Ultrasound-assisted simulated digestion method of milk protein active peptide and application thereof in health foods

Patent metadata

Field	Value
Publication number	US-11524977-B2
Application number	US-201816632546-A
Country	US
Kind code	B2
Filing date	Nov 12, 2018
Priority date	Nov 13, 2017
Publication date	Dec 13, 2022
Grant date	Dec 13, 2022

Abstract

An ultrasound-assisted simulated digestion method of a milk protein active peptide and an application thereof in health foods, pertaining to the technical field of intensive processing of dairy products and preparation of health foods. The method firstly employs ultrasonic pretreatment of casein and β-lactoglobulin, followed by enzymatic hydrolysis with a protease to prepare casein and ρ-lactoglobulin polypeptide, and traces the activity of the polypeptide by simulating gastrointestinal digestion, and then simulates absorption by intestinal epithelial cells with Caco-2 cells, to characterize a highly active milk protein polypeptide digested by the gastrointestinal tract and absorbed by the Caco-2 cells simulating absorption by the inner wall of the small intestine. The method has identified five such highly active milk protein polypeptides.

First claim

The invention claimed is:

1. A method for preparing a β-lactoglobulin (β-LG)-derived anti-inflammatory peptides, which is characterized by the following steps:
(1) extraction of β-LG: a concentration of 7% (w/v) whey protein solution is prepared by adding whey protein to 7% NaCl water, adjusting the pH to 2 with HCl, and centrifuging at 5000 rpm for 20 minutes to collect supernatant, then the supernatant is dialyzed using a dialysis bag with a molecular weight cut off of 14,000 Da, placed in 30 volumes of distilled water for 20 hours and retentate in the dialysis bag, β-LG, is collected;
(2) ultrasound treatment of β-LG: the β-LG with a concentration of 1 g/mL-4 g/mL is treated by an ultrasound equipment;
(3) enzymolysis of β-LG: after ultrasound treatment, the β-LG suspensions are preheated to 50° C.-70° C. and adjusted to pH 7.5-8.0 with 1.0 M NaOH, a protease (the ratio of protease to β-LG is 1:20-1:50) (w/w) is added to initiate the reaction and the enzymolysis time is 2-4 hours, the mixture is heated and maintained at 100° C. for 10 minutes to terminate the reaction, then the mixture is adjusted to pH 7.0 and centrifuged; the supernatant is collected, desalted, concentrated, and freeze-dried to a powder; wherein said protease is selected from the group consisting of alcalase, neutral protease and thermolysin,
(4) simulated GI digestion: β-LG-derived hydrolysate is subjected to simulated gastric and intestinal digestion, β-LG-derived hydrolysate is digested with gastric fluid at 1:20-1:50 (w/v) for 2-4 hours in a shaking incubator with 120-180 rpm at 37-° C. then the pH is adjusted to 6.8 and pancreatin is added at 1:100 (w/v) to form an intestinal fluid, the mixture is incubated for 4-6 h to mimic intestinal digestion, the digestion is terminated in boiling water for 10 minutes, the digest is cooled down and centrifuged at 10,000 g for 10 minutes to collect the supernatant, which is further centrifuged, desalted, concentrated, and freeze-dried to a powder;
(5) simulated intestinal epithelium absorption: a Caco-2 cells transport model is constructed, a concentration of 20 mg/mL β-LG hydrolysate digest is prepared by dissolving in an HBSS buffer, absorption of the casein hydrolysate digests is evaluated by adding the digests to an apical (AP) surface, basal (BL) surface samples at 0.5-4 hours are collected, desalted, concentrated, and freeze-dried;
(6) characterization of the β-LG derived peptides: the β-LG derived peptides absorbed by the Caco-2 cells transport model in step (5) are subjected to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis, the peptides with less than 500 Da are selected;
(7) the selected peptides in step (6) are synthesized and assayed for their anti-inflammatory activity, and the β-LG-derived peptide showing anti-inflammatory activity is:
Phe-Tyr-Gln-Ala (SU ID NO:4).

2. The method for preparing the β-lactoglobulin-derived anti-inflammatory peptide according to claim 1 , wherein the ultrasound treatment conditions in the step (2) are as follows: treatment time of 10 minutes-30 minutes; intermittent ratio of 10 seconds on/3 seconds off; temperature 25° C., wherein the ultrasound frequency used is selected from a single-frequency ultrasound of 20, 28 or 40 kHz, a dual-frequency simultaneous ultrasound of 20/40, 20/28 or 28/40 kHz and a triple-frequency simultaneous ultrasound of 20/28/40 kHz wherein the protease of step (3) is neutral protease, or thermolysin.

3. The method for preparing the β-lactoglobulin-derived anti-inflammatory peptide according to claim 1 , wherein the protease of the step (3) is thermolysin; and the ultrasound is triple-frequency simultaneous ultrasound of 20/28/40 kHz.

4. The method for preparing the β-lactoglobulin-derived anti-inflammatory peptide according to claim 1 , wherein the protease of the step (3) is alcalase.

5. An anti-inflammatory peptide consisting of the amino acid sequence Phe-Tyr-Gln-Ala (SEQ ID NO:4).

Assignees

• Univ Jiangsu

Inventors

• Ren Xiaofeng
• Liang Qiufang
• Ma Haile
• Duan Yuqing
• He Ronghai
• Yang Xiaoming
• Zhang Xi
• Hou Ting
• Chen Xinxiang

Classifications

• A23V2002/00

  Food compositions, function of food ingredients or processes for food or foodstuffs · CPC title

• C07K14/4717

  Plasma globulins, lactoglobulin · CPC title

• C07K5/1016

  and aromatic or cycloaliphatic · CPC title

• C07K5/0808

  the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu · CPC title

• C07K5/101 Primary

  the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu · CPC title