A strain of Pediococcus acidilactici ZKPa1222, its astragalus fermentation liquid and its application in improving immunity

Abstract

The present invention relates to the technical field of probiotics, and in particular to a strain of Pediococcus acidilactici ZKPa1222, its astragalus fermentation liquid and its application in improving immunity. Pediococcus acidilactici ) ZKPa1222 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on May 13, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30548. The present invention uses lactic acid bacteria ZKPa1222 to directly ferment astragalus extract, which simplifies the fermentation process and improves the fermentation efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to a Pediococcus acidilactici ZKPa1222, an astragalus fermentation liquid thereof and application thereof in improving immunity. Background Art

[0002] Astragalus is a common Chinese herbal medicine and one of the oldest Chinese medicines for invigorating the middle qi. It was first recorded in the Shennong's Herbal Classic of the Eastern Han Dynasty. The Chinese Pharmacopoeia stipulates that the medicinal Astragalus is the dried root of the leguminous plants Astragalus mongolica and Astragalus membranaceus. It tastes sweet and slightly warm, enters the lung and spleen meridians, and has the effects of invigorating qi and raising yang, consolidating the exterior and stopping sweating, strengthening the spleen and replenishing the middle, promoting diuresis and reducing swelling, promoting fluid and nourishing blood, relieving stagnation and relieving arthritis, expelling toxins and draining pus, and healing sores and promoting tissue regeneration. Astragalus contains a variety of active ingredients, including polysaccharides, flavonoids, saponins, amino acids and trace elements. Modern medicine has found that its pharmacological effects are very wide, and it has the functions of increasing the body's immune function, anti-tumor, consolidating the kidney and lowering blood pressure, protecting the liver and anti-inflammatory, and treating cardiovascular diseases.

[0003] Microorganisms have rich enzyme systems that can decompose and transform a variety of substances and produce rich secondary metabolites. Using microbial fermentation of traditional Chinese medicine can not only promote the release of active ingredients of traditional Chinese medicine, enzymolyze the active ingredients into small molecules, enhance the efficacy to facilitate digestion and absorption by the body, but also reduce the toxicity of some traditional Chinese medicines, reduce toxic and side effects, and even produce new active substances. The prior art usually uses lactobacillus and lactobacillus or enterococci to co-ferment astragalus for more than 24 hours in the presence of carbon and nitrogen sources to prepare microecological preparations. The above method introduces carbon and nitrogen sources into the astragalus fermentation system, and the fermentation process is cumbersome and difficult to control the fermentation process; the stability of astragalus co-fermented by mixed strains is poor, and when the mixed strains contain pathogenic strains, the application of microecological preparations will be limited. Summary of the invention

[0004] In view of the technical problems that the prior art uses mixed strains to co-ferment astragalus in the presence of a carbon source and a nitrogen source to prepare a microbial preparation, such as complicated fermentation process and low fermentation efficiency, the present invention provides a strain of Pediococcus acidilactici ZKPa1222, its astragalus fermentation liquid and application in improving immunity. The astragalus extract is directly fermented by Pediococcus acidilactici ZKPa1222, which simplifies the fermentation process and improves the fermentation efficiency.

[0005] In a first aspect, the present invention provides a strain of Pediococcus acidilactici ZKPa1222, Pediococcus acidilactici ( Pediococcus acidilactici ) ZKPa1222 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 13, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30548.

[0006] In a second aspect, the present invention provides a Pediococcus acidilactici ZKPa1222 Astragalus membranaceus fermentation liquid, which is prepared from the Pediococcus acidilactici ZKPa1222 fermentation Astragalus membranaceus extract as described above.

[0007] Furthermore, the preparation method of the astragalus extract comprises the following steps:

[0008] Step S1: adding 1 part by weight of astragalus to 8 parts by weight of water for soaking to obtain an astragalus soaking liquid;

[0009] Step S2: boiling the astragalus soaking liquid and continuing to boil for 40 minutes to separate the first decoction liquid and the first decoction residue;

[0010] Step S3: adding 4 times the weight of water to the first fry residue for second frying, and separating the second fry liquid and the second fry residue;

[0011] Step S4: adding 4 times the weight of water to the second decocted residue for a third decocting, and separating the third decocted liquid and the third decocted residue;

[0012] Step S5: combining the first decoction liquid, the second decoction liquid and the third decoction liquid, and concentrating them to obtain an astragalus extract having a crude drug content of 1.0 g / mL.

[0013] Furthermore, in step S1, the soaking time is 1 hour. In summer, water below the air temperature is used to soak the astragalus, and in winter, water above the air temperature is used to soak the astragalus. The astragalus soaking liquid includes the astragalus and the water for soaking the astragalus.

[0014] Furthermore, in step S3 and step S4, the method of decocting the medicine for the second decoction and the third decoction is to boil the medicine first, and then continue to decoct for 30 minutes, and continue to decoct slowly over low heat.

[0015] Furthermore, the preparation method of the astragalus fermentation broth of Pediococcus acidilactici ZKPa1222 comprises the following steps:

[0016] Step 1: sterilize the astragalus extract at 121° C. for 15 min to obtain a sterilized astragalus extract;

[0017] Step 2: inoculate Pediococcus acidilactici ZKPa1222 into the sterilized Astragalus extract, mix well, and culture at 37°C for 11 hours to obtain Pediococcus acidilactici ZKPa1222 Astragalus fermentation liquid.

[0018] Furthermore, in step 2, the inoculation amount of Pediococcus acidilactici ZKPa1222 is 8×10 6 CFU / mL. Pediococcus acidilactici ZKPa1222 is in freeze-dried powder form.

[0019] In a third aspect, the present invention also provides a use of the above-mentioned Pediococcus acidilactici ZKPa1222 Astragalus membranaceus fermentation liquid in the preparation of products for improving immunity, including food, medicine and health care products.

[0020] Furthermore, the product can increase spleen index and thymus index and improve the level of immunoglobulins, including but not limited to immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA).

[0021] The beneficial effects of the present invention are:

[0022] The invention provides a strain of Pediococcus acidilactici ZKPa1222, an astragalus fermentation liquid thereof and an application thereof in improving immunity. The astragalus extract is fermented by Pediococcus acidilactici ZKPa1222 to improve spleen and stomach disorders, promote the development of immune organs such as the spleen and thymus, and enhance humoral immune function and mucosal immune function while improving fermentation efficiency and simplifying fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a picture of the colony morphology on the enriched culture medium plate in Example 1.

[0024] Figure 2 This is a Gram staining microscopic examination of the colonies on the enriched medium plate in Example 1.

[0025] Figure 3 It is a statistical graph of pH and viable bacterial count of fermentation broth 1 to fermentation broth 6 in Example 3.

[0026] Figure 4 The inoculum size in Example 3 is 4×10 6 Statistical graph of pH and viable bacterial count of CFU / mL fermentation broth at different fermentation times.

[0027] Figure 5 It is a statistical graph of pH and viable bacterial count of fermentation broth 1' to fermentation broth 4' in Example 3. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0029] Example 1 Isolation, screening and identification of bacterial strains

[0030] 1. Strain screening and purification

[0031] (1) Sampling: Naturally fermented kimchi was collected in Songbai Town, Shennongjia Forest District, Hubei Province in August 2013.

[0032] (2) Sample preparation: The collected samples were diluted with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, and label them as 1# sample, 2# sample, 3# sample, 4# sample, and 5# sample in sequence for use.

[0033] (3) Preparation of MRS-CaCO3 solid culture medium: Add 1.5 wt% CaCO3 to MRS culture medium and sterilize at 121°C for 15 min to obtain MRS-CaCO3 solid culture medium for later use.

[0034] (4) Cultivation: Take 100 µL of sample #1, sample #2, sample #3, sample #4 and sample #5 and spread them evenly on MRS-CaCO3 solid culture medium, invert and culture at 37°C for 24-48 hours, and observe the growth of colonies.

[0035] (5) Colony selection: After the colonies are formed, single colonies with calcium rings are selected for three-zone streaking, and the streaking separation is repeated multiple times until the colonies are purified. The purified single colonies are inoculated into MRS liquid culture medium for culture. The culture medium of different single colonies and 50% sterile glycerol are mixed in a ratio of 1:1 (v / v) and stored in a -80℃ refrigerator. MRS liquid culture medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0036] 2. Identification and preservation

[0037] 1. Colony morphology identification

[0038] (1) Culture the purified single colony on MRS solid culture medium plate for 48 hours, and observe and record the morphology and color of the colony. Figure 1 As shown by Figure 1 It can be seen that the colonies are round, smooth, convex, milky white, occasionally light yellow or dark yellow, with neat edges and opaque. MRS solid medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0039] (2) Gram staining

[0040] The colonies on the enriched medium plate were selected for smearing, fixation, crystal violet primary staining, mordanting, decolorization, washing, safranin counterstaining, drying and microscopic examination. The microscopic examination results were as follows: Figure 2 shown.

[0041] 2. 16S rDNA sequence determination

[0042] (1) Extraction of genomic DNA of strains: The culture medium of the purified single colony was centrifuged at 5000 r / min for 10 min. The supernatant was discarded after centrifugation, and the strain to be tested was collected. The genomic DNA of the strain to be tested was extracted using a DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) and the operation was performed according to the instructions.

[0043] (2) PCR amplification

[0044] Based on the conserved region of lactic acid bacteria 16S rDNA sequence, bacterial universal primers 27F / 1492R were used.

[0045] The primer sequences used are as follows:

[0046] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (as shown in sequence 1);

[0047] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (as shown in sequence 2).

[0048] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 55°C for 35 s; extension at 72°C for 90 s; after 32 cycles, the final extension was at 72°C for 5 min.

[0049] The PCR products were identified by 1.0% agarose gel electrophoresis, and the amplified products were sequenced. The sequencing work was completed by Beijing Qingke Biotechnology Co., Ltd. The 16S rDNA sequence of the strain to be tested is as follows:

[0050]

[0051] The sequencing results were compared with the NCBI database by BLAST, and a phylogenetic tree was constructed to determine that the strain to be tested was Pediococcus acidilactici ( Pediococcus acidilactici ), therefore, the strain to be tested was named ZKPa1222. Pediococcus acidilactici ) ZKPa1222 was sent to the General Microbiology Center of China Microorganism Culture Collection Administration for preservation. The preservation date is May 13, 2024, the preservation number is CGMCC No.30548, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0052] Example 2 Preparation of Astragalus Extract

[0053] The preparation method of the astragalus extract comprises the following steps:

[0054] Step S1: adding 1 part by weight of Astragalus to 8 parts by weight of water and soaking the mixture for 1 hour to obtain an Astragalus soaking liquid, which includes Astragalus and water for soaking the Astragalus.

[0055] Step S2: first boil the Astragalus soaking liquid over high heat, then fry it over low heat, keep it slightly boiling for 40 minutes, and filter and separate the first frying liquid and the first frying residue.

[0056] Step S3: Add water 4 times the weight of the first frying residue to the first frying residue for second frying, first boil, then fry slowly over low heat for 30 minutes, and filter to separate the second frying liquid and the second frying residue.

[0057] Step S4: add water 4 times the weight of the second decocted residue to the second decocted residue for a third decocting, first boil, then decoct slowly over low heat for 30 minutes, and filter to separate the third decocted liquid and the third decocted residue.

[0058] During the decocting process of steps S2-S4, the mixture may be stirred appropriately to prevent the pot from sticking, but frequent opening of the lid is prohibited to prevent the loss of volatile components.

[0059] Step S5: combining the first decoction liquid, the second decoction liquid and the third decoction liquid, and performing steaming and concentration to obtain an astragalus extract having a crude drug content of 1.0 g / mL.

[0060] Example 3 Optimization of Preparation Conditions of Astragalus Fermentation Broth by Pediococcus acidilactici ZKPa1222

[0061] Optimization of the inoculation amount of Pediococcus acidilactici ZKPa1222

[0062] The preparation method of the astragalus fermentation liquid of Pediococcus acidilactici ZKPa1222 comprises the following steps:

[0063] Step 1: sterilize the astragalus extract prepared in Example 2 at 121° C. for 15 min to obtain a sterilized astragalus extract, and divide the sterilized astragalus extract into six equal portions for later use.

[0064] Step 2: According to 1×10 6 CFU / mL, 2×10 6 CFU / mL, 4×10 6 CFU / mL, 6×10 6 CFU / mL, 8×10 6 CFU / mL, 1.0×10 7 The inoculation amount of CFU / mL was 40.4% and 1.2% respectively. The lyophilized powder of lactic acid Pediococcus ZKPa1222 was inoculated into six portions of sterilized astragalus extract, and the mixture was shaken and placed in a constant temperature incubator at 37°C for 24 h. After the fermentation was completed, the six portions of lactic acid Pediococcus ZKPa1222 astragalus fermentation broth were marked as fermentation broth 1, fermentation broth 2, fermentation broth 3, fermentation broth 4, fermentation broth 5, and fermentation broth 6 in sequence, and the pH and viable count of fermentation broth 1 to fermentation broth 6 were measured. The pH and viable count of fermentation broth 1 to fermentation broth 6 were as follows: Figure 3 shown.

[0065] Depend on Figure 3 It can be seen that with the increase of the inoculation amount of lactic acid Pediococcus ZKPa1222, the number of viable bacteria in the fermentation broth showed an upward trend. 6 CFU / mL increased to 4×10 6 CFU / mL, the pH of the fermentation broth decreased rapidly and the number of viable bacteria increased rapidly. When the inoculation amount of Pediococcus acidilactici ZKPa1222 exceeded 4×10 6 CFU / mL, the number of live bacteria in the fermentation broth increased slowly and the pH changed slightly, indicating that when the inoculation amount of Pediococcus acidilactici ZKPa1222 exceeded 4×10 6 CFU / mL, too much lactic acid Pediococcus ZKPa1222 could not play a fermentation role, and the pH and viable count of the fermentation liquid changed little. Considering the economic benefits, when the crude drug content of Astragalus extract was 1.0 g / mL, 4×10 6 CFU / mL is the optimal inoculum size of Pediococcus acidilactici ZKPa1222.

[0066] Optimization of fermentation time of Pediococcus acidilactici ZKPa1222

[0067] The preparation method of the astragalus fermentation liquid of Pediococcus acidilactici ZKPa1222 comprises the following steps:

[0068] Step 1: sterilize the astragalus extract prepared in Example 2 at 121° C. for 15 min to obtain a sterilized astragalus extract for later use.

[0069] Step 2: According to 4×10 6 The inoculation amount of CFU / mL was 40.37 mL. The lyophilized powder of Pediococcus acidilactici ZKPa1222 was inoculated into the sterilized Astragalus extract, and the mixture was shaken and placed in a constant temperature incubator at 37°C for 18 h. The pH and viable count of the fermentation liquid were measured every two hours. The results were as follows: Figure 4 shown.

[0070] Depend on Figure 4 It can be seen that the hysteresis period of lactic acid Pediococcus ZKPa1222 fermenting astragalus extract is 2h, and fermentation begins 2h after lactic acid Pediococcus ZKPa1222 is inoculated into the sterilized astragalus extract; the 2nd to 10th hour after lactic acid Pediococcus ZKPa1222 is inoculated into the sterilized astragalus extract is the logarithmic phase, and the logarithmic phase fermentation proceeds rapidly, and enters the stable phase at the 12th hour. The number of live bacteria in the fermentation reaches the maximum value at the 12th hour after lactic acid Pediococcus ZKPa1222 is inoculated into the sterilized astragalus extract, and the number of live bacteria will not increase thereafter, so the optimal fermentation time is 12h.

[0071] Optimization of the crude drug content of Astragalus membranaceus extract

[0072] (1) The preparation method of astragalus extract with different crude drug contents comprises the following steps:

[0073] Step S1: adding 1 part by weight of Astragalus to 8 parts by weight of water and soaking the mixture for 1 hour to obtain an Astragalus soaking liquid, which includes Astragalus and water for soaking the Astragalus.

[0074] Step S2: first boil the Astragalus soaking liquid over high heat, then fry it over low heat, keep it slightly boiling for 40 minutes, and filter and separate the first frying liquid and the first frying residue.

[0075] Step S3: Add water 4 times the weight of the first frying residue to the first frying residue for second frying, first boil, then fry slowly over low heat for 30 minutes, and filter to separate the second frying liquid and the second frying residue.

[0076] Step S4: add water 4 times the weight of the second decocted residue to the second decocted residue for a third decocting, first boil, then decoct slowly over low heat for 30 minutes, and filter to separate the third decocted liquid and the third decocted residue.

[0077] During the decocting process of steps S2-S4, the mixture may be stirred appropriately to prevent the pot from sticking, but frequent opening of the lid is prohibited to prevent the loss of volatile components.

[0078] Step S5: combining the first decoction liquid, the second decoction liquid and the third decoction liquid, and performing steaming and concentration to obtain astragalus extracts with crude drug contents of 0.25 g / mL, 0.5 g / mL, 0.75 g / mL and 1.0 g / mL respectively.

[0079] (2) The method for preparing the fermentation liquid of Astragalus membranaceus ZKPa1222 comprises the following steps:

[0080] Step 1: sterilize the astragalus extracts with crude drug contents of 0.25 g / mL, 0.5 g / mL, 0.75 g / mL, and 1.0 g / mL at 121° C. for 15 min to obtain sterilized astragalus extracts;

[0081] Step 2: According to 4×10 6 The inoculation amount of CFU / mL was 40.477 g / mL. The lactic acid Pediococcus ZKPa1222 was inoculated into sterilized Astragalus extracts with crude drug contents of 0.25 g / mL, 0.5 g / mL, 0.75 g / mL and 1.0 g / mL, respectively. After shaking, the mixture was placed in a constant temperature incubator at 37°C for 12 h. After the fermentation was completed, fermentation broth 1', fermentation broth 2', fermentation broth 3' and fermentation broth 4' were obtained. The pH and viable count of fermentation broth 1'-fermentation broth 4' were measured. The measurement results are shown in FIG. Figure 5 shown.

[0082] Depend on Figure 5 It can be seen that when the crude drug content of the Astragalus extract gradually increases from 0.25g / mL to 0.75g / mL, the number of live bacteria in the fermentation broth gradually increases. When the crude drug content of the Astragalus extract rises to 0.75g / mL, the number of live bacteria in the fermentation broth increases to the maximum value; when the crude drug content of the Astragalus extract exceeds 0.75g / mL, the number of live bacteria in the fermentation broth decreases. Astragalus contains nutrients necessary for the growth of Pediococcus acidilactici ZKPa1222. When the crude drug content of the Astragalus extract gradually increases, the nutrients in the Astragalus extract gradually increase, and the number of live bacteria also gradually increases. However, when the nutrients in the Astragalus extract reach the level necessary for the growth of Pediococcus acidilactici ZKPa1222, further increasing the crude drug content of the Astragalus extract will cause the Astragalus extract to be too thick, affecting the strain growth of Pediococcus acidilactici ZKPa1222. Therefore, the crude drug content of the Astragalus extract is preferably 0.75g / mL.

[0083] (IV) Orthogonal experiment

[0084] An orthogonal experiment was designed based on the inoculation amount of Pediococcus acidilactici ZKPa1222, fermentation time and the crude drug content of Astragalus extract, and the number of viable bacteria of Pediococcus acidilactici ZKPa1222 in the fermentation broth was detected. The orthogonal experimental factor levels of the number of viable bacteria of Pediococcus acidilactici ZKPa1222 in the fermentation broth are shown in Table 1.

[0085] Table 1. Orthogonal experimental factor levels

[0086]

[0087] The L9(33) orthogonal experiment was used to test the level combinations of the orthogonal experimental factors in Table 1. The orthogonal experimental data were analyzed using the Orthogonal Design Assistant II software. The orthogonal analysis of the above orthogonal experimental factors and their results are shown in Table 2.

[0088] Table 2 Orthogonal analysis and its results

[0089]

[0090] From the statistical analysis results of the orthogonal experiment in Table 2, it can be seen that the order of the fermentation conditions that affect the fermentation of Astragalus extract by Pediococcus acidilactici ZKPa1222 is: inoculation amount > crude drug content of Astragalus extract > fermentation time. The optimal factor level combination is based on the maximum K value, which is 8×10 6 CFU / mL, the fermentation time was 11h, and the crude drug content of Astragalus extract was 1g / mL.

[0091] Example 4 Preparation of Astragalus Extract Fermented by Pediococcus acidilactici ZKPa1222

[0092] The preparation method of the astragalus fermentation liquid of Pediococcus acidilactici ZKPa1222 comprises the following steps:

[0093] Step 1: sterilize the astragalus extract prepared in Example 2 at 121° C. for 15 min to obtain a sterilized astragalus extract.

[0094] Step 2: According to 8×10 6 The lyophilized powder of lactic acid Pediococcus ZKPa1222 was inoculated into the sterilized Astragalus extract, and the mixture was shaken and placed in a 37°C constant temperature incubator for 11 h to obtain the lactic acid Pediococcus ZKPa1222 Astragalus fermentation liquid. The number of viable bacteria in the lactic acid Pediococcus ZKPa1222 Astragalus fermentation liquid was 3.0×10 9 CFU / mL, pH 3.20.

[0095] Example 5 Effects of Astragalus Extract Fermented by Pediococcus acidilactici ZKPa1222 on Immune Organs and Immune Factors in Mice

[0096] 1. Grouping and drug administration

[0097] Grouping: After one week of adaptive feeding with ordinary feed, 50 male C57BL / 6J mice were equally divided into five groups, with 10 mice in each group, and marked as control group (NC group), Pediococcus acidilactici ZKPa1222 fermented Astragalus extract group (FAS group), Astragalus extract + Pediococcus acidilactici ZKPa1222 group (PAS group), Astragalus extract group (AS group) and Pediococcus acidilactici group (PA group).

[0098] The dosing method for each group of mice is as follows:

[0099] NC group: Each mouse was intragastrically administered with 0.2 mL of sterile saline every day.

[0100] FAS group: Each mouse was intragastrically administered with 0.2 mL of the Astragalus extract fermented by Pediococcus acidilactici ZKPa1222 prepared in Example 4 every day.

[0101] PAS group: Each mouse was intragastrically administered with 0.2 mL of a mixture of astragalus extract and Pediococcus acidilactici ZKPa1222 every day. The preparation method of the mixture of astragalus extract and Pediococcus acidilactici ZKPa1222 was as follows: freeze-dried powder of Pediococcus acidilactici ZKPa1222 was added to the astragalus extract prepared in Example 2 and mixed evenly to obtain a viable cell count of 3.00 × 10 9 CFU / mL of the mixture.

[0102] AS group: Each mouse was intragastrically administered with 0.2 mL of the astragalus extract prepared in Example 2 every day.

[0103] PA group: Each mouse was gavaged with 0.2 mL of the solution every day, and the number of viable bacteria was 3.00×10 9 CFU / mL of lactic acid Pediococcus solution, the lactic acid Pediococcus solution is prepared as follows: 0.1g of lactic acid Pediococcus solution with a viable count of 1×10 11 The lyophilized powder of Pediococcus acidilactici ZKPa1222 with a CFU / g was diluted to 10 mL in a 10 mL volumetric flask.

[0104] The mice in each group were gavaged for four consecutive weeks.

[0105] 2. Sample collection and index testing

[0106] (1) Body weight: During the administration period, the body weight of each group of mice was recorded every week. The body weight test results of each group of mice after four consecutive weeks of gavage are shown in Table 3.

[0107] Table 3 Body weight of mice in each group before and after oral administration

[0108]

[0109] Note: Different letters in the table represent significant differences. P <0.05.

[0110] As shown in Table 3, after the Astragalus extract was fermented by Pediococcus acidilactici ZKPa1222, the content of effective ingredients was increased, which was beneficial to the growth and development of mice, and the weight of mice in the FAS group increased accordingly. Before oral administration, there was no significant difference in the weight of mice in each group (P>0.05). After oral administration, the FAS group, PAS group, AS group, PA group and NC group showed significant differences (P<0.05). Among them, the weight of mice in the FAS group was significantly higher than that in the PAS group, AS group and PA group (P<0.05). The above results show that astragalus extract, Pediococcus acidilactici ZKPa1222, a mixture of astragalus extract and Pediococcus acidilactici ZKPa1222, and astragalus extract fermented by Pediococcus acidilactici ZKPa1222 can all increase the body weight of mice, but the effect of astragalus extract fermented by Pediococcus acidilactici ZKPa1222 is more obvious than that of astragalus extract, Pediococcus acidilactici ZKPa1222, and a mixture of astragalus extract and Pediococcus acidilactici ZKPa1222.

[0111] (2) Organ / body weight ratio: After the administration, the mice in each group were killed by cervical dislocation, and the thymus and spleen were immediately removed. The blood was absorbed with filter paper and then weighed separately. The thymus index and spleen index of each group of mice were calculated. The calculation formulas of the thymus index and spleen index are as follows:

[0112] Thymus index (mg / g) = thymus weight / mouse body weight;

[0113] Spleen index (mg / g) = spleen weight / mouse body weight;

[0114] The mouse weight refers to the weight of mice in each group after continuous gavage for four weeks. The calculation results of the thymus index and spleen index of mice in each group are shown in Table 4.

[0115] Table 4 Calculation results of thymus index and spleen index of mice in each group

[0116]

[0117] The weight of thymus and spleen can be used to evaluate the immune status of mice. The thymus is an important lymphoid organ of the body, and its function is closely related to immunity. The spleen is the largest peripheral immune organ in the body and is involved in the humoral immunity and cellular immunity of the whole body. As can be seen from Table 4, the thymus index of the FAS group, PAS group, AS group and PA group was increased by about 45.37% (P < 0.05), 28.57%, 15.97% and 13.03% respectively compared with the thymus index of the NC group; the spleen index of the FAS group, PAS group, AS group and PA group was increased by about 43.91% (P < 0.05), 19.23%, 9.62% and 10.58% compared with the spleen index of the NC group. The results showed that astragalus extract, Pediococcus acidilactici ZKPa1222, a mixture of astragalus extract and Pediococcus acidilactici ZKPa1222, and astragalus extract fermented by Pediococcus acidilactici ZKPa1222 can promote the development of mouse immune organs. The promoting effect of astragalus extract fermented by Pediococcus acidilactici ZKPa1222 is better than that of astragalus extract, Pediococcus acidilactici ZKPa1222, and a mixture of astragalus extract and Pediococcus acidilactici ZKPa1222.

[0118] (3) Immune factors: After anesthetizing the mice in each group, the eyeballs were removed and blood was collected. The blood was centrifuged to obtain serum, and the serum IgG and sIgA were measured. The test method was carried out according to the operating steps in the instructions of the immunoglobulin G (IgG) kit (Nanjing Jiancheng) and the secretory immunoglobulin A (sIgA) kit (Nanjing Jiancheng). The serum IgG and sIgA concentrations of mice in each group are shown in Table 5.

[0119] Table 5 Serum IgG and sIgA concentrations of mice in each group

[0120]

[0121] Note: Different letters in the table represent significant differences, P < 0.05.

[0122] IgG is the main immunoglobulin in body fluids, accounting for about 70%-75% of the total immunoglobulin in the blood. It plays an important role in binding complement, enhancing immune cell phagocytosis of pathogenic microorganisms and neutralizing bacterial toxins. Secretory IgA, or sIgA, is the main antibody for local anti-infection immunity of the body's mucosa, so it is also called mucosal local antibody. As shown in Table 5, the IgG and sIgA of the AS group and the PA group were higher than those of the NC group, but there was no significant difference (P>0.05). The IgG and sIgA of the FAS group and the PAS group were significantly higher than those of the NC group (P<0.05); the IgG and sIgA of the FAS group were significantly higher than those of the PAS group, the AS group and the PA group (P<0.05). The results showed that both the Astragalus extract and the Astragalus extract fermented by Pediococcus acidilactici ZKPa1222 could increase the release of immune factors, and the Astragalus extract fermented by Pediococcus acidilactici ZKPa1222 had a better effect.

[0123] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. An application of a fermented liquid of Pediococcus acidilactici ZKPa1222 and Astragalus membranaceus in the preparation of a health food for improving immunity, characterized in that: The astragalus fermented liquid of Pediococcus acidilactici ZKPa1222 is prepared by fermenting the astragalus extract of Pediococcus acidilactici ZKPa1222; Pediococcus acidilactici ( Pediococcus acidilactici ) ZKPa1222 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 13, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30548.

2. The use according to claim 1, characterized in that The preparation method of the astragalus extract comprises the following steps: Step S1: adding 1 part by weight of astragalus to 8 parts by weight of water for soaking to obtain an astragalus soaking liquid; Step S2: boiling the astragalus soaking liquid and continuing to boil for 40 minutes to separate the first decoction liquid and the first decoction residue; Step S3: adding 4 times the weight of water to the first fry residue for second frying, and separating the second fry liquid and the second fry residue; Step S4: adding 4 times the weight of water to the second decocted residue for a third decocting, and separating the third decocted liquid and the third decocted residue; Step S5: combining the first decoction liquid, the second decoction liquid and the third decoction liquid, and concentrating them to obtain an astragalus extract having a crude drug content of 1.0 g / mL.

3. The use according to claim 2, characterized in that In step S1, the soaking time is 1 hour.

4. The use according to claim 2, characterized in that In step S3 and step S4, the decoction method of the second decoction and the third decoction is to boil the medicine first and then continue to decoct for 30 minutes.

5. The use according to claim 1, characterized in that The preparation method of the astragalus fermentation liquid of Pediococcus acidilactici ZKPa1222 comprises the following steps: Step 1: sterilize the astragalus extract at 121° C. for 15 min to obtain a sterilized astragalus extract; Step 2: inoculate Pediococcus acidilactici ZKPa1222 into the sterilized Astragalus extract, mix well, and culture at 37°C for 11 hours to obtain Pediococcus acidilactici ZKPa1222 Astragalus fermentation liquid.

6. The use according to claim 5, characterized in that In step 2, the inoculation amount of Pediococcus acidilactici ZKPa1222 was 8×10 6 CFU / mL.

7. The use according to claim 1, characterized in that Health foods can increase spleen index and thymus index and improve immunoglobulin levels.

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