A method for extracting sialic acid oligosaccharides from yak milk

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a method for extracting sialic acid oligosaccharides from yak milk, characterized in that it comprises the following steps: step 1, defatting and deproteinizing yak milk to obtain crude yak milk oligosaccharides; step 2, removing glucose from the crude yak milk oligosaccharides to obtain a fermentation broth; step 3, filtering the fermentation broth to remove polyvalent anions and cations from the filtrate, and drying to obtain sialic acid oligosaccharides. The extraction method of the present invention is simple, the process is stable, the operability is strong, the production cost is low, the product quality is stable, the preparation process meets the requirements of industrial-scale production and processing, and can play a positive role in promoting the scale development and utilization of low-lactose yak milk and its products.

Description

Technical Field

[0001] The invention relates to a method for extracting oligosaccharides, in particular to sialic acid oligosaccharides extracted from yak milk and a method thereof, belonging to the technical field of bioengineering. Background Art

[0002] Yak milk is rich in protein, fat, carbohydrates and other nutrients, and has extremely high nutritional value. It is an important source of nutrition for herders. Yak milk is rich in oligosaccharides (also known as "oligosaccharides"), which have multiple biological activities such as anti-hypertension, antioxidant, anti-tumor, and anti-cancer. Oligosaccharides isolated from various milk sources are divided into two categories: sialylated and non-sialylated. These two types of oligosaccharides have different biological activities. Fucosylated structures have been found in yak milk, which promote the growth of bifidobacteria, thereby promoting intestinal health and playing an important regulatory role in the intestinal flora of newborns.

[0003] Sialyl oligosaccharide (SL) is an acidic human milk oligosaccharide. It can be divided into 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) according to the position of sialic acid and lactose. In recent years, scholars have been exploring new oligosaccharides in yak milk. Singh et al. used gel filtration high performance liquid chromatography and capillary electrophoresis to separate two new types of milk oligosaccharides, namely groniose and vacosose, from yak milk, and elucidated the structure of purified milk oligosaccharides. At the same time, the optimization of its geometric structure is helpful to further refine and quantify the oligosaccharide compounds in yak milk and reveal the potential impact of yak milk on human health. However, there is currently no extraction method for yak milk oligosaccharides to make its extraction efficiency higher, among which oligosaccharides also include the improvement of the purity of sialylated oligosaccharides and fucosylated neutral oligosaccharides. The present invention obtains the optimal factors for removing lactose and glucose by combining enzyme hydrolysis and yeast fermentation with response surface optimization experiments, which greatly improves the problem of lactose intolerance in some people after drinking yak milk. Summary of the invention

[0004] In order to achieve these purposes and other advantages according to the present invention, the technical scheme of the present invention is as follows: A method for extracting sialic acid oligosaccharides from yak milk comprises the following steps:

[0005] Step 1, defatting and deproteinizing yak milk to obtain crude yak milk oligosaccharide;

[0006] Step 2, removing glucose from crude yak milk oligosaccharide to obtain a fermentation liquid;

[0007] Step 3: Filter the fermentation broth to remove polyvalent anions and cations in the filtrate, and dry to obtain sialic acid oligosaccharides.

[0008] In the above-mentioned method for extracting sialic acid oligosaccharides from yak milk, in step 1, the specific method of defatting and deproteinizing is: centrifuging the yak milk once to remove the fat on the upper layer and a small amount of protein at the bottom, taking out the middle layer and adding anhydrous ethanol to react, centrifuging again after the reaction is completed, taking the supernatant, and drying to obtain crude yak milk oligosaccharides.

[0009] The above-mentioned method for extracting sialic acid oligosaccharides from yak milk comprises the following conditions: centrifugation for 15 to 30 minutes at 8000 to 10000 rpm at 3 to 8°C, and centrifugation for 2 minutes at 4000×g at 3 to 8°C.

[0010] The above-mentioned method for extracting sialic acid oligosaccharides from yak milk has a volume ratio of middle layer: anhydrous ethanol = 1:2 to 1:6.

[0011] The above-mentioned method for extracting sialic acid oligosaccharides from yak milk has a reaction temperature of 0 to 4° C. and a reaction time of 2 to 12 hours.

[0012] In the above-mentioned method for extracting sialic acid oligosaccharides from yak milk, the drying is performed by using a vacuum concentrator for concentration and drying.

[0013] In the above-mentioned method for extracting sialic acid oligosaccharides from yak milk, in step three, a combination of anion and cation columns and gel columns is used to remove multivalent anions and cations.

[0014] The above-mentioned method for extracting sialic acid oligosaccharides from yak milk comprises desalting the retained concentrate by using a cation exchange resin and an anion exchange resin until the conductivity of the retained concentrate is less than 400 μs / cm.

[0015] In the above-mentioned method for extracting sialic acid oligosaccharides from yak milk, the anion exchange resin is a macroporous weakly basic anion exchange resin with a polyacrylic acid skeleton; and the cation exchange resin is a strongly acidic styrene-based cation exchange resin.

[0016] The present invention has at least the following beneficial effects:

[0017] The present invention optimizes the conditions for hydrolyzing lactose by enzymatic hydrolysis by using a single factor test, uses β-N-acetylglucosidase to hydrolyze lactose, and then separates and purifies defatted and deproteinized yak milk by using a Sephadex-G20 gel column, effectively removes the influencing substances lactose and glucose, and then performs qualitative and quantitative analysis on oligosaccharide isomers by optimizing the liquid phase conditions. The present invention adopts the above technical scheme, and combines membrane separation and enzymatic hydrolysis to prepare low-lactose yak milk, and the lactose removal rate can reach more than 90%. It gives full play to the respective technical advantages of membrane separation and enzymatic hydrolysis, and while removing lactose from yak milk, it completely retains the nutritional components, sensory quality and functional characteristics of yak milk. It has a simple preparation method, stable process, strong operability, low production cost, stable product quality, and a preparation process that meets the requirements of industrial-scale production and processing, and can play a positive role in promoting the scale development and utilization of low-lactose yak milk and its products. The technical problem to be solved by the present invention is to propose a solution with simple process, strong operability, low production cost and optimal oligosaccharide removal rate in yak milk, so as to solve the problem of lactose intolerance in some people after drinking yak milk.

[0018] The present invention conducts single factor investigation by processing samples respectively, and investigates three independent variables, namely, temperature, dilution multiple and yeast addition amount, wherein the temperature, dilution multiple and yeast addition amount are represented by A, B and C respectively;

[0019] Furthermore, on the basis of the single factor experiment, the Box-Behnken Design (BBD) central composite design was used to investigate the effects of three independent variables on the extraction yield, taking the glucose fermentation rate Y (%) as the response value;

[0020] Furthermore, regression analysis was performed on the response surface experimental results of glucose fermentation rate, and the regression equation was obtained after regression fitting of the three factors:

[0021] Y=90.116+2.5025A+1.85125B+2.83875C+0.3850AB-0.49AC+0.0975BC-14.02925A 2 -4.65675B 2 -8.04175C 2。

[0022] By comparing the ultrafiltration method and the chromatography column method, it was found that the chromatography column method was more effective in removing lactose and was more suitable for yak milk oligosaccharides, with a removal rate of 88.64-91.58%, while the ultrafiltration lactose removal rate was only 60.02-67.38%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The effect of temperature on glucose fermentation rate obtained in Example;

[0024] Figure 2 The effect of the dilution multiple on the glucose fermentation rate obtained in the embodiment is shown in FIG.

[0025] Figure 3 The effect of yeast addition amount on glucose fermentation rate obtained in Example;

[0026] Figure 4 The response surface diagram and contour diagram of the interaction between factor A and factor B on the glucose hydrolysis rate obtained in the embodiment;

[0027] Figure 5 The response surface diagram and contour diagram of the interaction between factor A and factor C on the glucose hydrolysis rate obtained in the embodiment;

[0028] Figure 6 The response surface diagram and contour diagram of the interaction between factor B and factor C on the glucose hydrolysis rate obtained in the embodiment;

[0029] Figure 7 The DPPH free radical scavenging experimental results of polysaccharides extracted by the extraction method of the present invention are shown;

[0030] Figure 8 The results of the superoxide anion free radical scavenging experiment of the polysaccharide extracted by the extraction method of the present invention are as follows;

[0031] Fig. 9 The ABTS free radicals of the polysaccharide extracted by the extraction method of the present invention are clear experimental results;

[0032] Fig.10 The hydroxyl radical clearing experimental results of the polysaccharide extracted by the extraction method of the present invention are shown in FIG.

[0033] Fig.11 Process flow chart obtained for the embodiment

[0034] Example 1: Defatting and deproteinizing yak milk to extract crude YMOs

[0035] Yak milk was centrifuged at 3-8°C and 8000-10000 rpm for 15-30 min to remove the fat on the upper layer and a small amount of protein on the bottom. The middle layer was taken out and added with chloroform / methanol solution in a volume ratio of 1:2-1:6 and mixed. The mixture was reacted at 0-4°C. After the reaction, the mixture was centrifuged at 4000 rpm for 15-30 min at 3-8°C to remove the protein and retain the supernatant. The supernatant was concentrated and dried by a vacuum concentrator to obtain the YMOs crude product. At this time, the protein removal rate was 87.159±1.62043%.

[0036] Comparative Example 1

[0037] Acetonitrile method for protein removal: yak milk is centrifuged at 3-8°C and 8000-10000rpm for 15-30min to remove the fat on the upper layer and a small amount of protein on the bottom, the middle layer is taken out and added with acetonitrile solution in a volume ratio of 1:2-1:6 and mixed, and the mixture is centrifuged at 10000rpm for 15-30min at room temperature to remove the protein and retain the supernatant, which is concentrated and dried by a vacuum concentrator to obtain the YMOs crude product. At this time, the protein removal rate is 69.2578±2.51261%.

[0038] Repeated freeze-thaw protein removal: Take yak milk and centrifuge it at 3-8℃ and 8000-10000rpm for 15-30min to remove the fat on the upper layer and a small amount of protein at the bottom. Place the skimmed yak milk sample in a -20--30℃ refrigerator and freeze-thaw it 5-10 times, then centrifuge it at 3-5℃ and 4000rpm for 15-30min. Centrifuge it several times to discard the precipitate. The protein removal rate was determined to be 70.7695±0.19809%.

[0039] NaCl method: take yak milk and centrifuge it at 3-8°C and 8000-10000rpm for 15-30min to remove the fat on the upper layer and a small amount of protein at the bottom. Under boiling water bath conditions, adjust the pH value of the skimmed human milk sample to 9.0-10.0, add 3-6% (m / v) NaCl while stirring, boil it for 15-30min after fully mixing, cool to room temperature, centrifuge it at 10000rpm for 8-15min, discard the precipitate, retain the supernatant, and obtain the YMOs crude product. At this time, the protein removal rate is 76.9852±1.16013.

[0040] Protease method: Take yak milk and centrifuge it at 3-8℃ and 8000-10000rpm for 15-30min to remove the upper fat and a small amount of protein at the bottom. Adjust the pH value of the skim milk sample to 6.0, take 50mL of skim milk sample, add 2-4% papain, the enzyme activity is 10000U / g, enzymolysis in a water bath at 45-55℃, and then use boiling water bath to inactivate the enzyme for 7-12min after 1h, cool to room temperature, and centrifuge at 10000rpm for 10min to obtain YMOs crude product. At this time, the protein removal rate is 75.2807±1.23385. Example 2: The YMOs crude product is treated with Sephadex-G20 gel column to remove lactose to obtain YMOs sample

[0041] The crude YMOs product is first decomposed by lactase and then purified by Sephadex-G20 gel column to remove lactose. First, the yak milk sample is placed in a 50-55°C water bath for 15-20 minutes, and when the temperature drops to about 25-30°C, β-N-acetyl hexosaminidase is added thereto under aseptic conditions, and the sample is placed in a shaker (100-150 rpm) for constant temperature treatment; the hydrolyzate after the reaction is placed in boiling water to inactivate the enzyme for 10-15 minutes, and then the lactose content in the hydrolyzate is determined by direct titration, and the hydrolysis time range is selected to be 3-5 hours; the hydrolysis temperature range is selected to be 40°C-60°C; and the pH range is selected to decompose lactose at 3-5. Then place the Sephadex-G20 gel in a beaker and soak it in ultrapure water for 12 hours, remove bubbles for 2 hours, and then add it to a chromatography column with a size of 3.6×60cm. Then use ultrapure water to pre-press for 12 hours at a flow rate of 2.5mL / min. Load the YMOs crude product onto the Sephadex-G20 gel column, use ultrapure water as the eluent, and the flow rate is 2.5mL / min. Use an automatic collector to collect the column liquid every 2 minutes, and use the phenol-sulfuric acid method to detect the column liquid. When the color changes when the 12th tube is collected, the column liquid that changes color after the 12th tube is placed in a -80°C refrigerator for use. The lactose removal rate was determined to be 90.84±1.24%

[0042] Comparative Example 2

[0043] Ultrafiltration method for lactose removal: Add 10-15 ml of enriched yak milk sample to a 30 kDa ultrafiltration centrifuge tube and centrifuge at 6000 rpm for 15-30 min at 3-7°C. The lactose removal rate was determined to be 61.02±5.36%.

[0044] Dialysis method: The enriched yak milk sample solution was transferred to a dialysis bag and dialyzed with running water and distilled water for 48 hours. The distilled water dialysis process was changed every 5-8 hours. After dialysis, the sample was freeze-dried for later use. The measured lactose removal rate was 70.86±3.98%

[0045] Single factor experiment of lactase method: Yak milk sample was heated in a water bath at 50-55℃ for 15-20min, and β-N-acetylhexosaminidase was added to it under aseptic conditions when the temperature dropped to about 25-30℃, and then placed in a shaker (100-150rpm) for constant temperature treatment; the hydrolyzate after the reaction was placed in boiling water to inactivate the enzyme for 10-15min, and then the lactose content in the hydrolyzate was determined by direct titration, and the lactose hydrolysis rate was calculated according to the formula. According to the single factor experiment, the factors that have the greatest impact on the hydrolysis rate of β-N-acetylhexosaminidase are determined as hydrolysis time, hydrolysis temperature and pH value. The hydrolysis time range is selected as 3-5h; the hydrolysis temperature range is selected as 40℃-60℃; when the pH value range is selected as 3-5, the lactose removal rate is 83.79±2.75%. According to the existing methods, it is found that lactose is mostly hydrolyzed by lactase. After improvement, it is found that the hydrolysis effect of β-N-acetylhexosaminidase is better. Compared with other lactose hydrolysis methods, it is found that this method is simple to operate and has optimized conditions, which is also one of the innovations of this project.

[0046] pH Lactose removal rate (%) Enzymatic hydrolysis temperature Lactose removal rate (%) Enzyme addition amount Lactose removal rate (%) 3 55.6233±0.45938 35 46.3667±0.63129 0.1 50.3267±0.99042 5 71.86±0.70491 45 72.68±0.31607 0.2 71.0467±0.75501 7 34.8833±0.85337 55 59.6767±0.92338 0.3 75.2033±0.62804

[0047] Example 3: YMOs crude product is fermented to remove glucose to obtain YMOs sample

[0048] Saccharomyces cerevisiae BF16 was used to remove glucose from YMOs concentrate after lactose fermentation. First, active dry yeast was activated and cultured, and Saccharomyces cerevisiae BF16 was inoculated into 20 mL of lactose-free milk oligosaccharide solution, fermented in a 50 mL conical flask, and slowly shaken at 100 rpm. The effects of the addition amount of brewer's yeast and fermentation time on the fermentation of glucose in human milk oligosaccharide concentrate after lactose removal were studied.

[0049] The Box-Behnken central composite design principle was used to design a response surface optimization method for glucose fermentation conditions. The fermentation temperature (℃), dilution factor of fermentation liquid (times), and amount of brewer's yeast added (g / L) were set as independent variables, and the glucose fermentation rate was used as the response value. The three factors and three levels were determined, and a total of 17 groups of experimental points were used for the response surface optimization experiment to determine the optimal fermentation conditions for glucose in the YMOs enrichment liquid after lactose removal by brewer's yeast BF16 fermentation. It is known that the concentration of 1g / L of commercial brewer's yeast BF16 corresponds to approximately 6×10 6 In order to obtain fast and reliable glucose fermentation, commercial brewer's yeast BF16 selected a fermentation temperature of 16-20°C based on the results of single-factor experiments and the conditions recommended by the manufacturer; selected a brewer's yeast BF16 addition range of 2-4g / L; selected a dilution multiple range of 12-16 times; and selected a fermentation time range of 20-30h.

[0050] The single factor experiment was carried out by changing the fermentation temperature, the dilution multiple of the fermentation solution, and the amount of brewer's yeast. The glucose fermentation rate was used as the response value to calculate the optimal extraction conditions of YMOs by fermentation. Each experiment was conducted in parallel three times. According to the results of the single factor experiment, the three factors of fermentation temperature, fermentation solution dilution multiple, and brewer's yeast addition that significantly affected the polysaccharide extraction effect were selected for the response surface method optimization design. The software was used to design the experiment according to the one-design principle. The fermentation temperature, fermentation solution dilution multiple, and brewer's yeast addition were selected for the setting of experimental factors and levels. The setting results are shown in Table 1.

[0051] Measurement results:

[0052] Table 1 Experimental factors and level settings

[0053]

[0054] The experimental scheme and results are shown in Table 2, with fermentation temperature (A), dilution multiple of fermentation solution (B), amount of brewer's yeast added (C), and lactose hydrolysis rate as the response value (Y).

[0055] Table 2

[0056] serial number A B C Y (glucose fermentation rate / %) 1 18 14 4 69.22 2 19 12 4 77.93 3 19 16 2 76.71 4 19 12 2 74.25 5 18 14 2 60.76 6 19 14 3 88.64 7 20 14 2 67.85 8 19 14 3 89.04 9 18 16 3 71.47 10 19 14 3 89.06 11 19 16 3 80.78 12 19 14 4 91.63 13 20 12 3 70.62 14 18 12 3 67.49 15 19 14 3 92.21 16 20 14 4 74.35 17 20 16 3 76.14

[0057] The obtained data were subjected to multiple regression analysis to obtain a binary linear equation between the corresponding fermentation temperature, dilution multiple of fermentation liquid, amount of brewer's yeast added and the response value glucose fermentation rate.

[0058] Y=90.116+2.5025A+1.85125B+2.83875C+0.3850AB-0.49AC+0.0975BC-14.02925A 2 -4.65675B 2 -8.04175C 2 Model multivariate correlation coefficient R 2 =98.48%, indicating that 98.48% of the change in glucose fermentation rate comes from fermentation temperature, dilution multiple and bacterial addition amount. The F value can reflect the influence of various factors on the lactose hydrolysis rate, and the F value is positively correlated with the influence on the lactose hydrolysis rate. According to Table 3, the order of influence of various factors on the hydrolysis rate of lactose is: C (bacteria addition amount) > A (fermentation temperature) > B (dilution multiple). It can also be seen from Table 3 that the interaction terms AB and BC have a very significant effect on the fermentation rate (P<0.001), and the first-order terms A, B, C and the interaction term AC have a more significant effect on the fermentation rate (P<0.05), which shows that the various factors do not have a simple linear relationship on the glucose fermentation rate.

[0059] Table 3 Results of variance analysis of experimental data

[0060]

[0061]

[0062] Use Design Expert 12 to analyze and optimize the test results. Use the software to draw and analyze according to the regression equation to obtain the response surface of the regression equation and its contour map, such as Figure 1 , Figure 2 and Figure 3 As shown. According to the model analysis, the optimal conditions for glucose fermentation are: fermentation temperature 19°C, dilution multiple 14.41 times, and bacteria addition amount 3.18g / L. Under these conditions, the glucose fermentation rate is 90.67%±0.52% and the relative deviation of the extraction rate is relatively high, and the process conditions are stable. The addition range is relatively close to that of comparative example 3

[0063] Fermentation time Glucose removal rate (%) Fermentation temperature Glucose removal rate (%) 15 46.1667±0.53529 16 65.66±0.46808 25 76.8633±0.8766 18 81.6567±0.3691 35 78.2867±0.2558 20 80.2033±0.49339 Enzyme addition amount Glucose removal rate (%) Dilution multiple Glucose removal rate (%) 0.1 50.3267±0.99042 10 67.25±0.46808 0.2 71.0467±0.75501 14 80.7867±0.68661 0.3 73.2033±0.62804 18 63.2233±0.72418

[0064] Example 4 Nanofiltration membrane technology

[0065] Step 4: Pass the filtrate through a 500Da-700Da membrane and collect the retained concentrate. The specific process is as follows: add 4-6 times the weight of water to the filtrate filtered through a 1000-3000Da membrane, and then pass it through a 500Da-700Da membrane to collect the retained concentrate. Filter through a 1000-3000Da membrane to further remove macromolecular impurities and small molecular impurities, and then collect the filtrate. When filtering through a 500Da-700Da membrane, add enough water to the filtrate, and use excess water to dissolve water-soluble substances in the filtrate, such as small molecular galactose, glucose and some salts, and filter them through a 500Da-700Da membrane, and collect the retained concentrate on the membrane.

[0066] Example 5 Optimization of fermentation broth treatment by combining anion and cation columns with gel columns to remove multivalent anions and cations

[0067] The retained concentrate is desalted by using a cation exchange resin and an anion exchange resin until the conductivity of the retained concentrate is lower than 400 μs / cm.

[0068] Furthermore, the anion exchange resin is a macroporous weakly basic anion exchange resin with a polyacrylic acid skeleton; and the cation exchange resin is a strongly acidic styrene-based cation exchange resin.

[0069] Example 6 Antioxidant activity test of the polysaccharide obtained in Example 5

[0070] DPPH free radical scavenging experiment

[0071] Add 1 mL of DPPH ethanol (0.1 mM) solution to HMOs and YMOs of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL), shake well, and place at room temperature in the dark for 30 minutes. Measure the absorbance at 517 nm. This test uses ethanol as a negative control, VC as a positive control, and distilled water as a blank control. The calculation formula is as follows:

[0072] DPPH free radical scavenging rate (%) = [1-(A 1 -A 0 ) / A 2 ]x100%

[0073] A 0 is the absorbance value of the blank DPPH solution; A 1 is the absorbance value of the mixed solution of sample and DPPH; A 2 is the absorbance value of the sample solution without DPPH.

[0074] (1) Superoxide anion free radical scavenging experiment:

[0075] Add 2 mL of 5 mM Tris-HCl buffer to 1 mL of samples of different concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL), and then add 0.2 mL of 6 mM pyrogallol. Shake the mixture vigorously, react for 5 min at 25 °C, and finally add 0.5 mL of HCl to terminate the reaction (0.1 M). Measure the absorbance value at 320 nm at this moment. VC is used as a positive control in this test. The calculation formula for the superoxide free radical scavenging rate of HMOs and YMOs is as follows:

[0076]

[0077] A 0 is the absorbance value of superoxide and distilled water; A 1 is the absorbance value of the solvent blank group; A 2 is the absorbance value of superoxide and sample; A 3 is the absorbance value of Tris-HCl buffer and sample solution.

[0078] (3) ABTS free radical scavenging activity experiment:

[0079] Mix 5 mL of ABTS solution (7 mM) with 5 mL of potassium persulfate (2.45 mM), and incubate for 12 hours at room temperature. Dilute the solution appropriately with phosphate buffer (pH = 6.6) to adjust the absorbance value at 734 nm to 0.70. Next, add 0.1 mL of HMOs and YMOs samples with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL to 3.9 mL of ABTS radical solution. After shaking well, measure the absorbance value at 734 nm. In this experiment, VC is used as the positive control. The scavenging rate of ABTS radicals is calculated as follows:

[0080]

[0081] A 0 is the absorbance value of the blank control without samples; A 1 is the absorbance value of the sample and ABTS sample; A 2 is the absorbance value without the ABTS sample.

[0082] (4) Hydroxyl radical scavenging activity

[0083] Add 1 mL of 9 mM FeSO4 solution, 9 mM salicylic acid-ethanol solution, and 1 mg / mL H2O2 solution to the HMOs and YMOs solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL in sequence. Shake the mixture well and react at 37 °C for 30 min. Measure the absorbance value at 510 nm. The activity of hydroxyl radical scavenging is calculated as follows:

[0084]

[0085] A 0 is the absorbance value of the blank control without samples; A 1 is the absorbance value of the sample solution; A 2 is without H 2 O 2 the absorbance value of the sample.

[0086] Conclusion: Yak milk oligosaccharides at different concentrations have a certain scavenging ability for the free radical DPPH·. As the concentration of yak milk oligosaccharides increases, the scavenging rate gradually increases, showing an obvious dose-effect relationship, and its scavenging rate is 65.88%.

[0087] Yak milk oligosaccharides at different concentrations have a certain scavenging ability for superoxide anions. As the concentration of yak milk oligosaccharides increases, the scavenging rate gradually increases, showing an obvious dose-effect relationship, and its scavenging rate is 41.08%

[0088] Different concentrations of yak milk oligosaccharides have a certain ability to scavenge the free radical ABTS+·. With the increase of polysaccharide concentration, the scavenging rate gradually increases, showing an obvious dose-effect relationship, and the scavenging rate is 78.67%.

[0089] Yak milk oligosaccharides of different concentrations have a certain ability to scavenge hydroxyl free radicals. With the increase of polysaccharide concentration, the scavenging rate gradually increases, showing an obvious dose-effect relationship. The scavenging rate is 63.91%, which has a certain antioxidant capacity.

Claims

1. A method for extracting sialic acid oligosaccharides from yak milk, It is characterized in that The steps include: Step 1, defatting and deproteinizing yak milk to obtain crude yak milk oligosaccharide; Step 2, removing glucose from the crude yak milk oligosaccharide, specifically, placing the crude yak milk oligosaccharide in a 50-55°C water bath for 15-20 minutes, adding β-N-acetyl hexosaminidase to hydrolyze it under aseptic conditions when the temperature drops to 25-30°C, and placing it in a shaker at 100-150 rpm for constant temperature treatment; placing the hydrolyzate after the reaction in boiling water to inactivate the enzyme for 10-15 minutes, and then determining the lactose content in the hydrolyzate by direct titration, the hydrolysis time range is selected to be 3-5 hours; the hydrolysis temperature range is selected to be 40°C-60°C; The pH value range is selected to decompose lactose when it is 3-5; the hydrolyzate is loaded onto a Sephadex-G20 gel column, ultrapure water is used as an eluent, the flow rate is 2.5 mL / min, the column liquid is collected every 2 minutes by an automatic collector, the column liquid is detected by a phenol-sulfuric acid method, and a color change occurs when the 12th tube is collected, and the column liquid that changes color after the 12th tube is placed in a -80°C refrigerator for standby use to obtain lactose-free oligosaccharides; active dry yeast is activated and cultured, and cerevisiae BF16 is inoculated into 20 mL of the lactose-free oligosaccharide solution, fermented in a 50 mL conical flask, and slowly shaken at a speed of 100 rpm to obtain a fermentation liquid; Step 3: Filter the fermentation broth, pass the filtrate through a 500Da-700Da membrane, and desalt the retained concentrate with a cation exchange resin and anion exchange resin until the conductivity of the retained concentrate is less than 400μs / cm, remove the polyvalent anions and cations in the filtrate, and dry to obtain sialic acid oligosaccharides.

2. A method for extracting sialic acid oligosaccharides from yak milk according to claim 1, It is characterized in that In step 1, the specific method of defatting and deproteinizing is: centrifuge the yak milk once to remove the fat on the upper layer and a small amount of protein at the bottom, take out the middle layer and add anhydrous ethanol to react, centrifuge it twice after the reaction is completed, take the supernatant, dry it, and obtain the crude yak milk oligosaccharide.

3. A method for extracting sialic acid oligosaccharides from yak milk according to claim 2, It is characterized in that The conditions for the first centrifugation are: 3-8°C, 8000-10000 rpm for 15-30 min, and the conditions for the second centrifugation are: 3-8°C, 4000×g for 15-30 min.

4. The method for extracting sialic acid oligosaccharides from yak milk according to claim 2, It is characterized in that By volume ratio, middle layer: anhydrous ethanol = 1:2 ~ 1:

6.

5. The method for extracting sialic acid oligosaccharides from yak milk according to claim 2, It is characterized in that The reaction temperature is 0-4°C and the reaction time is 2-12h.

6. The method for extracting sialic acid oligosaccharides from yak milk according to claim 2, It is characterized in that The drying is performed by using a vacuum concentrator for concentrated drying.

7. The method for extracting sialic acid oligosaccharides from yak milk according to claim 1, It is characterized in that The anion exchange resin is a macroporous weakly basic anion exchange resin with a polyacrylic acid skeleton; the cation exchange resin is a strongly acidic styrene-based cation exchange resin.

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