Sheep milk with activity of reducing blood sugar and uric acid and preparation method of sheep milk

Abstract

The invention relates to the technical field of enzyme engineering, in particular to sheep milk with the activity of reducing blood sugar and uric acid and a preparation method of the sheep milk. The invention provides pasteurized sheep milk, high-temperature sterilized sheep milk and sheep milk powder with the activity of reducing blood sugar and uric acid. The alpha-glucosidase inhibition rate of the pasteurized sheep milk is 64.75% + / -2.92%-78.92% + / -2.80%, and the xanthine oxidase inhibition rate of the pasteurized sheep milk is 61.64% + / -1.32%-81.68% + / -1.20%. The peptide for reducing blood sugar and uric acid is prepared by hydrolyzing sheep milk with compound enzyme, the sheep milk powder with the activity of reducing blood sugar and uric acid is developed, the amino acid sequence and source of the peptide and the position of the peptide in sheep milk protein are separated, purified and identified, and a theoretical basis and a technical support are provided for subsequent development of dairy products for reducing blood sugar and uric acid.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a sheep milk with hypoglycemic and uric acid-lowering activities and its preparation method. Specifically, it relates to a method for obtaining sheep milk with hypoglycemic and uric acid-lowering activities by hydrolyzing sheep milk protein with protease. Background Technology

[0002] In 2024, the total number of people with diabetes aged 20-79 worldwide reached 589 million. This not only causes serious complications such as kidney failure and cardiovascular and cerebrovascular diseases, leading to a decline in quality of life and a shortened lifespan, but also results in high medical expenses due to drug and treatment costs. Furthermore, it leads to decreased patient productivity and premature death, severely increasing the burden on families and society.

[0003] Diabetes and hyperuricemia are mainly treated with medication and dietary intervention. However, medication has significant side effects. For example, hypoglycemic drugs can cause significant weight changes, osteoporosis, gastrointestinal disturbances, and increased cardiovascular risk. Uric acid-lowering drugs can cause skin rashes, gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal pain), and headaches. In recent years, research on food-derived bioactive peptides has increased, discovering several hypoglycemic and uric acid-lowering peptides. These peptides are safe, easily absorbed, and have few toxic side effects, making them a hot research topic. Hypoglycemic peptides, such as α-glucosidase inhibitory peptides, can inhibit intestinal α-glucosidase, delaying the breakdown of carbohydrates into glucose and lowering postprandial blood glucose. Uric acid-lowering peptides can inhibit xanthine oxidase, reducing the formation of hypoxanthine → xanthine → uric acid.

[0004] Sheep milk is rich in protein, including α-lactalbumin, β-lactoglobulin, lactoferrin, and α-lactalbumin. s1 -Casein, α s2 Casein, β-casein, κ-casein, etc., are mainly used in the production of infant formula goat milk powder. There are few product types and few deep-processed products. There is also little research on preparing high-value sheep milk products by hydrolyzing sheep milk using enzymatic technology. Summary of the Invention

[0005] The purpose of this invention is to provide sheep milk with hypoglycemic and uric acid-lowering activities and its preparation method, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides sheep milk with hypoglycemic and uric acid-lowering activities. The sheep milk contains hypoglycemic and uric acid-lowering peptides, which are α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides. The amino acid sequences of the hypoglycemic and uric acid-lowering peptides are LDL, LRF, ILLQ, LTLP, ITMP, LFQI, LLILT, LHLPLPL, VVVPPF, LGSRYL, VVAPFPE, and GLDPYKL.

[0008] Preferably, the sheep milk is pasteurized sheep milk, sterilized sheep milk, or formulated sheep milk powder.

[0009] Preferably, the pasteurized sheep milk has an α-glucosidase inhibition rate of 64.75%±2.92%~78.92%±2.80% and a xanthine oxidase inhibition rate of 61.64%±1.32%~81.68%±1.20%.

[0010] Preferably, the α-glucosidase inhibition rate of the sterilized modified sheep milk is 37.66%±2.13%~82.83%±3.70%, and the xanthine oxidase inhibition rate is 44.79%±1.97%~80.46%±1.98%.

[0011] Preferably, the α-glucosidase inhibition rate of the reconstituted sheep milk powder is 58.83%±2.13%~72.56%±2.53%, and the xanthine oxidase inhibition rate is 45.11%±3.13%~80.46%±1.98%.

[0012] The present invention provides a method for preparing the above-mentioned sheep milk, comprising the steps of sterilizing sheep milk, cooling it, adding a complex protease for enzymatic hydrolysis, adding a nutrient fortifier after enzymatic hydrolysis, pasteurizing or high-temperature sterilizing to obtain pasteurized modified sheep milk or high-temperature sterilized modified sheep milk, concentrating it and drying it to obtain the modified sheep milk powder.

[0013] Preferably, the enzymes used in the enzymatic hydrolysis are a complex protease and a neutral protease, with an enzyme activity ratio of 1:1 to 1:5.

[0014] Preferably, the enzymatic hydrolysis time is 2 h-4 h, the temperature is 50℃-60℃, and the amount of the complex protease added is 3500 U / g~6500 U / g.

[0015] Preferably, the pasteurization conditions are 65°C for 25-30 minutes and the high-temperature sterilization conditions are 105°C-115°C for 5-10 minutes.

[0016] Preferably, the nutrient fortifier includes one or more of the following: inulin, isomaltooligosaccharide, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, stachyose, mannose, chitosan oligosaccharide, phytosterol esters, phosphatidylserine, lutein esters, resistant dextrin, casein phosphopeptide, cholecalciferol, retinyl acetate, tocopherol acetate, pyridoxine hydrochloride, sodium L-ascorbate, calcium carbonate, ferrous sulfate, zinc sulfate, and taurine.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides sheep milk with hypoglycemic and uric acid-lowering activities, including pasteurized and high-temperature sterilized sheep milk. The sheep milk is prepared by enzymatic hydrolysis of sheep milk using neutral protease and complex protease. The prepared sheep milk has a high α-glucosidase inhibition rate and xanthine oxidase inhibition rate. It can be used by consumers as an adjunct to lower hypoglycemia and uric acid, reducing the dosage of hypoglycemic and uric acid-lowering drugs and thus reducing side effects.

[0019] This invention provides a sheep milk powder with hypoglycemic and uric acid-lowering activities. The production process adopts conventional spray drying, which can utilize the company's existing production equipment, making it easy for industrial production. It does not require cold chain to maintain low temperature during transportation and sales, and has a long shelf life, reducing transportation and sales costs. The sheep milk powder is convenient for consumers to carry and use as an adjunct to lowering blood sugar and uric acid. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The effects of five proteases on the inhibition rates of α-glucosidase (a) and xanthine oxidase (b) in sheep milk were investigated.

[0022] Figure 2 The effect of protease complex on the hypoglycemic and hypouric acid-lowering activity of sheep milk;

[0023] Figure 3 The effect of the ratio of two proteases on the hypoglycemic and hypouric acid-lowering activities of sheep milk was investigated; the α-G inhibition rate was the α-glucosidase inhibition rate, and the XO inhibition rate was the xanthine oxidase inhibition rate.

[0024] Figure 4 Standard curves for the positive control drugs acarbose (a) and allopurinol (b) are shown.

[0025] Figure 5The changes in α-glucosidase inhibition rate (a) and xanthine oxidase inhibition rate (xanthine inhibition rate, b) of pasteurized sheep milk and hypoglycemic and hypouric acid-lowering sheep milk during refrigeration;

[0026] Figure 6 The changes in α-glucosidase inhibition rate (a) and xanthine oxidase inhibition rate (b) of high-temperature sterilized sheep milk and hypoglycemic and hypouric acid-lowering sheep milk during storage;

[0027] Figure 7 Particle size changes of pasteurized sheep milk (a) and hypoglycemic and hypouric acid sheep milk (b) during 12 days of refrigeration;

[0028] Figure 8 The particle size changes of high-temperature sterilized sheep milk (a) and hypoglycemic and hypouric acid-lowering sheep milk (b) during 90 days of storage;

[0029] Figure 9 Changes in sedimentation rate of pasteurized (a) and high-temperature sterilized (b) sheep milk and hypoglycemic and hypouric acid-lowering sheep milk during storage;

[0030] Figure 10 Color changes of pasteurized sheep milk and hypoglycemic and hypouric acid sheep milk during 12 days of refrigeration; where (a) is L*; (b) is a*; (c) is b*; and (d) is ΔE.

[0031] Figure 11 Color changes of high-temperature sterilized sheep milk and hypoglycemic and hypouric acid-lowering sheep milk during 90 days of storage; where (a) is L*; (b) is a*; (c) is b*; and (d) is ΔE.

[0032] Figure 12 The changes in the inhibition rates of α-glucosidase (a) and xanthine oxidase (b) of sheep milk powder with hypoglycemic and uric acid-lowering activities at different storage temperatures;

[0033] Figure 13 Preparative chromatogram for isolating and purifying hypoglycemic and hypouric acid peptides;

[0034] Figure 14 To prepare the hypoglycemic and uric acid-lowering activities of each component obtained by chromatography;

[0035] Figure 15 This is the secondary mass spectrum of LDL;

[0036] Figure 16 This is a secondary mass spectrum of LRF;

[0037] Figure 17 This is a secondary mass spectrum of ILLQ;

[0038] Figure 18 This is a secondary mass spectrum of LTLP;

[0039] Figure 19 This is a secondary mass spectrum of ITMP;

[0040] Figure 20 This is a secondary mass spectrum of LFQI;

[0041] Figure 21 This is the secondary mass spectrum of LLILT;

[0042] Figure 22 The mass spectrum of VVVPPF is shown.

[0043] Figure 23 This is the second-order mass spectrum of LHLPLPL;

[0044] Figure 24 This is the secondary mass spectrum of LGSRYL;

[0045] Figure 25 The mass spectrum of VVAPFPE is shown below.

[0046] Figure 26 This is the second-order mass spectrum of GLDPYKL. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0048] Example 1: Protease screening and preparation of sheep milk with hypoglycemic and uric acid-lowering activities

[0049] 1.1 Effects of a single protease on the hypoglycemic and hypouric acid-lowering activities of sheep milk

[0050] Five food-grade proteases were added to sheep milk, and the optimal conditions for each protease are shown in Table 1. The enzyme addition amount was 5000 U / g for each sample. The temperature of each sample was adjusted to the optimal value for the protease, and enzymatic hydrolysis was performed for 5 hours. After the enzymatic hydrolysis reaction, the enzymes were inactivated by boiling water for 10 minutes, cooled to room temperature, and centrifuged at 8000 r / min for 10 minutes. The supernatant was then collected, and the inhibition rates of α-glucosidase and xanthine oxidase were measured. The hypoglycemic and hypouricemic activities of the enzymatically hydrolyzed sheep milk are shown in the table below. Figure 1 As shown.

[0051] Table 1. Sources of protease and enzymatic hydrolysis temperature

[0052]

[0053] Depend on Figure 1It can be seen that the top four inhibitors of α-glucosidase in enzymatic hydrolysis of sheep milk were NZ (61.58%±2.27%), NF (56.02%±0.88%), XZ (47.87%±2.50%), and PZ (39.35%±2.49%), while the top four inhibitors of xanthine oxidase were PZ (72.93%±2.88%), NF (71.53%±1.58%), NZ (66.78%±2.68%), and XZ (54.56%±0.28%).

[0054] 1.2 Effects of complex protease on the hypoglycemic and hypouric acid-lowering activities of sheep milk

[0055] Based on the hypoglycemic and uric acid-lowering activities of single proteases, NF(A), PZ(B), XZ(C), and NZ(D) were selected to form a complex enzyme. For the two-enzyme combination, each enzyme was added at a dosage of 2500 U / g; for the three-enzyme combination, each enzyme was added at a dosage of 1667 U / g; and for the four-enzyme combination, each enzyme was added at a dosage of 1250 U / g. Samples were taken after enzymatic hydrolysis to determine their hypoglycemic and uric acid-lowering activities. The results are as follows: Figure 2 As shown.

[0056] Depend on Figure 2 It can be seen that the enzymatically hydrolyzed sheep milk obtained by NF+NZ(AD) combination hydrolysis has the highest hypoglycemic and uric acid-lowering activity (p<0.05), with α-glucosidase and xanthine oxidase inhibition rates of 63.85%±0.81% and 75.25%±0.57%, respectively. Therefore, this complex protease was selected for the preparation of hypoglycemic and uric acid-lowering sheep milk.

[0057] 1.3 Effect of the ratio of two proteases on the hypoglycemic and hypouric acid-lowering activities of sheep milk

[0058] NFH(A) and NZ(D) were selected as catalysts to study the effect of the ratio of the two proteases on the hypoglycemic and hypouric acid-lowering activities of sheep milk. The total amount of NFH and NZ added was 5000 U / g. The results are as follows: Figure 3 As shown.

[0059] Depend on Figure 3 It can be seen that when the enzyme activity ratio of NFH:NZ is within the range of 1:1 to 1:5, the hypoglycemic and uric acid-lowering activities of enzymatic hydrolysis of sheep milk are all higher than 60%. When NFH:NZ=1:3, the inhibition rates of α-glucosidase and xanthine oxidase in enzymatic hydrolysis of sheep milk reach the maximum (p < 0.05), which are 70.35%±2.82% and 74.99%±1.81%, respectively. Therefore, the suitable ratio of NF to NZ is 1:1 to 1:5.

[0060] 1.4 Effects of enzymatic hydrolysis conditions on the hypoglycemic and hypouric acid-lowering activities of sheep milk

[0061] The effects of compound enzyme dosage (3500 U / g, 5000 U / g and 6500 U / g), enzymatic hydrolysis temperature (50℃, 55℃ and 60℃) and enzymatic hydrolysis time (2h, 3h and 4h) on the hypoglycemic and hypouric acid-lowering activities of sheep milk were studied. The experimental design and results are shown in Table 2.

[0062] Table 2. Effects of enzymatic hydrolysis conditions on the hypoglycemic and hypouric acid-lowering activities of sheep milk.

[0063]

[0064] As shown in Table 2, within the experimental range of compound enzyme dosage (3500 U / g, 5000 U / g and 6500 U / g), enzymatic hydrolysis temperature (50℃, 55℃ and 60℃), and enzymatic hydrolysis time (2h, 3h and 4h), the inhibition rates of α-glucosidase and xanthine oxidase were 61.44%~74.61% and 72.27%~82.75%, respectively; the highest hypoglycemic and uric acid-lowering activities were observed at a compound enzyme dosage of 4800 U / g and enzymatic hydrolysis at 60℃ for 2h.

[0065] Example 2: Comparison of the uric acid-lowering effects of the enzymatically hydrolyzed sheep milk prepared in Example 1 with the positive control drug.

[0066] The standard curves for the positive control hypoglycemic drug acarbose and the uric acid-lowering drug allopurinol are as follows: Figure 4 As shown, the linear fitting equations for acarbose and allopurinol are y = 0.7093x + 24.806 (R²). 2 =0.9868) and y=0.4126x+53.983 (R 2 =0.9653). Calculations show that the enzymatically hydrolyzed sheep milk prepared using Example 1 at a compound enzyme dosage of 4800 U / g and enzymatic hydrolysis at 60℃ for 2 hours has an inhibitory effect on α-glucosidase that is approximately equivalent to 70.14 μg / ml acarbose, and an inhibitory effect on xanthine oxidase that is approximately equivalent to 69.72 μg / mL allopurinol.

[0067] Example 3: Changes in pasteurized and UHT sheep milk with hypoglycemic and uric acid-lowering activities during storage.

[0068] 1. Experimental Methods

[0069] Pasteurization and high-temperature sterilization are commonly used sterilization methods for liquid milk. Pasteurized milk is suitable for low-temperature refrigeration, while high-temperature sterilized milk is suitable for room temperature storage. The enzymatically hydrolyzed sheep milk with hypoglycemic and uric acid-lowering activities prepared in Example 1 was pasteurized (65℃, 30 min) and stored at 4℃ for 12 days (SME65, i.e., hypoglycemic and uric acid-lowering sheep milk). Unhydrolyzed and pasteurized sheep milk served as a control, denoted as SM65 or sheep milk. The enzymatically hydrolyzed sheep milk with hypoglycemic and uric acid-lowering activities prepared in Example 1 was also sterilized at high temperature (115℃, 10 min) and stored at 25℃ for 90 days (SME115, i.e., hypoglycemic and uric acid-lowering sheep milk). Unhydrolyzed and high-temperature sterilized sheep milk served as a control, denoted as SM115 or sheep milk. During storage, samples were taken to determine the hypoglycemic and uric acid-lowering activities, particle size, precipitation rate, and color of the samples.

[0070] 2. Experimental Methods

[0071] 2.1 Determination of α-glucosidase inhibition rate

[0072] As shown in Table 3, each reagent was added to a 96-well microtiter plate. After reacting at 37℃ for 30 min, 100 μL of 0.2 mol / L Na₂CO₃ solution was added to terminate the reaction. The absorbance (A) of the reaction solution was measured at a wavelength of 405 nm using an ELISA reader. Acarbose, a hypoglycemic drug, was used as a positive control. The inhibition rate was calculated using the following formula:

[0073] .

[0074] Table 3 Experimental design for α-glucosidase inhibition rate determination

[0075]

[0076] 2.2 Determination of xanthine oxidase inhibition rate

[0077] As shown in Table 4, each reagent was added to a 96-well microtiter plate, and the reaction was carried out at 37 °C for 30 min. After the reaction, the absorbance (A) of the reaction solution was measured at a wavelength of 295 nm using an ELISA reader.

[58] Using allopurinol, a uric acid-lowering drug, as a positive control, the inhibition rate was calculated using the following formula:

[0078] .

[0079] Table 4 Experimental Design for Xanthine Oxidase Inhibition Rate Determination

[0080]

[0081] 2.3 Sedimentation rate determination

[0082] First, weigh the centrifuge tube (M0). Then, add 10 mL of liquid milk and weigh it again (M1). Centrifuge at 7000 r / min for 15 min, discard the supernatant, and weigh it again (M2). The formula for calculating the centrifugation sedimentation rate is as follows:

[0083] .

[0084] 2.4 Particle size determination

[0085] The particle size distribution and average particle size of sheep milk were determined using a laser particle size analyzer. The sample was added to the sample cell, and the shading rate was stabilized at approximately 12%. The parameters were set as follows: a general analytical model was used, water was used as the dispersant, and the particle refractive index was adjusted to 1.53, while the medium refractive index was adjusted to 1.33.

[0086] 2.5 Colorimetric Measurement

[0087] The colorimetry of the samples was measured using a spectrophotometer. The color difference between the sample and the control group can be represented by ΔE, and the formula for calculating ΔE is as follows:

[0088] .

[0089] 3. Experimental Results

[0090] 3.1 Blood sugar and uric acid lowering activity

[0091] The changes in the hypoglycemic and uric acid-lowering activities of pasteurized and UHT sheep milk during storage are as follows: Figure 5 and Figure 6 As shown, the hypoglycemic and hypouric acid-lowering activities of both types of sterilized enzymatic hydrolysed sheep milk decreased with prolonged storage time (p<0.05). Figure 5 and Figure 6 It can be seen that within 12 days, the α-glucosidase inhibition rate (α-G inhibition rate) of SME65 decreased from 78.92%±2.80% to 64.75%±2.92%, and the xanthine oxidase inhibition rate decreased from 81.68%±1.20% to 61.64%±1.32%; within 90 days, the α-G inhibition rate of SME115 decreased from 82.83%±3.7% to 37.66%±2.13%, and the xanthine oxidase inhibition rate decreased from 80.46%±1.98% to 44.79%±1.97%.

[0092] 3.2 Particle size

[0093] The particle size of sheep milk affects the stability of dairy products; smaller particles settle more slowly, resulting in a more stable system. The particle size changes of pasteurized milk and UHT milk during storage are shown below. Figures 7-8 As shown in Tables 5 and 6.

[0094] Table 5. Effect of storage time on particle size of pasteurized sheep milk with hypoglycemic and hypouricemic activities.

[0095]

[0096] Table 6. Effect of storage time on particle size of high-temperature sterilized sheep milk with hypoglycemic and hypouric acid-lowering activities.

[0097]

[0098] Depend on Figure 7 It can be seen that within 12 days, the particle size of sheep milk was mainly distributed between 0.43 and 7.51 μm, with two particle size peaks, neither of which showed obvious aggregation. The particle size distribution curves showed little difference within 12 days, indicating good stability of the system. Figure 8 It was found that after sterilization at 115℃, both types of sheep milk exhibited two particle size peaks within 90 days. Specifically, the particle size of SM115 was mainly distributed between 0.43 and 7.51 μm, while that of SME115 was mainly distributed between 0.63 and 7.51 μm. After 90 days of storage, the particle size peaks of the sheep milk shifted to the right, and the particle size increased to varying degrees in both types.

[0099] As shown in Tables 5 and 6, enzymatic hydrolysis alters the particle size distribution of sheep milk to some extent. After sterilization at 65℃, compared with unhydrolyzed sheep milk, the enzymatically hydrolyzed sheep milk with hypoglycemic and uric acid-lowering activities exhibits increased D10, while D50, D90, D(4,3), and D(3,2) decreased, and SSA increased. However, after sterilization at 115℃, the enzymatically hydrolyzed sheep milk with hypoglycemic and uric acid-lowering activities showed increased D50, D90, D(4,3), and D(3,2), and decreased SSA, exhibiting the opposite trend. This demonstrates that sterilization temperature also affects the particle size distribution of dairy products.

[0100] Different sterilization temperatures have different effects on the particle size of sheep milk. Comparing the D(4,3) and D(3,2) of the two sterilized milks, it can be seen that as the sterilization temperature increases, D(4,3) and D(3,2) also increase, indicating that the substances in the sheep milk system have aggregated and formed larger particles.

[0101] The increase in sheep milk particle size due to increased sterilization temperature may be due to the high protein content of sheep milk. Heat treatment causes the whey protein in it to denature. As the sterilization temperature increases, the denaturation of whey protein intensifies. The denatured whey protein can attach to casein micelles and aggregate with κ-casein on the micelle surface through intermolecular disulfide bonds, increasing the volume of casein micelles and forming a larger protein gel network. This ultimately leads to protein precipitation, increased particle size, and compromises the stability of sheep milk.

[0102] 3.3 Sedimentation rate

[0103] Pasteurized milk was stored at low temperature (4℃) for 12 days (sheep milk, hypoglycemic and hypouric acid sheep milk), and UHT milk was stored at room temperature (25℃) for 3 months (sheep milk, hypoglycemic and hypouric acid sheep milk). The change in precipitation rate during the storage period was measured.

[0104] Sedimentation rate reflects the stability of sheep milk; the smaller the change in sedimentation rate during storage, the better the stability of the sheep milk. The sedimentation rate of sheep milk during storage is as follows: Figure 9 As shown. By Figure 9 It can be seen that the sedimentation rate of both pasteurized milk and UHT milk increases with the extension of time during storage, and the sedimentation rate of sheep milk, which has hypoglycemic and uric acid-lowering activities after enzymatic hydrolysis, changes even less. Figure 9 In (a), the centrifugal sedimentation rate of pasteurized sheep milk increased from 3.75% to 4.72%, and the centrifugal sedimentation rate of hypoglycemic and hypouricemic pasteurized sheep milk increased from 1.96% to 3.22% (p>0.05). Figure 9 In (b), the centrifugal sedimentation rate of high-temperature sterilized sheep milk increased from 2.5% to 6.27%, and the centrifugal sedimentation rate of high-temperature sterilized sheep milk for lowering blood sugar and uric acid increased from 1.8% to 3.99% (p<0.05).

[0105] The increased precipitation rate may be due to changes in the quality of sheep milk during storage, such as increased acidity and increased positive charge on casein particles, which causes the protein particles to repel each other and precipitate.

[0106] 3.4, Colorimetry

[0107] Color is one of the sensory attributes of dairy products and is also the first quality parameter that consumers observe. It affects the quality, freshness, and food safety of dairy products. The stability of dairy products during storage depends on several factors: temperature, time, relative humidity, light, and dairy product composition.

[0108] Pasteurized milk (sheep milk, hypoglycemic and hypouricemic sheep milk) was stored at low temperature (4℃) for 12 days, and UHT milk (sheep milk, hypoglycemic and hypouricemic sheep milk) was stored at room temperature (25℃) for 3 months. Changes in color during the storage period were measured, and the results are as follows: Figure 10 and Figure 11 As shown in the figure. Where L* represents luminance, a* represents red-green, b* represents yellow-blue, and ΔE represents total color difference.

[0109] Depend on Figures 10-11It was found that compared with unenzymatically hydrolyzed sheep milk, the L* value of enzymatically hydrolyzed sheep milk increased, the a* value decreased, and the b* value increased, with significant differences (p<0.05). This indicates that the color of sheep milk gradually brightens, with the red color becoming lighter and the yellow color deepening. The change in the brightness value of sheep milk during storage can be caused by colored substances released by non-enzymatic browning reactions (Maillard reaction, lipid peroxidation, ascorbic acid degradation, or sugar-sugar caramelization), or it may be due to the increased release of peptides after enzymatic hydrolysis, with more free amino groups reacting more readily with the reducing sugars in sheep milk, thus producing color changes.

[0110] Depend on Figure 10 It was found that after 12 days of storage, the L* value of pasteurized sheep milk increased significantly (p<0.05), the a* value decreased but not significantly (p>0.05), and the b* value increased significantly (p<0.05). Conversely, the hypoglycemic and uric acid-lowering sheep milk showed the opposite trend: the L* value decreased significantly (p<0.05), the a* value increased but not significantly (p>0.05), and the b* value decreased significantly (p<0.05). Using sheep milk from day 0 as a control, after 12 days of storage, the total color difference ΔE was less than 3. When 0 < ΔE < 3, the color change was considered small; when ΔE > 3, the color change was considered perceptible to the naked eye. Therefore, it can be concluded that the color of sheep milk remained essentially unchanged after 12 days of storage.

[0111] Depend on Figure 11 It was found that after 90 days of storage, the L* and b* values ​​of UHT milk gradually increased, while the a* value gradually increased in the early stage of storage and then gradually decreased. After enzymatic hydrolysis, the L* and b* values ​​of the hypoglycemic and uric acid-lowering sheep milk gradually decreased with the extension of storage time, while the a* value gradually increased. The total color difference ΔE of both samples increased with the extension of storage time, and ΔE>3, the reason for which requires further investigation.

[0112] Depend on Figure 11 (b) and Figure 10 As shown in (b), at 0d, the a* value of UHT sheep milk was significantly higher than that of pasteurized sheep milk (p<0.05). This may be because high temperature leads to an intensified Maillard reaction, increasing the degree of browning and thus increasing its redness value; Figure 11 (d) and Figure 7 As shown in (d), the color value ΔE of sheep milk changes more significantly after high-temperature sterilization. This may be because pasteurization has a lower temperature and slower reaction rate, while high-temperature sterilization has a faster reaction rate and produces more color-developing substances, leading to a significant color change. This indicates that high-temperature sterilization has a significant impact on the quality of sheep milk, and the quality can be improved by shortening the heating time or lowering the heating temperature.

[0113] Example 4: Preparation and storage of sheep milk powder with hypoglycemic and uric acid-lowering activities

[0114] Using sheep milk with hypoglycemic and uric acid-lowering activities prepared under the optimal complex enzyme conditions obtained in the previous examples as raw material, it was concentrated to a solid content of 50%, and then spray-dried to obtain sheep milk powder with hypoglycemic and uric acid-lowering activities. The obtained sheep milk powder was stored at 4℃ (SME4), 25℃ (SME25), and 45℃ (SME45) for 3 months, respectively, and the changes in its hypoglycemic and uric acid-lowering activities during the storage process were measured. The results are as follows. Figure 12 As shown.

[0115] Depend on Figure 12 It was found that the hypoglycemic and hypouric acid-lowering activities of sheep milk powder stored at different temperatures decreased with the extension of storage time, and the higher the storage temperature, the faster the activity decreased. Within 90 days, the inhibition rates of α-glucosidase and xanthine oxidase in sheep milk powder stored at three different temperatures decreased by 11.54%-13.75% and 28.19%-31.46%, respectively. Among them, SME45 and SME4 showed the greatest and least loss of inhibition rate, respectively. At the end of storage, the inhibition rates of α-glucosidase and xanthine oxidase in SME45 were 58.83% and 45.11%, respectively, while those in SME4 were 61.04% and 48.37%, respectively.

[0116] Example 5: Isolation, purification, and identification of hypoglycemic and uric acid-lowering peptides

[0117] 5.1 Isolation and purification of hypoglycemic and uric acid-lowering peptides

[0118] The sheep milk with hypoglycemic and hypouric acid-lowering activities obtained in Example 1 was subjected to ultrafiltration to obtain fractions with <1 kDa, which were then desalted and analyzed by mass spectrometry. Preparative chromatography (Gemini-NX 10μ C18 100A column, 4.6*250 mm) was used to separate the ultrafiltration fractions. The relevant liquid phase gradient settings were as follows: 0 min–30 min, linear gradient of solution A (acetonitrile containing 0.1% trifluoroacetic acid) from 30% to 70%; linear gradient of solution B (water containing 0.1% trifluoroacetic acid) from 70% to 30%. The preparative chromatograms of each fraction are shown below. Figure 13 As shown.

[0119] Depend on Figure 13 It was found that the ultrafiltration fraction of enzymatically hydrolyzed sheep milk with hypoglycemic and uric acid-lowering activities (<1K) was purified by preparative chromatography, yielding five fractions: A, B, C, D, and E. These fractions were then freeze-dried under vacuum and rehydrated to a concentration of 2 mg / mL. Their hypoglycemic and uric acid-lowering activities were then determined, and the results are as follows: Figure 14 As shown.

[0120] Depend on Figure 14It was found that enzymatic hydrolysis of sheep milk fraction C had a good effect on lowering blood sugar and uric acid, with α-glucosidase inhibition rates of 51.28%±2.47% and xanthine oxidase inhibition rates of 54.94%±2.80%. The inhibition rates obtained were lower than those of the original sheep milk enzymatic hydrolysate before ultrafiltration and chromatographic separation, possibly due to the low rehydration concentration.

[0121] 5.2 Identification of hypoglycemic and hypouric acid-lowering peptides by HPLC-MS / MS

[0122] The amino acid sequence and sheep milk protein origin of the obtained component C were identified by HPLC-MS / MS. The liquid chromatography column (0.15mm*150mm, RP-C18, Column Technology Inc.) was equilibrated with 95% solution A (0.1% formic acid aqueous solution). The sample was loaded onto a Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) via an autosampler and then separated by the liquid chromatography column. The relevant liquid chromatography gradient settings were as follows: 0 min–50 min, linear gradient of solution B (0.1% formic acid acetonitrile aqueous solution (acetonitrile 84%)) from 4% to 50%; 50 min–54 min, linear gradient of solution B from 50% to 100%; 54 min–60 min, solution B maintained at 100%. The sample was then separated using a Q Exactive HF-X mass spectrometer (Thermo). Mass spectrometry analysis was performed using Fisher (positive ion detection method). Data was acquired, and the corresponding sheep milk protein database was searched using MaxQuant 1.5.5.1 software. The obtained hypoglycemic and uric acid-lowering peptides were compared and identified. The results are shown in Table 7. The secondary mass spectrum of the hypoglycemic and uric acid-lowering peptides is shown in the figure. Figures 15-26 As shown

[0123] Table 7. Amino acid sequences and sources of sheep milk hypoglycemic and uric acid-lowering peptides

[0124]

[0125] Table 7 shows that 12 hypoglycemic and hypouric acid-lowering peptides were identified from component C, with molecular weights ranging from 359.2 to 804.4 Da. These bioactive peptides mainly originated from osteopontin and α-hydroxylamine. s1 -Casein, β-lactoglobulin, κ-casein, lactoferrin, α-lactalbumin, β-casein, and κ-casein, of which α-casein... s1Casein and β-lactoglobulin are allergenic proteins. Through enzymatic hydrolysis with a complex protease, they can be converted into hypoglycemic and uric acid-lowering peptides, which can reduce the allergenicity of sheep milk and increase its hypoglycemic and uric acid-lowering activity. The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of sheep milk with hypoglycemic and uric acid-lowering activity, characterized in that, The sheep milk contains hypoglycemic and uric acid-lowering peptides, which are α-glucosidase inhibitory peptides and xanthine oxidase inhibitory peptides. The amino acid sequences of the hypoglycemic and uric acid-lowering peptides are LDL, LRF, ILLQ, LTLP, ITMP, LFQI, LLILT, LHLPLPL, VVVPPF, LGSRYL, VVAPFPE, and GLDPYKL.

2. The sheep milk according to claim 1, characterized in that, The sheep milk is pasteurized sheep milk, sterilized sheep milk, and formulated sheep milk powder.

3. The sheep milk according to claim 2, characterized in that, The α-glucosidase inhibition rate of the pasteurized sheep milk was 64.75%±2.92%~78.92%±2.80%, and the xanthine oxidase inhibition rate was 61.64%±1.32%~81.68%±1.20%.

4. The sheep milk according to claim 2, characterized in that, The α-glucosidase inhibition rate of the sterilized sheep milk was 37.66%±2.13%~82.83%±3.70%, and the xanthine oxidase inhibition rate was 44.79%±1.97%~80.46%±1.98%.

5. The sheep milk according to claim 2, characterized in that, The modified sheep milk powder showed an α-glucosidase inhibition rate of 58.83%±2.13%~72.56%±2.53% and a xanthine oxidase inhibition rate of 45.11%±3.13%~80.46%±1.98% after reconstitution.

6. The method for preparing sheep milk according to claim 2, characterized in that, The process includes sterilizing sheep milk, cooling it, adding a complex protease for enzymatic hydrolysis, adding a nutrient fortifier after enzymatic hydrolysis, pasteurizing or high-temperature sterilizing to obtain pasteurized or high-temperature sterilized sheep milk, concentrating it, and then drying it to obtain the prepared sheep milk powder.

7. The preparation method according to claim 6, characterized in that, When performing the enzymatic hydrolysis, the enzymes used are a complex protease and a neutral protease, with an enzyme activity ratio of 1:1 to 1:

5.

8. The preparation method according to claim 6, characterized in that, The enzymatic hydrolysis time is 2 h-4 h, the temperature is 50℃-60℃, and the amount of the compound protease added is 3500 U / g~6500 U / g.

9. The preparation method according to claim 4, characterized in that, The pasteurization conditions are 65℃ for 25-30 minutes, and the high-temperature sterilization conditions are 105℃-115℃ for 5-10 minutes.

10. The preparation method according to claim 4, characterized in that, The nutritional fortifiers include one or more of the following: inulin, isomaltooligosaccharide, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, stachyose, mannose, chitosan oligosaccharide, phytosterol esters, phosphatidylserine, lutein esters, resistant dextrin, casein phosphopeptide, cholecalciferol, retinyl acetate, tocopherol acetate, pyridoxine hydrochloride, sodium L-ascorbate, calcium carbonate, ferrous sulfate, zinc sulfate, and taurine.

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