at 22℃ and 8000 rpm for 15 min, and the supernatant was collected to obtain the Lactobacillus rhamnosus fermentation composition.

[0053] Example 3 Optimization of Fermentation Conditions The fermentation compositions of Lactobacillus rhamnosus collected in Example 2 at 0, 6, 12, 18, 24, 30, and 36 hours were selected for pH value, viable cell count, and total sugar content determination and comparison. The fermentation endpoint was analyzed and the optimal fermentation conditions were determined.

[0054] This invention conducts a fractionation experiment to construct seven differentiated culture medium systems, namely: Culture medium A: contains extracts of raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel; Culture medium B: contains extracts of raspberry, poria cocos, gardenia, rehmannia glutinosa, and licorice. Culture medium C: containing extracts of raspberry, poria cocos, gardenia, rehmannia glutinosa, and tangerine peel; Culture medium D: contains extracts of raspberry, poria cocos, gardenia, licorice, and tangerine peel; Culture medium E: contains extracts of raspberry, poria cocos, rehmannia glutinosa, licorice, and tangerine peel; Culture medium F: containing extracts of raspberry, gardenia, rehmannia, licorice, and tangerine peel; Culture medium G: contains extracts of Poria cocos, Gardenia jasminoides, Rehmannia glutinosa, Glycyrrhiza uralensis, and Citrus reticulata peel.

[0055] Subsequently, using changes in pH, viable cell count, total sugar content, and characteristic metabolites during fermentation as core evaluation indicators, fermentation experiments were conducted using three inoculum sizes: 1%, 5%, and 10%. The system was used to optimize and screen fermentation conditions, and the screening results are as follows: Figure 1-3 As shown.

[0056] During fermentation, Lactobacillus rhamnosus, which has strong acid resistance, begins to adapt to the environment in the initial stage of fermentation. It grows rapidly by utilizing soluble sugars and amino acids in the culture medium, producing a small amount of organic acids or CO2, which causes the pH to decrease. As the carbon source in the culture medium is depleted, it enters a stable period, the acid production rate slows down, and the pH becomes more stable. Figure 1 The results showed that the pH value generally decreased during fermentation, with a more significant decrease in the first 0-6 hours and a gradual decrease in the second 6-36 hours. The viable cell count showed an initial increase followed by a decrease as fermentation progressed, but the viable cell count basically dropped to 0 CFU / mL by 18 hours.

[0057] Polysaccharides possess various functions, including enhancing both specific and non-specific immune functions in immunomodulation; and inhibiting tumor cell proliferation and inducing apoptosis. Results are as follows... Figure 2-3 As shown, during fermentation, sugars are consumed in large quantities as both carbon and energy sources, and the total sugar content initially shows a decreasing trend. However, under the action of microorganisms, the polysaccharides in the fermentation of *Lactobacillus rhamnosus* are degraded, leading to an increase in total sugar content, which reaches its highest value at 18 hours. The total sugar content first decreases during fermentation and then reaches its highest value at 18 hours. Based on these three indicators, the fermentation endpoint is preliminarily determined to be 18 hours, and the fermentation conditions are A-1%, A-5%, A-10%, E-5%, F-1%, F-5%, F-10%, G-1%. Differential metabolite identification results as follows Figure 4-6 As shown, the total content of amino acids and related metabolites, flavonoids, and polypeptides increased after fermentation of the complete formula. Among them, the content of immunosuppressant precursors such as methotrexate, arachidonic acid with anti-inflammatory effects, and D-gluconic acid derivatives with effects such as regulating cell apoptosis were significantly increased. At the same time, the content of neuroregulatory hormones and regulatory factors such as serotonin, melatonin, and L-tryptophan were also significantly increased. It can be seen that the fermentation process increases the potential effects in anti-inflammation, immune enhancement, promotion of tumor cell apoptosis, and relief of neuroendocrine disorders in tumor-bearing states.

[0058] Example 4 The above-mentioned fermentation conditions were selected as A-1%, A-5%, A-10%, E-5%, F-1%, F-5%, F-10%, G-1%, and the fermentation time was 18h to obtain the fermentation broth. The concentration of the fermentation broth was 1g / mL. The fermentation broth was then diluted to 25, 50, and 100μg / mL for later use.

[0059] Cellular experiments were conducted using mouse breast cancer 4T1 cells in a dedicated 4T1 cell culture medium. After three consecutive passages, the third generation cells were used for subsequent experiments: cell viability was assessed using a CCK-8 assay, cell proliferation was evaluated using a colony formation assay, anti-inflammatory factor expression levels were measured, cell migration was analyzed using a scratch assay, and the occurrence and degree of apoptosis were detected using flow cytometry. Through these experiments, the effects of different fermentation broths and concentration gradients on the activity, proliferation, invasion, migration, and apoptosis processes of mouse breast cancer 4T1 cells were systematically analyzed.

[0060] 4.1 CCK-8 cell viability assay Mouse breast cancer 4T1 cells were treated with fermentation broth at three concentration gradients of 25, 50, and 100 μg / mL, and cell viability was detected by CCK-8 assay.

[0061] As the fermentation broth concentration increased from 25 μg / mL to 100 μg / mL, the viability of 4T1 cells gradually decreased; combined with Figure 7 Data analysis shows that the fermentation broth prepared using culture medium A as the fermentation system has a significantly better inhibitory effect than the fermentation broths of groups E, F, and G, and its highest inhibition rate can reach about 20%.

[0062] 4.2 Assessing cell proliferation capacity using colony formation assays This invention evaluates the effects of different fermentation broth treatments on the clone formation ability of mouse breast cancer 4T1 cells through a cell colony formation experiment, and then assesses the cell proliferation level.

[0063] 4T1 cells were seeded at a low density of 1000 cells / mL in 24-well plates and pre-cultured in a suitable environment for 24 hours until the cells completely covered the bottom of the wells. Then, 25 and 50 μg / mL of the fermentation broth of each experimental group were added for intervention. After continuous culture for 7 days, the cell colonies were fixed and stained using crystal violet fixation staining. The morphological characteristics and formation of cell colonies in different treatment groups were then observed and recorded.

[0064] The results are as follows Figure 8 As shown, cells in the low-dose fermentation broth intervention group formed numerous dense, uniformly sized colonies. With increasing dose, the number of cell colonies decreased and their morphology became looser. Specifically, 50 μg / mL fermentation broth significantly inhibited long-term cell proliferation and colony formation. Figure 7 It was also found that the fermentation broth of the full formula A group had the best overall inhibitory effect on cells, with the full formula inoculation amount of 5% showing the best effect.

[0065] 4.3 Scratch Test This study simulated the migration of mouse breast cancer cells 4T1 under fermentation broth treatment, observing the speed and efficiency of cell filling of scratched areas. Cells were seeded at a density of 5000 cells / mL in 12-well plates and cultured for 24 hours until the cells completely covered the bottom of the plates. A representative fermentation formulation A-5% (total inoculum of 5%) was selected and prepared into three concentrations (25, 50, and 100 μg / mL) for cell treatment. Microscopic images were taken at 0, 12, and 24 hours.

[0066] The results are as follows Figure 9 As shown, compared with the control group, the scratch closure rate of cells treated with fermentation broth was significantly reduced; and the scratch closure efficiency showed a decreasing trend in dependence with increasing fermentation broth concentration; to a certain extent, this can be explained that fermentation broth has an inhibitory effect on cell migration and can slow down the migration speed of cells.

[0067] 4.4 Apoptosis After intervening 4T1 cells with different concentrations of the full-formulation fermentation broth as described above, the cells were collected, washed, counted, and resuspended. Using the Annexin V-PE apoptosis detection kit, the 4T1 cell suspensions after intervention with different concentrations of fermentation broth were labeled using Annexin V-PE / PI. The proportion of apoptotic cells was detected by flow cytometry. After routine voltage and compensation adjustments, the total number of apoptotic cells was detected and counted. The experimental results are as follows: Figure 10-11 As shown, the total apoptosis rate was significantly increased after intervention in the high-concentration fermentation broth group, with a significant increase in the proportion of early and late apoptotic cells. The total apoptosis rate at medium and low concentrations showed an increasing trend, but there was no statistical difference.

[0068] Example 5: Determination of anti-inflammatory factor content The levels of anti-inflammatory factors NF-κB and TNF-α were measured. NF-κB (Nuclear Factor-kappa B) is a transcription factor widely distributed in eukaryotic cells, playing a core regulatory role in immune responses, inflammation, cell survival, proliferation, and stress responses. It can regulate the expression of pro-inflammatory factors such as TNF-α, IL-6, and IL-8, and participate in the development and activation of B cells and T cells. TNF-α (tumor necrosis factor) has anti-infection and anti-tumor immune-boosting effects. During bacterial or viral infections, TNF-α is secreted by macrophages, T cells, etc., activating the inflammatory response to clear pathogens. It can also directly kill certain tumor cells and enhance the activity of immune cells (such as NK cells and CTLs).

[0069] like Figure 12-13As shown, compared with the control group (cultured in 4T1 special medium), the cell supernatant collected after fermentation broth intervention showed significantly increased levels of NF-κB and TNF-α. However, the levels decreased slightly with increasing fermentation broth concentration. Since excessively high concentrations of TNF-α may promote cancer cell survival, metastasis, and angiogenesis by activating NF-κB in the tumor microenvironment, and considering the results showed that the NF-κB and TNF-α levels in the full-formula group with an inoculation amount of 5% were relatively moderate, it is inferred that the fermentation broth intervention with an inoculation amount of 5% in the full-formula group had the best effect in the anti-inflammatory factor assay.

[0070] In summary, this invention has screened a *Lactobacillus rhamnosus* fermentation composition, consisting of raw materials and *Lactobacillus rhamnosus* NKU ML1-2. The raw materials include raspberry, poria cocos, gardenia, rehmannia glutinosa, licorice, and tangerine peel, with an inoculum size of 5% and a fermentation time of 18 hours. A series of experiments have demonstrated that this fermentation broth can inhibit the proliferation of mouse breast cancer tumor cells, promote their apoptosis, reduce tumor cell invasion and migration, and improve the level of inflammation induced by tumor cells.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation composition of Lactobacillus rhamnosus, characterized in that, The Lactobacillus rhamnosus fermentation composition consists of raw materials and Lactobacillus rhamnosus NKU ML1-2; The raw materials, by weight, consist of the following components: 15-20 parts raspberry, 5-15 parts poria cocos, 5-10 parts gardenia, 1-5 parts rehmannia glutinosa, 1-5 parts licorice, and 5 parts tangerine peel; the preservation number of Lactobacillus rhamnosus NKU ML1-2 is GDMCC No: 66837.

2. The Lactobacillus rhamnosus fermentation composition according to claim 1, characterized in that, The concentration of *Lactobacillus rhamnosus* NKUML1-2 was 1×10⁻⁶. 6 -5×10 8 CFU / mL.

3. A method for preparing the Lactobacillus rhamnosus fermentation composition as described in claim 1, characterized in that, Includes the following steps: Raspberry, Poria cocos, Gardenia jasminoides, Rehmannia glutinosa, Glycyrrhiza uralensis, and Citrus reticulata peel extracts were mixed in proportion, fully dissolved, homogenized, filtered, and concentrated for later use to obtain concentrated extract. Lactobacillus rhamnosus NKU ML1-2 was inoculated into the concentrated extract for fermentation to obtain fermentation product. The fermentation product was centrifuged, and the supernatant was collected to obtain Lactobacillus rhamnosus fermentation composition. The concentrated extract is a concentrated water extract.

4. The preparation method according to claim 3, characterized in that, The concentration of the Lactobacillus rhamnosus fermentation composition is 1 mg / mL.

5. The preparation method according to claim 3, characterized in that, The inoculation amount of Lactobacillus rhamnosus NKU ML1-2 is 1%-10%.

6. The preparation method according to claim 3, characterized in that, The fermentation time is 20-36 hours, and the fermentation temperature is 35-37°C; the centrifugation time is 10-15 minutes, and the centrifugation speed is 7000-9000 rpm.

7. The use of the Lactobacillus rhamnosus fermentation composition according to claim 1 or 2, or the Lactobacillus rhamnosus fermentation composition prepared by any one of the preparation methods according to claims 3-6, in the preparation of a medicament for preventing and / or inhibiting the proliferation, invasion and migration of breast tumor cells.

8. The use of a food-medicine homology composition in the preparation of products that help enhance immunity and / or regulate intestinal flora, characterized in that, The composition comprises the Lactobacillus rhamnosus fermentation composition of claim 1 or 2.

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