Preparation method of high-activity fruit and vegetable probiotic fermented beverage

Abstract

The invention relates to the technical field of fermented beverages, in particular to a preparation method of a high-activity fruit and vegetable probiotic fermented beverage, which comprises the following steps: adding composite fruit and vegetable juice after enzymolysis, mineral water, xylooligosaccharide, grape seed extract, yeast extract and xanthan gum into a fermentation tank, and stirring until the materials are completely dissolved; then carrying out sterilization treatment by adopting high-temperature instantaneous sterilization; adding probiotic microcapsules into the sterilized mixed liquid, and performing staged temperature-controlled fermentation to obtain fermentation liquid; and performing homogenization treatment on the fermentation liquid, and finally performing vacuum degassing to obtain the high-activity fruit and vegetable probiotic fermented beverage. A composite wall material (sodium alginate-eucheuma powder-gellan gum) of the probiotic microcapsule forms a compact gel network to effectively isolate phenols, organic acids and other antibacterial components in fruits and vegetables, so that the damage of pH fluctuation and temperature change to thalli is resisted.

Description

Technical Field

[0001] The present invention relates to the technical field of fermented beverages, and in particular to a method for preparing a highly active fruit and vegetable probiotic fermented beverage. Background Art

[0002] As a new type of functional food that combines the nutrients of fruits and vegetables with the functions of probiotics, fruit and vegetable probiotic fermented drinks have attracted more and more attention from consumers in recent years. Traditional fermented drinks such as yogurt and kimchi mainly rely on strains such as lactic acid bacteria. However, the synergistic fermentation of fruit and vegetable extracts with specific probiotics (especially strains that are beneficial to the intestines) can not only improve the nutritional value of the product and improve the taste and flavor, but also enhance the survival rate of probiotics and their ability to colonize the intestines.

[0003] However, the activity of probiotics is easily reduced by factors such as raw material ingredients (phenolics, organic acids), fermentation environment (pH, temperature, oxygen) and post-processing. In view of this, we propose a method for preparing highly active fruit and vegetable probiotic fermented beverages. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a highly active fruit and vegetable probiotic fermented beverage, so as to solve the problem raised in the above background technology that the activity of probiotics is easily reduced by factors such as raw material ingredients (phenolic substances, organic acids), fermentation environment (pH, temperature, oxygen) and post-processing.

[0005] The present invention provides a method for preparing a high-activity fruit and vegetable probiotic fermented beverage, comprising the following steps: S1.1. Weigh the following ingredients separately: compound fruit and vegetable juice, mineral water, probiotic microcapsules, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum; S1.2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 40°C at 200-300 rpm for 15-20 minutes until completely dissolved; then sterilize by high-temperature instantaneous sterilization to obtain a mixed solution. S1.3. Add probiotic microcapsules to the sterilized mixed solution and perform temperature-controlled fermentation in stages to obtain a fermentation liquid; S1.4. Homogenize the fermentation liquid at 4°C and a pressure of 20-25 MPa for 1-2 times; finally, perform vacuum degassing to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0006] Preferably, in S1.1, the following raw materials are weighed in parts by weight: 50-65 parts by weight of compound fruit and vegetable juice, 20-30 parts by weight of mineral water, 3-5 parts by weight of probiotic microcapsules, 1-2 parts by weight of xylo-oligosaccharides, 0.05-0.1 parts by weight of grape seed extract, 0.3-0.6 parts by weight of yeast extract, and 0.2-0.6 parts by weight of xanthan gum.

[0007] Preferably, in S1.2, the enzymatically hydrolyzed composite fruit and vegetable juice is obtained by adding 0.1% w / w cellulase to the composite fruit and vegetable juice, enzymatically hydrolyzing at 45°C for 1.5-2h, then inactivating the enzyme at 90°C for 5min, and finally cooling to 30°C.

[0008] Preferably, in S1.2, the temperature of the high-temperature instantaneous sterilization is 100-110° C., and the processing time is 3-5 s.

[0009] Preferably, in S1.3, the fermentation is temperature-controlled in stages: fermentation is performed at 38°C for 8 hours until the pH reaches 4.5-4.7, and then fermentation is performed at 36°C with stirring at 50 rpm for 16 hours until the pH reaches 4.5, maintained for 2 hours, and then cooled to 4°C.

[0010] Preferably, in S1.3, the probiotic microcapsules are prepared by embedding a probiotic suspension in a sodium alginate-wood unicorn powder-gellan gum composite wall material, loading a lauric acid-stearic acid eutectic, and then modifying the chitosan coating; The probiotic microcapsules were prepared by dissolving 1.8-2.2% w / v sodium alginate, 1.2-1.8% w / v wood unicorn powder, and 0.4-0.6% w / v gellan gum in purified water at 60-65°C, stirring at 300-400 rpm for 40-50 minutes until completely dissolved, cooling to 25-30°C, and allowing to stand for 20-30 minutes to degas, thereby obtaining a wall material solution. Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the electrostatic microcapsule generator reservoir to form uniform droplets, and then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 15-20 cm. The solution was kept in contact for 8-12 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed 2-3 times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 40-50 rpm for 3-5 minutes, drained, and air-dried. The probiotic microcapsules were then rinsed 1-2 times with a 1% sodium bicarbonate solution.

[0011] Preferably, the concentrated probiotic suspension is prepared by compounding Lactobacillus plantarum and Bifidobacterium bifidum in a volume ratio of 8:2 in MRS medium, activating the probiotics, centrifuging at 6000-8000 rpm for 10 min at 4°C to collect the bacteria, and then resuspending the bacteria in a 10% w / v sterile skim milk solution to a viable bacterial concentration of more than 1×10 11 CFU / mL.

[0012] Preferably, the voltage of the electrostatic microcapsule generator is 8-10 kV, the needle diameter is 0.4-0.6 mm, and the dropping speed is 3-5 mL / min.

[0013] Preferably, the loading amount of the lauric acid-stearic acid eutectic is 3-5% of the total weight of the probiotic microcapsules.

[0014] Preferably, in S1.4, the pressure of vacuum degassing is , time is 5-10 minutes.

[0015] Compared with the prior art, the present invention has the following beneficial effects: In the preparation method of the highly active fruit and vegetable probiotic fermented beverage of the present invention, the composite wall material (sodium alginate-wood unicorn powder-gellan gum) of the probiotic microcapsule effectively isolates antibacterial components such as phenols and organic acids in fruits and vegetables by forming a dense gel network, thereby resisting damage to the bacteria caused by pH fluctuations and temperature changes; in addition, the chitosan coating further enhances the density of the microcapsule, inhibits oxygen permeation, and reduces oxidative stress; chitosan and polysaccharides such as sodium alginate act synergistically to form a hard and dense microcapsule shell, significantly improving the survival rate of probiotics; the loaded lauric acid-stearic acid eutectic slowly melts in an acidic environment to avoid inactivation of the probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a bar graph comparing the viable bacterial rates of the samples of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0018] The compound fruit and vegetable juice is made by mixing watermelon juice, papaya juice and red tangerine juice in a volume ratio of 5:3:2; the watermelon juice is concentrated in a ratio of 4:1, the papaya juice is concentrated in a ratio of 3.5:1, and the red tangerine juice is concentrated in a ratio of 6:1 Grape seed extract CAS number: 84929-27-1, comes from Shanghai Yuanye Biotechnology Co., Ltd., the main active ingredients include oligomeric proanthocyanidins, proanthocyanidins, polyphenols (catechin, epicatechin), resveratrol, tannins, flavonoids, etc.

[0019] Yeast extract CAS number: 8013-01-2, from Beijing Solebow Technology Co., Ltd., the main active ingredients include proteins, amino acids, peptides, nucleotides, vitamins (such as B vitamins), and trace elements (such as calcium, iron, zinc, etc.).

[0020] The preservation number of Lactobacillus plantarum is CGMCC No.1258, and the preservation number of Bifidobacterium bifidum is CGMCC No.11959.

[0021] Preparation method of lauric acid-stearic acid eutectic: lauric acid and stearic acid are mixed in a molar ratio of 1:1 in a reactor, slowly heated to 75°C under nitrogen protection, and mechanically stirred at 300 rpm for 40 minutes to form a uniform liquid mixture; slowly cooled to 56°C at a rate of 0.5°C / min, and maintained at a constant temperature for 2 hours; rapidly cooled to below 25°C to solidify, and crushed to pass through a 100-mesh sieve to obtain lauric acid-stearic acid eutectic.

[0022] The enzymatically hydrolyzed composite fruit and vegetable juice is obtained by adding 0.1% w / w cellulase to the composite fruit and vegetable juice, enzymatically hydrolyzing the juice at 45° C. for 2 hours, then inactivating the enzyme at 90° C. for 5 minutes, and finally cooling the juice to 30° C.

[0023] Probiotic microcapsules are prepared by encapsulating probiotic suspension in a sodium alginate-wooden chinensis powder-gellan gum composite wall material, loading lauric acid-stearic acid eutectic and then modifying the chitosan coating.

[0024] The preparation steps of wood angelica powder are as follows: select fresh wood angelica leaves and manually remove the sharp thorns at the base of the leaves; rinse the impurities on the surface of the leaves with running water and drain the water; spread the leaves flat on the drying equipment and ventilate and dry them at 40-50℃ for 12-24 hours until the moisture content is less than 10%; grind the dried leaves into powder with a crusher and pass them through an 80-100 mesh sieve to remove coarse fiber particles.

[0025] Concentrated probiotic suspension is prepared by mixing Lactobacillus plantarum and Bifidobacterium bifidum in a volume ratio of 8:2. After activation in MRS medium, the bacteria are collected by centrifugation at 8000 rpm for 10 min at 4°C and then resuspended in 10% w / v sterile skim milk solution to a viable bacterial concentration of more than 1×10 11 CFU / mL.

[0026] Example 1: A method for preparing a highly active fruit and vegetable probiotic fermented beverage, comprising the following steps: S1.1. Weigh the following raw materials in parts by weight: 50 parts by weight of compound fruit and vegetable juice, 20 parts by weight of mineral water, 3 parts by weight of probiotic microcapsules, 1 part by weight of xylo-oligosaccharide, 0.05 parts by weight of grape seed extract, 0.3 parts by weight of yeast extract, and 0.2 parts by weight of xanthan gum. S1.2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 300 rpm at 40°C for 20 min until completely dissolved. Subsequently, sterilize the mixture by high-temperature instantaneous sterilization (110°C for 5 s) to obtain a mixed solution. S1.3. Add probiotic microcapsules to the sterilized mixed solution, let it ferment at 38°C for 8 h until the pH reaches 4.7, then stir and ferment at 36°C at 50 rpm for 16 h until the pH reaches 4.5, maintain the mixture for 2 h, and then cool to 4°C to obtain a fermentation broth. S1.4. Homogenize the fermentation liquid twice at 4°C and a pressure of 20 MPa; finally, perform vacuum degassing at a pressure of -0.08 MPa for 10 min to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0027] Furthermore, the preparation method of probiotic microcapsules is as follows: 1.8% w / v sodium alginate, 1.2% w / v wood unicorn powder and 0.4% w / v gellan gum are dissolved in 60 ° C purified water, stirred at 300 rpm for 40 minutes until completely dissolved, cooled to 25 ° C and allowed to stand for 30 minutes to degas, to obtain a wall material solution; Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the reservoir of an electrostatic microcapsule generator (voltage 8 kV, needle diameter 0.5 mm, dropping speed 4 mL / min) to form uniform droplets, which were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 20 cm. The solution was kept in contact for 10 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed three times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic (with a loading of 3% of the total weight of the probiotic microcapsules) was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 50 rpm for 5 minutes, drained, and dried with cold air. The probiotic microcapsules were then rinsed twice with a 1% sodium bicarbonate solution.

[0028] Example 2: A method for preparing a highly active fruit and vegetable probiotic fermented beverage, comprising the following steps: S1.1. Weigh the following raw materials in parts by weight: 60 parts by weight of compound fruit and vegetable juice, 25 parts by weight of mineral water, 4 parts by weight of probiotic microcapsules, 1.5 parts by weight of xylo-oligosaccharides, 0.07 parts by weight of grape seed extract, 0.5 parts by weight of yeast extract, and 0.4 parts by weight of xanthan gum. S1.2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 300 rpm at 40°C for 20 min until completely dissolved. Subsequently, sterilize the mixture by high-temperature instantaneous sterilization (110°C for 5 s) to obtain a mixed solution. S1.3. Add probiotic microcapsules to the sterilized mixed solution, let it ferment at 38°C for 8 h until the pH reaches 4.7, then stir and ferment at 36°C at 50 rpm for 16 h until the pH reaches 4.5, maintain the mixture for 2 h, and then cool to 4°C to obtain a fermentation broth. S1.4. Homogenize the fermentation liquid twice at 4°C and a pressure of 20 MPa; finally, perform vacuum degassing at a pressure of -0.08 MPa for 10 min to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0029] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.0% w / v sodium alginate, 1.5% w / v wood unicorn powder and 0.5% w / v gellan gum are dissolved in 60°C purified water, stirred at 300 rpm for 40 minutes until completely dissolved, cooled to 25°C and allowed to stand for 30 minutes to degas, to obtain a wall material solution; Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the reservoir of an electrostatic microcapsule generator (voltage 8 kV, needle diameter 0.5 mm, dropping speed 4 mL / min) to form uniform droplets, which were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 20 cm. The solution was kept in contact for 10 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed three times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic (with a loading of 4% of the total weight of the probiotic microcapsules) was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 50 rpm for 5 minutes, drained, and dried with cold air. The probiotic microcapsules were then rinsed twice with a 1% sodium bicarbonate solution.

[0030] Example 3: A method for preparing a highly active fruit and vegetable probiotic fermented beverage, comprising the following steps: S1.1. Weigh the following raw materials in parts by weight: 65 parts by weight of compound fruit and vegetable juice, 30 parts by weight of mineral water, 5 parts by weight of probiotic microcapsules, 2 parts by weight of xylo-oligosaccharides, 0.1 parts by weight of grape seed extract, 0.6 parts by weight of yeast extract, and 0.6 parts by weight of xanthan gum. S1.2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 300 rpm at 40°C for 20 min until completely dissolved. Subsequently, sterilize the mixture by high-temperature instantaneous sterilization (110°C for 5 s) to obtain a mixed solution. S1.3. Add probiotic microcapsules to the sterilized mixed solution, let it ferment at 38°C for 8 h until the pH reaches 4.7, then stir and ferment at 36°C at 50 rpm for 16 h until the pH reaches 4.5, maintain the mixture for 2 h, and then cool to 4°C to obtain a fermentation broth. S1.4. Homogenize the fermentation liquid twice at 4°C and a pressure of 20 MPa; finally, perform vacuum degassing at a pressure of -0.08 MPa for 10 min to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0031] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.2% w / v sodium alginate, 1.8% w / v wood unicorn powder and 0.6% w / v gellan gum are dissolved in 60 ° C purified water, stirred at 300 rpm for 40 minutes until completely dissolved, cooled to 25 ° C and allowed to stand for 30 minutes to degas, to obtain a wall material solution; Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the reservoir of an electrostatic microcapsule generator (voltage 8 kV, needle diameter 0.5 mm, dropping speed 4 mL / min) to form uniform droplets, which were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 20 cm. The solution was kept in contact for 10 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed three times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic (with a loading of 5% of the total weight of the probiotic microcapsules) was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 50 rpm for 5 minutes, drained, and dried with cold air. The probiotic microcapsules were then rinsed twice with a 1% sodium bicarbonate solution.

[0032] Example 4: A method for preparing a highly active fruit and vegetable probiotic fermented beverage, comprising the following steps: S1.1. Weigh the following raw materials in parts by weight: 60 parts by weight of compound fruit and vegetable juice, 25 parts by weight of mineral water, 5 parts by weight of probiotic microcapsules, 1.5 parts by weight of xylo-oligosaccharides, 0.07 parts by weight of grape seed extract, 0.5 parts by weight of yeast extract, and 0.4 parts by weight of xanthan gum. S1.2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 300 rpm at 40°C for 20 min until completely dissolved. Subsequently, sterilize the mixture by high-temperature instantaneous sterilization (110°C for 5 s) to obtain a mixed solution. S1.3. Add probiotic microcapsules to the sterilized mixed solution, let it ferment at 38°C for 8 h until the pH reaches 4.7, then stir and ferment at 36°C at 50 rpm for 16 h until the pH reaches 4.5, maintain the mixture for 2 h, and then cool to 4°C to obtain a fermentation broth. S1.4. Homogenize the fermentation liquid twice at 4°C and a pressure of 20 MPa; finally, perform vacuum degassing at a pressure of -0.08 MPa for 10 min to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0033] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.0% w / v sodium alginate, 1.5% w / v wood unicorn powder and 0.5% w / v gellan gum are dissolved in 60°C purified water, stirred at 300 rpm for 40 minutes until completely dissolved, cooled to 25°C and allowed to stand for 30 minutes to degas, to obtain a wall material solution; Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the reservoir of an electrostatic microcapsule generator (voltage 8 kV, needle diameter 0.5 mm, dropping speed 4 mL / min) to form uniform droplets, which were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 20 cm. The solution was kept in contact for 10 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed three times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic (with a loading of 4% of the total weight of the probiotic microcapsules) was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 50 rpm for 5 minutes, drained, and dried with cold air. The probiotic microcapsules were then rinsed twice with a 1% sodium bicarbonate solution.

[0034] Example 5: A method for preparing a highly active fruit and vegetable probiotic fermented beverage, comprising the following steps: S1.1. Weigh the following raw materials in parts by weight: 60 parts by weight of compound fruit and vegetable juice, 25 parts by weight of mineral water, 6 parts by weight of probiotic microcapsules, 1.5 parts by weight of xylo-oligosaccharides, 0.07 parts by weight of grape seed extract, 0.5 parts by weight of yeast extract, and 0.4 parts by weight of xanthan gum. S1.2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 300 rpm at 40°C for 20 min until completely dissolved. Subsequently, sterilize the mixture by high-temperature instantaneous sterilization (110°C for 5 s) to obtain a mixed solution. S1.3. Add probiotic microcapsules to the sterilized mixed solution, let it ferment at 38°C for 8 h until the pH reaches 4.7, then stir and ferment at 36°C at 50 rpm for 16 h until the pH reaches 4.5, maintain the mixture for 2 h, and then cool to 4°C to obtain a fermentation broth. S1.4. Homogenize the fermentation liquid twice at 4°C and a pressure of 20 MPa; finally, perform vacuum degassing at a pressure of -0.08 MPa for 10 min to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0035] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.0% w / v sodium alginate, 1.5% w / v wood unicorn powder and 0.5% w / v gellan gum are dissolved in 60°C purified water, stirred at 300 rpm for 40 minutes until completely dissolved, cooled to 25°C and allowed to stand for 30 minutes to degas, to obtain a wall material solution; Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the reservoir of an electrostatic microcapsule generator (voltage 8 kV, needle diameter 0.5 mm, dropping speed 4 mL / min) to form uniform droplets, which were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 20 cm. The solution was kept in contact for 10 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed three times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic (with a loading of 4% of the total weight of the probiotic microcapsules) was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 50 rpm for 5 minutes, drained, and dried with cold air. The probiotic microcapsules were then rinsed twice with a 1% sodium bicarbonate solution.

[0036] Comparative Example 1: Compared with Example 2, this comparative example does not add probiotic microcapsules.

[0037] Comparative Example 2: Compared with Example 2, this comparative example does not add wood unicorn powder as wall material, and only uses sodium alginate and gellan gum as wall materials.

[0038] Comparative Example 3: Compared with Example 2, the probiotic microcapsules in this comparative example are not loaded with lauric acid-stearic acid eutectic.

[0039] The present invention provides a high-activity fruit and vegetable probiotic fermented beverage prepared by probiotic microcapsules. The performance index test items and test standards of the high-activity fruit and vegetable probiotic fermented beverage are as follows: Take 10 mL of the beverage, add 90 mL of sterile phosphate buffered saline (PBS, pH 7.0), and homogenize (4,000 rpm, 1 min); then add 0.5% w / v sodium citrate solution, and shake at 37°C for 30 min to release the bacteria; take 1 mL of the homogenate and perform a 10-fold gradient dilution with PBS (Lactobacillus plantarum was inoculated on MRS agar plates and anaerobically cultured at 37°C for 48 h; Bifidobacterium bifidum was inoculated on TPY agar + 0.05% cysteine ​​plates and strictly anaerobically cultured at 37°C for 72 h); select plates with colony counts between 30 and 300 for counting, and calculate the viable bacterial count (CFU / mL) according to the formula = average colony count × dilution factor × 10.

[0040] Within 24 hours after filling, determine the initial viable cell count as described above and use it as the baseline value. Take samples at the end of the shelf life (e.g., 21 days, refrigerated at 4°C) and determine them using the same method. For the accelerated test, store the product at 37°C for 14 days (simulating a 3-month shelf life), with samples taken and determined every 48 hours. Viable cell rate (%) = (viable cell count after storage ÷ initial viable cell count) × 100.

[0041] The highly active fruit and vegetable probiotic fermented beverages prepared in Examples 1-5 and Comparative Examples 1-3 were tested using the above standards, and the obtained data are shown in Table 1: Table 1 Performance data of high-activity fruit and vegetable probiotic fermented beverages of Examples 1-5 and Comparative Examples 1-3

[0042] It can be seen from Examples 1-3 that changes in the raw material components of the high-activity fruit and vegetable probiotic fermented beverage have an improving effect on the number of viable bacteria in the fermented beverage and the stability of the probiotics during the shelf life.

[0043] The sodium alginate-gellan gum gel network combined with the fiber skeleton of wood qilin powder can effectively shield the oxidative damage of phenolic substances (such as tannic acid) in fruit and vegetable juices to the bacterial cell membrane; the chitosan coating densifies the membrane structure through positive charge, reduces the dissolved oxygen permeability, inhibits the reproduction of aerobic bacteria and the aerobic metabolic death of probiotics; the loaded lauric acid-stearic acid eutectic melts in an acidic environment to reduce the killing of probiotics by acid, thereby increasing the live bacteria rate.

[0044] As a high-efficiency bioactive substance, oligoxylose is specifically fermented by Lactobacillus plantarum and Bifidobacterium bifidum during the fermentation stage, rapidly generating short-chain fatty acids, lowering the environmental pH, inhibiting the competitive growth of miscellaneous bacteria, and at the same time increasing the biomass of the target probiotics; in addition, the proanthocyanidins in grape seed extract can scavenge free radicals produced during fermentation and storage, reducing the risk of DNA oxidative damage; yeast extract provides endogenous antioxidant peptides such as glutathione, which precisely control the pH at the fermentation endpoint within an appropriate range, avoiding autolysis of the bacteria due to an over-acidic environment; xanthan gum forms a shear-thinning colloidal network during the homogenization stage, encapsulating the microcapsules and inhibiting their sedimentation, while reducing oxygen infiltration after filling.

[0045] Furthermore, by comparing Example 2, Example 4 and Example 5, it can be seen that when other components in the highly active fruit and vegetable probiotic fermented beverage remain unchanged and the weight of the probiotic microcapsules continues to increase, the number of viable bacteria in the fermented beverage and the stability of the probiotics during the shelf life continue to decrease.

[0046] Excess microcapsules aggregate in the fermentation broth due to the weakened steric hindrance effect, forming clumps, which make it impossible to fully disperse in the homogenization stage; an oxygen-deficient microenvironment is formed inside the clumps, hindering the metabolic activity of probiotics; at the same time, the sedimentation of the clumps is accelerated, causing the density of live bacteria at the bottom of the beverage to increase locally, and the dissolved oxygen penetration at the top to intensify, accelerating the death of surface bacteria; the microcapsule wall material (sodium alginate-gellan gum complex) is continuously dissolved in the acidic fermentation broth, and the release of calcium ions will consume key buffers, resulting in an uncontrolled decrease in pH in the middle and late stages of fermentation, exceeding the tolerance limit of Bifidobacterium bifidum; free polysaccharide chains compete with xanthan gum for water molecules, destroying the colloidal network structure and increasing the precipitation rate; excessive lauric acid-stearic acid eutectics undergo eutectic rearrangement during storage, and some are converted into solid crystals and precipitated; these crystals pierce the microcapsule wall, causing the probiotics to leak prematurely; adsorbed on the fermentation broth interface, they accelerate the diffusion of oxygen, triggering the reproduction of aerobic bacteria and oxidative damage to the probiotics.

[0047] According to the above test experiments and combined Figure 1 It can be seen that embodiment 2 is regarded as the best embodiment; It can be seen from Example 2 and Comparative Example 1 that although the natural pH of fruit and vegetable juice can inhibit the growth of miscellaneous bacteria, the continuous acidic environment will damage the metabolic enzyme activity of probiotics, accelerate ATP synthesis disorders, and lead to the death of viable bacteria (the number of viable bacteria decreases after storage at room temperature); antioxidant substances such as polyphenols and vitamin C in fruits and vegetables gradually lose their effectiveness during storage, and residual oxygen free radicals attack bacterial DNA and membrane proteins, causing irreversible damage; temperature fluctuations during room temperature storage cause imbalances in the expression of heat shock proteins in bacteria, abnormal cell membrane fluidity, and shortened half-life of viable bacteria; and the lactic acid concentration increases in the late fermentation stage, inhibiting the activity of proton transporters, blocking the transmembrane proton gradient, and stagnating the energy metabolism of the bacteria, thereby reducing the stability of probiotics during the shelf life.

[0048] It can be seen from Example 2 and Comparative Example 2 that: wood unicorn powder is rich in natural pectin and hemicellulose, which form a three-dimensional network structure with sodium alginate through hydrogen bonds and hydrophobic interactions, which can enhance the density of the microcapsule wall; while the pure sodium alginate-gellan gum system can form a gel, it lacks the filling support of plant polysaccharides, resulting in an increase in the porosity of the capsule wall, making it easier for hydrogen ions to penetrate in an acidic environment, thereby causing a large number of live bacteria to be inactivated; in addition, the polyphenols in wood unicorn powder (such as ellagic acid) have the ability to scavenge free radicals, which can reduce the damage of oxidative stress to the bacteria during storage; wood unicorn powder also contains natural antacid ingredients (such as pectin calcium), which can neutralize part of the acid and further protect the probiotics; at the same time, the soluble fibers such as arabinoxylan in wood unicorn powder can serve as metabolic substrates (prebiotics) for probiotics, continuously supplying carbon sources in the later stages of fermentation, helping to maintain bacterial activity and inhibiting the sudden drop in pH caused by post-acidification; when wood unicorn powder is not added, the probiotics accelerate their death due to the interruption of nutrient supply, and their half-life is significantly shortened at room temperature.

[0049] It can be seen from Example 2 and Comparative Example 3 that when unloaded, the microcapsule wall material (such as sodium alginate) tends to dissolve quickly in an acidic environment, causing the probiotics to be exposed to adverse conditions prematurely, resulting in a high loss rate of viable bacteria; in addition, the lauric acid-stearic acid eutectic forms a dense crystal layer on the surface of the microcapsule, which can effectively block oxygen penetration and prevent the generation of reactive oxygen species, thereby extending the half-life of the probiotics; if the eutectic is missing, oxygen in the environment will directly contact the bacteria, accelerate aerobic metabolism and induce membrane lipid peroxidation, further shortening the survival time of the viable bacteria; at the same time, the eutectic absorbs heat through solid-liquid phase transition, which helps to buffer the impact of temperature fluctuations on the bacteria and improve their stability; however, if the eutectic is missing, the probiotics will be more easily inactivated during storage or transportation, resulting in a decrease in the number of viable bacteria.

[0050] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a highly active fruit and vegetable probiotic fermented beverage, characterized in that: The following steps are involved: S1.

1. Weigh the following ingredients separately: compound fruit and vegetable juice, mineral water, probiotic microcapsules, xylo-oligosaccharides, grape seed extract, yeast extract, and xanthan gum; S1.

2. Add the enzymatically hydrolyzed composite fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 40°C at 200-300 rpm for 15-20 minutes until completely dissolved; then sterilize by high-temperature instantaneous sterilization to obtain a mixed solution. S1.

3. Add probiotic microcapsules to the sterilized mixed solution and perform temperature-controlled fermentation in stages to obtain a fermentation liquid; S1.

4. Homogenize the fermentation liquid at 4°C and a pressure of 20-25 MPa for 1-2 times; finally, perform vacuum degassing to obtain a highly active fruit and vegetable probiotic fermented beverage.

2. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that: In S1.1, the following raw materials are weighed in parts by weight: 50-65 parts by weight of compound fruit and vegetable juice, 20-30 parts by weight of mineral water, 3-5 parts by weight of probiotic microcapsules, 1-2 parts by weight of xylo-oligosaccharides, 0.05-0.1 parts by weight of grape seed extract, 0.3-0.6 parts by weight of yeast extract, and 0.2-0.6 parts by weight of xanthan gum.

3. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that: In S1.2, the enzymatically hydrolyzed composite fruit and vegetable juice is obtained by adding 0.1% w / w cellulase to the composite fruit and vegetable juice, enzymatically hydrolyzing it at 45°C for 1.5-2 hours, then inactivating the enzyme at 90°C for 5 minutes, and finally cooling it to 30°C.

4. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, wherein: In S1.2, the temperature of the high-temperature instantaneous sterilization is 100-110° C., and the processing time is 3-5 seconds.

5. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that: In S1.3, the fermentation was carried out in stages with controlled temperature: the fermentation was allowed to stand at 38°C for 8 hours until the pH value was 4.5-4.7, and then the fermentation was stirred at 36°C with a speed of 50 rpm for 16 hours until the pH value was 4.5, and the temperature was lowered to 4°C after maintaining the temperature for 2 hours.

6. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that: In S1.3, the probiotic microcapsules are prepared by embedding the probiotic suspension in a sodium alginate-wood unicorn powder-gellan gum composite wall material, loading the lauric acid-stearic acid eutectic and then modifying the chitosan coating; The probiotic microcapsules were prepared by dissolving 1.8-2.2% w / v sodium alginate, 1.2-1.8% w / v wood unicorn powder, and 0.4-0.6% w / v gellan gum in purified water at 60-65°C, stirring at 300-400 rpm for 40-50 minutes until completely dissolved, cooling to 25-30°C, and allowing to stand for 20-30 minutes to degas, thereby obtaining a wall material solution. Pour the concentrated probiotic suspension into a sealed tank and pass nitrogen for 5-10 minutes to reduce the dissolved oxygen to less than 0.3 mg / L; The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the electrostatic microcapsule generator reservoir to form uniform droplets, and then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80 at a liquid level of 15-20 cm. The solution was kept in contact for 8-12 minutes to form gel microspheres. The gel microspheres were collected using a sterile sieve, transferred to a 0.5% w / v calcium chloride solution, and secondary cured at 4°C for 10 minutes. The microspheres were then rinsed 2-3 times with a pH 7.0 phosphate buffer solution. The lauric acid-stearic acid eutectic was heated to a liquid state and vacuum impregnated onto the surface of the microcapsules. The microspheres were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 40-50 rpm for 3-5 minutes, drained, and then dried with cold air. The probiotic microcapsules were then rinsed 1-2 times with a 1% sodium bicarbonate solution.

7. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 6, characterized in that: The concentrated probiotic suspension is prepared by activating Lactobacillus plantarum and Bifidobacterium bifidum in a volume ratio of 8:2 in MRS medium, centrifuging at 6000-8000 rpm for 10 minutes at 4°C to collect the bacteria, and then resuspending them in a 10% w / v sterile skim milk solution to a viable bacteria concentration of more than 1×10 11 CFU / mL.

8. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 6, characterized in that: The voltage of the electrostatic microcapsule generator is 8-10 kV, the needle diameter is 0.4-0.6 mm, and the dropping speed is 3-5 mL / min.

9. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 6, characterized in that: The loading amount of the lauric acid-stearic acid eutectic is 3-5% of the total weight of the probiotic microcapsules.

10. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that: In S1.4, the pressure of vacuum degassing is , time is 5-10 minutes.

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