A method for inhibiting the digestion of lactoferrin in the human body

Abstract

The present invention discloses a method for inhibiting the digestion of lactoferrin in the human body. By preparing a complex of caffeine and lactoferrin and simulating in vitro digestion tests, it is found that caffeine increases the retention rate of lactoferrin in the oral cavity and intestine, while decreasing the retention rate in the stomach. This is beneficial for lactoferrin to bind with free oxygen radicals proximal to the pylorus of the small intestine, helping to protect the body from their harmful effects. The present invention prepares a complex of caffeine and lactoferrin, which can inhibit the digestion of lactoferrin in the human body, plays a positive role in enabling lactoferrin to better exert its functional characteristics, and at the same time, highly nutritious lactoferrin has broad application prospects in the development of functional foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of food, and particularly relates to a method for inhibiting the digestion of lactoferrin in the human body. Background Art

[0002] As is well known, polyphenols have a very high binding affinity with proteins, and are prone to interact with the active parts of protein molecules to form complexes, affecting the structure and functional properties of proteins. Bovine lactoferrin is an iron-binding glycoprotein found in milk and has a high iron-binding ability. Research shows that bovine lactoferrin can not only regulate the immune system of mammals, reduce gastrointestinal irritation, maintain iron balance in the body, promote the absorption of nutrients such as calcium and magnesium, but also has antibacterial, anti-inflammatory and anti-tumor activities. It is reported that intact bovine lactoferrin is transported to the proximal part of the pylorus of the small intestine, where it can bind to free oxygen radicals and help protect the body from their harmful effects. However, if bovine lactoferrin is stressed by physical and chemical factors such as temperature in the physiological environment and further degraded by gastric juice, the function of bovine lactoferrin will be damaged. Therefore, bovine lactoferrin must reach the gastrointestinal tract in a structurally intact state.

[0003] Caffeine is a stimulant for the activities of the central nervous system. It increases the circulation of chemicals such as cortisol and biologically active adrenaline in the body and increases brain activity. However, caffeine has an unpleasant taste, and excessive intake will have an adverse impact on the nervous and cardiovascular systems. Research has found that binding caffeine to proteins can overcome solubility problems and reduce the feeling of pain. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for inhibiting the digestion of lactoferrin in the human body.

[0005] For the above purpose, the technical solution adopted by the present invention is: compound lactoferrin with caffeine to prepare a caffeine-lactoferrin complex. The specific method is: add lactoferrin and caffeine into a PBS buffer solution so that the concentration ratio of caffeine to lactoferrin in the PBS buffer solution is 4:1 to 10:1, and vortex to completely dissolve lactoferrin and caffeine to obtain a caffeine-lactoferrin complex solution.

[0006] In the above method, when adding lactoferrin and caffeine into the PBS buffer solution, it is preferred that the concentration ratio of caffeine to lactoferrin in the PBS buffer solution is 8:1.

[0007] In the above method, it is further preferred that the concentration of lactoferrin in the PBS buffer solution is 0.5 - 5 mg / mL.

[0008] In the above method, the pH value of the PBS buffer solution is 6.0 - 8.3.

[0009] In the above method, the lactoferrin is obtained by extracting fresh cow milk through degreasing, precipitating casein, filtering out impurities, and then using ion exchange chromatography. Among them, the ion exchange column for the ion exchange chromatography is CM-Sepharose Fast Flow, and the mobile phase is a PBS buffer solution with a pH value of 7.4.

[0010] In the present invention, caffeine and lactoferrin are prepared into a complex. Through in vitro digestion experiments, it is found that caffeine increases the retention rate of lactoferrin in the oral cavity and intestine, while decreasing the retention rate in the stomach. This is beneficial for lactoferrin to bind with free oxygen radicals proximal to the pylorus of the small intestine, helping to protect the body from their harmful effects.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. The present invention can inhibit the digestion of lactoferrin in the human body, which plays a positive role in enabling lactoferrin to better exert its functional characteristics. At the same time, highly nutritious lactoferrin plays an important role in the development of functional dairy products and other aspects.

[0013] 2. The present invention has simple operation, strong functionality, and broad application prospects, and is of great significance for the application of lactoferrin in food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the quantitative experimental result of simulating the influence of caffeine on the in vitro digestion of lactoferrin. The abscissa represents each stage of digestion, and the ordinate represents the retention rate of lactoferrin or caffeine-lactoferrin complex. The retention rate of lactoferrin at each stage of digestion in the presence or absence of caffeine can be reflected in the figure.

[0015] Figure 2 It is the SDS-PAGE gel electrophoresis diagram of simulating the in vitro digestion of lactoferrin by caffeine, where A is bLF, B is CAF-bLF, C is orally digested bLF, D is orally digested CAF-bLF, E is gastric digested bLF, F is gastric digested CAF-bLF, G is intestinal digested bLF, and H is intestinal digested CAF-bLF. The degree of digestion at each stage and the molecular weight of the complex can be seen from the figure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The present invention will be further described in detail below in conjunction with the drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments only.

[0017] Example 1

[0018] 1. Extract lactoferrin from milk

[0019] Skim fresh milk with a high-speed refrigerated centrifuge, then adjust the pH to 4.6, precipitate casein again with a high-speed refrigerated centrifuge, and filter the impurities from the whey protein obtained from the upper layer with a 0.45 μm microporous membrane to obtain a crude lactoferrin product. Finally, obtain high-purity lactoferrin by ion exchange chromatography. The ion exchange column is CM-Sephadex FastFlow, and the mobile phase is PBS buffer with a pH of 7.4.

[0020] 2. Preparation of caffeine-lactoferrin complex

[0021] Add 88 mg of lactoferrin and 352 mg of caffeine to 110 mL of PBS buffer with a pH of 7.4, vortex for 30 s to completely dissolve lactoferrin and caffeine, obtain a caffeine-lactoferrin complex solution, and store it at 4 °C.

[0022] Example 2

[0023] In this example, lactoferrin in milk was extracted according to step 1 of Example 1. Add 88 mg of lactoferrin and 528 mg of caffeine to 110 mL of PBS buffer with a pH of 7.4, vortex for 30 s to completely dissolve lactoferrin and caffeine, obtain a caffeine-lactoferrin complex solution, and store it at 4 °C.

[0024] Example 3

[0025] In this example, lactoferrin in milk was extracted according to step 1 of Example 1. Add 88 mg of lactoferrin and 880 mg of caffeine to 110 mL of PBS buffer with a pH of 7.4, vortex for 30 s to completely dissolve lactoferrin and caffeine, obtain a caffeine-lactoferrin complex solution, and store it at 4 °C.

[0026] Measure the digestion characteristics of the caffeine-lactoferrin complex obtained in Example 1 above. The specific measurement methods and results are as follows:

[0027] (1) Measurement method

[0028] An in vitro digestive system (oral cavity, stomach, and intestine) was simulated to track the digestion patterns of pure lactoferrin (bLF) and caffeine-lactoferrin complex (CAF-bLF), and simulated digestive fluids for each digestion phase were obtained. The specific method is as follows: Add 1.195 g of Na 2 HPO 4 , 0.095 g of KH 2 PO 4 , 0.05 g of α-amylase (enzyme activity ≥ 4000 U g -1) Dissolve 4 g of NaCl in ultrapure water, and make up the volume to 500 mL with ultrapure water. Adjust the pH of the solution to 6.75 with PBS buffer at pH 7.4 to obtain artificial saliva for simulating oral digestion. Dissolve 0.21 g of NaCl and 0.087 g of pepsin in 100 mL of distilled water to maintain the final activity of pepsin at 3000 U mg -1 , and adjust the pH to 2.0 - 3.0 with 1.0 mol / L hydrochloric acid to obtain artificial gastric juice. Dissolve 0.88 g of NaH 2 PO 4 , 0.876 g of NaCl, and 0.1 g of trypsin in 100 mL of double-distilled water, and adjust the pH to 7.0 - 8.0 with 1.0 mol / L aqueous NaOH solution to obtain artificial intestinal juice.

[0029] Using PBS buffer at pH 7.4 as the solvent, prepare a lactoferrin solution at 0.8 mg / mL; add caffeine to the 0.8 mg / mL lactoferrin solution so that the added concentration of caffeine is 8 mg / mL, and vortex for 30 s to completely dissolve lactoferrin and caffeine to obtain a caffeine-lactoferrin complex solution. Mix 9 mL of artificial saliva with 1 mL of the above lactoferrin solution or caffeine-lactoferrin complex solution, and incubate in a constant-temperature shaking incubator at 37°C; after oral digestion, heat the sample to 100°C for 10 minutes to inactivate the enzyme; mix the oral digestive juice with artificial gastric juice at a volume ratio of 1:1 and digest in a 37°C water bath for 1 hour. To prevent the enzyme from converting the substrate, take out the gastric digestive juice, and then adjust the pH value to 8.0 with NaOH. Take an equal volume of artificial intestinal juice and mix it with the gastric digestive juice, and digest in a 37°C water bath for 1 hour; take out the intestinal digestive juice and heat it at 95°C for 5 minutes to inactivate trypsin. Collect the digestive juices at each stage for the following determinations:

[0030] a. BCA protein quantitative determination

[0031] Distribute 10% TCA evenly in 0.1 mL of the digestive juice of each system, and then soak it in ice water at 4°C for 30 minutes. Centrifuge at 4000 rpm at 4°C in a refrigerated centrifuge for 10 minutes. After removing the supernatant, evaluate the digestion rate of lactoferrin in each system using the BCA test. The experiment was carried out three times to obtain the average value.

[0032] b. SDS-polyacrylamide gel electrophoresis

[0033] The digestive juices of each system of the above-mentioned lactoferrin solution and caffeine-lactoferrin complex solution were mixed well with 5×SDS-PAGE loading buffer at a volume ratio of 4:1 and then loaded into EP tubes. They were heated in a 100°C water bath for 3 - 5 minutes and then cooled. 4 μL of indicator Marker was dropped into the leftmost well on the gel plate, and 10 - 15 μL of the following samples were dropped into the remaining sample wells: artificial saliva for digesting bLF, artificial saliva for digesting CAF-bLF, artificial gastric juice for digesting bLF, artificial gastric juice for digesting CAF-bLF, artificial intestinal juice for digesting bLF, and artificial intestinal juice for digesting CAF-bLF. Concentration was carried out at 80 V for 35 min, and then the voltage was adjusted to 120 V for separation for 1.5 h. When the indicator Maker was about 3 mm away from the bottom of the gel plate, electrophoresis was stopped. The gel plate was removed, the gel was scraped off, washed with deionized water, and stained with a staining solution (containing 0.25% Coomassie Brilliant Blue G-250, 10% acetic acid, 45% methanol, and the rest water) on a shaker for 2 hours. The gel was taken out, washed twice with distilled water, and then soaked in a decolorizing solution (containing 10% acetic acid and 25% methanol, and the rest water) overnight. The decolorizing solution should be changed regularly.

[0034] (2) Measurement results

[0035] As Figure 1 shown, after each stage of digestion was completed, the molecular weights of pure lactoferrin and caffeine-lactoferrin complex could be clearly seen. Caffeine effectively reduced the amount of lactoferrin digested in the stomach and ensured its physiological activity in the intestine. As Figure 2 shown, without oral and gastrointestinal processing, the molecular weights of caffeine-lactoferrin complex and pure lactoferrin were about 84 kDa. During oral and gastric digestion, pure lactoferrin and caffeine-lactoferrin complex were broken down into tiny peptide or amino acid fragments. However, when pure lactoferrin and caffeine-lactoferrin complex entered the intestinal digestion stage, they were broken down into smaller peptide segments and amino acid fragments. The lactoferrin bands in the gastric and intestinal digestion periods were lighter than those of the caffeine-lactoferrin complex, further indicating that the addition of caffeine could partially block the digestion of lactoferrin.

Claims

1. Use of a caffeine-lactoferrin complex for inhibiting the digestion of lactoferrin in the human body, Characterized in that: The preparation method of the caffeine-lactoferrin complex is as follows: Lactoferrin and caffeine are added to PBS buffer, and the concentration ratio of caffeine to lactoferrin in the PBS buffer is 4:1 to 10:

1. Vortex to completely dissolve lactoferrin and caffeine to obtain a caffeine-lactoferrin complex solution.

2. The use of the caffeine-lactoferrin complex according to claim 1 for inhibiting the digestion of lactoferrin in the human body, Characterized in that: Lactoferrin and caffeine are added to PBS buffer, and the concentration ratio of caffeine to lactoferrin in the PBS buffer is 8:

1.

3. The use of the caffeine-lactoferrin complex according to claim 1 or 2 for inhibiting the digestion of lactoferrin in the human body, Characterized in that: The concentration of lactoferrin in the PBS buffer is 0.5 to 5 mg / mL.

4. The use of the caffeine-lactoferrin complex according to claim 1 for inhibiting the digestion of lactoferrin in the human body, Characterized in that: The pH value of the PBS buffer is 6.0 to 8.

3.

5. The use of the caffeine-lactoferrin complex according to claim 1 for inhibiting the digestion of lactoferrin in the human body, Characterized in that: The lactoferrin is obtained by extraction using ion exchange chromatography after defatting fresh milk, precipitating casein, and filtering out impurities.

6. The use of the caffeine-lactoferrin complex according to claim 5 for inhibiting the digestion of lactoferrin in the human body, Characterized in that: The ion exchange column for the ion exchange chromatography is CM-Sepharose Fast Flow, and the mobile phase is PBS buffer with a pH value of 7.4.

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