A synbiotic fermented donkey milk beverage and a method for preparing the same

Abstract

The application relates to the field of food processing and discloses a synbiotic fermented donkey milk beverage and a preparation method thereof. The method takes freeze-dried donkey milk powder as raw material, adds water to a mass fraction of 12% for re-dissolution, adds a compound probiotic accounting for 4% of the total mass of the donkey milk liquid, uniformly mixes, homogenizes at 20 MPa for 40 s, sterilizes at 95 DEG C for 10 min, inoculates 3% mixed probiotic powder after the material liquid is cooled to 42 DEG C, and ferments at 42 DEG C for 7 h to obtain a fermented milk base. The base is diluted with distilled water at a mass ratio of 1:3, 6% sucrose and 0.4% food stabilizer are added and uniformly mixed, the mixture is subjected to secondary homogenization at 20 MPa and secondary sterilization at 95 DEG C for 15 min, and then the mixture is cooled to room temperature to obtain the synbiotic fermented donkey milk beverage. The application solves the problems of easy stratification and sedimentation, prominent fishy and rancid odor of natural donkey milk in the prior art, simultaneously makes up for the defects of low digestion utilization rate and insufficient functional activity of the product, and improves the high added-value industrial utilization level of donkey milk resources.

Description

Technical Field

[0001] This invention relates to the field of food processing, and in particular to a biosimilar fermented donkey milk beverage and its preparation method. Background Technology

[0002] Donkey farming has now formed a large-scale, standardized industry. The traditional donkey industry chain has long focused on two core areas: donkey meat consumption and the production of donkey hide gelatin (Ejiao). Donkey milk has largely been wasted as a byproduct of farming, resulting in a consistently low level of comprehensive industrial utilization. However, with the upgrading of domestic consumption of specialty dairy products and the popularization of healthy eating concepts, the market demand for specialty dairy products with rich nutrition and physiological activity continues to rise. Donkey milk, with its high proportion of whey protein, natural organic selenium, rich unsaturated fatty acid content, low cholesterol, calcium-to-phosphorus ratio close to that of breast milk, and naturally containing various endogenous physiologically active substances, is gradually becoming a core raw material for extending the entire donkey industry chain and improving industrial economic benefits. Its potential for developing functional deep-processed products is enormous.

[0003] However, the current industrialization of donkey milk deep processing is severely constrained by the inherent physicochemical properties of the raw materials. Firstly, the casein micelle structure of natural donkey milk is loose and lacks the ability to spontaneously form gels. Directly blending it into beverages easily leads to problems such as protein sedimentation, fat floating, and system stratification. Conventional single stabilizers are insufficient to improve the stability of the protein network at the molecular level, resulting in inadequate product quality stability during storage. Secondly, donkey milk raw materials contain volatile nitrogenous components and short-chain fatty acids, resulting in a prominent fishy and gamey odor. Simple heat treatment or single-strain fermentation cannot completely remove these unpleasant flavors, significantly reducing the sensory palatability and market acceptance of the product. Furthermore, the endogenous antioxidants in donkey milk have poor thermal stability, and their activity is significantly lost during processing and sterilization. Existing deep-processed donkey milk products are mostly based on raw milk and simply blended beverages, resulting in a limited product range and insufficient development of functional activities, making it difficult to fully realize the nutritional and functional value of donkey milk.

[0004] Existing research indicates that combining prebiotics such as inulin, galactooligosaccharides, and fructooligosaccharides with probiotics to form a synbiotic system for milk fermentation can, to some extent, optimize product rheological properties, enhance antioxidant activity, and improve sensory quality. Prebiotics can provide fermentation carbon sources for probiotics and participate in the cross-linking modification of milk proteins, assisting in optimizing gel structure; their synergistic effects are significantly superior to those of using prebiotics or probiotics alone. However, there are few publicly reported systematic studies on the synergistic fermentation of donkey milk using a combination of three prebiotics and multiple strains. Current technologies cannot simultaneously overcome the multiple industrialization challenges of poor stability, undesirable flavor quality, and weak functionality in donkey milk beverage systems, hindering the high-value-added industrial development and application promotion of donkey milk resources. Summary of the Invention

[0005] The present invention aims to provide a biosimilar fermented donkey milk beverage and its preparation method, in order to solve the problems of easy stratification and sedimentation and prominent fishy and muttony odor in the processing of natural donkey milk in the prior art, while making up for the defects of low product digestibility and insufficient functional activity, and improving the level of high-value industrial utilization of donkey milk resources.

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

[0007] The present invention provides a method for preparing a biosimilar fermented donkey milk beverage:

[0008] S1. Raw material reconstitution and prebiotic preparation: Reconstitute freeze-dried donkey milk powder with water to obtain donkey milk liquid, add compound prebiotics to the donkey milk liquid, stir until completely dissolved and mixed to obtain a mixed liquid;

[0009] S2, One-time homogenization sterilization and constant temperature fermentation: The mixed liquid is subjected to one homogenization treatment and one sterilization treatment in sequence. After sterilization, the liquid is cooled to the appropriate inoculation temperature, mixed probiotic powder is added to the liquid, sealed and fermented at a constant temperature to obtain fermented milk base.

[0010] S3. Base material dilution and auxiliary material preparation: The fermented milk base material is mixed and diluted with distilled water. Sucrose and food stabilizer are added to the diluted solution and stirred until well mixed to obtain the prepared solution.

[0011] S4. Secondary homogenization, sterilization and cooling of the finished product: The prepared liquid is subjected to secondary homogenization and secondary sterilization in sequence. After sterilization, it is cooled to room temperature to obtain the finished product of Biostime fermented donkey milk beverage.

[0012] Furthermore, in step S1, the reconstitution mass fraction of the freeze-dried donkey milk powder is 12%; the total amount of the compound prebiotic added is 4% of the total mass of the donkey milk, and the compound prebiotic is composed of inulin, galactooligosaccharide, and fructooligosaccharide in a mass ratio of 1:1:1.

[0013] Furthermore, in step S2, the pressure of the first homogenization treatment is 20 MPa, and the homogenization time is 40 s; the temperature of the first sterilization treatment is 95 ℃, and the heat preservation sterilization time is 10 min.

[0014] Furthermore, in step S2, the total inoculation amount of the mixed probiotic powder is 3% of the total mass of the liquid, and the mixed probiotic powder is composed of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus rhamnosus in a mass ratio of 1:1:1:1; the activity of each strain is... CFU / g.

[0015] Furthermore, in step S2, the temperature for constant temperature fermentation is 42°C, and the fermentation time is 7 hours.

[0016] Furthermore, in step S3, the mass ratio of the fermented milk base to distilled water is 1:3; the amount of sucrose added is 6% of the total mass of the diluted liquid; and the amount of food stabilizer added is 0.4% of the total mass of the diluted liquid.

[0017] Furthermore, in step S4, the pressure of the secondary homogenization process is 20 MPa, and the homogenization time is 40 s; the temperature of the secondary sterilization process is 95 ℃, and the heat preservation and sterilization time is 15 min.

[0018] The present invention provides a synbiotic fermented donkey milk beverage, characterized in that it is prepared by the aforementioned method for preparing a synbiotic fermented donkey milk beverage.

[0019] The beneficial effects of this invention are as follows: This invention employs a compound prebiotic and multi-strain probiotic system to construct a synbiotic fermentation system, combined with a two-stage homogenization process to prepare fermented donkey milk beverages. Long- and short-chain prebiotics synergistically provide a gradient carbon source for the probiotics, promoting strain proliferation and metabolism. Simultaneously, cross-linking with milk proteins optimizes the gel structure. Combined with homogenization to refine system particles, this significantly improves the stability of the product system and solves the problem of easy stratification and sedimentation in donkey milk beverages. The multi-strain system synergistically metabolizes and decomposes the fishy-smelling substances in donkey milk, weakening its inherent fishy and pungent odor, while simultaneously hydrolyzing large-molecule milk proteins, improving product digestibility and utilization. The active substances generated by probiotic metabolism synergistically enhance the product's antioxidant function with the endogenous active components of donkey milk. A gentle, temperature-controlled sterilization process reduces heat loss of functional components. This process has stable and controllable parameters, readily available commercial raw materials, and is suitable for large-scale production. It can effectively improve the resource utilization rate of donkey milk by-products and contribute to the high-value-added industrial development of donkey milk resources. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0021] Figure 1 The process flow diagram for preparing the Biostime fermented donkey milk beverage in this embodiment is shown.

[0022] Figure 2 The images show a comparison of the appearance of fermented donkey milk beverages prepared with different prebiotics in this embodiment, including a blank control group and four groups of prebiotic samples.

[0023] Figure 3 This was used to characterize the effects of different prebiotics on the pH and titratable acidity of fermented donkey milk beverages. Figure 3 A and 3B represent the comparison results of pH and titration acidity for each group.

[0024] Figure 4 This is a comparison diagram of the rheological properties of different prebiotic-fermented donkey milk beverages in this embodiment. Figure 4 A and 4B are the test results of elastic modulus G′ and viscous modulus G″, respectively.

[0025] Figure 5 Compare the centrifugation sedimentation rate of each group of prebiotic-treated samples in this embodiment.

[0026] Figure 6 This is a comparison chart of the degree of protein hydrolysis before and after in vitro simulated digestion of different prebiotic fermented donkey milk beverages in this embodiment. The difference in protein hydrolysis level before and after digestion is reflected by the concentration of free amino acids.

[0027] Figure 7 The effects of different prebiotics on the antioxidant activity of fermented donkey milk beverages were investigated. Figure 7 A~7D represent the intergroup comparison results for DPPH scavenging rate, ABTS scavenging rate, hydroxyl radical scavenging rate, and reducing power, respectively. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or end that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or ends.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] Existing research indicates that combining prebiotics such as inulin, galactooligosaccharides, and fructooligosaccharides with probiotics to form a synbiotic system for milk fermentation can, to some extent, optimize product rheological properties, enhance antioxidant activity, and improve sensory quality. Prebiotics can provide fermentation carbon sources for probiotics and participate in the cross-linking modification of milk proteins, assisting in optimizing gel structure; their synergistic effects are significantly superior to those of using prebiotics or probiotics alone. However, there are few publicly reported systematic studies on the synergistic fermentation of donkey milk using a combination of three prebiotics and multiple strains. Current technologies cannot simultaneously overcome the multiple industrialization challenges of poor stability, undesirable flavor quality, and weak functionality in donkey milk beverage systems, hindering the high-value-added industrial development and application promotion of donkey milk resources.

[0032] The present invention aims to provide a biosimilar fermented donkey milk beverage and its preparation method, in order to solve the problems of easy stratification and sedimentation and prominent fishy and muttony odor in the processing of natural donkey milk in the prior art, while making up for the defects of low product digestibility and insufficient functional activity, and improving the level of high-value industrial utilization of donkey milk resources.

[0033] Example 1

[0034] like Figure 1 As shown in the figure, a specific embodiment of the present invention provides a synbiotic fermented donkey milk beverage and its preparation method. This embodiment is the optimal embodiment of the present invention. The specific steps for preparing the synbiotic fermented donkey milk beverage are as follows:

[0035] S1. Raw material reconstitution and prebiotic preparation: Reconstitute freeze-dried donkey milk powder with water at a mass fraction of 12%, and preheat in a water bath to 45°C to obtain donkey milk liquid; add compound prebiotics at a mass fraction of 4% of the total mass of the donkey milk liquid. The compound prebiotics are composed of inulin, galactooligosaccharides (GOS), and fructooligosaccharides (FOS) in a mass ratio of 1:1:1. After adding the materials, continue stirring until all the prebiotics are dissolved and the system is mixed evenly to obtain a mixed liquid.

[0036] S2. Homogenization and Sterilization followed by Constant Temperature Fermentation: The mixed liquid is passed into a high-pressure homogenizer and homogenized at 20 MPa for 40 seconds to break up fat globules and loose casein aggregates in the system. After homogenization, the liquid is transferred to a water bath sterilizer and sterilized at 95°C for 10 minutes to kill contaminants and pathogens in the raw materials. After sterilization, the liquid is cooled to a suitable inoculation temperature of 42°C in an ice-water bath. 3% of the total mass of the cooled liquid is then inoculated with a mixed probiotic powder, which consists of *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, *Lactobacillus plantarum*, and *Lactobacillus rhamnosus* in a mass ratio of 1:1:1:1, with each strain having a viability of 10¹. 0 CFU / g; after inoculation and thorough mixing, the liquid was sealed and fermented in a constant temperature environment of 42℃ for 7 hours to obtain fermented milk base.

[0037] S3. Base material dilution and auxiliary material preparation: The fermented milk base material is mixed and diluted with distilled water at a mass ratio of 1:3 to adjust the solid content and viscosity of the system; 6% sucrose and 0.4% food stabilizer are added to the diluted liquid, and the mixture is stirred until the materials are completely dispersed and uniform to obtain the prepared liquid.

[0038] S4. Secondary homogenization, sterilization, and cooling of the finished product: The prepared liquid is subjected to secondary homogenization at 20MPa pressure for 40s to refine the protein flocculent particles and fat microparticles in the system; after homogenization, it is kept at 95℃ for secondary sterilization for 15min to kill exogenous bacteria mixed in during the preparation process; after sterilization, the liquid is naturally cooled to room temperature to obtain the Synbiotic fermented donkey milk beverage finished product.

[0039] Example 2 Example 4 (Single prebiotic comparison group)

[0040] The operation steps and parameters of this series of embodiments are completely the same as those of Embodiment 1, the only difference being the type of prebiotic added in step S1:

[0041] Example 2: Only 4% inulin by total mass was added, without adding galactooligosaccharides or fructooligosaccharides;

[0042] Example 3: Only 4% galactooligosaccharides by total mass were added, without inulin or fructooligosaccharides;

[0043] Example 4: Only 4% of the total mass of fructooligosaccharides were added, without inulin or galactooligosaccharides.

[0044] Example 5: Add 4% by total mass of inulin: galactooligosaccharide: fructooligosaccharide in a 1:1:1 ratio.

[0045] Example 6: The reconstitution mass fraction is 10%;

[0046] Example 7: The reconstitution mass fraction was 14%;

[0047] Example 8: The reconstitution mass fraction was 16%.

[0048] Example 9 Example 12 (Gradual group of sucrose addition)

[0049] The operation steps and parameters of this series of embodiments are completely the same as those of Embodiment 1, the only difference being the mass fraction of sucrose added in step S3:

[0050] Example 9: Sucrose addition amount is 3%;

[0051] Example 10: Sucrose addition amount was 4%;

[0052] Example 11: Sucrose addition amount is 5%;

[0053] Example 12: Sucrose addition was 7%.

[0054] Example 13 Example 16 (Probiotic Inoculation Gradient Group)

[0055] The operating steps and parameters of this series of embodiments are completely consistent with those of Embodiment 1, the only difference being the total inoculation mass fraction of the probiotic powder in step S2:

[0056] Example 13: Inoculation amount was 2%;

[0057] Example 14: Inoculation amount was 4%;

[0058] Example 15: Inoculation amount was 5%;

[0059] Example 16: Inoculation amount was 6%.

[0060] Example 17 Example 20 (Fermentation Temperature Gradient Group)

[0061] The operating steps and parameters of this series of embodiments are completely consistent with those of Embodiment 1, the only difference being the temperature of the isothermal fermentation in step S2:

[0062] Example 17: Fermentation temperature was 36℃;

[0063] Example 18: Fermentation temperature was 38℃;

[0064] Example 19: Fermentation temperature was 40℃;

[0065] Example 20: Fermentation temperature was 44℃.

[0066] Comparative Example 1 (Blank Control Group)

[0067] The operation steps and parameters of this comparative example are completely consistent with those of Example 1, except that no prebiotics are added in step S1, while the other process parameters remain unchanged.

[0068] Test Example 1: Product Performance Verification Test

[0069] Take the above Example 1 Example 4: The fermented donkey milk beverage sample prepared in Comparative Example 1 was subjected to performance tests on system appearance, pH and acidity, rheological properties, centrifugal sedimentation rate, in vitro digestibility, and antioxidant activity to verify the effectiveness of the technical solution of the present invention. The test results are described below in conjunction with the accompanying drawings:

[0070] 1. Comparison of sample appearance

[0071] like Figure 2As shown, the control group sample system showed obvious stratification, with a large amount of whey precipitate and significant sedimentation at the bottom; the stratification of the single prebiotic group sample was alleviated, but a small amount of whey precipitate and sedimentation still existed; the compound prebiotic group sample system of Example 1 was uniform and delicate, with no obvious stratification and sedimentation, and the overall state was stable, with the best sensory performance.

[0072] 2. pH and acidity detection

[0073] The pH value of each sample was directly measured using a pH meter, and the titration acidity of the samples was determined by titration with a 0.1 mol / L NaOH standard solution. The results are as follows: Figure 3 As shown, where Figure 3 A represents the pH value comparison results. Figure 3 B represents the comparison results of titration acidity.

[0074] The results show that adding prebiotics can promote acid production by probiotics and lower the pH of the system, with significant differences in the effects of different prebiotics. The highest pH in the control group (Comparative Example 1) was 4.52. The pH differences between the inulin group (Example 2), the GOS group (Example 3), and the FOS group (Example 4) and the control group were not significant. The compound group (Example 1) showed the largest pH decrease, dropping to 4.44. Regarding acidity, the control group was only 37.2°T, while the inulin group, GOS group, and FOS group were 38°T, 38.8°T, and 39.4°T respectively. The compound group had the highest acidity, reaching 40°T. This is because the combination of long- and short-chain prebiotics can synergistically provide energy, balancing rapid acid production with continuous fermentation, thus improving the system's acid production efficiency.

[0075] 3. Rheological property testing

[0076] The test was performed using a rotational rheometer, with a parallel plate spacing of 2 mm and a test temperature of 25℃, at a shear rate of... The elastic modulus G′ and viscous modulus G″ of the sample were measured by scanning within the range, and the results are as follows: Figure 3 As shown, where Figure 4 A represents the comparison results of elastic modulus. Figure 4 B represents the comparison results of viscous modulus.

[0077] The results show that G′ was consistently greater than G″ across all samples within the full frequency range, indicating that all fermented milk beverages exhibited typical gel-like systems with solid-like behavior. The G′ and G″ values ​​of the compound prebiotic group were consistently higher than those of the single prebiotic group and the control group. For example, at 10 Hz, the G′ of the compound prebiotic group reached 230 Pa, while the GOS, FOS, and inulin groups were only 178 Pa, 138 Pa, and 182 Pa, respectively. This is because the compound prebiotics can enhance the acidity of the system, strengthening the gelation process of milk proteins; simultaneously, the diversified molecular structure can form more complex intermolecular interactions with milk proteins, such as hydrogen bonds, van der Waals forces, and hydrophobic interactions, thereby constructing a denser protein network structure and improving the rheological properties of the system.

[0078] 3. Centrifugal sedimentation rate detection

[0079] Accurately weigh a certain mass of sample into a centrifuge tube, set the centrifugation speed to 3500 r / min, and centrifuge for 15 min. After centrifugation, discard the supernatant, invert the centrifuge tube to drain any residual liquid from the tube wall, weigh the total mass of the centrifuge tube and the precipitate, and calculate the centrifugation sedimentation rate using the following formula:

[0080] ×100%;

[0081] In the formula: The initial mass of the sample is expressed in grams. The mass of the empty centrifuge tube is in grams. The total mass of the centrifuge tube and the precipitate is expressed in grams.

[0082] Test results as follows Figure 5 As shown, the centrifugal sedimentation rate of the control group was 7.25%, significantly higher than that of the other groups, indicating that the particles in the system were prone to aggregation and had the strongest sedimentation tendency, resulting in the worst stability. The centrifugal sedimentation rate of the inulin group in Example 2 decreased to 4.95%, indicating that the addition of inulin can effectively inhibit the aggregation of milk fat globules and milk protein particles. The centrifugal sedimentation rates of the GOS group in Example 3 and the FOS group in Example 4 increased to 6.02% and decreased to 5.35%, respectively, indicating that the steric hindrance between the two prebiotics and milk proteins was weak, and the stabilizing effect was limited. The sedimentation rate of the compound prebiotic group in Example 1 decreased significantly to 4.32%, indicating that the compound prebiotic inhibited particle sedimentation and optimized the gel structure through a synergistic mechanism, resulting in the most significant improvement in system stability.

[0083] 5. In vitro digestive characteristics detection

[0084] Samples were treated using an in vitro simulated gastrointestinal digestion test, and the degree of protein hydrolysis was characterized by the free amino group content using the OPA method. The OPA reagent was prepared as follows: 7.620 g of sodium tetraborate decahydrate and 200 mg of SDS were dissolved in 150 mL of pure water, 160 mg of OPA dissolved in 4 mL of ethanol was added, and 200 μL of β-mercaptoethanol was added to bring the volume to 200 mL. For detection, 3 mL of OPA reagent was mixed with 0.4 mL of sample, allowed to stand for 2 min, and pure water was used as a blank control. The absorbance was measured at a wavelength of 340 nm. The results are as follows: Figure 6 As shown.

[0085] The results showed that, compared with the control group, the free amino acid concentration in fermented donkey milk with added prebiotics was significantly increased before and after simulated digestion (P<0.05), and the compound prebiotic group in Example 1 showed the best improvement. This is because inulin, galactooligosaccharides, and fructooligosaccharides can promote the proliferation of probiotics and enhance their extracellular protease activity; the synergistic effect of the compound prebiotics resulted in the strongest probiotic activity, leading to the hydrolysis of more polypeptide bonds in milk proteins, generating more free amino acids, thereby improving the digestibility of the product.

[0086] 6. Antioxidant activity detection

[0087] The four indicators of DPPH radical scavenging ability, ABTS radical scavenging ability, hydroxyl radical scavenging ability, and reducing power of the samples were tested respectively. The results are shown in Figure 7. Figure 7 A represents the DPPH clearance rate result. Figure 7 B represents the ABTS clearance rate result. Figure 7 C represents the hydroxyl radical scavenging rate. Figure 7 D represents the result of the reducing force.

[0088] (1) DPPH free radical scavenging ability: The pH of the sample was adjusted to 4.6 with 1.0 mol / L NaOH solution, and centrifuged at 4℃ and 10000 r / min for 15 min. The supernatant was filtered through a 0.45 μm aqueous microporous membrane to obtain the water-soluble extract (WSE). 800 μL of DPPH reagent (0.1 mmol / L DPPH dissolved in 95% methanol) was added to 200 μL of the sample extract. After thorough shaking and mixing, the sample was allowed to stand at room temperature in the dark for 30 min. Methanol was used as a blank control. The absorbance was measured at 517 nm and the scavenging rate was calculated.

[0089] ;

[0090] The results showed that the clearance rate of all prebiotic groups was better than that of the blank control, with the inulin group achieving the best clearance rate of 98.6%. This is because inulin, after being hydrolyzed by probiotics, promotes the decomposition of donkey milk protein to produce a large number of fat-soluble antioxidant peptides. The GOS group had the second best effect, while the FOS group and the compound group had weaker effects. In the compound system, multiple carbon sources competed for energy, inhibiting the generation of functional peptides and reducing the level of fat-soluble antioxidants, but it was still better than the control group.

[0091] (2) ABTS free radical scavenging ability: 7.4 mmol / L ABTS and 2.6 mmol / L potassium persulfate solution were mixed in equal volumes (molar ratio 1:0.35) and reacted at room temperature in the dark for 12 h to obtain a stock solution; 1 mL of the stock solution was mixed with 50 Prepare a fresh working solution using 60 mL of methanol buffer solution. After equilibration at 30 °C, measure the absorbance at a wavelength of 734 nm. Add 2 mL of the working solution to 20 μL of the sample to be tested. After reacting at 30 °C for 6 min, measure the absorbance. Use 20 μL of double-distilled water as a blank control to calculate the clearance rate.

[0092] ;

[0093] The results showed that the clearance rate in the control group was 43.9%, significantly higher than that in each prebiotic group; the clearance rates in the inulin group, GOS group, FOS group, and compound group were 41.8%, 36.7%, 35.8%, and 33.9%, respectively. Prebiotics are preferentially utilized by probiotics as a carbon source, delaying milk matrix fermentation, reducing the degree of protein hydrolysis, and decreasing the yield of water-soluble antioxidant peptides; the compound prebiotics intensified the competition for carbon sources, thus exhibiting the weakest water-soluble free radical scavenging ability.

[0094] (3) Hydroxyl radical scavenging ability: Accurately measure 1 mL of sample and place it in a 50 mL beaker. Add 9 mL of 95% ethanol and mix thoroughly. Take 4 mL of the above mixture and transfer it to a 25 mL test tube. Add 0.5 mL of 8.8 mmol / L H2O2 solution, 0.5 mL of 9 mmol / L FeSO4 solution and 0.5 mL of 9 mmol / L salicylic acid solution in sequence. Shake and mix well. React in a 37℃ water bath for 30 min. Measure the absorbance at a wavelength of 510 nm and calculate the scavenging rate.

[0095] ;

[0096] The results showed that the clearance rate of all prebiotic groups was higher than that of the control group (75.3%); the clearance rates of the inulin group, GOS group, and FOS group were 83.3%, 87.9%, and 84.2%, respectively, with the compound group reaching the highest at 92.7%. Prebiotics can stimulate probiotics to synthesize extracellular polysaccharides, short-chain fatty acids, and antioxidant peptides, and work synergistically with endogenous active proteins in donkey milk. They enhance the clearance effect through hydrogen donation and metal ion chelation, and the synergistic effect of the compound is superior to that of a single prebiotic.

[0097] (4) Reducing power test: Take 1 mL of sample and place it in a 50 mL beaker. Add 9 mL of 95% ethanol and mix well. Transfer 2 mL of the mixture and add 2 mL of 1% potassium ferricyanide solution and pH 6.6, 0.2 mol / L phosphate buffer solution in sequence. Incubate at 50 °C for 20 min. Then add 2 mL of 10% trichloroacetic acid solution and mix well. Centrifuge at 3000 r / min for 10 min. Take 2 mL of supernatant and add 2 mL of deionized water and 0.4 mL of 0.1% ferric chloride solution respectively. After standing at room temperature for 10 min, measure the absorbance at 700 nm wavelength. The higher the absorbance, the stronger the reducing power.

[0098] The results showed that the absorbance corresponding to the reducing power of each group was higher than that of the control group (36.8%). The absorbance values ​​for the inulin group, GOS group, and FOS group were 42.5%, 41.6%, and 44.1%, respectively, with the compound group reaching the highest at 48.8%, and the difference was statistically significant (P<0.05). Prebiotics promote the proliferation of probiotics and enhance enzyme activity, generating a large number of antioxidant metabolites. Simultaneously, prebiotics themselves possess reducing activity, and the synergistic effect after compounding optimizes the fermentation microenvironment, promotes the release of active peptides from protein hydrolysis, and significantly enhances the overall reducing and antioxidant capacity.

[0099] In summary, this invention employs a synbiotic fermentation system constructed from compound prebiotics and multiple strains, which can simultaneously improve the system stability, sensory flavor, digestibility, and antioxidant activity of donkey milk beverages. This effectively addresses the industrialization pain points of natural donkey milk processing, such as easy stratification, poor sensory quality, and weak functionality. The entire process parameters are stable and controllable, and the raw materials are readily available for commercialization, making it suitable for large-scale mass production and contributing to the high-value-added industrial development of donkey milk resources.

[0100] The beneficial effects of this invention are as follows: This invention employs a compound prebiotic and multi-strain probiotic system to construct a synbiotic fermentation system, combined with a two-stage homogenization process to prepare fermented donkey milk beverages. Long- and short-chain prebiotics synergistically provide a gradient carbon source for the probiotics, promoting strain proliferation and metabolism. Simultaneously, cross-linking with milk proteins optimizes the gel structure. Combined with homogenization to refine system particles, this significantly improves the stability of the product system and solves the problem of easy stratification and sedimentation in donkey milk beverages. The multi-strain system synergistically metabolizes and decomposes the fishy-smelling substances in donkey milk, weakening its inherent fishy and pungent odor, while simultaneously hydrolyzing large-molecule milk proteins, improving product digestibility and utilization. The active substances generated by probiotic metabolism synergistically enhance the product's antioxidant function with the endogenous active components of donkey milk. A gentle, temperature-controlled sterilization process reduces heat loss of functional components. This process has stable and controllable parameters, readily available commercial raw materials, and is suitable for large-scale production. It can effectively improve the resource utilization rate of donkey milk by-products and contribute to the high-value-added industrial development of donkey milk resources.

[0101] The above descriptions are merely embodiments of the present invention. Commonly known technical solutions or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a biosimilar fermented donkey milk beverage, characterized in that, The method includes: S1. Raw material reconstitution and prebiotic preparation: Reconstitute freeze-dried donkey milk powder with water to obtain donkey milk liquid, add compound prebiotics to the donkey milk liquid, stir until completely dissolved and mixed to obtain a mixed liquid; S2, One-time homogenization sterilization and constant temperature fermentation: The mixed liquid is subjected to one homogenization treatment and one sterilization treatment in sequence. After sterilization, the liquid is cooled to the appropriate inoculation temperature, mixed probiotic powder is added to the liquid, sealed and fermented at a constant temperature to obtain fermented milk base. S3. Base material dilution and auxiliary material preparation: The fermented milk base material is mixed and diluted with distilled water. Sucrose and food stabilizer are added to the diluted solution and stirred until well mixed to obtain the prepared solution. S4. Secondary homogenization, sterilization and cooling of the finished product: The prepared liquid is subjected to secondary homogenization and secondary sterilization in sequence. After sterilization, it is cooled to room temperature to obtain the finished product of Biostime fermented donkey milk beverage.

2. The method for preparing a synbiotic fermented donkey milk beverage according to claim 1, characterized in that: In step S1, the reconstitution mass fraction of the freeze-dried donkey milk powder is 12%; the total amount of the compound prebiotic added is 4% of the total mass of the donkey milk, and the compound prebiotic is composed of inulin, galactooligosaccharide and fructooligosaccharide in a mass ratio of 1:1:

1.

3. The method for preparing a synbiotic fermented donkey milk beverage according to claim 1, characterized in that: In step S2, the pressure of the first homogenization process is 20 MPa, and the homogenization time is 40 s; the temperature of the first sterilization process is 95 ℃, and the heat preservation and sterilization time is 10 min.

4. The method for preparing a synbiotic fermented donkey milk beverage according to claim 1, characterized in that: In step S2, the total inoculation amount of the mixed probiotic powder is 3% of the total mass of the liquid. The mixed probiotic powder is composed of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus rhamnosus in a mass ratio of 1:1:1:1; the activity of each strain is [missing information]. CFU / g.

5. The method for preparing a synbiotic fermented donkey milk beverage according to claim 4, characterized in that: In step S2, the temperature for constant temperature fermentation is 42°C, and the fermentation time is 7 hours.

6. The method for preparing a synbiotic fermented donkey milk beverage according to claim 1, characterized in that: In step S3, the mass ratio of the fermented milk base to distilled water is 1:3; the amount of sucrose added is 6% of the total mass of the diluted liquid; and the amount of food stabilizer added is 0.4% of the total mass of the diluted liquid.

7. The method for preparing a synbiotic fermented donkey milk beverage according to claim 1, characterized in that: In step S4, the pressure of the secondary homogenization process is 20 MPa, and the homogenization time is 40 s; the temperature of the secondary sterilization process is 95 ℃, and the heat preservation and sterilization time is 15 min.

8. A biosimilar fermented donkey milk beverage, characterized in that, The beverage is prepared using the method described in any one of claims 1 to 7 for the preparation of a synbiotic fermented donkey milk beverage.

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