Detection method of buffalo milk oligosaccharide and application thereof

Abstract

The invention provides a method for detecting buffalo milk oligosaccharide. According to the method, the relative abundance of oligosaccharide components with m / z of 385.153, 424.090, 547.212 and 708.261 is detected by using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method. The buffalo milk and the buffalo milk products can be accurately identified by calculating the proportion of several oligosaccharides, and a series of defects of nutrient content, PCR detection and other methods are avoided.

Description

Field of the Invention

[0001] The present application belongs to the field of dairy products and glycobiology. Specifically, the present application provides a method for detecting buffalo milk oligosaccharides and its application. Background Art

[0002] Globally, buffalo milk is the second largest source of milk after dairy cow milk. Its protein, fat and other nutritional content, as well as its taste, are superior to dairy cow milk. In recent years, the buffalo milk industry has also developed rapidly in my country.

[0003] India is currently the world's largest producer of buffalo milk, with the Murrah and Nili-Raffi buffaloes being the most common breeds for both meat and dairy production. Other common breeds include the Italian buffalo. The dairy buffalo landscape in my country is more complex, encompassing both imported and domestically produced varieties and hybrids. This mixed variety, coupled with a lack of apparent differentiation, results in widespread contamination of buffalo and dairy milk products on the market. Identification using indicators such as fat and protein content is susceptible to interference from added ingredients. PCR-based detection methods cannot accurately identify buffalo milk products due to long-term hybridization, and qualitatively determining the degree of adulteration is also difficult.

[0004] In addition, the types, contents and even functions of oligosaccharides in buffalo milk are different from those in dairy cow milk, and research, development and utilization in this area are still relatively rare.

[0005] Therefore, it is necessary to further study the detection method of buffalo milk oligosaccharides and apply it to buffalo milk identification and oligosaccharide research. Summary of the Invention

[0006] In response to the above problems, the present application discovered that among the several oligosaccharide components with higher abundance, several oligosaccharides contained in buffalo milk have specific ratios, and based on this, provided a method for identifying buffalo milk.

[0007] On the one hand, the present application provides a method for detecting buffalo milk oligosaccharides, in which ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry is used to detect the relative abundance of oligosaccharide components with m / z of 385.153, 424.090, 547.212 and 708.261.

[0008] On the one hand, the present application provides a method for identifying whether a milk sample is pure buffalo milk, the method comprising:

[0009] (1) Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to detect the relative abundance of oligosaccharides with m / z values ​​of 385.153, 424.090, 547.212, and 708.261;

[0010] (2) Calculate the relative abundance ratio of oligosaccharide components with m / z of 424.090 and m / z of 547.212, or calculate the relative abundance ratio of oligosaccharide components with m / z of 385.153 and m / z of 708.261;

[0011] (3) Identify whether the milk sample is buffalo milk based on the ratio obtained in step (2).

[0012] Furthermore, in step (3), if the relative abundance ratio of the oligosaccharide components with m / z of 424.090 and m / z of 547.212 is 0.4-0.6, the milk sample is determined to be pure buffalo milk; if the relative abundance ratio of the oligosaccharide components with m / z of 424.090 and m / z of 547.212 is greater than 0.6, the milk sample is determined to be non-pure buffalo milk; if other ratios occur, it cannot be determined whether the milk sample is pure buffalo milk.

[0013] Furthermore, in step (3), if the relative abundance ratio of the oligosaccharide components with m / z of 385.153 and m / z of 708.261 is 2.7-3.3, the milk sample is determined to be pure buffalo milk; if the relative abundance ratio of the oligosaccharide components with m / z of 385.153 and m / z of 708.261 is less than 2.7, the milk sample is determined to be non-pure buffalo milk; if other ratios occur, it cannot be determined whether the milk sample is pure buffalo milk.

[0014] Furthermore, step (1) includes: (1-1) removing fat and protein; (1-2) purifying oligosaccharide components; (1-3) derivatizing and modifying the oligosaccharide components; and (1-4) detecting by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

[0015] Furthermore, the buffalo milk is buffalo milk produced by Mora buffalo, Neri-Lafite buffalo, Italian buffalo, Fuzhong buffalo, Shanghai buffalo or their hybrid breeds.

[0016] Furthermore, the method further comprises (4) determining the protein content of the milk sample and / or determining the fat content of the milk sample.

[0017] Due to factors such as mass spectrometry equipment, analytical methods, and other groups, the above m / z values ​​may not be completely reproduced in various tests. Those skilled in the art can understand these differences or calculate and remove these differences to obtain the relative abundance of the "oligosaccharide components with m / z of 385.153, 424.090, 547.212 and 708.261".

[0018] In this application, pure buffalo milk refers to other dairy products, especially products with a cow's milk content below a certain limit, such as 5% or 10%.

[0019] Methods for removing fat and protein, purifying oligosaccharide components, and derivatizing and modifying oligosaccharide components are well known to those skilled in the art, including but not limited to removing protein and fat with organic solvents, purifying sugars with alcohol precipitation, purifying sugars with commercially available kits / chromatographic columns, and PMP derivatization.

[0020] Protein and fat content determination and PCR determination methods (including but not limited to the methods of CN102808025A and CN110221007A) can be used to further confirm the results of the identification method of this application, especially when the ratio alone cannot be used to determine the result. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The relative abundances of several highly abundant oligosaccharides in the milk of Mora buffalo, Nili-Laffitte buffalo, Fuzhong buffalo, Holstein cow, and Jersey cow are shown in Table 1. A, B, C, and D are oligosaccharides with m / z 385.153, 424.090, 506.184, 547.212, 635.230, and 708.261, respectively. DETAILED DESCRIPTION

[0022] Example 1 Milk Sample

[0023] All milk samples used in the examples are mature milk (not colostrum):

[0024] Buffalo milk from Murrah buffaloes and Nili-Rafi buffaloes was obtained from a buffalo breeding base in India.

[0025] The buffalo milk produced by Italian buffalo, Fuzhong buffalo, Shanghai buffalo, two other types of Murra buffalo hybridized with local Chinese buffalo (Mura Buffalo 1 and Murra Buffalo 2), and one type of Nili-Lafite buffalo hybridized with local Chinese buffalo (Nili-Lafite Buffalo 1) was obtained from the buffalo breeding base of the applicant's cooperative unit in Guangxi.

[0026] The milk produced by Holstein cows, Jersey cows, dairy shorthorn cattle, Sanhe cattle and three types of hybrid cows between Holstein cows and Chinese native dairy cows (hybrid 1, hybrid 2, hybrid 3) was obtained from the applicant's cooperative units in Guangxi and Inner Mongolia.

[0027] Example 2 Analysis Method of Oligosaccharides

[0028] Extraction and derivatization of oligosaccharides:

[0029] (1) Place milk in a centrifuge tube and centrifuge at 12,000 rpm for 30 min at 4°C. Take the whey from the middle of the tube.

[0030] (2) Add 3 times the volume of the whey liquid and a mixture of chloroform and methanol in a volume ratio of 2:1 to the whey liquid obtained in step (1); after mixing evenly, centrifuge at 12000 rpm for 30 minutes at 4°C, and aspirate the supernatant;

[0031] (3) Add 2 times the volume of anhydrous ethanol to the supernatant obtained in step (2); mix well and let stand at 4°C for 24 hours; centrifuge at 12000 rpm for 30 minutes at 4°C and aspirate the supernatant;

[0032] (4) The supernatant obtained in step (3) is rotary evaporated to dryness to obtain a crude oligosaccharide sample.

[0033] (5) dissolving the crude oligosaccharide sample obtained in step (4) in water;

[0034] (6) The solution obtained in step (5) was loaded onto an activated SPE solid phase extraction column (Shanghai Jinlan);

[0035] (7) Wash the SPE column with water and elute with 25% acetonitrile containing 0.5% TFA;

[0036] (8) concentrating and lyophilizing the eluate obtained in step (7) to obtain a purified oligosaccharide sample;

[0037] (9) Dissolve the purified oligosaccharide sample obtained in step (8) in ammonia water; add a 0.5 mol / L PMP solution with an equal volume of ammonia water, and react in a water bath at 80 °C for 40 min;

[0038] (10) The reaction product obtained in step (9) is cooled to room temperature and freeze-dried.

[0039] Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS, Agilent 6530 equipped with an amide column) analysis:

[0040] The extracted and purified oligosaccharide sample was dissolved in the starting mobile phase for detection (internal standard cyclodextrin was added for quantitative detection).

[0041] Chromatography part:

[0042] Mobile phase A was 1 mM ammonium acetate aqueous solution, and mobile phase B was acetonitrile; injection volume was 10 μL, column temperature was 30°C, and flow rate was 0.3 mL / min;

[0043] The elution program was as follows: 10% mobile phase A: 2 min; 10-25% mobile phase A: 10 min; 25% mobile phase A: 5 min; 25-35% mobile phase A: 10 min; 35-55% mobile phase A: 10 min; 70% mobile phase A: 5 min; 5% mobile phase A: 5 min.

[0044] Mass spectrometry part:

[0045] The positive ion mode was used, the capillary temperature was 300 °C, and the capillary voltage was 3.5 kV.

[0046] MRE scanning mode, frequency 0.25s: primary mass spectrometry: m / z 100-3000, collision energy 3eV; secondary mass spectrometry: m / z 100-3000, collision energy 20-30eV.

[0047] The data acquisition software was Agilent's supporting software, and the sugar mass spectrometry data analysis software used was SimGlycan.

[0048] Example 3 Experimental Results

[0049] Oligosaccharide analysis was conducted using samples from Murrah, Nili-Raffi, Fujong, Holstein, and Jersey buffaloes. Among the readily detectable and abundant oligosaccharide components, no oligosaccharides were found that were unique to buffalo or dairy cow milk and could be used for identification. (As shown in Table 1, although their abundances vary, several highly abundant oligosaccharides are commonly found in buffalo and dairy cow milk.)

[0050] Table 1 Highly abundant oligosaccharide components and their presence in typical bovine milk (●: present, ×: absent)

[0051] serial number Highly abundant components m / z Predicted monosaccharide composition Murrah Buffalo Neri-Lafite Buffalo Fuzhong Buffalo Holstein cows Jersey cattle A 385.153 GalNAcGlcGalGlcNAc ● ● ● ● ● B 424.090 <![CDATA[GalGlcPO4]]> ● ● ● ● ● C 506.184 GalGalGlc ● ● ● ● ● D 547.212 GalNAcGalGlcGalGalGlcNAcHexNAcGalGlc ● ● ● ● ● E 635.230 Neu5AcGalGlc ● ● ● ● ● F 708.261 GalGlcNAcGalGlcGalHexNAcGalGlc ● ● ● ● ●

[0052] The applicant further observed the ratio relationship of several high abundance components in different cow's milk, such as Figure 1 As shown in Table 2, the applicant found that the abundance ratio of component B to component D in buffalo milk (about 0.5) was significantly lower than that in dairy cow milk (about 0.8), while the abundance ratio of component A to component F in buffalo milk (about 3) was significantly higher than that in dairy cow milk (about 1.7), as shown in Table 2.

[0053] Table 2 B / D and A / F oligosaccharide ratios in several types of milk

[0054] Milk Source B / D abundance ratio A / F abundance ratio Murrah Buffalo 0.48 3.02 Neri-Lafite Buffalo 0.56 2.85 Fuzhong Buffalo 0.54 3.14 Holstein cows 0.82 1.76 Jersey cattle 0.74 1.60

[0055] Example 4 Verification of oligosaccharide ratios in different samples

[0056] In order to verify whether the ratio of Example 3 is applicable to a wider range of milk and detection equipment, the applicant used WATERS' UPLC-ESI-Q-TOF-MS to detect more milk samples. The results are shown in Table 3:

[0057] Table 3 B / D and A / F oligosaccharide ratios in different samples

[0058] Milk Source B / D abundance ratio A / F abundance ratio Murrah Buffalo 0.51 3.12 Neri-Lafite Buffalo 0.49 2.97 Fuzhong Buffalo 0.53 3.03 Holstein cows 0.85 1.82 Jersey cattle 0.81 1.75 Italian Buffalo 0.58 3.03 Shanghai Buffalo 0.55 3.23 Murrah Buffalo 1 0.57 3.35 Murrah Buffalo 2 0.48 3.07 Neri-Lafite Buffalo 1 0.46 2.85 Dairy Shorthorn cattle 0.93 1.51 Sanhe cattle 0.96 1.79 Hybrid 1 0.79 1.86 Hybrid 2 0.81 1.66 Hybrid 3 0.94 1.91

[0059] In summary, taking into account factors such as sample and equipment, the B / D abundance ratio for identifying buffalo milk was set at 0.4-0.6, and the A / F abundance ratio was set at 2.7-3.3. Based on these standards, multiple samples of buffalo milk produced by the applicant's partner units, as well as self-produced and commercially available dairy cow milk products, were tested. The results showed that both ratios could accurately identify buffalo milk.

[0060] The applicant further attempted to identify the situation where buffalo milk was adulterated with dairy cow milk. The results showed that due to the large difference in the ratios between buffalo milk and dairy cow milk, as long as the adulteration ratio in various adulteration methods (Mora buffalo milk + commercially available Holstein cow milk, Nili-Lafite buffalo milk + commercially available Holstein cow milk, Mora buffalo milk + commercially available Jersey cow milk, Nili-Lafite buffalo milk + commercially available Jersey cow milk, Shanghai buffalo milk + dairy Shorthorn cow milk, etc.) exceeded 20%, the B / D abundance ratio and the A / F abundance ratio would definitely exceed the above range (higher than 0.6 or lower than 2.7).

Claims

1. A method for detecting buffalo milk oligosaccharides, characterized in that: In the method, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry is used to detect the relative abundance of oligosaccharide components with m / z of 385.153, 424.090, 547.212 and 708.

261.

2. A method for identifying whether a milk sample is pure buffalo milk, characterized in that: The method comprises: (1) Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to detect the relative abundance of oligosaccharide components with m / z values ​​of 385.153, 424.090, 547.212, and 708.261; (2) Calculate the relative abundance ratio of oligosaccharide components with m / z of 424.090 and m / z of 547.212, or calculate the relative abundance ratio of oligosaccharide components with m / z of 385.153 and m / z of 708.261; (3) Identify whether the milk sample is buffalo milk based on the ratio obtained in step (2).

3. According to the method of claim 2, in step (3), if the relative abundance ratio of the oligosaccharide components with m / z of 424.090 and m / z of 547.212 is 0.4-0.6, the milk sample is determined to be pure buffalo milk; if the relative abundance ratio of the oligosaccharide components with m / z of 424.090 and m / z of 547.212 is greater than 0.6, the milk sample is determined to be non-pure buffalo milk; if other ratios occur, it cannot be determined whether the milk sample is pure buffalo milk.

4. According to the method of claim 2, in step (3), if the relative abundance ratio of the oligosaccharide components with m / z of 385.153 and m / z of 708.261 is 2.7-3.3, the milk sample is determined to be pure buffalo milk; if the relative abundance ratio of the oligosaccharide components with m / z of 385.153 and m / z of 708.261 is less than 2.7, the milk sample is determined to be non-pure buffalo milk; if other ratios occur, it cannot be determined whether the milk sample is pure buffalo milk.

5. The method according to claim 2, step (1) comprising: (1-1) Removal of fat and protein; (1-2) Purification of oligosaccharide components; (1-3) Derivatization and modification of oligosaccharide components; and (1-4) Detection by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

6. The method according to claim 2, wherein the buffalo milk is produced by Mora buffalo, Neri-Lafite buffalo, Italian buffalo, Fuzhong buffalo, Shanghai buffalo or their hybrid breeds.

7. The method according to claim 2, further comprising (4) determining the protein content of the milk sample and / or determining the fat content of the milk sample.

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