Anti-fatigue and immunity-enhancing compound probiotic fermentation liquor, preparation method and application thereof

Abstract

The present application relates to the technical field of probiotic fermentation products, in particular to an anti-fatigue and immunity-enhancing compound probiotic fermentation liquor as well as a preparation method and application thereof. The preparation method of the compound probiotic fermentation liquor is as follows: homogenize and sterilize the homologous medicinal and edible ingredients and water to obtain a fermentation substrate, inoculate the substrate with Lactobacillus plantarum Lp45 culture solution for fermentation, adjust the pH to 4.8-5.0 when the dissolved oxygen rate is below 20%, inoculate the substrate with Bifidobacterium bifidum B11 culture solution, continue the fermentation under stirring, and separate the solid and liquid after the fermentation is completed, thus obtaining the compound probiotic fermentation liquor. The homologous medicinal and edible ingredients include Rhizoma Polygonati, Red Ginseng, Fructus Lycii, Longan Arillus, Jujube and Gardenia. The homologous medicinal and edible ingredients are safe and effective, and after the synergistic fermentation of Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11, the postbiotic metabolites are enriched, which can produce significant anti-fatigue and immunity-enhancing effects, and the obtained fermentation liquor has a rich sweet and sour taste.

Description

Technical Field

[0001] This invention relates to the field of probiotic fermentation product technology, specifically to a compound probiotic fermentation liquid that combats fatigue and enhances immunity, its preparation method, and its application. Background Technology

[0002] Fatigue is a symptom caused by excessive accumulation of energy metabolism, immune system dysfunction, or weakened bodily functions, making it difficult to maintain mental and physical activity, and it is difficult to recover in a short period of time. Staying up late, working overtime, irregular eating habits, and the pressures of modern life and work prevent the effective replenishment and recovery of physical and mental energy, leading to a year-on-year increase in the number of people prone to fatigue and with weakened immunity, and a trend towards affecting younger people. Fatigue affects people's lives, work, and studies; relieving fatigue and improving fatigue symptoms are crucial for improving immunity and maintaining overall health. In addition, multiple studies have shown that certain viruses can cause patients to experience persistent fatigue, easy fatigue, or muscle weakness as sequelae.

[0003] Currently, most common products for relieving fatigue and boosting immunity mainly rely on supplementing electrolytes, trace elements, or cerebral cortex stimulants. However, these products only treat the symptoms, not the root cause, and cannot fundamentally improve the sub-health symptoms of fatigue. Moreover, the stimulant ingredients are mostly short-acting, and may even be addictive, causing side effects such as gastrointestinal discomfort and nervous system disorders. Although there are also anti-fatigue soft capsules, drops, or solid beverages made from traditional Chinese medicine formulas or food-medicine homology formulas with added excipients, the complex ingredients can increase the metabolic burden on the kidneys, and traditional Chinese medicine often has an unpleasant taste, usually requiring the addition of flavoring excipients to improve it. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this invention provides a compound probiotic fermentation liquid for anti-fatigue and immune enhancement, its preparation method, and its application. This fermentation liquid is obtained by fermenting raw materials such as Polygonatum sibiricum (a medicinal and edible plant) with specific strains, exhibiting significant anti-fatigue and immune-enhancing effects, and also possessing a superior taste.

[0005] To solve the above technical problems, the first aspect of the present invention provides a method for preparing a compound probiotic fermentation liquid for anti-fatigue, specifically including the following operations: mixing and slurrying the raw materials of the medicinal and edible formula with water, sterilizing the mixture, using it as a fermentation substrate, inoculating with Lactobacillus plantarum Lp45 culture medium for fermentation, adjusting the pH to 4.8-5.0 when the dissolved oxygen rate drops below 20%, and then inoculating with Bifidobacterium bifidum B11 culture medium, continuing fermentation under stirring, and separating the solid and liquid after fermentation, the resulting fermentation liquid is the compound probiotic fermentation liquid; wherein, the raw materials of the medicinal and edible formula include 35-45 parts of Polygonatum sibiricum, 1-5 parts of red ginseng, 20-30 parts of Lycium barbarum, 10-15 parts of longan pulp, 5-10 parts of jujube, and 5-10 parts of Gardenia jasminoides.

[0006] Among them, *Lactobacillus plantarum* Lp45 has been disclosed in patent application number CN201310587738.8 (A *Lactobacillus plantarum* and its application), with accession number CGMCC No. 8072; *Bifidobacterium bifidum* B11 has been disclosed in patent application number CN202210476137.9 (A *Bifidobacterium bifidum* strain that reduces fat, relieves hyperglycemia, and regulates intestinal immunity and its application), with accession number CGMCC No. 24381.

[0007] The *Danxi Xinfa* states that the heart governs blood, and the kidneys govern essence; depletion of essence and dryness of blood lead to fatigue. This invention, based on the principles of targeted qi and blood tonification, and kidney and spleen tonification, focuses on the spleen, kidneys, and liver, rationally utilizing a combination of traditional Chinese medicine formulas. Specifically: Polygonatum sibiricum tonifies qi and nourishes yin, strengthens the spleen, moistens the lungs, benefits the kidneys, and calms the five internal organs; red ginseng greatly tonifies vital energy, restores pulse and consolidates the body, and benefits qi and blood; wolfberry focuses on nourishing the liver and kidneys, moistening the lungs and generating fluids; longan pulp tonifies the heart and spleen, nourishes blood and calms the mind; jujube tonifies the spleen and stomach, and tonifies the middle jiao and benefits qi; gardenia jasminoides clears heat and dampness, drains heat from the twelve meridians, and reduces the tendency of tonifying formulas to cause heatiness. The traditional Chinese medicine components in this compound probiotic fermented product, through the above combination, can supplement the body's required nutrients, enhance bodily functions, and replenish fatigued and damaged qi and blood by promoting the biochemical circulation of qi and blood. It strengthens the spleen and kidneys, nourishes yin and consolidates the foundation, thus achieving the effects of anti-fatigue and improved immunity. From the perspective of modern medicine, the polysaccharides, saponins, flavonoids, and other active ingredients in the aforementioned traditional Chinese medicines can improve the body's utilization of oxygen and eliminate lactic acid produced after exercise, exhibiting significant effects in resisting hypoxia, combating fatigue, and providing antioxidant benefits. Furthermore, most of these ingredients are sweet in taste, and when combined in the above proportions, they do not produce an unpleasant palatability.

[0008] This invention utilizes *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 to ferment the aforementioned traditional Chinese medicines. This biotransformation alters the active ingredients in these medicinal and edible raw materials, increasing or forming new compounds. Simultaneously, *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 metabolize and produce various active enzymes, promoting the release and decomposition of the active ingredients in the traditional Chinese medicines. Furthermore, *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 utilize the nutrients in the aforementioned traditional Chinese medicines as prebiotics for growth and metabolism, simultaneously producing abundant metabolites, thereby regulating the balance of intestinal flora. This invention, through the synergistic fermentation of the aforementioned qi-tonifying, blood-activating, liver-soothing, lung-moistening, spleen-strengthening, and kidney-tonifying medicinal and edible raw materials using *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11, can fundamentally improve the body's sub-health state, thereby achieving anti-fatigue and immune-enhancing effects. Experiments have shown that this compound probiotic fermentation liquid can prolong the swimming time of mice under load and relieve post-exercise fatigue; at the same time, it can promote the proliferation of macrophages RAW264.7 and activate the cell's phagocytic ability.

[0009] Furthermore, by using Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 to synergistically ferment the above-mentioned food and medicinal materials that have no unpleasant taste, the resulting fermentation liquid can also have a pleasantly sweet and sour taste, without the need to add other flavoring additives.

[0010] This invention expands the application of segmented co-fermentation of Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 in the field of anti-fatigue, and confirms the applicability of microbial preparations and their metabolites in anti-fatigue and immune-enhancing products.

[0011] In conjunction with the first aspect, the preferred weight ratio of the following ingredients is as follows: 38-42 parts of Polygonatum sibiricum, 2-4 parts of red ginseng, 23-27 parts of wolfberry, 12-13 parts of longan pulp, 7-8 parts of jujube, and 7-8 parts of gardenia.

[0012] Preferably, the weight ratio of the Polygonatum, red ginseng, wolfberry, longan pulp, jujube and gardenia is as follows: 40 parts Polygonatum, 3 parts red ginseng, 25 parts wolfberry, 12.5 parts longan pulp, 7.5 parts jujube and 7.5 parts gardenia.

[0013] In conjunction with the first aspect, the method for preparing the Lactobacillus plantarum Lp45 culture medium is as follows: the Lactobacillus plantarum Lp45 strain is cultured for two generations in a seed culture medium containing Polygonatum extract to obtain the second-generation seed culture of Lactobacillus plantarum Lp45, which is the Lactobacillus plantarum Lp45 culture medium.

[0014] In conjunction with the first aspect, the method for preparing the Bifidobacterium bifidum B11 culture medium is as follows: Bifidobacterium bifidum B11 strain is cultured for two generations in a seed culture medium containing Polygonatum sibiricum extract to obtain the second generation seed culture of Bifidobacterium bifidum B11, which is the Bifidobacterium bifidum B11 culture medium.

[0015] Polygonatum extract is rich in polysaccharides, oligosaccharides, monosaccharides, and other sugars, providing a rich carbon source for bacterial growth in seed culture media. Seed culture media containing Polygonatum extract also provide an adaptive growth environment for the later fermentation of traditional Chinese medicine formulas containing Polygonatum. Experimental studies have found that *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 can produce high levels of β-glucosidase and carboxymethyl cellulase in seed culture media containing Polygonatum extract. This reveals that the *Lactobacillus plantarum* Lp45 culture media and *Bifidobacterium bifidum* B11 culture media obtained in this seed culture medium have excellent cellulose degradation capabilities, effectively promoting the release of active ingredients from the aforementioned medicinal and edible raw materials.

[0016] Preferably, the preparation method of the Polygonatum extract is as follows: extract Polygonatum with water at 100-105℃ at least twice, and combine the extracts to obtain the extract.

[0017] More preferably, the preparation method of the Polygonatum extract can be as follows: Polygonatum and water are mixed and pulped, extracted at 100-105℃ for 40-80 min, and after solid-liquid separation, water is added to the resulting residue, and extracted at 100-105℃ for 20-30 min, and after solid-liquid separation, the extracts obtained from the two extractions are combined to obtain the final extract. More preferably, when mixing Polygonatum and water, the mass of water is 5-8 times the mass of Polygonatum; the mass of water added to the resulting residue is 5-10 times the mass of Polygonatum.

[0018] Preferably, the seed culture medium comprises: 3.5–4.5 g / L yeast extract, 8–12 g / L peptone, 4.5–5.5 g / L beef meal, 1.8–2.2 g / L dipotassium hydrogen phosphate, 4.5–5.5 g / L sodium acetate, 1.8–2.2 g / L triammonium citrate, 0.18–0.22 g / L magnesium sulfate, 0.045–0.055 g / L manganese sulfate, 0.8–1.2 mL / L Tween 80, and 150–350 mL / L Polygonatum extract, with water as the solvent. This seed culture medium is a modified version of liquid MRS medium, replacing glucose as the carbon source with Polygonatum extract, which is more conducive to providing an adaptive growth environment for the later fermentation of traditional Chinese medicine formulas containing Polygonatum. Before use, the seed culture medium should be sterilized at 121°C for 15–25 minutes.

[0019] A more preferred composition is: 4 g / L yeast powder, 10 g / L peptone, 5 g / L beef powder, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1 mL / L Tween 80, 250 mL / L Polygonatum extract, and water as the solvent.

[0020] Preferably, when culturing Bifidobacterium bifidum B11 with the seed culture medium, the seed culture medium further comprises 0.45-0.55 g / mL cysteine ​​hydrochloride solution.

[0021] Preferably, the *Lactobacillus plantarum* Lp45 strain is inoculated at 3%–5% in the seed culture medium at 37±1℃ for 14–18 h to obtain a first-generation seed culture of *Lactobacillus plantarum* Lp45; the first-generation seed culture of *Lactobacillus plantarum* Lp45 is then inoculated at 3±0.5% in the seed culture medium and incubated at 37±1℃ for 12–16 h to obtain a second-generation seed culture of *Lactobacillus plantarum* Lp45.

[0022] Preferably, the Bifidobacterium bifidum B11 strain is inoculated at 3%–5% in the seed culture medium and anaerobically cultured at 37±1℃ for 22–26 h to obtain a first-generation seed culture of Bifidobacterium bifidum B11; the first-generation seed culture of Bifidobacterium bifidum B11 is inoculated at 3±0.5% in the seed culture medium and cultured at 37±1℃ for 12–16 h to obtain a second-generation seed culture of Bifidobacterium bifidum B11.

[0023] Preferably, the inoculum amount of the second-generation Lactobacillus plantarum seed culture in the fermentation substrate is 3% to 5%.

[0024] Preferably, the inoculation amount of the second-generation Bifidobacterium bifidum B11 seed culture in the fermentation substrate is 5% to 10%, and the fermentation time is 72 to 96 hours.

[0025] In conjunction with the first aspect, the mass ratio of the medicinal and edible ingredients to water in the formula is 1:8 to 10.

[0026] In conjunction with the first aspect, the sterilization is performed at 105–110°C for 25–40 minutes.

[0027] In conjunction with the first aspect, the pH was adjusted using a citrate-dipotassium hydrogen phosphate buffer solution.

[0028] In conjunction with the first aspect, the stirring speed is 30 to 40 rpm.

[0029] In conjunction with the first aspect, the solid-liquid separation method can be centrifugation, filtration, or a combination of both.

[0030] Secondly, the present invention also provides a compound probiotic fermentation liquid that has the effects of relieving fatigue and improving immunity, which is prepared by the above method.

[0031] Thirdly, the present invention also provides a compound probiotic fermentation product that combats fatigue and enhances immunity, containing the above-mentioned compound probiotic fermentation liquid and other ingredients acceptable in food or medicine.

[0032] In conjunction with the third aspect, the compound probiotic fermented products include, but are not limited to, liquid preparations, granules, and compressed candies. Attached Figure Description

[0033] Figure 1 To examine the effect of each group of samples in Example 3 of this invention on the proliferation ability of RAW264.7 cells;

[0034] Figure 2 To examine the effect of each group of samples in Example 3 of this invention on the phagocytic activity of RAW264.7 cells; Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Fatigue affects people's lives, work, and studies. Relieving fatigue and improving fatigue symptoms are crucial for boosting immunity and maintaining overall health. In recent years, research on fatigue and gut microbiota has increased. Studies have found that patients prone to fatigue have reduced gut microbiota diversity and increased variability. It is possible that probiotics or their metabolites can regulate the gut microbiota structure, thereby affecting the gut-brain axis and alleviating fatigue through regulation of the central nervous system. However, while probiotics help regulate gut microbiota structure, not all probiotics are effective enough to relieve fatigue.

[0037] This invention uses food-medicine homology raw materials as probiotic fermentation substrate. Through the screening of food-medicine homology raw materials and probiotic strains, combined with a specific fermentation process, a compound probiotic fermentation liquid with anti-fatigue and immune-enhancing effects is obtained.

[0038] The embodiments of the present invention will be further described below with reference to several examples.

[0039] The Polygonatum, red ginseng, wolfberry, longan pulp, jujube, and gardenia in the following examples all meet the requirements of Part I of the Chinese Pharmacopoeia (2020 edition).

[0040] Example 1

[0041] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0042] 1. Preparation of seed culture medium

[0043] Seed culture medium preparation: 4g yeast powder, 10g peptone, 5g beef meal, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1mL Tween 80, 250mL Polygonatum extract, and water to 1000mL. The culture medium was sterilized at 121℃ for 20min. The Polygonatum extract was prepared as follows: Polygonatum was pulped at a ratio of 1g raw material to 6.5mL water. After pulping, it was extracted at 100-105℃ for 60min. The filtrate was filtered and stored. Then, 7.5mL of water was added, and the mixture was extracted at 100-105℃ for 25min. The filtrate was filtered again to obtain a second filtrate. The two filtrates were combined to obtain the Polygonatum extract.

[0044] 2. Preparation of seed solution

[0045] Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 were separately inoculated into the above-mentioned seed culture medium (the seed culture medium for Bifidobacterium bifidum B11 was supplemented with cysteine ​​hydrochloride to a concentration of 0.5 g / L), with an inoculation amount of 4%. Lactobacillus plantarum Lp45 was fermented in a sealed container at 37±1℃ for 16 h, and Bifidobacterium bifidum B11 was cultured anaerobically at 37±1℃ for 24 h, to obtain first-generation seed culture of Lactobacillus plantarum Lp45 and first-generation seed culture of Bifidobacterium bifidum B11, respectively. The first-generation seed culture was inoculated into the seed culture medium at an inoculation amount of 3% and cultured at 37±1℃ for 14 h to obtain second-generation seed culture of Lactobacillus plantarum Lp45 and second-generation seed culture of Bifidobacterium bifidum B11.

[0046] 3. Preparation of fermentation substrate

[0047] Weigh the ingredients for the medicinal and edible formula according to the following proportions: 40 parts Polygonatum sibiricum, 3 parts red ginseng, 25 parts wolfberry, 12.5 parts longan pulp, 7.5 parts jujube, and 7.5 parts gardenia. Mix the above ingredients with water in a 1:9 ratio and slurry. Sterilize at 105-110℃ for 35 minutes to prepare the fermentation substrate.

[0048] 4. Fermentation

[0049] The sterilized fermentation substrate was first inoculated with 4% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 8% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, centrifuging and filtering, the anti-fatigue compound probiotic fermentation broth was obtained.

[0050] Example 2

[0051] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0052] 1. Preparation of seed culture medium: Same as in Example 1

[0053] 2. Preparation of seed liquid: Same as in Example 1

[0054] 3. Preparation of fermentation substrate

[0055] Weigh the ingredients for the medicinal and edible formula according to the following proportions: 38 parts Polygonatum sibiricum, 2 parts red ginseng, 23 parts Lycium barbarum, 12 parts longan pulp, 7 parts jujube, and 7 parts Gardenia jasminoides. Mix the above ingredients with water in a 1:9 ratio and slurry. Sterilize at 105-110℃ for 35 minutes to prepare the fermentation substrate.

[0056] 4. Fermentation

[0057] The sterilized fermentation substrate was first inoculated with 4% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 8% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, centrifuging and filtering, the anti-fatigue compound probiotic fermentation broth was obtained.

[0058] Example 3

[0059] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0060] 1. Preparation of seed culture medium: Same as in Example 1

[0061] 2. Preparation of seed liquid: Same as in Example 1

[0062] 3. Preparation of fermentation substrate

[0063] Weigh the ingredients for the food-medicine homology formula according to the following proportions: 42 parts Polygonatum sibiricum, 4 parts red ginseng, 27 parts wolfberry, 13 parts longan pulp, 8 parts jujube, and 8 parts gardenia. Mix the above ingredients with water in a 1:9 ratio and slurry. Sterilize at 105-110℃ for 35 minutes to use as a fermentation substrate.

[0064] 4. Fermentation

[0065] The sterilized fermentation substrate was first inoculated with 4% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 8% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, centrifuging and filtering, the anti-fatigue compound probiotic fermentation broth was obtained.

[0066] Example 4

[0067] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0068] 1. Preparation of seed culture medium: Same as in Example 1

[0069] 2. Preparation of seed liquid: Same as in Example 1

[0070] 3. Preparation of fermentation substrate

[0071] Weigh the ingredients for the food-medicine homology formula according to the following proportions: 35 parts Polygonatum sibiricum, 1 part red ginseng, 20 parts wolfberry, 10 parts longan pulp, 5 parts jujube, and 5 parts gardenia. Mix the above ingredients with water in a 1:8 ratio and slurry. Sterilize at 105-110℃ for 40 minutes to use as a fermentation substrate.

[0072] 4. Fermentation

[0073] The sterilized fermentation substrate was first inoculated with 4% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 8% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, centrifuging and filtering, the anti-fatigue compound probiotic fermentation broth was obtained.

[0074] Example 5

[0075] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0076] 1. Preparation of seed culture medium: Same as in Example 1

[0077] 2. Preparation of seed liquid: Same as in Example 1

[0078] 3. Preparation of fermentation substrate

[0079] Weigh the ingredients for the food-medicine homology formula according to the following proportions: 45 parts Polygonatum sibiricum, 5 parts red ginseng, 30 parts wolfberry, 15 parts longan pulp, 10 parts jujube, and 10 parts gardenia. Mix the above ingredients with water at a ratio of 1:10 and slurry. Sterilize at 105-110℃ for 25 minutes to prepare the fermentation substrate.

[0080] 4. Fermentation

[0081] The sterilized fermentation substrate was first inoculated with 4% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 8% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, centrifuging and filtering, the anti-fatigue compound probiotic fermentation broth was obtained.

[0082] Example 6

[0083] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0084] 1. Preparation of seed culture medium

[0085] Seed culture medium preparation: 4g yeast powder, 10g peptone, 5g beef meal, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1mL Tween 80, 150mL Polygonatum extract, and water to a final volume of 1000mL. The culture medium was sterilized at 121℃ for 20 minutes. The Polygonatum extract was prepared as follows: Polygonatum was pulped at a ratio of 1g raw material to 5mL water. After pulping, it was extracted at 100-105℃ for 40 minutes. The filtrate was filtered and stored. Then, 5mL of water was added, and the mixture was extracted at 100-105℃ for 20 minutes. The filtrate was filtered again to obtain a second filtrate. The two filtrates were combined to obtain the Polygonatum extract.

[0086] 2. Preparation of seed solution

[0087] Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 were separately inoculated into the above seed culture medium (B11 medium was supplemented with 0.45 g / mL cysteine ​​hydrochloride solution) at an inoculation amount of 3%. Lactobacillus plantarum Lp45 was fermented in a sealed container at 37±1℃ for 18 h, and Bifidobacterium bifidum B11 was cultured anaerobically at 37±1℃ for 26 h, to obtain first-generation seed culture of Lactobacillus plantarum Lp45 and first-generation seed culture of Bifidobacterium bifidum B11, respectively. The first-generation seed culture was inoculated into the seed culture medium at an inoculation amount of 2.5% and cultured at 37±1℃ for 16 h to obtain second-generation seed culture of Lactobacillus plantarum Lp45 and second-generation seed culture of Bifidobacterium bifidum B11.

[0088] 3. Preparation of fermentation substrate: Same as in Example 1

[0089] 4. Fermentation

[0090] The sterilized fermentation substrate was first inoculated with 3% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 5% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 30 rpm, and the fermentation was carried out at 37±1℃ for 96 hours. After removing the residue, centrifugation and filtration were performed to obtain the anti-fatigue compound probiotic fermentation broth.

[0091] Example 7

[0092] This embodiment provides a compound probiotic fermentation broth for combating fatigue, and its preparation method is as follows:

[0093] 1. Preparation of seed culture medium

[0094] Seed culture medium preparation: 4g yeast powder, 10g peptone, 5g beef meal, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1mL Tween 80, 350mL Polygonatum extract, and water to 1000mL. The culture medium was sterilized at 121℃ for 20min. The Polygonatum extract was prepared as follows: Polygonatum was pulped at a ratio of 1g raw material to 8mL water. After pulping, it was extracted at 100-105℃ for 80min. The filtrate was filtered and stored. Then, 10mL of water was added, and the mixture was extracted at 100-105℃ for 30min. The filtrate was filtered again to obtain a second filtrate. The two filtrates were combined to obtain the Polygonatum extract.

[0095] 2. Preparation of seed solution

[0096] Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 were separately inoculated into the above seed culture medium (B11 medium was supplemented with 0.55 g / mL cysteine ​​hydrochloride solution) at an inoculation amount of 5%. Lactobacillus plantarum Lp45 was fermented in a sealed container at 37±1℃ for 14 h, and Bifidobacterium bifidum B11 was cultured anaerobically at 37±1℃ for 22 h, to obtain first-generation seed culture of Lactobacillus plantarum Lp45 and first-generation seed culture of Bifidobacterium bifidum B11, respectively. The first-generation seed culture was inoculated into the seed culture medium at an inoculation amount of 3.5% and cultured at 37±1℃ for 12 h to obtain second-generation seed culture of Lactobacillus plantarum Lp45 and second-generation seed culture of Bifidobacterium bifidum B11.

[0097] 3. Preparation of fermentation substrate: Same as in Example 1

[0098] 4. Fermentation

[0099] The sterilized fermentation substrate was first inoculated with 5% of Lactobacillus plantarum Lp45 second-generation seed liquid for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 10% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated. The stirring speed was 40 rpm, and the fermentation was carried out at 37±1℃ for 72 hours. After removing the residue, centrifugation and filtration were performed to obtain the anti-fatigue compound probiotic fermentation broth.

[0100] Comparative Example 1

[0101] This comparative example provides a compound probiotic fermentation broth, the preparation method of which is basically the same as that of Example 1, the difference being the fermentation process in step 4 as follows:

[0102] The sterilized fermentation substrate was first inoculated with 4% of Lactobacillus plantarum Lp45 second-generation seed liquid and sealed for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 5-10% of Lactobacillus plantarum Lp45 second-generation seed liquid was inoculated, the stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, the mixture was centrifuged and filtered to obtain the compound probiotic fermentation broth.

[0103] Comparative Example 2

[0104] This comparative example provides a compound probiotic fermentation broth, the preparation method of which is basically the same as that of Example 1, the difference being the fermentation process in step 4 as follows:

[0105] The sterilized fermentation substrate was first inoculated with 4% of Bifidobacterium bifidum B11 second-generation seed liquid and sealed for fermentation. When the dissolved oxygen rate dropped below 20%, the pH value was adjusted to 4.8-5.0 with citrate-dipotassium hydrogen phosphate buffer. Then, 5-10% of Bifidobacterium bifidum B11 second-generation seed liquid was inoculated again. The stirring speed was 35 rpm, and the fermentation was carried out at 37±1℃ for 84 hours. After removing the residue, the mixture was centrifuged and filtered to obtain the compound probiotic fermentation broth.

[0106] Test Example 1

[0107] This test case investigated the cellulase activity of the second-generation seed culture obtained in Example 1, as well as the carboxymethyl cellulase and β-glucosidase activities of *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 in different culture media.

[0108] (1) Cellulase activity

[0109] Carboxymethyl cellulase and β-glucosidase both belong to the cellulase class and are highly active biocatalysts with the ability to degrade cellulose. They can hydrolyze plant cell walls to promote the release of effective components from traditional Chinese medicines. Simultaneously, β-glucosidase can hydrolyze saponins, flavonoids, and other glycosides to generate secondary metabolites or aglycones, thereby improving biological activity and bioavailability. For cellulase-producing microorganisms, the carbon source is the most important factor affecting enzyme yield. As an inducible enzyme, cellulase production requires the induction of polysaccharides. In this invention, the seed culture medium uses Polygonatum extract rich in Polygonatum polysaccharides as the carbon source, replacing glucose in liquid MRS medium. This experiment investigated the effect of this seed culture medium on the cellulase activity of Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 strains, and also examined the cellulase activity of second-generation seed cultures of other strains (prepared using the same method as the Lactobacillus plantarum Lp45 second-generation seed culture in Example 1) in this seed culture medium, to analyze that this seed culture medium enhances the cellulase activity of different strains.

[0110] Using Congo red medium, the formation of clear zones on the medium by second-generation seed cultures of different strains was observed. A higher bacterial diameter ratio, to some extent, reflects higher endoglucanase activity in the strain. Congo red medium: 1g potassium dihydrogen phosphate, 10g sodium chloride, 2g ammonium sulfate, 0.5g magnesium sulfate, 20g CMC-Na, 0.4g Congo red, 20g agar, 1000mL distilled water.

[0111] The experimental results are shown in Table 1. Compared with other strains, the second-generation seed cultures of Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 showed higher endoglucanase activity on Congo red plates.

[0112] Table 1. Growth of second-generation seed cultures of different strains in Congo red medium.

[0113]

[0114] (2) Carboxymethyl cellulase activity and β-glucosidase activity

[0115] This experiment investigated the carboxymethyl cellulase and β-glucosidase activities of *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 in different culture media. Carboxymethyl cellulase activity was determined according to QB 2583-2003, using a 2 g / L CMC-Na solution as the substrate. One unit of activity (IU / mL) of carboxymethyl cellulase activity was defined as the amount of enzyme required to catalyze the production of 1 μmol of glucose from the substrate in 1 minute. β-glucosidase activity was determined using a 1 mmol / L 4-nitrophenyl-D-pyranoside solution (pNPG) as the substrate. One unit of activity (IU / mL) of β-glucosidase activity was defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute from the substrate per unit volume of enzyme reaction solution.

[0116] The results are shown in Table 2. Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 grew well in the carbon source environment of Polygonatum extract. Compared with glucose as the carbon source, there was no significant difference in bacterial concentration OD value, but the activities of carboxymethyl cellulase and β-glucosidase were significantly increased (P<0.01).

[0117] Table 2. Growth and enzyme production of Lp45 and B11 under different carbon source media.

[0118]

[0119] Test Example 2

[0120] This test example analyzed the components of the compound probiotic fermentation broth obtained in Example 1, Comparative Example 1, and Comparative Example 2, as well as the aqueous extract of the food-medicine homology formula.

[0121] The preparation method of the aqueous extract of the food-medicine homology formula is as follows: Weigh the raw materials of the food-medicine homology formula according to the following weight ratio: 40 parts of Polygonatum sibiricum, 25 parts of Lycium barbarum, 12.5 parts of Longan pulp, 7.5 parts of Jujube, and 7.5 parts of Gardenia jasminoides. Mix the above raw materials with water at a ratio of 1:9 and slurry. Sterilize at 105-110℃ for 35 min, and then extract with water at 37±1℃ with a stirring speed of 35 rpm. The extraction time is consistent with the total fermentation time after inoculating the second-generation seed liquid of Lactobacillus plantarum Lp45 and the second-generation seed liquid of Bifidobacterium bifidum B11 in step 4 of Example 1. After removing the residue and filtering, the aqueous extract is obtained.

[0122] 1. Detection Method

[0123] (1) Detection of crude polysaccharide content

[0124] The phenol-sulfuric acid method was used to detect crude polysaccharides in the samples. The principle is as follows: after the polysaccharides are separated by precipitation with 70% ethanol to remove interference from other soluble sugars and impurities, they are hydrolyzed into monosaccharides under the action of concentrated sulfuric acid, and then rapidly dehydrated to generate uronic acid derivatives. These derivatives react with phenol to generate an orange-red compound with characteristic absorption at 485 nm.

[0125] A standard curve for glucose was plotted with glucose content on the x-axis and absorbance on the y-axis, yielding the linear regression equation Y = 29.4934X - 0.2025(R²). 2 Calculate the total polysaccharide content using (=0.9909).

[0126] (2) Detection of total flavonoid content

[0127] The sodium nitrite-aluminum nitrate method was used, with rutin as a reference standard. A standard curve was plotted based on absorbance, and the regression equation Y = 5.9563X - 0.0033(R) was applied. 2 Calculate the total flavonoid content in the test solution using the formula =0.9994).

[0128] (3) Total saponin content detection

[0129] The vanillin colorimetric method was used, with ginsenoside Re as the reference standard. A standard curve was plotted based on absorbance, and the regression equation for the total saponin standard curve was established as Y = 0.0745X - 0.0025(R). 2 =0.9964), calculate the total saponin content in the test solution.

[0130] 2. Test Results

[0131] The content analysis of each component is shown in Table 3. Fermentation of *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 alone can increase the content of total flavonoids and total saponins in the medicinal and edible homology formula. The increase in total flavonoid and total saponin content after staged combined fermentation of *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 is greater than that after fermentation by the strains alone, and also better than the aqueous extract of the unfermented medicinal and edible homology formula. The content of crude polysaccharides decreased after fermentation by the strains, which is related to the production of small-molecule monosaccharides from polysaccharides during the fermentation process. The content of polysaccharides released by plant cell wall disruption is less than the content from polysaccharide decomposition and metabolism, thus reducing the total polysaccharide content.

[0132] Table 3 Component Content Analysis

[0133]

[0134]

[0135] Test Example 3

[0136] This test case validated the functionality of the compound probiotic fermentation broths obtained in Example 1, Comparative Example 1, and Comparative Example 2.

[0137] 1. Verification of anti-fatigue effect

[0138] 1.1 Animal grouping

[0139] Male Kunming mice (SPF grade, body weight 20±2g) were selected and acclimatized for 7 days in an animal room at 25℃ and 50%–60% humidity. Six mice were randomly selected from each group and divided into 10 groups: a blank control group (saline group), control groups (water extract group, bacterial solution plus water extract group, ginseng fermentation liquid group, Cistanche deserticola fermentation liquid group, Lp45 fermented Polygonatum sibiricum formula group, B11 fermented Polygonatum sibiricum formula group), and experimental groups (low, medium, and high dose fermentation liquid groups). Each group was administered physiological saline, water extract, bacterial solution plus water extract, ginseng fermentation liquid, Cistanche deserticola fermentation liquid, Lp45 fermented Polygonatum sibiricum formula, B11 fermented Polygonatum sibiricum formula, low-dose Polygonatum sibiricum fermentation liquid, medium-dose Polygonatum sibiricum fermentation liquid, and high-dose Polygonatum sibiricum fermentation liquid via gavage, with a volume of 10 mL / kg per gavage, once daily for 30 consecutive days. The pH of all samples was adjusted to 6.8–7.0 before gavage.

[0140] The preparation methods for the samples used in the above gavage administration groups are as follows:

[0141] (1) Aqueous extract: Weigh the raw materials of the food-medicine homology formula according to the following weight ratio: 40 parts of Polygonatum sibiricum, 3 parts of red ginseng, 25 parts of wolfberry, 12.5 parts of longan pulp, 7.5 parts of jujube, and 7.5 parts of gardenia. Mix the above raw materials with water at a ratio of 1:9 and slurry. Sterilize at 105-110℃ for 35 min and then extract with water at room temperature of 37±1℃. Stir at 35 rpm. The extraction time is the same as the total fermentation time after inoculating Lactobacillus plantarum Lp45 second generation seed liquid and Bifidobacterium bifidum B11 second generation seed liquid in step 4 of Example 1. After removing the residue and filtering, the aqueous extract is obtained.

[0142] (2) Bacterial extract with water: Take the above water extract, add 5% of the Lactobacillus plantarum Lp45 second generation seed liquid and 10% of Bifidobacterium bifidum B11 second generation seed liquid, mix, and obtain bacterial extract with water.

[0143] (3) Ginseng fermentation liquid: Weigh the raw materials according to the following weight ratios: ginseng 43 parts, wolfberry 25 parts, longan pulp 12.5 parts, jujube 7.5 parts and gardenia 7.5 parts. Follow step (1) for subsequent pulping, sterilization, water extraction, and slag removal filtration.

[0144] (4) Cistanche fermentation liquid: Weigh the raw materials according to the following weight ratio: 40 parts of Cistanche, 3 parts of red ginseng, 25 parts of wolfberry, 12.5 parts of longan pulp, 7.5 parts of jujube and 7.5 parts of gardenia. Follow the steps in (1) for subsequent pulping, sterilization, water extraction, and slag removal and filtration.

[0145] (5) Lp45 fermented Polygonatum formulation: The preparation method is the same as that of comparative example 1;

[0146] (6) B11 fermented Polygonatum formulation: The preparation method is the same as that of comparative example 2;

[0147] (7) Low dose of Polygonatum sibiricum fermentation broth: The fermentation substrate in Example 1 was diluted with water to 25%, and the fermentation broth was prepared according to step 4 of Example 1.

[0148] (8) Medium dose of Polygonatum sibiricum fermentation broth: The fermentation substrate in Example 1 was diluted with water to 50%, and the fermentation broth was prepared according to step 4 of Example 1.

[0149] (9) High dose of Polygonatum fermentation liquid: Same as in Example 1.

[0150] 1.2 Weighted Swimming Experiment

[0151] Thirty minutes after the last gavage, the mice were placed in a swimming tank (water temperature 25℃±1℃) with a lead sheet bearing 5% of their body weight on the base of their tails. The time for each group of mice to swim to exhaustion (the time it took to sink to the surface and not float back up) was recorded.

[0152] The experimental results are shown in Table 4. The results indicate that, compared with the blank control group (physiological saline), both the control and experimental groups significantly increased the swimming time of mice (P<0.05). Compared with the control group (water extract group, bacterial solution plus water extract group, ginseng fermentation liquid group, Cistanche deserticola fermentation liquid group, Lp45 fermented Polygonatum sibiricum formula group, B11 fermented Polygonatum sibiricum formula group), the experimental groups (low, medium, and high dose Polygonatum sibiricum fermentation liquid groups) significantly prolonged the swimming time of mice (P<0.05). Furthermore, the swimming time increased with increasing fermentation liquid dosage, and the synergistic fermentation effect of *Lactobacillus plantarum* Lp45 and *Bifidobacterium bifidum* B11 was superior to single-strain fermentation.

[0153] Table 4. Swimming time of mice at exhaustion

[0154]

[0155]

[0156] 1.3 Impact of Biochemical Indicators

[0157] After the mice finished swimming with weight and rested for 60 minutes, blood was collected from their eyes and livers were harvested. The levels of blood lactate (LD), blood urea nitrogen (BUN), and liver glycogen (LG) were measured according to the kit instructions.

[0158] The biochemical results are shown in Table 5. Compared with the blank control group and the control groups (water extract group, bacterial solution plus water extract group, ginseng fermentation liquid group, Cistanche deserticola fermentation liquid group, Lp45 fermented Polygonatum sibiricum formula group, and B11 fermented Polygonatum sibiricum formula group), the medium and high concentration Polygonatum sibiricum fermentation liquid groups significantly reduced the levels of blood lactate and serum urea nitrogen in mice after swimming and significantly increased the levels of liver glycogen in mice after 30 days of gavage (P<0.05). There was no significant difference in the effects of the water extract group and the bacterial solution plus water extract group on the levels of blood lactate, serum urea nitrogen, and liver glycogen in mice after exercise, indicating that adding bacterial fermentation liquid is not as effective as using bacterial strains for fermentation, and that the synergistic fermentation effect of Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 is worse than that of single-strain fermentation.

[0159] Table 5. Effects of exercise on biochemical parameters in different groups of mice

[0160]

[0161] 2. Verification of cellular immune effects

[0162] 2.1 Culture of RAW264.7 cells

[0163] Macrophages are one of the important types of immune cells in the body and an important component of the immune system. The phagocytic and clearance capabilities of macrophages are one of the important pathways for initiating the body's immune response. RAW264.7 cells were revived in DMEM complete culture medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 cell culture incubator. When the cell density reached approximately 80%, the cells were passaged. Subsequent experiments were performed after three passages.

[0164] 2.2 CCK8 assay for cell proliferation rate

[0165] The cells were divided into four groups: a blank control group (physiological saline group), a model group (LPS group), a control group (water extract group, bacterial culture plus water extract group, ginseng fermentation broth group, Cistanche deserticola fermentation broth group, Lp45 fermented Polygonatum sibiricum formula group, B11 fermented Polygonatum sibiricum formula group), and experimental groups (low, medium, and high dose Polygonatum sibiricum fermentation broth groups), with five replicates per group. After removing the original culture medium, physiological saline, LPS, water extract, bacterial culture plus water extract, ginseng fermentation broth, Cistanche deserticola fermentation broth, Lp45 fermented Polygonatum sibiricum formula, B11 fermented Polygonatum sibiricum formula, low-dose Polygonatum sibiricum fermentation broth, medium-dose Polygonatum sibiricum fermentation broth, and high-dose Polygonatum sibiricum fermentation broth were added to each group, respectively. The sample preparation methods for each group were the same as in 1.1. The proliferation of RAW264.7 cells was detected according to the CCK8 kit instructions. The calculation formula is as follows:

[0166]

[0167] The effects of different groups of samples on the proliferation of RAW264.7 macrophages are as follows: Figure 1 As shown in the figure. Compared with the blank control group and the control group, both medium and high doses of Polygonatum sibiricum fermentation broth significantly promoted the proliferation of RAW264.7 macrophages, and the Polygonatum sibiricum fermentation broth fermented with Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 had a better effect than the water extract and fermentation with a single strain (P<0.05).

[0168] 2.3 Neutral red assay for cytophagy

[0169] The cells were grouped as described above, with 5 replicate wells per group. The phagocytic capacity of macrophages in each group was determined using the neutral red assay.

[0170]

[0171] The effects of different groups on the phagocytic activity of RAW264.7 macrophages, such as Figure 2As shown, the fermentation broth can enhance the phagocytic activity of macrophages by activating them and inducing their differentiation and maturation. Compared with the blank control group, both the water extract and the fermentation broth significantly improved the phagocytic capacity of macrophages, and the phagocytic effect of the Polygonatum sibiricum fermentation broth was superior to that of the water extract and other fermentation broth formulations (P<0.05). The synergistic fermentation effect of Lactobacillus plantarum Lp45 and Bifidobacterium bifidum B11 was superior to that of single-strain fermentation, but the stimulatory effect on cell phagocytosis was lower than that of the LPS model group. Therefore, the Polygonatum sibiricum fermentation broth can significantly improve the proliferation and phagocytic capacity of RAW264.7 cells, thereby activating immune cells and enhancing the body's immunity.

[0172] 3. Verification of the effects of human trials

[0173] Participants were recruited according to the following criteria: no serious medical history or major surgery history; and no consumption of health supplements, traditional Chinese medicine, or chemical drugs that might affect the evaluation during the trial period. A total of 30 participants were recruited, with an average age of 35.6 years, including 25 women and 5 men.

[0174] Trial period: 30 days, take 30mL of the same batch of compound probiotic fermentation liquid prepared according to the preparation method of Example 1 after breakfast and dinner every day;

[0175] Testing indicators: Fasting blood routine and biochemical indicators were tested before and after the food trial, and the red blood cell and immunoglobulin indicators were analyzed.

[0176] The results are shown in Table 6. It was found that after a 30-day trial, the compound probiotic fermented liquid prepared according to the method in Example 1 increased the average hemoglobin index and average immunoglobulin index in the population, thereby improving the population's immune level. According to population feedback, more than 70% of the participants experienced improved energy and stamina, reduced fatigue, and improved sleep quality after the trial.

[0177] Table 6. Population Trial Indicators

[0178]

[0179] Example 8

[0180] This embodiment provides an anti-fatigue compound probiotic granule, the preparation method of which is as follows:

[0181] The compound probiotic fermentation broth obtained in Example 1 was concentrated into a thick paste (solid content 40%). The paste was then mixed with corn starch at a ratio of 40-60%, sucralose at 35-55%, and 95% alcohol to reduce the viscosity of the materials. The mixture was stirred to form a soft mass, which was then extruded and sieved to form granules. After packaging, the compound probiotic granules were obtained.

[0182] According to this method, the compound probiotic fermentation liquid obtained in Examples 2 to 7 can also be made into granules.

[0183] Example 9

[0184] This embodiment provides an anti-fatigue compound probiotic compressed candy, the preparation method of which is as follows:

[0185] After drying the compound probiotic granules obtained in Example 8, they were compressed into tablets with 65-85% granules, 0.5-2.5% magnesium stearate, and 15-25% sorbitol to obtain compressed candy.

[0186] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a complex probiotic fermentation broth for anti-fatigue and immunity enhancement, characterized in that, The homologous medicinal and edible formula raw materials are mixed and beaten with water, sterilized, used as a fermentation substrate, inoculated with a Lactobacillus plantarum Lp45 culture solution for fermentation, the pH is adjusted to 4.8-5.0 when the dissolved oxygen rate is below 20%, then a Bifidobacterium bifidum B11 culture solution is inoculated, and the fermentation is continued under stirring until the fermentation is completed, then the fermentation liquid is obtained by solid-liquid separation, which is the compound probiotic fermentation liquid; wherein the homologous medicinal and edible formula raw materials include 35-45 parts of rhizoma polygonati, 1-5 parts of red ginseng, 20-30 parts of medlar, 10-15 parts of longan meat, 5-10 parts of jujube, and 5-10 parts of gardenia. The preparation method of the Lactobacillus plantarum Lp45 culture solution is that the Lactobacillus plantarum Lp45 strain is cultured in a seed culture medium containing a rhizoma polygonati extract for two generations to obtain a Lactobacillus plantarum Lp45 second-generation seed solution, which is the Lactobacillus plantarum Lp45 culture solution. The preparation method of the Bifidobacterium bifidum B11 culture solution is that the Bifidobacterium bifidum B11 strain is cultured in a seed culture medium containing a rhizoma polygonati extract for two generations to obtain a Bifidobacterium bifidum B11 second-generation seed solution, which is the Bifidobacterium bifidum B11 culture solution. The preparation method of the rhizoma polygonati extract is that the rhizoma polygonati is extracted with water at 100-105 DEG C for at least two times, and the extract is combined to obtain the rhizoma polygonati extract. The components of the seed culture medium include 3.5-4.5 g / L of yeast powder, 8-12 g / L of protein peptone, 4.5-5.5 g / L of beef powder, 1.8-2.2 g / L of potassium phosphate dibasic, 4.5-5.5 g / L of sodium acetate, 1.8-2.2 g / L of triammonium citrate, 0.18-0.22 g / L of magnesium sulfate, 0.045-0.055 g / L of manganese sulfate, 0.8-1.2 mL / L of Tween 80, 150-350 mL / L of the rhizoma polygonati extract, and water as a solvent. When the Bifidobacterium bifidum B11 is cultured with the seed culture medium, the components of the seed culture medium further include 0.45-0.55 g / L of cysteine hydrochloride.

2. The production method according to claim 1, characterized by, The weight ratio of the rhizoma polygonati, red ginseng, medlar, longan meat, jujube, and gardenia is 38-42 parts of rhizoma polygonati, 2-4 parts of red ginseng, 23-27 parts of medlar, 12-13 parts of longan meat, 7-8 parts of jujube, and 7-8 parts of gardenia.

3. The preparation method according to claim 2, characterized in that, The weight ratio of the rhizoma polygonati, red ginseng, medlar, longan meat, jujube, and gardenia is 40 parts of rhizoma polygonati, 3 parts of red ginseng, 25 parts of medlar, 12.5 parts of longan meat, 7.5 parts of jujube, and 7.5 parts of gardenia.

4. The method of claim 1, wherein, The Lactobacillus plantarum Lp45 strain is inoculated in the seed culture medium at an inoculation amount of 3%-5% and cultured at 37±1 DEG C for 14-18 h to obtain a Lactobacillus plantarum Lp45 first-generation seed solution; the Lactobacillus plantarum Lp45 first-generation seed solution is inoculated in the seed culture medium at an inoculation amount of 3±0.5% and cultured at 37±1 DEG C for 12-16 h to obtain the Lactobacillus plantarum Lp45 second-generation seed solution; and / or The Bifidobacterium B11 strain is inoculated into the seed culture medium at an inoculation amount of 3-5% and cultured anaerobically at 37±1℃ for 22-26h to obtain a first generation seed solution of the Bifidobacterium B11; the first generation seed solution of the Bifidobacterium B11 is inoculated into the seed culture medium at an inoculation amount of 3±0.5% and cultured at 37±1℃ for 12-16h to obtain a second generation seed solution of the Bifidobacterium B11.

5. The preparation method according to claim 4, characterized in that, The inoculation amount of the second generation seed solution of the Lactobacillus plantarum Lp45 in the fermentation medium is 3-5%; and / or The inoculation amount of the second generation seed solution of the Bifidobacterium B11 in the fermentation medium is 5-10% and the fermentation time is 72-96h.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The mass ratio of the medicinal and edible formula raw material to water is 1:8-10; and / or The sterilization is sterilization at 105-110℃ for 25-40min; and / or The pH is adjusted with a citric acid-dipotassium hydrogen phosphate buffer; and / or The stirring speed is 30-40rpm; and / or The solid-liquid separation method is centrifugation, filtration, or a combination thereof.

7. A compound probiotic fermentation broth for anti-fatigue and immunity enhancement, characterized in that, The compound probiotic fermentation solution is prepared by the preparation method of any one of claims 1-6.

8. A fatigue-resistant, immunity-enhancing, composite probiotic fermentation product, characterized by, The compound probiotic fermentation solution of claim 7 and other ingredients acceptable in food or medicine are contained.

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