Isolation of a surfactin-producing strain of Bacillus subtilis and evaluation of the probiotic potential and antioxidant activity of surfactin from fermented soybean meal.

BACKGROUND: Surfactin, usually produced by microbial metabolism, has many advantages including low toxicity, high biodegradability, and stability at extreme pH levels and temperatures, making it suitable for industry. However, its commercial production has not yet been achieved. RESULTS: A strain with a strong surfactin-producing ability was isolated and identified as Bacillus subtilis SOPC5, based on the appearance of colonies, microscopic observation, and 16S rDNA sequencing. The isolate exhibited significant tolerance to acid, bile, gastric, and intestinal juices, and was sufficiently susceptible to antibiotics. Bacillus subtilis SOPC5 showed high levels of auto-aggregation and surface hydrophobicity, and a strong capacity to secrete protease, amylase, and cellulase. The strain also exhibited antibacterial activity against Staphylococcus aureus 10 306 with a antibacterial circle diameter of 18.0 ± 1.1 mm. The maximal yield of surfactin (1.32 mg mL-1) was obtained by fermenting soybean meal (SBM) using the isolate under the following conditions: SBM 86 g L-1, inoculation 1.5 × 107 CFU mL-1, FeSO4 1.2 mg L-1, MnSO4 2.6 mg L-1, MgSO4 0.5 mg mL-1, L-Glu 4 mg L-1, temperature 33 °C, duration 120 h, and shaking at 210 rpm. The purity of surfactin was 97.42% as measured by high-performance liquid chromatography (HPLC). The half inhibitory concentration (IC50) values for surfactin to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS·+) were 1.275 ± 0.11 and 0.73 ± 0.08 mg mL-1, respectively. CONCLUSION: This study provides a scientific basis for the application of B. subtilis SOPC5 (as a potential probiotic) and the preparation of its metabolic product (surfactin). © 2024 Society of Chemical Industry.

Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin.

The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH1.5 shift, pH12 shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH 12 shift treatment. In addition, pH1.5 shift modified BSFLA tended to form ß-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH12/1.5 shifting did not cause irreversible denaturation of protein molecules. Therefore, pH12-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.

Effect of carbonyl-amino condensation, non-covalent cross-linking and conformational changes induced by ultrasound-assisted Maillard reaction on lysinoalanine formation in silkworm pupa protein.

The inhibition of cross-linked lysinoalanine (LAL) formation in silkworm pupa protein isolates (SPPI) by Maillard reaction (using varying xylose concentration) and ultrasound treatment was studied. Results showed that sonicated SPPI was effectively grafted with high concentration of xylose (5 %), resulting in the lowest LAL content, which was 48.75 % and 30.64 % lower than the control and ultrasound-treated samples, respectively. Chemical bond analysis showed that the combined treatment destroyed the ionic bonds, intrachain (g-g-t), and interchain (g-g-g) disulfide bonds, but stimulated the polymerization of hydrogen and hydrophobic bonds between SPPI and xylose, and as well enhanced the net negative charge between SPPI/Xylose complexes. The particles of the complexes were more loose, dispersed and rough, and had a stronger hydrophilic microenvironment, accompanied by alterations in microscopic, secondary and tertiary structures. Ultrasound treatment induced the breakdown of the oxidative cross-linking in SPPI, and promoted the sulfhydryl group-dehydroalanine binding and the carbonyl-amino condensation of the protein and xylose, and thus inhibited the formation of cross-linked LAL. Furthermore, the physicochemical and structural parameters were highly interrelated with cross-linked LAL content (|r| > 0.9). The outcomes provided a novel avenue and theoretical basis for minimizing LAL formation in SPPI and improving the nutrition and safety of SPPI.

Inhibition of cross-linked lysinoalanine formation in pH12.5-shifted silkworm pupa protein, and functionality thereof: Effect of ultrasonication and glycation.

We added three different carbohydrates (Xylose/Xyl, Maltose/Mal, and Sodium alginate/Sal) to pH12.5-shifted silkworm pupa protein isolates (SPPI), and examined the influence of multi-frequency ultrasound (US) on them, with reference to lysinoalanine (LAL) formation, changes in conformational characteristics and functionality. Results showed that, the LAL content of the glycoconjugates - SPPI-Xyl, SPPI-Mal, and SPPI-Sal decreased by 1.47, 1.39, and 1.54 times, respectively, compared with the control. Notably, ultrasonication further reduced the LAL content by 45.85 % and brought SPPI-Xyl highest graft degree (57.14 %). SPPI-Xyl and SPPI-Mal were polymerized by different non-covalent bonds, and SPPI-Sal were polymerized through ionic, hydrogen, and disulfide (covalent/non-covalent) bonds. Significant increase in turbidity, Maillard reaction products and the formation of new hydroxyl groups was detected in grafted SPPI (p < 0.05). US and glycation altered the structure and surface topography of SPPI, in which sugars with high molecular weight were more likely to aggregate with SPPI into enormous nanoparticles with high steric hindrance. Compared to control, the solubility at pH 7.0, emulsifying capacity and stability, and foaming capacity of SPPI-US-Xyl were respectively increased by 244.33 %, 86.5 %, 414.67 %, and 31.58 %. Thus, combined US and xylose-glycation could be an effective approach for minimizing LAL content and optimizing functionality of SPPI.

Hexagonal plate ultrasound pretreatment on the correlation between soy protein isolate structure and cholesterol-lowering activity of peptides, and protein's enzymolysis kinetics, thermodynamics.

In this study, a hexagonal plate ultrasound (HPU) pretreatment technology was employed to modify soy protein isolate (SPI) and enhance the hypocholesterolemic activity of enzymatic digests from SPI. Results demonstrated that under the condition of ultrasound power density of 40 W/L, the hypocholesterolemic activity of enzymatic digests from HPU-pretreated SPI (HPU-SPI) increased by 88.40 % compared to control group after gastrointestinal digestion. The sulfhydryl content of HPU-SPI increased by a maximum of 45.32 % compared to control group. Fourier transform infrared and scanning electron microscopy revealed that HPU pretreatment partially unfolded the SPI conformation, reduced the intermolecular interactions, and exposed the internal hydrophobic regions. Pearson correlation analysis showed that sulfhydryl groups (r = 0.860), disulfide bonds (r = -0.875) and random coil (r = 0.917) were strongly correlated with the cholesterol-lowering activity of soy protein hydrolysate (SPH), following a simulated gastrointestinal digestion. Finally, the effects of HPU pretreatment on enzymolysis kinetics and thermodynamics of the SPI enzymatic process showed that HPU pretreatment significantly reduced the Mie's constant, activation energy, activation enthalpy, activation entropy and Gibbs free energy. Overall, the study outcome suggested that HPU pretreatment could positively influence the hypocholesterolemic peptide activity, and thus, may be beneficial to the pharmaceutical/food industry.

Effect of pectin concentration on emulsifying properties of black soldier fly (Hermetia illucens) larvae albumin modified by pH-shifting and ultrasonication.

The effect of pectin concentration on the structural and emulsifying properties of black soldier fly larvae albumin (BSFLA) modified by pH-shifting (pH12) and ultrasound (US) was studied. The results (intrinsic fluorescence, surface hydrophobicity, Fourier transform infrared spectrum, and disulfide bonds) showed that modified BSFLA samples, especially pH12-US, were more likely to bind to pectin through hydrogen bonding, electrostatic interactions, and hydrophobic interactions due to the unfolding of BSFLA, the collapse of disulfide bonds and exposure of hydrophobic groups. Thus, a BSFLA-pectin complex with smaller particle size, more negative charges, and a relatively loose structure was formed. The emulsifying activity (EAI) and stability index (ESI) of pH12-US modified BSFLA were significantly enhanced by the addition of pectin, reaching the highest values (associated with 174.41 % and 643.22 % increase, respectively) at pectin concentration of 1.0 %. Furthermore, the interface modulus of the emulsion prepared by the modified BSFLA was mainly viscous, and had higher apparent viscosity, smaller particle size and droplet size, contributing to higher EAI and ESI. The study findings suggest the addition of pectin to pH12-US treated BSFLA could be used in industry to prepare BSFLA-pectin emulsion with exceptional/desirable properties.

Enhancing the growth of thermophilic Bacillus licheniformis YYC4 by low-intensity fixed-frequency continuous ultrasound.

The effect of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of Bacillus licheniformis YYC4 was investigated. The changes in morphology and activity of the organism, contributing to the growth were also explored. Compared with the control, a significant increase (48.95%) in the biomass of B. licheniformis YYC4 (at the logarithmic metaphase) was observed following the LIFFCU (28 kHz, 1.5 h and 120 W (equivalent to power density of 40 W/L)) treatment. SEM images showed that ultrasonication caused sonoporation, resulting in increased membrane permeability, evidenced by increase in cellular membrane potential, electrical conductivity of the culture, extracellular protein and nucleic acid, and intracellular Ca2+ content. Furthermore, LIFFCU action remarkably increased the extracellular protease activity, volatile components of the culture medium, microbial metabolic activity, and spore germination of the strain. Therefore, LIFFCU could be used to efficiently promote the growth of targeted microorganisms.

Modification of whey protein isolate by ultrasound-assisted pH shift for complexation with carboxymethylcellulose: Structure and interfacial properties.

The application of whey protein isolate (WPI) is limited because of its compact spherical structure. In this study, ultrasound-assisted pH shift was employed to modify WPI for complexation with carboxymethylcellulose (CMC). The foaming and emulsifying properties of WPI/CMC complexes were investigated. The results demonstrate that the pretreatment of ultrasound-assisted pH 12 shift increased the content of free sulfhydryl groups from 16.5 µmol/g to 34.7 µmol/g and enhanced protein hydrophobicity from 311.4 to 370.6 (p < 0.05). Compared to the complexes formed by untreated WPI and CMC, the complexes pretreated with ultrasound-assisted pH 12 shift had a smaller size of 293.4 nm and a more uniform distribution. Furthermore, WPI/CMC complexes pretreated by ultrasound-assisted pH 12 shift exhibited higher emulsifying activity and emulsion stability index, which were increased by 8.9 % and 42.6 % respectively, in comparison with the control group (p < 0.05). A positive correlation was found between the surface hydrophobicity of WPI and emulsifying activity of WPI/CMC complexes. Ultrasound-assisted pH 2 shift improved the foaming capacity of complexes by 28.3 % over the control (p < 0.05). All the results indicate that the interfacial properties of WPI/CMC complexes can be improved significantly by the combination of pH shift and ultrasound.

Characterization of the interaction between allicin and soy protein isolate and functional properties of the adducts.

BACKGROUND: Soybean meal, a by-product of the soybean oil production industry, has a high protein content but the compact globular structure of the protein from soybean meal limits its wide application in food processing. Allicin has been found to have numerous functional properties. In this study, allicin was interacted with soy protein isolate (SPI). The functional properties of the adducts were investigated. RESULTS: Binding with allicin significantly quenched the fluorescence intensity of SPI. Static quenching was the main quenching mechanism. The stability of adducts decreased with increasing temperature. The greatest extent of binding between allicin and sulfhydryl groups (SH) of SPI was obtained at an allicin/SH molar ratio of 1:2. The amino groups of SPI did not bind with allicin covalently. Soy protein isolate was modified by allicin through covalent and non-covalent interactions. Compared with SPI, the emulsifying activity index and foaming capacity of adducts with a ratio of 3:1 were improved by 39.91% and 64.29%, respectively. Soy protein isolate-allicin adducts also exhibited obvious antibacterial effects. The minimum inhibitory concentrations (MICs) of SPI-allicin adducts on Escherichia coli and Staphylococcus aureus were 200 and 160 µg mL-1 , respectively. CONCLUSION: The interaction of allicin with SPI is beneficial for the functional properties of SPI. These adducts can be used in different food formulations as emulsifiers, foamers, and transport carriers. © 2023 Society of Chemical Industry.

The structure, antioxidant and antibacterial properties of thiol-modified soy protein isolate induced by allicin.

This study investigated the effect of allicin binding on the structure, antioxidant and antibacterial properties of soy protein isolate (SPI). Results showed that allicin bound to 82.6 % free thiol groups of SPI at a molar ratio of 0.5. The combination of allicin and SPI significantly affected the structure of protein. Result of circular dichroism showed that the content of α-helix decreased by 26.9 % and the content of ß-sheet increased by 12.2 % over control when the molar ratio was 0.5. The result of surface hydrophobicity signified the unfolding of SPI with the action of allicin. These results implied that allicin binding might be a suitable method for the modification of SPI. Furthermore, the antibacterialand antioxidant experiments indicated that allicin-SPI conjugates not only had the capacity to inhibit the growth of Escherichia coli and Staphyloccocus aureus, but also had DPPH and ABTS radicals scavenging activities.

Identification of a thermophilic protease-producing strain and its application in solid-state fermentation of soybean meal.

BACKGROUND: Thermophiles can thrive at 50-80 °C and produce some enzymes with special promise for biocatalysis. A thermophilic protease-producing strain YYC4 was isolated from Yunyan cigarette and employed in solid-state fermentation (SSF) of unsterilized soybean meal (SBM). RESULTS: The isolate was identified as Bacillus licheniformis based on appearance of colonies, microscopic observation and 16S rDNA sequencing. After SSF, soluble and crude protein contents in SBM increased from 49.24 to 185.73 g kg-1 and from 404.18 to 479.46 g kg-1 , respectively, under the fermentation conditions of 107 cfu g-1 inoculation of strain YYC4, 1:1.8 (g mL-1 ) SBM to distilled water, 1.2 g kg-1 magnesium sulphate addition, 55 °C and 48 h. During fermentation, pH of the medium increased from 6.30 to 9.09 and protease activity especially neutral protease increased significantly from 13.5 to 181.31 U g-1 . Meanwhile, trypsin inhibitor (TI) activity was decreased from 8.19 to 3.19 mg g-1 . The safety of fermented SBM (FSBM) was verified by acute toxicity animal experiment. Analysis of microbial community in FSBM showed that Bacillus licheniformis YYC4 as a dominant strain inhibited most of the other microorganisms pre-existing in the materials during fermentation. CONCLUSION: Increments of soluble and crude protein by 277.19% and 18.63% and decrement of harmful TI by 61.05% in SBM were achieved using thermophilic SSF by Bacillus licheniformis YYC4, providing a basis for the application of thermophiles in fermentation industry in an environmentally friendly and energy-saving way. © 2021 Society of Chemical Industry.

Effect of solid-state fermentation by three different Bacillus species on composition and protein structure of soybean meal.

BACKGROUND: Fermentation efficiency of thermophiles of Bacillus licheniformis YYC4 and Geobacillus stearothermophilus A75, and mesophilic Bacillus subtilis 10 160 on soybean meal (SBM), was evaluated by examining the nutritional and protein structural changes. RESULTS: SBM fermentation by B. licheniformis YYC4, B. subtilis 10 160 and G. stearothemophilus A75 increased significantly the crude and soluble protein from 442.4 to 524.8, 516.1 and 499.9 g kg-1 , and from 53.9 to 203.3, 291.3 and 74.6 g kg-1 , and decreased trypsin inhibitor from 8.19 to 3.19, 2.14 and 5.10 mg g-1 , respectively. Bacillus licheniformis YYC4 and B. subtilis 10 160 significantly increased phenol and pyrazine content. Furthermore, B. licheniformis YYC4 fermentation could produce abundant alcohols, ketones, esters and acids. Surface hydrophobicity, sulfhydryl groups and disulfide bond contents of SBM protein were increased significantly from 98.27 to 166.13, 173.27 and 150.71, from 3.26 to 4.88, 5.03 and 4.21 µmol g-1 , and from 20.77 to 27.95, 29.53 and 25.5 µmol g-1 after their fermentation. Fermentation induced red shifts of the maximum absorption wavelength (λmax ) of fluorescence spectra from 353 to 362, 376 and 361 nm, while significantly reducing the fluorescence intensity of protein, especially when B. subtilis 10 160 was used. Moreover, fermentation markedly changed the secondary structure composition of SBM protein. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and atomic force microscopy showed that macromolecule protein was degraded into small-sized protein or peptide during fermentation of SBM. CONCLUSION: Bacillus licheniformis YYC4 fermentation (without sterilization) improved nutrition and protein structure of SBM as B. subtilis 10 160, suggesting its potential application in the SBM fermentation industry. © 2021 Society of Chemical Industry.

Influence of ultrasound-assisted ionic liquid pretreatments on the functional properties of soy protein hydrolysates.

In this work, the effect of dual-frequency ultrasound-assisted ionic liquids (ILs) pretreatment on the functional properties of soy protein isolate (SPI) hydrolysates was investigated. The degree of hydrolysis (DH) of SPI pretreated by ultrasound and [BMIM][PF6] increased by 12.53% as compared to control (P < 0.05). More peptides with low molecular weight were obtained, providing support for the changes in DH. The trichloroacetic acid-nitrogen soluble index presented an increase, suggesting a better protein hydrolysate property. The increase in the calcium-binding activity showed the ultrasound-assisted ILs pretreatment could potentially improve bone health. The foaming capacity and stability of SPI hydrolysates pretreated by ultrasound-assisted [BMIM][PF6] always increased remarkably as compared to ultrasound-assisted [BDMIM][Cl] pretreatment. However, the synergistic effect of ultrasound-assisted [BMIM][PF6] on the emulsifying activity and antioxidant activities (DPPH and hydroxyl radical scavenging activity) was not as ideal as ultrasound-assisted [BDMIM][Cl] pretreatment, which may be affected by the structure of peptide. In conclusion, these results indicated the combination of dual-frequency ultrasound and ionic liquids would be a promising method to improve the functional properties of SPI hydrolysates and broaden the application scope of compound modification in proteolysis industry.

Solubility and aggregation of soy protein isolate induced by different ionic liquids with the assistance of ultrasound.

The aim of this study was to evaluate the effects of ionic liquids (ILs) and ultrasound on the solubility and aggregation behavior of soy protein isolate (SPI). A variety of ILs were tested. Results showed that changes in cation or anion altered the physicochemical properties of ionic liquids, which in turn influenced the solubility of SPI. High concentration of ILs resulted in the formation of insoluble aggregates, which lead to the decrease of solubility. In most of the cases, ultrasound pretreatment had a considerable impact on the solubility and aggregation of SPI. The solubility of SPI processed by combination of 1 mg/mL 1-butyl-2,3-dimethyl imidazolium chloride ([BDMIM]Cl) and ultrasound changed remarkably compared with single ultrasound and single [BDMIM]Cl processing, which was increased by 71.8% compared with that of control (P < .05). Changes in particle size, intrinsic fluorescence spectra and free sulfhydryl (SH) groups indicated that the structure of SPI refolded and reaggregated after the ultrasound and ILs pretreatments. Combined ultrasound and 1 mg/mL [BDMIM]Cl pretreatment showed a synergistic effect on changing the SPI microstructure. In conclusion, ultrasound-assisted ILs could be an effective modification method for the globular proteins.

Preparation of allicin-whey protein isolate conjugates: Allicin extraction by water, conjugates' ultrasound-assisted binding and its stability, solubility and emulsibility analysis.

The instability of allicin makes it easily decomposed into various organic sulfur compounds, resulting in significant decrease in biological activity. In this study, allicin was firstly extracted with water, then bound with whey protein isolates (WPI) which were pretreated by ultrasound to form conjugates, and the stability, water solubility and emulsibility of conjugates were as well investigated. The research results showed that there were no significant differences in the extraction yields of allicin from water, 40% and 80% ethanol. Appropriate frequency (20/40 kHz), power (50 W/L) and time (20 min) of ultrasonic pretreatments significantly increased (P < 0.05) the sulfhydryl groups content of WPI by 35.05% over control, causing improvement in binding ability of protein to allicin. The binding process of allicin-WPI displayed good fit with Elovich kinetic model (R2 = 0.9781). The mass retention rate of the conjugates (in 60% combination rate) with ultrasonic pretreating kept at 95.97% after 14 days of storage at 25 °C, whereas allicin's mass retention rate was only 61.79% at same storage condition. The water solubility of the prepared conjugates was significantly higher than allicin. And with optimal condition ultrasonic pretreatment of WPI, the conjugates showed the highest emulsifying capacity and emulsion stability (49.56 m2/g, 10.06 min). In conclusion, the ultrasonically pretreated allicin-WPI conjugates exhibited better stability, water solubility and emulsifying properties compared to allicin, this expands the application field of allicin.

The aggregation, structures and emulsifying properties of soybean protein isolate induced by ultrasound and acid.

The effects of ultrasound and acid on the aggregation, structures and emulsifying properties of soybean protein isolate (SPI) were investigated. Results of solubility showed that ultrasonic treatments at 0.001 M HCl increased the content of soluble SPI. The particle size of soluble aggregates subjected to ultrasonication and acid was initially decreased and then increased with increasing ultrasonic time. Secondary structure analysis, by circular dichroism, indicated lower a-helix and higher random coil amounts in SPI treated with short ultrasonic time, in contrast to the higher a-helix and lower random coil in SPI treated with longer time (more than 20 min). Emulsions prepared with SPI by 10 min of ultrasonication demonstrated significantly (P < 0.05) small droplet sizes and long term stability in comparison with their untreated counterparts. These results highlight that the emulsifying properties of SPI can be significantly improved by the synergistic effect of ultrasound and acid.

Changes in the structure and dissociation of soybean protein isolate induced by ultrasound-assisted acid pretreatment.

Structure and dissociation properties of soybean protein isolate (SPI) induced by ultrasound and acid were investigated. Results of solubility showed that ultrasound-assisted acid had no effect on the content of soluble aggregates in SPI. Increase of fluorescence intensity and red-shift of maximum emission wavelength indicated that acid induced molecular unfolding of SPI and exposure of hydrophobic groups. Circular dichroism spectra showed that ultrasound-assisted acid pretreatment resulted in increases in the α-helix content by 29.2% and random coils content by 8.3%, while ß-sheet decreased by 13.4% (P<0.05), as compared with those of control. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and atomic force microscope revealed that the contents of small subunits and particle increased significantly when SPI treated by ultrasound-assisted acid comparing with the SPI treated by single acid and ultrasound treatment. This study illustrated the ultrasound and acid have synergistic effect on the structure unfolding and dissociation of SPI.

Pretreatment of garlic powder using sweep frequency ultrasound and single frequency countercurrent ultrasound: optimization and comparison for ACE inhibitory activities.

The sweep frequency ultrasound (SFU) and single frequency countercurrent ultrasound (SFCU) pretreatments were modeled and compared based on production of angiotensin I-converting enzyme (ACE) inhibitory peptides from garlic hydrolysates. Two mathematical models were developed to show the effect of each variable and their combinatorial interactions on ACE inhibitory activity. The optimum levels of the parameters in SFU were determined using uniform design, which revealed these as follows: total ultrasonic time 1.5 h, on-time of pulse 18 s and off-time of pulse 3 s. Under optimized conditions, the experimental values of SFU and SFCU were 65.88% and 67.78%, which agreed closely with the predicted values of 63.44% and 67.33%. The SFU and SFCU pretreatments both resulted in higher ACE inhibitory activity compared with untreated garlic (p<0.05). However, there were no significant differences in the ACE inhibitory activities and IC50 values obtained from SFCU and SFU pretreatments under optimum conditions (p>0.05).

Antihypertensive activity of recombinant peptide IYPR expressed in Escherichia coli as inclusion bodies.

To produce more angiotensin converting enzyme inhibitory peptides (ACEIP), we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the recombinant protein, which was subcloned into the vector pET-30a(+), has been expressed as inclusion bodies in E. coli BL21 (DE3). The influences of induction time and concentration of isopropyl-ß-D-thiogalactopyranoside (IPTG) on the expression of recombinant protein were studied. The resulting expression level of the protein accounted for about 31% of cellular protein at a temperature of 37°C, IPTG concentration of 0.6mM and induction time of 7h. The inclusion bodies were washed, separated from the cells, and solubilized with urea. After purification by affinity chromatography, the recombinant protein was recovered with a high purity of about 90%. Molecular weight of the recombinant protein was measured using Tricine-SDS-PAGE. Peptide IYPR was obtained by cleavage of the recombinant protein with trypsin and the IC(50) value was 61 mg/L. The antihypertensive activity in spontaneously hypertensive rats (SHRs) was also investigated. Single oral administration of this peptide in 10-week old SHRs resulted in a significant reduction of systolic blood pressure to 50 mm Hg at 4 h. The data obtained provide a good reference for further development of peptide Ile-Tyr-Pro-Arg into an effective antihypertensive agent for prevention and treatment of hypertension.