Probiotic fermented fruit and vegetable beverage with functions of resisting fatigue and enhancing immunity

Abstract

The invention relates to the technical field of preparation of probiotic fermented beverages, in particular to a probiotic fermented fruit and vegetable beverage with functions of resisting fatigue and enhancing immunity. The hawthorn fruit beverage specifically comprises the following components in parts by mass: 60-80 parts of Zhongtian big hawthorn fruits, 10-20 parts of autumn pears, 11-15 parts of grapes, 9-15 parts of kiwi fruits, 6-10 parts of pomegranates, 3-5 parts of figs, 3-5 parts of beet roots, 3-5 parts of pumpkins, 1-2 parts of kale, 1-2 parts of honey and 2-3 parts of zymophyte. The beverage product provided by the invention can significantly prolong exhaustive swimming time, improve liver glycogen reserve, reduce serum urea nitrogen level after exercises, and effectively relieve physical fatigue; meanwhile, the food can comprehensively enhance the immune function of the organism, including improving the activity of phagocytes, promoting the proliferation of lymphocytes and enhancing humoral immune response, and provides a natural and healthy solution for resisting fatigue and enhancing the immunity function through daily diet for consumers.

Description

Technical Field

[0001] This invention relates to the field of probiotic fermented beverage preparation technology, and in particular to a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions. Background Technology

[0002] With the fast pace of modern life and increasing work pressure, chronic fatigue and the resulting weakened immunity are becoming increasingly common, posing a significant threat to public health. Currently, consumers are increasingly seeking health solutions that are shifting from traditional medicines and health supplements to natural, delicious functional foods and beverages with comprehensive health benefits. Against this backdrop, functional beverages developed through microbial fermentation using fruits and vegetables as a base demonstrate enormous market potential because they combine the inherent nutrients of fruits and vegetables with the active substances produced during fermentation. They precisely meet people's urgent need to upgrade their daily diet for better health, providing a new approach to relieving fatigue and enhancing immunity through everyday consumption.

[0003] In existing technologies, traditional functional beverages mostly achieve their anti-fatigue effects by directly adding nutrients (such as B vitamins and taurine) or chemical extracts. Their forms are limited, and long-term reliance may lead to metabolic burdens. On the other hand, although some probiotic fermented drinks, such as yogurt and kvass, exist on the market, their raw materials are mostly limited to dairy products or grains, with a limited variety of fruits and vegetables, resulting in insufficient nutritional value. In particular, there is a lack of scientifically formulated compound fruit and vegetable fermented products specifically designed for anti-fatigue and immune regulation, leading to limited product functionality and failing to meet the market's pursuit of comprehensive health benefits.

[0004] From a technical perspective, developing such beverages faces two core challenges. First, flavor balance is crucial; fruit and vegetable fermentation can easily produce excessive sourness or unpleasant flavors, affecting palatability. Second, functional synergy is critical; how to ensure the added probiotics work synergistically with the scientifically formulated fruit and vegetable matrix, and how to truly enrich small-molecule active substances (such as short-chain fatty acids and polyphenol conversion products) with anti-fatigue and immune-enhancing functions through the fermentation process, are key to determining the product's ultimate efficacy. Existing technologies often focus on one aspect of the fermentation process or nutritional formulation, failing to systematically integrate both with the clearly defined dual health function goals, resulting in insufficient evidence of product efficacy and weak market competitiveness.

[0005] Therefore, there is an urgent need in this field for an innovative technical solution that can scientifically blend natural fruits and vegetables with clear potential for anti-fatigue and immune-enhancing effects, and then introduce specific probiotic strains for controlled fermentation. This design and process aims to develop a natural health beverage that not only has excellent taste and stability, but also provides consumers with definite anti-fatigue and immune-support functions through the combined action of fermentation metabolites and live bacteria. Summary of the Invention

[0006] The purpose of this invention is to provide a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions, comprising the following components in parts by weight: 60-80 parts of Nakata hawthorn, 10-20 parts of Akizuki pear, 11-15 parts of grape, 9-15 parts of kiwifruit, 6-10 parts of pomegranate, 3-5 parts of fig, 3-5 parts of beetroot, 3-5 parts of pumpkin, 1-2 parts of kale, 1-2 parts of honey, and 2-3 parts of fermentation bacteria; wherein the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides.

[0008] Preferably, the mass ratio of Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides is 20~25:6~12:18~23:7~14.

[0009] Preferably, the bacterial counts of *Lactobacillus plantarum* (Xi'an Shilin Biotechnology Co., Ltd.), *Lactobacillus paracasei* (Xi'an Shilin Biotechnology Co., Ltd.), *Bifidobacterium bifidum* (Shenzhen Lefu Biotechnology Co., Ltd.), and *Leuconostoc mesenteroides* (Xi'an Shilin Biotechnology Co., Ltd.) are all 400-600 million / g.

[0010] This invention provides a method for preparing a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions, comprising the following steps: (1) Mix Nakata hawthorn, Akizuki pear, grape, kiwi, pomegranate, fig, beetroot, pumpkin and kale, add 1 to 2 times the weight of water, and blend to obtain fruit and vegetable puree; (2) Mix the fruit and vegetable puree with enzyme solution for enzymatic hydrolysis, heat to inactivate the enzyme, and add honey to obtain enzymatically hydrolyzed fruit and vegetable puree; (3) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and sealed for fermentation to obtain primary fermented material; (4) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and sealed for fermentation to obtain secondary fermentation material; (5) The secondary fermented material is refrigerated and stored, and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0011] Preferably, the enzyme inactivation in step (2) is carried out at 85~90℃ for 15~20 minutes.

[0012] Preferably, the enzyme solution in step (2) is composed of hemicellulase, α-amylase and rhamnosidase (all purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.), with a mass ratio of 12~16:7~11:7~11.

[0013] Preferably, the mass ratio of the fruit and vegetable puree to the enzyme solution in step (2) is 100:0.3~0.6.

[0014] Preferably, the temperature of the sealed fermentation in step (3) is 36~38℃ and the time is 48~72h.

[0015] Preferably, the temperature of the sealed fermentation in step (4) is 36~38℃ and the time is 30~40h.

[0016] Preferably, the refrigeration temperature in step (5) is 3~5℃ and the time is 20~30h.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The various fruit and vegetable ingredients selected in this invention form a highly efficient synergistic system in terms of nutrition and function. Hawthorn berries, as the main ingredient, are rich in organic acids and flavonoids, laying the foundation for the product's antioxidant, anti-fatigue, and metabolism-boosting effects. Fruits such as Akizuki pears, grapes, and kiwis provide abundant vitamins, polyphenols, and natural sugars, enhancing the taste while further strengthening antioxidant defense and energy replenishment. The addition of pomegranates, figs, beets, pumpkins, and kale not only enriches the diversity of dietary fiber, trace elements, and phytochemicals but also prepares a high-quality and comprehensive substrate for probiotic fermentation. This scientific formulation ensures the complementarity and synergistic effect of various nutrients.

[0018] Furthermore, a deep functional synergy exists between the raw materials and probiotics. Honey, as a natural prebiotic, effectively promotes the colonization and activity of probiotics.

[0019] The combination of *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Bifidobacterium bifidum*, and *Leuconostoc mesenteroides* used in this invention is not a simple superposition of strains, but a carefully designed synergistic system based on their metabolic characteristics. *Lactobacillus plantarum* and *Lactobacillus paracasei*, as leader bacteria, possess strong protease and carbohydrate enzyme activity and acid resistance. In the early stages of fermentation, they rapidly proliferate and decompose proteins and complex polysaccharides in the fruit and vegetable substrate, producing organic acids (mainly lactic acid) and readily available intermediate metabolites such as oligopeptides and oligosaccharides. This process not only effectively lowers the system pH and inhibits the growth of other microorganisms, but also creates a suitable acidic microenvironment and a nutrient-rich substrate for subsequent colonization of the microbial community, completing a key step in the fermentation process.

[0020] In the secondary fermentation stage, where Bifidobacterium bifidum and Leuconostoc mesenteroides continue to ferment, the synergistic effect is further deepened. Bifidobacterium bifidum can efficiently utilize specific oligosaccharides produced in the preceding stage and convert them into short-chain fatty acids such as acetic acid and lactic acid. These substances are core functional factors that directly act on the intestines and regulate immunity and energy metabolism. At the same time, Leuconostoc mesenteroides, with its unique fermentation characteristics, can produce bioactive extracellular polysaccharides and further modify flavor compounds. These two bacteria work synergistically in the stable environment created by the initial microbiota, avoiding direct substrate competition with the pioneer bacteria, forming a synergistic effect along the metabolic pathway. Ultimately, this results in the enrichment of higher concentrations and more diverse anti-fatigue and immune-enhancing active substances, achieving a functional synergy of "1+1>2".

[0021] The fermentation method of this invention achieves synergy between time and microbial metabolism through a sophisticated two-step fermentation process. In the primary fermentation stage, the strong acid-producing and protease activities of *Lactobacillus plantarum* and *Lactobacillus paracasei* preferentially decompose macromolecules such as proteins and polysaccharides in fruit and vegetable purees within 48-72 hours, initially converting the complex matrix into more readily usable oligosaccharides, peptides, and organic acids, laying a solid foundation for subsequent fermentation. In the secondary fermentation stage, *Bifidobacterium bifidum* and *Leuconostoc mesenteroides* are introduced to further synthesize short-chain fatty acids, extracellular polysaccharides, and other small-molecule metabolites with clear anti-fatigue and immune-enhancing functions using the products from the previous stage. This stepwise strategy avoids direct competition between microbial strains, forming a metabolic "relay" and synergy.

[0022] Furthermore, the pre-fermentation enzymatic hydrolysis and the fermentation process itself constitute a crucial synergy. The complex enzyme system of hemicellulase, α-amylase, and rhamnosidase used in this invention achieves multi-target, synergistic breakdown of the cell wall structure of fruits and vegetables through precise formulation. Hemicellulase is primarily responsible for decomposing the complex hemicellulose network in the cell wall, disrupting its tough skeletal structure; α-amylase specifically hydrolyzes starch granules in the fruit and vegetable slurry, releasing fermentable sugars and providing a readily available carbon source for subsequent fermentation; and the key role of rhamnosidase is to specifically hydrolyze bound polyphenols, breaking the garnetoside bonds that bind them, thereby releasing free polyphenols with higher biological activity. These three enzymes complement each other in terms of substrate and mechanism, jointly dismantling the physical barriers and chemical bonds of plant cells, achieving a comprehensive and efficient release of intracellular nutrients.

[0023] The probiotic fermented fruit and vegetable beverage provided by this invention has definite dual effects of anti-fatigue and enhanced immunity. Animal experiments have demonstrated that this product can significantly prolong the time spent swimming to exhaustion, increase liver glycogen reserves, and reduce serum urea nitrogen levels after exercise, effectively relieving physical fatigue. At the same time, it can comprehensively enhance the body's immune function, including increasing phagocytic cell activity, promoting lymphocyte proliferation, and enhancing humoral immune responses, providing consumers with a natural health solution that can be achieved through daily diet.

[0024] This product uses all-natural fruits and vegetables as raw materials, fermented with specific probiotics. It contains no chemical additives, ensuring high safety and a natural flavor. Its innovative scientific formulation and two-step fermentation process achieve highly efficient synergy between the nutrients in the raw materials and the metabolism of the microorganisms, ultimately creating a natural health beverage with excellent taste, stable quality, and clearly defined functions, meeting the modern market's demand for natural, comprehensive functional beverages. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions is composed of the following components in parts by weight: 60 parts of Nakata hawthorn, 10 parts of Akizuki pear, 11 parts of grape, 9 parts of kiwi, 6 parts of pomegranate, 3 parts of fig, 3 parts of beetroot, 3 parts of pumpkin, 1 part of kale, 1 part of honey, and 2 parts of fermentation bacteria; the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides, in a mass ratio of 20:6:18:7, with a bacterial count of 400 million / g for each.

[0028] The preparation method of the probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions includes the following steps: (1) Fruits (Nakata hawthorn, Akizuki pear, grapes, kiwi, pomegranate, fig): Wash, remove pits / seeds, and cut into pieces. Vegetables: Wash and peel beetroot, steam pumpkin until soft. Wash and chop kale.

[0029] (2) Mix all the processed fruits and vegetables together, add water equal to the weight of all the fruits and vegetables, and blend them into a uniform fruit and vegetable puree using a blender; (3) Mix the fruit and vegetable puree with the enzyme solution and hydrolyze at 45°C for 1 hour, then heat at 85°C to inactivate the enzyme for 15 minutes, and add honey to obtain the hydrolyzed fruit and vegetable puree; the enzyme solution is hemicellulase, α-amylase and rhamnosidase, with a mass ratio of 12:7:7; the mass ratio of the fruit and vegetable puree to the enzyme solution is 100:0.3.

[0030] (4) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and fermented at 36°C for 48 hours, but with an exhaust hole to release the small amount of gas produced during fermentation, to obtain the primary fermented material; (5) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and fermented at 36°C for 30 hours to obtain the secondary fermentation material; (6) The secondary fermented material is stored at 3°C ​​for 20 hours and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0031] Example 2

[0032] A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions is composed of the following components in parts by weight: 80 parts of Nakata hawthorn, 20 parts of Akizuki pear, 15 parts of grape, 15 parts of kiwi, 10 parts of pomegranate, 5 parts of fig, 5 parts of beetroot, 5 parts of pumpkin, 2 parts of kale, 2 parts of honey, and 3 parts of fermentation bacteria; the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides, with a mass ratio of 25:12:23:14, and a bacterial count of 600 million / g for each.

[0033] The preparation method of the probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions includes the following steps: (1) Fruits (Nakata hawthorn, Akizuki pear, grapes, kiwi, pomegranate, fig): Wash, remove pits / seeds, and cut into pieces. Vegetables: Wash and peel beetroot, steam pumpkin until soft. Wash and chop kale.

[0034] (2) Mix all the processed fruits and vegetables together, add twice the amount of water, and blend into a uniform fruit and vegetable puree using a blender; (3) Mix the fruit and vegetable puree with the enzyme solution and hydrolyze at 50°C for 2 hours, then heat at 90°C to inactivate the enzyme for 20 minutes, and add honey to obtain the hydrolyzed fruit and vegetable puree; the enzyme solution is hemicellulase, α-amylase and rhamnosidase, with a mass ratio of 16:11:11; the mass ratio of the fruit and vegetable puree to the enzyme solution is 100:0.6.

[0035] (4) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and fermented at 38°C for 72 hours, but with an exhaust hole to release the small amount of gas produced during fermentation, to obtain the primary fermented material; (5) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and fermented at 38°C for 40 hours to obtain the secondary fermentation material; (6) The secondary fermented material is stored at 5°C for 30 hours and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0036] Example 3

[0037] A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions is composed of the following components in parts by weight: 65 parts of Nakata hawthorn, 12 parts of Akizuki pear, 12 parts of grape, 10 parts of kiwi, 7 parts of pomegranate, 3 parts of fig, 3 parts of beetroot, 3 parts of pumpkin, 1 part of kale, 1 part of honey, and 2 parts of fermentation bacteria; the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides, with a mass ratio of 21:7:19:9, and a bacterial count of 400 million / g for each.

[0038] The preparation method of the probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions includes the following steps: (1) Fruits (Nakata hawthorn, Akizuki pear, grapes, kiwi, pomegranate, fig): Wash, remove pits / seeds, and cut into pieces. Vegetables: Wash and peel beetroot, steam pumpkin until soft. Wash and chop kale.

[0039] (2) Mix all the processed fruits and vegetables together, add water of equal weight, and blend into a uniform fruit and vegetable puree using a blender; (3) Mix the fruit and vegetable puree with the enzyme solution and hydrolyze at 46°C for 1 hour, then heat at 86°C to inactivate the enzyme for 16 minutes, and add honey to obtain the hydrolyzed fruit and vegetable puree; the enzyme solution is hemicellulase, α-amylase and rhamnosidase, with a mass ratio of 13:8:8; the mass ratio of the fruit and vegetable puree to the enzyme solution is 100:0.4.

[0040] (4) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and fermented at 36°C for 55 hours, but with an exhaust hole to release the small amount of gas produced during fermentation, to obtain the primary fermented material; (5) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and fermented at 36°C for 32 hours to obtain the secondary fermentation material; (6) The secondary fermented material is stored at 3°C ​​for 22 hours and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0041] Example 4

[0042] A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions is composed of the following components in parts by weight: 75 parts of Nakata hawthorn, 18 parts of Akizuki pear, 13 parts of grape, 13 parts of kiwi, 7 parts of pomegranate, 3 parts of fig, 5 parts of beetroot, 4 parts of pumpkin, 1 part of kale, 2 parts of honey, and 3 parts of fermentation bacteria; the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides, in a mass ratio of 23:9:19:9, with a bacterial count of 600 million / g for each.

[0043] The preparation method of the probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions includes the following steps: (1) Fruits (Nakata hawthorn, Akizuki pear, grapes, kiwi, pomegranate, fig): Wash, remove pits / seeds, and cut into pieces. Vegetables: Wash and peel beetroot, steam pumpkin until soft. Wash and chop kale.

[0044] (2) Mix all the processed fruits and vegetables together, add twice the amount of water, and blend into a uniform fruit and vegetable puree using a blender; (3) Mix the fruit and vegetable puree with the enzyme solution and hydrolyze at 48°C for 1 hour, then heat at 88°C to inactivate the enzyme for 18 minutes, and add honey to obtain the hydrolyzed fruit and vegetable puree; the enzyme solution is hemicellulase, α-amylase and rhamnosidase, with a mass ratio of 15:10:10; the mass ratio of the fruit and vegetable puree to the enzyme solution is 100:0.6.

[0045] (4) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and fermented at 38°C for 60 hours, but with an exhaust hole to release the small amount of gas produced during fermentation, to obtain the primary fermented material; (5) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and fermented at 38°C for 37 hours to obtain the secondary fermentation material; (6) The secondary fermented material is stored at 5°C for 26 hours and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0046] Example 5

[0047] A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions is composed of the following components in parts by weight: 70 parts of Nakata hawthorn, 15 parts of Akizuki pear, 13 parts of grape, 12 parts of kiwi, 8 parts of pomegranate, 4 parts of fig, 4 parts of beetroot, 4 parts of pumpkin, 2 parts of kale, 2 parts of honey, and 3 parts of fermentation bacteria; the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides, in a mass ratio of 23:10:20:12, with a bacterial count of 500 million / g for each.

[0048] The preparation method of the probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions includes the following steps: (1) Fruits (Nakata hawthorn, Akizuki pear, grapes, kiwi, pomegranate, fig): Wash, remove pits / seeds, and cut into pieces. Vegetables: Wash and peel beetroot, steam pumpkin until soft. Wash and chop kale.

[0049] (2) Mix all the processed fruits and vegetables together, add water of equal weight, and blend into a uniform fruit and vegetable puree using a blender; (3) Mix the fruit and vegetable puree with the enzyme solution and hydrolyze at 45°C for 2 hours, then heat at 88°C to inactivate the enzyme for 20 minutes, and add honey to obtain the hydrolyzed fruit and vegetable puree; the enzyme solution is hemicellulase, α-amylase and rhamnosidase, with a mass ratio of 14:9:9; the mass ratio of the fruit and vegetable puree to the enzyme solution is 100:0.5.

[0050] (4) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and fermented at 37°C for 72 hours, but with an exhaust hole to release the small amount of gas produced during fermentation, to obtain the primary fermented material; (5) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and fermented at 37°C for 35 hours to obtain the secondary fermentation material; (6) The secondary fermented material is stored at 4°C for 25 hours and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

[0051] Control group 1

[0052] The other methods are the same as in Example 5, except that the Nakata hawthorn is replaced with an equal amount of apples.

[0053] Control group 2

[0054] The other methods are the same as in Example 5, except that Bifidobacterium bifidum and Leuconostoc mesenteroides are replaced with equal amounts of Bifidobacterium lactis and Lactobacillus acidophilus.

[0055] Control group 3

[0056] The other methods are the same as in Example 5, except that only one fermentation is performed. In step (4), the enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides and fermented at 37°C for 72 hours to obtain fermented material. After being stored at 4°C for 25 hours and homogenized, a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions is obtained.

[0057] Experimental Example 1

[0058] Materials and Methods

[0059] 1.1 Experimental animals: SPF-grade healthy male Kunming mice (Shandong Aileke Biotechnology Co., Ltd.). All mice were housed in a barrier environment. During the experiment, the ambient temperature was controlled at 25±2℃ and the relative humidity was 55±3%.

[0060] 1.2 Experimental Methods: Mice were randomly divided into 6 groups of 10 mice each: experimental group, control group 1, control group 2, control group 3, blank control group, and positive control group. The experimental group, control group 1, control group 2, and control group 3 were administered 0.12 mL of fruit and vegetable beverage prepared according to the corresponding method per 10 g body weight via gavage. The positive control group was administered 0.12 mL of Red Bull energy drink (Red Bull Vitamin Drink (Jiangsu) Co., Ltd.) per 10 g body weight via gavage. The blank control group was administered the same volume of physiological saline via gavage. The experiment lasted for 30 days. After the experiment, the exhaustive swimming time, blood urea nitrogen, and liver glycogen content of each group of mice were measured.

[0061] Weighted swimming experiment: During the experiment, mice were loaded with 5% weight (lead wire) on their tails and placed in a 40cm×60cm×36cm pool for swimming. The water depth was 30cm and the water temperature was maintained at 30±0.5℃. Ten mice were placed in each pool at the same time. The time from when the mice started swimming until they were exhausted (sinking in the water for more than 10 seconds and failing to float to the surface) was recorded using a stopwatch and was taken as the exhaustion swimming time.

[0062] Liver glycogen and serum urea nitrogen determination: After 30 consecutive days of gavage, 30 minutes after the last gavage, mice were placed in a pool of the same size (water depth 30 cm, water temperature 30 ± 0.5 ℃) and swam without load for 90 minutes. After swimming, the mice rested for 60 minutes, and blood was collected by enucleation. Serum urea nitrogen was measured using the appropriate kit. At the same time, 200 mg of liver tissue was accurately weighed from the mice, and liver glycogen content was determined using the anthrone method.

[0063] 1.3 Detection indicators: Exhaustion swimming time, blood urea nitrogen, and liver glycogen content.

[0064] Table 1 Statistical Results of Fatigue Resistance

[0065] Based on the comparative analysis of anti-fatigue function data shown in Table 1, the beverage (experimental group) provided by this invention exhibits significant advantages in all core indicators. Compared with the blank control group, the experimental group mice showed an approximately 223% longer exhaustion swimming time, an approximately 97% increase in liver glycogen reserves, and a 48% decrease in serum urea nitrogen levels, clearly demonstrating the product's potent anti-fatigue effect. Notably, the experimental group's effects were significantly superior to all control groups: compared to control group 1, which only replaced hawthorn, it had higher liver glycogen and lower blood urea nitrogen, indicating that specific components of Zhongtian hawthorn play a crucial role in energy metabolism; compared to control group 2, which replaced the bacterial strain, the experimental group showed a significant advantage in exercise endurance, confirming the necessity of the specific probiotic combination; and compared to control group 3, which used one-step fermentation, the experimental group significantly outperformed all indicators, powerfully validating the core contribution of the two-step fermentation process in enriching anti-fatigue active substances. The comprehensive anti-fatigue effect of the experimental group is close to that of commercially available functional beverages (positive control group), highlighting its outstanding functional potential as a natural beverage.

[0066] Experiment Example 2

[0067] 1.1 Experimental animals: SPF-grade healthy male Kunming mice (Shandong Aileke Biotechnology Co., Ltd.). All mice were housed in a barrier environment. During the experiment, the ambient temperature was controlled at 25±2℃ and the relative humidity was 55±3%.

[0068] 1.2 Experimental Methods: Mice were randomly divided into 6 groups of 10 mice each: experimental group, control group 1, control group 2, control group 3, blank control group, and positive control group. The experimental group, control group 1, control group 2, and control group 3 were administered 0.12 mL of fruit and vegetable beverage prepared according to the corresponding method per 10 g body weight via gavage. The positive control group was administered 0.12 g of Nutrilite Pine Cone Chrysanthemum Health Tablets (an Amway health product) per 10 g body weight. The blank control group was administered the same volume of physiological saline via gavage. The experiment lasted for 30 days. After the experiment, the following immune indicators were detected and evaluated according to the relevant methods in the National Health Commission's "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition): lymphocyte proliferation capacity (expressed as OD difference), paw edema, and half-maximal hemolysis value (HC). 50 The study also included a mouse carbon clearance test (expressed as the phagocytic index a). The results for each group were averaged, as shown in Table 2.

[0069] Table 2 Statistical results of immune function

[0070] As shown in Table 2, the immune function data comparison reveals that the beverage of this invention has a comprehensive enhancing effect on the body's immune system. The phagocytic index, plantar edema, lymphocyte proliferation capacity, and half-hemolysis value of the experimental group were significantly higher than those of the blank control group, increasing by approximately 52%, 82%, 201%, and 21%, respectively, indicating that it can simultaneously strengthen non-specific immunity, cellular immunity, and humoral immunity. Compared with each control group, the comprehensive advantages of the experimental group are further highlighted: Control group 1 (replaced hawthorn) showed a decrease in all immune indicators, indicating an immune synergy between hawthorn and other ingredients in the original formula; Control group 2 (replaced bacterial strain) showed a significant decrease in immune enhancement, particularly lymphocyte proliferation capacity, demonstrating the unique advantage of the bacterial strain combination selected in this invention in activating specific immunity; Control group 3 (one-step fermentation) had the lowest all immune indicators among all experimental groups, directly indicating that the two-step fermentation process is crucial for generating immunomodulatory substances (such as specific short-chain fatty acids and extracellular polysaccharides). The comprehensive immune enhancement effect of the experimental group is at the same level as that of the dedicated immune health product (positive control group), achieving the scientific goal of achieving significant immune support through natural fermentation.

[0071] Comparing the data from the three control groups reveals that any change in the formula leads to a decrease in product efficacy. Control group 1 (replacing hawthorn) showed lower scores in all indicators compared to the experimental group, indicating that Zhongtian hawthorn possesses unique functions. Control group 2 (replacing the bacterial strain) and control group 3 (changing the fermentation process) showed further reduced efficacy, confirming the importance of specific bacterial combinations and the two-step fermentation process. The experimental group's efficacy was close to that of the positive control group, indicating that this natural fermented beverage can achieve a level of efficacy comparable to commercially available functional beverages and immune-boosting supplements.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions, characterized in that, The product comprises the following components in parts by weight: 60-80 parts of Nakata hawthorn, 10-20 parts of Akizuki pear, 11-15 parts of grape, 9-15 parts of kiwifruit, 6-10 parts of pomegranate, 3-5 parts of fig, 3-5 parts of beetroot, 3-5 parts of pumpkin, 1-2 parts of kale, 1-2 parts of honey, and 2-3 parts of fermentation bacteria; the fermentation bacteria are: Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium bifidum, and Leuconostoc mesenteroides.

2. The probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions according to claim 1, characterized in that, The mass ratio of *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Bifidobacterium bifidum*, and *Leuconostoc mesenteroides* is 20-25:6-12:18-23:7-14.

3. A probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions according to claim 1 or 2, characterized in that, The bacterial counts of *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Bifidobacterium bifidum*, and *Leuconostoc mesenteroides* were all 400-600 million / g.

4. A method for preparing a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Mix Nakata hawthorn, Akizuki pear, grape, kiwi, pomegranate, fig, beetroot, pumpkin and kale, add 1 to 2 times the weight of water to the mixture, and blend to obtain fruit and vegetable puree; (2) Mix the fruit and vegetable puree with enzyme solution for enzymatic hydrolysis, heat to inactivate the enzyme, and add honey to obtain enzymatically hydrolyzed fruit and vegetable puree; (3) The enzymatically hydrolyzed fruit and vegetable puree is mixed with Lactobacillus plantarum and Lactobacillus paracasei and sealed for fermentation to obtain primary fermented material; (4) The primary fermentation material is mixed with Bifidobacterium bifidum and Leuconostoc mesenteroides and sealed for fermentation to obtain secondary fermentation material; (5) The secondary fermented material is refrigerated and stored, and homogenized to obtain a probiotic fermented fruit and vegetable beverage with anti-fatigue and immune-enhancing functions.

5. The preparation method according to claim 4, characterized in that, The enzyme inactivation step (2) involves heating at 85-90°C for 15-20 minutes.

6. The preparation method according to claim 4, characterized in that, The enzyme solution in step (2) consists of hemicellulase, α-amylase and rhamnosidase in a mass ratio of 12~16:7~11:7~11.

7. The preparation method according to claim 4, characterized in that, The mass ratio of the fruit and vegetable puree to the enzyme solution in step (2) is 100:0.3~0.

6.

8. The preparation method according to claim 4, characterized in that, The temperature for sealed fermentation in step (3) is 36~38℃ and the time is 48~72h.

9. The preparation method according to claim 4, characterized in that, The temperature for sealed fermentation in step (4) is 36~38℃ and the time is 30~40h.

10. The preparation method according to claim 4, characterized in that, The refrigeration storage in step (5) is carried out at a temperature of 3~5℃ for 20~30h.

Images