A fermented beverage of aronia melanocarpa juice with fat-reducing efficacy and a preparation method thereof
Patent Information
- Application Number
- CN202510182462.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2026-08-21
AI Technical Summary
[0005]本发明的目的是提供具有减脂功效的黑果腺肋花楸果汁发酵饮品，旨在解决当前市场中黑果腺肋花楸相关产品不够丰富，产品开发缺乏创新等问题，满足消费者对多元健康食品的需求
[0020]本发明的饮品相比较发酵前果汁中大多数黄酮糖苷下调，乳酸菌发酵使黑果腺肋花楸中的酚类物质发生转化，酚类物质种类和含量发生明显改变，果汁中功能活性物质明显提高。同时，在蜜蜂高脂模型中，黑果腺肋花楸发酵汁相对于未发酵汁能有效改善机体脂代谢紊乱以及有效降低高脂饮食蜜蜂血淋巴中海藻糖和葡萄糖含量，减少脂肪体脂滴聚集，降低血淋巴中甘油三酯(TG)、体成分中甘油三酯(TG)和总胆固醇(TC)含量，发挥显著的降脂效果，这意味着黑果腺肋花楸发酵汁为肥胖人群的健康管理带来新的可能。此外，本发明提供的饮品去除了黑果腺肋花楸果实中的酸涩口感，口感较好，丰富了饮品种类。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of food microbial fermentation technology, specifically relating to a fermented fruit juice beverage of black chokeberry with fat-reducing effects and its preparation method. Background Technology
[0002] Due to unhealthy lifestyle habits such as consuming high-sugar, high-fat, and high-salt foods and lack of exercise, the incidence of hyperglycemia and hyperlipidemia is increasing year by year worldwide, causing serious health problems. Black chokeberry contains a high concentration of phenolic substances, which have various effects such as anti-oxidation, delaying aging, regulating blood sugar and lipids, anti-inflammation, and lowering blood pressure, making it a good functional food. However, fresh black chokeberry fruit is rarely eaten directly due to its sour and astringent taste, and the thin peel of the fresh fruit is easily damaged and spoiled; at the same time, the phenolic substances in fresh fruit have low bioavailability after oral administration.
[0003] Currently, the production of black chokeberry in my country is increasing year by year, and the market prospects are broad. Further research should be conducted on the edible and economic value of black chokeberry, and related functional products should be vigorously developed. Therefore, it is urgent to develop processing techniques for black chokeberry to improve the fruit's taste, enhance the functional activity of phenolic compounds in the fruit, thereby increasing its economic value and promoting the sustainable development of the industry. Summary of the Invention
[0004] Technical issues
[0005] The purpose of this invention is to provide a fermented fruit juice beverage of black chokeberry with fat-reducing effects, aiming to solve the problems of insufficient variety of black chokeberry-related products and lack of innovation in product development in the current market, and to meet consumers' demand for diverse healthy foods.
[0006] Technical solution
[0007] The first aspect of this invention provides a fermented fruit juice beverage of black chokeberry with fat-reducing effects, prepared by fermentation with lactic acid bacteria using black chokeberry fruit as raw material; wherein the lactic acid bacteria is Lactiplantibacillus plantarum CAU808, with the preservation number CGMCC No. 28129. Lactic acid bacteria are safe to consume with low risk and can metabolize to produce nutrients and special flavor substances, and are widely used in fruit and vegetable fermentation processing. During fermentation, microorganisms utilize the nutrients in fruits and vegetables for their own metabolism. Through complex interactions between different microorganisms and between microorganisms and nutrients, a variety of metabolites are generated, affecting the shelf life, flavor, and nutritional value of fruits and vegetables. The fermentation process produces a large amount of organic acids, which lowers the pH of the system, interferes with the cell membrane potential of harmful microorganisms, and inhibits their metabolic function, thus exhibiting good antibacterial effects. Lactic acid bacteria metabolism can also produce a variety of antibacterial active ingredients, such as bacteriocins, phenolic compounds, hydrogen peroxide, antimicrobial peptides, and extracellular polysaccharides, which inhibit the growth of harmful microorganisms during the storage of fruits and vegetables. Meanwhile, during fermentation, the metabolic activities of microorganisms increase the content of bioactive substances such as polyphenols, extracellular polysaccharides, amino acids, and vitamins in fruits and vegetables, thereby improving their functional properties.
[0008] In some embodiments, the fermented black chokeberry juice beverage with fat-reducing effects comprises the following ingredients in parts by weight: 200-400 parts of black chokeberry fruit, 1-4 parts of histidine, and 3-6 parts of lactic acid bacteria.
[0009] In some embodiments, the preparation method of the fermented black chokeberry juice with fat-reducing effect includes the following specific steps: S1: Add water to black chokeberry fruit and blend until uniform to obtain black chokeberry juice; S2: Add histidine and mix evenly to obtain compound black chokeberry juice; S3: Pasteurize to obtain sterile compound black chokeberry juice; S4: Inoculate and ferment to obtain fermented black chokeberry juice; S5: Filter to obtain fermented black chokeberry juice beverage.
[0010] In some embodiments, step S1 specifically involves: washing the black chokeberry fruit, adding water at a mass ratio of 1:1 and blending until homogeneous to obtain black chokeberry juice.
[0011] In some embodiments, step S2 specifically involves adding histidine to the juice of black chokeberry and mixing thoroughly to obtain a compound black chokeberry juice. Lactic acid bacteria have difficulty growing and surviving in black chokeberry juice, and the viable cell count is low after fermentation. Amino acids can serve as nutritional additives in fermented foods, providing the nutrients needed for microbial growth and promoting the fermentation process.
[0012] In some embodiments, step S3 specifically involves: placing the compound black chokeberry juice in a constant temperature water bath and sterilizing it at a constant temperature of 80°C to 90°C for 10 to 15 minutes, then removing it and rapidly cooling it to 3°C to 5°C to obtain sterile compound black chokeberry juice.
[0013] In some embodiments, step S4 specifically involves: inoculating the fermentation seed liquid of the lactic acid bacteria (Lactobacillus plantarum CAU808) into sterile compound black chokeberry juice, and fermenting at 34–37°C for 32–48 hours, so that the final concentration of lactic acid bacteria is not less than 7.1 log CFU / mL; after fermentation, the fermented black chokeberry juice contains not less than 1.6 mg GAE / mL of total plant phenols. Lactic acid bacteria fermentation is an effective fruit and vegetable processing technology that can metabolize and transform nutrients in fruits and vegetables, improve the bioactive components and functional properties of products, and endow fermented fruits and vegetables with rich flavor. During fermentation, enzymes produced by microorganisms cause metabolic transformation of phenolic substances, which is beneficial to enhancing the bioactivity of polyphenols.
[0014] In some embodiments, the method for preparing the fermentation seed liquid of the lactic acid bacteria (Lactobacillus plantarum CAU808) is as follows: the Lactobacillus plantarum CAU808 lactic acid bacteria strain is inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 hours, and activated twice consecutively; then, the activated strain suspension is centrifuged at 5000g for 5 minutes at 4°C to collect the bacterial sludge, and washed twice with 0.9% physiological saline. Then, the absorbance of the bacterial suspension at 600nm is adjusted to about 1.0 to obtain the fermentation seed liquid.
[0015] In some embodiments, step S5 specifically involves: in a sterile environment, pouring the fermented black chokeberry juice into a clean 200-mesh gauze for filtration to obtain the finished black chokeberry fermented juice.
[0016] In some embodiments, step S5 is followed by an ultra-high temperature instantaneous sterilization step; the ultra-high temperature instantaneous sterilization step specifically involves placing the fermented juice of black chokeberry in an environment of 132-140°C for 2-4 seconds, and then rapidly cooling it to 4-8°C.
[0017] In some embodiments, step S5 is followed by a packaging coding step; the packaging coding specifically involves filling the product in a sterile environment using sterile bottles or sterile cans, then sealing and coding it to obtain the finished product of black chokeberry juice fermentation beverage.
[0018] In some embodiments, the fermented black chokeberry juice with fat-reducing effects can be stored for 1 to 2 months in a clean environment at 2 to 4°C; after ultra-high temperature instantaneous sterilization, the fermented black chokeberry juice with fat-reducing effects can be stored for 6 to 12 months in a dry environment at room temperature.
[0019] Technical effect
[0020] Compared to the unfermented juice, the beverage of this invention exhibits a downregulation of most flavonoid glycosides. Lactic acid bacteria fermentation transforms the phenolic substances in *Sorbus spp.*, significantly altering the types and content of phenolic substances and resulting in a marked increase in functional active substances in the juice. Simultaneously, in a high-fat bee model, fermented *Sorbus spp.* juice effectively improves lipid metabolism disorders and reduces trehalose and glucose levels in the hemolymph of bees on a high-fat diet, decreasing lipid droplet aggregation in fat bodies and lowering triglyceride (TG) levels in hemolymph, body components, and total cholesterol (TC), demonstrating a significant lipid-lowering effect. This suggests that fermented *Sorbus spp.* juice offers new possibilities for health management in obese individuals. Furthermore, the beverage provided by this invention removes the astringent taste of *Sorbus spp.* fruit, resulting in a better palatability and enriching the variety of beverages available. Attached Figure Description
[0021] Figure 1 The image shows the colony morphology of Lactobacillus plantarum CAU808.
[0022] Figure 2 The image shows the morphology of *Lactobacillus plantarum* CAU808 under a scanning electron microscope.
[0023] Figure 3 The image shows the finished product of a fermented fruit juice beverage made from black chokeberry.
[0024] Figure 4 The figure shows the effects of FJ, NFJ, and LP interventions on the body weight of bees on a high-fat diet;
[0025] Figure 5A The figure shows the effects of FJ, NFJ, and LP interventions on trehalose content in the hemolymph of bees on a high-fat diet.
[0026] Figure 5B The figure shows the effects of FJ, NFJ, and LP interventions on glucose content in the hemolymph of bees on a high-fat diet.
[0027] Figure 6 The figure shows the effects of FJ, NFJ, and LP interventions on TG content in the hemolymph of bees on a high-fat diet.
[0028] Figure 7A The figure shows the effects of FJ, NFJ, and LP interventions on the TG content in the body components of bees fed a high-fat diet;
[0029] Figure 7B The figure shows the effects of FJ, NFJ, and LP interventions on the TC content in the body components of bees on a high-fat diet;
[0030] Figure 8AThe image shows the effects of FJ, NFJ, and LP interventions on fat bodies in bees on a high-fat diet (H&E staining).
[0031] Figure 8B The effects of FJ, NFJ, and LP interventions on fat bodies in bees on a high-fat diet are shown (Oil Red O staining).
[0032] Preservation Information
[0033] Taxonomy: Lactobacillus plantarum;
[0034] Name: CAU808;
[0035] Latin name: *Lactiplantibacillus plantarum*;
[0036] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);
[0037] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;
[0038] Date of deposit: August 9, 2023;
[0039] Registration number at the Preservation Center: CGMCC No. 28129. Detailed Implementation
[0040] To facilitate the explanation of the technical solution of this application, the following is a general explanation and definition of the terms and expressions used in this application.
[0041] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0042] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials are kept consistent to ensure comparability.
[0043] Unless otherwise specified, all reagents and instruments used in the embodiments of this invention can be purchased from the market.
[0044] The following is a further description of the fermented fruit juice of black chokeberry with fat-reducing effects and its preparation method provided in this application.
[0045] Example 1: Isolation and identification of Lactobacillus plantarum CAU808
[0046] 1) Source of separation
[0047] Table 1 Basic Information on Lactobacillus plantarum CAU808
[0048]
[0049] Note: The isolation and purification medium was MRS medium.
[0050] 2) Passage and cryopreservation
[0051] Subculturing: Weigh the appropriate amount of MRS medium, add pure water to resuspend and dissolve; sterilize at 115℃ for 20 min, or at 121℃ for 15 min; inoculate the strain and place at 36±1℃ for anaerobic culture for 24 h.
[0052] Cryopreservation of bacterial strains: The glycerol cryopreservation method is used. The subcultured and activated bacterial solution is added to a bacterial culture preservation tube containing 50% glycerol. The ratio of bacterial solution to 50% glycerol is 1:1. The mixture is blown evenly with a pipette and then placed in an ultra-low temperature freezer or liquid nitrogen tank for cryopreservation (it can be stored at -70℃ or below for at least two years).
[0053] 3) Strain identification
[0054] ① 16S rDNA sequence (5'-3')
[0055] The 16S rDNA sequence is shown in SEQ ID NO.1, as follows:
[0056]
[0057] ②Strain morphology
[0058] The colony morphology of Lactobacillus plantarum CAU808 is as follows: Figure 1 As shown; the morphology of *Lactobacillus plantarum* CAU808 under scanning electron microscopy is as follows. Figure 2 As shown.
[0059] 4) Environmental tolerance test
[0060] Table 2 Survival rate of *Lactobacillus plantarum* CAU808 in environmental tolerance test
[0061]
[0062]
[0063] Note: Survival rate In the formula: Nt is the number of viable bacteria after treatment with pH=3.0 or 0.3% bile salts; N0 is the number of viable bacteria at 0h.
[0064] 5) Antibiotic sensitivity
[0065] Table 3. Susceptibility testing of Lactobacillus plantarum CAU808 to different antibiotics.
[0066]
[0067] Note: Resistance (R), Sensitivity (S), Intermediate Sensitivity (IS); Antimicrobial susceptibility testing is performed according to the Clinical Laboratory Standards Committee (CLC) standards.
[0068] Example 2: Quality differences of black chokeberry fermented with different lactic acid bacteria
[0069] Ingredients: purified water, black chokeberry fruit, lactic acid bacteria.
[0070] The juice of black chokeberry was fermented using 25 strains of lactic acid bacteria. Specific strain information is shown in Table 4.
[0071] Table 4 Information on Lactic Acid Bacteria Strains
[0072]
[0073]
[0074] Methods for preparing black chokeberry juice fermented with different lactic acid bacteria:
[0075] 1) Pretreatment: Weigh 50g of fresh black chokeberry fruit, 50g of purified water and 2g of yeast extract. Wash the black chokeberry fruit, add purified water and mix until uniform. Then add yeast extract and mix evenly to obtain compound black chokeberry juice.
[0076] 2) Pasteurization: Place the compound black chokeberry juice in a constant temperature water bath and sterilize it at 85℃ for 15 minutes. Then remove it and place it in an ice-water mixture to cool it rapidly to 3℃~5℃ to obtain sterile compound black chokeberry juice.
[0077] 3) Activation of bacterial strains: Twenty-five types of lactic acid bacteria were activated separately. The lactic acid bacteria strains were inoculated into MRS liquid medium and anaerobically cultured at 37℃ for 18 hours, with activation repeated twice. Subsequently, the activated bacterial suspension was centrifuged at 5000g for 5 minutes at 4℃ to collect the bacterial sludge, and washed twice with 0.9% physiological saline. Then, the absorbance of the bacterial suspension at 600nm was adjusted to about 1.0 to obtain the fermentation seed liquid.
[0078] 4) Inoculation and fermentation: Inoculate the fermentation seed liquid into the sterile compound black chokeberry juice at a mass percentage of 0.5% and anaerobic ferment at 37℃ for 24 hours to ensure that the final concentration of the strain is not less than 7 log CFU / mL.
[0079] 5) Filtration: In a sterile environment, the fermented black chokeberry juice is poured into a clean 200-mesh gauze for filtration to obtain the finished black chokeberry fermented juice.
[0080] The optimal fermentation strain was screened using viable cell count, pH value, and total phenol content as evaluation indicators. The results are shown in Table 5.
[0081] Table 5 Results of fermentation of black chokeberry with different lactic acid bacteria
[0082]
[0083] Note: Different lowercase letters in the same column represent significant differences (p<0.05). All means are arranged in descending order, and the largest mean is labeled 'a'. This is then compared to the following means. If the difference is not significant, 'a' is continued until a significantly different mean is found, which is then labeled 'b'. Similarly, the largest mean labeled 'b' is compared to the unlabeled means. If the difference is not significant, 'b' is continued until a significantly different mean is encountered, which is then labeled 'c'. In short, if two samples have the same letter, the difference between them is not significant (e.g., a and ab, b and bc); samples labeled with different letters indicate a significant difference between them.
[0084] Traditional fermented plant-based foods were collected from multiple provinces in my country, and 25 lactic acid bacteria strains isolated from them were selected for screening fermentation strains. These included 13 strains of *Lactobacillus plantarum*, 9 strains of *Lactobacillus fermentum*, 2 strains of *Lactobacillus paracasei*, and 1 strain of *Lactobacillus reuteri*. The results are shown in Table 5. After 24 hours of fermentation, the viable bacterial count in the juice of *Lactobacillus plantarum* LP-2 increased to 8.80 log CFU / mL, an increase of 2 log CFU / mL compared to before fermentation. The pH value of the juice decreased significantly more than other groups, from 4.60 before fermentation to 3.70, with a ΔpH of 0.90. Simultaneously, the total phenolic content in the juice fermented by *Lactobacillus plantarum* LP-2 was the highest, indicating that this bacterium has a strong ability to metabolize and transform phenolic substances. In conclusion, *Lactobacillus plantarum* LP-2 exhibits good growth and fermentation characteristics in *Sorbus niger*, and is therefore determined to be the optimal fermentation strain.
[0085] The *Lactobacillus plantarum* CAU808 used in this patent is *Lactobacillus plantarum* LP-2, isolated from fermented sauerkraut in Xiaogan City, Hubei Province. The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with the number CGMCC No. 28129.
[0086] Example 3: Preparation of fermented fruit juice beverage from black chokeberry
[0087] Ingredients: 100g purified water, 100g black chokeberry fruit, 0.6g histidine, 1.805g fermentation bacteria.
[0088] Preparation method:
[0089] 1) Pretreatment: Weigh 100g of fresh black chokeberry fruit, 100g of purified water and 0.6g of histidine. Wash the black chokeberry fruit, add purified water and mix until uniform. Then add histidine and mix evenly to obtain compound black chokeberry juice.
[0090] 2) Pasteurization: Place the compound black chokeberry juice in a constant temperature water bath and sterilize it at 85℃ for 15 minutes. Then remove it and place it in an ice-water mixture to cool it rapidly to 3℃~5℃ to obtain sterile compound black chokeberry juice.
[0091] 3) Activation of the bacterial strain: *Lactobacillus plantarum* CAU808 strain was inoculated into MRS liquid medium and anaerobically cultured at 37℃ for 18 h, with activation repeated twice. Subsequently, the activated bacterial suspension was centrifuged at 5000g for 5 min at 4℃ to collect the bacterial sludge, and washed twice with 0.9% physiological saline. The absorbance of the bacterial suspension at 600 nm was then adjusted to approximately 1.0 to obtain the fermentation seed liquid.
[0092] 4) Inoculation and Fermentation: The fermentation seed liquid was inoculated into the aseptic compound black chokeberry juice at a mass percentage of 0.9%, and anaerobic fermentation was carried out at 34℃ for 32 hours to ensure that the final concentration of the strain was not less than 8.4 log CFU / mL. Under these conditions, the pH value of the fermented black chokeberry juice was 4.2, and the total phenol content was 1.92 mg GAE / mL, which was 0.26 mg GAE / mL higher than that of the juice before fermentation. Among them, there were 67 phenolic differential metabolites, of which 29 phenolic substances were significantly upregulated and 38 phenolic substances were significantly downregulated after fermentation.
[0093] 5) Filtration: In a sterile environment, the fermented black chokeberry juice is poured into a clean 200-mesh gauze for filtration.
[0094] 6) Packaging coding: Bottle and package according to quality at around 4℃, and then code to obtain the finished product of black chokeberry juice fermented beverage.
[0095] 7) Storage: Can be stored for 1 to 2 months in a clean environment at 2 to 4°C.
[0096] Black chokeberry juice fermented beverage finished product Figure 3 As shown.
[0097] Example 4: Preparation of fermented fruit juice beverage from black chokeberry
[0098] Ingredients: 200g purified water, 200g black chokeberry fruit, 2g histidine, 2.814g fermentation bacteria.
[0099] Preparation method:
[0100] 1) Pretreatment: Weigh 200g of fresh black chokeberry fruit, 200g of purified water and 2g of histidine. Wash the black chokeberry fruit, add purified water and mix until uniform. Then add histidine and mix evenly to obtain compound black chokeberry juice.
[0101] 2) Pasteurization: Place the compound black chokeberry juice in a constant temperature water bath and sterilize it at 90℃ for 10 minutes. Then remove it and place it in an ice-water mixture to cool it rapidly to 3℃~5℃ to obtain sterile compound black chokeberry juice.
[0102] 3) Activation of the bacterial strain: *Lactobacillus plantarum* CAU808 strain was inoculated into MRS liquid medium and anaerobically cultured at 37℃ for 18 h, with activation repeated twice. Subsequently, the activated bacterial suspension was centrifuged at 5000g for 5 min at 4℃ to collect the bacterial sludge, and washed twice with 0.9% physiological saline. The absorbance of the bacterial suspension at 600 nm was then adjusted to approximately 1.0 to obtain the fermentation seed liquid.
[0103] 4) Inoculation and fermentation: The fermentation seed liquid was inoculated into the sterile compound black chokeberry juice at a mass percentage of 0.7%, and anaerobic fermentation was carried out at 37℃ for 48h, so that the final concentration of the strain was not less than 7.88 log CFU / mL; under these conditions, the pH value of the fermented black chokeberry juice was 3.85, and the total phenol content was 1.67 mg GAE / mL.
[0104] 5) Filtration: In a sterile environment, the fermented black chokeberry juice is poured into a clean 200-mesh gauze for filtration.
[0105] 6) Packaging coding: Bottle and package according to quality at around 4℃, and then code to obtain the finished product of black chokeberry juice fermented beverage.
[0106] 7) Storage: Can be stored for 1 to 2 months in a clean environment at 2 to 4°C.
[0107] Example 5: Preparation of fermented fruit juice beverage from black chokeberry
[0108] Preparation method:
[0109] The rest is the same as in Example 3, except that the following steps are added after the filtration step in step 5) and before the packaging and coding step in the preparation method.
[0110] Ultra-high temperature instantaneous sterilization: The homogenized solution is kept at 132-140℃ for 2-4 seconds to achieve commercial sterility, and then rapidly cooled to 4-8℃;
[0111] Packaging coding: Fill the packaging in a sterile environment using sterile bottles or cans, then seal and code the contents.
[0112] Storage: Store at room temperature in a dry environment for 6 to 12 months.
[0113] Example 6: Efficacy Test of Fermented Juice Beverage of Black Rowan Fruit
[0114] 1) Test subjects
[0115] Bees' unique social and metabolic mechanisms make them a novel model organism for obesity research. Scientists have discovered that bees, like humans, can become obese due to dietary imbalances. High-calorie diets lead to significant fat accumulation in bees through their fat bodies (a complex organ similar to the liver and adipose tissue in mammals). Furthermore, the physiological metabolic processes and gene regulation mechanisms of bees share similarities with those in humans. This miniaturized model allows for precise analysis of the synergistic effects of diet, genes, and environment on obesity. Therefore, studying bees can help us understand the impact of obesity on metabolism and lifespan, thus providing new insights for the prevention and treatment of obesity in humans.
[0116] This invention selects Italian worker bees (Apis mellifera) as the experimental subjects. After acclimatization, they are randomly divided into 5 groups, with 25 bees per cup and at least 3 cups per group for replication.
[0117] 2) Experimental Design
[0118] ① Adaptive feeding
[0119] Day 0 was defined as the day bees emerged from the hive. Healthy, vigorous newly emerged bees were selected and placed in clean, disposable 400mL transparent plastic cups. A 2mL centrifuge tube (with holes punched in the tube wall) containing food was inserted into the side of the cup. The cups containing the bees were placed in an incubator at 36℃ and 50%–60% humidity, allowing the bees free access to food during the rearing period. The normal diet included sucrose as a carbohydrate source and casein amino acids as a protein source. Day 0 bees were fed the normal diet (50% sucrose, 1% casein amino acids) for two days. After acclimatizing to the new environment, bees in good condition were selected for further experiments.
[0120] ② Experimental feeding
[0121] Adaptive feeding was conducted by grouping bees into groups, with the grouping date recorded as day 0, for a 7-day experiment. The bee diet consisted primarily of 50% sucrose and 1% casein amino acids, with other ingredients added according to experimental needs. Specific groupings and dietary designs are shown in Table 6. During the experiment, the bees were given a change of food every other day, and dead bees were removed.
[0122] ③ Testing and Experimentation
[0123] During the experiment, the weight changes of the bees were measured and recorded every other day. Each cup of bees was used as a culture unit, and the weights were measured using an analytical balance. The weight changes of 5 cups of bees in each group were measured, and the average weight of each bee was calculated.
[0124] At the end of the 7th day of the experiment, bee hemolymph, fat bodies, and intestines were dissected and collected. The levels of trehalose, glucose, and TG (triglycerides) in the bee hemolymph were measured, as well as the TG and TC (total cholesterol) content in the body components. The bee fat bodies were also stained with H&E and Oil Red O to detect lipid accumulation.
[0125] Table 6 Grouping of Animal Experiments
[0126]
[0127]
[0128] 3) Results
[0129] ① Changes in bee weight
[0130] Figure 4The study showed changes in bee weight during the feeding period. It can be seen that the changes in bee weight during the experiment were not significant, and there was no significant difference in bee weight among the five treatments. However, overall, the bees in the HFD group (high-fat diet) had slightly higher weight.
[0131] ② Trehalose and glucose levels in bee hemolymph
[0132] like Figure 5A and Figure 5B As shown, the trehalose content in the CK group (normal diet) and the HFD group was 39.26 mg / mL and 65.95 mg / mL, respectively. A high-fat diet significantly increased the trehalose level in bee hemolymph. After FJ (high-fat diet + fermented juice) and NFJ (high-fat diet + unfermented juice) dietary interventions, the trehalose content in bee hemolymph significantly decreased, and there was no significant difference in trehalose content between the FJ and NFJ groups and the CK group. The changes in glucose content in bee hemolymph were similar to those of trehalose content. Furthermore, high-fat intake also led to an increase in glucose content in bee hemolymph. FJ, NFJ, and LP (high-fat diet + *Lactobacillus plantarum* CAU808) interventions all significantly reduced glucose content in hemolymph, and there was no significant difference in glucose content among the three groups. Therefore, it can be seen that the three interventions, FJ, NFJ, and LP, can effectively improve the hyperglycemia in bees caused by a high-fat diet and maintain the body's glucose metabolism balance.
[0133] ③TG content in bee hemolymph
[0134] like Figure 6 As shown, compared with the CK group, the TG content in the hemolymph of the HFD group increased significantly from 1.08 mmol / mL to 2.13 mmol / mL. FJ intervention effectively alleviated the increase in TG in the hemolymph caused by a high-fat diet. Compared with the NFJ and LP groups, the FJ group had the lowest TG content in the hemolymph, and this difference was significant compared with the HFD group. This indicates that FJ intervention can better inhibit the increase in blood lipid levels caused by a high-fat diet and effectively alleviate lipid metabolism disorders.
[0135] ④ The content of TG and TC in the body components of bees
[0136] like Figure 7A and Figure 7BAs shown, compared with the CK group, a high-fat diet led to a significant increase in both TG and TC levels in the body components of bees in the HFD group. Compared with the HFD group, the TG content in the body components of bees in the FJ and NFJ groups was significantly reduced, and the TG content in the FJ group was significantly lower than that in the NFJ group. FJ had the best effect on improving the increase in TG levels in bee body components caused by a high-fat diet. FJ, NFJ, and LP interventions also had a significant effect on the TC content in bee body components. The TC content in the body components of the FJ, NFJ, and LP groups was significantly lower than that in the HFD group, and the TC content in the FJ and NFJ intervention groups was significantly lower than that in the CK group. Combined with the results of TG content in hemolymph, it can be seen that a high-fat diet causes an imbalance in lipid absorption and metabolism in bees, resulting in a significant increase in TG in hemolymph and TG and TC content in body components. FJ, NFJ, and LP interventions can all effectively reduce the increase in lipid content and improve the abnormal lipid metabolism in bees, with FJ having a better ability to regulate lipid metabolism.
[0137] ⑤ Lipid accumulation in the fat body of bees
[0138] Figure 8A H&E staining was performed, with round vacuoles representing lipid droplets and red areas representing cytoplasm. The staining results showed that, compared to the control (CK), bees fed a high-fat diet had larger lipid droplets in their fat bodies and more lipid storage droplets, resulting in significant lipid accumulation. In the FJ and NFJ intervention groups, lipid droplets in the fat bodies were smaller, and lipid accumulation was significantly improved; moreover, the FJ group had even smaller and fewer lipid droplets. Figure 8B The image shows oil red O staining. The red areas represent lipid droplets stained with oil red O dye. The denser and larger the red area, the more lipid droplets there are. The results of oil red O staining are similar to those of H&E staining. Figure 8B Oil Red O staining revealed that, compared to the CK group, the HFD group showed a higher density of red-stained lipid droplets on the fat bodies of bees. In contrast, the FJ, NFJ, and LP intervention groups all exhibited significantly reduced lipid droplet content in their fat bodies. All three interventions effectively reduced lipid droplet aggregation in the fat bodies induced by a high-fat diet, with the FJ group showing even less lipid droplet distribution, thus more effectively alleviating fat accumulation caused by a high-fat diet. These results indicate that fermented black chokeberry juice can reduce the size of lipid droplets in the fat body, decrease lipid droplet aggregation, effectively prevent lipid accumulation in the fat body caused by a high-fat diet, and has the potential to improve lipid metabolism disorders in bees.
[0139] In summary, the results of the FJ (high-fat diet + fermented juice) group showed that the fermented juice of *Sorbus chokesoniae* provided by this invention can improve lipid metabolism disorders in bees and effectively reduce the content of trehalose and glucose in the hemolymph of bees on a high-fat diet, reduce lipid droplet aggregation in fat bodies, and lower the content of triglycerides (TG) in hemolymph, body components, and total cholesterol (TC), thus exhibiting a significant lipid-lowering effect. Given the similarities between bees and humans in physiological metabolic processes and gene regulation mechanisms, this means that fermented juice of *Sorbus chokesoniae* is highly likely to play a similar role in humans, regulating lipid metabolism, reducing the content of related sugars and lipids, thereby improving obesity problems, preventing obesity-related diseases, and bringing new possibilities for health management of obese populations.
[0140] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.
Claims
1. A fermented fruit juice beverage of black chokeberry with fat-reducing effects, characterized in that, It is prepared by fermentation of black chokeberry fruit with lactic acid bacteria; wherein the lactic acid bacteria is Lactiplantibacillus plantarum CAU808, with the preservation number CGMCC No.28129.
2. The fermented fruit juice beverage of black chokeberry with fat-reducing effect according to claim 1, characterized in that, The ingredients include the following parts by weight: 200-400 parts of black chokeberry fruit, 1-4 parts of histidine, and 3-6 parts of lactic acid bacteria.
3. The fermented fruit juice beverage of black chokeberry with fat-reducing effect according to claim 2, characterized in that, The preparation method of the fermented juice beverage of black chokeberry with fat-reducing effects includes the following specific steps: S1: Add water to the fruit of *Sorbus nigra* and blend until homogeneous to obtain *Sorbus nigra* juice; S2: Add histidine, mix well, and obtain compound black chokeberry juice; S3: Pasteurization to obtain aseptic compound black chokeberry juice; S4: Inoculate and ferment to obtain fermented black chokeberry juice; S5: Filter to obtain a fermented juice beverage of black chokeberry.
4. The fermented black chokeberry juice beverage with fat-reducing effects according to claim 3, characterized in that, Step S1 is as follows: After washing the black chokeberry fruit, add water at a mass ratio of 1:1 and blend until uniform to obtain black chokeberry juice.
5. The fermented black chokeberry juice beverage with fat-reducing effects according to claim 3, characterized in that, Step S2 specifically involves adding histidine to the juice of black chokeberry and mixing it evenly to obtain a compound black chokeberry juice.
6. The fermented black chokeberry juice beverage with fat-reducing effects according to claim 3, characterized in that, Step S3 is as follows: Place the compound black chokeberry juice in a constant temperature water bath and sterilize it at a constant temperature of 80℃~90℃ for 10~15 minutes. Then remove it and cool it rapidly to 3℃~5℃ to obtain sterile compound black chokeberry juice.
7. The fermented black chokeberry juice beverage with fat-reducing effects according to claim 3, characterized in that, Step S4 specifically involves inoculating the fermentation seed liquid of the lactic acid bacteria into sterile compound black chokeberry juice and fermenting it at 34–37°C for 32–48 hours, so that the final concentration of lactic acid bacteria is not less than 7.1 log CFU / mL; after fermentation, the fermented black chokeberry juice contains not less than 1.6 mg GAE / mL of total plant phenols.
8. The fermented fruit juice beverage of black chokeberry with fat-reducing effect according to claim 3, characterized in that, Step S5 is as follows: In a sterile environment, the fermented black chokeberry juice is poured into a clean 200-mesh gauze for filtration to obtain a fermented black chokeberry juice beverage.
9. The fermented fruit juice beverage of black chokeberry with fat-reducing effect according to claim 3, characterized in that, Step S5 is followed by an ultra-high temperature instantaneous sterilization step; the ultra-high temperature instantaneous sterilization step specifically involves placing the fermented juice of black chokeberry in an environment of 132-140℃ for 2-4 seconds, and then rapidly cooling it to 4-8℃.
10. The fermented fruit juice beverage of black chokeberry with fat-reducing effect according to claim 9, characterized in that, The fermented fruit juice of black chokeberry with fat-reducing effects can be stored for 1 to 2 months in a clean environment at 2 to 4 degrees Celsius. The fermented fruit juice of black chokeberry with fat-reducing effects can be stored for 6 to 12 months in a dry environment at room temperature after ultra-high temperature instantaneous sterilization.