Liquid goat milk and goat milk powder with hypoglycemic activity and preparation method of liquid goat milk and goat milk powder

Abstract

According to the liquid goat milk with the hypoglycemic activity, the goat milk powder and the preparation method of the liquid goat milk, the functional polysaccharide is added into the goat milk, and the product is obtained after heating sterilization and cooling in sequence, so that the goat milk has the definite hypoglycemic activity. Wherein the functional polysaccharide is obtained by sequentially carrying out activation, enzymolysis, ultrasonic treatment and purification extraction on water kefir grains, and after the functional polysaccharide is added into the goat milk, the nutritional value and functional characteristics of the goat milk are remarkably improved, the goat milk is endowed with good hypoglycemic activity, the functionality of the goat milk is enriched, and a goat milk product with higher quality and higher functionality is provided for consumers; the method has the advantages of being low in implementation cost, simple in process, convenient to operate and convenient to popularize and implement.

Description

Technical Field

[0001] This application belongs to the field of food engineering technology, specifically relating to a liquid goat milk with hypoglycemic activity, goat milk powder, and its preparation method. Background Technology

[0002] In existing technologies, water kefir grains, as a natural complex microbial symbiotic system, possess unique physicochemical properties and potential biological activities in their main polysaccharide components. Currently, systematic research on water kefir grain polysaccharides remains lacking, and their composition and structural characteristics require further exploration.

[0003] In existing technologies, polysaccharides have broad application potential in the food industry. Adding them as functional ingredients to dairy products can significantly increase product viscosity and effectively improve system stability. Furthermore, they can act as prebiotics, effectively increasing the number of live bacteria in fermented milk and shortening fermentation time. Goat milk has advantages over cow milk, including easier digestibility and absorption, lower allergenicity, and higher nutritional value. However, current research on how to fully utilize the potential functions of polysaccharides, such as their hypoglycemic activity, in goat milk systems, and how to improve the stability of goat milk systems remains insufficient.

[0004] Therefore, in order to fully explore the nutritional value and functional characteristics of goat milk, enhance its effects in areas such as blood sugar reduction, and improve its quality defects, it is now urgent to make improvements to meet consumer demand for new dairy products that combine nutrition and functionality, and to promote the development of the goat milk industry. Summary of the Invention

[0005] In order to address the technical problems of limited variety, severe homogenization, and scarcity of functional goat milk products in the existing technology, this application proposes a liquid goat milk with hypoglycemic activity, goat milk powder, and its preparation method.

[0006] This application adopts the following scheme: a method for preparing liquid goat milk with hypoglycemic activity, comprising the following steps: Step 101. Add functional polysaccharides to goat milk, and then heat and sterilize and cool it to obtain liquid goat milk with hypoglycemic activity. In step 101, the α-glucosidase inhibition rate of the liquid goat milk with hypoglycemic activity was 67.54%-96.41%, and the DPP-IV inhibition rate was 37.52%-94.17%. In step 101, the functional polysaccharide is extracted from water kefir grains by sequential activation, enzymatic hydrolysis, and ultrasonic treatment.

[0007] In practice, the water kefir granules are produced in Anshan, Liaoning Province, and can be purchased from: https: / / shop34241062.taobao.com / .

[0008] 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).

[0009] In practice, to improve production efficiency, reconstituted sheep milk is selected.

[0010] In step 101, the amount of functional polysaccharide added is 2wt%-3wt%.

[0011] In some feasible embodiments, step 101, the method for extracting 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 granules and distilled water to a homogenizer and homogenize thoroughly to obtain the first-stage extract. Then add enzyme solution to the first-stage extract and fully enzymatically hydrolyze it under water bath heating conditions to obtain the second-stage extract. Step 203. Add the secondary extract to the ultrasonic reactor, sonicate at 80°C, and collect the supernatant to obtain the primary crude extract. Step 204. Add the primary crude extract and trichloroacetic acid to a centrifuge in sequence. Centrifuge at 4000rpm-1000rpm for 10min-20min and collect the supernatant to obtain the secondary crude extract. Step 205. 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 206. Transfer the tertiary crude extract to a centrifuge and centrifuge at 4000rpm-1000rpm for 10min-20min. Remove the supernatant to obtain the crude polysaccharide. Step 207. 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.

[0012] In actual implementation, in some feasible embodiments, step 201 includes the following steps: fermentation culture using brown sugar water as a base material. First, prepare 10% (w / v) brown sugar water, heat to boiling until completely dissolved, pour 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.

[0013] In practice, the growth of water kefir particles is due to the conversion of sucrose into glucan by extracellular glucanase. Water kefir particles can continuously proliferate in brown sugar water, making full use of the carbon source and other nutrients in the brown sugar water for proliferation.

[0014] In some feasible embodiments, in step 202, the enzyme solution is selected from pullulan enzyme solution, dextran enzyme solution, amylase solution, and thermostable amylase solution, or a combination of one or more of them.

[0015] In practice, pullulanase and dextranase were both purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. Both amylase and thermostable amylase were purchased from Novozymes Ltd.

[0016] In some feasible embodiments, in step 202, the amount of enzyme solution added is 0.2%-1% (v / v), and the enzymatic hydrolysis temperature is 45℃-65℃.

[0017] In some feasible embodiments, in step 202, the ultrasonic treatment power is 340W; the ultrasonic treatment time is 42min; and in step 202, the material-to-liquid ratio of water kefir granules to distilled water is 1:20g / mL.

[0018] In actual implementation, the decolorization step 207 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, controlling the treatment time to 60 min and maintaining the temperature at 60℃. After decolorization, the solution is filtered while hot using a Buchner funnel to remove the activated carbon from the polysaccharide extract.

[0019] In some feasible embodiments, in step 207, chromatography is performed using a DEAE-cellulose ion exchange column; In actual implementation, step 207, 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 is then equilibrated with pure water (1mL / min) until the pH is neutral. The crude polysaccharide solution, after decolorization and filtration in step 206, is adjusted to 20mg / mL. This solution is centrifuged (10000 r / 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 1 mL / 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.

[0020] During chromatography, deionized water, 0.05M NaCl solution, 0.1M NaCl solution, and 0.3M NaCl solution were used for elution in sequence.

[0021] In some feasible embodiments, in step 207, dialysis is performed using an ultrafiltration tube. During dialysis, the molecular cutoff of the ultrafiltration tube is 10 kDa-300 kDa.

[0022] In order to solve the technical problem proposed in this application, this application provides a liquid goat milk with hypoglycemic activity, which is prepared by the above-mentioned method for preparing a liquid goat milk with hypoglycemic activity.

[0023] In order to solve the technical problem proposed in this application, this application provides a goat milk powder with hypoglycemic activity, which is prepared by concentrating and spray drying the above-mentioned liquid goat milk with hypoglycemic activity.

[0024] In order to solve the technical problems raised in this application, this application also provides a functional polysaccharide with hypoglycemic activity, which can be applied to food.

[0025] Compared with the prior art, this application has the following beneficial effects: This application provides a liquid goat milk with hypoglycemic activity, goat milk powder, and a method for preparing the same. The method involves adding functional polysaccharides to goat milk, followed by heating for sterilization and cooling to obtain the product. This results in goat milk exhibiting a clear inhibition rate of α-glucosidase and DPP-IV. The functional polysaccharides are obtained from water kefir grains through activation, enzymatic hydrolysis, ultrasonic treatment, and purification extraction. Adding these functional polysaccharides to goat milk significantly enhances its nutritional value and functional properties, imparting good hypoglycemic activity, enriching its functionality, and optimizing the stability of the goat milk system. This provides consumers with a higher-quality, more functional goat milk product, meeting market demand. The method boasts advantages such as low implementation cost, simple process, ease of operation, and ease of promotion and implementation. Attached Figure Description

[0026] Figure 1 The effect of different glycosidases on the extraction rate of functional polysaccharides; Figure 2 The effect of different combinations of glycosidases on the extraction rate of functional polysaccharides; Figure 3 The effect of different glycosidase ratios on the extraction rate of functional polysaccharides; Figure 4 The effect of enzyme addition on the extraction rate of functional polysaccharides; Figure 5 The effect of enzymatic hydrolysis temperature on the extraction rate of functional polysaccharides; Figure 6 The effect of enzymatic hydrolysis time on the extraction rate of functional polysaccharides; Figure 7 These are the chromatographic curves of functional polysaccharides; Figure 8 These are freeze-dried samples of functional polysaccharides (from left to right: WPE-1, WPE-2, WPE-3, WPE-4, WPE-5, WPE-6). Figure 9 The effects of WPE, WPE-4, WPE-5 and WPE-6 concentrations on the inhibition rate of dipeptidyl peptidase (DPP) IV; Figure 10 These are scanning electron microscope images of functional polysaccharides magnified at 200X, 1000X, and 5000X. Figure 11 This is the infrared spectrum of a functional polysaccharide; Figure 12 This is a standard monosaccharide ion chromatogram; Figure 13 This is an ion chromatogram of a functional polysaccharide; Figure 14 This is a graph showing the absolute molecular weight analysis of functional polysaccharides; Figure 15 This is a molecular configuration diagram of a functional polysaccharide. Detailed Implementation

[0027] Combination Figures 1-15 The technical solutions provided in this application are further illustrated by the descriptions in Examples 1-4. Example 1: Extraction of functional polysaccharides using ultrasound-assisted extraction The ultrasound-assisted extraction of functional polysaccharides includes the following steps: 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. Step 202. Add activated water kefir granules and distilled water to the ultrasonic reactor in sequence at 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. 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. 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. 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. 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.

[0028] 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). Formula (A): ; 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 weighed.

[0029] 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 weighed.

[0030] The functional polysaccharide extraction rate was 27.64±0.92% when the material-to-liquid ratio was 1:20 g / mL, the ultrasonic power was 340 W, the ultrasonic treatment time was 42 min, and the ultrasonic treatment temperature was 80℃.

[0031] Example 2: Ultrasound-enzyme-assisted extraction of functional polysaccharides The difference between Example 2 and Example 1 is that the functional polysaccharide is extracted using an ultrasound-enzyme-assisted method. Specifically, based on the ultrasonic treatment process parameters obtained in Example 1, the activated water kefir particles prepared in step 202 are transferred to a homogenizer for thorough homogenization to obtain the first-stage extract. Enzyme solution is then added to the first-stage extract, and after thorough enzymatic hydrolysis under water bath heating conditions, the second-stage extract is obtained. The second-stage extract is then transferred to an ultrasonic reactor for ultrasonic treatment, and the supernatant is collected to obtain the first-stage crude extract. Subsequent steps are the same as in Example 1 and will not be repeated here.

[0032] Based on the above-mentioned preparation method of functional polysaccharides, a single-factor control method was adopted. Under fixed parameters (liquid-to-solid ratio 1:15 g / mL, ultrasonic power 320 W, ultrasonic time 42 min, ultrasonic temperature 80℃, enzyme addition 10 μL / mL, enzymatic hydrolysis temperature 55℃, enzymatic hydrolysis time 1 h), functional polysaccharides were extracted under conditions of no enzyme addition and with the addition of different types of enzyme solutions. The extraction rates of the corresponding functional polysaccharides were calculated according to the above formula (A). The effects of different enzyme solutions on the extraction rates of functional polysaccharides are as follows: Figure 1 As shown.

[0033] Depend on Figure 1 It can be seen that the extraction rate using the ultrasound-enzyme-assisted method is significantly better than that of the ultrasound-assisted method in Example 1, and the extraction effects of pullulanase and dextranase are significantly better than those of the two amylases. That is, pullulanase and dextranase can effectively degrade water kefir polysaccharides. This is because the polysaccharide molecular weight in water kefir granules is too large, and its solubility is low. Therefore, the polysaccharide extraction rate in Example 1 is low. However, after adding glycosidase, the glycosidase hydrolyzes the glycosidic bonds of the polysaccharide, thereby reducing the molecular weight of the polysaccharide, making it easier to dissolve, and thus improving the polysaccharide yield.

[0034] Based on the above results, functional polysaccharides were extracted under the conditions of adding 0.2% pullulanase (experimental group 1), 0.2% dextranase (experimental group 2), 0.1% pullulanase + 0.1% dextranase (experimental group 3), and 0.2% pullulanase + 0.2% dextranase (experimental group 4), respectively. The extraction rates of the corresponding functional polysaccharides were calculated according to the above formula (A). The effect of different enzyme combinations on the extraction rate of functional polysaccharides is as follows: Figure 2 As shown.

[0035] Depend on Figure 2 It can be seen that the extraction rate of the dual-enzyme combination is significantly better than that of the single-enzyme combination, and the enzymatic hydrolysis effect is stronger. Although the amount of enzyme solution added in experimental group 4 was twice that of the first three groups, the increase in extraction rate was not significant. That is, under the combined action of dual enzymes, the total enzyme amount of 0.2w% had already reached its maximum hydrolysis effect on water kefir polysaccharide.

[0036] Based on the above research results, functional polysaccharides were extracted under the conditions of a total enzyme addition of 0.2% and pullulanase to dextranase ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, respectively. The extraction rates of the corresponding functional polysaccharides were calculated according to the above formula (A). The effects of different enzyme ratios on the extraction rates of functional polysaccharides are as follows: Figure 3 As shown.

[0037] Depend on Figure 3 It can be seen that the ratio of the two enzymes has no significant effect on the extraction rate, and the optimal ratio of pullulanase to dextranase is 1:1.

[0038] Based on the extraction conditions obtained in Examples 1-2, and with fixed parameters (liquid-to-solid ratio 1:20 g / mL, ultrasonic power 340 W, ultrasonic time 42 min, ultrasonic temperature 80 °C), the extraction of functional polysaccharides was studied under the following conditions: enzyme dosage (0.2% (v / v), 0.4% (v / v), 0.6% (v / v), 0.8% (v / v), 1% (v / v)), enzymatic hydrolysis temperature (45 °C, 50 °C, 55 °C, 60 °C, 65 °C), and enzymatic hydrolysis time (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h). The extraction rates of the corresponding functional polysaccharides were calculated according to the above formula (A). The effects of different enzymatic hydrolysis parameters on the extraction rate of functional polysaccharides are as follows: Figures 4-6 As shown.

[0039] Depend on Figures 4-6 It was found that the extraction rate of functional polysaccharides increased continuously with the increase of enzyme dosage, reaching its highest level when the enzyme dosage was 0.6%. As the enzyme dosage increased, the surface area of ​​the water kefir grains in contact with the enzyme increased, leading to a greater degradation effect of the enzyme on the cell wall. Further increases in enzyme dosage resulted in a gradual decrease in the polysaccharide extraction rate, indicating that the enzyme sites on the cell wall of the water kefir grains had reached saturation. Within the enzyme dosage range of 0.2%-1%, the extraction rate of functional polysaccharides was consistently above 40%, significantly higher than the extraction rate in Example 1.

[0040] Within the temperature range of 45-50℃, the extraction rate of water kefir granules increased with increasing enzymatic hydrolysis temperature. However, when the temperature exceeded 50℃, the yield of water kefir granules decreased, because higher temperatures can damage the enzyme structure, thus reducing its activity.

[0041] Under the synergistic effect of the two enzymes, the polysaccharide content in the extract showed a trend of first increasing and then stabilizing. The extraction rate reached its maximum when the enzymatic hydrolysis time was 2.5 h; further extending the extraction time had little effect on the extraction rate, indicating that the enzymatic hydrolysis reaction at 2.5 h was close to saturation, and the dissolution and diffusion of polysaccharides in water kefir grains reached a dynamic equilibrium.

[0042] Based on the results of the single-factor experiment on enzymatic hydrolysis conditions, the effects of enzyme dosage, hydrolysis temperature and hydrolysis time on the ultrasonic-enzyme-assisted extraction of functional polysaccharides were further studied. The experimental design and results are shown in Table 1.

[0043] Table 1. Experimental Design and Results of Ultrasonic-Assisted Enzymatic Method

[0044] As shown in Table 1, when the enzyme dosage is 0.5-0.7%, the enzymatic hydrolysis temperature is 45-55℃, and the enzymatic hydrolysis time is 2-3h, the extraction rate of functional polysaccharides is 43.90%-53.86%, which is significantly higher than that of the ultrasonic-assisted method.

[0045] Example 3: Isolation, purification, activity study and characterization of functional polysaccharides Based on Examples 1 and 2, a complex of pullulanase and dextranase at a mass ratio of 1:1 was selected as the enzyme solution. The enzyme dosage was 0.6% (v / v), the enzymatic hydrolysis temperature was 50℃, and the enzymatic hydrolysis time was 2.5 h. Functional polysaccharide WPE was extracted using an ultrasound-enzyme-assisted method under the conditions of a liquid-to-solid ratio of 1:20 g / mL, ultrasonic power of 340 W, ultrasonic time of 42 min, and ultrasonic temperature of 80 °C. First, trichloroacetic acid was used for deproteinization and activated carbon for decolorization. Then, DEAE-cellulose anion exchange resin was used to separate the polysaccharide using an ionic strength gradient elution method, with deionized water, 0.05 M, 0.1 M, and 0.3 M NaCl solutions as the eluents. The total sugar content of the eluent was measured intermittently using the phenol-sulfuric acid method. Elution curves were plotted based on the number of tubes and absorbance values. Specific results are shown below. Figure 7 As shown, the prepared functional polysaccharide product is labeled as WPE.

[0046] Depend on Figure 7 It can be seen that after collecting the eluents from tubes 6-16 and 50-56, they were combined and concentrated, dialyzed, and then freeze-dried to obtain two polysaccharide fractions, WPE-I and WPE-II, respectively. It can be seen that the fraction with the highest yield of both polysaccharides was the neutral polysaccharide WPE-I eluted with pure water. Both of them had single elution peaks with symmetrical peak shapes and good separation.

[0047] The lyophilized fraction collected after purification by ion exchange chromatography was dissolved to a concentration of 10 mg / ml. WPE-I was then separated sequentially using ultrafiltration tubes with molecular weight cutoffs of 300 kDa, 100 kDa, 50 kDa, and 10 kDa (5000 r / min, 30 min). Considering that enzymatic hydrolysis would reduce the molecular weight of the polysaccharides, an additional 5 kDa molecular weight cutoff ultrafiltration tube was used for further separation. The concentrates and filtrates were then collected, yielding six fractions of WPE-I: WPE-1, WPE-2, WPE-3, WPE-4, WPE-5, and WPE-6. Finally, the collected polysaccharide fractions were freeze-dried, and the freeze-dried samples are shown below. Figure 8 As shown, the yields of the six components were 1.61%, 1.47%, 2.08%, 29.06%, 27.42%, and 38.36%, respectively. WPE with the highest yield and WPE-4, WPE-5, and WPE-6 were selected for hypoglycemic activity studies.

[0048] The inhibition rates of different concentrations of WPE, WPE-4, WPE-5, and WPE-6 on dipeptidyl peptidase (DPP) IV are as follows: Figure 9 As shown.

[0049] As shown in Figure 9, within the experimental concentration range, the DPP-IV inhibitory activity of water kefir polysaccharides is directly proportional to their concentration. The DPP-IV inhibition rates of the polysaccharides obtained by ultrasonic-enzyme-assisted extraction at a final concentration of 5 mg / mL were 70.52%, 44.44%, 46.1%, and 54.39%, respectively for WPE, WPE-4, WPE-5, and WPE-6. The inhibition rate of WPE was significantly higher than the other three components, indicating that WPE has better hypoglycemic activity. Further characterization was performed, and its 200X, 1000X, and 5000X scanning electron microscope images are shown below. Figure 10 As shown, Figure 10 It is evident that the polysaccharide sample exhibits an overall membrane-like fusion morphology, and its surface structure is tight and smooth.

[0050] The infrared spectral results of WPE are as follows Figure 11 As shown.

[0051] Depend on Figure 11 It can be seen that some typical functional group signals exist in WPE, and WPE-6 is at 3390.9 cm⁻¹. -1 and 3330.5cm -1 The strong and broad absorption peak at 2928.3 cm⁻¹ is related to the OH stretching vibration. -1 2942.6 cm -1 The absorption peaks at these locations are related to the CH stretching vibration, and these two signals can be considered characteristic signals of carbohydrate compounds. Additionally, WPE occurs at 1658.4 cm⁻¹.-1 and 1672.7 cm -1 The absorption peak at 1422.8 cm⁻¹ indicates the stretching vibration of the C=O bond, which is associated with bound water. -1 and 1419.9 cm -1 The absorption peak is related to C-O, at 1276.2 cm⁻¹. -1 and 1238.9 cm -1 The absorption peak at 1020.5 cm⁻¹ is related to the stretching vibration of C. -1 and 1037.8cm -1 At this location, WPE exhibits a strong peak, which is related to the stretching vibration of the COC bond on the glycosidic bond and the COH side group, indicating that WPE contains a pyranose ring. Furthermore, the absorption peak at this location also represents the characteristic of α-1,6-glycosidic bond linkage. (911.4 cm⁻¹) -1 and 925.7 cm -1 The absorption peak at that point indicates the asymmetric vibration of the pyranose ring.

[0052] Figure 12 and Figure 13 The images show the ion chromatograms of the standard monosaccharide and WPE, respectively. Comparing the ion chromatogram of WPE with that of the monosaccharide standard, it can be seen that WPE-6 is composed only of glucose. This may be because enzymatic hydrolysis has hydrolyzed all the non-glucose sugars.

[0053] Polysaccharides typically exhibit certain molecular weight distribution characteristics, and this polydispersity is an important indicator for evaluating the heterogeneity of polysaccharide molecules. Molecular weight distribution directly affects the physicochemical properties and functional characteristics of polysaccharides, thus becoming one of the most critical research parameters in the synthesis and characterization of polysaccharides.

[0054] Figure 14 This is a graph showing the absolute molecular weight analysis of WPE. The red curve represents the multi-angle laser light scattering (LS) signal, the intensity of which is proportional to the size and molecular weight of the sample molecules. The blue curve represents the differential refractive index (RI) signal, the response value of which is related to the refractive index change of the column effluent, reflecting the chemical properties, concentration, and molecular weight information of the sample. The black curve is the molecular weight distribution curve obtained by fitting the LS and RI signals, and its value directly corresponds to the vertical axis. The solvent peak of the mobile phase is located around 37 min. Figure 14 The strong red signal and distinct peak shape at 26-27 min indicate that the components eluting near this position have larger molecular weights, and the large blue signal peak area also represents their higher content. The larger the blue signal peak area, the higher the sample content.

[0055] Figure 15The diagram shows the molecular configuration of WPE, and its slope can be used to determine the molecular morphology. Generally, when the slope = 1, the molecule has a rod-like structure; when the slope is in the range of 0.5-0.6, the molecule exhibits a random coil conformation; and when the slope = 1 / 3, the molecule has a spherical structure. Figure 15 The absence of a clear molecular configuration diagram indicates that WPE molecules are small in size and have a low molecular weight, resulting in weak light dispersion signals and no complete peak curves. Therefore, a fitted molecular configuration diagram could not be obtained.

[0056] The number-average molecular weight (Mn), weight-average molecular weight (Mw), peak molecular weight (Mp), z-average molecular weight (Mz), and polydispersity index (Mw / Mn) of WPE were calculated using software, and the specific results are presented in Table 2. Weight-average molecular weight is the most commonly used indicator to represent the molecular weight of polysaccharides. The weight-average molecular weight of WPE is 1.636 kDa, which proves the effectiveness of ultrafiltration separation. Furthermore, the polydispersity index (Mw / Mn) was used to represent the width of the molecular weight distribution. When the ratio is less than 2, it indicates good homogeneity of the polysaccharide. It can be seen that WPE has good homogeneity.

[0057] Molecular weight is an important parameter affecting the application of biopolymers in various fields such as pharmaceuticals, food, cosmetics, and chemical industries. Generally speaking, the smaller the molecular weight of a polysaccharide, the easier it is for its active site to bind to ligands, resulting in better reactions and stronger activity.

[0058] Table 2 Results of WPE Molecular Weight Determination

[0059] The total ion chromatogram and linkage analysis table of WPE methylation are shown below. Figure 15 And Table 3.

[0060] Table 3. Methylation analysis (linkage analysis) data of WPE

[0061] Table 3 shows that six derivatives were obtained from WPE: 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 glucitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol, 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 corresponds to six glycosidic bonds: t-Glcp-(1→, →3)-Glcp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→, →3,6)-Glcp-(1→ and 4,6)-Glcp-(1→), with molar ratios of 40.62%, 12.01%, 1.01%, 33.53%, 11.99%, and 0.83%, respectively. The results indicate that the WPE-6 polysaccharide component consists only of glucose (Glc), with glucose residues existing in the forms of t-Glcp, 3-Glcp, 4-Glcp, 6-Glcp, 3,6-Glcp, and 4,6-Glcp, with a molar ratio of 40.62:12.01:1.01:33.53:11.99:0.83.

[0062] Example 4: Preparation method of liquid goat milk and goat milk powder with hypoglycemic activity (1) The preparation method of liquid goat milk with hypoglycemic activity includes the following steps: Step 101. Add 2.0%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, and 3.0wt% of the functional polysaccharide (WPE) prepared in Example 3 to fresh goat milk or reconstituted goat milk with a concentration of 12.5% ​​(w / v), respectively. After heating and sterilizing and cooling, liquid goat milk with hypoglycemic activity is obtained. Liquid goat milk with hypoglycemic activity is concentrated to a solid content of 40-50%, and then spray-dried to obtain goat milk powder with hypoglycemic activity.

[0063] (2) The following tests were performed during the preparation of goat milk in (1): Test 1: α-glucosidase inhibition rate 25 μL of the liquid sheep milk prepared in (1) was mixed with 50 μL of α-glucosidase solution (2 U / mL) and 25 μL of PNPG (2.5 mmol / L). After reacting at 37℃ for 30 min, 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).

[0064] Formula (E): ; In formula (E), A1 is the absorbance of the sample well; A2 is the absorbance of the sample control well (with PBS instead of α-glucosidase); A3 is the absorbance of the blank well (with PBS instead of the sample); and A4 is the absorbance of the blank control well (with PBS instead of the sample and α-glucosidase).

[0065] Test 2: DPP-IV inhibition rate Take 25 μL of the liquid sheep milk prepared in (1) 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℃ 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).

[0066] Formula (F): ; 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). The test results are shown in Table 4.

[0067] Table 4. Effects of functional polysaccharide WPE on the hypoglycemic activity of liquid goat milk.

[0068] As shown in Table 4, the α-glucosidase inhibition rate of liquid goat milk with added 2.0%-3.0% functional polysaccharide WPE was 67.54%-96.10%, and the DPP-IV inhibition rate was 37.52%-94.17%, while the α-glucosidase inhibition rate and DPP-IV inhibition rate of liquid goat milk without added functional polysaccharide WPE were both 0. It can be seen that adding 2.0%-3.0% functional polysaccharide WPE to liquid goat milk can give it hypoglycemic activity.

[0069] In summary, this invention adds functional polysaccharides to liquid goat milk. These functional polysaccharides are extracted from water kefir grains, and their addition significantly enhances the hypoglycemic activity and storage stability of the goat milk. Specifically, when the amount of functional polysaccharides added is 2.0%-3.0 wt%, the hypoglycemic activity of the liquid goat milk is significantly improved, providing a reference for the production of liquid goat milk and goat milk powder with hypoglycemic activity.

[0070] This application provides a liquid goat milk with hypoglycemic activity, goat milk powder, and a method for preparing the same. The method involves adding functional polysaccharides to goat milk, followed by heating for sterilization and cooling to obtain the product. This results in goat milk exhibiting a clear inhibition rate of α-glucosidase and DPP-IV. The functional polysaccharides are obtained from water kefir grains through activation, enzymatic hydrolysis, ultrasonic treatment, and purification extraction. Adding these functional polysaccharides to goat milk significantly enhances its nutritional value and functional properties, endowing it with good hypoglycemic activity and enriching its functionality. This provides consumers with a higher-quality and more functional goat milk product, meeting market demand. The method boasts advantages such as low implementation cost, simple process, ease of operation, and ease of promotion and implementation.

[0071] 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 liquid goat milk with hypoglycemic activity, characterized in that, Includes the following steps: Step 101. Add functional polysaccharides to goat milk, and then heat and sterilize and cool it to obtain liquid goat milk with hypoglycemic activity. In step 101, the α-glucosidase inhibition rate of the liquid goat milk with hypoglycemic activity was 67.54%-96.41%, and the DPP-IV inhibition rate was 37.52%-94.17%. In step 101, the functional polysaccharide is extracted from water kefir grains by sequential activation, enzymatic hydrolysis, and ultrasonic treatment.

2. The method for preparing liquid goat milk with hypoglycemic 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). In step 101, the amount of functional polysaccharide added is 2wt%-3wt%.

3. The method for preparing liquid goat milk with hypoglycemic 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 granules and distilled water to a homogenizer and homogenize thoroughly to obtain the first-stage extract. Then add enzyme solution to the first-stage extract and fully enzymatically hydrolyze it under water bath heating conditions to obtain the second-stage extract. Step 203. Add the secondary extract to the ultrasonic reactor, sonicate at 80°C, and collect the supernatant to obtain the primary crude extract. Step 204. Add the primary crude extract and trichloroacetic acid to a centrifuge in sequence. Centrifuge at 4000rpm-1000rpm for 10min-20min and collect the supernatant to obtain the secondary crude extract. Step 205. 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 206. Transfer the tertiary crude extract to a centrifuge and centrifuge at 4000rpm-1000rpm for 10min-20min. Remove the supernatant to obtain the crude polysaccharide. Step 207. 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.

4. The method for preparing liquid goat milk with hypoglycemic activity according to claim 3, characterized in that, In step 202, the enzyme solution is selected from pullulan enzyme solution, dextran enzyme solution, amylase solution, and thermostable amylase solution, or a combination of one or more of them.

5. The method for preparing liquid goat milk with hypoglycemic activity according to claim 3, characterized in that, In step 202, the amount of enzyme solution added is 0.2%-1% (v / v), and the enzymatic hydrolysis temperature is 45℃-65℃.

6. The method for preparing liquid goat milk with hypoglycemic activity according to claim 3, characterized in that, In step 202, the ultrasonic treatment power is 340W; the ultrasonic treatment time is 42min; and the material-to-liquid ratio of water kefir granules to distilled water is 1:20g / mL.

7. The method for preparing liquid goat milk with hypoglycemic activity according to claim 1, characterized in that, In step 207, chromatography was performed using a DEAE-cellulose ion exchange column. During chromatography, deionized water, 0.05M NaCl solution, 0.1M NaCl solution, and 0.3M NaCl solution were used for elution sequentially. In step 207, dialysis was performed using an ultrafiltration tube with a molecular weight cutoff of 10kDa-300kDa. The functional polysaccharide product has a weight-average molecular weight of 1.636kDa, and its monosaccharide composition is glucose. The glucose residues exist in the forms of t-Glcp, 3-Glcp, 4-Glcp, 6-Glcp, 3,6-Glcp, and 4,6-Glcp, with a molar ratio of 40.62:12.01:1.01:33.53:11.99:0.

83.

8. A liquid goat milk with hypoglycemic activity, characterized in that, It is prepared by any one of the methods for preparing liquid goat milk with hypoglycemic activity according to claims 1-7.

9. A goat milk powder with hypoglycemic activity, characterized in that, It is prepared by concentrating and spray drying a liquid sheep milk with hypoglycemic activity as described in claim 8.

10. The functional polysaccharide according to claim 2, characterized in that, It is used in foods with hypoglycemic activity.

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