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Liquid milk in-vitro static digestion and intestinal cell culture method for simulating gastrointestinal environment of rats

A technology for intestinal cells and liquid milk, applied in the field of food digestion and cell biology, can solve the problems of limited accuracy and affect the digestion process, and achieve the effect of improving reliability and authenticity

Pending Publication Date: 2022-03-04
SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to differences in food state, texture and test subjects will significantly affect the digestion process, the values ​​of relevant digestion parameters (pH, time, enzyme amount, etc.) in this system are mainly based on the average or generalization of existing data. limited accuracy

Method used

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  • Liquid milk in-vitro static digestion and intestinal cell culture method for simulating gastrointestinal environment of rats
  • Liquid milk in-vitro static digestion and intestinal cell culture method for simulating gastrointestinal environment of rats
  • Liquid milk in-vitro static digestion and intestinal cell culture method for simulating gastrointestinal environment of rats

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Simulating the digestion process of liquid milk in the gastrointestinal tract of rats

[0074] Wistar male rats (SPF grade) of three age groups were purchased for the test. Among them, "group 1, young age" is 1-2 weeks old, 64 rats; "group 2, adult" is 6-8 weeks old, 64 rats; "group 3, old age" is 20-22 weeks old, 64 rats only. All rats were fasted for 12 hours in advance, and fresh skim milk (2.5mL, with a total milk protein content of about 75mg) was artificially administered to the rats (in order to ensure that the starting time was basically uniform, each group was operated by 4 skilled experimenters at the same time , with 2-person number and timing). Dissect rats at 8 time points such as 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, and 4 hours after gavage (for pH and enzyme activity tests, each time point Get 4; for the total nitrogen test, get 4 additionally at each time point), as follows:

[0075] a. Measure the pH of the gastri...

Embodiment 2

[0082] Using the parameters constructed in Example 1, taking adult Wistar mice as an example, the soluble digestion product was pretreated by high-temperature boiling to inactivate trypsin, and then used for intestinal cell culture.

[0083] Specifically:

[0084] ①Take 100mL of raw milk (the crude protein content of milk is determined to be 3200mg), and centrifuge to skim the fat.

[0085] ② In vitro simulated gastric digestion steps of cow's milk. Use a glass Erlenmeyer bottle to hold milk and adjust the pH to 2.8. Prepare intestinal digestion buffer (pH=2.8): KCl 6.9mM, KH 2 PO 4 0.9mM, NaHCO 3 25mM, NaCl 47.2mM, MgCl 2 0.1mM, (NH 4 ) 2 CO 3 0.5mM, CaCl 2 0.15mM, add milk volume 1 / 2 (50mL) gastric digestion buffer. Pepsin (Sigma, P6887, 40 mg) was weighed at 1 / 80 of the milk protein mass, and a small amount (1-2 mL) of gastric digestion buffer was used to dissolve the pepsin. Add the pepsin solution into milk (shake well), and quickly transfer the Erlenmeyer ...

Embodiment 3

[0092] Same as Steps ① to ④ of Example 2, step ⑤ adopts the ultrafiltration method to pretreat the milk digestate (50 mL) to obtain the lower layer filtrate (<3KD). Add 300 μL of saturated NaCl solution, adjust the osmotic pressure of the solution to 310 mOsm / L, and sterilize by microfiltration. Subsequent steps are the same as ⑦ in embodiment 2.

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Abstract

The invention discloses a liquid milk in-vitro static digestion and intestinal cell culture method for simulating the gastrointestinal environment of rats. The method comprises the following steps: (1) selecting defatted raw milk; (2) constructing stomach digestion parameters, and performing stomach digestion treatment to obtain stomach digestive juice; (3) constructing intestinal digestion parameters, and performing intestinal digestion treatment to obtain intestinal digestion liquid; (4) centrifuging the intestinal digestive juice, and taking supernate to obtain a soluble digestion product; (5) pretreating the soluble digestion product; (6) regulating the osmotic pressure of the product to be isotonic, and sterilizing; and (7) culturing intestinal cells. By simulating the gastrointestinal effect environment of the Wistar rat, the in-vitro digestion endpoint of the liquid milk is closer to the in-vivo digestion result of the Wistar rat, and the reliability and authenticity of in-vitro digestion are improved; the liquid milk digestible product can be used for in-vitro intestinal cell culture and can be used for carrying out various biological evaluation tests.

Description

technical field [0001] The invention belongs to the technical field of food digestion and cell biology, and specifically relates to a method for in vitro static digestion of liquid emulsion and intestinal cell culture that simulates the gastrointestinal environment of rats. Background technique [0002] Liquid milk contains various basic nutrients and functional active substances, and may also contain allergens, pesticide / drug residues and other substances. To understand or evaluate the nutritional value, physiological effects, and health and safety risks of each component in liquid milk on the human body, in addition to general chemical determination, the method of "biological digestion + biological evaluation" is also used. Animal models and human clinical trials are costly, complicated to operate, subject to environmental interference, and accompanied by ethical (animal welfare) issues. As a simple alternative to the above-mentioned process, simulated digestion in vitro ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0679C12N2500/84C12N2500/34
Inventor 杨怀谷王旭苹程镜蓉林耀盛邹金浩唐道邦刘学铭
Owner SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI