Production Of ACE-inhibitory Peptides Research Articles

Directed‐Vat‐Set starter producing ACE ‐inhibitory peptides: Optimization and evaluation of stability

Angiotensin convening enzyme (ACE) inhibitory peptides can inhibit ACE effectively, which is crucial to regulating blood pressure. Many studies on the production of ACE-inhibitory peptides by probiotics, but few works have been carried out on Directed-Vat-Set (DVS) starter producing ACE-inhibitory peptides. In the study, The DVS starter producing ACE-inhibitory peptides were optimized using Lactiplantibacillus plantarum L69 (L69) and starter with Lactobacillus bulgaricus and Streptococcus thermophilus (Starter LS) through the single factor and the Box–Behnken design. The optimum conditions were: Addition of DVS starter is 0.20%, which is composed of Lactiplantibacillus plantarum powder and starter in the ratio of 1:1, cultivation time of 4.5 hr, fermentation temperature 42°C, and the response value were pH of 4.63, optical density (OD) of 0.759, ACE-inhibition activity of 87.20% in fermented milk. The accelerated storage test demonstrated that the inactivation rate constant of the DVS starter was k−18 = 4.24 × 10−3 at −18°C, indicating good stability. Practical applications ACE-inhibitory peptides have a significant antihypertensive effect and are safe without toxic side effects compared with ordinary antihypertensive drugs. In this study, Single-factor and Box–Behnken design were used to optimize the conditions of producing ACE-inhibitory peptides from DVS starter and the acceleration test was used to evaluate its stability. The fermentation time of DVS starter is shorter and the storage property is better. This study provided technical support for the development of DVS starter for functional dairy products.

Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides

Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had Km values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC50 of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.

Milk Fermentation by Lacticaseibacillus rhamnosus GG and Streptococcus thermophilus SY-102: Proteolytic Profile and ACE-Inhibitory Activity

Health benefits of probiotics and production of inhibitors of angiotensin converting enzyme (ACE) released during milk fermentation are well known. That is why in this investigation the proteolytic profile and ACE inhibitory capacity of peptide fractions from protein hydrolysis of milk during fermentation processes was analyzed. Milk fermentation was carried out inoculating 106 CFU of L. rhamnosus GG, S. thermophilus SY-102 and with both bacteria. The proteolytic profile was determined using: TNBS, SDS-PAGE and SEC-HPLC techniques. In vitro ACE inhibition capacity was measured. The pH of 4.5 was reached at 56 h when the milk was fermented with L. rhamnosus, at 12 h with S. thermophillus and at 41 h in the co-culture. Production of free amino groups corresponded with the profile of low molecular weight peptides observed by SDS-PAGE and SEC-HPLC. Co-culture fermentation showed both the highest concentration of low molecular weight peptides and the ACE inhibitory activity (>80%). Results indicated that the combination of lactic cultures could be useful in manufacture of fermented milk with an added value that goes beyond basic nutrition, such as the production of ACE-inhibitory peptides.

Application of Plackett-Burman Design in Screening Casein and Prebiotics for the Production of ACE Inhibitory Peptides from Cow Milk Fermented by L. bulgaricus LB6

Abstract Prebiotics can play an important role in functional foods. In this paper, casein and five probiotics were selected to study the effects on ACE inhibitory peptides in fermented milk of L. bulgaricus LB6 through Plackett-Burman design, so as to improve the production of ACE inhibitory peptides. The results showed that xylooligosaccharides (XOS), fructosaccharide (FOS) and inulin had the most significant effect on the yield of ACE inhibitory peptides. Optimization added the amount of the three prebiotics added, that is, the amount of XOS added was 0.7%, the amount of FOS added was 1.1%, and the amount of inulin added was 0.7%. It provides a basis for subsequent optimization experiments.

The application of the Plackett-Burman design in investigating ACE inhibitory peptide-producing conditions and media for Lactobacillus bulgaricus LB6.

The intake of angiotensin converting enzyme (ACE, EC 3.4.15.1) inhibitory peptides from food has become a promising method for treating hypertension. Lactic acid producing bacteria are widely used to produce ACE inhibitors during fermentation. Several factors which promote the production of ACE inhibitory peptides in Lacto- bacillus bulgaricus LB6 (inoculum size, incubation temperature, goat’s milk powder, sterilization time, whey powder, casein hydrolyses and calcium lactate) and media (casein peptone, soybean peptone, glucose, casein, ascorbic acid, Ca(H2PO4)2 and lactose) were investigated to optimize and increase ACE inhibitory activity during the fermentation of goat’s milk by using a Plackett-Burman design, in accordance with previous tests. The results indicated that incubation temperature, whey powder, calcium lactate, soybean peptone,glucose and casein had significant effects on both ACE inhibition rate and viable counts. In addition, the effects of incubation temperature, whey powder, calcium lactate and glucose were found to increase ACE inhibition rate, while soybean peptone and casein caused it to decrease. This investigation of conditions which promote the production of ACE inhibitory peptides and media for LB6 was performed using a Plackett-Burman design. The results indicated that incubation tem- perature, whey powder, calcium lactate, soybean peptone, glucose and casein had a significant impact on the ACE inhibition rate and viable counts of LB6, which provide the basis and reference for further optimization.

In Vitro and In Silico Analysis of Novel ACE-Inhibitory Bioactive Peptides Derived from Fermented Goat Milk

In the study, two Lactobacillus cultures i.e. L. casei (NK9) and L. fermentum (LF) were studied for their proteolytic activity, di and tripeptidase activity, ACE-inhibitory activity and peptides production under optimized growth condition from fermented goat milk (Capra aegagrus hircus). NK9 and LF were found to be a strong proteolytic culture with 2.0% rate of inoculation after 48 h. LF (10 kDa retentate) produced maximum peptides among all the retentates of the fermented goat milk. Goat milk fermented with NK9 (10 kDa permeates) exhibited peptide sequence i.e. AFPEHK which had ACE inhibitory activity, matched with goat milk protein databases of AHTPDB. However, L. casei (NK9) and L. fermentum (LF) could be explored for the production of ACE inhibitory peptides from fermented goat milk.

Preparation and purification of angiotensin‐converting enzyme inhibitory peptides from hydrolysate of shrimp (Litopenaeus vannamei) shell waste

SummaryAngiotensin I‐converting enzyme (ACE) inhibitory peptides from the shrimp shell waste (SSW) were isolated using different proteases. The orthogonal test results showed alcalase hydrolysates with ACE inhibitory activity of 67.07% under the optimal hydrolysis conditions of 60 °C hydrolysis temperature, pH = 9.5, 25 g L−1 substrate and 4000 U g−1 of enzyme, whereas neutral protease hydrolysates had an ACE inhibitory activity of 84.04% under the hydrolysis temperature of 50 °C at pH = 7.0 with 25 g L−1 of substrate and in the presence of 2000 U g−1 of enzyme. Neutral protease was more suitable for the production of ACE inhibitory peptides from SSW, where peptides with MW <5 kDa were recommended. The results of this study indicated that peptides obtained from SSW are as beneficial as antihypertension compounds in the functional food resources.

Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study

In this study, sweet sorghum grain protein (SSGP) was hydrolyzed using alcalase yielding ACE inhibitory peptides. A kinetic model was proposed to describe the enzymolysis process of SSGP. The kinetic parameters, a and b, were determined according to experimental data. It was found that the model was reliable to describe the kinetic behaviour for SSGP hydrolysis by alcalase. After hydrolysis, the SSGP hydrolysate with DH of 19% exhibited the strongest ACE inhibitory activity and the hydrolysate was then used to isolate ACE inhibitory peptides. A novel ACE inhibitory peptide was successfully purified from this hydrolysate by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reversed-phased high performance liquid chromatography (RP-HPLC). The amino acid sequence of the purified peptide was identified as Thr–Leu–Ser (IC50=102.1μM). The molecular docking studies revealed that the ACE inhibition of the tripeptide was mainly attributed to its C-terminal Ser, which can effectively interact with the S1 and S2 pockets of ACE. Our studies suggest that the tripeptide from the SSGP hydrolysate can be utilized to develop functional food ingredients or pharmaceuticals for prevention of hypertension.

Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions

The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

Combined Effect of Ultrasound and Enzymatic Treatments on Production of ACE Inhibitory Peptides from Wheat Germ Protein

Combined Effect of Ultrasound and Enzymatic Treatments on Production of ACE Inhibitory Peptides from Wheat Germ Protein

Production of ace inhibitory peptides by digestion of chickpea legumin with alcalase

Short peptides from different sources have proved to be very efficient inhibitors of the angiotensin I-converting enzyme, an enzyme with a major role in the regulation of blood pressure. These peptides are of therapeutic value, so that the possibility of obtaining such peptides by treatment of chickpea legumin with the protease alcalase has been explored. Legumin is the main storage protein in chickpea seeds. Treatment of legumin with alcalase yielded a hydrolysate that inhibited the angiotensin I-converting enzyme with an IC50 of 0.18 mg/ml. Fractionation of this hydrolysate by reverse phase chromatography afforded six inhibitory peptides with IC50 values ranging from 0.011 to 0.021 mg/ml. All these peptides contain the amino acid methionine and are also rich in other hydrophobic amino acids. These results demonstrate that hydrolysates of chickpea legumin obtained by treatment with alcalase are a good source of peptides with angiotensin I-converting enzyme inhibitory activity.