Preparation method of apple water kefir beverage and application of apple water kefir beverage in lowering cholesterol

Abstract

The invention provides a preparation method of an apple water kefir beverage and application of the apple water kefir beverage in lowering cholesterol. The method comprises the following steps: preparing apple juice, activating water kefir grains, inoculating the activated water kefir grains into the apple juice, and fermenting to prepare the apple water kefir beverage. The volatile component change in the fermentation process is analyzed through GC-MS and GC-IMS systems, it is proved that the unique bouquet characteristic and flavor coordination of the apple water kefir beverage are improved through water kefir grain fermentation, and meanwhile the organic acid composition is optimized. A mouse model verifies that the apple water kefir beverage has the effect of reducing cholesterol, can reduce liver lipid droplets, total cholesterol, low-density lipoprotein and triglyceride levels, and also can regulate intestinal microbial communities (increase firmicobacteria, bacteroides and faecalis bacteria). The apple water kefir beverage disclosed by the invention has wide market potential and provides a scientific basis for the development of functional beverages.

Description

Technical Field

[0001] This invention belongs to the field of fermented fruit and vegetable beverage technology, specifically relating to a method for preparing an apple water kefir beverage and its application in lowering cholesterol. Background Technology

[0002] Kefir is a beverage made by fermenting sugars using a diverse symbiotic microbial community. It is low in alcohol, effervescent, and slightly acidic, and offers multiple health benefits, including regulating gut microbiota and lowering blood lipids, thus attracting increasing attention from academia and the market. Depending on the fermentation substrate, kefir is mainly divided into water kefir and milk kefir. Water kefir (WK) typically uses sugar water or fruit and vegetable juice as a substrate, inoculating it with water kefir grains (WKG, composed of yeast, lactic acid bacteria, and acetic acid bacteria encapsulated in polysaccharides) to obtain a clear, transparent, and refreshing product. This not only meets consumers' demand for diverse flavors but also provides an ideal choice for vegetarians and those with lactose intolerance. However, water kefir is currently in the early stages of research, with limited reports and relatively limited research content. Systematic studies on the formation mechanism of flavor compounds and its health-promoting potential (such as cholesterol reduction) are lacking.

[0003] CN104286173A discloses a raw material composition and preparation process for a kefir beverage, mainly using water, sweeteners (such as white sugar and fructose), and kefir grains for fermentation. However, the fermentation substrate is mostly limited to sugar water or whey, resulting in a single flavor and a lack of optimization for fruit juice substrates (such as apple juice). CN107156325A relates to a kefir-fermented whey beverage, adding concentrated fruit juice to introduce polyphenols, but does not analyze the systematic fermentation process for apple juice. CN1729803A describes a production method for lactic acid bacteria-fermented apple juice beverage, using plant-based lactic acid bacteria and Lactobacillus bulgaricus, but does not involve a complex microbial system of water kefir grains, and lacks in-depth verification of the dynamic changes of volatile components and health functions (such as cholesterol reduction). CN114747706A discloses a method for preparing lipid-lowering apple juice through co-fermentation of Monascus purpureus and Lactobacillus plantarum, focusing on the lipid-lowering function, but the strains are different, water kefir grains are not used, and the flavor formation mechanism is insufficiently studied. CN105062929A discloses a water kefir complex microbial community and its fermentation beverage preparation method, including inoculating the microbial community into fruit juice (such as apple juice) for fermentation, but does not systematically analyze the dynamic changes of volatile components or verify the cholesterol-lowering effect through animal models. CN101720967A involves fermenting fruit and vegetable juice beverages with kefir starter, using concentrated apple juice and other diluted products for fermentation, but the flavor and functional verification is limited. These existing technologies have the following disadvantages: (1) The fermentation substrate is mostly sugar water or whey, with poor flavor coordination and lack of the unique wine aroma and coordination characteristics brought by fruit juice substrate (such as apple juice); (2) There is a lack of systematic analysis of the dynamic changes of volatile components and differential markers (such as ester compounds) during fermentation, resulting in inaccurate flavor control; (3) Health benefits (such as cholesterol lowering) are mostly at the theoretical or preliminary reporting stage, lacking systematic verification through animal experiments such as mouse models induced by high-fat diets, and cannot provide scientific evidence to support functional applications; (4) The changes in organic acid and total phenol content have not been optimized, affecting the slightly acidic characteristics and antioxidant potential of the beverage. These shortcomings limit the functional development and market application of water kefir beverages, making it unable to meet consumers' demands for flavor diversity and health benefits. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for preparing an apple water kefir beverage and its application in lowering cholesterol. By using apple juice as the fermentation substrate and brown sugar water as a control, the dynamic changes of volatile components during the fermentation process are systematically analyzed, and its cholesterol-lowering effect is verified through animal models, thus providing an apple water kefir beverage with a harmonious flavor and clearly defined functions.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing an apple water kefir beverage, comprising the following steps: S1. Apple juice preparation First, select fresh apples with no obvious blemishes on the surface, rinse the peel with running water to remove dirt, cut the washed apples into pieces, juice them, then add potassium metabisulfite and pectinase to the juice, mix well, and place it at 35~45℃ for 2~6 hours for enzymatic hydrolysis. Then, centrifuge at 4℃, take the supernatant for sterilization, and cool to obtain apple juice. S2, Water Kefir Particle Activation Prepare brown sugar water, sterilize and cool it to obtain sterile brown sugar water; add water kefir granules to sterile brown sugar water, incubate at 25℃ for 24 hours, filter to obtain activated first-generation water kefir granules; rinse the surface of the water kefir granules with sterile water to make them translucent, then inoculate them into sterile brown sugar water for 24 hours of activation; the process of inoculation-filtration-washing-inoculation is one activation cycle, which is repeated 2-3 times to obtain activated water kefir granules; S3, Preparation of apple water kefir beverage The activated water kefir grains obtained in S2 were inoculated into the apple juice obtained in S1 and fermented at 18-37°C. When the soluble solids content remained unchanged, the fermentation endpoint was determined to obtain the apple water kefir beverage.

[0006] Preferably, the concentration of potassium metabisulfite in S1 is 40-55 mg / L, and the concentration of pectinase is 25-40 mg / L.

[0007] Preferably, the centrifugation speed in S1 is 7500~8500 r / min, and the time is 3~8 min.

[0008] Preferably, the sterilization temperature in S1 is 100~110℃ and the time is 10~20min.

[0009] Preferably, the concentration of the brown sugar water in S2 is 50~65g / L, and the sterilization temperature of the brown sugar water is 121℃ for 20min.

[0010] Preferably, the amount of water kefir grains inoculated into apple juice in S3 is 5-8%.

[0011] The present invention also provides an apple water kefir beverage prepared by the above method.

[0012] The present invention also provides the application of the apple water kefir beverage for lowering cholesterol or regulating the gut microbiota.

[0013] This invention has significant technical advantages compared to existing technologies: 1. This invention provides a method for preparing apple water kefir beverage, which uses fresh apple juice (pH 4.0-4.5, SSC 10-12°Brix) as a substrate, inoculates water kefir grains (WKG), and ferments to produce apple water kefir beverage (pH 3.6, alcohol 5% vol).

[0014] 2. This invention uses GC-MS to identify 77 volatile components in the final fermented product of apple water kefir beverage, and uses GC-IMS to construct a dynamic fingerprint spectrum of fermentation, precisely controlling the increase of esters and changes of aldehydes to achieve flavor optimization. It proves that fermentation of water kefir grains enhances the unique aroma characteristics and flavor harmony of apple water kefir beverage, while also optimizing the composition of organic acids (such as the increase of oxalic acid and lactic acid).

[0015] 3. This invention uses a mouse model of high cholesterol induced by a high-fat diet to systematically verify the application of apple water kefir beverage in reducing cholesterol and regulating the intestinal microbiota. It can reduce liver lipid droplets, total cholesterol (20-30%), low-density lipoprotein (15-25%) and triglyceride levels, and promote beneficial intestinal microorganisms (such as increasing Firmicutes, Bacteroides and Faecalibacterium, with an increase of 3-16 OTUs), quantifying health benefits.

[0016] 4. The process of this invention is simple and easy to industrialize, making it suitable for the development of functional beverages. It provides refreshing products with health benefits such as regulating intestinal flora and lowering blood lipids for vegetarians and lactose-intolerant individuals, and has broad market potential.

[0017] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 These are radar images of sensory evaluation of four groups of samples from Embodiment 1 and Comparative Examples 1-3 of the present invention; Figure 2 This invention provides a thermographic analysis of volatile substances in the final fermentation products of four groups of samples from Example 1 and Comparative Examples 1-3. Figure 3 This refers to the percentage of various volatile substances in four groups of samples from Example 1 and Comparative Examples 1-3 of the present invention, (a) WK-A, (b) CK-A, (c) CK-B, and (d) WK-B; Figure 4 These are PLS-DA diagrams of volatile substances in four groups of samples from Example 1 and Comparative Examples 1-3 of this invention; Figure 5 This is a fingerprint spectrum of flavor compounds in the apple water kefir (WK-A) fermented product of Example 1 of the present invention; Figure 6This is the fingerprint spectrum of flavor compounds in the fermented product of brown sugar water kefir (WK-B) of Comparative Example 2 of this invention; Figure 7 This is the effect of apple water kefir of Example 4 of the present invention on the body weight of high-fat mice, wherein (a) is the change in mouse body weight, and (b) is the final body weight after fasting; Figure 8 The following are the blood lipid levels of mice in each group in Example 4 of the present invention: (a) TC content, (b) TG content, (c) HDL-C content, and (d) LDL-C content. Figure 9 These are the AST (a) and ALT levels (b) of each group of mice in Example 4 of this invention. Figure 10 These are HE-stained sections of mouse livers from various groups in Example 4 of this invention; Figure 11 This refers to the levels of short-chain fatty acids in the colon contents of mice in each group of Example 4 of the present invention; Figure 12 This is an analysis of OTUs (Venn plot) of the microbial diversity of mice in each group in Example 4 of the present invention. Figure 13 This is an analysis of microbial diversity among mouse samples in Example 4 of the present invention; Figure 14 This is the abundance distribution of gut microbiota at the phylum level in each group of mice in Example 4 of the present invention; Figure 15 This is the distribution of the abundance of gut microbiota genera in each group of mice in Example 4 of the present invention; Figure 16 This is a heatmap showing the correlation between mouse body weight, blood lipid levels, AST and ALT content, and short-chain fatty acid content in Example 4 of this invention. Detailed Implementation

[0019] The water-kefir grains (WKG) used in this invention are produced by Feige Food Factory in Zhanjiang City, Guangdong Province. Example 1 This embodiment describes the preparation of an apple water kefir beverage, and the method is as follows: S1. Apple juice preparation Select fresh apples with no obvious blemishes and rinse the peels with running water to remove dirt. Cut the washed apples into pieces and juice them. Add 50 mg / L potassium metabisulfite and 30 mg / L pectinase to the juice according to the yield, stir to mix thoroughly, and place in a 37°C water bath for 3 hours of enzymatic hydrolysis. Then, centrifuge at 8000 rpm for 5 minutes at 4°C, collect the supernatant and sterilize (105°C, 15 minutes). After cooling to room temperature, obtain apple juice.

[0020] S2, Water Kefir Particle Activation Prepare 60 g / L brown sugar water, mix well, sterilize (121℃, 20 min), and allow to cool to obtain sterile brown sugar water. Add water kefir grains (WKG) to the sterile brown sugar water, incubate at 25℃ for 24 h, filter, and obtain activated generation I WKG; rinse the brown sugar on the surface of WKG with sterile water to make it translucent, and then inoculate it into sterile brown sugar water for activation for 24 h; the process of inoculation-filtration-washing-inoculation constitutes one activation cycle, and this cycle is repeated 3 times to achieve high activity of WKG, thus obtaining activated WKG.

[0021] S3, Preparation of apple water kefir beverage The activated WKG obtained in S2 was inoculated into the apple juice obtained in S1 at an inoculation rate of 6%, and fermented at 25°C. Samples were taken every 8 hours. When the soluble solids content remained unchanged for 3 consecutive times, the fermentation endpoint was determined, and the apple water kefir beverage was obtained. The fermented product was denoted as WK-A.

[0022] Comparative Example 1 Apple juice, left to stand at 25°C, is labeled CK-A.

[0023] Comparative Example 2 The preparation method for brown sugar kefir beverage is as follows: S1. Preparation of brown sugar water Prepare 60 g / L brown sugar water, mix well and sterilize (121℃, 20 min), cool to obtain sterile brown sugar water; S2, Water Kefir Particle Activation Water kefir grains (WKG) were added to sterile brown sugar water and incubated at 25°C for 24 hours. After filtration, activated generation WKG was obtained. The surface of the WKG was rinsed with sterile water to make it translucent, and then inoculated into sterile brown sugar water for activation for 24 hours. The process of inoculation-filtration-washing-inoculation constitutes one activation cycle, which is repeated 3 times to ensure that the activated WKG reaches a high activity level.

[0024] S3. Preparation of brown sugar water kefir beverage The activated WKG obtained from S2 was inoculated into the brown sugar water obtained from S1 at an inoculation rate of 6%, and fermented at 25°C. Samples were taken every 8 hours. When the soluble solids content remained unchanged for 3 consecutive times, the fermentation endpoint was determined, and the fermentation product was recorded as WK-B.

[0025] Comparative Example 3 Brown sugar water, left to stand at 25℃, is denoted as CK-B.

[0026] The physicochemical properties, sensory evaluation, and volatile flavor compounds of four groups of products—water kefir grain fermented apple juice (WK-A) of Example 1, apple juice of Comparative Example 1 (CK-A), water kefir grain fermented brown sugar water of Comparative Example 2 (WK-B), and brown sugar water of Comparative Example 3 (CK-B)—were studied. The results are as follows: 1. Determination of physicochemical indicators Table 1 lists the pH, soluble solids (SSC), total acid (calculated as malic acid), total phenols (gallic acid equivalent), and alcohol content of four groups of samples: apple juice (CK-A), brown sugar water (CK-B), fermented apple water kefir (WK-A), and brown sugar water kefir (WK-B). The samples fermented with WKG showed increased total acid, decreased pH, and significantly reduced SSC, with the WK-A group showing the largest decrease in SSC and the WK-B group showing the lowest SSC content. Alcohol content determination revealed that the WK-A group had an alcohol content of 5.08%, more than twice that of WK-B, while the alcohol volume fraction was below 7% vol, indicating low alcohol content. The total phenol content determination results indicate that the total phenol content of apple juice and brown sugar water decreased after WKG fermentation, which may be a result of phenolic structure degradation or the antibacterial and detoxification mechanisms of yeast and bacteria.

[0027] Table 1. Physicochemical properties of the samples after fermentation. Note: Numerical values ​​represent mean ± standard deviation. Significance was tested using Duncan's analysis (P < 0.05). Different letters represent the significance level of numerical differences between different treatment groups; ND, not detected. (The same applies below.) Table 2 shows the determination of the content of 10 organic acids in the four groups of samples. The content of organic acids after fermentation was richer than that of the unfermented group, and the content also increased to varying degrees, especially the content of oxalic acid, lactic acid, and fumaric acid. In addition, among the four newly generated organic acids in WK-A, the content of lactic acid and succinic acid was higher than that in WK-B. Succinic acid increased from undetectable to 580.84 mg / L, and lactic acid increased to 1379.40 mg / L, giving apple kefir a mellow sour taste and making the taste more refreshing. The malic acid unique to the apple group also made the overall flavor better.

[0028] Table 2. Content of different organic acids in the samples 2. Sensory evaluation Referencing the national standard GB / T15038-2006 "General Analytical Methods for Wine and Cider" REF _Ref199061828 \r \h \* MERGEFORMATThe process was conducted. A sensory evaluation panel of 30 individuals with sensitive taste and smell (15 men and 15 women, aged 20-30) was formed. A 5-point scale was used to describe and score the aromas of the apple juice and brown sugar water before and after fermentation in the categories of wine, fruit, floral, baking, sweet, nutty, and honey.

[0029] Figure 1 This is a radar chart of the sensory evaluation of the samples. Before and after fermentation, there was no significant difference in overall aroma between CK-A and WK-A (CK-B and WK-B), but the sweet aroma score decreased. This is presumably because yeast fermentation consumed sugars, resulting in yeast fermentation aromas and a refreshing, spicy flavor that masked the sweet aroma. However, the apple group scored significantly higher overall than the brown sugar water group, with better wine, fruit, floral, and honey aromas. The fermented WK-A had a more intense wine aroma and a higher overall aroma score, making it more popular.

[0030] 3. Determination of volatile flavor compounds by GC-MS The changes in volatile substances during the fermentation of apple juice (CK-A), brown sugar water (CK-B), apple water kefir (WK-A), and brown sugar water kefir (WK-B) were detected using an HP-5MS capillary column (30m×0.25mm×0.25μm), a 50 / 30μm DVB / DVB / PDMS (Supelco, Bellefonte, USA) extraction fiber head, and gas chromatography-mass spectrometry.

[0031] like Figure 2 The final sample contained 77 compounds, including 34 esters, 18 alcohols, 11 aldehydes, 3 ketones, 5 acids, and 6 other substances. Figure 3 This represents the percentage of various volatile substances in the four groups of samples from Example 1 and Comparative Examples 1-3. The percentages of esters in WK-A and WK-B increased to 61.5% and 35.1%, respectively. Compared to WK-B, WK-A showed increases in decanoic acid, nonanoic acid, and linoleic acid, with phenylethanol content reaching a high of 6691.96 μg / L, and also increased levels of eugenol and damascene. This not only enhanced the flavor with fruity, floral, and sweet notes but also gave the product a more refreshing, mellow, and sweet taste.

[0032] The volatile components of the four groups of samples were analyzed using the partial least squares discriminant analysis (PLS-DA), and the results are as follows: Figure 4 As shown: CK-B group and WK-B group are located on the positive axis of t[1]; apple group is distributed along the negative axis of t[1] along the axis of t[2], which confirms that the four groups of volatile substances are significantly different.

[0033] 4. GC-IMS determination of volatile flavor compounds Volatile components during fermentation were detected using FlavorSpec® gas chromatography-ion mobility spectrometry (GC-IMS) equipped with an MXT-WAX (30m × 0.53mm, 1μm) column. During WKG fermentation, four sets of samples were centrifuged every 8 hours, and the supernatant was used to calculate the retention index using C4-C9 n-ketones as external standards. Qualitative analysis of volatile compounds was achieved by comparing the retention indices and relative migration times of volatile components with standards in the GC-IMS database, and the results were displayed as fingerprint chromatograms. Figure 5 and Figure 6 The fingerprint spectroscopy revealed the dynamic changes of flavor compounds in the WK-A and WK-B groups during fermentation. A total of 94 substances were detected in all samples, with 77 in the WK-A group and 66 in the WK-B group, among which 4 substances showed dimerization. Compared to milk kefir, which is richer in alcohols, water kefir samples showed a greater influence of esters on its flavor. Precursors to acetate esters, fatty acid ethyl esters, and lactate esters, such as isoamyl alcohol and phenylethanol, were detected in the samples, which is conducive to the formation of water kefir flavor compounds. According to the fingerprint spectroscopy results, with the extension of WKG fermentation time, the contents of hexanal, 1,4-cineole, and tetrahydrofuran in apple juice and brown sugar water increased, adding grassy and minty aromas to water kefir. The contents of ethyl acetate, propyl propionate, 2-butanol, and α-pinene in the WK-A group were higher than those in the WK-B group, resulting in a more harmonious and varied aroma profile.

[0034] Example 2 This embodiment describes the preparation of an apple water kefir beverage, and the method is as follows: S1. Apple juice preparation Select fresh apples with no obvious blemishes and rinse the peels with running water to remove dirt. Cut the washed apples into pieces and juice them. Add 40 mg / L potassium metabisulfite and 25 mg / L pectinase to the juice according to the yield, stir to mix thoroughly, and let stand in a 45°C water bath for 2 hours for enzymatic hydrolysis. Then, centrifuge at 7500 rpm for 8 minutes at 4°C, collect the supernatant and sterilize (100°C, 20 minutes). After cooling to room temperature, obtain apple juice.

[0035] S2, Water Kefir Particle Activation Prepare 50 g / L brown sugar water, mix well, sterilize (121℃, 20 min), and allow to cool to obtain sterile brown sugar water. Add water kefir grains (WKG) to the sterile brown sugar water, incubate at 25℃ for 24 h, filter, and obtain activated generation WKG; rinse the brown sugar on the surface of WKG with sterile water to make it translucent, and then inoculate it into sterile brown sugar water for activation for 24 h; the process of inoculation-filtration-washing-inoculation constitutes one activation cycle, and this cycle is repeated 3 times to achieve high activity of WKG, thus obtaining activated WKG.

[0036] S3, Preparation of apple water kefir beverage The activated WKG obtained in S2 was inoculated into the apple juice obtained in S1 at an inoculation rate of 5%, and fermented at 37°C. Samples were taken every 8 hours. When the soluble solids content remained unchanged for 3 consecutive times, the fermentation endpoint was determined, and the apple water kefir beverage was obtained. The fermented product was denoted as WK-A.

[0037] Example 3 This embodiment describes the preparation of an apple water kefir beverage, and the method is as follows: S1. Apple juice preparation Select fresh apples with no obvious blemishes and rinse the peels with running water to remove dirt. Cut the washed apples into pieces and juice them. Add 55 mg / L potassium metabisulfite and 40 mg / L pectinase to the juice according to the yield, stir to mix thoroughly, and let stand in a 40°C water bath for 6 hours for enzymatic hydrolysis. Then, centrifuge at 8500 rpm for 3 minutes at 4°C, collect the supernatant and sterilize (110°C, 10 minutes). After cooling to room temperature, obtain apple juice.

[0038] S2, Water Kefir Particle Activation Prepare 65 g / L brown sugar water, mix well, sterilize (121℃, 20 min), and allow to cool to obtain sterile brown sugar water. Add water kefir grains (WKG) to the sterile brown sugar water, incubate at 25℃ for 24 h, filter, and obtain activated generation WKG; rinse the brown sugar on the surface of WKG with sterile water to make it translucent, and then inoculate it into sterile brown sugar water for activation for 24 h; the process of inoculation-filtration-washing-inoculation constitutes one activation cycle, and this cycle is repeated twice to achieve high activity of WKG, thus obtaining activated WKG.

[0039] S3, Preparation of apple water kefir beverage The activated WKG obtained in S2 was inoculated into the apple juice obtained in S1 at an inoculation rate of 8%, and fermented at 18°C. Samples were taken every 8 hours. When the soluble solids content remained unchanged for 3 consecutive times, the fermentation endpoint was determined, and the apple water kefir beverage was obtained. The fermented product was denoted as WK-A.

[0040] Example 4 This example is a study on the cholesterol-lowering effects of apple water kefir beverage.

[0041] 1. Animal experiments Eighty-four specific pathogen-free (SPF) grade male 6-week-old C57BL / 6J mice (18-22 g, Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.) were used in the experiment. Mice maintenance diet, 60% high-fat diet, and SPF grade corn cob bedding were purchased from Xi'an Huaren Biotechnology Co., Ltd. To comply with ethical guidelines, the experimental mice were authorized by the Northwest University Laboratory Animal Management and Use Committee ([SCXK(Su)2020-0009]), and all related procedures were strictly performed in accordance with the "National Laboratory Animal Welfare Guidelines".

[0042] Mice were housed in a constant temperature (25±2°C), humidity (55±5%), and 12h / 12h light-dark cycle environment. After 4 days of acclimatization, the mice were divided into seven different groups (n=12). The control group (CK) was fed a maintenance diet for 5 consecutive weeks, while the other six groups were fed a high-fat diet to establish a high-fat mouse model. Subsequently, the seven groups of mice were given different diets: the CK group and the high-fat group (MD) were administered 0.9% saline (200 μL / d) by gavage; the positive control group (PC) was controlled by gavage with simvastatin aqueous solution (3 mg / kg·d); the apple juice group (CK-A), brown sugar water group (CK-B), apple water kefir group (WK-A), and brown sugar water kefir group (WK-B) were administered apple juice, brown sugar water, apple water kefir, and brown sugar water kefir, respectively, by gavage, 200 μL / d. The patients were treated with a modeling and drug administration approach for 3 weeks. After a final 12-hour fast, they were euthanized with ether, and blood was collected from their eyes. Finally, they were euthanized by cervical dislocation, and organ samples (heart, kidney, liver, and colon) were collected. The blood was allowed to stand at room temperature for 2 hours, and then centrifuged at 4°C and 4000 r / min for 10 minutes to obtain serum, which was stored at -80°C for later analysis.

[0043] 2. Determination of mouse body weight and organ index The experiment lasted for 8 weeks, during which the weight of the mice was recorded every 4 days. After the 3-week sample intervention, mouse feces were collected and mouse weight was recorded. The organs obtained after dissection were rinsed with sterile PBS solution, wiped with filter paper to remove surface moisture, weighed, and used for organ index calculation. They were stored in a -80 °C freezer.

[0044] Organ Index (%) = (Organ weight / Body weight) × 100.

[0045] See results Figure 7(a) shows the change in mouse body weight, and (b) shows the final body weight after fasting. On day 36, the weight of the CK group was 26.12g, and the weight of the high-fat diet group exceeded that of the CK group by 20%, so gavage treatment could begin. After the first gavage, the body weight of mice in all groups decreased, and gradually increased during the gavage period; after the gavage was completed, the mice were fasted for 12 hours, and the last recorded body weight was ( ). Figure 7 (b) After fasting, the body weight of mice in all seven groups decreased. The average body weight of the CK group (24.99g) was significantly different from the other groups. The MD group had the highest average body weight, while the PC group's body weight was close to that of the CK group. The body weight differences among the four groups after sample intervention were relatively small.

[0046] Table 3 shows that a high-fat diet causes fat to accumulate in the liver of mice, but has no significant effect on the heart and kidneys.

[0047] Table 3. Effects of different sample treatments on organ indices in high-fat mice 3. Serum lipid analysis Collect the serum samples to be tested, and use the kits to measure the levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

[0048] Figure 8 The following data represent the blood lipid levels in each group of mice: (a) total cholesterol (TC), (b) total triglycerides (TG), (c) high-density lipoprotein cholesterol (HDL-C), and (d) low-density lipoprotein cholesterol (LDL-C). Results showed that the MD group had higher levels of TC, TG, and LDL-C than the other groups, while the PC group's levels were close to those of the CK group. In the TC content analysis, the WK-A and WK-B groups were between those of the PC and MD groups. Among the other three indicators, the WK-A group's levels were closer to those of the CK and MD groups, possibly because apples contain dietary fiber, which enhances the cholesterol-lowering effect. The CK-A and CK-B groups had higher HDL-C levels than the fermented group. There were no significant differences in TC and LDL-C levels between the apple juice / brown sugar water group and the high-fat model group, possibly because the apple juice / brown sugar water (SSC) had a higher sugar content, leading to increased blood glucose levels in both groups. Overall, the WK-A group played a more positive role in reducing blood lipid levels in high-fat mice.

[0049] Figure 9The AST (a) and ALT (b) levels in each group of mice are shown. The serum AST and ALT levels in the MD group were significantly higher than those in other groups, increasing by 303.82% and 102.27% respectively compared to the CK group. The AST and ALT levels in both the WK-A and WK-B groups were significantly lower, while the AST and ALT levels in the CK-A group were lower than those in the CK-B group, indicating that apple juice has a protective effect on the liver, and this protective effect is enhanced after fermentation by WKG.

[0050] 4. Liver pathological section analysis Liver specimens were fixed with 4% paraformaldehyde (purchased from Wuhan Serviccebio Biotechnology Co., Ltd.), embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed and recorded under a biological microscope for histological evaluation.

[0051] Figure 10 These are the observation results of HE-stained liver sections from seven groups of mice. In the CK group, the liver structure was intact, with hepatocytes arranged radially around the central vein and hepatocyte nuclei centrally and neatly arranged, representing a normal liver morphology. The remaining six groups of liver sections all showed round vacuoles of varying sizes, which are gaps left by dissolved lipid droplets and are an inherent characteristic of the high-fat diet model. In the MD group, a few cell nuclei were pushed to one side, indicating triglyceride accumulation due to lipid metabolism imbalance. Additionally, except for the PC group, a small amount of inflammatory factors were observed in the other images, while the MD group showed a significant increase in cell density in the left area, possibly due to inflammatory cell infiltration. The PC group showed a significant reduction in lipid droplets, and the cells were more neatly arranged compared to other high-fat diet groups. The CK-A and CK-B groups had more lipid droplets, possibly due to the high sugar content of apple juice and brown sugar water, leading to elevated blood glucose levels in the mice. The fermented group had fewer lipid droplets, indicating that water kefir helps improve lipid metabolism disorders in the liver.

[0052] 5. Analysis of intestinal short-chain fatty acids Take 0.1g of colon contents from each group, 0.1mL of 50% sulfuric acid, and 1mL of diethyl ether into a 2mL centrifuge tube. Vortex for 3 minutes to mix thoroughly, then centrifuge (12000×g, 15min). Collect the supernatant, add 0.25g of anhydrous sodium sulfate, and centrifuge again (12000×g, 15min). Collect the supernatant, filter it through a 0.22μm filter membrane, and determine the concentration of short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, n-valeric acid, and hexanoic acid) in the intestine using gas chromatography.

[0053] Figure 11This study reflects the effects of different sample interventions on the levels of short-chain fatty acids (SCFAs) in the colonic contents of mice in each group: a) acetic acid, b) propionic acid, c) butyric acid, d) isobutyric acid, e) valerate, f) isovaleric acid, and g) hexanoic acid. It can be seen that water kefir fermentation significantly increased the total SCFA level. Acetic acid had the largest proportion of total SCFAs and was also the most abundant substance in all groups. It is a precursor to palmitate and stearate production and can improve fatty acid metabolism in the liver. In the PC group, the acetic acid content reached 30.94 μmol / g, an increase of 273.67% compared to the MD group, followed by the water kefir fermentation group, indicating that water kefir can increase acetic acid content, thus having a positive effect on lowering cholesterol. Among the four sample treatment groups, the WK-A group had the highest propionic acid content, and the WK-B group had the highest butyric acid content. Propionic acid has the ability to inhibit HMGCR activity and mRNA expression, thereby controlling cholesterol metabolism. Isovalerate and valerate are mainly produced by intestinal microorganisms through protein / amino acid fermentation. Compared to other groups, the apple juice group had a higher content of valeric acid, which may be related to the presence of dietary fiber in the matrix.

[0054] 6. Intestinal microbiome analysis Before fasting, feces were collected from the CK, MD, PC, CK-A, WK-A, CK-B, and WK-B groups, respectively. After being flash-frozen in liquid nitrogen, the feces were stored at -80°C for later use. At least three replicates from each group were taken for microbial diversity assays. The sample analysis was commissioned to Wuhan BGI Genomics Co., Ltd. to perform paired-end sequencing of the V3-V4 regions of the 16S rRNA of archaea and bacteria.

[0055] (1) Analysis of microbial diversity OTUs Figure 12 The analysis of microbial diversity OTUs in different samples (Venn plot) shows that (a) is the apple juice group and (b) is the brown sugar water group. The results showed that the PC, CK, MD, CK-A, and WK-A groups shared 145 core OTUs, with 38 unique OTUs in the CK-A group and 41 unique OTUs in the WK-A group. The PC, CK, MD, CK-B, and WK-B groups shared 161 core OTUs, with 28 unique OTUs in the CK-B group and 44 unique OTUs in the WK-B group. This indicates that a high-fat diet alters the number of OTUs compared to the normal group, and the four interventions (CK-A, WK-A, CK-B, and WK-B) further developed unique gut microbiota characteristics distinct from the high-fat diet group.

[0056] (2) Analysis of microbial community diversity in samples Figure 13For the analysis of microbial diversity among different samples, (a) is the ACE index, (b) is the Chao index, (c) is the Shannon index, and (d) is the Simpson index. Compared with the CK group, the MD group showed a decrease in ACE and Chao indices, while the Shannon and Simpson indices increased, with the Shannon index being the highest. This indicates a decrease in the number of species and an increase in diversity of gut microbiota in high-fat mice. Compared with the MD group, the CK-A group had similar indices to the MD group except for a lower Shannon index. Compared with before WKG fermentation, the data from the WK-A and WK-B groups were more concentrated. The Simpson index of the WK-A group was higher than that of the CK-A group, indicating that apple water kefir increases the richness of gut microbiota. The ACE, Chao, and Shannon indices of the WK-B group were higher than those of the CK-B group, indicating that brown sugar water kefir can decrease the richness of the microbiota but increase its evenness, showing a trend of microbiota optimization.

[0057] (3) Changes in the relative abundance of gut microbiota in high-fat mice Figure 14 Analysis of the relative abundance of species at the phylum level in each group revealed that the gut microbiota of mice was mainly composed of Firmicutes-A and Firmicutes-B, and Bacteroidota. Compared with the CK group, the MD group showed an increased relative abundance of Firmicutes and a decreased abundance of Bacteroidota; the CK-A and WK-A groups showed an increased abundance of Firmicutes-B, while the CK-B and WK-B groups showed a significant increase in the abundance of Firmicutes-A and a decrease in the abundance of Bacteroidota. These trends suggest that water kefir intervention may have a specific regulatory effect on the gut microbiota structure of diabetic mice.

[0058] Figure 15 This study reflects the abundance distribution of gut microbiota genera in mice under different treatment groups. A total of 32 genera were detected at the genus level, with the most abundant being *Faecalibaculum*, *Peptococcus*, *Kineothrix*, *Dubosiella*, and *Acetatifactor*. *Kineothrix* has metabolic functions, participating in the production of short-chain fatty acids acetic acid and butyrate. *Dubosiella* is a newly named genus, associated with the Lactobacillus family, and possesses potential probiotic functions. Compared to the CK group, the MD group showed a higher abundance of *Faecalibaculum*, with some strains exhibiting bile acid metabolism capabilities. Excessive bile acid metabolism may lead to lipid digestion disorders, indirectly affecting barrier integrity. Compared to the MD group, the WK-A group showed increased abundance of *Peptococcus* and *Acetatifactor*.

[0059] (4) Correlation analysis Figure 16 This is a visualization of the correlation analysis of body weight, blood lipid levels, AST and ALT levels, and short-chain fatty acid content in seven groups of mice, presented using a heatmap method to more intuitively show the correlation between each indicator and cholesterol (* indicates p<0.05). In the graph, the redder the color and the larger the circle, the higher the correlation, and vice versa. The graph shows that HDL-C content is negatively correlated with high cholesterol, while TC, TG, and LDL-C are positively correlated and have significant effects. Acetic acid, propionic acid, and butyric acid play a more active role in lowering cholesterol.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing an apple water kefir beverage, characterized by, The method comprises the following steps: S1, apple juice preparation First, fresh apples without obvious surface scratches are selected, the fruit skin is washed with flowing clean water, the washed apples are cut and juiced, then potassium metabisulfite and pectinase are added to the juice, mixed, and enzymolysis is carried out at 35-45℃ for 2-6h, then centrifugation is carried out at 4℃, the supernatant is sterilized, and the apple juice is obtained after cooling; S2, water kefir grain activation Brown sugar water is prepared, sterilized and cooled to obtain sterile brown sugar water; water kefir grains are added to the sterile brown sugar water, and after 24h of static culture at 25℃, filtration is carried out to obtain the first generation of activated water kefir grains; the surface brown sugar of the water kefir grains is washed with sterile water until it is translucent, and then inoculated in sterile brown sugar water for 24h of activation; the process of inoculation, filtration, washing and inoculation is one activation cycle, and the cycle is repeated 2-3 times to obtain activated water kefir grains; S3, preparation of apple water kefir beverage The activated water kefir grains obtained in S2 are inoculated in the apple juice obtained in S1, and fermentation is carried out at 18-37℃, and the fermentation endpoint is determined when the soluble solid content is constant to obtain the apple water kefir beverage.

2. The method of claim 1, wherein, The concentration of the potassium metabisulfite in S1 is 40-55mg / L, and the concentration of the pectinase is 25-40mg / L.

3. The method of claim 1, wherein, The speed of the centrifugation in S1 is 7500-8500r / min, and the time is 3-8min.

4. The method of claim 1, wherein, The sterilization temperature in S1 is 100-110℃, and the time is 10-20min.

5. The method of claim 1, wherein, The concentration of the brown sugar water in S2 is 50-65g / L, and the sterilization temperature of the brown sugar water is 121℃ for 20min.

6. The method of claim 1, wherein, The inoculation amount of the water kefir grains in the apple juice in S3 is 5-8%.

7. An apple water kefir beverage prepared by the method of any one of claims 1-6.

8. Use of the apple water kefir beverage according to claim 7, characterized in that, The apple water kefir beverage is used for reducing cholesterol.

9. Use of the apple water kefir beverage according to claim 7, characterized in that, The apple water kefir beverage is used for regulating intestinal microbial community.

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