Method for determining bioaccessibility of triglyceride based on pH-stat combined with Caco-2 model
A triglyceride and biological technology, applied in the direction of biochemical equipment and methods, biological testing, microbial measurement/inspection, etc., can solve the problems of lack of research on the accessibility of disinfection organisms, and achieve moral superiority, high efficiency, low cost effect
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Embodiment 1
[0030] Example 1 Determination of digestibility of triglycerides by pH-stat model
[0031] Add 100 mg of triglycerides to the conical flask, then add 1.2 mL of simulated saliva with pH 6.8, incubate at 37°C for 5 min on a water bath shaker, and then add 2.4 mL of simulated gastric juice with pH 1.3, and incubate in a water bath shaker at 80 rpm Incubate for 2 h, and finally add 2.4 mL of simulated intestinal fluid pH 8.1.
[0032] The pH was maintained at 8.1 with a potentiometric titrator and titrated with 0.1 M NaOH. The temperature was controlled at 37 °C throughout the digestion process, and the amount of NaOH solution was recorded to calculate the digestibility of triglycerides.
[0033]
[0034]V NaOH is the amount of NaOH (mL) used to neutralize free fatty acids, C NaOH is the concentration of NaOH (0.1 M), M Lipid is the molar mass (g / mol) of the different lipids, m Lipid is the mass (g) of the triglyceride sample initially added.
[0035] Calculate the tri...
Embodiment 2
[0037] Example 2 pH-stat model to measure the digestion rate of triglycerides
[0038] Put 50 mg of triglycerides into a conical flask, add 1.2 mL of simulated saliva (pH 6.8), and incubate for 5 min at 37 °C on a water bath shaker. Then, 2.4 mL of simulated gastric juice (pH 1.3) was added, and the samples were incubated with a water bath shaker (80 rpm) for 2 h. Finally add and 2.4 mL of simulated intestinal fluid (pH 8.1). The pH was maintained at 8.1 using a potentiometric titrator and titrated with 0.05 M NaOH. The temperature during the entire digestion process was controlled at 37 °C, and the amount of NaOH solution was recorded to calculate the digestion rate of triglycerides.
[0039]
[0040] in k is the rate constant (s -1 ), t is the digestion time (min), y is the proportion (%) of remaining free fatty acids.
[0041] Calculate triglyceride digestibility according to formula (2) (result as image 3 ).
[0042] The results showed that the fatty acid hydro...
Embodiment 3
[0043] Example 3 Establishment of Caco-2 cell model
[0044] The Caco-2 cell culture protocol is as follows: Caco-2 cells (purchased from Shanghai Cell Bank-ATCC Cell Resource Center) are cultured in DMEM high-glucose medium containing 8%-20% fetal bovine serum, 1% double antibody and non-essential amino acids, cells at 25 cm 2 cultured in a flask, which was placed at 37 °C, 5% CO 2 In the incubator, the medium was changed every two days. When the cells grew to cover 80% of the bottom of the flask, EDTA-trypsin was added to digest, and the cells were passaged at a ratio of 1:3.
[0045] When the Caco-2 cells have grown to log phase, i.e. cover 80% of the flask, wash twice with 2 mL of PBS. Digest with 1 mL of EDTA-trypsin to ensure cells are single and make a cell suspension with medium. Count with a cell counter and add a volume of fresh medium to dilute the cells to 1×10 5 cells / mL and seeded on 6-well Transwell plates. Then place the Transwell plate at 37 °C, 5% CO 2 ...
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