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

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.

Claims

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).

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a technical route diagram of ultrasound-assisted enzymolysis coupled to simulated gastrointestinal digestion and absorption to prepare casein-derived peptides.

(2) FIG. 2 shows chromatographic profiles of digests derived from casein hydrolysate before and after transcellular transport at 0.5, 1.0, 2.0 and 4.0 h (A).

(3) FIG. 3 is a technical route diagram of ultrasound-assisted enzymolysis coupled to simulated gastrointestinal digestion and absorption to prepare β-LG-derived peptides.

EMBODIMENTS

(4) 1. Experimental Method

(5) 1.1. Degree of Hydrolysis (DH) and Protein Conversion Rate

(6) The DH of casein was determined using the pH-state method, DH is defined as the percentage of cleaved peptide bonds, which was calculated using the equation below:

(7) $\mathrm{DH}\left(\%\right)=\frac{V\times N}{\alpha \times M\times h_{\mathrm{tot}}}\times 100\%$

(8) Where, V is the titrant volume of NaOH (mL), N is the concentration of NaOH (mol/L), a is the degree of dissociation of α-NH.sub.2 (0.985 for casein), M is the mass of protein (g), and h.sub.tot is the number of peptide bonds in the substrate; different proteins had different values of h.sub.tot the empirical value of casein is h.sub.tot=7.35 mol/g.

(9) The total nitrogen content of the casein protein and its derived hydrolysate was determined by the Kjeldahl method, and the conversion rate of casein was calculated as follows:
Protein conversion rate (%)=hydrolysate nitrogen content/substrate protein nitrogen content*100%.

(10) 1.2 Measurement of ACE Inhibitory Activity

(11) The FAPGG was used as the substrate of ACE, each reaction component was added according to the Table, and the ACE inhibition rate of the sample was measured with a microtiter plate reader at 340 nm.

(12) Where X.sub.1 is the absorbance of the control group without protein hydrolysates before reaction, Y.sub.1 is the absorbance of the sample group before the reaction, X.sub.2 is the absorbance of the blank group after the reaction, and Y.sub.2 is the absorbance of the sample group after the reaction. The test was performed five times. The ACE inhibitory activity was calculated as follows:
The ACE inhibitory rate (%)=100−(ΔA.sub.sample)/(ΔA.sub.blank)×100%
ΔA.sub.sample=X.sub.1−X.sub.2,ΔA.sub.blank=Y.sub.1−Y.sub.2.

(13) TABLE-US-00001 Measurement of ACE inhibitory activity Blank Sample (μL) (μL) ACE (0.1 U/mL) 10 10 FAPGG (1 mmol/L) * 50 50 Matrix buffer ** 40 0 ACE inhibitor 0 40 Note: FAPGG (1.0 mmol/L) was prepared by taking 3.994 mg of FAPGG plus matrix buffer, making up to 10 mL, dissolving and mixing, and then storing at 4° C. in the dark. Matrix buffer ** was prepared by dissolving 1.910 g of HEPES and 1.755 g of NaCl in double distilled water, adjusting the pH to 8.3 with NaOH, and replenishing the water to 100 mL, and storing at 4° C. for later use.

(14) 1.3 Cell Culture

(15) The human colon adenocarcinoma cell line, Caco-2 (HTB-37™) was obtained from American-type culture collection (ATCC. Manassas. Va., USA). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2.5% HEPES, 1% non-essential amino acids and 1% antibiotics. Cells were incubated at 37° C. in a humidified atmosphere (5% CO.sub.2). The medium was replaced three times a week, and the cells were subcultured (at 80-90% confluence) by using 0.25% trypsin-EDTA treatment before use in the experiments.

(16) 1.4 Measurement of Cytotoxicity

(17) The cell cytotoxic properties were monitored using an Alamar Blue assay. Briefly, Caco-2 cells were grown in 96-well plates at a density of 1×10.sup.4 cell/well for 24 h. After 24 h, the medium was changed and the cells were treated with various concentrations (10-50 mg/nil) of casein hydrolysate for another 24 h. After 24 h treatment, the media was discarded, and the fresh medium with 10% Alamar Blue reagent was added and incubated for an additional 4 h at 37° C. The fluorescence intensity of the wells was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability is expressed as a percentage compared to untreated cells.

(18) 1.5 Simulated Intestinal Epithelium Absorption Using Caco-2 Cells

(19) The samples collected from the AP and BL surfaces of Caco-2 cells were analyzed on an Acquity Ultra-Performance Liquid Chromatograph (UPLC) system with an Acquity UPLC BEH C.sub.18 column (100 mm×2.1 mm i.d., 1.7 μm, Waters, Milford, Mass., USA) using an injection volume of 15 μL. Mobile phases were solvent A (1% TFA in Milli-Q water) and solvent B (1% TFA in acetonitrile). The peptides were eluted with a gradient of solvent A (100-75% in 25 min, 75-50% in 25-35 min) at a flow rate of 0.3 m/min. The elution was monitored at 220 nm. Absorption was expressed as the percentage of total peak area calculated at different time points (0.5 h, 1 h, 2 h and 4 h) in the BL surface as compared to 0 h in the AP surface.

(20) 1.6 Identification of Casein-Derived Peptides Using UPLC-MS

(21) The liquid chromatography column used in this study was nanoACQUITY BEH130 C.sub.18 (75 μm×50 mm, 1.7 μm. The solvent A was acetonitrile (ACN) containing 0.1% formic acid. The peptides were separated using the following gradient: increasing solvent B containing water with 0.1% formic acid from 1% to 6% B in 2 min, to 25% B in 23 min. to 45% B in 15 min. to 75% B in 5 min, to 95% B in 5 min, and keeping at 95% B for 5 min. The mass spectrometer was operated in a positive mode with a capillary voltage of 3.5 kV and a source temperature of 100° C. Spectra were recorded over the m/z ranges of 200-1000 in MS mode and in 50-1990 MS/MS mode. The amino acid sequences of peptides were analyzed using Mass Lynx software (Micromass U.K. Ltd.). Peaks Viewer 4.5 (Bioinformatics Solutions Inc., Waterloo, ON, Canada), in combination with manual de novo sequencing was used to process the MS/MS data. Identified peptide sequences were synthesized (>98% purity) by Genscript Corp (Piscataway, N.J.) and used for the bioactivity assays.

Example 1

(22) Ultrasound treatment of casein. Casein with a concentration of 1 g/100 mL was prepared by dissolving in a phosphate buffer (pH 7.8). The above concentration of casein suspension was treated by ultrasound equipment. The ultrasound treatment conditions are as follows: treatment time 30 min, intermittent ratio 10 s/3 s; temperature 25° C. Single-frequency ultrasound with 40 kHz was used for the sample treatment.

(23) Enzymolysis of casein. After ultrasound treatment, the casein suspensions were preheated to 50° C. and adjusted to pH 8.0 with 1.0 M NaOH. Alcalase (the ratio of E/S was 1:20)(w/w) was added to initialize the reaction, and the enzymolysis time was 2 h. The mixture was heated and maintained at 100° C. for 10 min to terminate the reaction. Then the mixture was adjusted to pH 7.0 and centrifuged; the supernatant was collected, desalted, concentrated, and freeze-dried to a powder. The DH and protein conversion rate (CR) of the casein, and the ACE inhibitory activity of the casein-derived hydrolysate were determined.

(24) Simulated gastrointestinal digestion. Simulated gastric and intestinal fluids were prepared according to the U.S. Pharmacopeia (USP30-NF25). Briefly, Casein-derived hydrolysates were digested with gastric fluid at 1:20 (w/v) for 4 h in a shaking incubator with 120 rpm at 37° C. Then the pH was adjusted to 6.8 and pancreatin was added at 1:100 (w/v) to form the intestinal fluid. The mixture was incubated for a further 6 h to mimic intestinal digestion. The digestion was terminated in boiling water for 10 min. The digests were allowed to cool down and were centrifuged at 10,000 g for 10 min to collect the supernatant, which was further centrifuged, desalted, concentrated, and freeze-dried to a powder. The ACE inhibitory activity of the 4 h's and 10 h's casein hydrolysate digest was measured.

(25) As shown in Table 1, after ultrasound pretreatment, the DH of casein increased from 10.02% to 16.54%, and the protein conversion rate increased from 30.10% to 44.08%. The ACE inhibitory activity of casein hydrolysate, as shown by IC.sub.50 value, was decreased from 64.21 μg/mL to 52.13 μg/mL, indicating ultrasound pretreatment largely improved the ACE inhibitory activity of casein hydrolysate. After simulated gastric digestion and simulated intestinal digestion, the casein hydrolysate digest showed good ACE inhibitory activity, while the IC.sub.50 values were 49.21 μg/mL and 55.19 μg/mL, respectively (Table. 2). The above results indicate that the casein-derived hydrolysate exhibits excellent ACE inhibitory activity after simulated GI digestion.

Example 2

(26) Ultrasound treatment of casein. Casein with a concentration of 2 g/100 mL was prepared by dissolving in a phosphate buffer (pH 7.8). The above concentration of casein suspension was treated by ultrasound equipment. The ultrasound treatment conditions are as follows: treatment time 20 min; intermittent ratio 10 s/3 s; temperature 30° C. Dual-frequency simultaneous ultrasound treatments of 20/40 kHz were used for the sample treatment.

(27) Enzymolysis of casein. After ultrasound treatment, the casein suspensions were preheated to 55° C. and adjusted to pH 8.0 with 1.0 M NaOH. Neutral protease (the ratio of E/S was 1:30) (w/w) was added to initialize the reaction, and the enzymolysis time was 4 h. The mixture was heated and maintained at 100° C. for 10 min to terminate the reaction. Then the mixture was adjusted to pH 7.0 and centrifuged; the supernatant was collected, desalted, concentrated, and freeze dried to a powder. The DH and CR of the casein, and the ACE inhibitory activity of the casein-derived hydrolysate was determined.

(28) Simulated gastrointestinal digestion. Simulated gastric and intestinal fluids were prepared according to the U.S. Pharmacopeia (USP30-NF25). Briefly. Casein-derived hydrolysates were digested with gastric fluid at 1:30 (w/v) for 3 h in a shaking incubator with 150 rpm at 37° C. Then the pH was adjusted to 6.8 and pancreatin was added at 1:100 (w/v) to form the intestinal fluid. The mixture was incubated for a further 4 h to mimic intestinal digestion. The digestion was terminated in boiling water for 10 min. The digests were allowed to cool down and centrifuged at 10,000 g for 10 min to collect the supernatant, which was further centrifuged, desalted, concentrated, and freeze-dried to a powder. The ACE inhibitory activity of the 4 h's and 10 h's casein hydrolysate digest was measured.

(29) As shown in Table 1, after dual-frequency simultaneous ultrasound pretreatment, the DH of casein increased from 5.21% to 9.45%, and the protein conversion rate increased from 18.11% to 22.39%. The ACE inhibitory activity of casein hydrolysate indicated by IC.sub.50 value was decreased from 100.23 μg/mL to 95.21 μg/mL, indicating ultrasound pretreatment largely improved the ACE inhibitory activity of casein hydrolysate. After simulated gastric digestion, the casein hydrolysate digest showed good ACE inhibitory activity; its IC.sub.50 value was 72.11 μg/mL; after simulated intestinal digestion, the casein hydrolysate digest still showed good ACE inhibitory activity, and its IC.sub.50 value was 79.03 μg/mL (Table. 2). The above results indicate that the casein-derived hydrolysate still exhibited excellent ACE inhibitory activity after simulated GI digestion.

Example 3

(30) Ultrasound treatment of casein. Casein with a concentration of 5 g/100 mL was prepared by dissolving in a phosphate buffer (pH 7.8). The above concentration of casein suspension was treated by ultrasound equipment. The ultrasound treatment conditions are as follows: treatment time 10 min; intermittent ratio 10 s/3 s; temperature 40° C. Triple-frequency simultaneous ultrasound treatments of 20/40/60 kHz were used for the sample treatment.

(31) Enzmylosis of casein. After ultrasound treatment, the casein suspensions were preheated to 70° C. and adjusted to pH 8.0 with 1.0 M NaOH. Papain (the ratio of E/S was 1:50) (w/w) was added to initialize the reaction, and the enzymolysis time was 2 h. The mixture was heated and maintained at 100° C. for 10 min to terminate the reaction. Then the mixture was adjusted to pH 7.0 and centrifuged; the supernatant was collected, desalted, concentrated, and freeze-dried to a powder. The DH and CR of the casein, and the ACE inhibitory activity of the casein-derived hydrolysate were determined.

(32) Simulated gastrointestinal digestion. Simulated gastric and intestinal fluids were prepared according to the U.S. Pharmacopeia (USP30-NF25). Briefly, Casein-derived hydrolysates were digested with gastric fluid at 1:50 (w/v) for 4 h in a shaking incubator with 180 rpm at 37° C. Then the pH was adjusted to 6.8 and pancreatin was added at 1:100 (w/v) to form the intestinal fluid. The mixture was incubated for a further 6 h to mimic intestinal digestion. The digestion was terminated in boiling water for 10 min. The digests were allowed to cool down and centrifuged at 10,000 g for 10 min to collect the supernatant, which was further centrifuged, desalted, concentrated, and freeze-dried to a powder. The ACE inhibitory activity of the 4 h's and 10 h's casein hydrolysate digest was measured.

(33) As shown in Table 1, after triple-frequency ultrasound pretreatment, the DH of casein increased from 7.21% to 11.36%, the protein conversion rate increased from 21.98% to 26.02%. The IC.sub.50 value of ACE inhibitory activity of casein hydrolysate was decreased from 97.32 μg/mL to 90.11 μg/mL, indicating ultrasound pretreatment largely improved the ACE inhibitory activity of casein hydrolysate.

(34) After simulated gastric digestion, the casein hydrolysate digest showed good ACE inhibitory activity, its IC.sub.50 value was 65.32 μg/mL; after simulated intestinal digestion, the casein hydrolysate digest still showed good ACE inhibitory activity, its IC.sub.50 value was 60.31 μg/mL (Table. 2). The above results indicate that the casein derived hydrolysate still exhibited excellent ACE inhibitory activity after simulated GI digestion.

(35) TABLE-US-00002 TABLE 1 Effects of ultrasound pretreatment on the DH, CR and ACE inhibitory activity of casein hydrolysates by different enzymatic hydrolysis Traditional hydrolysis Ultrasound assisted hydrolysis IC.sub.50 of ACE IC.sub.50 of ACE inhibitory Ultra- inhibitory activity sound activity Enzyme DH (%) CR (%) (μg/mL) mode DH (%) CR (%) (μg/mL) Alcalase 10.02 ± 1.78  30.10 ± 0.91 64.21 ± 9.12 40 kHz 16.54 ± 0.99 44.08 ± 0.59 52.13 ± 6.11 Neutral 5.21 ± 0.52 18.11 ± 0.37 100.23 ± 14.98 20/40  9.45 ± 0.27 22.39 ± 0.37 95.21 ± 8.0  protease kHz Papain 7.21 ± 0.49 21.98 ± 0.64  97.32 ± 10.43 20/40/60 11.36 ± 1.18 26.02 ± 0.64  90.11 ± 11.34 kHz

(36) TABLE-US-00003 TABLE 2 Effects of different casein hydrolysates before gastric digestion, after gastric digestion, intestinal digestion and Caco-2 cells absorption on ACE inhibitory activity IC.sub.50 of ACE inhibitory activity (μg/mL) After Before After After Caco-2 Casein-derived gastric gastric intestinal cells hydrolysates digestion digestion digestion absorption Alcalase 52.13 ± 6.11  49.21 ± 4.91 55.19 ± 5.72 21.37 ± 2.07 hydrolysates Neutral protease 95.21 ± 8.0   72.11 ± 6.42 79.03 ± 8.19 hydrolysates Papain 90.11 ± 11.34 65.32 ± 5.0  60.31 ± 6.23 hydrolysates

Example 4

(37) The casein-derived hydrolysate digest prepared in example 1 was subjected to Caco-2 mimicking intestinal endothelial cell absorption.

(38) The cytotoxicity of the casein-derived hydrolysate digest to Caco-2 cells was first detected. The absorption model of Caco-2 cells mimicking intestinal endothelial cells was built as follows: Caco-2 cells were grown in 12-well Transwell® plates at a concentration of 2×10.sup.5 cells/mL. The medium of the cell was replaced every other day. After 21 days of cell culture, some evaluation indicators of the Caco-2 cells were measured, including epithelial cell resistance, alkaline phosphatase activity, and sodium fluorescein leakage test. Before initiation of the transport experiments, the Caco-2 cells were washed by an HBSS buffer; and 0.5 mL of the casein hydrolysate digest (20 mg/mL, dissolved in an HBSS buffer) was added to the AP surface; 1.5 mL of HBSS buffer was added to the BL surface; finally, the Caco-2 cells were incubated for 4 h at 37° C. 0.2 mL of AP surface samples at 0 h and BL surface samples at 0.5, 1, 2, and 4 h were collected for the absorption detection. The AP (apical) surface samples and BL (basolateral) surface samples at 4 h were collected for the ACE inhibitory activity detection, respectively. The casein hydrolysates digests and their absorbed digests were subjected to the amino acid sequences analysis by UPLC-MC. Some small peptides with strong ionic strength were screened out, synthesized, and detected for ACE inhibitory activity.

(39) As shown in Table 3, the addition of casein-derived hydrolysate digest increased the viability of Caco-2 cells, indicating that casein-derived hydrolysate digest didn't have any toxicity in Caco-2 cells and helped the growth of Caco-2 cells. As shown in FIG. 1, the absorption of the casein-derived hydrolysate digest from the AP to BL surface increased with time and reached 2.31% at 4 h, indicating selective absorption of peptides in Caco-2 cells. The absorbed digest obtained from the BL surface at the end of 4-hour transport study was tested for its in vitro ACE inhibitory effect and compared to that of the casein hydrolysate digest. After absorption by Caco-2 cells, the IC.sub.50 value of the ACE inhibitory activity of the casein hydrolysate digest decreased from 55.19 μg/mL to 21.37 μg/mL (Table 2). The above results showed that the ACE inhibitory activity of casein polypeptide was significantly enhanced by Caco-2 cells mimicking intestinal endothelial cells absorption, which indicates that the absorbed polypeptide displayed stronger ACE inhibitory activity than the digest. The absorbed peptides were subjected to LC-ESI-MS/MS analysis. The small peptides with strong ionic strength were chosen out, synthesized and further assayed for their ACE inhibitory activity. Three peptides with high ACE inhibition, Leu-Gln-Pro-Pro (LQPP) (SEQ ID No. 1). Ala-Pro-Tyr (APY) (SEQ ID No. 2), Leu-Ser-Leu-Pro (LSLP) (SEQ ID No. 3) were chosen out; their IC.sub.50 were 14.21 μM, 19.12 μM, and 21.09 μM, respectively (Table 4).

(40) TABLE-US-00004 TABLE 3 Cytotoxicity of casein hydrolysate derived digests at different concentration in Caco-2 cells Concentration of digest Caco-2 (mg/Ml) viability Control — 100 Group 1 5 109.8 ± 5.3 Group 2 10 119.2 ± 8.5 Group 3 20  110.2 ± 10.6 Group 4 50 103.6 ± 9.8

(41) TABLE-US-00005 TABLE 4 The peptides that are absorbed by Caco-2 cells were sequenced, identified and chemically synthesized to validate the ACE inhibitory activity of the identified peptides. Peptide IC.sub.50 of ACE inhibitory sequence activity (μg/mL) LQPP 14.21 APY 19.12 LSLP 71.09

(42) Experimental method and examples of ultrasound-assisted simulated digestion and absorption method for β-LG derived inflammatory peptide

(43) 2. Experimental Method

(44) 2.1 Degree of Hydrolysis (DH) and Protein Conversion Rate

(45) The DH of β-LG determined using the pH-state method, DH is defined as the percentage of cleaved peptide bonds, which was calculated using the equation below:

(46) $\mathrm{DH}\left(\%\right)=\frac{V\times N}{\alpha \times M\times h_{\mathrm{tot}}}\times 100\%$

(47) Where, V is the titrant volume of NaOH (mL), N is the concentration of NaOH (mol/L), a is the degree of dissociation of α-NH.sub.2 (0.99 for β-LG), M is the mass of protein (g), and hr is the number of peptide bonds in the substrate, different proteins had different values of ha, the empirical value of β-LG is h.sub.tot=7.35 mol/g.

(48) The total nitrogen content of the β-LG and its derived hydrolysate was determined by the Kjeldahl method, and the conversion rate of β-LG was calculated as follows:
Protein conversion rate (%)=hydrolysate nitrogen content/substrate protein nitrogen content*100%

(49) 2.2 Cell Culture

(50) The endothelial cell line, EA.hy926 (CRL-2922™), and human colon adenocarcinoma cell line, Caco-2 (HTB-37), were purchased from American-type culture collection (ATCC, Manassas, Va., USA). DMEM supplemented with 10% FBS, 2.5% HEPES, 1% antibiotics, and 1% non-essential amino acids was used as the cell growth medium. The cells were incubated in a humidified atmosphere with 5% CO.sub.2 at 37° C. The medium was replaced for three times for every week, and the cells were subcultured using 0.25% trypsin-EDTA treatment.

(51) 2.3 Measurement of Cytotoxicity

(52) The cell cytotoxic properties were monitored using an Alamar Blue assay. Caco-2 cells were seeded in 96-well plates at a density of 1×10.sup.4 cell/well for 24 h. Then the cells were treated with various concentrations (10-50 mg/mL) of β-LG hydrolysate for another 24 h in a fresh medium. After the 24 h treatment, the media was discarded, and the fresh medium with 10% Alamar Blue reagent was added and incubated for 4 h at 37° C. The fluorescence intensity of the wells was measured at an emission wavelength of 590 nm and an excitation wavelength of 560 nm. The viability of the treated cells was expressed as a percentage as compared to untreated cells.

(53) 2.4 Measurement of Anti-Inflammatory Activity

(54) The peptides were subjected to study the anti-inflammatory activity; the levels of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) expressed in EA.hy926 cells were detected by Western blot as inflammatory biomarkers. The EA, hy926 cells with passage number <12 were grown in 48-well plates. The cells reaching 80-90% confluence were treated with various concentrations (2.5 mg/mL of hydrolysates and digests, 0.2 mM and 3.0 mM of synthetic peptides) of the samples for 18 h. Then the cells were stimulated with TNF-α at 10 ng/mL and incubated for an additional 6 h in order to induce inflammation.

(55) After the treatment period, the culture medium of the EA. hy926 cells was discarded and a boiling Laemmle buffer containing 0.2% TritonX-100 and 50 μM dithiothreitol was added to lysate the cells. The cell lysates were then run on 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were transferred onto nitrocellulose membranes, and immunoblotted with anti-ICAM-1/anti-VCAM-1 antibodies. The concentration of antibody to α-tubulin used was 0.4 μg/mL, while that for all other antibodies was 0.1 μg/mL. The protein bands were scanned using Licor Odyssey BioImager (Licor Biosciences, Lincoln, NB, USA) and quantified by densitometry using Image Studio Lite 5.2. All the data were expressed as the percentage change of the corresponding positive control (cells treated with TNF-α alone).

(56) 2.5 Simulated Intestinal Epithelium Absorption Using Caco-2 Cells

(57) The samples collected from the AP and BL surfaces of Caco-2 cells were analyzed on an Acquity Ultra-Performance Liquid Chromatograph (UPLC) system with an Acquity UPLC BEH Cis column (100 mm×2.1 mm i.d., 1.7 μm, Waters, Milford, Mass., USA) using an injection volume of 15 μL. Mobile phases were solvent A (1% TFA in Milli-Q water) and solvent B (1% TFA in acetonitrile). The peptides were eluted with a gradient of solvent A (100-75% in 25 min, 75-50% in 25-35 min) at a flow rate of 0.3 ml/min. The elution was monitored at 220 nm. Absorption was expressed as the percentage of total peak area calculated at different time points (0.5 h, 1 h, 2 h and 4 h) in the BL surface as compared to 0 h in the AP surface.

(58) 2.6 Identification of β-Lg-Derived Peptides

(59) The liquid chromatography column used in this study was nanoACQUITY BEH130 C.sub.18 (75 μm×150 mm, 1.7 μm). The solvent A was acetonitrile (ACN) containing 0.1% formic acid. The peptides were separated using the following gradient: solvent B was water with 0.1% formic acid increasing from 1% to 6% B in 2 min, to 25% B in 23 min, to 45% B in 15 min, to 75% B in 5 min, to 95% B in 5 min, and keeping at 95% B for 5 min. The mass spectrometer was operated in a positive mode with a capillary voltage of 3.5 kV and a source temperature of 100° C. Spectra were recorded over the m/z ranges of 200-1000 in MS mode and in 50-1990 MS/MS mode. The amino acid sequences of peptides were analyzed using Mass Lynx software (Micromass U.K. Ltd.). Peaks Viewer 4.5 (Bioinformatics Solutions Inc., Waterloo, ON, Canada), in combination with manual de novo sequencing was used to process the MS/MS data. Identified peptide sequences were synthesized (>98% purity) by Genscript Corp (Piscataway, N.J.) and used for the bioactivity assays.

(60) The method for extracting β-lactoglobulin with the present invention was as follows: A concentration of 7% (w/v) whey protein solution was prepared by adding whey protein to 7% NaCl water, adjusting the pH to 2 with HCl solution, and centrifuging at 5000 rpm for 20 min to collect the supernatant. Then the supernatant was dialyzed using a dialysis bag with a molecular weight cut off of 14000 Da, and placed in 30 volumes of distilled water for 20 h. Then the retentate in the dialysis bag, i.e. β-LG, was collected.

Example 5

(61) Ultrasound treatment of β-LG. The 200 mL of β-LG with a concentration of 1 g/mL was treated by ultrasound equipment. The ultrasound treatment conditions are as follows: treatment time 30 min; intermittent ratio 10 s/3 s; temperature 30° C. Single frequency ultrasound of 40 kHz was used for the sample treatment.

(62) Enzymolysis of β-LG. After ultrasound treatment, the β-LG suspensions were preheated to 50° C. and adjusted to pH 8.0 with 1.0 M NaOH. Alcalase (the ratio of E'S was 1:20) (w/w) was added to initialize the reaction, and the enzymolysis time was 2 h. The mixture was heated and maintained at 100° C. for 10 min to terminate the reaction. Then the mixture was adjusted to pH 7.0 and centrifuged; the supernatant was collected, desalted, concentrated, and freeze-dried to a powder. The DH and CR of the β-LG, and the anti-inflammatory activity of β-LG-derived hydrolysate, were determined.

(63) Simulated GI digestion. Simulated gastric and intestinal fluids were prepared according to the U.S. Pharmacopeia. Briefly, β-LG-derived hydrolysate was digested with gastric fluid at 1:20 (w/v) for 4 h in a shaking incubator with 120 rpm at 37° C. Then the pH was adjusted to 6.8 and pancreatin was added at 1:100 (w/v) to form the intestinal fluid. The mixture was incubated for a further 6 h to mimic intestinal digestion. The digestion was terminated in boiling water for 10 min. The digest was cooled down and centrifuged at 10.000 g for 10 min to collect the supernatant, which was further centrifuged, desalted, concentrated, and freeze-dried to a powder. The anti-inflammatory activity of the 4 h's and 10 h's β-LG hydrolysate digest was measured.

(64) As shown in Table 5, after single frequency ultrasound pretreatment, the DH of β-LG increased from 10.32% to 13.70%, the protein conversion rate increased from 30.27% to 35.17%. Alcalase hydrolysate showed good anti-inflammatory activity, the expression of the VCAM-1 and ICAM-1 were 42.3% and 62.7%, respectively (Table 6). After simulated gastric digestion, the β-LG hydrolysate digest showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 48.2% and 55.3%, respectively. After simulated intestinal digestion, the β-LG hydrolysate digest still showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 50.7% and 63.2%, respectively. Simulated gastrointestinal digestion appeared to have minimal effect on the anti-inflammatory activity of β-LG hydrolysates.

Example 6

(65) Ultrasound treatment of β-LG. The 200 mL of β-LG with a concentration of 4 g/mL was treated by ultrasound equipment. The ultrasound treatment conditions are as follows: treatment time 20 min; intermittent ratio 10 s/3 s; temperature 25° C. Dual-frequency simultaneous ultrasound with 20/28 kHz was used for the sample treatment.

(66) Enzymolysis of β-LG. After ultrasound treatment, the β-LG suspensions were preheated to 55° C. and adjusted to pH 7.5 with 1.0 M NaOH. Neutral protease (the ratio of E/S was 1:30) (w/w) was added to initial the reaction and the enzymolysis time was 4 h. The mixture was heated and maintained at 100° C. for 10 min to terminate the reaction. Then the mixture was adjusted to pH 7.0 and centrifuged; the supernatant was collected, desalted, concentrated, and freeze-dried to a powder. The DH and CR of the β-LG, and the anti-inflammatory activity of β-LG-derived hydrolysate, were determined.

(67) Simulated GI digestion. β-LG-derived hydrolysate was subjected to simulated gastric and intestinal digestion. Simulated gastric and intestinal fluids were prepared according to the U.S. Pharmacopeia. Briefly, β-LG-derived hydrolysate was digested with gastric fluid at 1:30 (w/v) for 3 h in a shaking incubator with 150 rpm at 37° C. Then the pH was adjusted to 6.8 and pancreatin was added at 1:100 (w/v) to form the intestinal fluid. The mixture was incubated for a further 4 h to mimic intestinal digestion. The digestion was terminated in boiling water for 10 min. The digest was cooled down and centrifuged at 10,000 g for 10 min to collect the supernatant, which was further centrifuged, desalted, concentrated, and freeze-dried to a powder. The anti-inflammatory activity of the 4 h's and 10 h's β-LG hydrolysate digest was measured.

(68) As shown in Table 5, after dual-frequency simultaneous ultrasound pretreatment, the DH of β-LG increased from 6.19% to 9.53%, the protein conversion rate increased from 15.11% to 22.34%. Neutral protease hydrolysate showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 63.2% and 52.3%, respectively. The simulated gastric digestion of the β-LG hydrolysate digest resulted in good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 50.3% and 47.3%, respectively. Also, the simulated intestinal digestion of the β-LG hydrolysate digest showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 53.4% and 53.8%, respectively. Simulated gastrointestinal digestion appeared to have minimal effect on the anti-inflammatory activity of β-LG hydrolysates.

Example 7

(69) Ultrasound treatment of β-LG. The 200 mL of β-LG with a concentration of 4 g/mL was treated by ultrasound equipment. The ultrasound treatment conditions are as follows: treatment time 10 min; intermittent ratio 10 s/3 s; temperature 25° C. Triple-frequency simultaneous ultrasound with 20/28/40 kHz was used for the sample treatment.

(70) Enzymolysis of β-LG. After ultrasound treatment, the β-LG suspensions were preheated to 70° C. and adjusted to pH 8 with 1.0 M NaOH. Thermolysin (the ratio of E/S was 1:50) (w/v) was added to initial the reaction and the enzymolysis time was 2 h. The mixture was heated and maintained at 100° C. for 10 min to terminate the reaction. Then the mixture was adjusted to pH 7.0 and centrifuged, the supernatant was collected, desalted, concentrated, and freeze-dried to a powder. The DH and CR of the β-LG, and the anti-inflammatory activity of β-LG-derived hydrolysate, were determined.

(71) Simulated GI digestion. β-LG-derived hydrolysate was subjected to simulated gastric and intestinal digestion. Simulated gastric and intestinal fluids were prepared according to the U.S. Pharmacopeia. Briefly, β-LG-derived hydrolysate was digested with gastric fluid at 1:50 (w/v) for 2 h in a shaking incubator with 180 rpm at 37° C. Then the pH was adjusted to 6.8 and pancreatin was added at 1:100 (w/v) to form the intestinal fluid. The mixture was incubated for a further 4 h to mimic intestinal digestion. The digestion was terminated in boiling water for 10 min. The digest was cooled down and centrifuged at 10,000 g for 10 min to collect the supernatant, which was further centrifuged, desalted, concentrated, and freeze-dried to a powder. The anti-inflammatory activity of the 4 h's and 10 h's β-LG hydrolysate digest was measured.

(72) As shown in Table 5, after triple-frequency simultaneous ultrasound pretreatment, the DH of β-LG increased from 13.20% to 21.41%, the protein conversion rate increased from 36.90% to 41.02%.

(73) TABLE-US-00006 TABLE 5 Effects of ultrasound pretreatment on the DH, CR of β-LG hydrolysates by different enzymatic hydrolysis Ultrasound assisted hydrolysis Traditional hydrolysis Ultrasound Enzyme DH (%) CR (%) mode DH (%) CR (%) Akalase 10.32 ± 1.73 30.27 ± 0.91 40 kHz 13.70 ± 1.09 35.17 ± 1.77 Neutral protease  6.19 ± 0.42 15.11 ± 0.37 20/28 kHz  9.53 ± 0.97 22.34 ± 1.45 Thermolysin 13.20 ± 0.54 36.90 ± 0.64 20/28140 kHz 21.41 ± 2.31 41.02 ± 2.00

(74) Thermolysin hydrolysate showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 48.9% and 36.5%, respectively. After simulated gastric digestion, the β-LG hydrolysate digest showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 43.3% and 30.1%, respectively. After simulated intestinal digestion, the β-LG hydrolysate digest still showed good anti-inflammatory activity, and the expression of the VCAM-1 and ICAM-1 was 49.1% and 36.2%, respectively (Table 6). Simulated gastrointestinal digestion appeared to have minimal effect on the anti-inflammatory activity of β-LG hydrolysates.

(75) TABLE-US-00007 TABLE 6 Effects of different β-LG hydrolysates before gastric digestion, after gastric digestion, intestinal digestion and Caco-2 cells absorption on TNF-α-induced VCAM-1 and ICAM-1 protein expression in EA.hy926 cells After intestinal After Caco-2 cells Before gastric digestion After gastric digestion digestion absorption β-LG- TNF- VCAM-1 ICAM-1 VCAM-1 ICAM-1 VCAM-1 ICAM-1 VCAM-1 ICAM-1 derived α (% TNF-α (% TNF-α (% TNF-α (% TNF-α (% TNF-α (% TNF-α (% TNF-α (% TNF-α hydrolysates (6h) alone) alone) alone) alone) alone) alone) alone) alone) — − 12.1 ± 1.1 13.3 ± 2.8 14.5 ± 0.9 16.2 ± 2.0 11.9 ± 1.2  12.3 ± 0.79 — + 100 100 100 100 100 100 Alcalase + 42.3 ± 7.1  62.7 ± 10.5 48.2 ± 8.6 55.3 ± 6.2 50.7 ± 7.0 63.2 ± 7.8 hydrolysate Neutral + 63.2 ± 7.5 52.3 ± 7.3 50.3 ± 7.0 47.3 ± 7.1 53.4 ± 6.5 53.8 ± 8.4 protease hydrolysate Thermolysin + 48.9 ± 8.8 36.5 ± 4.9 43.3 ± 6.3 30.1 ± 3.3  49.1 ± 13.2 36.2 ± 5.5 22.1 ± 1.7 16.9 ± 2.4 hydrolysate

Example 8

(76) The β-LG-derived hydrolysate digest prepared in example 7 was subjected to Caco-2 mimicking intestinal endothelial cell absorption.

(77) The cytotoxicity of the β-LG-derived hydrolysate digest to Caco-2 cells was first detected. The absorption model of Caco-2 cells mimicking intestinal endothelial cells was built as follows: Caco-2 cells were grown in 12-well Transwell® plates at a concentration of 2×10.sup.5 cells/mL. The medium of the cell was replaced every other day. After 21 days of cell culture, some evaluation indicators of the Caco-2 cells were measured, including epithelial cell resistance, alkaline phosphatase activity and sodium fluorescein leakage test. Before initiation of the transport experiments, the Caco-2 cells was washed by an HBSS buffer; and 0.5 mL of the β-LG hydrolysate digest (20 mg/mL, dissolved in an HBSS buffer) was adding to the AP surface; 1.5 mL of HBSS buffer was added to the BL surface; then the Caco-2 cells were incubated for 4 h at 37° C. 0.2 mL of AP surface samples at 0 h, and BL surface samples at 0.5, 1, 2, and 4 h were collected for the absorption detection. The AP (apical) surface samples and BL (basolateral) surface samples at 4 h were collected for the anti-inflammatory activity detection, respectively. The β-LG hydrolysates digests and its absorbed digests subjected to the amino acid sequences analysis by UPLC-MC. Some small peptides with strong ionic strength were screened out, synthesized, and detected for anti-inflammatory activity.

(78) TABLE-US-00008 TABLE 7 Cytotoxicity of β-LG hydrolysate derived digests at different concentration in Caco-2 cells Concentration of digest Caco-2 (mg/Ml) viability Control — 100 Group 1 5 125.8 ± 4.7 Group 2 10 124.3 ± 9.0 Group 3 20  110.2 ± 10.8 Group 4 50 106.5 ± 8.0

(79) As shown in Table 7, the addition of β-LG-derived hydrolysate digest increased the viability of Caco-2 cells, indicating that β-LG-derived hydrolysate digest didn't have any toxicity in Caco-2 cells. As shown in Table 8, the absorption of the β-LG-derived hydrolysate digest from the AP to BL surface increased with time and reached 2.67% at 4 h, indicating selective absorption of peptides in Caco-2 cells. After absorption by Caco-2 cells, the anti-inflammatory activity of the β-LG hydrolysate digest largely increased. The expression of the VCAM-1 and ICAM-1 of the absorption digest was 22.1% and 16.9%. Compared to the thermolysin hydrolysate digest, the expression of VCAM-1 and ICAM-1 of the absorption digest decreased by 17.0% and 19.3% (Table 6).

(80) TABLE-US-00009 TABLE 8 Transcellular absorption of digests derived from β-LG hydrolysates was monitored in Caco-2 cell monolayers at 0.5, 1.0, 2.0 and 4.0 h Absorption Absorption time percentage (h) (%) 0.5 0.22 ± 0.01 1 0.71 ± 0.02 2 1.35 ± 0.01 4 2.67 ± 0.03

(81) The above results showed that the anti-inflammatory activity of β-LG polypeptide was significantly enhanced by Caco-2 cells mimicking intestinal endothelial cells absorption, which indicated that the absorbed polypeptide displayed stronger anti-inflammatory activity than the digest. The β-LG hydrolysate digests and their absorbed digests were subjected to identification and analysis. Some small peptides with strong ionic strength were chosen out, synthesized and further assayed for their anti-inflammatory activity. Two peptides with high anti-inflammatory activity, Phe-Tyr-Gln-Ala (FYQA) (SEQ ID No. 4). Leu-Gln-Tyr (LQY) (SEQ ID No. 5) were chosen out. These two peptides strongly inhibited the expression of VCAM-1 and ICAM-1 of these two peptides, which was 41.3% and 56.6%, 33.7% and 48.2%, respectively (Table 9).

(82) TABLE-US-00010 TABLE 9 The β-LG derived peptides that are absorbed by Caco-2 cells were sequenced, identified and chemically synthesized to validate the anti-inflammatory activity of the identified peptides. Peptide Concentration VCAM-1 ICAM-1 sequence (μM) TNF-α (TNF-α) (%TNF-α) − 16.2 ± 7.2  12.6 ± 13.3 + 100 100 FYQA 100 +  41.3 ± 10.6 56.6 ± 9.0 LQY 100 + 33.7 ± 6.9 48.2 ± 6.3