Multifunctional compound probiotic composition and preparation process thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a multifunctional compound probiotic composition which comprises the following raw materials in percentage by mass: 40-50% of probiotics; 30%-40% of a microcapsule embedding material; prebiotics: 10%-25%; 1%-5% of auxiliary materials; the invention also provides a preparation process of the multifunctional compound probiotic composition. The preparation process comprises the following steps: S1, culturing the probiotics and detecting the activity; s2, a microencapsulation process: preparing a multi-layer microcapsule through a spray drying method and an emulsion crosslinking method, and embedding the probiotics; and S3, mixing the microcapsules with prebiotics and auxiliary materials to obtain the multifunctional compound probiotic composition. By adopting a composite probiotic compatibility technology and through the synergistic effect of different strains, the stability and functionality of intestinal microecology are enhanced, and the effects of improving the survival rate of probiotics in gastrointestinal tracts, promoting planting of beneficial bacteria and enhancing the immunoregulation ability are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a multifunctional composite probiotic composition and a preparation process thereof. Background Art

[0002] The accelerated pace of modern life and changes in dietary structure have led to increasingly prominent intestinal health problems. Problems such as indigestion, imbalance of intestinal flora, and decreased immunity have become a concern for many people. Probiotics, as an important means to regulate intestinal microecology and enhance immunity, have been widely used. However, simply supplementing probiotics often has problems such as low survival rate and insufficient intestinal colonization ability, making it difficult to achieve ideal health improvement effects.

[0003] Existing technologies have made many explorations in improving the survival rate and stability of probiotics. For example, the application of single-strain probiotics can supplement the lack of specific bacteria in the body to a certain extent and improve the short-term intestinal flora environment. Hydrogel encapsulation technology can provide certain protection for probiotics when they enter the digestive tract to prevent some strains from being inactivated by gastric acid. These technical solutions provide a basis for the widespread application of probiotics.

[0004] However, these technologies still have many limitations. After a single strain of probiotics enters the intestine, the effect is often limited, and it is difficult to form a synergistic effect of the flora, resulting in poor colonization ability. It may be competitively squeezed out by the original flora in a short period of time. Although hydrogel encapsulation provides protection, it has a loose structure and high gastric acid permeability, and the loss rate of probiotics is still high. In addition, the lack of a probiotic supplementation method supported by prebiotics makes the flora regulation effect relatively short-lived, and it is difficult to maintain a healthy ecology in the intestine for a long time. For this reason, those skilled in the art propose a multifunctional composite probiotic composition and its preparation process to solve the above problems. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a multifunctional composite probiotic composition and a preparation process thereof, which solves the problems in the prior art of low survival rate of probiotics, insufficient colonization ability, unsustainable regulating effect and easy inactivation of probiotics in the gastrointestinal environment.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a multifunctional composite probiotic composition, comprising the following raw materials in percentage by mass:

[0007] Probiotics: 40%-50%;

[0008] Microcapsule embedding material: 30%-40%;

[0009] Prebiotics: 10%-25%;

[0010] Excipients: 1%-5%.

[0011] Preferably, the microcapsule embedding material comprises:

[0012] The inner layer embedding material includes sodium alginate, calcium lactate and gelatin, wherein the mass fraction of sodium alginate accounts for 5%-18% of the total mass, the mass fraction of calcium lactate accounts for 1%-4% of the total mass, and the mass fraction of gelatin accounts for 3%-8% of the total mass;

[0013] The middle layer embedding material includes chitosan and polylactic acid, wherein the mass fraction of chitosan accounts for 2%-8% of the total mass, and the mass fraction of polylactic acid accounts for 3%-12% of the total mass;

[0014] The outer layer embedding material comprises polylactic acid and polyvinyl alcohol, wherein the mass fraction of the polylactic acid accounts for 5%-12% of the total mass, and the mass fraction of the polyvinyl alcohol accounts for 1%-8% of the total mass.

[0015] The core of microencapsulation technology is to form a protective layer through encapsulation materials to prevent probiotics from being killed by gastric acid in the gastric environment (low pH value). The gel formed by the combination of sodium alginate and calcium lactate can provide strong acid resistance in the stomach to prevent probiotics from direct contact with gastric acid. The use of gelatin helps to improve the structural stability of the encapsulation material and ensure that the microcapsules are not dissolved prematurely in the stomach. Chitosan, as the middle layer encapsulation material, can dissolve relatively slowly in the alkaline environment of the small intestine and release probiotics in the process. The outer layer of polylactic acid acts as a protective film, which can enhance the mechanical strength and dissolution control of the microcapsules, allowing them to release probiotics in a targeted manner in the intestine.

[0016] Preferably, the probiotics are a composite ratio of lactic acid bacteria, bifidobacteria and Lactobacillus acidophilus.

[0017] in:

[0018] Lactic acid bacteria account for 15%-45%;

[0019] Bifidobacterium accounts for 35%-50%;

[0020] Lactobacillus acidophilus accounts for 10%-35%.

[0021] Lactobacillus, bifidobacterium and Lactobacillus acidophilus are common types of intestinal probiotics, with good intestinal colonization ability and intestinal flora regulation function. Lactobacillus can produce lactic acid through fermentation, regulate intestinal pH value, and inhibit the growth of pathogens; Bifidobacterium helps promote intestinal immune function and inhibit harmful microorganisms; Lactobacillus acidophilus has the functions of enhancing intestinal barrier function and improving digestive function. Through the appropriate proportion, a synergistic probiotic complex is formed, thereby providing comprehensive health benefits in the gastrointestinal tract.

[0022] Preferably, the prebiotics include plant extracts, and the plant extracts include:

[0023] Wolfberry extract accounts for 5%-12% of the total mass;

[0024] Curcumin accounts for 1%-4% of the total mass;

[0025] Grape seed extract accounts for 2%-8% of the total mass.

[0026] The main role of plant extracts in this formula is antioxidant and immunomodulatory. Lycium barbarum extract is rich in carotenoids, flavonoids and polysaccharides, and has strong antioxidant capacity, which can reduce the risk of probiotics dying due to oxidative stress in the intestine. Curcumin has good anti-inflammatory effects and can reduce intestinal inflammatory responses, thereby promoting the colonization of probiotics. Grape seed extract is rich in polyphenolic compounds, which can further enhance the antioxidant and immunomodulatory effects, thereby protecting probiotics from adverse factors in the gastrointestinal environment.

[0027] Preferably, the prebiotics further include dietary fiber, and the dietary fiber is inulin, which accounts for 5%-10% of the total mass.

[0028] Inulin, as a water-soluble dietary fiber, can promote intestinal motility and help probiotics colonize in the intestines. In addition, as a low-calorie, high-fiber food ingredient, inulin can also promote the balance of intestinal microbiota, provide a suitable growth environment for probiotics, and enhance their functionality.

[0029] Preferably, the auxiliary material includes a natural sweetener, and the natural sweetener is one or more of erythritol and monk fruit extract.

[0030] Erythritol, as a low-calorie sweetener, not only provides sweetness, but also has certain antioxidant effects. Monk fruit extract is rich in natural sweet ingredients and has a low GI value, which helps to control blood sugar levels. The combined use of the two can not only improve the taste of the beverage, but also meet the needs of a low-sugar healthy diet without affecting the survival and functionality of probiotics.

[0031] A preparation process of a multifunctional composite probiotic composition comprises the following steps:

[0032] S1. Cultivation and activity detection of probiotics;

[0033] S2, microencapsulation process: preparing multilayer microcapsules by spray drying and emulsion cross-linking to encapsulate the probiotics;

[0034] S3, mixing the microcapsules with prebiotics and auxiliary materials to obtain a multifunctional composite probiotic composition;

[0035] S4, drying the obtained composition to obtain a final product.

[0036] The spray drying method can quickly evaporate the water in the liquid to form tiny particles, and at the same time, by controlling the temperature and spray flow rate, the efficient encapsulation of microcapsules can be achieved. Multi-layer microencapsulation technology can provide progressive release control through different levels of encapsulation materials, provide protection in the stomach, and gradually release probiotics in the small intestine, thereby improving their bioavailability.

[0037] Preferably, the microencapsulation process is controlled by the process parameters of the spray drying method as follows:

[0038] The spray temperature is 150-180℃;

[0039] Feed flow rate is 5-20mL / min;

[0040] Feed concentration is 10%-30%;

[0041] Drying time is 3-5 minutes.

[0042] Preferably, the microencapsulation process is carried out by an emulsion cross-linking method, and during the emulsification process, the concentration of the emulsifier is 1%-5%.

[0043] Preferably, the microencapsulated composition is dried by a combination of spray drying and freeze drying to ensure the stability of the final product, and the freeze drying temperature is controlled between -40°C and -20°C.

[0044] The present invention provides a multifunctional composite probiotic composition and a preparation process thereof. It has the following beneficial effects:

[0045] 1. The present invention adopts the technology of compound probiotic compatibility, and enhances the stability and functionality of intestinal microecology through the synergistic effect of different strains, thereby achieving the effect of improving the survival rate of probiotics in the gastrointestinal tract, promoting the colonization of beneficial bacteria, and enhancing the immune regulation ability. Compared with the technical solution of single strain probiotics in the prior art, it solves the problems of insufficient flora regulation ability, low survival rate, and limited stimulation to the host immune system.

[0046] 2. The present invention adopts microcapsule multi-layer embedding technology, combined with inner, middle and outer layer protection systems, so that probiotics can more effectively resist adverse environments such as gastric acid, bile and high temperature, thereby achieving the effects of improving the stability of probiotics, extending the storage period, and optimizing the release rate of probiotics. Compared with the single-layer embedding or non-embedding solutions in the prior art, it solves the problems of probiotics being easily destroyed by gastric acid, having a decreased survival rate during storage, and being largely inactivated before entering the intestine.

[0047] 3. The present invention adopts a synergistic combination strategy of probiotics and prebiotics, and utilizes plant extracts and dietary fiber to promote the proliferation and metabolic activity of probiotics, thereby achieving the effects of increasing the growth rate of probiotics, promoting the balance of intestinal flora, and enhancing the colonization ability of probiotics. Compared with the technical solution of only using probiotics in the prior art, it solves the shortcomings of limited growth of probiotics, low colonization rate, and limited intestinal regulation ability, making its effect more lasting and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the process flow of the present invention. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Please refer to the attached Figure 1 ,

[0051] Embodiment 1:

[0052] Formula and ingredients:

[0053] Probiotics: 45% (composite ratio: lactic acid bacteria 18%, bifidobacterium 55%, Lactobacillus acidophilus 27%);

[0054] Microcapsule embedding material: 30%; contains:

[0055] Inner layer embedding material (sodium alginate 8%, calcium lactate 2%, gelatin 4%);

[0056] Middle layer embedding material (chitosan 5%, polylactic acid 6%);

[0057] Outer layer embedding material (polylactic acid 3%, polyvinyl alcohol 2%);

[0058] Prebiotics: 20%; Contains: Plant extracts: 12% (wolfberry extract 7%, curcumin 3%, grape seed extract 2%); Dietary fiber: 8% (inulin);

[0059] Excipients: 5% (natural sweetener erythritol).

[0060] Process steps:

[0061] Probiotics are cultured to active standards, ensuring a survival rate of over 95%.

[0062] Microencapsulation was performed using emulsion crosslinking and spray drying.

[0063] The spray temperature was set at 160°C, the feed flow rate was controlled at 10 mL / min, and the feed concentration was 25%.

[0064] Thoroughly mix the plant extracts, dietary fiber and microencapsulated probiotics to ensure even distribution.

[0065] The terminal treatment is freeze-dried (-30°C) to maximize the preservation of activity.

[0066] Packaged into powdered composition to ensure product stability.

[0067] Embodiment 2:

[0068] Formula and ingredients:

[0069] Probiotics: 48% (composite ratio: lactic acid bacteria 28%, bifidobacterium 40%, Lactobacillus acidophilus 32%);

[0070] Microcapsule embedding material: 32%; contains:

[0071] Inner layer embedding material (sodium alginate 7%, calcium lactate 3%, gelatin 4%);

[0072] Middle layer embedding material (chitosan 5%, polylactic acid 7%);

[0073] Outer layer embedding material (polylactic acid 3%, polyvinyl alcohol 3%);

[0074] Prebiotics: 16%; Contains: Plant extracts: 10% (wolfberry extract 5%, curcumin 3%, grape seed extract 2%); Dietary fiber: 6% (inulin);

[0075] Excipients: 4% (natural sweetener monk fruit extract).

[0076] Process steps:

[0077] The probiotics are cultured to a stable active state, with a bacterial viability of not less than 90%.

[0078] Microencapsulation is carried out by combining spray drying with emulsion cross-linking:

[0079] The spray temperature was 170°C, the feed flow rate was 12 mL / min, and the feed concentration was 28%.

[0080] Add plant extracts and dietary fiber and mix well.

[0081] Spray drying (180°C) was used for secondary drying with a feed flow rate of 15 mL / min and a drying time of 4 minutes to improve powder uniformity.

[0082] Molding after packaging ensures the long-term survival of probiotics.

[0083] Embodiment three:

[0084] Formula and ingredients:

[0085] Probiotics: 42% (composite ratio: lactic acid bacteria 40%, bifidobacterium 35%, Lactobacillus acidophilus 25%);

[0086] Microcapsule embedding material: 35%; contains:

[0087] Inner layer embedding material (sodium alginate 8%, calcium lactate 3%, gelatin 5%);

[0088] middle layer embedding material (chitosan 6%, polylactic acid 7%);

[0089] Outer layer embedding material (polylactic acid 3%, polyvinyl alcohol 3%);

[0090] Prebiotics: 18%: Contains: Plant extracts: 12% (wolfberry extract 6%, curcumin 3%, grape seed extract 3%); Dietary fiber: 6% (inulin);

[0091] Excipients: 5% (natural sweetener erythritol).

[0092] Process steps:

[0093] Probiotics are cultured in a temperature-controlled environment and separated after reaching activity standards.

[0094] Microencapsulation of probiotics using spray drying:

[0095] The spray temperature was 175°C, the feed flow rate was 10 mL / min, and the feed concentration was 22%.

[0096] Mix plant extracts, dietary fiber and microencapsulated probiotics.

[0097] Freeze drying (-40°C) reduces water activity and improves stability.

[0098] The powder composition is packaged.

[0099] Embodiment 4:

[0100] Formula and ingredients:

[0101] Probiotics: 46% (composite ratio: lactic acid bacteria 25%, bifidobacterium 50%, Lactobacillus acidophilus 25%);

[0102] Microcapsule embedding material: 33%; contains:

[0103] Inner layer embedding material (sodium alginate 7%, calcium lactate 2%, gelatin 4%);

[0104] Middle layer embedding material (chitosan 5%, polylactic acid 6%);

[0105] Outer layer embedding material (polylactic acid 4%, polyvinyl alcohol 5%);

[0106] Prebiotics 16%; Contains: Plant extracts: 9%; (wolfberry extract 4%, curcumin 3%, grape seed extract 2%); Dietary fiber: 7% (inulin);

[0107] Excipients: 5% (natural sweetener monk fruit extract).

[0108] Process steps:

[0109] Probiotics are cultured to a stable state to ensure that their metabolic activity meets the requirements.

[0110] Microencapsulation is carried out by combining emulsion cross-linking and spray drying:

[0111] The spray temperature was 155°C, the feed flow rate was 8 mL / min, and the drying time was 4 minutes.

[0112] The compound formula is mixed so that all ingredients are evenly distributed to form a stable system.

[0113] The spray drying and freeze drying technology is combined (freeze drying temperature -25°C) to ensure long-term stability.

[0114] The mixture is packaged and finally made into a composition.

[0115] Comparative Example 1:

[0116] The method is basically the same as Example 1, except that only 45% of a single probiotic (100% of lactic acid bacteria) is used, and the combination of bifidobacterium and lactobacillus acidophilus is cancelled.

[0117] Comparative Example 2:

[0118] It is basically the same as Example 2, except that hydrogel embedding technology is used, 5% sodium alginate and 3% chitosan are used for embedding, instead of spray drying technology, and middle and outer layer embedding is not performed.

[0119] Comparative Example 3:

[0120] It is basically the same as Example 3, except that the microcapsule embedding process is not used, the probiotics are directly added to the powder formula without embedding materials, and only the freeze-dried probiotics are mixed with other ingredients.

[0121] Comparative Example 4:

[0122] It is basically the same as Example 2, except that no prebiotics (plant extracts and dietary fiber) are added, and only probiotics and microcapsule embedding materials are contained.

[0123] Experiment 1: Comparative test of compound probiotics and single probiotics

[0124] Experimental steps:

[0125] Probiotic survival rate test: Take 10 g of the probiotic products of Example 1 and Comparative Example 1, respectively, and dissolve them in 100 mL of artificial gastric juice (pH 2.0, 37° C.).

[0126] Samples were taken at 0 min, 30 min, 60 min, and 120 min, and 1 mL of culture medium was taken each time. After appropriate dilution with saline, the samples were spread on MRS agar plates.

[0127] After incubation at 37°C for 48 hours, the colony forming units (CFU / mL) were counted and the survival rate was calculated.

[0128] Immune enhancement ability test (mouse experiment): 6-week-old BALB / c mice were selected and divided into Example 1 group, Comparative Example 1 group and blank control group, with 10 mice in each group.

[0129] The daily dose was 1×10 9 CFU / piece.

[0130] On the 29th day, the tail vein blood of mice was collected, the serum was separated, and the concentrations of IL-10 and TNF-α were determined using ELISA kits.

[0131] Intestinal flora regulation ability test: SD rats were selected and divided into Example 1 group, Comparative Example 1 group and blank control group, 8 rats in each group, and 1×10 9 CFU / bird / day.

[0132] Feces were collected on the 29th day, intestinal flora DNA was extracted, and the relative abundance of the flora was analyzed using 16SrRNA high-throughput sequencing.

[0133] The relative abundance changes of Bifidobacterium, Lactobacillus, Escherichia coli, and Bacteroides were statistically analyzed.

[0134] Probiotic survival rate, immune factor levels and intestinal flora abundance

[0135]

[0136] Experimental summary:

[0137] The probiotic combination exhibits higher acid resistance. Experimental data show that in the gastric acid environment, the survival rate of the probiotics in Example 1 is significantly better than that in Comparative Example 1. This may be attributed to the synergistic effect of different strains. Some strains can provide a protective barrier and reduce acid damage. Single lactic acid bacteria lack this ability to support each other, resulting in a lower survival rate. In addition, fine-tuning of the microecological environment may also be one of the influencing factors. Interactions between strains may change the cell wall structure, making it more resistant.

[0138] The immune enhancement effect also showed differences. The IL-10 level of mice in Example 1 was generally higher, and the TNF-α level was also increased, which shows that the combination of multiple strains may play a more complex regulatory function in the immune system. Different strains may have different stimulating effects on immune cells. The synergistic effect may amplify the immune regulatory effect, while the immune response of a single strain is more limited and fails to provide the same degree of regulation. These differences may be used as the basis for developing personalized intestinal health products in the future.

[0139] The changes in the flora structure are more intuitive. The probiotic combination significantly increased the relative abundance of bifidobacteria and lactic acid bacteria, while inhibiting Escherichia coli and Bacteroides. The interaction between bacterial species may promote the accumulation of specific metabolites, such as short-chain fatty acids, which may affect the competitive relationship between microorganisms. In the single strain group, the flora regulation ability is weak and the balance ability of the intestinal microecology is relatively poor. The experimental results reveal that the combination of bacterial species may improve the level of intestinal health through competitive exclusion or metabolic regulation.

[0140] Experiment 2: Comparison of hydrogel encapsulation and spray drying encapsulation

[0141] Purpose:

[0142] This experiment aims to compare the effects of hydrogel encapsulation technology and spray drying encapsulation technology on the survival rate, gastric acid resistance and dissolution characteristics of probiotics. The focus is on analyzing the differences in the probiotic protection effects of the two technologies and exploring their applicability in functional foods.

[0143] Experimental methods:

[0144] Materials preparation:

[0145] Embodiment 2;

[0146] Comparative Example 2;

[0147] Simulated gastrointestinal fluid: pH 2.5 artificial gastric fluid, pH 7.4 artificial intestinal fluid.

[0148] Experimental steps:

[0149] (1) Preparation of probiotic embedding: Probiotics are cultured to 10 9 CFU / mL.

[0150] Spray drying embedding (Example 2):

[0151] The probiotics are suspended in a sodium alginate-calcium lactate solution and dispersed by ultrasonication.

[0152] The encapsulation was carried out by spray drying to form microcapsule powder.

[0153] Hydrogel embedding (Comparative Example 2):

[0154] Probiotics were suspended in sodium alginate solution and added dropwise to CaCl2 solution to form gel microspheres.

[0155] The outer layer was modified with chitosan and was not spray dried.

[0156] (2) Survival rate test in gastrointestinal environment: Take an equal amount of encapsulated probiotic samples, add pH 2.5 gastric juice, incubate at 37°C for 2 hours, and measure the survival rate.

[0157] The cells were transferred to intestinal fluid at pH 7.4 and incubated for another 4 hours to measure the probiotic release rate.

[0158] (3) Dissolution rate test: The probiotic-encapsulated samples were placed in simulated gastrointestinal fluid, and the probiotic release rates at different time points (10, 30, 60, and 120 minutes) were measured.

[0159] (4) Storage stability: The probiotic-encapsulated samples were stored at 25°C, 30°C, and 40°C, and the survival rate was measured weekly for 8 weeks.

[0160] Comparison of gastric acid resistance and release characteristics of probiotics

[0161]

[0162] Experimental summary:

[0163] Compared with spray drying encapsulation, the hydrogel encapsulation structure forms microspheres with higher internal moisture and looser structure, which increases gastric acid permeability and higher probiotic damage rate. Spray drying encapsulation forms a dense structure of microcapsule wall material by evaporating water instantly, effectively reducing the penetration rate of acidic liquids, thereby improving the gastric acid resistance of probiotics.

[0164] In simulated intestinal fluid, the probiotics encapsulated in hydrogel are released faster, indicating that the encapsulated structure disintegrates rapidly after entering the alkaline environment, which results in the release of a large number of probiotics in a short period of time, which may affect the sustained effect of the probiotics. The release curve of spray-dried encapsulation is flatter, and the release time of the probiotics is extended, which enables it to have a longer action time in the intestine, which is beneficial to the survival and colonization of the probiotics.

[0165] The results of the storage stability experiment show that the spray-dried encapsulated probiotics can still maintain a good survival rate at higher temperatures, while the hydrogel encapsulation is susceptible to microbial contamination due to its high water content, resulting in a significant decrease in survival rate. Therefore, from a practical application perspective, spray-drying encapsulation has more advantages in the long-term storage and transportation of probiotic foods.

[0166] Experiment 3: Comparison of non-encapsulated probiotics and microcapsule-encapsulated probiotics

[0167] Purpose:

[0168] To explore the differences in survival rate, gastrointestinal environment tolerance and dissolution rate between unencapsulated probiotics and microcapsule-encapsulated probiotics, and to evaluate the effect of encapsulation on the stability and functionality of probiotics.

[0169] Experimental methods:

[0170] Materials preparation:

[0171] Embodiment 3;

[0172] Comparative Example 3;

[0173] Culture medium: MRS medium;

[0174] Simulated gastrointestinal fluid: pH 2.5 artificial gastric fluid, pH 7.4 artificial intestinal fluid.

[0175] Experimental steps:

[0176] (1) Preparation of probiotic samples:

[0177] Microcapsule embedding (Example 3):

[0178] Probiotics cultured to 10 9 CFU / mL, embedded in sodium alginate-calcium lactate and then spray dried (150°C).

[0179] No embedding (Comparative Example 3):

[0180] Probiotics cultured to 10 9 CFU / mL, directly freeze-dried, no embedding.

[0181] (2) Survival rate test in gastrointestinal environment: Take an equal amount of probiotic sample, add pH 2.5 gastric juice, incubate at 37°C for 2 hours, and measure the survival rate.

[0182] The intestinal fluid with pH 7.4 was transferred and the incubation was continued for 4 hours, and the survival rate was measured.

[0183] (3) Dissolution rate test: Probiotic samples were placed in simulated gastrointestinal fluid, and the probiotic release rate was measured every 30 minutes.

[0184] (4) Storage stability: Probiotic samples were stored at 25°C, 30°C, and 40°C, and the survival rate was measured every week for 8 weeks.

[0185] Comparison of survival rate and dissolution rate of encapsulated and unencapsulated probiotics

[0186]

[0187] Experimental summary:

[0188] The survival rate of unencapsulated probiotics drops sharply in the gastric fluid environment. Under acidic conditions, the exposed probiotic cell wall is directly exposed to the low pH environment, resulting in protein denaturation, DNA damage, and rapid cell death. In contrast, microcapsule encapsulation forms a physical barrier that effectively isolates the erosion of gastric acid, allowing probiotics to maintain a high survival rate in the stomach and successfully enter the intestine to exert their effects.

[0189] The comparison of dissolution rates also clearly demonstrates the value of encapsulation. Unencapsulated probiotics are almost completely released in a short period of time, and a large number of active bacteria have been lost in the gastric fluid environment. Microcapsule encapsulation gradually releases probiotics in the intestinal fluid environment through a sustained release mechanism, which improves the intestinal colonization ability of probiotics. The uniform release pattern makes the probiotics last longer and is more conducive to the regulation of intestinal microecological balance.

[0190] The results of the storage stability experiment further prove the advantage of encapsulation. Non-encapsulated probiotics are extremely sensitive to environmental humidity and temperature, and their survival rate is extremely low under high temperature conditions. However, the encapsulated probiotics have a relatively stable survival rate because the microcapsules form a protective layer, which reduces the impact of oxidation, humidity and temperature fluctuations. This shows that when used in commercial products, microcapsule-encapsulated probiotics have a longer shelf life and higher functional stability.

[0191] Experiment 4: Comparison of adding prebiotics and not adding prebiotics

[0192] Experimental steps:

[0193] Probiotic proliferation ability test: Take the probiotic products of Example 2 and Comparative Example 2, and dissolve 10 g of each in 100 mL of MRS culture medium (37° C., anaerobic environment).

[0194] Sampling was performed at different time points (0 h, 6 h, 12 h, 24 h, 48 h), and 1 mL of bacterial solution was taken each time.

[0195] The turbidity of the bacterial solution was measured by a spectrophotometer (OD600), and the number of viable bacteria was further determined by the plate count method (CFU / mL).

[0196] Probiotic intestinal colonization ability test: 8-week-old SD rats were selected and randomly divided into groups (Example 2, Comparative Example 2, blank control), with 8 rats in each group.

[0197] Probiotics (1×10 9 CFU / bird / day).

[0198] On the 29th day, feces were collected, intestinal flora DNA was extracted, and the colonization of lactic acid bacteria and bifidobacteria was determined by qPCR.

[0199] Test of intestinal flora regulation ability: Fecal samples of experimental rats were selected, microbial DNA was extracted, and 16SrRNA sequencing was performed to analyze the flora structure.

[0200] The relative abundance changes of beneficial bacteria (lactic acid bacteria, bifidobacterium) and harmful bacteria (Escherichia coli, Bacteroides) were statistically analyzed.

[0201] Probiotic proliferation ability, intestinal colonization rate and relative abundance of bacterial flora

[0202]

[0203]

[0204] Experimental summary:

[0205] The addition of prebiotics significantly promoted the proliferation of probiotics. The experimental data clearly showed that after 12 hours of culture, the colony count of Example 4 far exceeded that of Comparative Example 4, while the colony count of Comparative Example 4 grew slowly and did not reach the level of Example 4 after 48 hours. This shows that prebiotics are not only a source of energy, but also likely to accelerate the expansion of the flora by regulating osmotic pressure and improving the growth environment. In particular, in the absence of a fermentable carbon source, the growth of probiotics will be limited, and the presence of prebiotics seems to have changed this. The interaction between bacterial species may be more complex, and the generation of short-chain fatty acids and the transmission of metabolic signals may be key factors in improving proliferation capacity.

[0206] The comparison of colonization ability is more significant. The colonization rates of lactic acid bacteria and bifidobacteria in Example 4 in the rat intestine are much higher than those in Comparative Example 4, which shows that prebiotics not only provide a basis for growth, but also may promote the expression of colonization-related proteins. For example, some prebiotics may stimulate the synthesis of adhesion proteins, making it easier for probiotics to adhere to intestinal epithelial cells. At the same time, the metabolites of prebiotics, such as acetic acid and lactic acid, may also inhibit harmful flora and create a more stable ecological niche for probiotics. In the absence of prebiotics, probiotics are more easily excreted and cannot form a stable flora in the intestine. This phenomenon may involve host-microorganism interactions, and perhaps more in-depth mechanism research is needed.

[0207] The changes in the flora structure are also very intuitive. The abundance of Escherichia coli and Bacteroides in Example 4 group is low, while the proportion of Bifidobacterium and Lactobacillus is greatly increased. Prebiotics may play an important role in this process. Through selective fermentation, prebiotics may promote the growth of beneficial bacteria while inhibiting the competitiveness of harmful bacteria. In addition, prebiotic metabolites, such as butyrate, may affect intestinal pH and inhibit the growth of specific pathogenic bacteria. This competitive advantage may be more obvious after long-term use, and the comparative example 4 is limited in its flora regulation ability due to the lack of prebiotics, and the same effect cannot be achieved. This also shows again that simple probiotic supplementation is not enough, and the addition of prebiotics is the key factor in truly stabilizing and balancing the flora.

[0208] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multifunctional composite probiotic composition, characterized in that: The following raw materials are included in percentage by mass: Probiotics: 40%-50%; Microcapsule embedding material: 30%-40%; Prebiotics: 10%-25%; Excipients: 1%-5%.

2. A multifunctional composite probiotic composition according to claim 1, characterized in that: The microcapsule embedding material comprises: The inner layer embedding material includes sodium alginate, calcium lactate and gelatin, wherein the mass fraction of sodium alginate accounts for 5%-18% of the total mass, the mass fraction of calcium lactate accounts for 1%-4% of the total mass, and the mass fraction of gelatin accounts for 3%-8% of the total mass; The middle layer embedding material includes chitosan and polylactic acid, wherein the mass fraction of chitosan accounts for 2%-8% of the total mass, and the mass fraction of polylactic acid accounts for 3%-12% of the total mass; The outer layer embedding material comprises polylactic acid and polyvinyl alcohol, wherein the mass fraction of the polylactic acid accounts for 5%-12% of the total mass, and the mass fraction of the polyvinyl alcohol accounts for 1%-8% of the total mass.

3. A multifunctional composite probiotic composition according to claim 1, characterized in that: The probiotics are a composite ratio of lactic acid bacteria, bifidobacteria, and Lactobacillus acidophilus, wherein: Lactic acid bacteria account for 15%-45%; Bifidobacterium accounts for 35%-50%; Lactobacillus acidophilus accounts for 10%-35%.

4. The multifunctional composite probiotic composition according to claim 1, characterized in that: The prebiotics include plant extracts, and the plant extracts include: Wolfberry extract accounts for 5%-12% of the total mass; Curcumin accounts for 1%-4% of the total mass; Grape seed extract accounts for 2%-8% of the total mass.

5. The multifunctional composite probiotic composition according to claim 1, characterized in that: The prebiotics also include dietary fiber, which is inulin, accounting for 5%-10% of the total mass.

6. The multifunctional composite probiotic composition according to claim 1, characterized in that: The auxiliary materials include natural sweeteners, and the natural sweeteners are one or more of erythritol and monk fruit extract.

7. A preparation process of a multifunctional composite probiotic composition, used for preparing a multifunctional composite probiotic composition according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Cultivation and activity detection of probiotics; S2, microencapsulation process: preparing multilayer microcapsules by spray drying and emulsion cross-linking to encapsulate the probiotics; S3, mixing the microcapsules with prebiotics and auxiliary materials to obtain a multifunctional composite probiotic composition; S4, drying the obtained composition to obtain a final product.

8. The preparation process of a multifunctional composite probiotic composition according to claim 7, characterized in that: The microencapsulation process is controlled by the process parameters of the spray drying method as follows: The spray temperature is 150-180℃; Feed flow rate is 5-20mL / min; Feed concentration is 10%-30%; Drying time is 3-5 minutes.

9. The preparation process of a multifunctional composite probiotic composition according to claim 7, characterized in that: The microencapsulation process is carried out by an emulsion cross-linking method, and the concentration of the emulsifier in the emulsification process is 1%-5%.

10. The preparation process of a multifunctional composite probiotic composition according to claim 7, characterized in that: The microencapsulated composition is dried by a combination of spray drying and freeze drying to ensure the stability of the final product, and the freeze drying temperature is controlled between -40°C and -20°C.

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