Preparation method of yak milk fat globule membrane and application of yak milk fat globule membrane in inhibition of pseudomonas aeruginosa

Abstract

The invention provides a preparation method of a yak milk fat globule membrane and application of the yak milk fat globule membrane in inhibition of pseudomonas aeruginosa, and belongs to the technical field of biology. The preparation method comprises the following steps: centrifuging yak milk to obtain coarsely separated milk fat, washing the milk fat with a potassium phosphate buffer solution and ultrapure water in sequence, refrigerating, grinding with a tissue grinder, further crushing with a cell crusher, and finally performing ultracentrifugation on a sample to obtain the milk fat globule membrane. According to the application, the yak milk fat globule membrane is used for inhibiting pseudomonas aeruginosa, in-vitro inhibition experiments prove that the yak milk fat globule membrane has an inhibition effect on the pseudomonas aeruginosa, and the inhibition effect is better than that of Holstein milk fat globule membrane and buffalo milk fat globule membrane. According to the method, the milk consumption is small, the stability is good, the operation difficulty is low, introduction of chemical impurities in the extraction process and the influence of the chemical impurities on membrane components are reduced, the new function of the yak milk fat globule membrane is developed, and the method has good application and popularization prospects and economic value in the aspects of yak milk fat globule membrane extraction and pseudomonas aeruginosa inhibition research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method of yak milk fat globule membrane and its application in inhibiting Pseudomonas aeruginosa. Background Art

[0002] The milk fat globule membrane is a thin film surrounding the milk fat globule, with a cross-sectional diameter of about 10 - 20 nm. It acts as an emulsifier and prevents the aggregation and enzymatic degradation of fat globules. The milk fat globule membrane is mainly composed of membrane-specific proteins such as glycoproteins and membrane lipid components such as phospholipids and sphingolipids. Although the milk fat globule membrane has a very low content in milk, it has various biological activities such as preventing cardiovascular diseases, immunization, and maintaining intestinal health.

[0003] Compared with the extraction of other common components in milk, the extraction of milk fat globule membrane is more difficult. In industrial production, skimmed buttermilk is generally used for the production of milk fat globule membrane. Because of its low cost, its commercial utilization value is limited, and the content of membrane substances is relatively rich. In the laboratory, untreated cow milk is generally used as the raw material, and it is prepared through steps such as fat globule separation, milk fat washing, releasing the milk fat globule membrane from the fat globules, and collecting the milk fat globule membrane, or the milk is prepared into butter, and the buttermilk produced is used as the material to separate the milk fat globule membrane. The difficulty in extracting the milk fat globule membrane mainly comes from the release and collection of the milk fat globule membrane. During the extraction process, extracting the membrane through butter or buttermilk requires a large amount of raw materials, which is not suitable for dairy products with small collection amounts and difficult collection, such as yak milk and yak colostrum. Membrane separation by ultrafiltration is difficult to achieve for general laboratories. Collecting the membrane by the method of protein isoelectric point precipitation will damage the membrane lipid components, and chemically lysing the membrane will introduce more salt components, which is not conducive to analysis with high purity requirements such as mass spectrometry and chromatography. Therefore, more extraction methods need to be developed to meet the requirements of less usage, low instrument requirements, less loss of membrane components, and less chemical impurities.

[0004] Pseudomonas aeruginosa is a Gram-negative bacterium widely distributed in nature. Because it produces green pigments during its metabolic process, making the wound surface appear green, it is also known as pyocyanic bacillus. Pseudomonas aeruginosa is usually colonized as a normal strain in parts of the human body such as the skin, respiratory tract, and intestine, and it is also a common opportunistic pathogen in clinical practice, posing a greater health risk to people with weaker resistance. When the human immune system is not sound or there is immunodeficiency, for example, newborn infants, the elderly, the weak, the sick, pregnant women, and patients with extensive burns on the body, they are extremely vulnerable to the infection of Pseudomonas aeruginosa. Infected patients usually show symptoms such as fever, jaundice, splenomegaly, wound ulceration, etc., and secondary diseases such as pneumonia, urinary tract infection, meningitis, and septicemia will occur. In recent years, the isolation rate of Pseudomonas aeruginosa has shown an increasing trend, and it has become an important type of pathogenic bacterium causing hospital infections.

[0005] Due to the low outer membrane permeability of Pseudomonas aeruginosa, this type of pathogenic bacterium has a certain degree of natural resistance to commonly used clinical antibacterial drugs, and it is relatively easy to derive multi-drug resistant strains. In addition, it also has strong resistance to physical and chemical factors such as disinfectants, drying, and ultraviolet rays. Ultraviolet rays have poor bactericidal effects on it, so it is listed in the ranks of "super bacteria".

[0006] For combating bacterial infections, the use of chemically synthesized antibacterial agents is a common method, but it often has problems such as toxicity and drug resistance. Therefore, antibacterial components of natural origin have received extensive attention in recent years. Antibacterial components of natural origin have high safety, few side effects, can reduce the risk of drug resistance, and have some additional health benefits. Against the background of severe drug resistance problems and the improvement of health awareness, their application background is broad. In recent years, with the in-depth study of milk fat globule membrane, its components and functions have been further developed. It is reported that components such as xanthine dehydrogenase, xanthine oxidase, and sphingomyelin in milk fat globule membrane play a certain role in inhibiting the growth of Staphylococcus aureus, Escherichia coli, and Salmonella, exerting antibacterial effects in the gastrointestinal tract, regulating the intestinal flora, and promoting the intestinal development of infants. The contents of various nutritional components such as protein, fat, vitamins, inorganic salts, and lactose in yak milk are higher than those in ordinary cow milk. Yak milk is natural concentrated milk, and some important components in its milk fat globule membrane, such as phospholipids, are also higher than those in ordinary cow milk fat globule membrane. Therefore, the use of milk fat globule membrane, especially yak milk fat globule membrane, may become a new way to inhibit Pseudomonas aeruginosa. Summary of the Invention

[0007] The technical problem to be solved by the present invention is: to overcome the problems of inconvenient operation, difficult extraction, large dosage, low stability, and easy introduction of chemical reagent impurities in the existing extraction process of yak milk fat globule membrane, and to provide a preparation method of yak milk fat globule membrane with less milk consumption, good stability, low operation difficulty, and reduced introduction of chemical impurities and their influence on membrane components during the extraction process. At the same time, it is confirmed that it has an inhibitory effect on Pseudomonas aeruginosa, and the inhibitory effect is better than that of Holstein cow milk fat globule membrane and buffalo milk fat globule membrane.

[0008] To achieve the above object, the present invention includes the following contents:

[0009] In the first aspect of the present invention, a preparation method of yak milk fat globule membrane is provided, which has the following technological steps;

[0010] 1) Take yak milk as the whole milk sample;

[0011] 2) Centrifuge the yak milk at 4 °C for 0.5 h with a centrifugal force of 5000 - 10000 g, and collect the upper milk fat layer;

[0012] 3) Wash the milk fat layer with potassium phosphate buffer solution, take the upper milk fat, and repeat the washing with potassium phosphate buffer solution once; then wash it once with ultrapure water, and centrifuge to obtain the crude extract of milk fat; the volume of potassium phosphate buffer solution and ultrapure water used for each washing is 10 times that of the upper milk fat by volume;

[0013] 4) Let the crude extract of milk fat stand at 4 °C until the milk fat solidifies;

[0014] 5) Grind the milk fat in a tissue grinder;

[0015] 6) Further break the ground milk fat with a cell disruptor;

[0016] 7) Ultracentrifuge the sample at 4 °C for 1 h at a centrifugal force of 20,000 - 100,000 g, and collect the lower milk fat globule membrane;

[0017] In step 1), the whole milk sample is fresh yak milk taken; or yak milk transported back at -20 - 4 °C within 1 - 3 hours; or stored in an environment of -80 °C and thawed in a 4 °C refrigerator before extracting the lipid of yak milk fat globule membrane.

[0018] In step 5), the grinding in the tissue grinder is carried out with a single grinding time of 30 s, grinding 3 times, and the time interval is 10 s.

[0019] In step 6), the further breaking of the ground milk fat with a cell disruptor is carried out with a breaking power of 650 ± 50 W, a time of 60 s, and a frequency of 25 kHz.

[0020] In the second aspect of the present invention, an antibacterial method for inhibiting Pseudomonas aeruginosa is provided, which includes using yak milk fat globule membrane.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: First, grinding the yak milk fat with a tissue grinder can better break the milk fat globule membrane. Secondly, further breaking with a cell disruptor can reduce the separation difficulty of the milk fat globule membrane and effectively improve the extraction rate of the lipid of the milk fat globule membrane. Finally, a new application of using yak milk fat globule membrane to inhibit Pseudomonas aeruginosa is provided. Description of the Drawings

[0022] Figure 1 The preparation flow chart of the yak milk fat globule membrane described in the present invention;

[0023] Figure 2 The extraction rates of yak milk fat globule membranes with different comparative examples;

[0024] Figure 3 The comparison chart of the inhibitory effects of yak milk fat globule membranes with different concentrations on Pseudomonas aeruginosa;

[0025] Figure 4 Comparison chart of the inhibitory effects of different yak milk fat globule membranes on Pseudomonas aeruginosa. Detailed implementation manners

[0026] The present invention provides a preparation method of yak milk fat globule membrane and its application in inhibiting Pseudomonas aeruginosa. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0027] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with embodiments.

[0028] Example 1 A preparation method of yak milk fat globule membrane

[0029] 1) Select fresh yak milk from healthy yaks. To eliminate individual differences, the yak milk from 30 yaks is taken as the whole milk sample, and 100 mL of milk is taken from each yak. The fresh yak milk taken from the pasture must be transported back to the laboratory at 4°C or -20°C as soon as possible. If it cannot be processed in time, it needs to be stored at -80°C. Before use, it is thawed in a 4°C refrigerator. This method must use fresh or ultra-low temperature condition (-80°C) stored yak milk. 4°C and -20°C are only suitable for short-term transportation and cannot be used for the storage of yak milk samples, otherwise protein flocculation or degradation and lipid stratification or oxidation will occur, affecting the measurement results.

[0030] 2) Centrifuge the 30 yak milk samples in step 1) at 4°C for 0.5 h with a centrifugal force of 10,000 g, and carefully collect the upper milk fat layer. The centrifugation temperature in this step should not be increased. In addition, when collecting the upper fat at room temperature, the operation should be carried out as soon as possible, and the unoperated samples should be placed in a 4°C environment and taken out when needed. The above precautions are to prevent the fat layer from redissolving due to temperature increase.

[0031] 3) Wash the yak milk fat obtained in step 2) with 10 times potassium phosphate buffer solution. After washing, centrifuge at 4°C for 0.5 h with a centrifugal force of 10,000 g, take the upper milk fat, and repeat the potassium phosphate buffer solution washing once; after washing, wash with 10 times ultrapure water once to remove the residue of potassium phosphate buffer solution, and the crude extract of milk fat is obtained after centrifugal separation.

[0032] 4) Leave the crude extract of milk fat standing at 4 °C overnight (8 - 12 h) to solidify the milk fat.

[0033] 5) Grind the milk fat in a tissue grinder, with a single grinding time of 30 s, grind 3 times, and the time interval is 10 s.

[0034] 6) Further break up the ground milk fat with a cell disruptor, with a frequency of 25 kHz, a power of 600 W, and a time of 60 s.

[0035] 7) Ultracentrifuge the sample in step 6) at 4 °C for 1 h, with a centrifugal force of 100000 g, and collect the lower milk fat globule membrane.

[0036] In this example, a tissue grinder was used to grind yak milk fat, and a cell disruptor was used for further disruption. The extraction rate of yak milk fat globule membrane was 53.4 mg / 100 mL (see Figure 2 ).

[0037] Comparative example 1: Yak milk fat was not ground with a tissue grinder

[0038] 1) Select fresh yak milk from healthy yaks. To eliminate individual differences, select the yak milk from 30 yaks as the whole milk sample, take 100 mL of milk from each yak. The fresh yak milk taken from the pasture must be transported back to the laboratory at 4 °C or -20 °C as soon as possible. If it cannot be processed in time, it needs to be stored at -80 °C. Before use, thaw it in a 4 °C refrigerator. This method must use yak milk stored fresh or at ultra-low temperature (-80 °C). 4 °C and -20 °C are only suitable for short-term transportation and cannot be used for the storage of yak milk samples. Otherwise, protein flocculation or degradation and lipid stratification or oxidation will occur, affecting the measurement results.

[0039] 2) Centrifuge the 30 portions of yak milk samples in step 1) at 4 °C for 0.5 h, with a centrifugal force of 10000 g, and carefully collect the upper milk fat layer. The centrifugation temperature in this step should not be increased. In addition, when collecting the upper fat at room temperature, it needs to be operated as soon as possible, and the unoperated samples need to be placed in a 4 °C environment and taken out when needed. The above precautions are all to prevent the fat layer from redissolving due to temperature increase.

[0040] 3) Wash the yak milk fat obtained in step 2) with 10-fold potassium phosphate buffer solution. After washing, centrifuge at 4 °C for 0.5 h, with a centrifugal force of 10000 g, and take the upper milk fat. Repeat the potassium phosphate buffer solution washing 1 time; after washing, wash 1 time with 10-fold ultrapure water to remove the residual potassium phosphate buffer solution, and obtain the crude extract of milk fat after centrifugal separation.

[0041] 4) Leave the crude extract of milk fat standing at 4 °C overnight (8 - 12 h) to solidify the milk fat.

[0042] 5) Further break the milk fat with a cell disruptor at a frequency of 25 kHz, a power of 500 W, and a time of 60 s.

[0043] 6) Ultracentrifuge the sample in step 5) at 4 °C for 1 h at a centrifugal force of 100,000 g, and collect the lower milk fat globule membrane.

[0044] After measurement, the extraction rate of yak milk fat globule membrane is 35.2 mg / 100 mL (see Figure 2 ).

[0045] Comparative Example 2: The yak milk fat layer was not broken with a cell disruptor.

[0046] In this comparative example, in step 6) of Example 1, the ground milk fat was not further broken with a cell disruptor at a frequency of 25 kHz, a power of 500 W, and a time of 60 s. The remaining steps were the same as those in Example 1. After measurement, the extraction rate of yak milk fat globule membrane is 41.8 mg / 100 mL (see Figure 2 ).

[0047] Comparative Example 3: The yak milk fat was neither ground with a tissue grinder nor broken with a cell disruptor.

[0048] In this comparative example, in step 5) of Example 1, the milk fat was not ground in a tissue grinder for 30 s each time, 3 times with a 10 s interval, and in step 6) of Example 1, the ground milk fat was not further broken with a cell disruptor at a frequency of 25 kHz, a power of 500 W, and a time of 60 s. The remaining steps were the same as those in Comparative Example 1. After measurement, the extraction rate concentration of yak milk fat globule membrane was the lowest, at 18.1 mg / 100 mL (see Figure 2 ).

[0049] Comparative Example 4: The milk fat was ground by a traditional grinding method.

[0050] In this comparative example, step 5) used a traditional mortar grinding method instead of the tissue grinder in the present invention, and the remaining steps were the same as those in Example 1. After measurement, the extraction rate concentration of yak milk fat globule membrane was about 35.4 mg / 100 mL (see Figure 2 ).

[0051] Analysis of the extraction rate results of yak milk fat globule membrane in Example 1 and Comparative Examples 1-4 shows that: the concentration of milk fat globule membrane lipid obtained in Example 1 is 53.4 mg / 100 mL, which is higher than that obtained in Comparative Examples 1-4. Among them, the concentration of milk fat globule membrane lipid obtained in Comparative Example 3 is the lowest, only 18.1 mg / 100 mL, indicating that the use of tissue grinders and cell breakers in the examples can effectively improve the extraction rate of yak milk fat globule membrane.

[0052] Example 2. Determination of the minimum inhibitory concentration of yak milk fat globule membrane

[0053] Dissolve the yak milk fat globule membrane with 1×PBS solution to prepare solutions with concentrations of 0 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL, and water bath at 60 °C in a water bath for 30 min. Add 100 μL of Pseudomonas aeruginosa bacterial solution with a concentration of 1×10 5 CFU / mL that has been cultured at 37 °C for 6 h, 100 μL of each concentration of membrane solution, and 100 μL of BHI liquid medium, and culture at 37 °C for 10 h to 12 h.

[0054] Use the plate counting method to calculate the number of Pseudomonas aeruginosa in each well.

[0055] Calculate the inhibition rate according to the following formula

[0056] Bacterial inhibition rate = (number of colonies in negative control group - number of colonies in experimental group) / number of colonies in negative control group x 100%

[0057] After determination, the minimum inhibitory concentration of yak milk fat globule membrane is 10 mg / mL (see Figure 3 ).

[0058] Example 3. Comparison of the antibacterial abilities of milk fat globule membranes from different cows

[0059] Dissolve the milk fat globule membranes of yak, buffalo, and Holstein cows with 1×PBS solution to prepare solutions with concentrations of 5 mg / mL, 10 mg / mL, and 15 mg / mL, and water bath at 60 °C in a water bath for 30 min. Add 100 μL of Pseudomonas aeruginosa bacterial solution with a concentration of 1×10 5 CFU / mL that has been cultured at 37 °C for 6 h, 100 μL of each membrane solution, and 100 μL of BHI liquid medium, and culture at 37 °C for 10 - 12 h.

[0060] Use the plate counting method to calculate the number of Pseudomonas aeruginosa in each well.

[0061] Calculate the inhibition rate according to the following formula

[0062] Bacterial inhibition rate = (number of colonies in the negative control group - number of colonies in the experimental group) / number of colonies in the negative control group × 100%

[0063] It was determined that the antibacterial effect of yak milk fat globule membrane was the best (see Figure 4 ).

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing yak milk fat globule membrane, characterized in that: Extracted by a method comprising the following steps: 1) Taking yak milk as whole milk sample; 2) Centrifuge the yak milk at 4°C for 0.5 h at a centrifugal force of 5000-10000 g, and collect the upper milk fat layer; 3) washing the milk fat layer with potassium phosphate buffer, taking the upper layer of milk fat, and repeating the washing with potassium phosphate buffer once; washing once with ultrapure water, and centrifuging to obtain a crude extract of milk fat; the potassium phosphate buffer and ultrapure water used in each washing are 8 to 10 times the volume of the upper layer of milk fat; 4) The crude milk fat extract is allowed to stand at 4°C until the milk fat solidifies; 5) Grinding the milk fat in a tissue grinder; 6) further crushing the ground milk fat using a cell crusher; 7) The sample was ultracentrifuged at 4°C for 1 h with a centrifugal force of 20,000-100,000 g, and the lower layer was collected to obtain the yak milk fat globule membrane.

2. The method for preparing yak milk fat globule membrane according to claim 1, characterized in that: In step 1), the whole milk sample is freshly collected yak milk; or yak milk transported back at -20-4°C within 1-3 hours; or fresh yak milk stored at -80°C and thawed in a 4°C refrigerator before extracting the yak milk fat globule membrane.

3. The method for preparing yak milk fat globule membrane according to claim 1, characterized in that: In step 5), the tissue is ground in a tissue grinder, with a single grinding time of 30 seconds, and ground three times with a time interval of 10 seconds.

4. The method for preparing yak milk fat globule membrane according to claim 1, characterized in that: In step 6), the ground milk fat is further crushed by a cell crusher with a crushing power of 650±50W, a time of 60s, and a frequency of 25kHz.

5. Application of yak milk fat globule membrane in inhibiting Pseudomonas aeruginosa, characterized in that: Includes the use of yak milk fat globule membrane.

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