Inhibitory Activity Of Hydrolysates Research Articles

Bioproduction of Chitin Hydrolysate Containing N-Acetylglucosamine by Serratia marcescens PT6 Crude Chitinase and Its Effects on Bacterial Growth Inhibition in Various Temperature

N-acetylglucosamine (GlcNAc), a chitin monomer, can be used as a natural preservative to ensure food quality and safety. Combining natural preservatives with low storage temperature offers physical hurdles to bacterial growth in food. This study aimed to produce chitin hydrolysate containing GlcNAc using Serratia marcescens PT6 crude chitinase and investigate its effect on bacterial growth rate as a function of temperature. Crude chitinase from partial purification was used to hydrolyze 1.3% colloidal chitin. The optimal enzymatic conditions were pH 6 and 45˚C for 120 min, at an enzyme:substrate ratio of 1:1, yielding a 65.6 µg/mL GlcNAc. Inhibitory activity of hydrolysate containing 2.5-7.5 ppm GlcNAc on Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus was measured at 4, 15, and 30oC in nutrient broth. Bacterial growth was measured using of optical density for each combination of GlcNAc concentration and temperature. Growth curves fitted by the Baranyi and Roberts model were developed using DMFit software. The growth rate was converted to the square root and then modeled as a function of temperature using the Ratkowsky square root model. Incubation temperature exerted a pronounced effect on the inhibition of all bacterial species (P<0.0001), with the greatest effect observed for E. coli at 30°C (P<0.0001), and the least effect for V. parahaemolyticus (P=0.0878). The inhibitory effect of GlcNAc in chitin hydrolysate was only significant for E. coli (P<0.0001) and S. aureus (P=0.0041). This study revealed that the effect of temperature in growth inhibition was more significant than GlcNAc addition. However, a reduction in bacterial growth with the addition of GlcNAc at 30°C was observed, which may be effective for food encountered thermal abuse conditions. Further investigation of the effect of GlcNAc on bacteria structure and metabolism is required to elucidate the mechanism of GlcNAc as a food preservative.

Effects of dual-frequency slit ultrasound on the enzymolysis of high-concentration hydrolyzed feather meal: Biological activities and structural characteristics of hydrolysates

Ultrasound-assisted enzymolysis has been applied to improve conventional enzymolysis, while there are rare reports on the application of ultrasound to high-concentration feather protein enzymolysis. Therefore, the feasibility of dual-frequency slit ultrasound (DFSU) for enzymolysis of high-concentration hydrolyzed feather meal (HFM), as well as the biological activities and structural characteristics of hydrolysates were investigated. The single-factor test was used to optimize the ultrasonic processing parameters: substrate concentration, frequency mode, intermittent ratio, power density, and time. The results showed that protein recovery rate and conversion rate increased by 6.08% and 18.63% under the optimal conditions (200 g/L, 28/80 kHz, 5:2 s/s, 600 W/L, and 3 h) compared with conventional enzymolysis, respectively. The macromolecular proteins in hydrolysates were converted into micromolecular peptides (< 500 Da) when treated by DFSU, and antioxidant activity and angiotensin-I-converting enzyme (ACE) inhibitory activity of hydrolysates were increased. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images illustrated the microstructure changes of feather protein particles in the ultrasound-assisted enzymatic hydrolysates of HFM (UEH), including more porous, smaller, and more uniform. Additionally, the conformation of protein molecules was significantly affected (P < 0.05), including the increase in free sulfhydryl (SH), the decrease in disulfide bond (SS) and surface hydrophobicity (H0). Fourier transform infrared (FTIR) spectra analysis further showed that the secondary structure of feather proteins was modified with a reduction in α-helix, β-turn, and β-sheet, while an increase in random coil content was observed. These results indicated that DFSU could be a promising method to enhance high-concentration HFM for preparing peptide-rich hydrolysates with high antioxidant activity and ACE inhibitory activity.

In-situ and real-time monitoring of enzymatic process of wheat gluten by miniature fiber NIR spectrometer

The degree of hydrolysis (DH), peptide content and angiotensin I-converting enzyme (ACE) inhibitory activity of hydrolysates are the three most important indexes for process control in proteins enzymatic hydrolysis and ACE inhibitory peptides preparation. In this study, a miniature fiber optical spectrometer in conjunction with multivariate analysis for in-situ and real-time monitoring of the parameters (DH, peptide content and ACE inhibitory activity) in wheat gluten (WG) hydrolysis process. Synergy interval partial least square (Si-PLS) algorithm was performed to variables selection and the regression model was calibrated. The performance of a model was evaluated by the correlation coefficient (Rp) and the root mean square error (RMSEP) in the prediction set. The results showed that Rp=0.9270, RMSEP=1.73% for DH; Rp=0.9673, RMSEP=0.79mg/ml for peptide content; Rp=0.9507, RMSEP=5.12% for ACE inhibitory. This work demonstrated that miniature fiber optical spectrometer combined Si-PLS model can be used as an alternative method for in-situ and real-time monitoring the enzymatic process of WG.

Rice bran protein hydrolysates exhibit strong in vitro α-amylase, β-glucosidase and ACE-inhibition activities.

The objective of this study was to systematically examine the in vitro health-promotion activities of rice bran protein hydrolysates. Rice bran proteins were fractioned into albumin, globulin, prolamin and glutelin, which were subjected to hydrolysis by four protease preparations, namely Alcalase, Neutrase, Flavourzyme and Protamax, and the inhibitory activities of the hydrolysates against α-amylase, α-glucosidase and angiotensin converting enzyme (ACE), were monitored over a hydrolysis period of 240 min. Active peptides in the hydrolysates were isolated by ultra-filtration and ion-exchange chromatography and the peptide sequences of the active fractions were identified by LC-MS/MS. Hydrolysis of the proteins resulted in significant increases in these bioactivities, which were generally correlated with the degree of protein hydrolysis. In general, the highest bioactivities were found with albumin and glutelin hydrolysates, followed by globulin hydrolysates, while prolamin hydrolysates showed the lowest activities. Of the four enzymes used, Alcalase- and Protamax-catalysed hydrolysates generally had the highest activities while Flavourzyme-produced hydrolysates had the lowest activity. The MW < 3 kDa fraction of the Alcalase-catalysed glutelin hydrolysates had the highest β-glucosidase inhibition activity, which was identified to contain 13 peptides with six to 32 amino acid residues. The α-amylase and α-glucosidase inhibitory activities of albumin and glutelin hydrolysates produced by Alcalase and Protamax were comparable in magnitude to those of the standard anti-diabetic drug acarbose, and had the potential to be developed into a dietary or nutraceutical supplement for the management of diabetes.

Angiotensin I‐converting enzyme inhibitory activity of protein hydrolysates prepared from three freshwater carps (Catla catla, Labeo rohita and Cirrhinus mrigala) using Flavorzyme

SummaryFish protein hydrolysates from three freshwater carps, Catla catla, Labeo rohita and Cirrhinus mrigala with different degree of hydrolysis (DH) (5%, 10%, 15% and 20%), were prepared using Flavorzyme enzyme and designated as HCF, HRF and HMF, respectively. The angiotensin I‐converting enzyme (ACE) inhibitory activity of hydrolysates was found to vary from 43 ± 2% to 71 ± 3%. Based on ACE inhibitory activity, HRF with DH‐15% was taken up for further study. The mode of ACE activity inhibition by HRF‐DH 15% was mixed type as revealed by Lineweaver–Burk plot. Sequential digestion of HRF‐DH 15% using pepsin and pancreatin decreased the ACE inhibitory activity from 76% to 63%. Partial purification of HRF‐DH 15% by size exclusion chromatography gave three different fractions designated as F‐1, F‐2 and F‐3 with the molecular mass in the range of 6456–407 Da. Fraction 2 had significantly higher ACE inhibitory activity than the other fractions.

Ultrasonic pretreatment of corn gluten meal proteins and neutrase: Effect on protein conformation and preparation of ACE (angiotensin converting enzyme) inhibitory peptides

The impact of ultrasound pretreatment of corn gluten meal (CGM) protein and neutrase on the ACE (angiotensin converting enzyme) inhibitory activity of hydrolysate was studied. The UV–vis differential spectra and fluorescence spectra of the CGM protein and neutrase were determined. The neutrase activity and the ACE inhibitory activity of the CGM protein hydrolysates were analyzed. The highest ACE inhibitory activity was obtained at 10min hydrolysis when the CGM protein was pretreated at 20kHz for 10–30min and at a power of 1000W. The neutrase activity and ACE inhibitory activity were at their maxima at sonication of 250W and sonication time of 15min. The fluorescence and UV differential spectra of the proteins of CGM and neutrase reflected molecular unfolding due to sonication. Sonication of the protein substrate and the enzyme before hydrolysis may be an effective way to enhance the ACE inhibition attributes of the hydrolysate.

Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 proteases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment significantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5min resulted in a significantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release of ACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improved the DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes. The major peptides obtained with Alcalase were identified; all were released from the triple helix structure of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory peptides. The present results suggest that collagen-rich food materials are good substrates for the release of potent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.

COMPARATIVE STUDY ON ANGIOTENSIN CONVERTING ENZYME INHIBITORY ACTIVITY OF HYDROLYSATE OF MEAT PROTEIN OF INDONESIAN LOCAL LIVESTOCKS

The experiment was conducted to investigate the angiotensin converting enzyme (ACE) inhibitoryactivity of hydrolysate in meat protein of Bali cattle, Kacang goat, native chicken, and local duck. Themeats of Bali cattle, Kacang goat, native chicken, and local duck were used in this study. The meatswere ground using food processor added with aquadest to obtain meat extract. The meat extracts werethen hydrolyzed using protease enzymes to obtain hydrolysate of meat protein. Protein concentration ofmeat extract and hydrolysate of meat protein were determined, and were confirmed by sodium dodecylsulfate - poly acrylamide gel electrophoresis (SDS-PAGE). ACE inhibitory activity of hydrolysate ofmeat protein derived from Bali cattle, Kacang goat, native chicken, and local duck was also determined.The results showed that protein concentration of hydrolysate of meat protein of Bali cattle, Kacang goat,native chicken, and local duck meat was significantly higher than their meat extracts. SDS-PAGEanalysis indicated that hydrolysate of meat protein of Bali cattle, Kacang goat, native chicken, and localduck had more peptides with lower molecular weight, compared to their meat extracts. Hydrolysate ofmeat protein of Bali cattle, Kacang goat, native chicken, and local duck had potencies in inhibiting ACEactivity, so it will potentially reduce blood pressure.

Production of enzymatic hydrolysates with antioxidant and angiotensin-I converting enzyme inhibitory activity from pumpkin oil cake protein isolate

Protein isolate from pumpkin oil cake (PuOC PI) was hydrolysed by alcalase, flavourzyme and by sequential use of these enzymes, respectively, and the antioxidant properties and angiotensin-I converting enzyme (ACE) inhibitory activities of hydrolysates were evaluated. Under the same reaction conditions, alcalase hydrolysates showed a higher degree of hydrolysis (DH) than did flavourzyme hydrolysates. The highest DH’s by individual enzymes were 53.23 ± 0.7% and 37.17 ± 1.05%, respectively, both at 60 min. The increase of radical scavenging activity (RSA) in hydrolysates was positively correlated with the increase of DH, for both enzymes, though hydrolysates with flavourzyme showed two- or three-fold lower RSA than with alcalase. The highest bioactive potential was determined in the alcalase hydrolysate at 60 min, with RSA being 7.59 ± 0.081 mM TEAC/mg and ACE-inhibitory activity 71.05 ± 7.5% (IC 50 = 0.422 mg/ml). When this hydrolysate was further hydrolysed by flavourzyme, DH increased up to 69.29 ± 0.9%, but lower RSA (4.82 ± 0.21 mM TEAC/mg) and ACE-inhibitory activity (55.81 ± 6.196%) were determined in the final hydrolysate. This study suggested that the PuOC proteins could be converted into protein hydrolysates with antioxidant and ACE-inhibitory activities by enzymatic hydrolysis. Alcalase was shown as promising enzyme in further development of bioprocesses for the production of new bioactive food ingredients.

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate, enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC 50 values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL −1, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.