3.7.1. Ultrafiltration Separation

The hydrolysate solution was passed through ultrafiltration membranes with molecular weight (MW) cutoffs of 10 and 3 kDa to obtain three fractions (MW < 3 kDa; MW 3–10 kDa; MW > 10 kDa). Each fraction was assessed for ACE inhibitory activity and IC50.

#### 3.7.2. Gel Filtration Chromatography Analysis

The fraction with the highest ACE inhibition rate was applied to a Sephadex G-25 column ( ϕ2.5 × 60 cm) at a flow rate of 0.5 mL/min using an MC99-3 automatic liquid chromatograph (Shanghai Huxi Analysis Instrument Factory Co., Ltd., Shanghai, China). Elution was measured at 220 nm, and 3-mL fractions were collected. Each major peak was pooled and lyophilized for ACE inhibitory activity assays. Fraction C with the highest activity was further fractionated using a Sephadex G-15 gel filtration column ( ϕ1.0 × 60 cm) with the same operation as described above for Sephadex G-25.

#### 3.7.3. RP-HPLC Analysis of ACE Inhibitory Peptides

The selected fraction C2 was fractionated using semi-preparatory RP-HPLC. Briefly, 7.5 mg of the peptide fraction was dissolved in 1 mL 0.05% TFA (*v*/*v*) and passed through a 0.2-μm filter. The solution was purified using an AKTA pure system (GE Healthcare, Uppsala, Sweden) with an Inertsil ODS-3 C18 column ( ϕ10 × 250 mm). Ultra-pure water containing 0.1% TFA (*v*/*v*) was used as mobile phase A, and acetonitrile containing 0.1% TFA (*v*/*v*) was used as mobile phase B. Separation of the peptides was carried out at a flow rate of 1.5 mL/min using the following linear gradient: 0 to 50% eluent B from 0 to 65 min. Elution peaks were monitored at 220 nm. The purity of the fraction with the highest activity was further analyzed using a ZORBAX Eclipse XDB-C18 column (5 μm, ϕ4.6 mm × 150 mm; Agilent, USA). The column was eluted with a linear gradient of 0–35% mobile phase B from 0 to 25 min at a flow rate of 1 mL/min. Collected peaks were concentrated using a rotary evaporator system and lyophilized for later use.

#### *3.8. Identification of the Amino Acid Sequence by UPLC-MS/MS*

Identification of amino acid sequences was achieved using an Acquity UPLC system (Waters, USA) equipped with an Eksigent ChromXP C18 column (3 μm, ϕ250 nm × 75 μm). The mobile phase consisted of solvent A (0.1% formic acid in acetonitrile, *v*/*v*) and solvent B (0.1% formic acid in water, *v*/*v*), and the elution conditions were as follows: 0.0–45.0 min, 95–70% A; 45.0–52.0 min, 70–20% A; 52.0–53.0 min, 20–95% A; 53.0–60.0 min, 95% A. The flow rate was set at 300 nL/min. The injection volume was 4 μL.

A Q-Exactive Orbitrap mass spectrometry instrument (Thermo Fisher Scientific, USA) was employed for the identification and quantification of ACEIPs from *U. intestinalis.* The conditions were set as follows: full MS, resolution 70,000, AGC 1e6, scan range 350–1600 *m*/*z*; dd-MS2, resolution 17,500, AGC 2e5, isolation window 2.0 *<sup>m</sup>*/*<sup>z</sup>*. The amino acid sequences of the peptides were determined by de novo sequencing using PEAKS Studio 6. Using a solid-phase method, synthetic peptides (purity >95% by HPLC) were obtained from China Peptides Co., Ltd (Shanghai, China).

#### *3.9. In Vitro Digestion*

In vitro digestion was performed according to the method of Cing et.al [50]. The purified peptide sample was mixed with 0.1 M phosphate buffer (pH 2.0) and pepsin (E/S 4%, *w*/*w*); the reaction was allowed to proceed for 2 h at 37 ◦C and stopped by heating at 100 ◦C for 10 min. Subsequently, the remaining suspension was adjusted to pH 8.0 with 5 M NaOH solution and further digested with trypsin (E/S 4%, *w*/*w*); the solution was incubated at 37 ◦C for 2 h and then heated at 100 ◦C for 10 min. The protein hydrolysate was centrifuged at 8000 rpm/min for 15 min, and the supernatant was assayed for ACE inhibitory activity.
