Composite prebiotic embedded probiotic microcapsule as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food, and particularly discloses a compound prebiotics embedded probiotic microcapsule and a preparation method and application thereof.The green tea leaves serve as main raw materials, water extraction is conducted on the green tea leaves, meanwhile, ultrasonic-assisted treatment is adopted, active ingredients in the green tea leaves can be more effectively released, the extraction time is shortened, and the content of probiotics in the green tea leaves is increased; the extraction rate of active ingredients in the green tea leaves is improved; then, lactobacillus acidophilus is adopted to ferment the extract liquid, so that the green tea fermentation liquid contains more antioxidant active ingredients, combined polyphenol in the extract liquid can release micromolecular polyphenol in the fermentation process, the content of substances such as flavone and polyphenol in the green tea fermentation liquid is increased, and the antioxidant activity is further improved; and then the modified dietary fiber is used as a main wall material of the microcapsule to embed the green tea fermentation liquor and the probiotics, so that the stability of active substances and the probiotics in the green tea fermentation liquor is improved, and the green tea fermentation liquor can play a role in protection and slow release in the simulated digestion process.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and particularly relates to a composite prebiotic-embedded probiotic microcapsule, its preparation method and application. Background Art

[0002] Green tea is an important part of traditional Chinese tea culture. As one of the main tea categories, its unique manufacturing process and rich nutritional components have earned it a high reputation worldwide. The tea polyphenols and catechins rich in green tea have been widely studied and shown significant effects in aspects such as antioxidant, anti-aging, antibacterial, lipid-lowering, and promoting metabolism. As a powerful antioxidant, tea polyphenols can effectively neutralize free radicals and slow down the process of cell aging, thus contributing to enhancing the body's immunity.

[0003] With the increasing emphasis on healthy diets, the application of green tea extract has gradually expanded to multiple fields such as food, health products, medicine, and daily chemical engineering. Especially in the development of functional foods, green tea extract has been widely used due to its rich bioactive components. However, due to the extreme sensitivity of these active substances to environmental factors such as temperature, light, pH value, and oxygen, many challenges are faced in practical applications. For example, green tea extract may produce unpleasant tastes during certain food processing processes, affecting the taste and quality of the final product.

[0004] To overcome these problems, researchers have been continuously exploring new technical means, and among them, microencapsulation technology has become an effective solution. By embedding active ingredients in tiny capsules, microencapsulation technology can effectively protect them from the external environment, thereby maintaining their stability and bioactivity. This technology can not only prevent the degradation of active ingredients but also control their release in foods, thus improving the overall quality of products. During the microencapsulation process, the selection of embedding materials is crucial. Commonly used embedding materials include natural polymer substances such as gelatin, gum arabic, and starch. They not only have good biocompatibility but also can provide sufficient protection. In addition, the optimization of embedding techniques, such as adjusting the embedding ratio, temperature, and time, will also directly affect the stability of microcapsules and the release efficiency of active ingredients.

[0005] Chinese patent document CN202310845151.6 discloses a microcapsule containing green tea extract, its preparation method and application. By compounding green tea extract with other plant extracts to prepare a whitening composition, and then preparing microcapsules from the whitening composition, and embedding green tea extract, etc. through spray drying embedding technology, it can effectively control the release of its active substances, improve its bioavailability and stability, and ensure the product quality. However, the stability of the microcapsules still needs to be improved. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a composite prebiotic-embedded probiotic microcapsule, its preparation method and application.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a preparation method of a composite prebiotic-embedded probiotic microcapsule, comprising the following steps: S1. Crush and sieve green tea leaves, add them to deionized water, then add an amino acid surfactant, and perform ultrasonic-assisted extraction. After the extraction is completed, filter to obtain an extract and filter residue; S2. Add the extract to sterilized coconut water, then inoculate Lactobacillus acidophilus, and perform fermentation culture. After the fermentation culture is completed, filter and collect the filtrate to obtain a green tea fermentation broth; S3. Add the filter residue to a hydrogen peroxide solution for treatment. After the treatment is completed, perform suction filtration, washing, drying, and grinding and sieving to obtain modified dietary fiber; S4. Add inulin, sodium alginate, and modified dietary fiber to deionized water, stir evenly, then add the green tea fermentation broth and Lactobacillus rhamnosus powder to it, mix evenly, then add a zinc lactate solution, and continue to stir to obtain a colloidal solution. After freeze-drying, grind it into powder to obtain the composite prebiotic-embedded probiotic microcapsule.

[0008] Preferably, in step S1, the mass ratio of green tea leaves, deionized water, and amino acid surfactant is 15 - 25:300 - 400:0.5 - 1.

[0009] Preferably, in step S1, the ultrasonic power is 300 - 600w, the extraction temperature is 70 - 80°C, and the extraction time is 10 - 20min.

[0010] Preferably, in step S2, based on the volume of sterilized coconut water, the addition amount of the extract is 20 - 30%, and the inoculation amount of Lactobacillus acidophilus is 1 - 3%.

[0011] Preferably, in step S2, during the fermentation culture, the fermentation temperature is 28 - 32°C, the fermentation time is 48 - 72h, and the stirring speed is 100 - 200r / min.

[0012] Preferably, in step S3, the mass ratio of the filter residue to the hydrogen peroxide solution is 10 - 20:100, and the mass fraction of the hydrogen peroxide solution is 10 - 20%.

[0013] Preferably, in step S4, the mass ratio of inulin, sodium alginate, modified dietary fiber, deionized water, green tea fermentation broth, Lactobacillus rhamnosus powder, and zinc lactate solution is 0.4 - 0.8:1 - 1.5:2 - 3:10 - 20:10 - 15:0.3 - 0.6:10 - 20.

[0014] Preferably, in step S4, the mass fraction of the zinc lactate solution is 3-6%.

[0015] In a second aspect, the present invention provides a composite prebiotic-embedded probiotic microcapsule prepared by the above preparation method.

[0016] In a third aspect, the present invention also provides the application of the above composite prebiotic-embedded probiotic microcapsule in the preparation of solid beverage products.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses green tea leaves as the main raw material and performs water extraction on them. During the water extraction process, an amino acid surfactant is added, which can effectively improve the cell wall permeability and enable the effective dissolution of flavonoid compounds and polyphenol compounds inside the cells. At the same time, ultrasonic-assisted treatment is adopted. Ultrasonic waves can generate tiny bubbles, and through the cavitation effect of ultrasonic waves, the cell wall is broken, thereby more effectively releasing the active ingredients in green tea leaves, shortening the extraction time, and improving the extraction rate of the active ingredients in green tea leaves.

[0018] (2) The present invention uses Lactobacillus acidophilus to ferment the leaching solution, and at the same time supplements coconut water as the fermentation substrate. Coconut water contains a large amount of nutrients, reducing the utilization of effective substances in the leaching solution by Lactobacillus acidophilus. As a result, the green tea fermentation broth contains more antioxidant active ingredients, and during the fermentation process, the bound polyphenols in the leaching solution can be released to form small molecule polyphenols, increasing the contents of substances such as flavonoids and polyphenols in the green tea fermentation broth, thereby further improving the antioxidant activity. At the same time, Lactobacillus acidophilus can ferment to produce some bioactive peptide substances, which act together with prebiotics such as inulin and sodium alginate to promote the growth and reproduction of beneficial bacteria such as Lactobacillus, Bifidobacterium, and Acetobacter in the intestine. Lactobacillus acidophilus can also be used as a probiotic species and can play a role in regulating gastrointestinal health.

[0019] (3) The present invention uses modified dietary fiber as the main wall material of the microcapsule. First, hydrogen peroxide is used to perform surface treatment on the filter residue, aiming to improve the activity of the surface groups of the filter residue, increase the specific surface area of the filter residue, and improve the binding force between the filter residue and inulin and sodium alginate. Subsequently, a zinc lactate solution is added. Zinc ions form a gel through chelation with the hydroxyl groups in inulin, sodium alginate, and modified dietary fiber, encapsulating the green tea fermentation broth and Lactobacillus rhamnosus, improving the stability of the active substances in the green tea fermentation broth and Lactobacillus rhamnosus, and enabling them to play a role in protection and slow release during the simulated digestion process. At the same time, zinc also has certain antioxidant properties, which can protect intestinal cells from oxidative damage and, acting together with the active ingredients in the green tea fermentation broth, further improves the antioxidant effect of the composite prebiotic-embedded probiotic microcapsule.

[0020] (4)The composite prebiotic-embedded probiotic microcapsules provided by the present invention form a protective barrier on the outer layer of the probiotics, helping the probiotics resist the destruction of digestive juices such as gastric acid and bile, and ensuring that more live bacteria reach the intestine. At the same time, the prebiotics provide nutrition for the probiotics after fermentation in the intestine, further enhancing their colonization and reproduction abilities. This synergistic effect not only improves the survival rate of the probiotics, but also can more effectively regulate the balance of the intestinal flora, improve digestive function, enhance immunity, and relieve intestinal inflammation and other problems. The composite prebiotic-embedded probiotic microcapsules provided by the present invention have broad application prospects in functional foods and health products. Description of the Drawings

[0021] Figure 1 It is a comparative diagram of the effects of the DPPH free radical scavenging rate of microcapsules in different groups in vitro; Figure 2 It is a comparative diagram of the effects of the storage stability of microcapsules in different groups; Figure 3 It is a comparative diagram of the effects of the polyphenol extraction rate and flavonoid extraction rate of different groups. Detailed Embodiments

[0022] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0023] It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are all purchased through commercial channels.

[0024] A preparation method of composite prebiotic-embedded probiotic microcapsules includes the following steps: S1. Prepare the leaching solution and filter residue Crush and sieve green tea leaves, then add them to deionized water, and then add an amino acid surfactant, and perform ultrasonic-assisted extraction. After the extraction is completed, filter to obtain the leaching solution and filter residue.

[0025] In this step, first crush the green tea leaves and sieve them through a 40-80 mesh sieve for treatment.

[0026] In this step, the mass ratio of green tea leaves, deionized water and amino acid surfactant is 15-25:300-400:0.5-1. In some embodiments of the present invention, for example, 15:300:0.5, 15:400:1, 20:300:0.5, 20:300:1, 25:300:0.5, 25:400:1 can be selected, but it is not limited to the listed values, and other unlisted values within the numerical range are equally applicable.

[0027] Among them, the amino acid surfactant can be selected from proline or glycine.

[0028] In this step, the ultrasonic power is 300 - 600 w. For example, 300 w, 350 w, 400 w, 450 w, 500 w, 550 w, 600 w can be selected; the extraction temperature is 70 - 80 °C. For example, 70 °C, 72 °C, 74 °C, 75 °C, 76 °C, 78 °C, 80 °C can be selected; the extraction time is 10 - 20 min. For example, 10 min, 12 min, 14 min, 15 min, 16 min, 18 min, 20 min can be selected; however, it is not limited to the listed values, and other unlisted values within the numerical range are equally applicable.

[0029] In this step, taking green tea leaves as the main raw material, performing water extraction on them, and adding an amino acid surfactant during the water extraction process can effectively improve the permeability of the cell wall, enabling the effective dissolution of flavonoid compounds and polyphenol compounds inside the cells; at the same time, ultrasonic-assisted treatment is adopted. Ultrasonic waves can generate tiny bubbles, and through the cavitation effect of ultrasonic waves, the cell wall is broken, thereby more effectively releasing the active ingredients in green tea leaves, shortening the extraction time, and increasing the extraction rate of the active ingredients in green tea leaves.

[0030] S2. Prepare green tea fermentation broth Add the extraction solution to sterilized coconut water, then inoculate Lactobacillus acidophilus, and perform fermentation culture. After the fermentation culture is completed, filter and collect the filtrate to obtain green tea fermentation broth.

[0031] In this step, based on the volume of sterilized coconut water, the addition amount of the extraction solution is 20 - 30% (v / v), and the inoculation amount of Lactobacillus acidophilus is 1 - 3% (v / v). Among them, the viable count of Lactobacillus acidophilus ≥ 1.0×10 11 cfu / mL.

[0032] In this step, the fermentation temperature is 28 - 32 °C. For example, 28 °C, 30 °C, 32 °C can be selected; the fermentation time is 48 - 72 h. For example, 48 h, 54 h, 60 h, 66 h, 72 h can be selected; the stirring speed is 100 - 200 r / min. For example, 100 r / min, 150 r / min, 200 r / min can be selected; however, it is not limited to the listed values, and other unlisted values within the numerical range are equally applicable.

[0033] In this step, Lactobacillus acidophilus is used to ferment the leaching solution, and coconut water is used as the fermentation substrate. Coconut water contains a large amount of nutrients, which reduces the utilization of effective substances in the leaching solution by Lactobacillus acidophilus, so that the green tea fermentation liquor contains more antioxidant active ingredients. And during the fermentation process, the bound polyphenols in the leaching solution can be released into small molecule polyphenols, increasing the contents of substances such as flavonoids and polyphenols in the green tea fermentation liquor, thereby further enhancing the antioxidant activity. At the same time, Lactobacillus acidophilus can ferment to produce some bioactive peptide substances, which act together with prebiotics such as inulin and sodium alginate to promote the growth and reproduction of beneficial bacteria such as Lactobacillus, Bifidobacterium and Acetobacterium in the intestine. Lactobacillus acidophilus can also be used as a probiotic species, which can play a role in regulating gastrointestinal health.

[0034] S3. Preparation of modified dietary fiber The filter residue is added to the hydrogen peroxide solution for treatment. After the treatment is completed, suction filtration, washing, drying and grinding through a sieve are carried out to obtain the modified dietary fiber.

[0035] In this step, the mass ratio of the filter residue to the hydrogen peroxide solution is 10-20:100, for example, 10:100, 12:100, 15:100, 18:100, 20:100 can be selected; among them, the mass fraction of the hydrogen peroxide solution is 10-20%, for example, 10%, 12%, 15%, 18%, 20% can be selected; but it is not limited to the listed values, and other unlisted values within the numerical range are equally applicable.

[0036] In this step, by using hydrogen peroxide to perform surface treatment on the filter residue, the purpose is to improve the activity of the surface groups of the filter residue and increase the specific surface area of the filter residue.

[0037] S4. Preparation of composite prebiotic-embedded probiotic microcapsules Inulin, sodium alginate and modified dietary fiber are added to deionized water and stirred evenly. Then, green tea fermentation liquor and Lactobacillus rhamnosus powder are added thereto and mixed evenly. Subsequently, zinc lactate solution is added and stirring is continued to obtain a colloidal solution. After freeze-drying and grinding into powder, the composite prebiotic-embedded probiotic microcapsules are obtained.

[0038] In this step, the mass ratio of inulin, sodium alginate, modified dietary fiber, deionized water, green tea fermentation liquor, Lactobacillus rhamnosus powder and zinc lactate solution is 0.4-0.8:1-1.5:2-3:10-20:10-15:0.3-0.6:10-20. Among them, the mass fraction of the zinc lactate solution is 3-6%, for example, 3%, 4%, 5%, 6% can be selected.

[0039] In this step, zinc ions in the zinc lactate solution form a gel with the hydroxyl groups in inulin, sodium alginate, and modified dietary fiber through chelation, encapsulating the green tea fermentation broth, improving the stability of the active substances and Lactobacillus acidophilus in the green tea fermentation broth, and enabling it to play a role in protection and slow release during the simulated digestion process; at the same time, zinc also has certain antioxidant properties, which can protect intestinal cells from oxidative damage and, in conjunction with the active ingredients in the green tea fermentation broth, further enhance the antioxidant effect of the composite prebiotic-encapsulated probiotic microcapsules.

[0040] For the composite prebiotic-encapsulated probiotic microcapsules provided by the present invention, the prebiotic forms a protective barrier on the outer layer of the probiotic, helping the probiotic resist the destruction of digestive juices such as gastric acid and bile, ensuring that more live bacteria reach the intestine; at the same time, the prebiotic provides nutrients for the probiotic after fermentation in the intestine, further enhancing its colonization and reproduction ability. This synergistic effect not only improves the survival rate of the probiotic but also more effectively regulates the balance of the intestinal flora, improves digestive function, enhances immunity, and alleviates problems such as intestinal inflammation.

[0041] The following further illustrates the present invention through specific examples. The green tea leaves provided by the present invention are produced in Enshi City, Hubei Province; the CAS number of Lactobacillus acidophilus is 308084-36-8, purchased from Shanghai Ruiyingying Biotechnology Co., Ltd., and the viable count ≥ 1.0×10 11 cfu / mL; the Lactobacillus rhamnosus GG powder is purchased from Shanghai Ruiyingying Biotechnology Co., Ltd., and the viable count ≥ 1.0×10 10 cfu / g.

[0042] Example 1 A preparation method of a composite prebiotic-encapsulated probiotic microcapsule, comprising the following steps: S1. Crush 15 g of green tea leaves, pass through an 80-mesh sieve, add them to 300 g of deionized water, then add 0.5 g of glycine, and perform ultrasonic-assisted extraction. The ultrasonic power is 300 w, the extraction temperature is 80 °C, and the extraction time is 10 min. After the extraction is completed, filter to obtain an extract and filter residue; S2. Add the extract to sterilized coconut water, then inoculate Lactobacillus acidophilus. Based on the volume of the sterilized coconut water, the addition amount of the extract is 20%, and the inoculation amount of Lactobacillus acidophilus is 1%. Ferment and culture at 30 °C for 60 h, and the stirring speed is 150 r / min. After the fermentation and culture are completed, filter and collect the filtrate to obtain the green tea fermentation broth; S3. Add 10 g of the filter residue to 100 g of a 10 wt% hydrogen peroxide solution for treatment for 3 h. After the treatment is completed, perform suction filtration, washing, drying, and grinding through a 400-mesh sieve to obtain the modified dietary fiber; S4. Add 0.4 g of inulin, 1 g of sodium alginate, and 2 g of modified dietary fiber to 10 g of deionized water, stir evenly, then add 10 g of green tea fermentation broth and 0.5 g of Lactobacillus rhamnosus powder, mix well, and then add 10 g of 3 wt% zinc lactate solution, continue to stir for 2 h to obtain a colloidal solution, which is ground into powder after freeze-drying to obtain the composite prebiotic-embedded probiotic microcapsules.

[0043] Example 2 A preparation method of composite prebiotic-embedded probiotic microcapsules includes the following steps: S1. Crush 25 g of green tea leaves, pass through an 80-mesh sieve, add them to 400 g of deionized water, then add 1 g of glycine, carry out ultrasonic-assisted extraction with an ultrasonic power of 400 w, an extraction temperature of 70 °C, and an extraction time of 20 min. After the extraction is completed, filter to obtain the extract and the filter residue; S2. Add the extract to sterilized coconut water, then inoculate Lactobacillus acidophilus. Based on the volume of sterilized coconut water, the addition amount of the extract is 30%, and the inoculation amount of Lactobacillus acidophilus is 3%. Ferment and culture at 30 °C for 60 h with a stirring speed of 150 r / min. After the fermentation and culture are completed, filter and collect the filtrate to obtain the green tea fermentation broth; S3. Add 20 g of the filter residue to 100 g of 20 wt% hydrogen peroxide solution for treatment for 3 h. After the treatment is completed, carry out suction filtration, washing, drying, and grinding through a 400-mesh sieve to obtain the modified dietary fiber; S4. Add 0.8 g of inulin, 1.5 g of sodium alginate, and 3 g of modified dietary fiber to 20 g of deionized water, stir evenly, then add 15 g of green tea fermentation broth and 0.6 g of Lactobacillus rhamnosus powder, mix well, and then add 10 g of 5 wt% zinc lactate solution, continue to stir for 2 h to obtain a colloidal solution, which is ground into powder after freeze-drying to obtain the composite prebiotic-embedded probiotic microcapsules.

[0044] Example 3 A preparation method of composite prebiotic-embedded probiotic microcapsules includes the following steps: S1. Crush 20 g of green tea leaves, pass through an 80-mesh sieve, add them to 400 g of deionized water, then add 0.8 g of glycine, carry out ultrasonic-assisted extraction with an ultrasonic power of 600 w, an extraction temperature of 70 °C, and an extraction time of 15 min. After the extraction is completed, filter to obtain the extract and the filter residue; S2. Add the extract to sterilized coconut water, then inoculate Lactobacillus acidophilus. Based on the volume of sterilized coconut water, the addition amount of the extract is 25%, and the inoculation amount of Lactobacillus acidophilus is 2%. Ferment and culture at 30 °C for 48 h with a stirring speed of 150 r / min. After the fermentation and culture are completed, filter and collect the filtrate to obtain the green tea fermentation broth; S3. Add 15 g of the filter residue to 100 g of a 20 wt% hydrogen peroxide solution and process for 3 h. After the treatment is completed, perform suction filtration, washing, drying, and grinding through a 400-mesh sieve to obtain modified dietary fiber. S4. Add 0.5 g of inulin, 1.2 g of sodium alginate, and 2.5 g of modified dietary fiber to 15 g of deionized water, stir evenly, then add 15 g of green tea fermentation broth and 0.3 g of Lactobacillus rhamnosus powder, mix evenly, and then add 20 g of a 3 wt% zinc lactate solution. Continue to stir for 2 h to obtain a colloidal solution. After freeze-drying, grind it into powder to obtain the composite prebiotic-embedded probiotic microcapsules.

[0045] Comparative Example 1 A method for preparing composite prebiotic-embedded probiotic microcapsules includes the following steps: S1. Crush 15 g of green tea leaves, pass them through an 80-mesh sieve, add them to 300 g of deionized water, then add 0.5 g of glycine, and perform ultrasonic-assisted extraction with an ultrasonic power of 300 w, an extraction temperature of 80 °C, and an extraction time of 10 min. After the extraction is completed, filter to obtain the extract and the filter residue. S2. Add 10 g of the filter residue to 100 g of a 10 wt% hydrogen peroxide solution and process for 3 h. After the treatment is completed, perform suction filtration, washing, drying, and grinding through a 400-mesh sieve to obtain modified dietary fiber. S3. Add 0.4 g of inulin, 1 g of sodium alginate, and 2 g of modified dietary fiber to 10 g of deionized water, stir evenly, then add 10 g of the extract and 0.5 g of Lactobacillus rhamnosus powder, mix evenly, and then add 10 g of a 3 wt% zinc lactate solution. Continue to stir for 2 h to obtain a colloidal solution. After freeze-drying, grind it into powder to obtain the composite prebiotic-embedded probiotic microcapsules.

[0046] Compared with Example 1, in Comparative Example 1, the extract was not fermented.

[0047] Comparative Example 2 A method for preparing composite prebiotic-embedded probiotic microcapsules includes the following steps: S1. Crush 15 g of green tea leaves, pass them through an 80-mesh sieve, add them to 300 g of deionized water, then add 0.5 g of glycine, and perform ultrasonic-assisted extraction with an ultrasonic power of 300 w, an extraction temperature of 80 °C, and an extraction time of 10 min. After the extraction is completed, filter to obtain the extract and the filter residue. S2. Add the leaching solution to sterilized coconut water, then inoculate Lactobacillus acidophilus. Based on the volume of the sterilized coconut water, the addition amount of the leaching solution is 20%, and the inoculation amount of Lactobacillus acidophilus is 1%. Ferment and culture at 30 °C for 60 h, with a stirring speed of 150 r / min. After the fermentation and culture are completed, filter to collect the filtrate to obtain the green tea fermentation broth; S3. Add 0.4 g of inulin and 1 g of sodium alginate to 10 g of deionized water, stir evenly, then add 10 g of the green tea fermentation broth and 0.5 g of Lactobacillus rhamnosus powder thereto, mix evenly, and then add 10 g of a 3 wt% zinc lactate solution, and continue to stir for 2 h to obtain a colloidal solution. After freeze-drying, grind it into powder to obtain the composite prebiotic-embedded probiotic microcapsules.

[0048] Compared with Example 1, in Comparative Example 2, no filter residue was added to the capsule wall material.

[0049] Comparative Example 3 A preparation method of composite prebiotic-embedded probiotic microcapsules includes the following steps: S1. Crush 15 g of green tea leaves, pass through an 80-mesh sieve, add them to 300 g of deionized water, then add 0.5 g of glycine, and perform ultrasonic-assisted leaching. The ultrasonic power is 300 w, the leaching temperature is 80 °C, and the leaching time is 10 min. After the leaching is completed, filter to obtain the leaching solution and filter residue; S2. Add the leaching solution to sterilized coconut water, then inoculate Lactobacillus acidophilus. Based on the volume of the sterilized coconut water, the addition amount of the leaching solution is 20%, and the inoculation amount of Lactobacillus acidophilus is 1%. Ferment and culture at 30 °C for 60 h, with a stirring speed of 150 r / min. After the fermentation and culture are completed, filter to collect the filtrate to obtain the green tea fermentation broth; S3. Add 0.4 g of inulin, 1 g of sodium alginate and 2 g of filter residue (400 mesh) to 10 g of deionized water, stir evenly, then add 10 g of the green tea fermentation broth and 0.5 g of Lactobacillus rhamnosus powder thereto, mix evenly, and then add 10 g of a 3 wt% zinc lactate solution, and continue to stir for 2 h to obtain a colloidal solution. After freeze-drying, grind it into powder to obtain the composite prebiotic-embedded probiotic microcapsules.

[0050] Compared with Example 1, in Comparative Example 3, the filter residue was not modified.

[0051] Perform an in vitro DPPH free radical scavenging test on the composite prebiotic-embedded probiotic microcapsules prepared in Example 1 and Comparative Examples 1-3. The specific steps are as follows: Release media of 40% ethanol - normal saline with different pH values (3, 7.4, 8.5) were respectively prepared. 5 mL of each medium was measured and put into a 10 mL centrifuge tube. Then, 10 mg of the composite prebiotic - encapsulated probiotic microcapsules prepared in Example 1 and Comparative Examples 1 - 3 were respectively added. The mixture was shaken in the dark at 37°C in a constant temperature shaker (180 r / min) for 3 h. Subsequently, it was centrifuged at 2000×g for 5 min. The supernatant obtained was the solution of the released active substances. 100 μL of the supernatant was added to 300 μL of DPPH ethanol solution (0.04 mg / mL), shaken well, incubated in the dark for 30 min, then centrifuged at 4000 r / min for 10 min. The absorbance of the supernatant was measured at 517 nm.

[0052] The DPPH radical scavenging rate was calculated according to the formula: DPPH radical scavenging rate / % = [1 - (A - A0) / A1] × 100; Where, A is the absorbance value after adding the active substance solution, A0 is the background absorbance value of the active substance solution, and A1 is the absorbance value of the blank control group.

[0053] All experiments were carried out with 3 independent replicates, and the test results are as Figure 1 shown. From Figure 1It can be seen that the DPPH radical scavenging rate of the composite prebiotic-encapsulated probiotic microcapsules prepared in Example 1 and Comparative Examples 1-3 gradually increases with the increase of pH. The reason is that with the increase of pH, the release amount of active substances in the composite prebiotic-encapsulated probiotic microcapsules gradually increases. Therefore, the DPPH radical scavenging rate gradually increases. From the data in Example 1, it can be seen that when the pH is 3, the DPPH radical scavenging rate of the composite prebiotic-encapsulated probiotic microcapsules is about 43%, while when the pH is 8.5, the DPPH radical scavenging rate can reach 87%. The reason may be that when the pH is 8.5, the hydrogen bond interaction between the substances in the capsule wall material weakens, the structure becomes loose, and a large amount of the encapsulated active substances are released. This shows that the release rate of the active substances in the composite prebiotic-encapsulated probiotic microcapsules prepared in the present invention is low in the gastric stage and can be released in large amounts in the intestinal stage, enabling the intestinal sustained and controlled release function. In Comparative Example 1, the leaching solution was not fermented, and its DPPH radical scavenging rate was lower than that of Example 1, indicating that the antioxidant active ingredient content can be increased by fermenting the leaching solution; in Comparative Example 2, no filter residue was added to the capsule wall material, and in Comparative Example 3, the filter residue was not modified. The DPPH radical scavenging rates of the composite prebiotic-encapsulated probiotic microcapsules prepared in Comparative Example 2 and Comparative Example 3 were both higher than that of Example 1. The reason may be that by adding modified dietary fiber to the capsule wall material, the active substances in the green tea fermentation broth are embedded in the lamellar structure and pores of the modified dietary fiber, enhancing the encapsulation effect of the active substances and delaying the release of the active substances, thereby achieving the purpose of slow release.

[0054] The storage stability test was carried out on the composite prebiotic-encapsulated probiotic microcapsules prepared in Example 1 and Comparative Examples 2-3. The specific experimental steps are as follows: The composite prebiotic-encapsulated probiotic microcapsules were stored at room temperature for 4 weeks. The viable bacteria counts at the start of storage (recorded as week 0) and after 4 weeks of storage were tested. When measuring the viable bacteria count, each time 0.1 g of microcapsules was placed in 10 mL of sterile PBS and shaken at a constant temperature of 37 °C until completely dissolved, and then plated and counted by the dilution gradient method. All experiments were carried out in 3 independent replicates. The test results are as Figure 2 shown. In Comparative Example 2, no filter residue was added to the capsule wall material, and the viable bacteria count decreased by about 2.8 (lg(CFU / g)). In Comparative Example 3, the filter residue was not modified, and the viable bacteria count decreased by about 1.5 (lg(CFU / g)). However, the viable bacteria count in Example 1 of the present invention decreased by about 0.7 (lg(CFU / g)). It can be seen that the composite prebiotic-encapsulated probiotic microcapsules prepared in the present invention can maintain good stability during storage at room temperature.

[0055] Polyphenol extraction rate test: The leaching solution obtained in step S1 of Examples 1-3 was freeze-dried to obtain a freeze-dried powder, and then a test solution with a concentration of 2 mg / ml was prepared. At the same time, a control group was set up. The experimental steps of the control group were as follows: 15 g of green tea leaves were crushed, passed through an 80-mesh sieve, and then added to 300 g of deionized water. Ultrasonic-assisted extraction was carried out with an ultrasonic power of 300 w, an extraction temperature of 80 °C, and an extraction time of 10 min. After the extraction was completed, filtration was carried out to obtain the leaching solution and the filter residue. The leaching solution was freeze-dried to obtain a freeze-dried powder, and then a test solution with a concentration of 2 mg / ml was prepared for standby. 1 mL of the test solutions of Examples 1-3 and the control group were respectively taken into test tubes. 5.00 mL of 10% Folin-Ciocalteu reagent was added to the test tubes. After standing for 5 min, 4 mL of 7.5% sodium carbonate solution was added, and the mixture was shaken well. After reacting at room temperature for 60 min, the absorbance at 765 nm was measured using a UV-visible spectrophotometer. Finally, the absorbance value was compared with the absorbance measured at 765 nm after the aqueous solution of gallic acid reference substance with a concentration of 2 mg / mL was treated through the above steps, and the extraction rate of polyphenols was calculated. The polyphenol extraction rate (%) = the mass of polyphenols in the freeze-dried powder / the mass of green tea leaves × 100%. The test was carried out 3 times, and the results were averaged.

[0056] Flavonoid extraction rate test: 1 mL of the leaching solutions obtained in Examples 1-3 and the control group were respectively taken and placed in 10-mL volumetric flasks. 0.3 mL of 5% NaNO2 solution was added, and the mixture was shaken well. After standing for 6 min, 0.3 mL of 10% aluminum nitrate solution was added, and the mixture was shaken well and then stood for 6 min. 4.0 mL of 4% NaOH solution was added, and the volume was made up to the scale with 75% ethanol, and the mixture was shaken well and then stood for 12 min. The absorbance was measured at 510 nm, and the mass of flavonoids in the leaching solution was calculated. The flavonoid extraction rate (%) = the mass of flavonoids in the leaching solution / the mass of green tea leaves × 100%. The test was carried out 3 times, and the results were averaged.

[0057] The test results are as Figure 3 shown. It can be seen from Figure 3 that glycine was not added during the extraction process in the control group. Compared with the examples of the present invention, the polyphenol extraction rate and the flavonoid extraction rate were significantly reduced. It can be seen that adding an amino acid surfactant during the water extraction process can effectively dissolve the flavonoid compounds and polyphenol compounds in the cells.

[0058] Finally, it should be noted that the above examples do not limit the present invention in any form. For those skilled in the art, based on the present invention, some modifications and improvements can be made. Therefore, any modification or improvement made without departing from the spirit of the present invention falls within the scope of protection required by the present invention.

Claims

1. A preparation method of a composite prebiotic-embedded probiotic microcapsule, characterized in that, It includes the following steps: S1. Crush and sieve green tea leaves, add them to deionized water, then add an amino acid surfactant, and perform ultrasonic-assisted extraction. After the extraction is completed, filter to obtain an extract and filter residue; S2. Add the extract to sterilized coconut water, then inoculate Lactobacillus acidophilus, and perform fermentation culture. After the fermentation culture is completed, filter and collect the filtrate to obtain a green tea fermentation broth; S3. Add the filter residue to a hydrogen peroxide solution for treatment. After the treatment is completed, perform suction filtration, washing, drying, and grinding and sieving to obtain modified dietary fiber; S4. Add inulin, sodium alginate, and modified dietary fiber to deionized water, stir evenly, then add the green tea fermentation broth and Lactobacillus rhamnosus powder to it, mix evenly, then add a zinc lactate solution, and continue to stir to obtain a colloidal solution. After freeze-drying, grind it into powder to obtain a composite prebiotic-embedded probiotic microcapsule.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of green tea leaves, deionized water, and amino acid surfactant is 15-25:300-400:0.5-1.

3. The preparation method according to claim 1, wherein, In step S1, the ultrasonic power is 300-600 w, the extraction temperature is 70-80 °C, and the extraction time is 10-20 min.

4. The preparation method according to claim 1, wherein, In step S2, based on the volume of sterilized coconut water, the addition amount of the extract is 20-30%, and the inoculation amount of Lactobacillus acidophilus is 1-3%.

5. The preparation method according to claim 1, characterized in that In step S2, during the fermentation culture process, the fermentation temperature is 28-32 °C, the fermentation time is 48-72 h, and the stirring speed is 100-200 r / min.

6. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of the filter residue to the hydrogen peroxide solution is 10-20:100, and the mass fraction of the hydrogen peroxide solution is 10-20%.

7. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of inulin, sodium alginate, modified dietary fiber, deionized water, green tea fermentation broth, Lactobacillus rhamnosus powder, and zinc lactate solution is 0.4-0.8:1-1.5:2-3:10-20:10-15:0.3-0.6:10-20.

8. The preparation method according to claim 1, wherein, In step S4, the mass fraction of the zinc lactate solution is 3-6%.

9. The composite prebiotic-embedded probiotic microcapsule prepared by the preparation method according to any one of claims 1-8.

10. The application of the composite prebiotic-embedded probiotic microcapsule according to claim 9 in the preparation of a solid beverage product.

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