Uric acid-reducing peptide goat milk powder and preparation method thereof

Abstract

The invention discloses uric acid-reducing peptide goat milk powder and a preparation method thereof, and belongs to the technical field of enzymes. According to the process for preparing the uric acid lowering peptide by hydrolyzing goat milk through the compound enzyme, the total addition amount of the compound enzyme (the enzyme activity ratio of neutral protease N to neutral protease X is 1: 1) is 4000 U / g-6000 U / g, enzymolysis is conducted for 2-3 h at the temperature of 45-55 DEG C, and under the condition, the xanthine oxidase inhibition rate is 71.67 + / -1.32%-83.64 + / -0.98%. The enzymolysis goat milk containing the uric acid-reducing peptide is subjected to spray drying to prepare the uric acid-reducing peptide goat milk powder, and the storage stability of the goat milk powder is relatively good. The amino acid sequence and the source of the uric acid lowering peptide are separated and identified, the in-vitro uric acid lowering activity of the uric acid lowering peptide is verified through the solid-phase synthesized uric acid lowering peptide, and a theoretical basis and technical support are provided for development of new uric acid lowering peptide goat milk products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of dairy products, in particular to a goat milk powder containing uric acid-lowering peptides and a preparation method thereof. BACKGROUND

[0002] Uric acid is one of the metabolic products of the human body, mainly produced by the metabolism of dietary intake and in vivo decomposition of purine compounds in the liver, and excreted through the kidney and digestive tract. Under normal circumstances, the production and excretion of uric acid in the body remain in a balanced state, and when there is a purine metabolism disorder, hyperuricemia occurs. The domestic diagnosis of hyperuricemia is defined as: under normal dietary conditions, regardless of gender, the blood uric acid level of two different days is more than 420 μmol / L.

[0003] At present, the treatment methods for hyperuricemia can be divided into drug therapy and dietary therapy, and the treatment drugs mainly include uric acid generation inhibitors, uric acid excretion promoters and uricase drugs: among them, the uric acid generation inhibitors mainly include xanthine oxidase inhibitors such as allopurinol, colchicine and febuxostat, which mainly take xanthine oxidase as a target, inhibit the activity of xanthine oxidase, reduce the generation of uric acid, and thus effectively reduce the uric acid level. However, drug therapy has obvious side effects, such as hepatitis, nephropathy, abdominal pain, abdominal distension, diarrhea and skin rash. At the same time, the uric acid-lowering peptides prepared from food source proteins have attracted widespread attention of researchers due to their high bioavailability, obvious inhibitory effect and no toxic side effects.

[0004] The preparation methods of uric acid-lowering peptides mainly include fermentation or enzymolysis, and the enzymolysis method has attracted widespread attention due to its mild reaction conditions, strong specificity and easy control. Different proteases have different cleavage sites, action modes and hydrolysis degrees of proteins, and the types, sizes and biological activities of the prepared uric acid-lowering peptides are different, so it is very important to select a suitable protease and reasonable process conditions for obtaining uric acid-lowering peptides. Previous literatures have reported that bovine milk and its casein or whey protein are used as raw materials to prepare antihypertensive peptides and antioxidant peptides, but there are few studies on the preparation of uric acid-lowering peptides from goat milk and the direct development of goat milk products. SUMMARY

[0005] The present application aims to provide a goat milk powder containing uric acid-lowering peptides and a preparation method thereof, to solve the problems existing in the prior art. The liquid goat milk containing uric acid-lowering peptides is obtained by enzymolysis of goat milk with a compound protease, and the goat milk powder with uric acid-lowering function is further prepared. The storage stability of the goat milk powder is good, which provides a theoretical basis and technical support for the deep development of local goat milk products in Shaanxi.

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

[0007] This invention provides a urate-lowering peptide goat milk powder, wherein the goat milk powder contains a urate-lowering peptide, which is a xanthine oxidase inhibitory peptide with the amino acid sequence LTL, GPFP, LRF, FLSW, LTQTP, LFRQ, FNPTQ, VVVPPF, ILPLTQ, IASAEEPT, AGPFTPT, LTDVEK, STPTTEA, QTPVVVP, PYKLRP, and FYPQLFR. The xanthine oxidase inhibition rate of the urate-lowering peptide goat milk powder after reconstitution is 50.04%-72.66%.

[0008] Preferably, the amino acid sequence of the urate-lowering peptide is GPFP, LRF, LTDVEK, LTQTP, and VVVPPF, with molecular weights of 416 Da, 434 Da, 558 Da, 703 Da, and 656 Da, respectively, and its xanthine oxidase inhibition IC50 is [value missing]. 50 The values ​​were 30.13 mM, 33.76 mM, 18.93 mM, 29.14 mM and 26.24 mM, respectively.

[0009] Preferably, the uric acid-lowering peptide goat milk powder includes uric acid-lowering peptide goat milk powder for middle-aged and elderly people and uric acid-lowering peptide goat milk powder for teenagers.

[0010] Preferably, the urate-lowering peptide goat milk powder further includes a nutritional fortifier; the nutritional fortifier added to the urate-lowering peptide aged goat milk powder includes one or more of the following: phytosterol esters, Acer truncatum seed oil, galactooligosaccharides, inulin, and fructooligosaccharides.

[0011] The nutritional fortifiers added to the uric acid-lowering peptide juvenile goat milk powder include one or more of lutein, lutein ester, DHA, phosphatidylserine, isomaltooligosaccharide, xylooligosaccharide, and stachyose.

[0012] This invention also provides a method for preparing uric acid-lowering peptide goat milk powder, comprising the following steps:

[0013] After sterilization and cooling, goat milk is mixed with a compound protease, kept warm for enzymatic hydrolysis, and then heated to inactivate the enzyme to obtain uric acid-lowering peptide liquid goat milk.

[0014] With or without the addition of nutritional fortifiers, the uric acid-lowering peptide liquid goat milk is concentrated and then spray-dried to obtain uric acid-lowering peptide goat milk powder.

[0015] Preferably, the complex protease is neutral protease N and neutral protease X;

[0016] The addition amounts of neutral protease N and neutral protease X are both 4000 U / g-6000 U / g;

[0017] When two proteases are added, the ratio of the enzyme activities of neutral protease N to neutral protease X is 1:1.

[0018] Preferably, the enzymatic hydrolysis conditions are: 45℃-55℃ for 2-3 hours.

[0019] Preferably, the goat milk is fresh goat milk or reconstituted goat milk. The preparation method of reconstituted goat milk includes: mixing water and goat milk powder to obtain goat milk with a mass percentage of 11.5%-12.5%, which is the reconstituted goat milk.

[0020] Fresh or reconstituted goat milk is sterilized at 95°C for 5 minutes or at 90°C for 10 minutes and then cooled to 45°C-55°C.

[0021] Preferably, the xanthine oxidase inhibition rate of the urate-lowering peptide liquid goat milk is 71.67±1.32%~83.64±0.98%.

[0022] This invention also provides the use of the described uric acid-lowering peptide in the preparation of dairy products or pharmaceuticals that help maintain healthy uric acid levels. More specifically, it can also be a product, such as a dairy product comprising the aforementioned uric acid-lowering peptide or the described uric acid-lowering peptide milk powder. As another example, it can be a pharmaceutical comprising the aforementioned uric acid-lowering peptide.

[0023] The present invention discloses the following technical effects:

[0024] This invention provides a uric acid-lowering peptide goat milk powder, which utilizes a complex protease to enzymatically hydrolyze goat milk. The enzymatically hydrolyzed goat milk has a high xanthine oxidase inhibition rate and can be used by consumers as an adjunct to lower uric acid.

[0025] This invention also provides a uric acid-lowering peptide derived from goat milk protein, which is a food-derived bioactive peptide. It can be used as a dietary supplement to assist in lowering uric acid, reducing the dosage of uric acid-lowering drugs to decrease side effects, and can also be used in the development of uric acid-lowering drugs.

[0026] The present invention uses a compound enzyme to hydrolyze goat milk to prepare uric acid-lowering peptides. The total amount of compound enzyme (neutral protease N and neutral protease X with an activity ratio of 1:1) added is 4000 U / g-6000 U / g, and the enzymatic hydrolysis is carried out at 45-55 ℃ for 2-3 h. Under these conditions, the xanthine oxidase inhibition rate is 71.67±1.32%~83.64±0.98%. Goat milk powder containing uric acid-lowering activity, after spray drying, was stored for 2.5 months under low temperature (4 ℃), room temperature (25 ℃), and high temperature (45 ℃) conditions. The results showed that with the extension of storage time, the inhibition rate of xanthine oxidase in goat milk powder decreased, the particle size changed little, the brightness gradually decreased, and it gradually darkened and turned yellow. The total color difference and diffraction peak intensity increased, the bound water content in the milk powder increased, and the free water content decreased. With the increase of storage temperature, the activity decreased faster, the change in total color difference and the damage of goat milk particles intensified, and the impact on the crystal structure of goat milk powder was small. The enzymatic reaction destroyed the particle structure of goat milk powder. The diffraction patterns of goat milk powder before and after enzymatic hydrolysis were both amorphous broad peaks. Attached Figure Description

[0027] 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.

[0028] Figure 1 The effects of single and complex proteases on the inhibitory effect and degree of hydrolysis of goat lactoxine oxidase;

[0029] Figure 2 Effects of compound enzyme dosage (a), hydrolysis temperature (b), and hydrolysis time (c) on the activity of urate-lowering peptides in goat milk.

[0030] Figure 3 The changes in the uric acid-lowering activity of goat milk powder under different storage temperatures;

[0031] Figure 4 The particle size variation of goat milk powder under different storage temperatures: (a) GM particle size (GM4) at 4℃, (b) GM particle size (GM25) at 25℃, (c) GM particle size (GM45) at 45℃, (d) GME particle size (GME4) at 4℃, (e) GME particle size (GME25) at 25℃, (f) GME particle size (GME45) at 45℃.

[0032] Figure 5 The color change of goat milk powder under different storage temperatures; (a) L*, (b) a*, (c) b*, (d) ΔE;

[0033] Figure 6 A scanning electron microscope image of goat milk powder containing urate-lowering peptides;

[0034] Figure 7 X-ray diffraction patterns of different goat milk powders; (a) goat milk; (b) goat milk powder;

[0035] Figure 8 T2 relaxation spectra of goat milk powder at different temperatures during storage period;

[0036] Figure 9 The preparative chromatogram for the separation of urate-lowering peptides;

[0037] Figure 10 To prepare the uric acid-lowering activity of each component obtained by chromatography;

[0038] Figure 11 The images show the secondary mass spectra of urate-lowering peptides LTL and GPFP.

[0039] Figure 12 The images show the secondary mass spectra of the urate-lowering peptides LRF and FLSW.

[0040] Figure 13 The images show the secondary mass spectra of urate-lowering peptides LTQTP and LFRQ.

[0041] Figure 14 The images show the secondary mass spectra of the urate-lowering peptides FNPTQ and VVVPPF.

[0042] Figure 15 The images show the secondary mass spectra of the urate-lowering peptides ILPLTQ and IASAEPT.

[0043] Figure 16 The mass spectra of the urate-lowering peptides AGPFTPT and LTDVEK are shown.

[0044] Figure 17 The mass spectra of the urate-lowering peptides STPTTEA and QTPVVVP are shown.

[0045] Figure 18 The images show the secondary mass spectra of the urate-lowering peptides PYKLRP and FYPQLFR.

[0046] Figure 19 HPLC purity analysis of urate-lowering peptide GPFP (a) and primary mass spectrum (b);

[0047] Figure 20 HPLC purity analysis of urate-lowering peptide LRF (a) and primary mass spectrum (b);

[0048] Figure 21 HPLC purity analysis and primary mass spectrum of urate-lowering peptide LTDVEK (a);

[0049] Figure 22 HPLC purity analysis of LTQTP (a) and primary mass spectrum (b).

[0050] Figure 23 HPLC purity analysis of urate-lowering peptide VVVPPF (a) and primary mass spectrum (b). Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] The following examples involve the explanation of some abbreviations: Goat milk: GM; Goat milk hydrolysate: GME.

[0054] The analytical methods involved in the following examples are as follows:

[0055] (1) Determination of protein hydrolysis

[0056] To maintain a constant pH, 0.1 mol / L NaOH solution was added dropwise to the enzymatically hydrolyzed sheep milk solution after a certain period of time, and the volume of NaOH solution used was recorded. Then, the degree of hydrolysis (DH) of the protein was calculated using the pH-Stat method, as shown in formula (1).

[0057] Equation (1)

[0058] In the above formula (1): B represents the volume of NaOH (mL); Mb represents the concentration of NaOH (mol / L); 1 / α represents the reciprocal of the degree of α-amino dissociation; Mp represents the protein mass (g); 8.35 is the number of millimoles of peptide bonds in sheep milk (mmol).

[0059] (2) Determination of xanthine oxidase inhibition rate

[0060] As shown in Table 1, 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. Allopurinol was used as a positive control, and the inhibition rate was calculated using the formula (2).

[0061] Equation (2)

[0062] Table 1. Experimental design for determining xanthine oxidase inhibition rate.

[0063]

[0064] (3) Particle size determination

[0065] 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.

[0066] (4) Scanning electron microscopy determination

[0067] The microstructure of the sample was determined using a scanning electron microscope. The sample was placed on a stage of conductive adhesive of appropriate size, excess sample was blown away with nitrogen gas, gold was sprayed on, and then the sample was observed.

[0068] (5) Colorimetric determination

[0069] The colorimetry of the sample was measured using a spectrophotometer. The color difference between the sample and the control group can be represented by ΔE. The formula for calculating ΔE is shown in equation (3) below:

[0070] Equation (3)

[0071] (6) XRD diffraction

[0072] The structure of the goat milk powder was determined by referring to the method in Wang Zifei's "Preparation and Storage Study of Functional Iron-Fortified Goat Milk Powder".

[0073] (7) Moisture content determination

[0074] The water activity of the goat milk powder was determined by referring to the method in Wang Zifei's "Preparation and Storage Study of Functional Iron-Fortified Goat Milk Powder".

[0075] Example 1: Study on the enzymatic hydrolysis process of goat milk containing uric acid-lowering peptides

[0076] 1.1 Screening of suitable proteases for the preparation of uric acid-lowering peptides by enzymatic hydrolysis of goat milk

[0077] Using single neutral protease N (Neutrase 0.8L, Novozymes, Denmark), bromelain (Nanning Pangbo Biotechnology Co., Ltd., 100,000 U), and neutral protease X (Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., 400,000 U), as well as their complex proteases, as biocatalysts, they were added to sterilized (90℃, 10 min) goat milk cooled to 50℃. The total enzyme addition was 4500 U / g, the addition of each enzyme in the two-enzyme combination was 2250 U / g, and the addition of each enzyme in the three-enzyme combination was 1500 U / g. Enzymatic hydrolysis was carried out at 50℃ for 2 h, followed by boiling for 5 min, cooling to room temperature, and centrifugation at 7000 r / min for 15 min. The supernatant was obtained, and samples were taken to determine the xanthine oxidase inhibition rate and degree of hydrolysis. The results are shown in [Figure number missing]. Figure 1 .

[0078] Depend on Figure 1 The results showed that neutral protease X exhibited the highest inhibition rate and degree of hydrolysis of xanthine oxidase in goat milk, at 68.05±2.71% and 6.93±0.09%, respectively, followed by neutral protease N at 63.56±3.01% and 5.11±0.06%, respectively. Bromelain yielded the lowest inhibition rate and degree of hydrolysis of xanthine oxidase in goat milk, at 61.56±2.45% and 2.10±0.03%, respectively. The combined enzyme of neutral protease N and neutral protease X showed the highest inhibition rate of xanthine oxidase in goat milk, at 76.38±1.57%, significantly higher than other two- or three-enzyme combinations. The combination of neutral protease N and bromelain resulted in the highest degree of hydrolysis of goat milk, but its xanthine oxidase inhibition rate was only 60.18±2.11%, indicating that the degree of hydrolysis and xanthine oxidase inhibition rate were not positively correlated.

[0079] 1.2 Effects of enzymatic hydrolysis conditions on the activity of urate-lowering peptides in goat milk

[0080] Using neutral protease N and neutral protease X as biocatalysts, the effects of the amount of compound protease added (3000 U, 4000 U, 5000 U, 6000 U, and 7000 U, with an enzyme activity ratio of 1:1), the enzymatic hydrolysis temperature (40℃, 45℃, 50℃, 55℃, and 60℃), and the enzymatic hydrolysis time (1 h, 1.5 h, 2 h, 2.5 h, and 3.0 h) on the inhibition rate of xanthine oxidase in goat milk were investigated. The results are shown in [Figure number missing]. Figure 2 .

[0081] Depend on Figure 2(a) It can be seen that with the increase of enzyme addition, the uric acid-lowering activity of enzymatic hydrolysis of goat milk showed a trend of first increasing and then decreasing. The uric acid-lowering inhibitory activity of enzymatic hydrolysis of goat milk reached its maximum (p < 0.05), at an addition of 5000 U / g, which was 77.51±2.23%. When the addition was 4000-6000 U / g, the inhibition rate of xanthine oxidase was greater than 70%. When the enzyme addition was low, the high concentration of substrate may lead to aggregation at the active site of the substrate group, hindering the full interaction between enzyme and substrate, resulting in a lower inhibitory effect. With the increase of enzyme addition, the substrate was more evenly dispersed around the enzyme, improving the interaction between them and increasing the hydrolysis rate. Further increasing the enzyme addition will reduce the substrate-to-enzyme ratio. When the available amount of substrate is limited, excessive enzyme will lead to excessive hydrolysis of proteins, destroying peptide chains and generating free amino acids, thereby reducing the inhibitory effect. Adding a certain amount of enzyme is beneficial for the hydrolysis of proteins to produce peptides, thereby increasing the hypoglycemic and uric acid-lowering activities. However, when the amount of enzyme added is too high, it may lead to the further degradation of the hypoglycemic and uric acid-lowering peptides that have already been produced, generating inactive peptide fragments, thereby reducing the inhibitory activity of sheep milk hydrolysate and also causing a waste of resources.

[0082] Depend on Figure 2 (b) It can be seen that with the increase of enzymatic hydrolysis temperature, the uric acid-lowering activity of enzymatic hydrolysis of goat milk showed a trend of first increasing and then decreasing. The xanthine oxidase inhibitory activity of enzymatic hydrolysis of goat milk reached its maximum at 55℃ (p < 0.05), and the xanthine oxidase inhibition rate was greater than 70% when the enzymatic hydrolysis temperature was 45-55℃. This is because higher temperature is conducive to diffusion, accelerates the binding of enzyme to substrate and improves enzyme catalytic efficiency, thereby releasing more xanthine oxidase inhibitory peptides. However, too high a temperature will have a negative impact on enzyme activity, causing enzyme denaturation and leading to a decrease in catalytic efficiency.

[0083] Depend on Figure 2 (c) It can be seen that as the enzymatic hydrolysis time is extended, the uric acid-lowering activity in enzymatically hydrolyzed goat milk gradually increases. When the enzymatic hydrolysis time is ≥2h, the xanthine oxidase inhibition rate is higher than 70%. When the enzymatic hydrolysis time is 3h, the xanthine oxidase inhibition rate reaches 83.64±0.98%, and its inhibitory effect on xanthine oxidase is approximately equivalent to 71.88μg / mL allopurinol.

[0084] In summary, when the target is an inhibition rate of xanthine oxidase greater than 70%, the enzyme addition range of 4000-6000 U / g, the enzymatic hydrolysis temperature of 45-55℃, and the enzymatic hydrolysis time of 2-3h all meet the requirements.

[0085] Example 2: Preparation and characterization of uric acid-lowering peptide goat milk powder

[0086] 1. Experimental Methods

[0087] Using goat milk (GME) with uric acid-lowering activity prepared in Example 1 and unhydrolyzed goat milk (GM) as raw materials, goat milk powder containing uric acid-lowering peptides was obtained by spray drying. The obtained goat milk powder was stored at low temperature (4°C), room temperature (25°C) and high temperature (45°C) for 2.5 months, respectively. The changes of milk powder under low temperature, room temperature and high temperature storage were investigated by using uric acid-lowering activity, particle size, color, particle shape, crystallization and water content as indicators.

[0088] 2. Results and Analysis

[0089] 2.1 Uric acid-lowering activity

[0090] Changes in the uric acid-lowering activity of goat milk powder at different storage temperatures and time periods, as follows: Figure 3 As shown. By Figure 3 It was found that the uric acid-lowering activity of goat milk powder stored at different temperatures decreased with prolonged storage time, and the higher the storage temperature, the faster the activity decreased. At the end of storage, the xanthine oxidase inhibition rate of goat milk powder stored at the three different temperatures was higher than 50%.

[0091] 2.2 Particle size

[0092] The particle size distribution changes of goat milk powder at different storage temperatures and time periods are as follows: Figure 4 As shown, the particles of goat milk powder are mainly distributed between 0.43 and 6.21 μm, with a single particle size peak, and the particle distribution range remains unchanged before and after protease hydrolysis. At day 0, the peak intensity of goat milk GM is centered at 1.63 μm, and the peak intensity of GME is centered at 1.97 μm. Within 75 days of storage at different temperatures, the peak intensity center of GM remains unchanged, while the peak intensity center of GME changes from 1.97 μm to 1.63 μm at day 60.

[0093] As shown in Table 2, after enzymatic hydrolysis, the D10, D50, D90, D(4,3), and D(3,2) of goat milk powder all increased, while the SSA decreased. Within 75 days of storage at different temperatures, the changes in D(4,3) and D(3,2) of goat milk powder were relatively small, demonstrating good particle stability.

[0094] Table 2. Effect of storage time on particle size of goat milk powder containing urate-lowering peptides

[0095]

[0096] 2.3 Chromaticity

[0097] Color parameter L of goat milk powder containing uric acid-lowering peptides at different storage temperatures and time periods * a * ,b* The changes in ΔE value are as follows Figure 5 As shown in (a)-(c). From Figure 5 It can be seen that the total color difference value of goat milk increases after enzymatic hydrolysis, and the L of goat milk powder increases. * The larger value indicates that the goat milk powder has a lighter color. Compared with the blank control (0 days), the L value increased with prolonged storage time. * The values ​​generally showed a downward trend (p<0.05), indicating that the brightness of goat milk gradually decreased. * The data showing fluctuations within a small range and being negative indicates that the redness of the goat milk powder is relatively weak. * The value increased slightly, meaning the goat milk powder gradually darkened and turned yellow.

[0098] Using the goat milk powder from day 0 as a control, from Figure 5 As shown in Figure (d), the ΔE values ​​of GM4 and GME4 after 75 days of storage at 4℃ were 1.3±0.18 and 3.28±0.11, respectively, with relatively small color changes. The ΔE values ​​of GM25 and GME25 after 75 days of storage at 25℃ were 2.19±0.61 and 3.44±1.51, respectively. The ΔE values ​​of GM45 and GME45 after 75 days of storage at 45℃ were 3.01±0.71 and 6.44±0.17, respectively. The higher the storage temperature, the more significant the change in color value (p<0.05). This may be because as the storage temperature increases, lipid oxidation and Maillard reaction intensify, thereby producing more colored substances.

[0099] 2.4 Particle shape

[0100] Particle shape changes of goat milk powder at different storage temperatures and time periods, such as Figure 6 As shown in the figure, scanning electron microscopy results of goat milk and its enzymatically hydrolyzed samples revealed that the initial goat milk particles were spherical of varying sizes, with most having smooth surfaces, aggregated particles, and low dispersion. After enzymatic hydrolysis, the goat milk particles were broken down, the surfaces became rough, and the microstructure changed, indicating that the enzymatic hydrolysis reaction destroyed the structure of the goat milk to some extent. After storage at different temperatures for 75 days, it was found that the breakage of goat milk particles intensified with increasing storage temperature.

[0101] 2.5 Crystallization Analysis

[0102] XRD is commonly used to evaluate the crystallization state and structural changes of molecular crystals after physicochemical treatment, thereby determining the crystal form of protein molecules. Clumping during storage is one of the main problems causing changes in milk powder quality. Clumping is primarily caused by lactose crystallization in the milk powder during the spray drying process. Lactose in milk powder can absorb water and transform from an amorphous state to a crystalline state. Figure 7It can be seen that the diffraction patterns of different goat milk powders are similar, with only one diffraction peak, and the highest diffraction peak appears at around 2θ 20°. The shape of the diffraction pattern of the enzymatically hydrolyzed goat milk powder does not change, and all of them are amorphous diffraction broad peaks without the formation of crystalline peaks, indicating that the temperature selected during the spray drying of goat milk powder is appropriate. After storage at different temperatures for 75 days, the intensity of the diffraction peaks increases, indicating that the storage and transportation process will affect the crystal structure of goat milk powder, but the effect of different temperatures on the crystal structure of goat milk powder is not significant.

[0103] 2.6 Moisture content

[0104] Low-field nuclear magnetic resonance (LF-NMR) is often used to assess the molecular mobility of water and the interactions between water and biopolymers in food systems.

[0105] In the T2 relaxation spectrum obtained using LF-NMR, the transverse relaxation time reflects the water state and can be divided into three regions: T2 relaxation time between 0 ms and 1 ms. 21 The region represents the relaxation of hydrogen protons in a layer of water molecules tightly bound to polar groups (i.e., bound water), where their mobility is minimal; the relaxation time T is between 1 ms and 100 ms. 22 The region represents stationary water, i.e., non-flowing water; the relaxation time T is between 100 ms and 1000 ms. 23 The region represents free water. The shorter the transverse relaxation time, the tighter the binding between water and material. The corresponding peak area can represent the relative water content in each phase.

[0106] T2 relaxation spectra of uricosuric peptide goat milk powder obtained by LF-NMR at different storage temperatures and time periods are as follows: Figure 8 As shown, it mainly consists of three peaks. During storage, the moisture migration pattern of the uric acid-lowering peptide goat milk powder remains the same over time, i.e., T... 23 The peak gradually decreases, T 21 The peak gradually increased and shifted to the left, with a shortened transverse relaxation time, indicating that the water in the urate-lowering peptide goat milk powder bound more tightly to the substrate, resulting in a decrease in free water content. Furthermore, after 75 days, the urate-lowering peptide goat milk powder stored at 45℃ showed a higher T... 23 The peak area is the smallest, T 21 The largest peak area indicates the highest bound water content in the urate-lowering peptide goat milk powder. The T2 relaxation spectra obtained before and after enzymatic hydrolysis of goat milk are similar, with only the peak intensity changing, indicating that the enzymatic hydrolysis reaction has little effect on the water content of goat milk.

[0107] Example 3: Isolation and identification of urate-lowering peptides from enzymatic hydrolysis of goat milk

[0108] The enzymatically hydrolyzed goat milk containing urate-lowering peptides prepared in Example 1 was subjected to low-temperature freeze centrifugation to obtain the supernatant. This supernatant was then subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 10K, 5K, 3K, and 1K, respectively. The resulting fractions (F1, F2, F3, and F4) were freeze-dried and prepared to the same concentration for xanthine oxidase inhibition rate determination. It was found that the permeate with a molecular weight cutoff of 1K exhibited the highest xanthine oxidase inhibition rate, indicating that the urate-lowering peptides were mainly present in this fraction. This fraction was further separated using preparative chromatography. The results of the preparative chromatographic separation are shown below. Each fraction was freeze-dried and prepared to the same concentration for xanthine oxidase inhibition rate determination. Figure 9 As shown.

[0109] Fractions F1, F2, F3, and F4 of the goat milk enzymatic hydrolysate obtained from the preparation of the chromatogram were collected, freeze-dried, and rehydrated to 5 mg / mL. Their uric acid-lowering activity was then determined, and the results are as follows: Figure 10 As shown in the figure, the goat milk hydrolysate component F1 had a better uric acid-lowering effect (p<0.05), with a xanthine oxidase inhibition rate of 55.61±3.27%.

[0110] The amino acid sequence and position in goat milk protein of the component F1 with the highest uric acid-lowering activity were identified by LC-MS / MS, and the results are shown in Table 3.

[0111] Table 3 shows that 16 uric acid-lowering peptides were identified from the F1 fraction, with molecular weights ranging from 345 to 970 Da. These bioactive peptides mainly originated from β-casein and α-casein. s1 - Casein, lactoferrin, β-lactoglobulin, κ-casein, and α s2 -Casein. The secondary mass spectrum of the urate-lowering peptide is shown below. Figures 11-18 .

[0112] Table 3. Amino acid sequence and source of goat milk urate-lowering peptides

[0113]

[0114] Example 4: Synthesis and Activity Verification of Uric Acid-Lowering Peptides

[0115] Uric acid-lowering peptides GPFP, LRF, LTDVEK, LTQTP, and VVVPPF were selected for solid-phase synthesis. The HPLC purity analysis and primary mass spectrometry results of the synthesized uricosuric peptides are shown below. Figures 19~23 The results showed that the purities of the synthesized GPFP, LRF, LTDVEK, LTQTP, and VVVPPF were 95.11%, 96.18%, 96.51%, 95.76%, and 96.61%, respectively. Different concentrations were prepared, and their inhibition rates against xanthine oxidase were measured. The IC50 values ​​for xanthine oxidase inhibition were then calculated. 50 The values ​​are shown in Table 4.

[0116] Table 4. IC50 of the five urate-lowering peptides on xanthine oxidase inhibition 50 value

[0117]

[0118] Table 4 shows that the five urate-lowering peptides exhibited significant differences in their inhibition rates against xanthine oxidase. LTDVEK showed the strongest inhibitory effect on xanthine oxidase, with an IC50 value of [missing value]. 50 The value was 18.93±0.42 mM, indicating that LRF had the worst inhibitory effect on xanthine oxidase, with an IC50 value of 18.93±0.42 mM. 50 The value is 33.76 ± 1.53 mM.

[0119] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A uric acid-lowering peptide goat milk powder, characterized in that, The goat milk powder contains a urate-lowering peptide, which is a xanthine oxidase inhibitory peptide with the amino acid sequence LTL, GPFP, LRF, FLSW, LTQTP, LFRQ, FNPTQ, VVVPPF, ILPLTQ, IASAEEPT, AGPFTPT, LTDVEK, STPTTEA, QTPVVVP, PYKLRP, and FYPQLFR. The xanthine oxidase inhibition rate of the goat milk powder containing the urate-lowering peptide after reconstitution is 50.04%-72.66%.

2. The uric acid-lowering peptide goat milk powder as described in claim 1, characterized in that, The amino acid sequence of the urate-lowering peptide is GPFP, LRF, LTDVEK, LTQTP, and VVVPPF, with molecular weights of 416 Da, 434 Da, 558 Da, 703 Da, and 656 Da, respectively. Its xanthine oxidase inhibition IC50 value is [not specified]. 50 The values ​​were 30.13 mM, 33.76 mM, 18.93 mM, 29.14 mM and 26.24 mM, respectively.

3. The uric acid-lowering peptide goat milk powder as described in claim 1, characterized in that, The uric acid-lowering peptide goat milk powder includes uric acid-lowering peptide goat milk powder for middle-aged and elderly goats and uric acid-lowering peptide goat milk powder for teenagers.

4. The uric acid-lowering peptide goat milk powder as described in claim 3, characterized in that, The uric acid-lowering peptide goat milk powder also includes nutritional fortifiers; in the uric acid-lowering peptide aged goat milk powder, the added nutritional fortifiers include one or more of the following: phytosterol esters, Acer truncatum seed oil, galactooligosaccharides, inulin, and fructooligosaccharides. The nutritional fortifiers added to the uric acid-lowering peptide juvenile goat milk powder include one or more of lutein, lutein ester, DHA, phosphatidylserine, isomaltooligosaccharide, xylooligosaccharide, and stachyose.

5. A method for preparing uric acid-lowering peptide goat milk powder, characterized in that, Includes the following steps: After sterilization and cooling, goat milk is mixed with a compound protease, kept warm for enzymatic hydrolysis, and then heated to inactivate the enzyme to obtain uric acid-lowering peptide liquid goat milk. With or without the addition of nutritional fortifiers, the uric acid-lowering peptide liquid goat milk is concentrated and then spray-dried to obtain uric acid-lowering peptide goat milk powder.

6. The preparation method according to claim 5, characterized in that, The complex protease is neutral protease N and neutral protease X; The addition amount of both neutral protease N and neutral protease X is 4000 U / g-6000 U / g; When two proteases are added, the ratio of the enzyme activities of neutral protease N to neutral protease X is 1:

1.

7. The preparation method according to claim 5, characterized in that, The enzymatic hydrolysis conditions are: 45℃-55℃ for 2-3 hours.

8. The preparation method according to claim 5, characterized in that, The goat milk is either fresh goat milk or reconstituted goat milk. The preparation method of reconstituted goat milk includes: mixing water and goat milk powder to obtain goat milk with a mass percentage of 11.5%-12.5%, which is the reconstituted goat milk. Fresh or reconstituted goat milk is sterilized at 95°C for 5 minutes or at 90°C for 10 minutes and then cooled to 45°C-55°C.

9. The preparation method according to claim 5, characterized in that, The xanthine oxidase inhibition rate of the urate-lowering peptide liquid goat milk was 71.67±1.32%~83.64±0.98%.

10. The use of the urate-lowering peptide as described in claim 1 or 2 in the preparation of dairy products or pharmaceuticals that help maintain healthy uric acid levels.

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