Fermentation composition helpful for enhancing immunity and preparation method thereof

Abstract

The invention belongs to the technical field of health-care products, and particularly relates to a fermented composition beneficial to enhancing immunity and a preparation method thereof. The fermentation composition for improving immunity is prepared by taking yeast beta-glucan, an elderberry extract, glucoraphanin, sea cucumber peptide, inulin and a traditional Chinese medicine zymolyte as fermentation raw materials, firstly inoculating bacillus coagulans and staphylococcus xylosus for aerobic fermentation, and then inoculating bifidobacterium longum for anaerobic fermentation, the aerobic fermentation stage and the anaerobic fermentation stage have a synergistic effect, so that the fermentation speed can be increased, the fermentation time can be shortened, and richer nutrient substances can be provided, and therefore, the composition has an excellent effect of enhancing the immunity; the fermentation composition is applied to preparation of capsules helpful for enhancing immunity, is helpful for enhancing human immunity, and can solve the problem that active substances in existing health-care products are difficult to effectively absorb by human bodies, so that the effect of enhancing immunity is influenced.

Description

Technical Field

[0001] This invention belongs to the field of health product technology, specifically relating to a fermented composition that helps enhance immunity and its preparation method. Background Technology

[0002] The aging population and the fast pace of modern life have led to imbalances in the body's immune homeostasis and a decline in immune function. The immune system, the core defense system against invading pathogens, the elimination of aging or abnormal cells, and the monitoring and removal of mutated or virus-infected cells, becomes more susceptible to infectious diseases and even increases the risk of chronic diseases such as cancer when its immune response weakens. Therefore, strengthening immune function has become an important direction for resisting unknown diseases.

[0003] From the perspective of Traditional Chinese Medicine (TCM), weakened immune function is often attributed to insufficient vital energy (Qi), the causes of which are complex and frequently related to congenital deficiencies, improper diet, overwork, and emotional fluctuations. TCM contains various functional components; for example, polysaccharides have been widely proven to have significant immune-enhancing activity, as well as anti-tumor, anti-inflammatory, lipid-lowering, and liver-protective activities. Therefore, most products on the market for boosting immunity use TCM or food-medicine homologous ingredients. However, polysaccharides typically have large molecular weights and complex structures, and some polysaccharides have poor water solubility, making them difficult to absorb effectively and resulting in low bioavailability, thus limiting their efficacy.

[0004] With the increasing public awareness of health, consumers are increasingly demanding immune-modulating health products that combine safety and functionality. Therefore, breaking through the absorption bottleneck of active ingredients in existing products has become the key to developing highly effective immune-enhancing agents. Summary of the Invention

[0005] In view of the above-mentioned defects or improvement needs of the prior art, the present invention provides a fermentation composition that helps to enhance immunity and its preparation method, which solves the problem that the active ingredients in the existing products are difficult to be effectively absorbed by the human body, thus affecting the efficacy of enhancing immunity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a fermented composition that helps enhance immunity, the method comprising the following steps: A1. Dry yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of (3-7):(3-7):(2-5):(2-5) to obtain a traditional Chinese medicine mixture. Deionized water is added and mixed evenly to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme is inactivated by heating to obtain the enzymatic hydrolysate of traditional Chinese medicine. The compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.2-1.7. A2. Weigh each fermentation raw material, mix them evenly, and sterilize them to obtain the fermentation substrate; wherein, the fermentation raw materials include, by mass, 13-17 parts of yeast β-glucan, 10-15 parts of elderberry extract, 4-7 parts of glucosinolate, 13-17 parts of sea cucumber peptide, 8-12 parts of inulin, and 11-17 parts of traditional Chinese medicine enzymatic hydrolysate. A3. After aerobic fermentation by inoculating Bacillus coagulans and Staphylococcus xylose into the fermentation substrate, Bifidobacterium longum is inoculated for anaerobic fermentation. After fermentation, the substrate is centrifuged and the supernatant is retained to obtain the fermentation broth. The preservation number of Bacillus coagulans is CCTCC NO: M 2019722, the preservation number of Staphylococcus xylose is CGMCC NO.26373, and the preservation number of Bifidobacterium longum is GDMCC NO.62129. A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition.

[0007] In this invention, yeast β-glucan is a water-insoluble polymer with antibacterial and immunomodulatory effects. Due to its unique targeting effect, it can selectively remove toxins from the body, enhance the phagocytic capacity of macrophages, and enable the human immune system to quickly reach the optimal balance without any drug toxic side effects. At the same time, it also has unique biological activities in tumors, hepatitis, cardiovascular diseases, diabetes, lowering blood lipids, and anti-aging.

[0008] Elderberry extract is rich in bioactive polyphenols, which are highly antioxidant. Its antioxidant capacity is 20 times that of vitamin C and 50 times that of vitamin E. At the same time, it can significantly enhance the activity of endothelial nitric oxide synthase, boost immunity, and has antibacterial, antioxidant, and collagenase-inhibiting properties.

[0009] Glucosamine is a type of glucosinolate that can inhibit the expression of phase I enzymes and induce the expression of phase II enzymes. It has a significant blocking effect on liver cancer, breast cancer, lung cancer, esophageal cancer, prostate cancer, and gastric cancer. Glucosamine is hydrolyzed by intracellular glucosinolate hydrolase (also known as myrosinase) to produce sulforaphane, which also has protective effects on the skin, antibacterial, anti-inflammatory, antioxidant, and immunomodulatory effects.

[0010] Sea cucumber peptides are small molecule peptides obtained by hydrolysis and purification of proteases. They have multiple effects such as enhancing immunity, moisturizing, lowering uric acid, and anti-oxidation, and play an irreplaceable role in human physiological functions. Furthermore, due to their special physicochemical properties such as good solubility, stability, low viscosity, easy digestion and absorption, non-antigenicity, and food safety, sea cucumber peptides have a higher biological value than other ordinary sea cucumber products.

[0011] Inulin is a natural polysaccharide with good water solubility and stability. It can be fermented and utilized by intestinal bacteria to produce short-chain fatty acids, lowering the intestinal pH, inhibiting the growth of harmful bacteria, and thus promoting the reproduction of beneficial bacteria and improving the intestinal microecological environment. In addition, inulin can significantly increase the number of beneficial bacteria such as Bifidobacteria and reduce the proportion of harmful bacteria, thereby improving intestinal immunity and disease resistance by altering the intestinal flora structure.

[0012] Yam is sweet and neutral in nature, and enters the spleen, lung, and kidney meridians. It has the effects of tonifying the spleen and stomach, promoting body fluid production and benefiting the lungs, and tonifying the kidneys and astringing essence.

[0013] Goji berries are sweet in taste and enter the liver and kidney meridians. They have the effects of nourishing the liver and kidneys, and benefiting the essence and improving eyesight.

[0014] Astragalus has a sweet taste and enters the lung and spleen meridians. It has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, generating fluids and nourishing blood, promoting circulation and relieving pain, promoting detoxification and draining pus, and promoting wound healing and tissue regeneration.

[0015] Poria cocos is sweet, bland, and neutral in nature. It enters the heart, lung, spleen, and kidney meridians and has the effects of promoting diuresis and eliminating dampness, strengthening the spleen, and calming the mind.

[0016] Preferably, the ratio of the herbal medicine mixture to deionized water in step A1 is 1g:10-20mL.

[0017] Preferably, the amount of the compound enzyme added in step A1 is 2-4 wt% of the enzymatic hydrolysis substrate, and the ultrasonic enzymatic hydrolysis temperature is 55-60℃, pH is 4.5-5.5, ultrasonic frequency is 30-40 kHz, and time is 30-40 min.

[0018] Preferably, the temperature for inactivating the enzyme in step A1 is 95-105°C and the time is 25-35 min.

[0019] Preferably, the fermentation raw materials in step A2 include the following components by mass: 15 parts yeast β-glucan, 12 parts elderberry extract, 6 parts glucosinolate, 15 parts sea cucumber peptide, 10 parts inulin, and 14 parts enzymatic hydrolysate of traditional Chinese medicine.

[0020] Preferably, the total inoculum amount of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum in step A3 is 4-6% v / v of the fermentation substrate.

[0021] Preferably, the inoculation ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum in step A3 is 1:1.5-2:0.8-1.3.

[0022] Preferably, the viable counts of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum mentioned in step A3 are all 5.0 × 10⁻⁶. 7 CFU / mL.

[0023] More preferably, the inoculation ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum in step A3 is 1:1.7:1.1.

[0024] Preferably, the aerobic fermentation in step A3 is carried out at 34-40°C by introducing sterile air with an aeration ratio of 0.4-0.7 vvm for 12-18 hours.

[0025] Preferably, the anaerobic fermentation in step A3 is performed at 34-40℃ for 12-18 hours.

[0026] Preferably, the centrifugation speed in step A3 is 2500-4500 rpm and the time is 18-30 min.

[0027] Preferably, the filtration sterilization in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm.

[0028] Preferably, the concentration in step A4 is achieved by rotary evaporation at 55-65°C to 60-70% of the original volume.

[0029] In a second aspect, the present invention provides the use of the fermentation composition prepared by the preparation method described in the first aspect in the preparation of health products that help enhance immunity.

[0030] Preferably, the dosage form of the health product is any one of tablets, hard capsules, soft capsules, ointments, granules, or powders.

[0031] The beneficial effects of this invention are: 1. The immune-boosting fermentation composition of this invention uses yeast β-glucan, elderberry extract, sulforaphane, sea cucumber peptide, inulin, and enzymatic hydrolysate of traditional Chinese medicine as fermentation raw materials. It is prepared by first inoculating *Bacillus coagulans* and *Staphylococcus xylose* for aerobic fermentation, and then inoculating *Bifidobacterium longum* for anaerobic fermentation. The *Bifidobacterium longum* selected in this application has sulforaphane enzyme activity, which can convert sulforaphane into sulforaphane. Sulforaphane has multiple effects such as anti-inflammatory, antioxidant, and immunomodulatory properties. The spores produced by *Bacillus coagulans* have high tolerance to temperature, acidity, and bile, and their formation... The spores of *Bifidobacterium longum* can rapidly germinate into vegetative cells in the gastrointestinal tract, and its main metabolite, L-lactic acid, can lower the pH of the gastrointestinal tract, creating a more favorable growth environment for *Bifidobacterium longum*, thereby promoting its growth and colonization. *Staphylococcus xylose* consumes oxygen, providing an anaerobic environment for *Bifidobacterium longum*, while its decomposition of proteins to produce various amino acids provides abundant nutrients for *Bifidobacterium longum*, all of which are beneficial to the proliferation of *Bifidobacterium longum*. There is a synergistic effect between the two fermentations, which can increase the fermentation speed, shorten the fermentation time, and provide richer nutrients, thereby achieving an excellent effect of enhancing immunity.

[0032] 2. Applying the fermentation composition obtained by this invention to the preparation of health products with the effect of improving immunity is beneficial to restoring and enhancing human immune function, and can solve the problem that the active substances in existing products are difficult to be effectively absorbed by the human body, thus affecting the effect of improving immunity. Detailed Implementation

[0033] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific embodiments, structure, features and effects of the present invention, in conjunction with the composition, is provided below.

[0034] For the following compositions, unless otherwise specified, experimental methods are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all materials and reagents used are commercially available.

[0035] Some of the raw materials and their sources are as follows: Yeast β-glucan was purchased from KAIRE Ingredients Trading (Shanghai) Co., Ltd.; Elderberry extract was purchased from IPRONA, Italy. Glucosamine was purchased from Brassica, USA. Sea cucumber peptides were purchased from Shengminuo Company; Inulin was purchased from Villeroy & Boch. The yam was purchased from Bozhou Linbo Pharmaceutical Co., Ltd. The goji berries were purchased from Bozhou Fuyongtang Pharmaceutical Co., Ltd. Astragalus was purchased from Ningxia Xiangcao Biotechnology Co., Ltd. Poria cocos was purchased from Bozhou Linbo Pharmaceutical Co., Ltd. α-Amylase was purchased from Hubei Haijia Biotechnology Co., Ltd., with an enzyme activity of 2000 U / g; Cellulase was purchased from Shandong Aicai Biotechnology Co., Ltd., with an enzyme activity of 200,000 U / g.

[0036] Example 1 A method for preparing a fermented composition that helps enhance immunity includes the following steps: A1. The dried yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of 3:7:2:5 to obtain a Chinese medicine mixture. Deionized water is added and mixed evenly to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the temperature is raised to inactivate the enzyme to obtain the Chinese medicine enzymatic hydrolysate. A2. Weigh each fermentation raw material, mix them evenly, and then sterilize to obtain the fermentation substrate; A3. After aerobic fermentation by inoculating Bacillus coagulans and Staphylococcus xylose into the fermentation substrate, Bifidobacterium longum was inoculated for anaerobic fermentation. After fermentation, the substrate was centrifuged and the supernatant was retained to obtain the fermentation broth. A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition. In step A1, the ratio of the herbal mixture to deionized water is 1g:10mL; the compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.7, and the amount of the compound enzyme added is 2wt% of the enzymatic hydrolysis substrate; the ultrasonic enzymatic hydrolysis temperature is 60℃, pH is 5.5, ultrasonic frequency is 30kHz, and time is 40min; the temperature for inactivating the enzyme is 95℃ and time is 35min. The fermentation raw materials mentioned in step A2, by weight, include: 13 parts yeast β-glucan, 10 parts elderberry extract, 4 parts glucosinolate, 13 parts sea cucumber peptide, 8 parts inulin, and 11 parts enzymatic hydrolysate of traditional Chinese medicine; the sterilization temperature is 121℃ and the time is 10s. The Bacillus coagulans described in step A3 has the accession number CCTCC NO: M 2019722, the Staphylococcus xylose has the accession number CGMCC NO.26373, and the Bifidobacterium longum has the accession number GDMCC NO.62129. The total inoculum amount of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 4% v / v of the fermentation substrate. The inoculum ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 1:2:1.3, and the viable count of each of the three bacteria is 5.0 × 10⁻⁶. 7CFU / mL; the aerobic fermentation was carried out at 34°C with sterile air at an aeration ratio of 0.4 vvm for 18 hours; the anaerobic fermentation was carried out at 34°C for 18 hours; the centrifugation speed was 2500 rpm and the time was 30 min; The filtration sterilization mentioned in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm; the concentration is performed by rotary evaporation at 65°C to concentrate to 70% of the original volume.

[0037] Example 2 A method for preparing a fermented composition that helps enhance immunity includes the following steps: A1. The dried yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of 5:4:3:3 to obtain a Chinese medicine mixture. Deionized water is added and mixed evenly to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the temperature is raised to inactivate the enzyme to obtain the Chinese medicine enzymatic hydrolysate. A2. Weigh each fermentation raw material, mix them evenly, and then sterilize to obtain the fermentation substrate; A3. After aerobic fermentation by inoculating Bacillus coagulans and Staphylococcus xylose into the fermentation substrate, Bifidobacterium longum was inoculated for anaerobic fermentation. After fermentation, the substrate was centrifuged and the supernatant was retained to obtain the fermentation broth. A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition. In step A1, the ratio of the herbal mixture to deionized water is 1g:15mL; the compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.5, and the amount of the compound enzyme added is 3wt% of the enzymatic hydrolysis substrate; the ultrasonic enzymatic hydrolysis temperature is 57℃, pH is 4.7, ultrasonic frequency is 35kHz, and time is 35min; the temperature for inactivating the enzyme is 100℃ and time is 30min. The fermentation raw materials mentioned in step A2, by weight, include: 15 parts yeast β-glucan, 12 parts elderberry extract, 6 parts glucosinolate, 15 parts sea cucumber peptide, 10 parts inulin, and 14 parts enzymatic hydrolysate of traditional Chinese medicine; the sterilization temperature is 121℃ and the time is 10s. The Bacillus coagulans described in step A3 has the accession number CCTCC NO: M 2019722, the Staphylococcus xylose has the accession number CGMCC NO.26373, and the Bifidobacterium longum has the accession number GDMCC NO.62129. The total inoculum amount of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 5% v / v of the fermentation substrate. The inoculum ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 1:1.7:1.1, and the viable count of each of the three bacteria is 5.0 × 10⁻⁶. 7 CFU / mL; the aerobic fermentation was carried out at 37°C with sterile air at an aeration ratio of 0.5 vvm for 15 h; the anaerobic fermentation was carried out at 37°C for 15 h; the centrifugation speed was 2500 rpm and the time was 30 min; The filtration sterilization mentioned in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm; the concentration is performed by rotary evaporation at 60°C to concentrate to 65% of the original volume.

[0038] Example 3 A method for preparing a fermented composition that helps enhance immunity includes the following steps: A1. The dried yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of 7:3:5:2 to obtain a Chinese medicine mixture. Deionized water is added and mixed well to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the temperature is raised to inactivate the enzyme to obtain the Chinese medicine enzymatic hydrolysate. A2. Weigh each fermentation raw material, mix them evenly, and then sterilize to obtain the fermentation substrate; A3. After aerobic fermentation by inoculating Bacillus coagulans and Staphylococcus xylose into the fermentation substrate, Bifidobacterium longum was inoculated for anaerobic fermentation. After fermentation, the substrate was centrifuged and the supernatant was retained to obtain the fermentation broth. A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition.

[0039] In step A1, the ratio of the herbal mixture to deionized water is 1g:20mL; the compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.2, and the amount of the compound enzyme added is 4wt% of the enzymatic hydrolysis substrate; the ultrasonic enzymatic hydrolysis temperature is 55℃, pH is 4.5, ultrasonic frequency is 40kHz, and time is 30min; the temperature for inactivating the enzyme by heating is 105℃ and time is 25min. The fermentation raw materials mentioned in step A2, by weight, include: 17 parts yeast β-glucan, 15 parts elderberry extract, 7 parts glucosinolate, 17 parts sea cucumber peptide, 12 parts inulin, and 17 parts enzymatic hydrolysate of traditional Chinese medicine; the sterilization temperature is 121℃ and the time is 10s. The Bacillus coagulans described in step A3 has the accession number CCTCC NO: M 2019722, the Staphylococcus xylose has the accession number CGMCC NO.26373, and the Bifidobacterium longum has the accession number GDMCC NO.62129. The total inoculum amount of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 6% v / v of the fermentation substrate. The inoculum ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 1:1.5:0.8, and the viable count of each of the three bacteria is 5.0 × 10⁻⁶. 7 CFU / mL; the aerobic fermentation was carried out at 40°C with sterile air at an aeration ratio of 0.7 vvm for 12 hours; the anaerobic fermentation was carried out at 40°C for 12 hours; the centrifugation speed was 2500 rpm and the time was 30 min; The filtration sterilization mentioned in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm; the concentration is performed by rotary evaporation at 55°C to concentrate to 60% of the original volume.

[0040] Comparative Example 1 Compared with Example 2, the difference is that Bacillus coagulans was not inoculated in Comparative Example 1, and Staphylococcus xylose with the same inoculation amount and the same number of viable bacteria was used to supplement it. All other steps and parameters are the same as in Example 2.

[0041] Comparative Example 2 Compared with Example 2, the difference is that Staphylococcus xylose was not inoculated in Comparative Example 2, and Bacillus coagulans with the same inoculation amount and the same number of viable bacteria was used to supplement it. All other steps and parameters are the same as in Example 2.

[0042] Comparative Example 3 Compared with Example 2, the difference is that Comparative Example 3 does not involve inoculation with Bifidobacterium longum for anaerobic fermentation, and its preparation method includes the following steps: A1. The dried yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of 5:4:3:3 to obtain a Chinese medicine mixture. Deionized water is added and mixed evenly to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the temperature is raised to inactivate the enzyme to obtain the Chinese medicine enzymatic hydrolysate. A2. Weigh each fermentation raw material, mix them evenly, and then sterilize to obtain the fermentation substrate; A3. After inoculating the fermentation substrate with Bacillus coagulans and Staphylococcus xylose for aerobic fermentation, centrifuge after fermentation and retain the supernatant to obtain the fermentation broth; A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition. In step A1, the ratio of the herbal mixture to deionized water is 1g:15mL; the compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.5, and the amount of the compound enzyme added is 3wt% of the enzymatic hydrolysis substrate; the ultrasonic enzymatic hydrolysis temperature is 57℃, pH is 4.7, ultrasonic frequency is 35kHz, and time is 35min; the temperature for inactivating the enzyme is 100℃ and time is 30min. The fermentation raw materials mentioned in step A2, by weight, include: 15 parts yeast β-glucan, 12 parts elderberry extract, 6 parts glucosinolate, 15 parts sea cucumber peptide, 10 parts inulin, and 14 parts enzymatic hydrolysate of traditional Chinese medicine; the sterilization temperature is 121℃ and the time is 10s. The Bacillus coagulans described in step A3 has the accession number CCTCC NO: M 2019722, and the Staphylococcus xylose has the accession number CGMCC NO. 26373; the total inoculum size of Bacillus coagulans and Staphylococcus xylose is 5% v / v of the fermentation substrate, the inoculum ratio of Bacillus coagulans to Staphylococcus xylose is 1:1.7, and the viable count of both Bacillus coagulans and Staphylococcus xylose is 5.0 × 10⁻⁶. 7 CFU / mL; the aerobic fermentation was carried out at 37°C with sterile air at an aeration ratio of 0.5 vvm for 30 h; the centrifugation speed was 2500 rpm and the time was 30 min; The filtration sterilization mentioned in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm; the concentration is performed by rotary evaporation at 60°C to concentrate to 65% of the original volume.

[0043] Comparative Example 4 Compared with Example 2, the difference is that Comparative Example 3 does not involve aerobic fermentation with Bacillus coagulans and Staphylococcus xylose, and its preparation method includes the following steps: A1. The dried yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of 5:4:3:3 to obtain a Chinese medicine mixture. Deionized water is added and mixed evenly to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the temperature is raised to inactivate the enzyme to obtain the Chinese medicine enzymatic hydrolysate. A2. Weigh each fermentation raw material, mix them evenly, and then sterilize to obtain the fermentation substrate; A3. Inoculate the fermentation substrate with Bifidobacterium longum for anaerobic fermentation. After fermentation, centrifuge and retain the supernatant to obtain the fermentation broth. A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition. In step A1, the ratio of the herbal mixture to deionized water is 1g:15mL; the compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.5, and the amount of the compound enzyme added is 3wt% of the enzymatic hydrolysis substrate; the ultrasonic enzymatic hydrolysis temperature is 57℃, pH is 4.7, ultrasonic frequency is 35kHz, and time is 35min; the temperature for inactivating the enzyme is 100℃ and time is 30min. The fermentation raw materials mentioned in step A2, by weight, include: 15 parts yeast β-glucan, 12 parts elderberry extract, 6 parts glucosinolate, 15 parts sea cucumber peptide, 10 parts inulin, and 14 parts enzymatic hydrolysate of traditional Chinese medicine; the sterilization temperature is 121℃ and the time is 10s. The Bifidobacterium longum mentioned in step A3 has the preservation number GDMCC NO.62129; the inoculum size of the Bifidobacterium longum is 5% v / v of the fermentation substrate, and the viable count of the Bifidobacterium longum is 5.0 × 10⁻⁶. 7 CFU / mL; the anaerobic fermentation was carried out at 37°C for 30 h; the centrifugation speed was 2500 rpm and the time was 30 min; The filtration sterilization mentioned in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm; the concentration is performed by rotary evaporation at 60°C to concentrate to 65% of the original volume.

[0044] Comparative Example 5 Compared with Example 2, the difference is that in Comparative Example 5, Staphylococcus xylose with accession number CGMCC NO.3437 was used instead of Staphylococcus xylose with accession number CGMCC NO.26373. All other steps and parameters are the same as in Example 2.

[0045] Comparative Example 6 Compared with Example 2, the difference is that Bifidobacterium longum with accession number GDMCC NO.61618 was used instead of Bifidobacterium longum with accession number GDMCC NO.62129 in Comparative Example 6. All other steps and parameters are the same as in Example 2.

[0046] Comparative Example 7 Compared with Example 2, the difference is that the inoculation ratio of Bacillus coagulans, Staphylococcus xylose and Bifidobacterium longum in Comparative Example 7 is 1:1.2:1.5, while the other steps and parameters are the same as in Example 2.

[0047] Comparative Example 8 Compared with Example 2, the difference is that the inoculation ratio of Bacillus coagulans, Staphylococcus xylose and Bifidobacterium longum in Comparative Example 8 is 1:2.2:0.6, while the other steps and parameters are the same as in Example 2.

[0048] Example 1: Measurement of Immune Indicators This efficacy example demonstrates the determination of the following immune indicators according to the test methods described in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)": sheep erythrocyte (SRBC) induced delayed-type hypersensitivity (DTH) test in mice (using the paw thickening method); and the determination of the half-maximal hemolytic value (HC). 50 Serum hemolysin was measured; mouse carbon clearance assay was used to determine mouse monocyte-macrophage function; NK cell activity was measured (using lactate dehydrogenase (LDH) assay). The specific experimental preparation and procedures are as follows: 1) Test substance treatment: The compositions prepared in Examples 1-3 and Comparative Examples 1-8 were diluted with physiological saline to prepare test solutions with a mass concentration of 1.5%; 2) Experimental animals: 528 healthy adult ICR mice of SPF grade, weighing 18-22g, were acclimatized in an SPF-grade environment for one week before the experiment. The rearing conditions were a temperature of 20±2℃ and a relative humidity of 55±5%. During the experiment, the mice had free access to food and water. The mice were fed standard pelleted feed every day. The experimental observation room was open, and the lighting, noise, ventilation and other conditions were controlled within the specified range. The room was cleaned regularly every day. 3) Experiment: The experiment was divided into 4 immunization groups, each with 1 blank control group and 11 test groups (Examples 1-3 and Comparative Examples 1-8, respectively). Each blank control group and each test group contained 12 mice. Immunization group 1: DTH test; Immunization group 2: serum hemolysin assay; Immunization group 3: mouse carbon clearance test; Immunization group 4: NK cell activity test. The test groups were administered the compositions prepared in Examples 1-3 and Comparative Examples 1-8, respectively. The blank control group was replaced with an equal volume of physiological saline. The dosage was 0.3 mL / kg, administered by gavage at 9:00 AM every day for 30 consecutive days. 4) Data processing: All measurement data are expressed as mean ± standard deviation. Statistical analysis was performed using t-tests. P < 0.05 was considered statistically significant. The specific results are shown in Table 1.

[0049] Table 1. Data on immune marker measurements Group Difference in plantar swelling (mm) <![CDATA[Half hemolysis value / HC 50 > Phagocytosis Index / α NK cell activity (%) Blank control group 0.52±0.01 69.42±2.73 4.72±0.20 40.36±0.99 Example 1 <![CDATA[0.84±0.05 ▲ ]]> <![CDATA[102.45±4.06 ▲ ]]> <![CDATA[7.01±0.18 ▲ ]]> <![CDATA[64.32±2.25 ▲ ]]> Example 2 <![CDATA[0.87±0.04 ▲ ]]> <![CDATA[106.72±3.89 ▲ ]]> <![CDATA[7.18±0.17 ▲ ]]> <![CDATA[66.41±2.11 ▲ ]]> Example 3 <![CDATA[0.85±0.06 ▲ ]]> <![CDATA[105.08±4.21 ▲ ]]> <![CDATA[6.92±0.14 ▲ ]]> <![CDATA[63.55±2.49 ▲ ]]> Comparative Example 1 <![CDATA[0.67±0.03 ▲# ]]> <![CDATA[84.73±3.23 ▲# ]]> <![CDATA[6.14±0.19 ▲# ]]> <![CDATA[52.56±1.02 ▲# ]]> Comparative Example 2 <![CDATA[0.66±0.04 ▲# ]]> <![CDATA[83.25±3.02 ▲# ]]> <![CDATA[6.19±0.11 ▲# ]]> <![CDATA[53.49±1.14 ▲# ]]> Comparative Example 3 <![CDATA[0.60±0.03 ▲# ]]> <![CDATA[77.84±2.68 ▲# ]]> <![CDATA[5.80±0.23 ▲# ]]> <![CDATA[48.24±1.23 ▲# ]]> Comparative Example 4 <![CDATA[0.62±0.02 ▲# ]]> <![CDATA[79.36±3.10 ▲# ]]> <![CDATA[5.91±0.15 ▲# ]]> <![CDATA[49.32±1.45 ▲# ]]> Comparative Example 5 <![CDATA[0.72±0.05 ▲# ]]> <![CDATA[89.76±3.67 ▲# ]]> <![CDATA[6.42±0.13 ▲# ]]> <![CDATA[56.21±1.33 ▲# ]]> Comparative Example 6 <![CDATA[0.70±0.03 ▲# ]]> <![CDATA[87.35±3.09 ▲# ]]> <![CDATA[6.31±0.16 ▲# ]]> <![CDATA[54.73±1.48 ▲# ]]> Comparative Example 7 <![CDATA[0.77±0.01 ▲ ]]> <![CDATA[94.01±2.38 ▲# ]]> <![CDATA[6.61±0.10 ▲# ]]> <![CDATA[60.52±2.14 ▲# ]]> Comparative Example 8 <![CDATA[0.74±0.04 ▲# ]]> <![CDATA[93.87±3.42 ▲# ]]> <![CDATA[6.52±0.12 ▲# ]]> <![CDATA[59.34±2.01 ▲# ]]> Note:" ▲ "This indicates that compared with the blank control group, P < 0.05;" # "" indicates that compared with Example 2, Comparative Examples 1-8, P < 0.05.

[0050] Compared with Example 2, Comparative Examples 1-2 did not inoculate either Bacillus coagulans or Staphylococcus xylose in the preparation process of the compositions, Comparative Examples 3-4 involved only aerobic fermentation or only anaerobic fermentation in the preparation process of the compositions, Comparative Examples 5-6 involved replacing Staphylococcus xylose and Bifidobacterium longum with different preservation numbers in the preparation process of the compositions, and Comparative Examples 7-8 involved the ratio of viable Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum in the preparation process of the compositions exceeding the scope of this application.

[0051] The data in Table 1 regarding the difference in plantar edema, half-maximal hemolysis value, phagocytic index, and NK cell activity between Example 2 and Comparative Examples 1-2 show that Example 2 is more effective than Comparative Examples 1-2. This indicates that the composition of the fermentation strains affects the immune-enhancing effect of the composition. This may be due to the synergistic effect between Bacillus coagulans and Staphylococcus xylose, which can increase the fermentation speed, shorten the fermentation time, and provide richer nutrients, thereby enhancing the immune-enhancing effect of the composition. The data in Table 1 regarding Example 2 and Comparative Examples 3-4 show that Example 2 is more effective than Comparative Examples 3-4. This may be due to the synergistic effect between aerobic and anaerobic fermentation, which affects the efficacy of the composition. The data in Table 1 regarding Example 2 and Comparative Examples 5-6 show that xylose with different preservation numbers... Staphylococcus and Bifidobacterium longum have a certain impact on the fermentation effect. Even for the same species, the differences in properties between different strains can affect the fermentation effect. The Staphylococcus xylose and Bifidobacterium longum selected in this invention are the best strains that can have a synergistic effect with Bacillus coagulans, obtained by the inventors through extensive experimental screening in the early stage. According to the data of Example 2 and Comparative Examples 7-8 in Table 1, the inoculation ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum also affects the effect of the composition. When the inoculation ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 1:1.5-2.0:0.8-1.3, the composition has the effect of enhancing immunity. Among them, when the inoculation ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum is 1:1.7:1.1, the composition has the best effect.

[0052] Application example: A capsule that enhances immunity. The fermented compositions prepared in Examples 1-3 that help improve immunity were added to capsules to obtain capsules with immunity-enhancing effects as described in Examples 1-3.

[0053] In the capsule that enhances immunity, the amount of maltodextrin added is 20% and the amount of maltitol added is 19% based on the mass of the fermentation composition.

[0054] The preparation method of the capsule with the effect of improving immunity specifically includes the following steps: mixing the fermentation composition and maltodextrin at 50°C, stirring evenly, and then spray drying, wherein the inlet air temperature is 180°C and the outlet air temperature is 100°C; after complete drying, adding maltitol, mixing evenly, dry granulation, encapsulating the obtained granules in porcine gelatin capsules, and coating with an enteric coating to obtain the capsules; wherein the feeding speed of dry granulation is 200 r / min, the stirring speed of dry granulation is 25 r / min, and the pressure roller speed of dry granulation is 35 r / min; Blank application example: Compared with application examples 1-3, the difference is that the capsules of the blank application example do not contain the fermentation composition, and maltodextrin and maltitol in a mass ratio of 20:19 are used instead of the fermentation composition. The preparation method is the same as that of application examples 1-3.

[0055] Example 2: Toxicology Test One hundred healthy adult female ICR mice weighing 20-24g (SPF grade) were selected and divided into a control group and an experimental group, with 20 mice in each group. The experimental group was administered 45mg / g of the powder from the capsules prepared in Examples 1-3 and the blank application example by gavage. The powder was diluted with physiological saline to a mass concentration of 1% before gavage. The gavage was performed twice a day. The control group was administered the same amount of physiological saline by gavage daily. The gavage was performed for 2 consecutive days. The condition of the mice was observed after each gavage, and the number of dead mice was counted. Mice that did not die after 48 hours were observed for 2 weeks. The results showed that all members of both the control and experimental groups survived and were in good condition, indicating that the composition prepared in this application has no toxicity issues when applied to capsules and can be safely used as a health product.

[0056] Example 3: Human Efficacy Test The main immune cells in the human body are T cells and B cells. T cells are primarily responsible for cell-mediated immunity, while CD4+ cells are responsible for immune cell-mediated immunity. 4+ The cells are induced cells, a marker on the surface of helper T cells; CD4+ 8+ The cells are cytotoxic cells and are a marker of suppressor T cells; CD4+ 4+ / CD 8+ An elevated CD4 count indicates that helper T cells are higher than suppressor T cells, suggesting good immunity. 3+ It is all total T cells, CD 3+ A high level indicates a higher total number of T cells and CD4+. 4+ / CD 8+ and CD 3+ The high levels of both indicate an increase in helper CD4+ cells, which in turn increases the total number of T cells, suggesting good immunity. This efficacy example demonstrates this by testing the CD4+ levels of capsules prepared in Application Examples 1-3 and the blank application example. 3+ CD 4+CD 8+ and CD 4+ / CD 8+ The ratio of [value] to assess its effectiveness in boosting immunity; Thirty-two sub-healthy women aged 25-35, prone to colds and fatigue, were selected as subjects and randomly divided into four groups of eight per group. They took two capsules once daily with 150 mL of warm water for 30 consecutive days. After a 12-hour fast, a suitable amount of whole blood was collected in an EDTA anticoagulant tube. 100 μL of whole blood was transferred to a flow cytometry tube, and antibodies conjugated with three fluorescent dyes were added. The mixture was then pipetted and tapped to mix thoroughly. After incubation at room temperature in the dark for 15 minutes, 1 mL of lysis buffer was added, followed by 1 mL of PBS. The cells were centrifuged at 350 × g for 5 minutes, the supernatant was discarded, and the cells were dispersed. The cells were washed with 1 mL of PBS, centrifuged at 350 × g for 5 minutes, the supernatant was discarded, and CD4+ was detected by flow cytometry. 3+ Cells, CD 4+ Cells, CD 8+ Cell subset percentages and CD were calculated. 4+ / CD 8+ The ratios are presented as averages, and the results are shown in Table 2. Application Examples 1-3 and Blank Application Example CD in Table 2 3+ CD 4+ CD 8+ and CD 4+ / CD 8+ The data shows that, compared with the blank application example, the CD of application examples 1-3 is... 3+ and CD 4+ / CD 8+ All data showed significant improvement, CD 4+ Increased cell ratio, CD 8+ The decrease in cell ratio indicates that the capsules prepared using Examples 1-3 all help to enhance human immune function and can be used to prepare health products that help enhance immunity.

[0057] Table 2 Human Efficacy Data Group <![CDATA[CD 3+ (%)]]> <![CDATA[CD 4+ (%)]]> <![CDATA[CD 8+ (%)]]> <![CDATA[CD 4+ / CD 8+ ]]> Blank application example 56.34 27.82 33.57 0.83 Application Example 1 62.55 33.41 24.15 1.38 Application Example 2 66.48 35.09 24.61 1.43 Application Example 3 63.13 32.36 25.42 1.27 The above description is merely a preferred composition of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with preferred compositions, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent compositions without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above compositions based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a fermented composition that helps enhance immunity, characterized in that, The preparation method includes the following steps: A1. Dry yam, wolfberry, astragalus and poria are pulverized and passed through a 30-mesh sieve. The resulting yam powder, wolfberry powder, astragalus powder and poria powder are mixed in a mass ratio of (3-7):(3-7):(2-5):(2-5) to obtain a traditional Chinese medicine mixture. Deionized water is added and mixed evenly to obtain an enzymatic hydrolysis substrate. A compound enzyme is added for ultrasonic-assisted enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme is inactivated by heating to obtain the enzymatic hydrolysate of traditional Chinese medicine. The compound enzyme is α-amylase and cellulase in a mass ratio of 1:1.2-1.

7. A2. Weigh each fermentation raw material, mix them evenly, and sterilize them to obtain the fermentation substrate; wherein, the fermentation raw materials include, by mass, 13-17 parts of yeast β-glucan, 10-15 parts of elderberry extract, 4-7 parts of glucosinolate, 13-17 parts of sea cucumber peptide, 8-12 parts of inulin, and 11-17 parts of traditional Chinese medicine enzymatic hydrolysate. A3. After aerobic fermentation by inoculating Bacillus coagulans and Staphylococcus xylose into the fermentation substrate, Bifidobacterium longum is inoculated for anaerobic fermentation. After fermentation, the substrate is centrifuged and the supernatant is retained to obtain the fermentation broth. The preservation number of Bacillus coagulans is CCTCC NO: M 2019722, the preservation number of Staphylococcus xylose is CGMCC NO.26373, and the preservation number of Bifidobacterium longum is GDMCC NO.62129. A4. The fermentation broth is filtered, sterilized, and concentrated to obtain the fermentation composition.

2. The preparation method according to claim 1, characterized in that, The ratio of the herbal medicine mixture to deionized water in step A1 is 1g:10-20mL.

3. The preparation method according to claim 1, characterized in that, The amount of the compound enzyme added in step A1 is 2-4 wt% of the enzymatic hydrolysis substrate. The ultrasonic enzymatic hydrolysis temperature is 55-60℃, the pH is 4.5-5.5, the ultrasonic frequency is 30-40kHz, and the time is 30-40min. The temperature for inactivating the enzyme by heating is 95-105℃, and the time is 25-35min.

4. The preparation method according to claim 1, characterized in that, The fermentation raw materials mentioned in step A2 include the following components by weight: 15 parts yeast β-glucan, 12 parts elderberry extract, 6 parts glucosinolate, 15 parts sea cucumber peptide, 10 parts inulin, and 14 parts enzymatic hydrolysate of traditional Chinese medicine.

5. The preparation method according to claim 1, characterized in that, In step A3, the total inoculum size of *Bacillus coagulans*, *Staphylococcus xylose*, and *Bifidobacterium longum* is 4-6% v / v of the fermentation substrate. The inoculum ratio of *Bacillus coagulans*, *Staphylococcus xylose*, and *Bifidobacterium longum* is 1:1.5-2:0.8-1.3, and the viable count of each of *Bacillus coagulans*, *Staphylococcus xylose*, and *Bifidobacterium longum* is 5.0 × 10⁻⁶. 7 CFU / mL.

6. The preparation method according to claim 5, characterized in that, The inoculation ratio of Bacillus coagulans, Staphylococcus xylose, and Bifidobacterium longum in step A3 is 1:1.7:1.

1.

7. The preparation method according to claim 1, characterized in that, The aerobic fermentation in step A3 is as follows: at 34-40℃, sterile air with an aeration ratio of 0.4-0.7 vvm is introduced for aerobic fermentation for 12-18 hours; the anaerobic fermentation is as follows: at 34-40℃, anaerobic fermentation is carried out for 12-18 hours.

8. The preparation method according to claim 1, characterized in that, The centrifugation speed in step A3 is 2500-4500 rpm and the time is 18-30 min.

9. The preparation method according to claim 1, characterized in that, The filtration sterilization in step A4 is performed using a sterile filter membrane with a pore size of 0.22 μm, and the concentration is performed by rotary evaporation at 55-65℃ to concentrate to 60-70% of the original volume.

10. The use of the fermented composition prepared by any one of claims 1-9 in the preparation of health products that help enhance immunity, characterized in that, The dosage form of the health product is any one of tablets, hard capsules, soft capsules, ointments, granules, or powders.