Fermented ginseng-probiotic-natural antibacterial peptide composite oral liquid with active immune defense function and preparation method thereof

Abstract

The invention discloses a fermented ginseng-probiotic-natural antibacterial peptide composite oral liquid with an active immune defense function and a preparation method thereof, and belongs to the technical field of functional health food. The oral liquid is prepared by compounding a fermented ginseng matrix, an active probiotic preparation, a fly maggot source natural antibacterial peptide extract and auxiliary materials, and the activity of the components is guaranteed through a whole-course cold chain preparation process. On the basis of a microbial counterbalance theory, a human body active immune defense system is constructed by utilizing the synergistic effect of the fermented ginseng, the probiotics and the antibacterial peptide, and the limitation of traditional passive medical treatment is broken through; the shelf life of the product at 0-5 DEG C reaches 12 months, and the product is convenient to take and high in safety. The invention has the advantages of simple preparation process, wide raw material source and suitability for large-scale production, and has extremely high practical value in the field of human health protection.

Description

Technical Field

[0001] This invention relates to the field of functional health food preparation technology, specifically to a compound oral liquid based on the theory of microbial balance, integrating the conditioning effects of fermented traditional Chinese medicine, the balancing effect of probiotic flora, and the antibacterial activity of natural antimicrobial peptides. It is particularly suitable for building the human body's active immune defense system, enhancing resistance to pathogens, and reducing the probability of infection and disease. Background Technology

[0002] Microbiological studies have confirmed that the symbiotic balance between pathogenic bacteria and probiotics in an organism directly determines the organism's health level. Probiotics inhibit the proliferation of pathogenic bacteria by colonizing the gut, competing for nutrients, and secreting antibacterial substances, thus forming a natural immune barrier.

[0003] In large-scale animal husbandry, the feeding model of fermented traditional Chinese medicine combined with active probiotics has been successfully applied. It can build an active defense immune system for animals and achieve the effect of preventing the invasion and disease of pathogens throughout the entire process. Its core mechanism lies in the exogenous supplementation of probiotics and active ingredients of traditional Chinese medicine, which regulates the balance of the in vivo microbial community and strengthens the immune barrier function.

[0004] Currently, human health protection is still mainly based on passive medical intervention, that is, when pathogens invade and cause illness, treatment is carried out through drugs, surgery and other means. There is a lack of food or drugs that can build an active immune defense system before pathogens invade.

[0005] With the increasing awareness of health, the demand for proactively maintaining health through nutritional intervention is growing. Probiotic preparations and traditional Chinese medicines (such as ginseng) are widely used in regulating immunity and enhancing resistance. However, most existing probiotic preparations and ginseng health products are single-ingredient or simply compounded products, suffering from low activity, limited target sites, and inability to maintain gut microbiota balance in a long-term manner. Natural antimicrobial peptide products, on the other hand, suffer from high extraction costs, poor stability, and susceptibility to decomposition by human digestive enzymes, making large-scale application and daily consumption difficult.

[0006] Existing related products have significant technical defects: 1. Probiotic preparations (such as CN114524321A, US20230054981A1) are mostly single probiotic compound or probiotic + simple nutrient combination. They have a single target of action, no active defense synergistic mechanism, and the shelf life of live bacteria is only 6-8 months, with poor activity stability. Ordinary probiotic preparations are prone to activity decay during storage, transportation and passage through the human digestive tract, and the intestinal colonization rate is unstable.

[0007] 2. Fermented ginseng-related products (such as CN113813522B and KR1020220087654A) only focus on optimizing the ginseng fermentation process, without introducing probiotics and antimicrobial peptides. They cannot achieve dual protection of "microbial balance + direct antibacterial action", and their immune-regulating effect is limited to the single effect of ginsenosides. The bioavailability of the main active ingredients in ginseng (such as saponins) is not high when taken orally.

[0008] 3. Natural antimicrobial peptide products are used in aquaculture or external applications (such as CN112724793A, EP3890123B1), with very few oral products for humans. Oral antimicrobial peptides (such as CN115040318A) have problems such as high extraction costs (costs reach 500 yuan / gram when purity ≥85%) and easy decomposition by digestive enzymes (activity retention rate <40% after oral administration), making it difficult to apply on a large scale.

[0009] 4. Some existing compound formulations have failed to achieve deep synergistic effects. For example, Chinese patent CN118745452A discloses a method for fermenting ginseng with compound microbial communities to convert saponins and improve flavor. However, its core technology lies in component conversion and taste optimization, and does not involve the construction of a systematic active immune defense system.

[0010] Therefore, developing compound preparations that integrate the activity of probiotics, the conditioning effects of traditional Chinese medicine, and the antibacterial effects of antimicrobial peptides, and can solve the problems of unstable activity, short shelf life, and high cost, to build an active immune defense system for the human body, has become a key technological requirement to break through the passive situation of human health protection. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function and its preparation method. The oral liquid combines fermented Chinese medicine, probiotics and natural antimicrobial peptides. By synergistically exerting the immunomodulatory effect of fermented ginseng, the flora balancing effect of active probiotics and the antibacterial effect of natural antimicrobial peptides, it constructs an active immune defense system for the human body, breaking through the limitations of "passive treatment" or "single protection", and achieving the health protection effect of "prevention before the disease occurs". At the same time, it solves the problems of unstable activity, short shelf life and inconvenient use of existing products.

[0012] The technical solution of the present invention is: a fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function. The formula composition of the compound oral liquid by weight is: 27-37 parts of fermented ginseng matrix, 5-13 parts of active probiotic preparation, 1-3 parts of natural antimicrobial peptide component, 2.5-6 parts of excipients, and the balance being purified water.

[0013] The fermented ginseng matrix is ​​prepared by constant-temperature fermentation of ginseng extract, fermentation strain, and fermentation excipients; the fermentation strain is Lactobacillus plantarum + Bifidobacterium; the fermentation excipient is brown sugar + wolfberry juice; the active probiotic preparation is a mixture of freeze-dried lactic acid bacteria powder and freeze-dried Bifidobacterium powder; the natural antimicrobial peptide component is fly larvae-derived antimicrobial peptide extract; and the excipients are xylitol and citric acid.

[0014] Furthermore, the formula of this compound oral liquid, by weight, consists of: 20-30 parts ginseng extract, 2-5 parts fermentation strains (Lactobacillus plantarum + Bifidobacterium), 5-10 parts fermentation excipients (brown sugar + goji berry juice), and freeze-dried lactic acid bacteria powder (live count ≥10). 10 3-8 parts (CFU / g), freeze-dried Bifidobacterium powder (live count ≥10) 10 2-5 parts of CFU / g antimicrobial peptide extract (purity ≥90%), 1-3 parts of xylitol, 0.5-1 part of citric acid, and the remainder purified water.

[0015] Furthermore, the ratio of *Lactobacillus plantarum* + *Bifidobacterium* is 1:1, the ratio of brown sugar to goji berry juice is 2:1, and the weight ratio of the mixture of freeze-dried lactic acid bacteria powder and freeze-dried bifidobacterium powder is 4-6:3-4.

[0016] The preparation method of the above-mentioned compound oral liquid includes the following steps: Step 1: Preparation of fermented ginseng substrate (1) Take 5-year-old forest ginseng, rinse it with clean water 3 times to remove mud and sand, slice it to a thickness of 2mm, add 3 times the volume of purified water, place it in a 300W, 40℃ ultrasonic extraction device for 2 hours, filter it with a 200 mesh filter cloth, and collect the ginseng extract (saponin content ≥0.9mg / mL). (2) Add fermentation adjuvants and fermentation strains to the ginseng extract, and adjust the pH value to 1 mol / L citric acid. 6.0-6.5 Transfer to a 30-35℃ constant temperature fermentation tank, seal and ferment for 42-50 hours, stirring once every 12 hours for 10 minutes each time; (3) After fermentation, the ginseng substrate was refrigerated at 4°C for 24 hours and filtered through a 400-mesh filter cloth to remove the precipitate, and then the fermented ginseng substrate was prepared for use. Step 2: Extraction of natural antimicrobial peptides (1) Using shrimp shell waste (crushed to a particle size of 5 mm) as a culture medium, fly eggs were inoculated (inoculation amount 5 g / m²), cultured at 28℃ for 72 hours, fly larvae were collected, freeze-dried and crushed to 100 mesh powder; (2) Add phosphate buffer solution with pH 7.0 and concentration of 0.05mol / L at a material-to-liquid ratio of 1:5 (g / mL), and pretreat it at 40℃ for 30 minutes in a 200W ultrasonic device; (3) Add trypsin (enzyme-substrate ratio 1:45), and hydrolyze in a constant temperature water bath at 37°C for 3-3.5 hours, stirring for 5 minutes every 30 minutes during the process; if it is necessary to improve the purity, use trypsin + papain (1:1) for secondary hydrolysis. (4) The enzymatic hydrolysate was centrifuged at 8000 r / min and 4℃ for 20 minutes. The supernatant was filtered through a 10 kDa ultrafiltration membrane. The retentate was freeze-dried at -50℃ and 0.01 MPa to obtain fly maggot-derived antimicrobial peptide extract with a purity ≥92%, which was then used for later use. Step 3: Compound preparation of oral liquid (1) Add purified water to the fermented ginseng substrate, place it in a low-temperature stirring tank at 4℃, stir at 800-1200r / min for 10 minutes, add xylitol and citric acid in sequence, stir until completely dissolved, and adjust the pH of the solution to 5.2-5.4 with 1mol / L NaOH; (2) Maintain a low temperature environment of 4°C, add freeze-dried lactic acid bacteria powder, freeze-dried bifidobacteria powder and fly larvae-derived antimicrobial peptide extract to the above solution, adjust the speed to 1200-1500 r / min, stir for 30 minutes to ensure that the probiotics and antimicrobial peptides are evenly dispersed; (3) The mixture was aseptically filtered through a 0.22μm polyethersulfone filter membrane and then filled into brown oral liquid bottles using a fully automatic aseptic filling machine (aluminum foil gasket + screw cap seal). (4) After filling, seal and store at a low temperature of 0-5℃ to obtain the finished compound oral liquid.

[0017] The technical parameters of the above-mentioned finished compound oral liquid are as follows: - Probiotic live bacteria count: ≥10 8 CFU / mL; - Antimicrobial peptide activity: Inhibition rate against Escherichia coli and Staphylococcus aureus ≥90%; - Ginsenoside content: ≥0.9mg / mL; - Storage conditions: Refrigerate at a low temperature of 0-5℃; -Shelf life: 12 months; Dosage: Adults take 5-10 mL daily, children take half the dose, either directly or diluted with warm water.

[0018] The beneficial effects of this invention are: 1. Constructing an active immune defense system: This invention is the first to combine fermented ginseng, active probiotics, and natural antimicrobial peptides derived from fly larvae. It constructs a synergistic protection network from three dimensions: regulating the balance of intestinal flora, enhancing the activity of immune cells, and directly inhibiting pathogenic bacteria. This breaks through the limitations of traditional passive medical treatment and achieves the health protection goal of "prevention is better than cure". Animal experiments have shown that the incidence rate in the experimental group is only 3%-5% (compared to 80%-82% in the control group).

[0019] 2. Synergistic effect of components: The fermented ginseng matrix is ​​made from ginseng extract through a specific ratio of Lactobacillus plantarum (…). Lactobacillus plantarum ) and Bifidobacteria ( Bifidobacterium It is made through symbiotic fermentation, and its hallmark feature is a significantly increased content of rare ginsenoside Rg3, which can regulate human immune function and provide a good intestinal environment for probiotic colonization. The compound probiotic freeze-dried powder preferably uses a mixed powder of Lactobacillus plantarum and Bifidobacterium that has been microencapsulated. It occupies the intestinal ecological niche through proliferation and inhibits the growth of pathogenic bacteria, so as to ensure its storage resistance and gastrointestinal passage rate. The fly larvae-derived antimicrobial peptide extract is made from fly larvae raised from kitchen waste such as shrimp and crab shells. It is obtained by ultrasonic-assisted enzymatic hydrolysis, membrane separation and chromatography purification, with a purity of not less than 95%. The natural antimicrobial peptides directly target and kill pathogenic bacteria. The three work synergistically to enhance the immune defense effect.

[0020] 3. High product stability: The entire cold chain process, including low-temperature fermentation, low-temperature extraction, low-temperature compounding, and low-temperature storage, ensures the activity of probiotics and antimicrobial peptides. Through formula optimization, the product has a shelf life of 12 months at 0-5℃, solving the problem of easy loss of activity and short shelf life (6-8 months) of similar products.

[0021] 4. Controllable cost and excellent safety: Antimicrobial peptides are extracted by using shrimp shell waste to raise fly larvae through ecological circular agriculture technology, reducing the cost to below 120 yuan / gram (76% lower than existing technologies), and the raw materials are widely available; the product contains no chemical preservatives, antibiotics or other additives, and is flavored with xylitol and citric acid, with a sweet and sour taste that is easily accepted by adults and children and suitable for long-term use.

[0022] 5. The process is scalable: the preparation process steps are clear, the equipment requirements are conventional, and the parameters are easy to control, enabling large-scale industrial production. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the embodiments, but it should not be construed as a limitation on the technical solution.

[0024] Example 1: Preparation of compound oral liquid according to the following steps (1) Raw material preparation (by weight): 25 parts of 5-year-old forest ginseng extract, 3.5 parts of Lactobacillus plantarum + Bifidobacterium (1:1) mixed fermentation strain, 7 parts of brown sugar + wolfberry juice (2:1) mixture, 5 parts of freeze-dried lactic acid bacteria powder, 3.5 parts of freeze-dried Bifidobacterium powder, 2 parts of fly larvae antimicrobial peptide extract (purity 93%), 3.5 parts of xylitol, 0.7 parts of citric acid, and the remainder purified water; (2) Preparation of compound oral liquid: Prepare fermented ginseng matrix according to step 1 of the technical scheme (fermented at 32℃ for 48 hours, saponin content 1.2mg / mL); extract fly larvae antimicrobial peptide extract according to step 2 of the technical scheme (single enzymatic hydrolysis for 3.5 hours, purity 93%); compound according to step 3 of the technical scheme: dissolve excipients by stirring at 800r / min at 4℃, mix probiotics and antimicrobial peptides by stirring at 1500r / min, filter aseptically and fill (10mL per bottle); store at 0-5℃ to obtain the finished product.

[0025] (3) Product test results: Live probiotic count 1.8 × 10⁻⁶ 8 CFU / mL; 95% inhibition rate against Escherichia coli and 96% inhibition rate against Staphylococcus aureus; pH 5.3; sweet and sour taste with no off-flavor; viable count of 1.3 × 10⁻⁶ after 12 months of storage at 0-5℃. 8 The CFU / mL level did not significantly decrease the antibacterial rate.

[0026] Example 2: Preparation of compound oral liquid according to the following steps (1) Raw material preparation (by weight): 22 parts of 5-year-old forest ginseng extract, 3 parts of Lactobacillus plantarum + Bifidobacterium (1:1) mixed fermentation strain, 6 parts of brown sugar + wolfberry juice (2:1) mixture, 4 parts of freeze-dried lactic acid bacteria powder, 3 parts of freeze-dried Bifidobacterium powder, 1.5 parts of fly larvae antimicrobial peptide extract, 3 parts of xylitol, 0.6 parts of citric acid, and the remainder purified water; (2) Preparation of compound oral liquid: Prepare fermented ginseng matrix according to step 1 of the technical scheme (fermented at 30℃ for 42 hours, saponin content 0.9mg / mL); extract fly larvae antimicrobial peptide extract according to step 2 of the technical scheme (single enzymatic hydrolysis for 3 hours, purity 92%); compound according to step 3 of the technical scheme: dissolve excipients by stirring at 800r / min at 4℃, mix probiotics and antimicrobial peptides by stirring at 1200r / min, filter aseptically and fill (10mL per bottle); store at 0-5℃ to obtain the finished product.

[0027] (3) Product test results: Live probiotic count 1.1×10⁻⁶ 8CFU / mL; inhibition rate against Escherichia coli 90%, inhibition rate against Staphylococcus aureus 91%; viable count 0.9 × 10⁻⁶ after 12 months of storage at 0-5℃. 8 CFU / mL, meeting the technical parameter requirements; cost reduced by 18% compared to Example 1.

[0028] Example 3: Preparation of compound oral liquid according to the following steps (1) Raw material preparation (by weight): 28 parts of 5-year-old forest ginseng extract, 4 parts of mixed fermentation strain of Lactobacillus plantarum + Bifidobacterium (1:1), 8 parts of mixed excipient of brown sugar + wolfberry juice (2:1), 6 parts of freeze-dried lactic acid bacteria powder, 4 parts of freeze-dried Bifidobacterium powder, 2.5 parts of fly larvae antimicrobial peptide extract, 4 parts of xylitol, 0.9 parts of citric acid, and the remainder purified water; (2) Preparation of compound oral liquid: Prepare fermented ginseng matrix according to step 1 of the technical scheme (fermented at 35℃ for 50 hours, ultrasonic extraction time extended to 2.5 hours, saponin content 1.5mg / mL); extract fly larvae antimicrobial peptide extract according to step 2 of the technical scheme (trypsin + papain (1:1) secondary enzymatic hydrolysis, purity 95%); compound according to step 3 of the technical scheme: dissolve excipients by stirring at 1000r / min at 4℃, mix probiotics and antimicrobial peptides by stirring at 1500r / min, filter aseptically and fill (10mL per bottle); store at 0-5℃ to obtain the finished product.

[0029] (3) Product test results: Live probiotic count 2.3 × 10⁻⁶ 8 CFU / mL; 97% inhibition rate against Escherichia coli and 98% inhibition rate against Staphylococcus aureus; In animal experiments, the morbidity rate in the experimental group mice was only 3% (compared to 82% in the control group); Suitable for people with weakened immunity (such as the elderly and those recovering from surgery).

Claims

1. A fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function. The composition of this compound oral liquid by weight includes the following components: 27-37 parts fermented ginseng matrix, 5-13 parts active probiotic preparation, 1-3 parts natural antimicrobial peptide component, 2.5-6 parts excipients, and the balance purified water.

2. The fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 1, characterized in that: The fermented ginseng matrix is ​​prepared by constant-temperature fermentation of ginseng extract, fermentation strains, and fermentation excipients; the fermentation strains are Lactobacillus plantarum and Bifidobacterium; the fermentation excipients are brown sugar and wolfberry juice; the active probiotic preparation is a mixture of freeze-dried lactic acid bacteria powder and freeze-dried Bifidobacterium powder; the natural antimicrobial peptide component is fly larvae-derived antimicrobial peptide extract; and the excipients are xylitol and citric acid.

3. The fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 2, characterized in that: The formula of this compound oral liquid, by weight, consists of: 20-30 parts ginseng extract, 2-5 parts fermentation strains (Lactobacillus plantarum + Bifidobacterium), 5-10 parts fermentation excipients (brown sugar + goji berry juice), and freeze-dried lactic acid bacteria powder (live count ≥10). 10 3-8 parts (CFU / g), freeze-dried Bifidobacterium powder (live count ≥10) 10 2-5 parts of CFU / g antimicrobial peptide extract (purity ≥90%), 1-3 parts of xylitol, 0.5-1 part of citric acid, and the remainder purified water.

4. The fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 3, characterized in that: Lactobacillus plantarum + Bifidobacterium = 1:1, brown sugar: goji berry juice = 2:1, the weight ratio of the mixture of freeze-dried lactic acid bacteria powder and freeze-dried Bifidobacterium powder is 4-6:3-4.

5. The preparation method of the fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 1, characterized in that: The preparation method of this compound oral liquid includes the following steps: Step 1: preparation of fermented ginseng matrix; Step 2: extraction of natural antimicrobial peptides; Step 3: compounding of the compound oral liquid.

6. The preparation method of the fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 5, characterized in that: The preparation process of the fermented ginseng matrix is ​​as follows: (1) Take 5-year-old forest ginseng, rinse it with clean water 3 times to remove mud and sand, slice it to a thickness of 2mm, add 3 times the volume of purified water, place it in a 300W, 40℃ ultrasonic extraction device for 2 hours, filter it with a 200 mesh filter cloth, and collect the ginseng extract (saponin content ≥0.9mg / mL). (2) Add fermentation adjuvants and fermentation strains to ginseng extract, adjust the pH value to 6.0-6.5 with 1mol / L citric acid, transfer to a constant temperature fermentation tank at 30-35℃, seal and ferment for 42-50 hours, stirring once every 12 hours for 10 minutes each time; (3) After fermentation, the ginseng substrate was refrigerated at 4°C for 24 hours and filtered through a 400-mesh filter cloth to remove the precipitate and obtain the fermented ginseng substrate for later use.

7. The preparation method of the fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 5, characterized in that: The extraction process of the natural antimicrobial peptides is as follows: (1) Using shrimp shell waste (crushed to a particle size of 5 mm) as a culture medium, fly eggs were inoculated (inoculation amount 5 g / m²), cultured at 28℃ for 72 hours, fly larvae were collected, freeze-dried and crushed to 100 mesh powder; (2) Add phosphate buffer solution with pH 7.0 and concentration of 0.05mol / L at a material-to-liquid ratio of 1:5 (g / mL), and pretreat it at 40℃ for 30 minutes in a 200W ultrasonic device; (3) Add trypsin (enzyme-substrate ratio 1:45), and hydrolyze in a constant temperature water bath at 37°C for 3-3.5 hours, stirring for 5 minutes every 30 minutes during the process; if it is necessary to improve the purity, use trypsin + papain (1:1) for secondary hydrolysis. (4) The enzymatic hydrolysate was centrifuged at 8000 r / min and 4℃ for 20 minutes. The supernatant was filtered through a 10 kDa ultrafiltration membrane. The retentate was freeze-dried at -50℃ and 0.01 MPa to obtain fly larvae-derived antimicrobial peptide extract with a purity ≥92%, which was then used for later use.

8. The preparation method of the fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 5, characterized in that: The compounding process of the compound oral liquid is as follows: (1) Add purified water to the fermented ginseng substrate, place it in a low-temperature stirring tank at 4℃, stir at 800-1200r / min for 10 minutes, add xylitol and citric acid in sequence, stir until completely dissolved, and adjust the pH of the solution to 5.2-5.4 with 1mol / L NaOH; (2) Maintain a low temperature environment of 4°C, add freeze-dried lactic acid bacteria powder, freeze-dried bifidobacteria powder and fly larvae-derived antimicrobial peptide extract to the above solution, adjust the speed to 1200-1500 r / min, stir for 30 minutes to ensure that the probiotics and antimicrobial peptides are evenly dispersed; (3) The mixture was aseptically filtered through a 0.22μm polyethersulfone filter membrane and then filled into brown oral liquid bottles using a fully automatic aseptic filling machine (aluminum foil gasket + screw cap seal). (4) After filling, seal and store at a low temperature of 0-5℃ to obtain the finished compound oral liquid.

9. The application of the fermented ginseng-probiotic-natural antimicrobial peptide compound oral liquid with active immune defense function according to claim 1 in the preparation of health food with active immune defense function.