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Binding buffer solution for extracting lactoferrin, preparation method and application of binding buffer solution and detection method of bovine lactoferrin

A technology combining buffer solution and bovine lactoferrin, which is applied in the field of protein detection, can solve the problems of inaccurate lactoferrin determination results and low recovery rate of lactoferrin, and achieve stable results, good applicability, and long detection time Effect

Pending Publication Date: 2022-04-29
BEIJING MEIZHENG BIOLOGICAL TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, using the method of the existing group standard "T / TDSTIA006-2019", the recovery rate of lactoferrin in dairy products is low, and the value of lactoferrin is not accurate

Method used

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  • Binding buffer solution for extracting lactoferrin, preparation method and application of binding buffer solution and detection method of bovine lactoferrin
  • Binding buffer solution for extracting lactoferrin, preparation method and application of binding buffer solution and detection method of bovine lactoferrin
  • Binding buffer solution for extracting lactoferrin, preparation method and application of binding buffer solution and detection method of bovine lactoferrin

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preparation example Construction

[0033] In the present invention, there is no special limitation on the preparation process of the extraction reagent, and the extraction reagent with the above-mentioned concentration requirements can be prepared according to the well-known process in the art. In an embodiment of the present invention, the preparation process of the extraction reagent specifically includes: weighing 2.922g of sodium chloride, dissolving it in 10mL of deionized water, adding 5mL of Tween-20, and distilling the volume to 20mL.

[0034] In the present invention, it is sufficient that the amount of the water meets the concentration requirements of the above-mentioned components in the binding buffer.

[0035] The present invention provides a method for preparing a binding buffer for extracting lactoferrin described in the above technical scheme, comprising the following steps:

[0036] According to the concentration ratio of the preparation raw materials, disodium hydrogen phosphate dodecahydrate, s...

Embodiment 1

[0096] Different brands of commercially available sterilized milk (Jindian skim pure milk, Yili skim pure milk, Wandashan skim pure milk, Mengniu pure milk, Deluxe skim pure milk, Jindian whole milk) were weighed 10g (accurate to 0.01g) sterilized milk in a 50mL centrifuge tube, add binding buffer to make up to 50mL and mix well; then centrifuge at 4°C, 12000r / min for 10min, and use a large-range pipette to remove the middle layer after centrifugation Aspirate the liquid part, take 10mL as the sample solution to load the heparin affinity column, rinse the heparin affinity column with 10mL binding buffer after loading, then elute with 4mL eluent, centrifuge at 4°C, 12000r / min for 5min , carry out HPLC detection to the purified liquid obtained; each type of sterilized milk is provided with a blank control (the spiked amount is 0, that is, the actual sample is detected, no additional lactoferrin standard is added) and a spiked experiment (50 μg / g spiked amount, added Lactoferrin ...

Embodiment 2

[0102] Different brands of commercially available solid milk powder (Yilijin Lingguan 1st stage, Aoyou Aiyou 2nd stage, Beingmei Tongxiang 1st stage, Feihe Zhenaibeihuo 3rd stage, Junle Baolezhen 1st stage) were weighed 2.5g respectively (accurate to 0.01g) sample in a 50mL centrifuge tube, first add 30mL of binding buffer, vortex until the sample is completely dissolved, then add binding buffer to 50mL and mix well; then at 4°C, 12000r / min Centrifuge for 10min. After centrifugation, use a large-capacity pipette to suck out the middle liquid part. Take 10mL as the sample solution to load the heparin affinity column. The eluent was eluted, and after centrifugation at 4°C and 12000r / min for 5min, the resulting purified solution was subjected to HPLC detection; each brand of milk powder was set up with a blank control group (scaling amount of 0) and a spiked experiment (scaling amount of 500ppm, adding lactoferrin standard stock solution).

[0103] Table 2 Test results of differ...

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Abstract

The invention provides a binding buffer solution for extracting lactoferrin, a preparation method and application of the binding buffer solution and a detection method of bovine lactoferrin, and belongs to the technical field of protein detection. The binding buffer solution serves as an extracting solution of lactoferrin, the binding capacity of an affinity column on lactoferrin in a dairy product can be guaranteed, enrichment and detection of lactoferrin in a sample can be achieved, the extraction rate and the recovery rate of lactoferrin in the product are increased, the influence of a sample matrix on a target substance is overcome, and the detection accuracy is improved. The method can be applied to extraction of lactoferrin in different types of dairy products, quantitative detection of lactoferrin in different types of dairy products can be met, the recovery rate can reach 90% or above, the result is stable, and after elution, the peak shape is good, and impurity peaks are few.

Description

technical field [0001] The invention relates to the technical field of protein detection, in particular to a binding buffer for extracting lactoferrin, a preparation method and application thereof, and a detection method for bovine lactoferrin. Background technique [0002] Lactoferrin (LF) is an important iron-binding glycoprotein in milk, with a relative molecular mass of 70-80 kDa, mainly expressed and secreted by mammary epithelial cells, and widely distributed in mammalian milk, especially in colostrum , Lactoferrin is also contained in various other tissue secretions (saliva, tears or bile, etc.), which provides the first and most important line of defense for the mucosal system invaded by a large number of pathogenic microorganisms. Lactoferrin not only participates in the transport of iron, but also has powerful biological functions such as broad-spectrum antibacterial, antioxidative, anticancer and immune system regulation. It is considered to be a new type of antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/86
CPCG01N30/02G01N30/06G01N30/8634C07K14/79G01N2030/062G01N2030/8813G01N30/88
Inventor 田秀梅武冬霞顾浩柳家鹏
Owner BEIJING MEIZHENG BIOLOGICAL TECH
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