Rhamnosus lactis GG fermentum with function of promoting immunity of patient and preparation method thereof

Abstract

The application discloses a Lactobacillus rhamnosus GG ferment with the function of promoting the immunity of patients and a preparation method thereof. The ferment is obtained by fermenting a composition by Lactobacillus rhamnosus GG, and the composition comprises the following components in parts by weight: 8-15 parts of full-fat milk powder, 1-4 parts of concentrated milk protein, 2-7 parts of soybean milk powder, 3-13 parts of glucose, 1-4 parts of fructose glucose syrup, 0.2-0.6 parts of yeast beta-glucan and 60-80 parts of water. The Lactobacillus rhamnosus GG ferment with the function of promoting the immunity of patients contains a high concentration of active Lactobacillus rhamnosus GG in particular. After patients who are taking medicine eat the ferment, the number of platelets in the blood of the patients can be significantly increased, so that the immunity of the patients is improved and the recovery of the patients is promoted.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and in particular to a Lactobacillus rhamnosus GG ferment that promotes the enhancement of patients' immunity and its preparation method. Background Technology

[0002] When the human body is sick, the body's immune system is also damaged, causing one or more symptoms. Patients with low immunity will not only experience a worsening of their symptoms, but also a prolonged course of illness, which can lead to chronic inflammation or even a deterioration of the condition, seriously endangering human health.

[0003] Most immune-boosting products on the market are preventative tonics, designed to enhance the body's immunity before illness occurs, thus preventing the onset and progression of disease. However, these products are generally not suitable for use when the body is already ill, as they can easily lead to drug resistance in pathogens, render existing medications ineffective, and prolong the treatment course. Therefore, how to improve the immunity of patients currently taking medication, shorten the course of illness, and promote recovery has become an urgent problem to be solved.

[0004] The gut of a healthy person is rich in lactic acid bacteria. During the proliferation of lactic acid bacteria, the gut can promote the absorption of nutrients, ensure the supply of nutrients to the body's immune cells, and maintain the integrity of the body's immune barrier. Patients often take medication for a long time, and many medications can damage the gut microbiota, resulting in a significant reduction in the types and quantities of lactic acid bacteria in the gut. By supplementing with lactic acid bacteria or lactic acid bacteria products, the gut microecology can be rebuilt to improve the immunity of patients who are taking medication from the root. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a Lactobacillus rhamnosus GG ferment that promotes the enhancement of patients' immunity.

[0006] The second objective of this invention is to provide a method for preparing Lactobacillus rhamnosus GG ferment that promotes the enhancement of patients' immunity.

[0007] A fermented product of *Lactobacillus rhamnosus* GG that promotes the enhancement of patients' immunity is produced by fermenting a composition with *Lactobacillus rhamnosus* GG. The composition comprises the following components in parts by weight: 8-15 parts whole milk powder, 1-4 parts concentrated milk protein, 2-7 parts soy milk powder, 3-13 parts glucose, 1-4 parts fructose syrup, 0.2-0.6 parts yeast β-glucan, and 60-80 parts water.

[0008] In this invention, the Lactobacillus rhamnosus GG is beneficial for improving immunity, with significant effects and is safe and healthy.

[0009] As a preferred embodiment of the present invention, the composition comprises the following components in parts by weight: 9-13 parts whole milk powder, 1-3 parts concentrated milk protein, 2-5 parts soy milk powder, 5-10 parts glucose, 1-3 parts fructose syrup, 0.2-0.5 parts yeast β-glucan, and 60-70 parts water.

[0010] As a preferred embodiment of the present invention, the composition comprises the following components in parts by weight: 11 parts whole milk powder, 2 parts concentrated milk protein, 4 parts soy milk powder, 8 parts glucose, 2 parts fructose syrup, 0.3 parts yeast β-glucan, and 72.7 parts water.

[0011] In a preferred embodiment of the present invention, during the preparation of the ferment, *Lactobacillus rhamnosus* GG activated bacterial solution is inoculated into the composition, and the concentration of the *Lactobacillus rhamnosus* GG activated bacterial solution is 2.0 × 10⁻⁶. 11 CFU / mL, inoculation ratio was (1-9):100.

[0012] As a preferred embodiment of the present invention, the inoculation amount of Lactobacillus rhamnosus GG activated bacterial solution is (3-7):100.

[0013] As a preferred embodiment of the present invention, the inoculation amount of Lactobacillus rhamnosus GG activated bacterial solution is 5:100.

[0014] As a preferred embodiment of the present invention, the fermentation product exerts its effect of enhancing the patient's immunity through one or more of the following (I) to (IV):

[0015] (I) Increase the number of platelets in the patient's blood;

[0016] (II) Increase the number of macrophages in patients;

[0017] (III) Increase the number of T cells in patients;

[0018] (IV) Enhance the phagocytic function of macrophages in patients.

[0019] The second objective of this invention is achieved by the following technical solution:

[0020] A method for preparing Lactobacillus rhamnosus GG ferment with the function of promoting the immunity of patients includes the following steps:

[0021] Dissolving: Boil the first part of water, add the prescribed amount of yeast β-glucan, stir to dissolve, and obtain a yeast β-glucan solution;

[0022] Mixing: Mix the whole milk powder, concentrated milk protein, soy milk powder and glucose in the formula amount evenly, add the second part of water and stir to dissolve, then add the fructose syrup and the yeast β-glucan solution in the formula amount, and continue stirring to obtain base material A;

[0023] Homogenization: Heat base material A and homogenize it to obtain base material B;

[0024] Sterilization: Heat base material B and then cool it to obtain base material C;

[0025] Strain activation: Lactobacillus rhamnosus GG was activated to obtain bacterial culture D;

[0026] Low-temperature fermentation: Weigh bacterial solution D, place it in substrate C, and ferment to obtain fermented product E; the weight ratio of bacterial solution D to substrate C is (1-9):100;

[0027] Maturation: Refrigerate fermented product E to obtain the desired product.

[0028] As a preferred embodiment of the present invention, the method for preparing the Lactobacillus rhamnosus GG ferment with the function of promoting the enhancement of patients' immunity includes the following steps:

[0029] Dissolving: Boil 5-15 parts of water, add 0.2-0.6 parts of yeast β-glucan, stir to dissolve, and obtain yeast β-glucan solution;

[0030] Mixing: Mix 8-15 parts whole milk powder, 1-4 parts concentrated milk protein, 2-7 parts soy milk powder, and 3-13 parts glucose evenly. Add 45-72 parts water at 50-60℃ and stir to dissolve. Stir at 450-600 r / min for 12-18 minutes. Then add 1-4 parts fructose syrup and the yeast β-glucan solution. Continue stirring for 12-18 minutes to obtain base material A.

[0031] Homogenization: Heat base material A to 55~65℃, place it in a homogenizer, and homogenize it 1~4 times under the condition of 15~20MPa to obtain base material B;

[0032] Sterilization: Heat base material B to 85~100℃, maintain for 4~6 minutes, and cool to 30~35℃ to obtain base material C;

[0033] Strain activation: Lactobacillus rhamnosus GG was activated, and the lactic acid bacteria concentration of the bacterial solution was adjusted to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture D;

[0034] Low-temperature fermentation: Weigh 3-7 parts by weight of bacterial solution D, place it in substrate C, and ferment at 30-35℃ for 8-11 hours to obtain fermented product E;

[0035] Maturation: Place fermented product E in an environment of 3~5℃ and refrigerate for 10~14 hours to obtain the product.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The Lactobacillus rhamnosus GG fermentation product provided by the present invention, which has the function of promoting the improvement of patients' immunity, can increase the number of platelets in the blood of patients and shorten the course of the disease.

[0038] (2) The Lactobacillus rhamnosus GG fermentation product provided by the present invention has the function of promoting the improvement of patients' immunity. It contains a high concentration of active Lactobacillus rhamnosus GG. After patients, including those who are taking medication, take it, the number of platelets in their blood can be significantly increased, thereby improving immunity and promoting physical recovery.

[0039] (3) The preparation method of Lactobacillus rhamnosus GG fermentation product with the function of promoting the improvement of patients' immunity provided by the present invention utilizes low temperature and long time fermentation technology. The fermentation effect is better than the traditional 37-40℃ high temperature fermentation. It can not only better extend the life cycle of Lactobacillus rhamnosus GG and extend the fermentation time, but also make the entire fermentation process smooth and stable, promote the complete conversion of nutrients such as sugar, protein and lipid in the composition, produce rich short chain fatty acids and small molecule peptides, and better preserve volatile products and aromatic substances, improve the nutritional value of the formula complex. After patients take it, the number of platelets in their blood can be significantly increased, thereby improving immunity and promoting physical recovery. Detailed Implementation

[0040] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the raw materials, equipment, etc., used in the following embodiments can all be obtained through commercial channels.

[0041] System 1: Screening of bacterial strains to enhance immunity

[0042] 1. Experimental strains: Lactobacillus rhamnosus CICC 6137 (accession number: CICC 6137), Lactobacillus rhamnosus CICC 6161 (accession number: CICC 6161), and Lactobacillus rhamnosus GG.

[0043] 2. Experimental animals: transgenic macrophage fluorescent zebrafish, wild-type AB strain zebrafish, and transgenic T lymphocyte fluorescent zebrafish.

[0044] 3. Sample preparation:

[0045] Formulation compound A:

[0046] (1) Dissolving: Boil 10 parts of purified water, add 0.3 parts of yeast β-glucan, stir at high speed to dissolve, and obtain yeast β-glucan solution;

[0047] (2) Mixing: Mix 11 parts whole milk powder, 2 parts concentrated milk protein, 4 parts soy milk powder and 8 parts glucose evenly, add 73 parts purified water at 55℃ and stir to dissolve, with a speed of 500 r / min, keep for 15 min, then add 2 parts fructose syrup and yeast β-glucan solution, continue stirring, keep for 15 min, to obtain base material A;

[0048] (3) Homogenization: Heat the base material A to 60°C, place it in a homogenizer, and homogenize it three times under the condition of 18MPa to obtain base material B;

[0049] (4) Sterilization: Heat base material B to 95°C, hold for 5 minutes, and cool to 33°C to obtain base material C;

[0050] (5) Culture medium preparation:

[0051] 1) Culture medium A: peptone 10g / L, beef extract 8g / L, yeast extract 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.01g / L, Tween-80 1g / L, dissolved and diluted with distilled water, pH=6.8, sterilized at 121℃ for 20min;

[0052] 2) Culture medium B: peptone 10g / L, beef extract 8g / L, yeast extract 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.01g / L, Tween-80 1g / L, dissolved and diluted with distilled water, pH=6.8, agar powder 20g / L, sterilized at 121℃ for 20min;

[0053] (6) Activation of bacterial strain: Take out the glycerol cryovial of Lactobacillus rhamnosus CICC 6137 stored at -80℃, shake it rapidly in a 35℃ water bath for 3 minutes, and use an inoculation loop to take 2 loops and spread them on culture medium B. Incubate at 35℃ for 48 hours; pick up colonies with an inoculation loop and place them in culture medium A. Incubate at 35℃ for 48 hours. Repeat twice, and adjust the lactic acid bacteria concentration of the bacterial solution to 2.0×10⁻⁶. 11 CFU / mL, yielding bacterial culture D;

[0054] (7) Low-temperature fermentation: Weigh 5 parts by weight of bacterial solution D, place it in substrate C, and ferment at 33℃ for 10 hours to obtain fermented product E;

[0055] (8) Maturation: Place the fermented product E in an environment of 4°C for 12 hours to obtain the formulation complex A.

[0056] Formula complex B: The preparation method is the same as that of formula complex A, except that the strain "Lactobacillus rhamnosus CICC 6137" in step (5) is replaced with "Lactobacillus rhamnosus CICC 6161".

[0057] Formula complex C: The preparation method is the same as that of formula complex A, except that the strain “Lactobacillus rhamnosus CICC 6137” in step (5) is replaced with “Lactobacillus rhamnosus GG”.

[0058] 4. Zebrafish Culture: Zebrafish are bred through natural pair mating. Zebrafish of the same strain are selected and placed in a spawning tank at a female-to-male ratio of 1:2. Four pairs of adult zebrafish are prepared for each mating. A partition is used to separate the males and females. The partition is removed before the next day's light exposure to allow for free fertilization. Timing begins from the removal of the partition, and fertilized eggs are collected 2 hours later. On average, each pair produces 300 embryos. Dead embryos are removed 6 hours and 24 hours after fertilization, and suitable embryos are selected based on their developmental stage. The zebrafish are raised in aquarium water at 28℃ (water quality: 200 mg of readily soluble sea salt per 1L of reverse osmosis water; conductivity 450 µS / cm; pH 7.5; hardness 60 mg / L CaCO3).

[0059] 5. Experimental methods:

[0060] (1) Experimental grouping: After the zebrafish embryos naturally molted 3 days after fertilization, zebrafish that were developing normally and had started feeding were selected and randomly divided into 5 groups, denoted as group A to group E. All zebrafish in all groups were placed in 6-well culture plates, with 1 group per well and 30 zebrafish per group. The volume of zebrafish culture water was 20 mL, and 0.5 mg of 0.01% sodium percarbonate was added to each group for oxygenation.

[0061] (2) Modeling: Zebrafish from groups B to E were intravenously injected with 10 nL of 0.2 mg / mL vinorelbine tartrate injection via microinjection to establish a zebrafish immunodeficiency model.

[0062] (3) Culture medium preparation: The fermentation supernatant was diluted with fresh zebrafish culture water to a concentration of 8333 μg / mL. The types and dosages of culture medium added to each group are detailed in Table 1. The culture was carried out at a constant temperature of 28 ℃, and the culture medium was changed every 24 h.

[0063] Table 1. Types and dosages of zebrafish culture medium for each group

[0064] Group Culture medium conditions Group A 3mL of fresh zebrafish culture water Group B 3mL of fresh zebrafish culture water Group C 3mL formulation complex A Group D 3mL formulation of compound B Group E 3mL formulation complex C

[0065] (4) Determination of macrophage number changes: Transgenic macrophage fluorescent zebrafish 3 days after fertilization were selected as experimental subjects. After being treated with culture medium continuously for 4 days according to the grouping, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. The images were analyzed using the image processing software NIDS-Element's. The total fluorescence intensity (S) of macrophages from the cloaca to the tail vein of the zebrafish was counted, and the improvement effect of macrophage number reduction was calculated and expressed as W. The results are shown in Table 2.

[0066]

[0067] In the formula, S0 is the sum of fluorescence intensity of macrophages in group A; S1 is the sum of fluorescence intensity of macrophages in the experimental group; and S2 is the sum of fluorescence intensity of macrophages in group B.

[0068] (5) Determination of changes in macrophage phagocytic function: Wild-type AB strain zebrafish 3 days after fertilization were selected as experimental subjects. Fluorescent microspheres were used as foreign bodies. A mixture of 0.4 mg / mL vinorelbine and fluorescent microspheres at a volume ratio of 1:1 was prepared and injected intravenously into zebrafish 3 days after fertilization. The injection volume was 10 nL / fish. After continuous treatment with culture medium for 4 days according to the grouping, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. The number of residual fluorescent particles (N) in the zebrafish was counted using ImageJ image analysis software. The improvement effect of macrophage phagocytic function was calculated and expressed as V. The results are shown in Table 2.

[0069]

[0070] In the formula, N0 is the number of remaining fluorescent particles in group A; N1 is the number of remaining fluorescent particles in the experimental group; and N2 is the number of remaining fluorescent particles in group B.

[0071] (6) Changes in T cell number: Transgenic T lymphocyte fluorescent zebrafish 4 days after fertilization were selected as experimental subjects. After being treated with culture medium continuously for 1 day according to the grouping, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. The images were analyzed using the image processing software NIDS-Element's. The total fluorescence intensity of zebrafish T cells (S) was counted, and the improvement effect of the reduction in T cell number was calculated and represented by T. The results are shown in Table 2.

[0072]

[0073] In the formula, S0 is the sum of T cell fluorescence intensity in group A; S1 is the sum of T cell fluorescence intensity in the experimental group; and S2 is the sum of T cell fluorescence intensity in group B.

[0074] 6. Results and Analysis:

[0075] Table 2. Changes in Zebrafish Indicators

[0076] Group W（%） V（%） T（%） Group A 100.00±0.00 100.00±0.00 100.00±0.00 Group B 0.00±0.00 0.00±0.00 0.00±0.00 Group C 45.18±6.15 37.17±7.30 115.36±11.45 Group D 31.53±3.46 25.45±7.76 60.80±9.17 Group E 89.74±4.01 78.93±10.15 147.57±10.68

[0077] Vinorelbine tartrate is a semi-synthetic vinca alkaloid with broad-spectrum antitumor activity and low toxicity. It exerts cytotoxic effects by interfering with microtubule aggregation during cell mitosis. Studies have shown that high-dose vinorelbine significantly suppresses bone marrow, leading to a decrease in platelet, erythrocyte, and leukocyte (neutrophils, macrophages, T cells, etc.) numbers, ultimately inducing immunodeficiency. Furthermore, the combined use of vinorelbine tartrate and fluorescent microspheres can simultaneously induce macrophage phagocytosis as a foreign substance while establishing an immune function impairment model.

[0078] Macrophages are an important component of the human innate immune system, possessing powerful functions in recognizing, phagocytizing, and clearing pathogens and cellular debris. As the first line of defense in the immune response, they maintain immune homeostasis through functions such as identifying and clearing pathogens, killing target cells, antigen presentation, and immune regulation. T cells can specifically bind to target cells and directly kill them, assist or inhibit B cells in producing antibodies, and can also enhance immune effects by releasing cytokines.

[0079] Table 2 shows that the W, V, and T values ​​of groups C to E were all increased, indicating that the formulation complexes of different strains improved the number of macrophages, macrophage phagocytic function, and T cell count in immunocompromised zebrafish models, thus enhancing the immunity of low-immunity zebrafish. Among them, group E had the highest W, V, and T values, indicating that the formulation complex in group E had the best improvement effect on low-immunity zebrafish, significantly increasing the number of macrophages and T cells and enhancing the phagocytic function of macrophages, thereby enhancing immunity. Therefore, the strain in group E, namely *Lactobacillus rhamnosus* GG, was selected as the optimal strain for improving immunity.

[0080] System 2: Screening of Composition Formulations

[0081] Table 3. Formulation of the composition (parts by weight)

[0082]

[0083] I. Sample Preparation

[0084] 1. Mixing: Weigh each component according to the formula in Table 3, mix them evenly, add purified water at 55℃ and stir to dissolve, stirring at 500 r / min for 15 min, then add fructose syrup and continue stirring for 15 min to obtain base material A;

[0085] 2. Homogenization: Heat base material A to 60℃, place it in a homogenizer, and homogenize it three times under 18MPa conditions to obtain base material B;

[0086] 3. Sterilization: Heat base material B to 95℃, hold for 5 minutes, and cool to 35℃ to obtain base material C;

[0087] 4. Culture medium preparation:

[0088] (1) Culture medium A: peptone 10g / L, beef extract 8g / L, yeast extract 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.01g / L, Tween-80 1g / L, dissolved and diluted with distilled water, pH=6.8, sterilized at 121℃ for 20min;

[0089] (2) Culture medium B: peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.01 g / L, Tween-80 1 g / L, dissolved and diluted with distilled water, pH=6.8, agar powder 20 g / L, sterilized at 121℃ for 20 min;

[0090] 5. Strain activation: Remove the *Lactobacillus rhamnosus* GG glycerol cryovials stored at -80℃, and rapidly shake them in a 35℃ water bath for 3 minutes. Use an inoculation loop to apply two loops of the solution to culture medium B, and incubate at 35℃ for 48 hours. Use an inoculation loop to pick up colonies and place them in culture medium A, incubating at 35℃ for 48 hours. Repeat this process twice, adjusting the lactic acid bacteria concentration to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture D;

[0091] 6. Fermentation: Weigh 5 parts by weight of bacterial solution D, place it in substrate C, and ferment at 33℃ for 10 hours to obtain fermented product E;

[0092] 7. Maturation: Place fermented product E in an environment of 4°C for 12 hours to obtain the formulation complex.

[0093] II. Viable Bacterial Count Determination

[0094] The lactic acid bacteria in the formulation complexes of different embodiments were counted according to the National Food Safety Standard GB 4789.35, and the results are shown in Table 4.

[0095] III. Results and Analysis

[0096] Table 4. Lactic acid bacteria content in the formulation complexes of different embodiments (×10⁹ CFU / mL)

[0097] Example 1 Example 2 Example 3 Example 4 Example 5 Example 11 Example 12 Example 13 0.15 1.01 2.57 0.38 0.63 0.58 0.70 1.34

[0098] Table 4 shows that the lactic acid bacteria content in the formulation complexes of different embodiments varies, indicating that the different components affect the fermentation effect of *Lactobacillus rhamnosus* GG. Among them, the formulation complex of Example 3 has the highest lactic acid bacteria content, indicating that the formulation complex of Example 3 has the best fermentation effect. The formulation complex contains a high concentration of *Lactobacillus rhamnosus* GG. After human consumption of this formulation complex, more *Lactobacillus rhamnosus* GG can colonize and proliferate in the intestine, exerting an immune-enhancing effect. Therefore, the formulation of Example 3 was selected as the optimal formulation of the composition.

[0099] System 3: Screening of Fermentation Ratios

[0100] Table 5. Amount of bacterial solution added (parts by weight)

[0101] Example 6 Example 7 Example 8 Example 9 Example 10 1 3 5 7 9

[0102] I. Sample Preparation

[0103] 1. Mixing: Mix 11 parts whole milk powder, 2 parts concentrated milk protein, 4 parts soy milk powder and 8 parts glucose evenly, add 73 parts purified water at 55℃ and stir to dissolve, stirring at 500 r / min for 15 min, then add 2 parts fructose syrup and continue stirring for 15 min to obtain base material A.

[0104] 2. Homogenization: Heat base material A to 60℃, place it in a homogenizer, and homogenize it three times under 18MPa conditions to obtain base material B;

[0105] 3. Sterilization: Heat base material B to 95℃, hold for 5 minutes, and cool to 35℃ to obtain base material C;

[0106] 4. Culture medium preparation:

[0107] (1) Culture medium A: peptone 10g / L, beef extract 8g / L, yeast extract 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.01g / L, Tween-80 1g / L, dissolved and diluted with distilled water, pH=6.8, sterilized at 121℃ for 20min;

[0108] (2) Culture medium B: peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.01 g / L, Tween-80 1 g / L, dissolved and diluted with distilled water, pH=6.8, agar powder 20 g / L, sterilized at 121℃ for 20 min;

[0109] 5. Strain activation: Remove the *Lactobacillus rhamnosus* GG glycerol cryovials stored at -80℃, and rapidly shake them in a 35℃ water bath for 3 minutes. Use an inoculation loop to apply two loops of the solution to culture medium B, and incubate at 35℃ for 48 hours. Use an inoculation loop to pick up colonies and place them in culture medium A, incubating at 35℃ for 48 hours. Repeat this process twice, adjusting the lactic acid bacteria concentration to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture D;

[0110] 6. Fermentation: Weigh out the bacterial solution D according to the weight parts in Table 5, place it in the substrate C, and ferment at 33℃ for 10 hours to obtain fermented product E;

[0111] 7. Maturation: Place fermented product E in an environment of 4°C for 12 hours to obtain the formulation complex.

[0112] II. Population Testing

[0113] 1. Selection of Patients: Select patients with aplastic anemia who are currently receiving treatment. (Inclusion criteria: (1) Meet the diagnostic criteria for AA in the Chinese Expert Consensus on the Diagnosis and Treatment of Aplastic Anemia; (2) Be over 18 years of age with complete data, be able to cooperate actively, and sign an informed consent form. Exclusion criteria: (1) Have congenital mental abnormalities, communication disorders, cognitive impairments, be pregnant or breastfeeding; (2) Have blood diseases, myelofibrosis, congenital immune diseases, or malignant tumors; (3) Have received hormone therapy within 3 months)

[0114] 2. Grouping: The patients were randomly divided into 5 groups of 5 people each, which were designated as Group A to Group E.

[0115] 3. Administration method: Take the formula compound half an hour after meals, 3 times a day, 150mL each time, for 30 consecutive days. The types of formula compounds taken by patients in each group are shown in Table 6.

[0116] Table 6. Types of formulation compounds taken by patients in each group

[0117] Group Types of formulation compounds Group A Formulation compound of Example 6 Group B Formulation complex of Example 7 Group C Formulation complex of Example 8 Group D Formulation complex of Example 9 Group E Formulation compound of Example 10

[0118] 4. Testing Method:

[0119] At 8:00 AM on Day 1 and Day 31, 5 mL of fasting peripheral venous blood was drawn from each group of patients. The blood routine test was performed using an automated blood analyzer, and the platelet count before and after taking the formula compound was recorded. The results are shown in Table 7.

[0120] Table 7. Changes in platelet count (×10⁹ / L) in each group of patients.

[0121] Group Before taking After taking Group A 35.50±19.74 63.14±35.67 Group B 36.11±16.41 157.85±58.31 Group C 36.36±13.58 246.73±60.15 Group D 34.84±18.35 189.46±61.06 Group E 35.45±15.63 161.38±55.44

[0122] Aplastic anemia is a syndrome of bone marrow hematopoietic failure, characterized by pancytopenia and decreased bone marrow proliferation, typically manifesting as anemia, bleeding, and infection. The pathogenesis of aplastic anemia mainly involves immune dysfunction, abnormal hematopoietic microenvironment, and hematopoietic stem cell defects. Current treatment options primarily include hematopoietic stem cell transplantation and immunosuppressant therapy, but these still have drawbacks such as high relapse rates and numerous complications. This study evaluated the therapeutic effect of the formulated compound by measuring changes in platelet counts before and after administration.

[0123] As shown in Table 7, compared with before administration, the platelet counts of patients in different groups increased after 30 days of taking the formulation complex. This indicates that even with different amounts of bacterial solution added, the formulation complexes in different embodiments can all improve the platelet count of patients with aplastic anemia. Among them, the platelet count of patients in group C was the highest after 30 days of taking the formulation complex, indicating that the formulation complex in group C can improve the patient's immunity and has the best effect on improving the platelet count of patients with aplastic anemia, effectively shortening the treatment course. Therefore, the amount of bacterial solution added in Example 8 was selected as the optimal fermentation ratio.

[0124] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A fermented product of *Lactobacillus rhamnosus* GG that promotes the enhancement of patients' immunity, characterized in that, The composition is prepared by fermenting the composition with Lactobacillus rhamnosus GG, and the composition comprises the following components in parts by weight: 8-15 parts whole milk powder, 1-4 parts concentrated milk protein, 2-7 parts soy milk powder, 3-13 parts glucose, 1-4 parts fructose syrup, 0.2-0.6 parts yeast β-glucan, and 60-80 parts water. The ferment is prepared by a method comprising the following steps: Dissolving: Boil the first part of water, add the prescribed amount of yeast β-glucan, stir to dissolve, and obtain a yeast β-glucan solution; Mixing: Mix the whole milk powder, concentrated milk protein, soy milk powder and glucose in the formula amount evenly, add the second part of water and stir to dissolve, then add the fructose syrup and the yeast β-glucan solution in the formula amount, and continue stirring to obtain base material A; Homogenization: Heat base material A and homogenize it to obtain base material B; Sterilization: Heat base material B and then cool it to obtain base material C; Strain activation: Lactobacillus rhamnosus GG was activated to obtain bacterial culture D; Low-temperature fermentation: Weigh bacterial solution D, place it in substrate C, and ferment at 30-35℃ for 8-11 hours to obtain fermented product E; the weight ratio of bacterial solution D to substrate C is (1-9):

100. Maturation: Place the fermented material in an environment of 3-5℃ and refrigerate for 10-14 hours to obtain the product.

2. The *Lactobacillus rhamnosus* GG ferment as described in claim 1, characterized in that, The composition comprises the following components in parts by weight: 9-13 parts whole milk powder, 1-3 parts concentrated milk protein, 2-5 parts soy milk powder, 5-10 parts glucose, 1-3 parts fructose syrup, 0.2-0.5 parts yeast β-glucan, and 60-70 parts water.

3. The *Lactobacillus rhamnosus* GG ferment as described in claim 1, characterized in that, The composition comprises the following components in parts by weight: 11 parts whole milk powder, 2 parts concentrated milk protein, 4 parts soy milk powder, 8 parts glucose, 2 parts fructose syrup, 0.3 parts yeast β-glucan, and 72.7 parts water.

4. The *Lactobacillus rhamnosus* GG ferment as described in claim 1, characterized in that, During the preparation of the ferment, *Lactobacillus rhamnosus* GG activated bacterial solution was inoculated into the composition, with a concentration of 2.0 × 10⁻⁶. 11 CFU / mL, inoculation ratio was (1-9):

100.

5. The *Lactobacillus rhamnosus* GG ferment as described in claim 4, characterized in that, The inoculation ratio of activated Lactobacillus rhamnosus GG culture was (3-7):

100.

6. The *Lactobacillus rhamnosus* GG ferment as described in claim 4, characterized in that, The inoculation ratio of activated Lactobacillus rhamnosus GG culture was 5:

100.

7. The *Lactobacillus rhamnosus* GG ferment as described in claim 1, characterized in that, The fermented product enhances the patient's immunity by increasing the number of platelets in the blood.

8. A method for preparing Lactobacillus rhamnosus GG ferment with the function of promoting the enhancement of patients' immunity, characterized in that, Includes the following steps: Dissolving: Boil 5-15 parts of water, add 0.2-0.6 parts of yeast β-glucan, stir to dissolve, and obtain yeast β-glucan solution; Mixing: Mix 8-15 parts whole milk powder, 1-4 parts concentrated milk protein, 2-7 parts soy milk powder, and 3-13 parts glucose evenly. Add 45-72 parts water at 50-60℃ and stir to dissolve. Stir at 450-600 r / min for 12-18 minutes. Then add 1-4 parts fructose syrup and the yeast β-glucan solution. Continue stirring for 12-18 minutes to obtain base material A. Homogenization: Heat base material A to 55~65℃, place it in a homogenizer, and homogenize it 1~4 times under the condition of 15~20MPa to obtain base material B; Sterilization: Heat base material B to 85~100℃, maintain for 4~6 minutes, and cool to 30~35℃ to obtain base material C; Strain activation: Lactobacillus rhamnosus GG was activated, and the lactic acid bacteria concentration of the bacterial solution was adjusted to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture D; Low-temperature fermentation: Weigh 3-7 parts by weight of bacterial solution D, place it in substrate C, and ferment at 30-35℃ for 8-11 hours to obtain fermented product E; Maturation: Place the fermented product E in an environment of 3~5℃ and refrigerate for 10~14 hours to obtain the product.

9. The method for preparing *Lactobacillus rhamnosus* GG ferment as described in claim 8, characterized in that, Includes the following steps: Dissolving: Boil 10 parts of water, add 0.3 parts of yeast β-glucan, stir to dissolve, and obtain yeast β-glucan solution; Mixing: Mix 11 parts whole milk powder, 2 parts concentrated milk protein, 4 parts soy milk powder and 8 parts glucose evenly, add 62.7 parts water at 55℃ and stir to dissolve at 500 r / min for 15 min, then add 2 parts fructose syrup and the yeast β-glucan solution, continue stirring and keep for 15 min to obtain base material A; Homogenization: Heat base material A to 60°C, place it in a homogenizer, and homogenize it three times under 18MPa conditions to obtain base material B; Sterilization: Heat base material B to 95°C, hold for 5 minutes, and cool to 33°C to obtain base material C; Strain activation: Remove the *Lactobacillus rhamnosus* GG glycerol cryovials stored at -80℃, and rapidly shake them in a 35℃ water bath for 3 minutes. Use an inoculation loop to apply two loops of the solution to solid culture medium and incubate at 35℃ for 48 hours. Then, use an inoculation loop to pick up colonies and place them in liquid culture medium, incubating at 35℃ for 48 hours. Repeat this process twice, adjusting the lactic acid bacteria concentration to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture D; Low-temperature fermentation: Weigh 5 parts by weight of bacterial solution D, place it in substrate C, and ferment at 33℃ for 10 hours to obtain fermented product E; Maturation: Place fermented product E in an environment of 4°C for 12 hours to obtain the product.

Images