Goat yogurt with activity of reducing blood sugar and blood fat and preparation method of goat yogurt

Abstract

According to the sour goat milk with the activity of reducing blood sugar and blood fat and the preparation method of the sour goat milk, functional polysaccharide ultrasonically extracted from kefir grains in water and a fermentation system of lactobacillus delbrueckii subsp. Bulgaricus and streptococcus salivarius subsp. Thermophilus are adopted for fermentation for 4-5 h at the constant temperature of 41-43 DEG C, a polysaccharide-strain synergistic system is constructed, and the sour goat milk with the activity of reducing blood sugar and blood fat is obtained. The blood sugar and lipid lowering activity, texture characteristics and sensory evaluation of the prepared goat yogurt are remarkably improved; the leavening agent is fermented in the goat milk containing functional polysaccharides, so that the coagulability of the sour goat milk is effectively enhanced, whey precipitation is reduced, the curding time is shortened, and the stability of a system is greatly improved. The goat yogurt prepared by the process has the advantages of high activity of reducing blood sugar and blood fat, good coagulability, difficulty in precipitation of whey, high system stability and good taste.

Description

Technical Field

[0001] This application belongs to the field of food engineering technology, specifically relating to a type of acidic goat milk with hypoglycemic and lipid-lowering activities and its preparation method. Background Art

[0002] In the current technology, yogurt goat milk, as a dairy product with unique flavor and nutritional value, has a certain consumer base in the market, and it has many health benefits, such as being easier to digest and absorb, and having lower allergenicity. However, there are still some issues with yogurt goat milk currently on the market that need improvement.

[0003] While traditional methods for preparing fermented goat milk can improve its flavor and nutritional value to some extent, they cannot effectively solve problems such as poor coagulation, easy whey separation, and long curdling time. These problems directly affect the quality and market competitiveness of fermented goat milk, limiting its further development and promotion.

[0004] Furthermore, existing technologies have shortcomings in enhancing the functionality of yogurt. While some studies have highlighted the application potential of polysaccharides in the food industry, systematic research on functional polysaccharides in yogurt remains lacking. The composition, structural characteristics, and compatibility of water kefir grains with yogurt beverages require further exploration. Currently, there is a lack of in-depth and mature research and application on how to optimize the extraction efficiency of functional polysaccharides and introduce them as natural additives into the fermentation system to increase the bioactivity and improve the texture of yogurt, thereby developing novel yogurts that combine nutrition and functionality.

[0005] Therefore, in order to comprehensively improve the quality, stability, and functionality of yogurt goat milk, improvements are urgently needed to meet consumer demand for high-quality functional dairy products and promote the further development of the goat milk industry. Summary of the Invention

[0006] In order to address the technical problems of traditional fermented goat milk having limited functionality and nutritional value, poor coagulation properties, easy whey separation, long coagulation time, poor system stability, and a lack of functional fermented goat milk products, this application proposes a fermented goat milk with hypoglycemic and lipid-lowering activities.

[0007] In order to solve the technical problems raised in this application, this application also provides a method for preparing yogurt with hypoglycemic and lipid-lowering activities.

[0008] This application adopts the following scheme: a method for preparing acidic goat milk with hypoglycemic and lipid-lowering activities, comprising the following steps:

[0009] Step 101. Add functional polysaccharides to goat milk, and then heat and cool it to obtain sterilized goat milk.

[0010] Step 102. After adding the direct-inoculation starter culture to the sterilized goat milk prepared in step 101, place it at a constant temperature of 41℃-43℃ for 4h-5h to obtain the acid goat milk with hypoglycemic and lipid-lowering activities.

[0011] In step 102, the α-glucosidase inhibition rate of the acidic goat milk with hypoglycemic and lipid-lowering activities was 77.46%-84.40%, the DPP-IV inhibition rate was 52.68%-61.01%, and the pancreatic lipase inhibition rate was 78.25%-86.43%.

[0012] In step 101, the functional polysaccharide was extracted from water kefir grains;

[0013] In step 102, the fermentation agent is a combination of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus.

[0014] In practice, the water kefir granules are produced in Anshan, Liaoning Province, and can be purchased from: https: / / shop34241062.taobao.com / .

[0015] In practice, the direct-inoculation fermentation agent for yogurt was purchased from Shenghe Biotechnology Co., Ltd.

[0016] In some feasible embodiments, in step 101, the goat milk is selected from fresh goat milk or reconstituted goat milk, and the concentration of the reconstituted goat milk is 12.5% ​​(w / v).

[0017] In practice, to improve production efficiency, reconstituted sheep milk is selected.

[0018] In some feasible embodiments, in step 101, the amount of functional polysaccharide added is 0.05wt%-0.15wt%.

[0019] The amount of starter culture added in step 102 is 0.005 wt%.

[0020] In some feasible embodiments, step 101, the method for extracting functional polysaccharides, includes the following steps:

[0021] Step 201. Under room temperature conditions, water kefir grains are continuously passaged and cultured using sterile brown sugar water as a substrate to obtain activated water kefir grains.

[0022] Step 202. Add activated water kefir particles and distilled water to an ultrasonic reactor in sequence. After ultrasonic treatment at 40℃-80℃, collect the supernatant to obtain the first-stage crude extract.

[0023] Step 203. Add the primary crude extract and trichloroacetic acid to a centrifuge in sequence. Centrifuge at 1000 rpm to 4000 rpm for 10 min to 20 min, and collect the supernatant to obtain the secondary crude extract.

[0024] Step 204. After adding anhydrous ethanol to the secondary crude extract, refrigerate it at 4°C for 12-48 hours to obtain the tertiary crude extract.

[0025] Step 205. Transfer the tertiary crude extract to a centrifuge and centrifuge at 1000rpm-4000rpm for 10min-20min. Remove the supernatant to obtain the crude polysaccharide.

[0026] Step 206. After completely dissolving the crude polysaccharide in deionized water, the solution is then subjected to decolorization, filtration, chromatography, dialysis, and freeze-drying in sequence to obtain the functional polysaccharide product.

[0027] In some feasible embodiments, in step 202, the ultrasonic processing power is 240W-320W; the ultrasonic processing time is 30min-50min.

[0028] In some feasible embodiments, in step 202, the ratio of water kefir granules to distilled water is 1:(10-30).

[0029] In some feasible embodiments, step 201 includes the following steps: fermentation culture using brown sugar water as a base. First, prepare 10% (w / v) brown sugar water, heat it to boiling until completely dissolved, pour it into a glass bottle, and after the brown sugar water cools to room temperature, add water kefir grains at an inoculum rate of 5% (w / v). After static culture for 24 hours, filter out the water kefir grains with a sieve, and then add fresh brown sugar water to continue the activation culture twice more to obtain activated water kefir grains.

[0030] In practice, the growth of water kefir grains is due to the conversion of sucrose into glucan by extracellular glucanase. Water kefir grains can continuously proliferate in brown sugar water, making full use of the carbon source and other nutrients in the brown sugar water for proliferation.

[0031] In actual implementation, the decolorization step 206 includes the following steps: the crude polysaccharide is completely dissolved in deionized water, 1.5 wt% activated carbon powder is added to the solution, and decolorization is carried out under constant temperature water bath conditions, with the treatment time controlled at 60 min and the temperature maintained at 60℃. After decolorization, the solution is filtered while hot using a Buchner funnel to remove the activated carbon from the polysaccharide extract.

[0032] In some feasible embodiments, in step 206, chromatography is performed using a DEAE-cellulose ion exchange column;

[0033] During chromatography, deionized water, 0.05M NaCl solution, 0.1M NaCl solution, and 0.3M NaCl solution were used for elution in sequence.

[0034] In actual implementation, step 206, using a DEAE-cellulose ion exchange column for chromatography, includes the following steps: First, DEAE-cellulose is swollen with 0.1M acetic acid (containing 0.5M sodium chloride) solution, washed multiple times to remove debris, equilibrated with buffer, and then packed into a chromatographic column (Φ1.6cm×30cm). Air bubbles are gently tapped to eliminate them, and a constant flow pump is used to pressurize until the packing material is stable. The effluent pH is then equilibrated with pure water (1mL / min) until neutral. The crude polysaccharide solution, after decolorization and filtration in step 206, is adjusted to 20mg / mL. This solution is centrifuged (10000r / min, 10min), passed through a 0.45μm membrane, and loaded in a circular motion around the column wall. Once the sample level is flush with the column surface, gradient elution is performed sequentially with deionized water, 0.05M, 0.1M, and 0.3M NaCl solutions (2 column volumes each, flow rate 1mL / min). Elution buffer was collected in separate tubes, with 10 mL collected in each tube. The eluent was replaced every 20 tubes. The total sugar content was determined using the phenol-sulfuric acid method, and elution curves were plotted. The eluents corresponding to the target peak were combined, concentrated, and then dialyzed. Finally, the target functional polysaccharide product was obtained by freeze-drying.

[0035] In some feasible embodiments, in step 206, dialysis is performed using an ultrafiltration tube; during dialysis, the molecular cutoff of the ultrafiltration tube is 10 kDa-300 kDa.

[0036] In order to solve the technical problems raised in this application, this application also provides a type of fermented goat milk with hypoglycemic and lipid-lowering activities, which is prepared by the above-mentioned method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activities.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] This application provides a fermented goat milk with hypoglycemic and lipid-lowering activities and its preparation method. A fermentation system using functional polysaccharides extracted by ultrasonic extraction from water kefir grains and *Lactobacillus delbrueckii* subsp. *bulgaricus* and *Streptococcus salivarius* subsp. *thermophilus* is constructed by fermenting at a constant temperature of 41℃-43℃ for 4-5 hours, establishing a polysaccharide-strain synergistic system. The resulting fermented goat milk exhibits significantly improved hypoglycemic and lipid-lowering activities, textural properties, and sensory evaluation. The fermentation agent in the goat milk containing the functional polysaccharides effectively enhances the coagulation properties of the fermented goat milk, reduces whey separation, shortens coagulation time, and significantly improves the system's stability. The fermented goat milk prepared by this process has the advantages of high hypoglycemic and lipid-lowering activities, good coagulation properties, minimal whey separation, high system stability, and good taste. Attached Figure Description

[0039] Figure 1 This is a line graph showing the relationship between the extraction rate of functional polysaccharides and the material-liquid ratio under the basic extraction conditions in Example 1 of this application;

[0040] Figure 2 This is a line graph showing the relationship between the functional polysaccharide extraction rate and the change in ultrasonic power under the basic extraction conditions in Example 1 of this application;

[0041] Figure 3 This is a line graph showing the relationship between the extraction rate of functional polysaccharides and the change in ultrasonic time under the basic extraction conditions in Example 1 of this application;

[0042] Figure 4 This is a line graph showing the relationship between the extraction rate of functional polysaccharides and the change in ultrasonic temperature under the basic extraction conditions in Example 1 of this application;

[0043] Figure 5 This is the chromatographic curve of the functional polysaccharide in step 206 of Example 2 of this application;

[0044] Figure 6 This is the infrared spectrum of the functional polysaccharide prepared in Example 2 of this application;

[0045] Figure 7 This is an ion chromatogram of the functional polysaccharide prepared in Example 2 of this application;

[0046] Figure 8 This is the total methylation ion chromatogram of the functional polysaccharide prepared in Example 2 of this application;

[0047] Figure 9 This is a 200X scanning electron microscope image of the functional polysaccharide prepared in Example 2 of this application;

[0048] Figure 10 This is a 1000X scanning electron microscope image of the functional polysaccharide prepared in Example 2 of this application;

[0049] Figure 11 This is a 5000X scanning electron microscope image of the functional polysaccharide prepared in Example 2 of this application;

[0050] Figure 12 This is the result of the determination of α-glucosidase inhibition rate in Example 3 of this application;

[0051] Figure 13 This is the result of the DPP-IV inhibition rate measurement in Example 3 of this application;

[0052] Figure 14 This is the result of the determination of pancreatic lipase inhibition rate in Example 3 of this application;

[0053] Figure 15 This is the result of the cholesterol micelle binding rate determination in Example 3 of this application;

[0054] Figure 16 This refers to the pH value measurement result in Example 3 of this application;

[0055] Figure 17 This is the result of acidity measurement in Example 3 of this application;

[0056] Figure 18 This is the sensory evaluation of the refrigerated goat milk in Example 3 of this application;

[0057] Figure 19 This refers to the textural characteristics of the refrigerated sheep milk in Example 3 of this application. Detailed Implementation

[0058] Combination Figure 1-19 The embodiments 1-3 further illustrate the technical solutions provided in this application.

[0059] Example 1: Ultrasound-assisted extraction of functional polysaccharides

[0060] (1) The extraction method of functional polysaccharides includes the following steps:

[0061] Step 201. Under room temperature conditions, water kefir grains are continuously subcultured using sterile brown sugar water with a mass fraction of 10% (w / v) as the substrate to obtain activated water kefir grains. The single subculture time is 24 hours.

[0062] Step 202. Add activated water kefir granules and distilled water to the ultrasonic reactor in a material-to-liquid ratio of 1:20 g / mL. After ultrasonic treatment at an ultrasonic temperature of 60℃, an ultrasonic power of 320W, and an ultrasonic time of 30min, collect the supernatant to obtain the first-stage crude extract.

[0063] Step 203. Add 40 wt% trichloroacetic acid and the primary crude extract to a centrifuge in sequence. When the mass fraction of trichloroacetic acid is measured to be 4 wt%, stop adding the primary crude extract. Centrifuge at 4000 rpm for 15 min and collect the supernatant to obtain the secondary crude extract.

[0064] Step 204. Add three times the volume of anhydrous ethanol to the secondary crude extract, and then refrigerate at 4°C for 24 hours to obtain the tertiary crude extract.

[0065] Step 205. Transfer the tertiary crude extract to a centrifuge and centrifuge at 4000 rpm for 15 min. Remove the supernatant to obtain the crude polysaccharide.

[0066] Step 206. After completely dissolving the crude polysaccharide in deionized water, the solution is then subjected to decolorization, filtration, chromatography, dialysis, and freeze-drying in sequence to obtain the functional polysaccharide product.

[0067] Based on the above preparation method of functional polysaccharides, a single-factor control method was adopted. With fixed parameters (liquid-to-solid ratio 1:20 (w / v), ultrasonic power 320W, ultrasonic time 30min, ultrasonic temperature 60℃), the single parameters in the ultrasonic treatment process of activated water kefir particles in step 202 were changed respectively. Functional polysaccharides were extracted under the following conditions: liquid-to-solid ratio (1:10, 1:15, 1:20, 1:25, 1:30 g / mL), ultrasonic power (240W, 280W, 320W, 360W, 400W), ultrasonic time (20min, 30min, 40min, 50min, 60min), and ultrasonic temperature (40℃, 50℃, 60℃, 70℃, 80℃).

[0068] After extraction, the functional polysaccharide products were accurately weighed and diluted to the preset concentration. The content of functional polysaccharides was determined by the phenol-sulfuric acid method, and the extraction rate of the corresponding functional polysaccharides was calculated according to the following formula (A).

[0069] Formula (A):

[0070] In formula (A): C is the concentration of functional polysaccharides; V is the volume of the extract; N is the dilution factor of the extract; and m is the dry weight of the water kefir grains.

[0071] The effects of different process parameters on the extraction rate of functional polysaccharides, such as Figure 1-4 As shown, by Figure 1-4 It can be seen that as the material-liquid ratio increases from 1:10 to 1:15, the extraction efficiency of functional polysaccharides shows an upward trend, reaching its peak at a material-liquid ratio of 1:15. However, as the material-liquid ratio continues to increase to 1:30, the polysaccharide extraction rate gradually decreases.

[0072] As the ultrasonic power gradually increases, the extraction rate of functional polysaccharides also increases. The extraction rate reaches its peak at 320W, but begins to decline beyond this value. This phenomenon can be attributed to the following: within the lower ultrasonic power range, increasing the ultrasonic power produces a cavitation effect, which helps break down cell walls and promotes mass transfer, thereby improving extraction efficiency. However, when the critical intensity is exceeded, although the enhanced cavitation effect is beneficial for polysaccharide dissolution, excessively high energy leads to violent particle collisions, causing a rapid increase in system temperature, damaging the structure of water kefir particles, and ultimately resulting in a decrease in the extraction rate.

[0073] During ultrasonic treatment, the extraction rate of functional polysaccharides showed a trend of first increasing and then decreasing. The extraction rate significantly increased within the 20-40 min treatment period, reaching its maximum at 40 min. This phenomenon may be attributed to the increased cell disruption with prolonged ultrasonic treatment, which facilitates polysaccharide dissolution. However, after 40 min, the extraction rate began to decline. This could be due to two reasons: firstly, prolonged ultrasonic treatment might promote the dissolution of other impurities; secondly, the mechanical shearing effect of ultrasound might damage some of the dissolved polysaccharide molecules, leading to a decrease in extraction efficiency.

[0074] With increasing ultrasonic temperature, the extraction rate of functional polysaccharides showed a continuous upward trend, reaching its maximum at 80℃. This indicates that during ultrasonic extraction, higher temperatures can enhance substance transfer, increase the mobility of polysaccharide molecules, and facilitate the diffusion of intracellular polysaccharide molecules into the solvent.

[0075] Based on the single-factor experiments, a combined experiment of ultrasound-assisted extraction conditions was further studied. The experimental design and results are shown in Table 1.

[0076] Table 1. Effects of ultrasound-assisted extraction combination experiments on polysaccharide extraction rate

[0077]

[0078]

[0079] As shown in Table 1, the extraction rate of functional polysaccharides was 15.87%-26.99% within the range of ultrasonic power of 280-320W, ultrasonic time of 30-50min, and material-liquid ratio of 1:10-1:30. Regression analysis of the data further yielded the optimal extraction parameters for functional polysaccharides as follows: material-liquid ratio of 1:15 g / mL, ultrasonic power of 320W, ultrasonic treatment time of 40min, and ultrasonic treatment temperature of 80℃.

[0080] Example 2: Extraction, separation, purification and characterization of functional polysaccharides

[0081] (1) Functional polysaccharide extraction was performed using the optimal ultrasonic treatment process parameters obtained in Example 1, wherein the crude polysaccharide chromatography curve in step 206 is as follows: Figure 5 As shown.

[0082] (2) The functional polysaccharides extracted in (1) above were subjected to infrared spectroscopy, ion chromatography, methylation analysis and scanning electron microscopy analysis, respectively. The analysis results are as follows: Figures 6-11 And as shown in Table 2;

[0083] Table 2. Methylation analysis (linkage analysis) data of WPU

[0084]

[0085]

[0086] Depend on Figure 7 As shown in Table 2, after complete methylation, hydrolysis, reduction, and derivatization, six derivatives were obtained from WPU: 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl mannitol, 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol, 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl glucitol, and 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol. glucitol and 1,2,5,6-tetra-O-acetyl-3,4-di-O-methylglucitol correspond to six glycosidic bonds: t-Glcp-(1→, →3)-Manp-(1→, →6)-Glcp-(1→, →3,6)-Glcp-(1→, →4,6)-Glcp-(1→ and 2,6)-Glcp-(1→), with molar ratios of 15.33%, 1.33%, 72.33%, and 7.80%, respectively. The percentages were 1.43% and 11.78%. This indicates that the main chain is primarily composed of 6-Glcp, with t-Glcp as the terminal group. The results show that the WPU polysaccharide component mainly consists of glucose (Glc) and mannose (Man). Glucose residues exist in the forms of t-Glcp, 6-Glcp, and 3,6-Glcp, 4,6-Glcp, and 2,6-Glcp, with a molar ratio of 15.33:72.33:7.8:1.43:1.78. mannose residues exist as 3-Manp.

[0087] Example 3: Preparation and refrigeration study of fermented goat milk with hypoglycemic and lipid-lowering activities.

[0088] (1) The preparation method of yogurt with hypoglycemic and lipid-lowering activities includes the following steps:

[0089] Step 101. Add 0 wt%, 0.05 wt%, 0.10 wt%, and 0.15 wt% of the functional polysaccharides prepared in Example 2 to reconstituted goat milk with a concentration of 12.5% ​​(w / v), respectively. After heating and sterilizing and cooling, respectively, sterilized goat milk is obtained.

[0090] Step 102. Add 0.005 wt% of a direct-inoculation starter culture TW (Shenghe Biotechnology Co., Ltd.) composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus to the sterilized goat milk prepared in Step 101, and then place it at a constant temperature of 42℃ for 4.5 h to obtain acidic goat milk with hypoglycemic and lipid-lowering activities, and label it as TW, WPU (0.05 wt%), WPU (0.1 wt%), and WPU (0.15 wt%), respectively.

[0091] The starter culture is a mixture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus in a mass ratio of 1:1.

[0092] (2) The following tests were conducted during the preparation of yogurt in (1):

[0093] Test 1: α-glucosidase inhibition rate

[0094] 25 μL of the acidified sheep milk prepared in (1) [TW, WPU (0.05 wt%), WPU (0.1 wt%), WPU (0.15 wt%)] was mixed with 50 μL of α-glucosidase solution (2 U / mL) and 25 μL of PNPG (2.5 mmol / L). The mixture was reacted at 37 °C for 30 min. Then, 100 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance was measured at 405 nm. Acarbose was used as a positive control. The α-glucosidase inhibition rate was calculated according to formula (E).

[0095] Formula (E):

[0096] In formula (E), A1 is the absorbance of the sample well; A2 is the absorbance of the sample control well (with PBS replacing α-glucosidase); A3 is the absorbance of the blank well (with PBS replacing the sample); and A4 is the absorbance of the blank control well (with PBS replacing both the sample and α-glucosidase).

[0097] Test 2: DPP-IV inhibition rate

[0098] Take 25 μL of the acidified sheep milk prepared in (1) [TW, WPU (0.05 wt%), WPU (0.1 wt%), WPU (0.15 wt%)] and mix it with 50 μL of DPP-IV solution (0.01 U / mL) and 25 μL of Gly-Pro-pNA substrate solution (1.6 mmol / L). After reacting at 37 °C for 1 h, add 100 μL of CH3COONa solution (1 mol / L, pH 4.0) to terminate the reaction. Measure the absorbance at 405 nm. Use dipeptidyl peptidase (IPI) as a positive control and calculate the DPP-IV inhibition rate according to formula (F).

[0099] Formula (F):

[0100] In formula (F), A1 is the absorbance of the sample well, A2 is the absorbance of the sample control well (with Tris-HCl replacing DPP-IV solution), A3 is the absorbance of the blank well (with Tris-HCl replacing the sample), and A4 is the absorbance of the blank control well (with Tris-HCl buffer replacing both the sample and DPP-IV).

[0101] Test 3: Pancreatic lipase inhibition rate

[0102] 40 μL of the acidified sheep milk prepared in (1) [TW, WPU (0.05 wt%), WPU (0.1 wt%), WPU (0.15 wt%)], 160 μL of PBS buffer solution (0.1 mol / L, pH 7.4), and 60 μL of pancreatic lipase (30 U / ml) were added sequentially to centrifuge tubes. The tubes were incubated at 37 °C for 10 min, followed by the addition of 180 μL of 4-nitrobenzene laurate solution (P-PNL). The reaction was carried out at 37 °C in the dark for 1 h, followed by centrifugation (12000 rpm, 5 min). The supernatant was then measured at 405 nm. Simvastatin was used as a positive control, and the pancreatic lipase inhibition rate was calculated according to formula (G).

[0103] Formula (G):

[0104] In formula (G), A1 is the absorbance of the sample tube; A2 is the absorbance of the sample control tube (with PBS replacing pancreatic lipase); A3 is the absorbance of the blank tube (with PBS replacing the sample); and A4 is the absorbance of the blank control tube (with PBS replacing both the sample and pancreatic lipase).

[0105] Test 4: Cholesterol micelle inhibition rate

[0106] A standard curve was established using the concentration of phthalaldehyde as the independent variable and the absorbance at 550 nm as the dependent variable. The resulting linear regression equation was y = 4.0093x + 0.0026(R²). 2 =0.9997). Cholesterol micelle solutions were prepared by ultrasonic disruption, with each 100 mL of PBS (15 mmol / L) buffer containing 0.077 g cholesterol, 0.538 g sodium taurocholate, 158.7 μL oleic acid, and 0.772 g sodium chloride. After ultrasonic emulsification for 30 min, the solutions were incubated at 37 °C with shaking for 24 h.

[0107] Add 3 times the volume of the above cholesterol micelle solution to the acidic sheep milk [TW, WPU (0.05wt%), WPU (0.1wt%), WPU (0.15wt%)] prepared in (1) at a predetermined volume, shake in a shaker at 37°C for 1 h, centrifuge at 12000 rpm for 20 min, take the supernatant and determine the cholesterol content, and calculate the pancreatic lipase inhibition rate according to formula (H).

[0108] Formula (H):

[0109] In formula (H), A1 is the cholesterol solubility (mg / mL) of the sample tube; A0 is the cholesterol solubility (mg / mL) of the blank tube (PBS buffer instead of sample).

[0110] Test 5: pH Measurement

[0111] pH values ​​were measured using a pH meter.

[0112] Test 6: Acidity Measurement

[0113] The acidity of the sample was determined according to the national standard (GB5009.239-2016): the acidified goat milk sample was thoroughly mixed with distilled water at a volume ratio of 1:2, and titrated to pH 8.3 using 0.1 mol / L sodium hydroxide standard solution. Nitrogen gas was continuously purged during the titration to prevent the solution from absorbing carbon dioxide from the air. The acidity was calculated according to formula (J).

[0114] Formula (J):

[0115] In the formula: X is the acidity of the sample; C is the concentration of the NaOH standard titration solution; V1 is the volume of NaOH standard solution consumed during titration; V2 is the volume of NaOH standard solution consumed in the blank group; m is the solution volume.

[0116] Test results are as follows Figure 12-17 As shown.

[0117] Depend on Figure 12-17 It can be seen that an appropriate amount of functional polysaccharides has a significant effect on enhancing the hypoglycemic activity of goat milk during fermentation. The α-glucosidase inhibition rate and dipeptidyl peptidase IV (DPP-IV) inhibition rate of fermented goat milk with added polysaccharides [WPU (0.05wt%), WPU (0.1wt%), WPU (0.15wt%)] increased rapidly with the extension of fermentation time.

[0118] Added polysaccharides to yogurt [WPU (0.05wt%), WPU (0.1wt%), WPU (0.15wt%)] can enhance the lipid-lowering activity of yogurt. On one hand, the functional polysaccharides, acting as prebiotics, may promote the proliferation of beneficial bacteria in the yogurt, leading to an increase in metabolites such as peptides and organic acids, thereby inhibiting pancreatic lipase activity. On the other hand, casein can form a stable complex with polysaccharides, enhancing the physical adsorption of cholesterol. Additionally, some probiotic-produced extracellular polysaccharides possess micellar bonding capabilities, forming a dual network structure with the added exogenous polysaccharides, thereby enhancing the ability to encapsulate micelles.

[0119] As fermentation time increased, the pH value of samples with different amounts of functional polysaccharides added decreased continuously, while the acidity increased continuously. In the early stages of fermentation, there was no significant difference in pH changes between the polysaccharide-added and control groups. However, after 3 and 4.5 hours of fermentation, the pH of the polysaccharide-added yogurt was lower than that of the control group, indicating that the addition of polysaccharides was beneficial for acid production by the yogurt fermentation strains. Regarding acidity, the WPU group reached an acidity of 74°T at 4.5 hours, which was higher than that of the TW group (70.3°T).

[0120] A cold storage study was conducted on yogurt with 0.1% added WPU, using polysaccharides without added WPU as a control. The sensory evaluation and textural properties during the cold storage process are as follows: Figure 18-19 As shown.

[0121] Depend on Figures 18-19 It was found that the addition of polysaccharides gave the fermented goat milk a better taste, resulting in a higher sensory score than the TW group. Adding functional polysaccharides to fermented dairy products can promote lactic acid bacteria fermentation, inhibit whey separation during storage, enhance product stability, and extend shelf life. During refrigeration, the sensory scores of all groups of fermented goat milk showed an increasing trend from day 1 to day 7, with the highest scores for the two groups reaching 86.35 and 89.95 respectively on day 7. Subsequently, with prolonged refrigeration, the metabolic products of organic acids and other substances produced by probiotics in the fermented goat milk continued to accumulate, the pH value decreased, affecting the taste, and the sensory scores gradually decreased, reaching 75.65, 80.8, and 81 respectively on day 21. However, the sensory evaluation of the fermented goat milk with added 0.1% WPU was significantly higher than the control group from 3 to 21 days of refrigeration, indicating that adding WPU can significantly improve the sensory evaluation of fermented goat milk.

[0122] The hardness, consistency, and cohesiveness of fermented milk all showed a trend of first increasing and then gradually decreasing. Regarding viscosity index, both TW and WPU decreased with increasing refrigeration time. Specifically, the maximum hardness of TW and WPU on day 3 was 12.80g and 13.43g, respectively; the maximum consistency on day 3 was 83.16gs and 87.88gs, respectively; and the maximum cohesiveness of TW and WPU on day 7 was 7.43g and 7.80g, respectively. Figures 18-19This indicates that polysaccharides have a significant effect on enhancing the hardness and consistency of fermented milk.

[0123] During the process, polysaccharides can increase the content of soluble solids in fermented milk, thus increasing its hardness. Furthermore, polysaccharides have a certain degree of water absorption, especially small-molecule polysaccharides after enzymatic hydrolysis. When polysaccharides combine with water molecules, they can strengthen the intermolecular interactions within the fermented milk system, thereby promoting the aggregation of macromolecules. In addition, water kefir polysaccharides may improve the hardness, viscosity, adhesiveness, and cohesiveness of fermented milk by promoting the formation of extracellular polysaccharides in yogurt, enhancing their interaction with milk proteins, and increasing the degree of polymerization, thus giving the product a better taste. Alternatively, active polysaccharides may directly interact with proteins to form a dense structural network, thereby optimizing the texture of fermented milk. Therefore, water kefir polysaccharide (WPU) can improve the texture of fermented goat milk and enhance its refrigeration stability.

[0124] In summary, this application introduces a functional polysaccharide during the fermentation of goat milk. This functional polysaccharide is extracted from water kefir grains. The addition of the functional polysaccharide significantly enhances the hypoglycemic and lipid-lowering activities, storage stability, and sensory evaluation of fermented goat milk.

[0125] This application provides a fermented goat milk with hypoglycemic and lipid-lowering activities and its preparation method. A fermentation system using functional polysaccharides extracted by ultrasonic extraction from water kefir grains and *Lactobacillus delbrueckii* subsp. *bulgaricus* and *Streptococcus salivarius* subsp. *thermophilus* is constructed by fermenting at a constant temperature of 41℃-43℃ for 4-5 hours, establishing a polysaccharide-strain synergistic system. The resulting fermented goat milk exhibits significantly improved hypoglycemic and lipid-lowering activities, textural properties, and sensory evaluation. The fermentation agent in the goat milk containing the functional polysaccharides effectively enhances the coagulation properties of the fermented goat milk, reduces whey separation, shortens coagulation time, and significantly improves the system's stability. The fermented goat milk prepared by this process has the advantages of high hypoglycemic and lipid-lowering activities, good coagulation properties, minimal whey separation, high system stability, and good taste.

[0126] The embodiments provided by the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing acidic goat milk with hypoglycemic and lipid-lowering activities, characterized in that, The following steps are involved: Step 101. Add functional polysaccharides to goat milk, and then heat and cool it to obtain sterilized goat milk. Step 102. After adding the direct-inoculation starter culture to the sterilized goat milk prepared in step 101, place it at a constant temperature of 41℃-43℃ for 4h-5h to obtain the acid goat milk with hypoglycemic and lipid-lowering activities. In step 102, the α-glucosidase inhibition rate of the acidic goat milk with hypoglycemic and lipid-lowering activities was 77.46%-84.40%, the DPP-IV inhibition rate was 52.68%-61.01%, and the pancreatic lipase inhibition rate was 78.25%-86.43%. In step 101, the functional polysaccharide was extracted from water kefir grains; In step 102, the fermentation agent is a combination of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus.

2. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 1, characterized in that, In step 101, the goat milk is selected from fresh goat milk or reconstituted goat milk, and the concentration of the reconstituted goat milk is 12.5% ​​(w / v).

3. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 1, characterized in that, In step 101, the amount of functional polysaccharide added is 0.05wt%-0.15wt%. The amount of starter culture added in step 102 is 0.005 wt%.

4. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 1, characterized in that, In step 101, the extraction method for functional polysaccharides includes the following steps: Step 201. Under room temperature conditions, water kefir grains are continuously passaged and cultured using sterile brown sugar water as a substrate to obtain activated water kefir grains. Step 202. Add activated water kefir particles and distilled water to an ultrasonic reactor in sequence. After ultrasonic treatment at 40℃-80℃, collect the supernatant to obtain the first-stage crude extract. Step 203. Add the primary crude extract and trichloroacetic acid to a centrifuge in sequence. Centrifuge at 1000 rpm to 4000 rpm for 10 min to 20 min, and collect the supernatant to obtain the secondary crude extract. Step 204. After adding anhydrous ethanol to the secondary crude extract, refrigerate it at 4°C for 12-48 hours to obtain the tertiary crude extract. Step 205. Transfer the tertiary crude extract to a centrifuge and centrifuge at 1000rpm-4000rpm for 10min-20min. Remove the supernatant to obtain the crude polysaccharide. Step 206. After completely dissolving the crude polysaccharide in deionized water, the solution is then subjected to decolorization, filtration, chromatography, dialysis, and freeze-drying in sequence to obtain the functional polysaccharide product.

5. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 4, characterized in that, In step 202, the ultrasonic treatment power is 240W-320W; the ultrasonic treatment time is 30min-50min.

6. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 4, characterized in that, In step 202, the ratio of water kefir granules to distilled water is 1:(10-30).

7. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 4, characterized in that, Step 201 includes the following steps: inoculating water kefir grains into 10% (w / v) brown sugar water, allowing it to stand for 24 hours, filtering out the brown sugar water, and adding an equal amount of brown sugar water again. After repeating the culture twice, activated water kefir grains are obtained; the brown sugar water is then boiled and cooled.

8. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 1, characterized in that, In step 206, chromatography was performed using a DEAE-cellulose ion exchange column; during chromatography, elution was carried out sequentially with deionized water, 0.05M NaCl solution, 0.1M NaCl solution, and 0.3M NaCl solution. Dialysis was then performed using an ultrafiltration tube; during dialysis, the molecular cutoff of the ultrafiltration tube was 10 kDa-300 kDa.

9. The method for preparing a type of fermented goat milk with hypoglycemic and lipid-lowering activity according to claim 1, characterized in that, In step 206, the functional polysaccharide is a neutral polysaccharide with a weight-average molecular weight of 27.303 kDa. Its monosaccharides are mainly composed of glucose (Glc) and mannose (Man). The glucose residues exist in the form of t-Glcp, 6-Glcp, 3,6-Glcp, 4,6-Glcp, and 2,6-Glcp, with a molar ratio of 15.33:72.33:7.8:1.43:1.

78. The mannose residues exist in the form of 3-Manp.

10. A type of fermented goat milk with hypoglycemic and lipid-lowering activities, characterized in that, It is prepared by any one of the methods for preparing acidic goat milk with hypoglycemic and lipid-lowering activities according to claims 1-9.

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