Ultrasound-assisted thawing of frozen white yak meat: Effects on thawing rate,meat quality,nutrients, and microstructure

The objective of this study was to assess the effects of ultrasound-assisted thawing (UAT) on the quality of longissimus dorsi muscles from white yak meat (WYM). Ultrasonic power levels of 0, 200, 400, and 600 W (frequency of 20 kHz) were used to assist thawing. The thawing rate, meat quality, nutrient substances, volatile compounds, and microstructure of the WYM were determined. The results showed that ultrasonic thawing treatment reduced thawing times by 30.95–64.28% compared to control. The meat quality results revealed that the thawing loss, cooking loss, L* and b* values, and pH values decreased significantly while the a* value and cutting force increased significantly (P < 0.05) at the lower 400 W power level compared with the control. In addition, the free amino acid (FAA), mineral, and vitamin (especially water-soluble vitamins) contents were significantly (P < 0.05) increased with the ultrasound treatment. UAT significantly (P < 0.05) increased the content of volatile compounds, an effect that was highest in the UAT-400 W group. Partial least squares discrimination analysis (PLS-DA) showed that 2,4-heptadienal was critical in distinguishing the UAT groups from the control. When the ultrasonic power was lower than 400 W, the muscle cell area was significantly (P < 0.05) increased but decreased when higher power was used. Therefore, UAT improves the thawing efficiency and quality of frozen WYM, particularly at a power level of 400 W, and these findings have potential applications in the meat industry.     

Study of ultrasonic treatment on the structural characteristics of gluten protein and the quality of steamed bread with potato pulp

Physicochemical properties and microstructure of gluten protein, and the structural characteristics of steamed bread with 30 % potato pulp (SBPP) were investigated by ultrasonic treatments. Results showed that 400 W ultrasonic treatment significantly (P < 0.05) increased the combination of water and substrate in the dough with 30 % potato pulp (DPP). The contents of wet gluten, free sulfhydryl (SH), and disulfide bond (SS) were influenced by ultrasonic treatment. Moreover, UV-visible and fluorescence spectroscopy demonstrated that the conformation of gluten protein was changed by ultrasonic treatment (400 W). Fourier transform infrared (FT-IR) illustrated that the β-sheet content was significantly (P < 0.05) increased (42 %) after 400 W ultrasonic treatment, and the surface hydrophobicity of gluten protein in SBPP increased from 1225.37 (0 W ultrasonic treatment) to 4588.74 (400 W ultrasonic treatment). Ultrasonic treatment facilitated the generation of a continuous gluten network and stabilized crumb structure, further increased the specific volume and springiness of SBPP to 18.9 % and 6.9 %, respectively. Those findings suggested that ultrasonic treatment would be an efficient method to modify gluten protein and improve the quality of SBPP.     

Effect of ultrasound-assisted resting on the quality of surimi-wheat dough and noodles

In this study, the influence of ultrasound-assisted resting at different power on the rheological properties, water distribution and structural characteristics of dough with 50 % surimi as well as the texture, cooking and microstructure characteristics of the surimi-wheat noodles were investigated. Compared with the fermentation control (FC) noodles, the microstructure, cooking and texture characteristics of noodles (≤24.00 W/L) were significantly (p < 0.05) improved after ultrasonic treating. As the increasing of ultrasonic power, compared to FC, the creep strain, recovery strain, semi-bound water, and free sulfhydryl (SH) contents of surimi-wheat dough decreased at first and then increased significantly (p < 0.05). The α-helix and β-turn content of dough increased at first and then decreased after ultrasonic treatment, while the β-sheet was reversed. The surimi-wheat dough network structure was improved by ultrasonic treatment, with the densest and continuous pore size in 21.33 W/L, but the dough structure was broken and loose (>21.33 W/L), which consisted of the hardness, elasticity, chewiness, resistant and cooked quality of surimi-wheat noodles. This work elucidated the effect of ultrasonic power on the performance of surimi-wheat dough, and the optimal ultrasound power was obtained, thereby improving the nutritional properties and the quality of surimi-wheat noodles.     

Effects of ultrasound-assisted vacuum tumbling on the oxidation and physicochemical properties of pork myofibrillar proteins

The present research aimed to investigate the effects of high-intensity ultrasound (HIU, 20 kHz, 0 W, 100 W, 300 W and 500 W)-assisted vacuum tumbling (UVT) for 60 min and 120 min on the oxidation and physicochemical properties of the pork myofibrillar proteins (MPs). Compared with the vacuum tumbling (VT) groups without the HIU assistance, the carbonyl content increased, while the total sulfhydryl (SH) content was reduced with the increase of HIU power and treatment time (P < 0.05). The reactive SH content was increased significantly after treated by UVT with 300 W compared with the VT group (P < 0.05) regardless of the treatment time. Similarly, the surface hydrophobicity (S₀), the intrinsic tryptophan intensity, and the solubility in the UVT group (300 W) were remarkably higher than those of the VT group (P < 0.05). In contrast, the α-helix content and the particle size of MPs significantly decreased when the HIU power was at 100 W and 300 W (P < 0.05). The results suggest that UVT treatment could change the structure and physicochemical properties of MPs accompanied by protein oxidation.     

Influence of ultrasound-assisted tumbling on NaCl transport and the quality of pork

The present study aimed to investigate the impact of ultrasound-assisted tumbling (UAT; 20 kHz, 100, 300, 500 and 700 W) with different treatment time (30, 60, 90 and 120 min) on the diffusion and distribution of NaCl as well as the change of pork texture properties during curing. Results showed that in comparison with the single tumbling (ST), the NaCl content and the NaCl diffusion coefficient were increased along with UAT treatment (P < 0.05). The scanning electron microscopy and the energy dispersive X-ray analysis showed that UAT treatment changed the microstructure of pork which may facilitate the NaCl dispersion homogeneously. In addition, the moderate UAT treatment of 300 W with 60 min could significantly improve the tumbling yield, water-holding capacity and textural properties of pork compared with the ST treatment (P < 0.05). Meanwhile, in comparison with the ST group, protein extraction was considerably increased after UAT (300 and 500 W) treated for 120 min (P < 0.05). Our study demonstrated that UAT treatment could effectively promote the penetration and distribution of NaCl and improve pork meat quality via facilitating the extraction of meat protein.     

Improving the gel properties of salted egg white/cooked soybean protein isolate composite gels by ultrasound treatment: Study on the gelling properties and structure

In this study, the effects of ultrasound treatment on the texture, physicochemical properties and protein structure of composite gels prepared by salted egg white (SEW) and cooked soybean protein isolate (CSPI) at different ratios were investigated. With the increased SEW addition, the ζ-potential absolute values, soluble protein content, surface hydrophobicity and swelling ratio of composite gels showed overall declining trends (P < 0.05), while the free sulfhydryl (SH) contents and hardness of exhibited overall increasing trends (P < 0.05). Microstructural results revealed that composite gels exhibited denser structure with the increased SEW addition. After ultrasound treatment, the particle size of composite protein solutions significantly decreased (P < 0.05), and the free SH contents of ultrasound-treated composite gels were lower than that of untreated composite gels. Moreover, ultrasound treatment enhanced the hardness of composite gels, and promoted the conversion of free water into non-flowable water. However, when ultrasonic power exceeded 150 W, the hardness of composite gels could not be further enhanced. FTIR results indicated that ultrasound treatment facilitated the composite protein aggregates to form a more stable gel structure. The improvement of ultrasound treatment on the properties of composite gels was mainly by promoting the dissociation of protein aggregates, and the dissociated protein particles further interacted to form denser aggregates through disulfide bond, thus facilitating the crosslinking and reaggregation of protein aggregates to form denser gel structure. Overall, ultrasound treatment is an effective approach to improve the properties of SEW-CSPI composite gels, which can improve the potential utilization of SEW and SPI in food processing.     

Influence of ultrasound treatment on the physicochemical and antioxidant properties of mung bean protein hydrolysate

This study aimed to investigate influence of ultrasonic treatment on physicochemical and antioxidant properties of mung bean protein hydrolysate (MPH). Physicochemical properties of MPH were evaluated by Tricine-SDS-PAGE, particle size distribution, fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy, among others. Radicals scavenging activities of ABTS, hydroxyl, superoxide anion, Fe²⁺ chelating ability and reducing power characterized antioxidant activities of MPH. MPH contained four bands of 25.6, 12.8, 10.6 and 4.9 kDa, in which 4.9 kDa was the most abundant. Ultrasonic treatment increased the contents of aromatic and hydrophobic amino acids in MPH. Ultrasonic treatment decreased the content of α-helix of MPH and increased β-sheet and β-turn compared to MPH. MPH-546 W (ultrasonic treatment 546 W, 20 min) had the lowest average particle size (290.13 nm), zeta potential (-36.37 mV) and surface hydrophobicity (367.95 A.U.). Antioxidant activities of ultrasonicated-MPH increased with the ultrasonic power, achieving the lowest IC₅₀ (mg/mL) of 0.1087 (ABTS), 1.796 (hydroxyl), 1.003 (superoxide anion) and 0.185 (Fe²⁺ chelating ability) in 546 W power. These results indicated ultrasonic treatment would be a promising method to improve the antioxidant properties of MPH, which would broaden the application scope of MPH as bioactive components in the food industry.     

Effect of ultrasound-assisted dough fermentation on the quality of dough and steamed bread with 50% sweet potato pulp

Ultrasound at an intensity of 17.5, 20.0, 22.5, 25.0 and 27.5 W/L was used to assist dough fermentation to prepare steamed bread with 50% sweet potato pulp (SB-50% SPP), which was compared with SB-50% SPP without ultrasonic treatment. The dough rheology, starch–gluten network, texture characteristics and sensory quality of steamed bread with different ultrasonic power densities (UPDs) were investigated. Dough samples at UPD of 22.5 W/L showed optimal viscoelasticity. The microstructure images exhibited that the content of starch particles wrapped in the gluten network increased significantly after sonication. In addition, the reduction in free sulfhydryl (SH) content and increase in wet gluten content after ultrasonic treatment led to significantly improved dough extensibility (p < 0.05). Results exhibited that the specific volume of SB-50% SPP increased by 13.93% and the hardness decreased by 21.96% compared with the control under UPD of 22.5 W/L. This study suggested that the application of ultrasound as a green technology to dough fermentation could lead to SB-50% SPP with good quality and sensory characteristics.     

Underlying formation mechanisms of ultrasound-assisted brined porcine meat: The role of physicochemical modification,myofiber fragmentation and histological organization

Ultrasound treatment has been a good hurdle technique for meat curing processing, where both physical and chemical consequences can be involved towards final quality of obtained products. However, the specific correlation between ultrasound parameters and muscle fiber fragmentation and myofibrillar microstructural changes during curing deserve further evaluation. In present study, we comparatively studied the effect of ultrasound-assisted brining (UAB) and static brining (SB) on the muscle proteolysis events and microstructural/morphological variation of porcine meat as well as the physicochemical indices and histological characteristics. The results showed that UAB (20 kHz, 315 W for 1 h) could markedly enhance the muscle proteolysis with higher free-/peptide-bound alpha-amino-nitrogen (α-NH2-N) content (P < 0.05) than SB treatment and greatly improved the fragmentation of muscle fiber tissues of cured meat. Meanwhile, UAB processing favored more opening structures of myofibrillar proteins with more hydrophobic groups being exposed. The quantitative histological analysis revealed that, compared with SB treatment, UAB could significantly increase the gap between muscle fibers and the swelling of the perimysium (P < 0.01), proving an efficient curing process with better textural and water holding properties.     

Effects of low-frequency ultrasonic pre-treatment in water/oil medium simulated system on the improved processing efficiency and quality of microwave-assisted vacuum fried potato chips

The effects of low-frequency ultrasonic pre-treatment in water/oil medium simulated system on the improved processing efficiency and quality of microwave-assisted vacuum fried potato chips were investigated. The water medium system (distilled water and 5% NaCl osmotic solution) and oil medium system (90 °C) were designed with different power levels of ultrasound to simulate the ultrasonic conditions. Results showed that the changes of moisture content, water loss, solid gain and dielectric properties of potato slices were facilitated by the ultrasonic treatment. LF-NMR analysis showed the binding force between the moisture and structure in the material was significantly (p < 0.05) weakened. The changes become greater with the increase of ultrasonic power levels. Microscopic channels and disruptions were induced on the microstructure by the ultrasonic treatment. The effective moisture diffusivity of vacuum fried (VF) potato chips was increased by about 56.2%-67.0% and 53.9% with the combination of microwave energy and the ultrasonic pre-treatment in water and oil medium simulated system, respectively. The oil uptake, hardness, shrinkage, total color change and water activity of vacuum fried samples were significantly (p < 0.05) decreased by the assist of microwave energy combined ultrasonic pre-treatment.     

Ultrasonic structural modification of myofibrillar proteins from Coregonus peled improves emulsification properties

This study evaluated the effects of high intensity ultrasonication (HIU, 100, 150, 200, and 250 W) and treatment time (0, 3, 6, 9, and 12 min) on the structure and emulsification properties of myofibrillar proteins (MPs) from Coregonus peled. These investigations were conducted using an ultrasonic generator at a frequency of 20 kHz (ultrasonic probe). Analysis of the carbonyl content and total number of sulfhydryl groups showed that HIU significantly improved the oxidative modification of MPs (P < 0.05). SDS-PAGE profiling showed significant degradation of the myosin heavy chain (P < 0.05). In addition, Fourier transformed infrared spectroscopy (FTIR) revealed that HIU altered these treated MP secondary structures, this was due to molecular unfolding and stretching, exposing interior hydrophobic groups. Particle size analysis showed that HIU treatment reduced particle sizes. Solubility, emulsification capacity, and emulsion stability were improved significantly, and each decreased with an increase in treatment time (up to 12 min), indicating aggregation with prolonged sonication. These results indicate that HIU could improve the emulsification properties of MPs from C. peled, demonstrating a promising method for fish protein processing.     

Effect of ultrasound on the structural characteristics and oxidative stability of walnut oil oleogel coated with soy protein isolate-phosphatidylserine

In this study, the three-dimensional network system formed by rice bran wax (RBW) was used as the internal structure, and the external structure formed by soybean protein isolate (SPI) and phosphatidylserine (PS) was added on the basis of the internal structure to prepare walnut oil oleogel (SPI-PS-WOG). Ultrasonic treatment was applied to the mixed solution to make SPI-PS-WOG, on the basis, the effects of ultrasonic treatment on SPI-PS-WOG were investigated. The results showed that both β and β’ crystalline forms were present in all SPI-PS-WOG samples. When the ultrasonic power was 450 W, the first weight loss peak in the thermogravimetric (TGA) curve appeared at 326 °C, which was shifted to the right compared to the peak that occurred when the ultrasonic power was 0 W, indicating that the thermal stability of the SPI-PS-WOG was improved by the ultrasonic treatment. Moreover, when the ultrasonic power was 450 W, the oil holding capacity (OHC) reached 95.3 %, which was the best compared with other groups. Both confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) showed that the ultrasonic treatment of appropriate power succeeded in making the SPI-PS-WOG samples more evenly dispersed in the internal structure and denser in the external structure. In terms of oxidative stability, it was found that the peroxide value of SPI-PS-WOG remained at 9.8 mmol/kg oil for 50 days under 450 W ultrasonic power treatment, which was significantly improved compared with liquid walnut oil (WO). These results provide a new idea for the preparation of oleogels, and also lay a theoretical foundation for the application of ultrasonic treatment in oleogels.     

Optimisation of the ultrasound-assisted extraction of betalains and polyphenols from Amaranthus hypochondriacus var. Nutrisol

The present study optimised the ultrasound-assisted extraction (UAE) of bioactive compounds from Amaranthus hypochondriacus var. Nutrisol. Influence of temperature (25.86–54.14 °C) and ultrasonic power densities (UPD) (76.01–273.99 mW/mL) on total betalains (BT), betacyanins (BC), betaxanthins (BX), total polyphenols (TP), antioxidant activity (AA), colour parameters (L*, a*, and b*), amaranthine (A), and isoamaranthine (IA) were evaluated using response surface methodology. Moreover, betalain extraction kinetics and mass transfer coefficients (K_La) were determined for each experimental condition. BT, BC, BX, TP, AA, b*, K_La, and A were significantly affected (p < 0.05) by temperature extraction and UPD, whereas L*, a*, and IA were only affected (p < 0.05) by temperature. All response models were significantly validated with regression coefficients (R²) ranging from 87.46 to 99.29%. BT, A, IA, and K_La in UAE were 1.38, 1.65, 1.50, and 29.93 times higher than determined using conventional extraction, respectively. Optimal UAE conditions were obtained at 41.80 °C and 188.84 mW/mL using the desired function methodology. Under these conditions, the experimental values for BC, BX, BT, TP, AA, L*, a*, b*, K_La, A, and IA were closely related to the predicted values, indicating the suitability of the developed quadratic models. This study proposes a simple and efficient UAE method to obtain betalains and polyphenols with high antioxidant activity, which can be used in several applications within the food industry.     

Fabrication of emulsions prepared by rice bran protein hydrolysate and ferulic acid covalent conjugate: Focus on ultrasonic emulsification

The aim of the paper was to investigate the effect of ultrasonic emulsification treatment on the fabrication mechanism and stability of the emulsion. The covalent conjugate made with rice bran protein hydrolysate (RBPH) and ferulic acid (FA) was used as the emulsifier. The effects of high intensity ultrasound (HIU) power with different level (0 W, 150 W, 300 W, 450 W and 600 W) on the stability of emulsion were evaluated. The results showed that ultrasonic emulsification can significantly improve the stability of the emulsions (p < 0.05). The emulsion gained better stability and emulsifying property at 300 W. It was able to fabricate emulsion with smaller particle size, more uniform distribution and higher interfacial protein content. It was confirmed by fluorescent microscopy and cryo-scanning electron microscopy (cryo-SEM) furtherly. And it was also proved that the emulsion treated by proper HIU treatment at 300 W had better storage stability. Excessive HIU treatment (450 W, 600 W) had negative effects on the stability of emulsion. The stability of emulsion (300 W) against different environmental stresses was further explored, which established a theoretical basis for the industrial application of emulsion in food industry.     

Ultrasound-assisted immersion freezing reduces the structure and gel property deterioration of myofibrillar protein from chicken breast

The effects of air freezing (AF), immersion freezing (IF) and ultrasound-assisted immersion freezing (UF) at different power levels (125, 165, 205 and 245 W) on the structure and gel properties of the myofibrillar protein (MP) of chicken breast were investigated. UF at 165 W (UF-165) had no obvious negative impact on the primary structure of the MP and effectively reduced the change in the secondary and tertiary structure. In addition, UF-165 significantly reduced the losses in the elastic modulus (G′), gel strength, and gel water holding capacity (P < 0.05). According to low field nuclear magnetic resonance analysis, the T₂₁ and T₂₂ of the UF-165 MP gels were shorter than those of the AF and IF samples, which meant that the UF-165 reduced the mobility of the immobilized water and free water in MP gel. A scanning electron microscopy analysis showed that the appropriate ultrasonic power promoted the formation of a compact and homogeneous protein gel network. These results suggested that the appropriate ultrasonic power maintained the MP structure and reduced the loss of gel quality.     

Effect of high intensity ultrasound on the fermentation profile of Lactobacillus sakei in a meat model system

The objective of this study was to investigate the efficacy of high intensity ultrasound on the fermentation profile of Lactobacillus sakei in a meat model system. Ultrasound power level (0–68.5 W) and sonication time (0–9 min) at 20 °C were assessed against the growth of L. sakei using a Microplate reader over a period of 24 h. The L. sakei growth data showed a good fit with the Gompertz model (R² > 0.90; SE < 0.042). Second order polynomial models demonstrated the effect of ultrasonic power and sonication time on the specific growth rate (SGR, μ, h⁻¹) and lag phase (λ, h). A higher SGR and a shorter lag phase were observed at low power (2.99 W for 5 min) compared to control. Conversely, a decrease (p < 0.05) in SGR with an increase in lag phase was observed with an increase in ultrasonic power level. Cell-free extracts obtained after 24 h fermentation of ultrasound treated samples showed antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium at lower concentrations compared to control. No significant difference (p < 0.05) among treatments was observed for lactic acid content after a 24 h fermentation period. This study showed that both stimulation and retardation of L. sakei is possible, depending on the ultrasonic power and sonication time employed. Hence, fermentation process involving probiotics to develop functional food products can be tailored by selection of ultrasound processing parameters.     

Effects of ultrasound on the beef structure and water distribution during curing through protein degradation and modification

The objective of this study was to explore the mechanisms of power ultrasound (PUS, 150 and 300 W) and treatment time (30 and 120 min) on the water-holding capacity (WHC) and tenderness of beef during curing. Beef muscle at 48 h post mortem was subjected to PUS treatment at a frequency of 20 kHz. Analysis of compression loss and shear force showed that PUS-assisted curing significantly increased the WHC and the tenderness of beef compared to static brining (p < 0.05). According to the analysis of LF-NMR, PUS treatment could increase the P₂₁ values which indicated an improvement in water-binding ability of beef muscle. SDS-PAGE and LC-ESI-MS/MS analysis suggested that PUS induced moderate oxidation of myosin causing polymerization, which may contribute to increased water retention. On the other hand, an increased tenderness of beef is suggested by the increased MFI values and proteolysis of desmin and troponin-T. Transmission electron microscopy (TEM) further supported the effects of PUS on WHC and tenderness changes due to the swelling and disruption of myofibrils. Thus, these results provide knowledge about the mechanism for improving WHC and tenderness of beef by PUS curing, which could be employed as an emerging technology for various meat curing processes.     

Effects of power ultrasound on oxidation and structure of beef proteins during curing processing

The aim of this study was to evaluate the effects of power ultrasound intensity (PUS, 2.39, 6.23, 11.32 and 20.96 W cm⁻²) and treatment time (30, 60, 90 and 120 min) on the oxidation and structure of beef proteins during the brining procedure with 6% NaCl concentration. The investigation was conducted with an ultrasonic generator with the frequency of 20 kHz and fresh beef at 48 h after slaughter. Analysis of TBARS (Thiobarbituric acid reactive substances) contents showed that PUS treatment significantly increased the extent of lipid oxidation compared to static brining (P < 0.05). As indicators of protein oxidation, the carbonyl contents were significantly affected by PUS (P < 0.05). SDS–PAGE analysis showed that PUS treatment increased protein aggregation through disulfide cross-linking, indicated by the decreasing content of total sulfhydryl groups which would contribute to protein oxidation. In addition, changes in protein structure after PUS treatment are suggested by the increases in free sulfhydryl residues and protein surface hydrophobicity. Fourier transformed infrared spectroscopy (FTIR) provided further information about the changes in protein secondary structures with increases in β-sheet and decreases in α-helix contents after PUS processing. These results indicate that PUS leads to changes in structures and oxidation of beef proteins caused by mechanical effects of cavitation and the resultant generation of free radicals.     

Effects of multi-frequency ultrasound pretreatment under low power density on the enzymolysis and the structure characterization of defatted wheat germ protein

The effects of ultrasonic frequency mode, power density, pretreatment time and other parameters under low power density on the degree of hydrolysis (DH) of defatted wheat germ protein (DWGP) and angiotensin-I-converting enzyme (ACE) inhibitory activity of DWGP hydrolysate were studied in this research. Ultraviolet–visible (UV–Vis) spectra, free sulfhydryl (SH), disulfide bond (SS), surface hydrophobicity and hydrophobic protein content of ultrasound-pretreated protein and hydrophobic amino acid (HAA) content of alcalase-hydrolysate of DWGP were measured under optimized ultrasonic condition. The ultrasonic frequency mode with dual-fixed frequency combination of 28/40 kHz showed higher ACE inhibitory activity of DWGP hydrolysate compared with that of other ultrasound frequency modes and all the ultrasonic frequency combinations involving in 28 kHz showed higher ACE inhibitory activity. Under the dual-fixed frequency ultrasound mode of 28/40 kHz, ultrasonic power density of 60 W/L, pretreatment time of 70 min, temperature of 60°C and substrate concentration of 60 g/L, the ACE inhibitory activity of DWGP hydrolysate was the highest with its value of 74.75% (increased by 62.30% compared to control). However, all the ultrasonic pretreatment did not increase the DH of DWGP significantly (p > 0.05). The changes in UV–Vis spectra, SH and SS groups, surface hydrophobicity and hydrophobic protein content indicated that the structure of DWGP unfolded after ultrasound pretreatment. The HAA content of hydrolysate from the pretreated DWGP increased significantly (p < 0.05). The results proved that ultrasound pretreatment loosed the protein structure and exposed more HAA residues of protein to be attacked easily by alcalase. This resulted in the increase in the HAA content which related to the ACE inhibitory activity.