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

Abstract

The present application relates to the technical field of fermented drinks, in particular to a preparation method of high-activity fruit and vegetable probiotic fermented drinks, which comprises the following steps: adding enzyme-decomposed compound fruit and vegetable juice, mineral water, xylo-oligosaccharide, grape seed extract, yeast extract and xanthan gum into a fermentation tank and stirring until completely dissolved; then sterilizing by high-temperature instantaneous sterilization; adding probiotic microcapsules into the sterilized mixture and performing stage-controlled temperature fermentation to obtain a fermentation liquor; homogenizing the fermentation liquor and finally performing vacuum degassing to obtain the high-activity fruit and vegetable probiotic fermented drink. The composite wall material (sodium alginate-wood kylin powder-konjac gum) of the probiotic microcapsules forms a dense gel network, effectively isolates the bacteriostatic components such as phenols and organic acids in fruits and vegetables, and thus resists the damage of pH fluctuation and temperature change to the bacterial body.

Description

Technical Field

[0001] This invention relates to the field of fermented beverage technology, and more specifically, to a method for preparing a fermented beverage with highly active fruit and vegetable probiotics. Background Technology

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

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

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

[0005] This invention provides a method for preparing a fermented beverage with highly active fruit and vegetable probiotics, comprising the following steps:

[0006] S1.1 Weigh the following ingredients separately: compound fruit and vegetable juice, mineral water, probiotic microcapsules, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum;

[0007] S1.2 Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum to a fermenter, and stir at 200-300 rpm for 15-20 minutes at 40°C until completely dissolved; then sterilize by high-temperature instantaneous sterilization to obtain the mixture.

[0008] S1.3 Add probiotic microcapsules to the sterilized mixture and carry out staged temperature-controlled fermentation to obtain fermentation liquid;

[0009] S1.4 Homogenize the fermentation liquid at 4℃ and 20-25MPa pressure 1-2 times; finally, perform vacuum degassing to obtain a highly active fruit and vegetable probiotic fermented beverage.

[0010] Preferably, in step 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 xylooligosaccharides, 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.

[0011] Preferably, in step S1.2, the enzymatically hydrolyzed compound fruit and vegetable juice is obtained by adding 0.1% w / w cellulase to the compound 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.

[0012] Preferably, in step S1.2, the temperature for high-temperature instantaneous sterilization is 100-110℃, and the processing time is 3-5 seconds.

[0013] Preferably, in step S1.3, the fermentation is carried out in stages with controlled temperature: the fermentation is carried out at 38°C for 8 hours until the pH is 4.5-4.7, and then at 36°C with stirring at 50 rpm for 16 hours until the pH is 4.5. After maintaining this temperature for 2 hours, the temperature is lowered to 4°C.

[0014] Preferably, in S1.3, the probiotic microcapsules are prepared by embedding a probiotic suspension in a sodium alginate-wood powder-gellan gum composite wall material, and then modifying it with a chitosan coating after loading a lauric acid-stearic acid eutectic.

[0015] The preparation method of probiotic microcapsules is as follows: Dissolve 1.8-2.2% w / v sodium alginate, 1.2-1.8% w / v Euphorbia lactea powder and 0.4-0.6% w / v gellan gum in purified water at 60-65℃, stir at 300-400 rpm for 40-50 min until completely dissolved, cool to 25-30℃ and let stand for 20-30 min to degas, to obtain the wall material solution;

[0016] Inject the concentrated probiotic suspension into a sealed container and purge with nitrogen for 5-10 minutes to reduce dissolved oxygen to less than 0.3 mg / L.

[0017] The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3, placed in the storage tank of the electrostatic microcapsule generator to form uniform droplets, and then dropped vertically into a coagulation bath containing 0.05% v / v Tween-80 and 2.0% w / v calcium chloride. The liquid level was 15-20 cm high, and the gel microspheres were formed after 8-12 minutes of contact.

[0018] Gel microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution. The mixture was then subjected to secondary curing at 4°C for 10 min, followed by rinsing 2-3 times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules 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 min, drained, and then air-dried. Finally, the microcapsules were rinsed 1-2 times with a 1% sodium bicarbonate solution to obtain the probiotic microcapsules.

[0019] Preferably, the concentrated probiotic suspension is prepared by activating a probiotic mixture of *Lactobacillus plantarum* and *Bifidobacterium bifidum* at a volume ratio of 8:2 in MRS medium, collecting the bacterial cells by centrifugation at 6000-8000 rpm for 10 min at 4°C, and then resuspending the suspension in 10% w / v sterile skim milk solution until the viable cell concentration is greater than 1×10⁻⁶. 11 CFU / mL.

[0020] Preferably, the voltage of the electrostatic microcapsule generator is 8-10kV, the needle diameter is 0.4-0.6mm, and the dripping speed is 3-5mL / min.

[0021] Preferably, the loading of the lauric acid-stearic acid eutectic is 3-5% of the total weight of the probiotic microcapsules.

[0022] Preferably, in step S1.4, the pressure of vacuum degassing is... The time is 5-10 minutes.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] In the preparation method of the high-activity fruit and vegetable probiotic fermented beverage of the present invention, the composite wall material (sodium alginate-Kirin powder-gellan gum) of the probiotic microcapsules forms a dense gel network, which effectively isolates antibacterial components such as phenols and organic acids in fruits and vegetables, thereby resisting the damage to the bacteria caused by pH fluctuations and temperature changes. In addition, the chitosan coating further enhances the density of the microcapsules, inhibits oxygen penetration, and reduces oxidative stress. Chitosan and polysaccharides such as sodium alginate work synergistically to form a hard and dense microcapsule shell, which significantly improves the survival rate of probiotics. The loaded lauric acid-stearic acid eutectic melts slowly in an acidic environment, avoiding the inactivation of probiotics. Attached Figure Description

[0025] Figure 1 This is a bar chart comparing the viability of different samples in this invention. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] The compound fruit and vegetable juice is made by blending watermelon juice, papaya juice, and red orange juice in a volume ratio of 5:3:2; the watermelon juice concentration ratio is 4:1, the papaya juice concentration ratio is 3.5:1, and the red orange juice concentration ratio is 6:1.

[0028] Grape seed extract CAS No.: 84929-27-1, sourced from Shanghai Yuanye Biotechnology Co., Ltd., with main active ingredients including oligomeric proanthocyanidins, proanthocyanidins, polyphenols (catechins, epicatechins), resveratrol, tannins, flavonoids, etc.

[0029] Yeast extract CAS number: 8013-01-2, sourced from Beijing Solarbio Technology Co., Ltd., with main active ingredients including proteins, amino acids, peptides, nucleotides, vitamins (such as B vitamins), and trace elements (such as calcium, iron, zinc, etc.).

[0030] The preservation numbers for *Lactobacillus plantarum* and *Bifidobacterium bifidum* are CGMCC No. 1258 and CGMCC No. 11959, respectively.

[0031] Preparation method of lauric acid-stearic acid eutectic: Lauric acid and stearic acid are mixed in a molar ratio of 1:1 into a reaction vessel, and the mixture is slowly heated to 75°C under nitrogen protection. The mixture is mechanically stirred at 300 rpm for 40 min to form a homogeneous liquid mixture. The mixture is then slowly cooled to 56°C at a rate of 0.5°C / min and kept at a constant temperature for 2 h. The mixture is then rapidly cooled to below 25°C to solidify, and then pulverized through a 100-mesh sieve to obtain the lauric acid-stearic acid eutectic.

[0032] The enzymatically hydrolyzed compound fruit and vegetable juice is obtained by adding 0.1% w / w cellulase to the compound fruit and vegetable juice, enzymatically hydrolyzing at 45℃ for 2 hours, then inactivating the enzyme at 90℃ for 5 minutes, and finally cooling to 30℃.

[0033] Probiotic microcapsules are prepared by encapsulating probiotic suspension in a sodium alginate-wood powder-gellan gum composite wall material, and then modifying it with a chitosan coating after loading a lauric acid-stearic acid eutectic.

[0034] Preparation steps of wood euphorbia powder: Select fresh wood euphorbia leaves and manually remove the sharp thorns at the leaf base; rinse the leaf surface with running water to remove impurities and drain the water; lay the leaves flat on a drying device and dry them in a ventilated environment at 40-50℃ for 12-24 hours until the moisture content is less than 10%; grind the dried leaves into powder using a pulverizer and pass them through an 80-100 mesh sieve to remove coarse fiber particles.

[0035] Concentrated probiotic suspension is produced by activating probiotics (Lactobacillus plantarum and Bifidobacterium bifidum) in MRS medium at a volume ratio of 8:2, collecting the bacterial cells by centrifugation at 8000 rpm for 10 min at 4°C, and then resuspending them in 10% w / v sterile skim milk solution until the viable cell concentration is greater than 1×10⁻⁶. 11 CFU / mL.

[0036] Example 1: A method for preparing a fermented beverage with highly active fruit and vegetable probiotics, comprising the following steps:

[0037] S1.1 Weigh the following ingredients by weight: 50 parts of compound fruit and vegetable juice, 20 parts of mineral water, 3 parts of probiotic microcapsules, 1 part of xylooligosaccharide, 0.05 parts of grape seed extract, 0.3 parts of yeast extract, and 0.2 parts of xanthan gum.

[0038] S1.2 Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum to the fermenter, and stir at 300 rpm for 20 minutes at 40°C until completely dissolved; then sterilize by high-temperature instantaneous sterilization (temperature is 110°C, treatment time is 5s) to obtain the mixture.

[0039] S1.3 Add probiotic microcapsules to the sterilized mixture, let it stand at 38℃ for 8 hours to ferment until the pH is 4.7, then stir at 36℃ at 50 rpm for 16 hours to ferment until the pH is 4.5, maintain for 2 hours and then cool down to 4℃ to obtain the fermentation liquid.

[0040] S1.4 The fermentation broth was homogenized twice at 4℃ and 20MPa; finally, it was degassed under vacuum at -0.08MPa for 10 minutes to obtain a fermented beverage with high-activity fruit and vegetable probiotics.

[0041] Furthermore, the preparation method of probiotic microcapsules is as follows: 1.8% w / v sodium alginate, 1.2% w / v Euphorbia lactea powder and 0.4% w / v gellan gum are dissolved in pure water at 60℃, stirred at 300 rpm for 40 min until completely dissolved, cooled to 25℃ and allowed to stand for degassing for 30 min to obtain the wall material solution;

[0042] Inject the concentrated probiotic suspension into a sealed container and purge with nitrogen for 5-10 minutes to reduce dissolved oxygen to less than 0.3 mg / L.

[0043] The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3 and placed in the storage tank of an electrostatic microcapsule generator (voltage 8kV, needle diameter 0.5mm, dripping speed 4mL / min) to form uniform droplets. The droplets were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80, with a liquid level of 20cm. After 10 minutes of contact, gel microspheres were formed.

[0044] Gel microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution. They were then subjected to a second curing process at 4°C for 10 min, followed by rinsing three times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic (3% of the total weight of the probiotic microcapsules) was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, agitated at 50 rpm for 5 min, drained, and then air-dried. Finally, the microcapsules were obtained by rinsing twice with a 1% sodium bicarbonate solution.

[0045] Example 2: A method for preparing a fermented beverage with highly active fruit and vegetable probiotics, comprising the following steps:

[0046] S1.1 Weigh the following ingredients by weight: 60 parts of compound fruit and vegetable juice, 25 parts of mineral water, 4 parts of probiotic microcapsules, 1.5 parts of xylooligosaccharide, 0.07 parts of grape seed extract, 0.5 parts of yeast extract, and 0.4 parts of xanthan gum.

[0047] S1.2 Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum to the fermenter, and stir at 300 rpm for 20 minutes at 40°C until completely dissolved; then sterilize by high-temperature instantaneous sterilization (temperature is 110°C, treatment time is 5s) to obtain the mixture.

[0048] S1.3 Add probiotic microcapsules to the sterilized mixture, let it stand at 38℃ for 8 hours to ferment until the pH is 4.7, then stir at 36℃ at 50 rpm for 16 hours to ferment until the pH is 4.5, maintain for 2 hours and then cool down to 4℃ to obtain the fermentation liquid.

[0049] S1.4 The fermentation broth was homogenized twice at 4℃ and 20MPa; finally, it was degassed under vacuum at -0.08MPa for 10 minutes to obtain a fermented beverage with high-activity fruit and vegetable probiotics.

[0050] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.0% w / v sodium alginate, 1.5% w / v euphorbia milii powder and 0.5% w / v gellan gum are dissolved in purified water at 60℃, stirred at 300 rpm for 40 min until completely dissolved, cooled to 25℃ and allowed to stand for degassing for 30 min to obtain the wall material solution;

[0051] Inject the concentrated probiotic suspension into a sealed container and purge with nitrogen for 5-10 minutes to reduce dissolved oxygen to less than 0.3 mg / L.

[0052] The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3 and placed in the storage tank of an electrostatic microcapsule generator (voltage 8kV, needle diameter 0.5mm, dripping speed 4mL / min) to form uniform droplets. The droplets were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80, with a liquid level of 20cm. After 10 minutes of contact, gel microspheres were formed.

[0053] Gel microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution. They were then subjected to secondary curing at 4°C for 10 min, followed by rinsing three times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic (4% of the total weight of the probiotic microcapsules) was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, agitated at 50 rpm for 5 min, drained, and then air-dried. Finally, the microcapsules were obtained by rinsing twice with a 1% sodium bicarbonate solution.

[0054] Example 3: A method for preparing a fermented beverage with highly active fruit and vegetable probiotics, comprising the following steps:

[0055] S1.1 Weigh the following ingredients by weight: 65 parts of compound fruit and vegetable juice, 30 parts of mineral water, 5 parts of probiotic microcapsules, 2 parts of xylooligosaccharide, 0.1 parts of grape seed extract, 0.6 parts of yeast extract, and 0.6 parts of xanthan gum.

[0056] S1.2 Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum to the fermenter, and stir at 300 rpm for 20 minutes at 40°C until completely dissolved; then sterilize by high-temperature instantaneous sterilization (temperature is 110°C, treatment time is 5s) to obtain the mixture.

[0057] S1.3 Add probiotic microcapsules to the sterilized mixture, let it stand at 38℃ for 8 hours to ferment until the pH is 4.7, then stir at 36℃ at 50 rpm for 16 hours to ferment until the pH is 4.5, maintain for 2 hours and then cool down to 4℃ to obtain the fermentation liquid.

[0058] S1.4 The fermentation broth was homogenized twice at 4℃ and 20MPa; finally, it was degassed under vacuum at -0.08MPa for 10 minutes to obtain a fermented beverage with high-activity fruit and vegetable probiotics.

[0059] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.2% w / v sodium alginate, 1.8% w / v Euphorbia lactea powder and 0.6% w / v gellan gum are dissolved in purified water at 60℃, stirred at 300 rpm for 40 min until completely dissolved, cooled to 25℃ and allowed to stand for degassing for 30 min to obtain the wall material solution;

[0060] Inject the concentrated probiotic suspension into a sealed container and purge with nitrogen for 5-10 minutes to reduce dissolved oxygen to less than 0.3 mg / L.

[0061] The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3 and placed in the storage tank of an electrostatic microcapsule generator (voltage 8kV, needle diameter 0.5mm, dripping speed 4mL / min) to form uniform droplets. The droplets were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80, with a liquid level of 20cm. After 10 minutes of contact, gel microspheres were formed.

[0062] Gel microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution. They were then subjected to secondary curing at 4°C for 10 min, followed by rinsing three times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic (5% of the total weight of the probiotic microcapsules) was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, shaken at 50 rpm for 5 min, drained, and then air-dried. Finally, the microcapsules were obtained by rinsing twice with a 1% sodium bicarbonate solution.

[0063] Example 4: A method for preparing a fermented beverage with highly active fruit and vegetable probiotics, comprising the following steps:

[0064] S1.1 Weigh the following ingredients by weight: 60 parts of compound fruit and vegetable juice, 25 parts of mineral water, 5 parts of probiotic microcapsules, 1.5 parts of xylooligosaccharide, 0.07 parts of grape seed extract, 0.5 parts of yeast extract, and 0.4 parts of xanthan gum.

[0065] S1.2 Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum to the fermenter, and stir at 300 rpm for 20 minutes at 40°C until completely dissolved; then sterilize by high-temperature instantaneous sterilization (temperature is 110°C, treatment time is 5s) to obtain the mixture.

[0066] S1.3 Add probiotic microcapsules to the sterilized mixture, let it stand at 38℃ for 8 hours to ferment until the pH is 4.7, then stir at 36℃ at 50 rpm for 16 hours to ferment until the pH is 4.5, maintain for 2 hours and then cool down to 4℃ to obtain the fermentation liquid.

[0067] S1.4 The fermentation broth was homogenized twice at 4℃ and 20MPa; finally, it was degassed under vacuum at -0.08MPa for 10 minutes to obtain a fermented beverage with high-activity fruit and vegetable probiotics.

[0068] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.0% w / v sodium alginate, 1.5% w / v euphorbia milii powder and 0.5% w / v gellan gum are dissolved in purified water at 60℃, stirred at 300 rpm for 40 min until completely dissolved, cooled to 25℃ and allowed to stand for degassing for 30 min to obtain the wall material solution;

[0069] Inject the concentrated probiotic suspension into a sealed container and purge with nitrogen for 5-10 minutes to reduce dissolved oxygen to less than 0.3 mg / L.

[0070] The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3 and placed in the storage tank of an electrostatic microcapsule generator (voltage 8kV, needle diameter 0.5mm, dripping speed 4mL / min) to form uniform droplets. The droplets were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80, with a liquid level of 20cm. After 10 minutes of contact, gel microspheres were formed.

[0071] Gel microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution. They were then subjected to secondary curing at 4°C for 10 min, followed by rinsing three times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic (4% of the total weight of the probiotic microcapsules) was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, agitated at 50 rpm for 5 min, drained, and then air-dried. Finally, the microcapsules were obtained by rinsing twice with a 1% sodium bicarbonate solution.

[0072] Example 5: A method for preparing a fermented beverage with highly active fruit and vegetable probiotics, comprising the following steps:

[0073] S1.1 Weigh the following ingredients by weight: 60 parts of compound fruit and vegetable juice, 25 parts of mineral water, 6 parts of probiotic microcapsules, 1.5 parts of xylooligosaccharide, 0.07 parts of grape seed extract, 0.5 parts of yeast extract, and 0.4 parts of xanthan gum.

[0074] S1.2 Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract and xanthan gum to the fermenter, and stir at 300 rpm for 20 minutes at 40°C until completely dissolved; then sterilize by high-temperature instantaneous sterilization (temperature is 110°C, treatment time is 5s) to obtain the mixture.

[0075] S1.3 Add probiotic microcapsules to the sterilized mixture, let it stand at 38℃ for 8 hours to ferment until the pH is 4.7, then stir at 36℃ at 50 rpm for 16 hours to ferment until the pH is 4.5, maintain for 2 hours and then cool down to 4℃ to obtain the fermentation liquid.

[0076] S1.4 The fermentation broth was homogenized twice at 4℃ and 20MPa; finally, it was degassed under vacuum at -0.08MPa for 10 minutes to obtain a fermented beverage with high-activity fruit and vegetable probiotics.

[0077] Furthermore, the preparation method of probiotic microcapsules is as follows: 2.0% w / v sodium alginate, 1.5% w / v euphorbia milii powder and 0.5% w / v gellan gum are dissolved in purified water at 60℃, stirred at 300 rpm for 40 min until completely dissolved, cooled to 25℃ and allowed to stand for degassing for 30 min to obtain the wall material solution;

[0078] Inject the concentrated probiotic suspension into a sealed container and purge with nitrogen for 5-10 minutes to reduce dissolved oxygen to less than 0.3 mg / L.

[0079] The deoxygenated concentrated probiotic suspension was added to the wall material solution at a volume ratio of 1:3 and placed in the storage tank of an electrostatic microcapsule generator (voltage 8kV, needle diameter 0.5mm, dripping speed 4mL / min) to form uniform droplets. The droplets were then vertically dropped into a 2.0% w / v calcium chloride coagulation bath containing 0.05% v / v Tween-80, with a liquid level of 20cm. After 10 minutes of contact, gel microspheres were formed.

[0080] Gel microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution. They were then subjected to secondary curing at 4°C for 10 min, followed by rinsing three times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic (4% of the total weight of the probiotic microcapsules) was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid, agitated at 50 rpm for 5 min, drained, and then air-dried. Finally, the microcapsules were obtained by rinsing twice with a 1% sodium bicarbonate solution.

[0081] Comparative Example 1: This comparative example, compared to Example 2, did not contain probiotic microcapsules.

[0082] Comparative Example 2: Compared with Example 2, this comparative example did not add wood unicorn powder as a wall material, but only used sodium alginate and gellan gum as wall materials.

[0083] Comparative Example 3: Compared to Example 2, the probiotic microcapsules in this comparative example were not loaded with lauric acid-stearic acid cocrystal.

[0084] This invention relates to a highly active fruit and vegetable probiotic fermented beverage prepared via probiotic microcapsules. The performance indicators and testing standards for this beverage are as follows:

[0085] Take 10 mL of the beverage and add 90 mL of sterile phosphate-buffered saline (PBS, pH 7.0), 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 10-fold serial dilutions 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 anaerobic cultured at 37°C for 72 h); select plates with colony counts between 30 and 300 for counting, and calculate the viable count (CFU / mL) according to the formula = average colony count × dilution factor × 10.

[0086] Within 24 hours after filling, the initial viable count was determined using the above method as the baseline value; samples at the end of the shelf life (e.g., 21 days, refrigerated at 4℃) were measured using the same method; accelerated testing was conducted by storing at 37℃ for 14 days (simulating a 3-month shelf life), with samples taken and measured every 48 hours; viable count (%) = (number of viable bacteria after storage ÷ initial number of viable bacteria) × 100.

[0087] The highly active fruit and vegetable probiotic fermented beverages prepared in Examples 1-5 and Comparative Examples 1-3 were tested according to the above standards, and the data obtained are shown in Table 1:

[0088] Table 1 Performance data of fermented beverages with high-activity fruit and vegetable probiotics in Examples 1-5 and Comparative Examples 1-3

[0089]

[0090] As can be seen from Examples 1-3, changes in the raw material composition of highly active fruit and vegetable probiotic fermented beverages can improve the number of live bacteria and the stability of probiotics during the shelf life of the fermented beverage.

[0091] Sodium alginate-gellan gel network combined with the fibrous skeleton of wood unicorn powder effectively shields the bacterial cell membrane from oxidative damage caused by phenolic substances (such as tannic acid) in fruit and vegetable juices; the chitosan coating reduces dissolved oxygen permeability by positively charged densifying the membrane structure, inhibiting the growth of aerobic bacteria and the aerobic metabolic death of probiotics; the loaded lauric acid-stearic acid eutectic melts in an acidic environment, which can reduce the killing effect of acid on probiotics, thereby increasing the viability rate.

[0092] Xylooligosaccharides, as a highly effective bioactive component, are 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 other bacteria, and simultaneously increasing the biomass of the target probiotics. In addition, proanthocyanidins in grape seed extract can scavenge free radicals generated during fermentation and storage, reducing the risk of DNA oxidative damage. Yeast extract provides endogenous antioxidant peptides such as glutathione, precisely controlling the pH at the fermentation endpoint within a suitable range, avoiding autolysis of the cells due to an overly acidic environment. Xanthan gum forms a shear-thinned colloidal network during the homogenization stage, encapsulating microcapsules and inhibiting their sedimentation, while also reducing oxygen penetration after filling.

[0093] Furthermore, a comparison of Examples 2, 4, and 5 shows that when other components in the high-activity fruit and vegetable probiotic fermented beverage remain unchanged, and the weight of probiotic microcapsules is continuously increased, the number of live bacteria in the fermented beverage and the stability of probiotics during the shelf life continuously decrease.

[0094] Excessive microcapsules aggregate in the fermentation broth due to weakened steric hindrance, forming clumps that prevent adequate dispersion during homogenization. The clumps create an anaerobic microenvironment, hindering the metabolic activity of probiotics. Simultaneously, accelerated clump settling leads to a localized increase in live bacteria density at the bottom of the beverage and increased dissolved oxygen penetration at the top, accelerating surface bacterial death. The microcapsule wall material (sodium alginate-gellan gum complex) continuously dissolves in the acidic fermentation broth, and the release of calcium ions consumes key buffers, causing an uncontrolled pH drop in the later stages of fermentation, exceeding the tolerance limit of Bifidobacterium bifidum. Free polysaccharide chains compete with xanthan gum for water molecules, disrupting the colloidal network structure and increasing the precipitation rate. Excess lauric acid-stearic acid eutectic rearrangement occurs during storage, with some precipitating as solid crystals. These crystals puncture the microcapsule walls, causing premature leakage of probiotics. Adsorption at the fermentation broth interface accelerates oxygen diffusion, triggering the proliferation of aerobic bacteria and oxidative damage to probiotics.

[0095] Based on the above test experiments and combined with Figure 1 Therefore, Example 2 is considered the optimal example.

[0096] As can be seen from Example 2 and Comparative Example 1: Although the natural pH of fruit and vegetable juice can inhibit the growth of miscellaneous bacteria, a continuously acidic environment will damage the metabolic enzyme activity of probiotics, accelerate ATP synthesis disorders, and lead to the death of live bacteria (the number of live bacteria decreases after storage at room temperature); antioxidants such as polyphenols and vitamin C in fruits and vegetables gradually become ineffective during storage, and residual oxygen free radicals attack bacterial DNA and membrane proteins, causing irreversible damage; temperature fluctuations during storage at room temperature cause an imbalance in the expression of heat shock proteins in bacteria, abnormal cell membrane fluidity, and a shortened half-life of live bacteria; the increased lactic acid concentration in the later stage of fermentation inhibits the activity of proton transport enzymes, blocks the transmembrane proton gradient, and causes the energy metabolism of bacteria to stagnate, thereby reducing the stability of probiotics during the shelf life.

[0097] As can be seen from Example 2 and Comparative Example 2, the *Euphorbia lactea* powder is rich in natural pectin and hemicellulose, which form a three-dimensional network structure with sodium alginate through hydrogen bonds and hydrophobic interactions, enhancing the density of the microcapsule walls. While the sodium alginate-gellan gum system can form a gel, it lacks the support of plant polysaccharides, leading to increased porosity of the capsule walls and making it easier for hydrogen ions to penetrate in acidic environments, resulting in the inactivation of a large number of live bacteria. Furthermore, the polyphenols (such as ellagic acid) in *Euphorbia lactea* powder have the ability to scavenge free radicals, reducing the damage to the bacteria caused by oxidative stress during storage. *Euphorbia lactea* powder also contains natural anti-acid components (such as pectin calcium), which can neutralize some acid and further protect probiotics. Simultaneously, the soluble fibers such as arabinoxylan in *Euphorbia lactea* 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. Without the addition of *Euphorbia lactea* powder, probiotics die off rapidly due to the interruption of nutrient supply, and their half-life at room temperature is significantly shortened.

[0098] As can be seen from Examples 2 and 3, when unloaded, the microcapsule wall material (such as sodium alginate) dissolves rapidly in an acidic environment, causing probiotics to be exposed to adverse conditions prematurely, resulting in a high loss rate of live bacteria. In addition, the lauric acid-stearic acid co-crystal forms a dense crystalline layer on the surface of the microcapsules, which can effectively block oxygen permeation and prevent the generation of reactive oxygen species, thereby prolonging the half-life of probiotics. If the co-crystal is missing, oxygen in the environment will directly contact the bacteria, accelerating aerobic metabolism and triggering membrane lipid peroxidation, further shortening the survival time of live bacteria. At the same time, the co-crystal 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 co-crystal is missing, probiotics will be more easily inactivated during storage or transportation, resulting in a decrease in the number of live bacteria.

[0099] The foregoing has shown and described 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 embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a fermented beverage with highly active fruit and vegetable probiotics, characterized in that, Includes the following steps: S1.1 Weigh the following ingredients by weight: 50-65 parts of compound fruit and vegetable juice, 20-30 parts of mineral water, 3-5 parts of probiotic microcapsules, 1-2 parts of xylooligosaccharides, 0.05-0.1 parts of grape seed extract, 0.3-0.6 parts of yeast extract, and 0.2-0.6 parts of xanthan gum. S1.2 Add 0.1% w / w cellulase to the compound fruit and vegetable juice, and enzymatically hydrolyze it at 45℃ for 1.5-2 hours. Then, inactivate the enzyme at 90℃ for 5 minutes and cool it to 30℃ to obtain the enzymatically hydrolyzed compound fruit and vegetable juice. Add the enzymatically hydrolyzed compound fruit and vegetable juice, mineral water, xylooligosaccharides, grape seed extract, yeast extract, and xanthan gum to a fermenter and stir at 200-300 rpm at 40℃ for 15-20 minutes until completely dissolved. Then, sterilize it by high-temperature instantaneous sterilization to obtain the mixture. S1.3 Add probiotic microcapsules to the sterilized mixture and carry out staged temperature-controlled fermentation: let it stand at 38℃ for 8 hours until the pH is 4.5-4.7, then stir at 36℃ at 50 rpm for 16 hours until the pH is 4.5, maintain for 2 hours and then cool down to 4℃ to obtain the fermentation liquid. S1.

4. Homogenize the fermentation liquid at 4℃ and 20-25MPa pressure 1-2 times; finally, degas it under vacuum to obtain a highly active fruit and vegetable probiotic fermented beverage. The preparation method of the probiotic microcapsules is as follows: 1.8-2.2% w / v sodium alginate, 1.2-1.8% w / v Euphorbia lactea powder, and 0.4-0.6% w / v gellan gum are dissolved in pure water at 60-65℃, stirred at 300-400 rpm for 40-50 minutes until completely dissolved, cooled to 25-30℃, and allowed to stand for degassing for 20-30 minutes to obtain a wall material solution; the concentrated probiotic suspension is injected into a sealed container, and nitrogen is purged for 5-10 minutes to ensure dissolved oxygen is less than 0.3 mg / L; the deoxygenated concentrated probiotic suspension is added to the wall material solution at a volume ratio of 1:3, placed in the storage tank of an electrostatic microcapsule generator to form uniform droplets, and vertically dripped into a solution containing 0. Gel microspheres were formed in a 2.0% w / v calcium chloride coagulation bath at 0.5% v / v Tween-80 for 8-12 minutes at a liquid level of 15-20 cm. The microspheres were collected using a sterile sieve and transferred to a 0.5% w / v calcium chloride solution for secondary solidification at 4°C for 10 minutes. The microspheres were then rinsed 2-3 times with pH 7.0 phosphate buffer. Lauric acid-stearic acid eutectic was heated to a liquid state and impregnated onto the microcapsule surface using vacuum. The microcapsules were then immersed in a 0.10% w / v chitosan acetate solution containing 1% v / v acetic acid and shaken at 40-50 rpm for 3-5 minutes. After draining, the microcapsules were dried with cold air. Finally, the microcapsules were rinsed 1-2 times with a 1% sodium bicarbonate solution to obtain the probiotic microcapsules. The concentrated probiotic suspension is prepared by activating probiotics, namely Lactobacillus plantarum and Bifidobacterium bifidum, in an 8:2 volume ratio in MRS medium, centrifuging at 6000-8000 rpm for 10 min at 4°C to collect the cells, and then resuspending them in 10% w / v sterile skim milk solution until the viable cell concentration is greater than 1×10¹¹ CFU / mL.

2. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that, In step S1.2, the temperature for high-temperature instantaneous sterilization is 100-110℃, and the processing time is 3-5 seconds.

3. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that, The voltage of the electrostatic microcapsule generator is 8-10kV, the needle diameter is 0.4-0.6mm, and the dripping speed is 3-5mL / min.

4. The method for preparing a fermented beverage with highly active fruit and vegetable probiotics according to claim 1, characterized in that, The loading of the lauric acid-stearic acid eutectic is 3-5% of the total weight of the probiotic microcapsules.

5. The method for preparing a highly active fruit and vegetable probiotic fermented beverage according to claim 1, characterized in that, In step S1.4, the vacuum degassing pressure is -0.08 to -0.1 MPa, and the time is 5 to 10 minutes.

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