Most commercial fermented milks showed moderate ACE-inhibitory activity, though some exceptions had higher activity that could relate to the milk origin. Two fermented milks were subjected to simulated gastrointestinal digestion, which increased their ACE-inhibitory activity, suggesting physiological digestion promotes formation of active peptides. Peptides generated from one product during digestion were sequenced, and most formed after 30 minutes of pancreatic enzyme incubation, indicating digestion facilitates peptide release from proteins in these fermented products.
This document summarizes a study that investigated substituting an ethanol extract of alfalfa for antibiotics in broiler chicken feed. Sixty broiler chicks were divided into three treatment groups: a control group and two groups fed diets with 0.1% or 0.15% alfalfa extract. Liver enzyme levels and weight gain were measured. While alkaline phosphatase levels increased significantly in the treatment groups, other liver enzymes did not change significantly. All treatment groups showed significant weight gain compared to the control. The study concluded that the alfalfa extract can promote weight gain in broilers without antibiotics and does not negatively impact liver function.
This study assessed the antioxidant properties and polyphenol, caffeine, and chlorogenic acid content of extracts from spent coffee grounds using different extraction methods. Shorter ultrasound-assisted extraction (15 minutes) yielded extracts with lower total polyphenol content but higher levels of caffeine and chlorogenic acid compared to longer extraction times, and greater antioxidant activity. While extraction yields were low at 6-8% for caffeine and 0.8-0.9% for chlorogenic acid, applying this to the annual global spent coffee grounds waste of 0.63 billion kg could recover significant amounts of these compounds. The results support the feasibility of utilizing spent coffee grounds as a source of natural antioxidants.
The document describes optimization of protein extraction from sacha inchi (Plukenetia volubilis) kernel cake using alkaline and enzyme-assisted methods. Response surface methodology was used to optimize extraction parameters. For alkaline extraction, optimal conditions were 54.2°C, solvent/meal ratio of 42/1, 1.65M NaCl concentration at pH 9.5 for 30 minutes, yielding 29.7% protein. For enzyme extraction, optimal conditions were 5.6% enzyme concentration, 40.4 minutes extraction at pH 9.0 and 50°C, solvent/meal ratio of 50/1, yielding 44.7% protein and 7.8% hydrolysis degree. The enzyme method produced higher protein recovery
This document discusses protein adducts as biomarkers of exposure to alkylating agents. It begins by introducing protein adducts and globin adducts specifically as surrogate biomarkers for DNA adducts. The document then details a research plan to test the hypothesis that globin adducts undergo degradation, producing free amino acid adducts that are excreted in urine and could serve as non-invasive biomarkers of exposure. Initial results are presented demonstrating the successful synthesis of model amino acid adducts and detection of some administered adducts and their metabolites in rat urine. Further research is planned to study the fate of native globin adducts in vivo.
This document discusses enzymatic tailoring of hydrolysate properties. It provides an overview of enzymes, including enzyme classes and nomenclature. It describes how enzymatic hydrolysis can be used to customize the chemical and biological properties of protein hydrolysates, such as solubility, molecular weight distribution, and amino acid profile. Several examples are given of how different proteases can be used to tailor the degree of hydrolysis, antioxidant activity, foam stability, and amino acid composition of soy flour hydrolysates. The document concludes that enzymatic tailoring provides opportunities to engineer product specifications, improve yields and recoveries, customize protein properties, and develop new active ingredients and product paradigms from plant sources.
Hydrolysate Characterization Technical Presentation Webinar11 2009Mason Williams
1) The document discusses developing a chemically defined hydrolysate alternative to mitigate risks associated with undefined natural hydrolysates.
2) The approach involves fractionating multiple hydrolysates using RP-HPLC and identifying bioactive fractions that stimulate cell growth and protein production in CHO cells.
3) A new product called EX-CELL CD Hydrolysate Fusion was developed that contains synthetic components identified from bioactive fractions and provides 80-100% of the performance of natural hydrolysates.
This document summarizes a study that investigated the antidiabetic and antioxidant effects of the hydro-alcoholic stem bark extract of Callicarpa arborea Roxb. in diabetic rats. Key findings include:
1) The extract showed significant antioxidant activity in various in vitro assays and was found to be non-toxic up to 2000 mg/kg.
2) In diabetic rats, the extract significantly reduced blood glucose levels, improved glucose tolerance, increased serum insulin levels, and positively affected lipid profiles and liver function markers.
3) Histopathological analysis of pancreas and liver tissues showed protective effects of the extract against diabetes-induced damage.
4) The results suggest that the stem bark
This document summarizes a study that investigated substituting an ethanol extract of alfalfa for antibiotics in broiler chicken feed. Sixty broiler chicks were divided into three treatment groups: a control group and two groups fed diets with 0.1% or 0.15% alfalfa extract. Liver enzyme levels and weight gain were measured. While alkaline phosphatase levels increased significantly in the treatment groups, other liver enzymes did not change significantly. All treatment groups showed significant weight gain compared to the control. The study concluded that the alfalfa extract can promote weight gain in broilers without antibiotics and does not negatively impact liver function.
This study assessed the antioxidant properties and polyphenol, caffeine, and chlorogenic acid content of extracts from spent coffee grounds using different extraction methods. Shorter ultrasound-assisted extraction (15 minutes) yielded extracts with lower total polyphenol content but higher levels of caffeine and chlorogenic acid compared to longer extraction times, and greater antioxidant activity. While extraction yields were low at 6-8% for caffeine and 0.8-0.9% for chlorogenic acid, applying this to the annual global spent coffee grounds waste of 0.63 billion kg could recover significant amounts of these compounds. The results support the feasibility of utilizing spent coffee grounds as a source of natural antioxidants.
The document describes optimization of protein extraction from sacha inchi (Plukenetia volubilis) kernel cake using alkaline and enzyme-assisted methods. Response surface methodology was used to optimize extraction parameters. For alkaline extraction, optimal conditions were 54.2°C, solvent/meal ratio of 42/1, 1.65M NaCl concentration at pH 9.5 for 30 minutes, yielding 29.7% protein. For enzyme extraction, optimal conditions were 5.6% enzyme concentration, 40.4 minutes extraction at pH 9.0 and 50°C, solvent/meal ratio of 50/1, yielding 44.7% protein and 7.8% hydrolysis degree. The enzyme method produced higher protein recovery
This document discusses protein adducts as biomarkers of exposure to alkylating agents. It begins by introducing protein adducts and globin adducts specifically as surrogate biomarkers for DNA adducts. The document then details a research plan to test the hypothesis that globin adducts undergo degradation, producing free amino acid adducts that are excreted in urine and could serve as non-invasive biomarkers of exposure. Initial results are presented demonstrating the successful synthesis of model amino acid adducts and detection of some administered adducts and their metabolites in rat urine. Further research is planned to study the fate of native globin adducts in vivo.
This document discusses enzymatic tailoring of hydrolysate properties. It provides an overview of enzymes, including enzyme classes and nomenclature. It describes how enzymatic hydrolysis can be used to customize the chemical and biological properties of protein hydrolysates, such as solubility, molecular weight distribution, and amino acid profile. Several examples are given of how different proteases can be used to tailor the degree of hydrolysis, antioxidant activity, foam stability, and amino acid composition of soy flour hydrolysates. The document concludes that enzymatic tailoring provides opportunities to engineer product specifications, improve yields and recoveries, customize protein properties, and develop new active ingredients and product paradigms from plant sources.
Hydrolysate Characterization Technical Presentation Webinar11 2009Mason Williams
1) The document discusses developing a chemically defined hydrolysate alternative to mitigate risks associated with undefined natural hydrolysates.
2) The approach involves fractionating multiple hydrolysates using RP-HPLC and identifying bioactive fractions that stimulate cell growth and protein production in CHO cells.
3) A new product called EX-CELL CD Hydrolysate Fusion was developed that contains synthetic components identified from bioactive fractions and provides 80-100% of the performance of natural hydrolysates.
This document summarizes a study that investigated the antidiabetic and antioxidant effects of the hydro-alcoholic stem bark extract of Callicarpa arborea Roxb. in diabetic rats. Key findings include:
1) The extract showed significant antioxidant activity in various in vitro assays and was found to be non-toxic up to 2000 mg/kg.
2) In diabetic rats, the extract significantly reduced blood glucose levels, improved glucose tolerance, increased serum insulin levels, and positively affected lipid profiles and liver function markers.
3) Histopathological analysis of pancreas and liver tissues showed protective effects of the extract against diabetes-induced damage.
4) The results suggest that the stem bark
This method confirms the presence of the tranquilizer azaperone and its metabolite azaperol in swine liver tissue using gas chromatography/mass spectrometry (GC/MS). Swine liver is ground and extracted using acetonitrile and sodium chloride buffer. Solid phase extraction is used to isolate azaperone and azaperol, which are then eluted and analyzed by GC/MS. The method was validated by analyzing fortified tissue samples containing 10 ppb of both compounds and incurred tissue from pigs dosed with azaperone, confirming the presence of the parent drug and metabolite.
CYP2A6_HPLC_PK_2015 New Simple Method for Coumarin in Liver Cytochrome of RatsWael Ebied
This document describes the development and validation of a new liquid chromatography method for determining coumarin and its 7-hydroxy metabolite as a marker of cytochrome P450 2A6 activity in rats. The method uses a C18 column and isocratic mobile phase to separate coumarin, 7-hydroxycoumarin, and an internal standard. The method was validated and showed good linearity, precision, and accuracy. This validated method was then applied to study the effect of resveratrol, sulforaphane, and thymoquinone on hepatic CYP2A6 activity in spontaneously hypertensive rats.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Hepatoprotective activity of aqueous extract of Hibiscus Sabdariffa on alcoho...Bhavana Gundavarapu
The aim of present study was to investigate the Hepatoprotective activity of aqueous extract of Hibiscus sabdariffa (Malvaceace) leaves in albino rats on alcohol induced hepatotoxic activity. .
Strategie nutraceutiche per ridurre l'infiammazione.CreAgri Europe
I polifenoli estratti dalle olive sono in grado di ridurre l'infiammazione mediata da TNF alfa. Esiste inoltre un effetto sinergico nella riduzione dello stato infiammatorio tra idrossitirosolo e glucosammina.
- inglese - testo scientifico -
This study examined the secretion and intracellular trafficking of apolipoprotein A-V (apoA-V) using transfected cell lines. The key findings were:
1) Only about 20% of newly synthesized apoA-V was secreted from cells within 3 hours, with about 65% undergoing degradation before secretion.
2) When cells were treated with oleic acid to stimulate triglyceride synthesis, apoA-V secretion decreased while cell-associated apoA-V increased and moved to cytosolic lipid droplets.
3) Expression of apoA-V in hepatoma cells inhibited triglyceride secretion by 50% and reduced the diameter of secreted VLDL particles, but did not impact apolip
This study investigated the effects of antioxidants such as alpha-lipoic acid, vitamin C, and resveratrol on the overexpression of cytochrome P450 2E1 in streptozotocin-induced diabetic rat tissues. Real-time PCR and RT-PCR analysis showed that administration of vitamin C to diabetic rats had the most significant effect in reducing CYP2E1 overexpression, while administration of vitamin C together with alpha-lipoic acid caused the second most decrease. Administration of alpha-lipoic acid alone resulted in lower reduction of CYP2E1 expression compared to vitamin C and the combination of vitamins.
Purification optimization and characterization of protease from Bacillus va...Vaibhav Maurya
This presentation is a research work carried out by me in B.Tech 8 semester. and gives an idea about purification, optimization and characterization of protease from Bacillus Valismortis
This document summarizes various experiments analyzing mitochondrial ribosome interacting proteins and overexpression of EF-Tu and EF-G proteins in E. coli. Figure 1 shows a SDS-PAGE gel with labeled mitochondrial ribosome subunit samples. The second section provides criteria for database analysis of mitochondrial ribosome associated proteins. Figures 2-4 show SDS-PAGE gels and western blots analyzing overexpression of EF-Tu and purification of EF-Tu and EF-G. Figures 7-8 again examine overexpression of EF-Tu and EF-G using SDS-PAGE and western blot.
1) The document discusses measuring enzyme activity, which is important for understanding cellular functions and diagnosing diseases. Enzyme activity is determined by measuring the rate of substrate conversion to product.
2) Common methods to measure enzyme activity include spectrophotometric, fluorescence, manometric, and electrode methods. Spectrophotometric methods are often used to monitor the appearance of product that absorbs light.
3) Factors that affect enzyme activity include substrate and enzyme concentration, temperature, pH, and inhibitors. Activity is typically measured under conditions where less than 20% of substrate is consumed to determine the initial reaction rate.
Isolation and analytical characterization of local malaysian leech saliva ext...Younis I Munshi
This study isolated and characterized proteins and peptides from the saliva of local Malaysian leeches. A modified extraction method was used to obtain leech saliva without harming the leeches. The saliva extract was found to contain high concentrations of proteins using UV and Bradford assays. Gel electrophoresis revealed over 25 protein and peptide bands between 3.7-80 kDa. RP-HPLC showed over 30 distinct peaks in the saliva extract. Some proteins matched known proteins from related leech species, while others were potentially novel biologically active compounds. This research provides a basis for further investigation of proteins in Malaysian leech saliva that may have applications in medicine.
Glutathione S-transferase enzymes (GSTs) play central roles in phase II detoxification of both xenobiotics and endogenous compounds in almost all living organisms. The enzyme was extracted and partially purified from wheat leaves through a procedure including ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures yielded a 7.14-fold purification with 71% recovery. Optimum activity conditions-pH, temperature and ionic strength-of the enzyme were determined. Its some kinetic properties such as Vmax, KM, and kcat were calculated for GSH and CDNB substrates. The kcat/KM values of the enzyme were 603.5 for GSH and 385.3 for CDNB. The native molecular weight of the enzyme was estimated to be 52 kDa based on its mobility in gel filtration column.
1) The study investigated the potential protective effects of honey against paracetamol-induced liver toxicity in rats.
2) Administration of paracetamol caused significant increases in liver enzymes and oxidative stress markers along with liver damage.
3) Pretreatment with honey or the reference drug silymarin prevented the paracetamol-induced increases in liver enzymes and oxidative stress and reduced liver damage based on histological examination.
This project involves the structural analysis of various starch debranching enzymes from Bacillus species and their production. The objectives are to perform comparative sequence and structural analysis of active sites, analyze activity of debranching enzymes from Bacillus, and optimize production of these enzymes. Experiments will include culturing Bacillus organisms, qualitative and quantitative analysis of amylase and pullulanase activity, protein purification, DNA isolation, sequence alignment and structural modeling of enzymes. Preliminary results show highest amylase and pullulanase activity in Bacillus strains 121 and 1790.
This study assessed the enzymatic hydrolysis of proteins from the microalgae Chlorella pyrenoidosa and Spirulina sp. LEB 18 to produce protein hydrolysates. Three commercial proteases were used under different conditions to hydrolyze the microalgae proteins. The highest degrees of hydrolysis for Spirulina and Chlorella, respectively, were 55.31% and 52.9% and were obtained with 4 hours of reaction time using Protemax N200 protease. Statistical analysis showed that enzyme concentration, substrate concentration, and reaction time significantly affected the degree of hydrolysis. The results indicate it is possible to obtain protein hydrolysates with varying degrees of hydrolysis from microalgae
This study investigated whether BAR704, a potent and selective FXR agonist, protects against liver fibrosis in a mouse model. Mice were administered carbon tetrachloride to induce liver fibrosis over 8 weeks. Treatment with BAR704 reversed biochemical and histological signs of fibrosis, reducing markers of liver damage and collagen deposition. BAR704 also decreased expression of profibrogenic genes and normalized levels of genes related to endothelial function. The results suggest that selective FXR agonists like BAR704 may be an attractive treatment for liver fibrosis.
1) BAR501, a selective agonist of Gpbar1, promoted adipose tissue browning and autophagy in mice fed a high fat diet, improving their lipid metabolism and reducing features of non-alcoholic steatohepatitis (NASH).
2) In mice, BAR501 treatment attenuated weight gain, insulin resistance, liver steatosis and fibrosis induced by the high fat diet. It also increased the expression of browning and autophagy markers in white adipose tissue.
3) In cultured adipocytes, BAR501 treatment induced autophagy and the expression of genes related to browning and adipogenesis.
ANTIOXIDANT ACTIVITY AND HEPATOPROTECTIVE EFFECTOF POMEGRANATE PEEL AND WHEY...Anurag Raghuvanshi
The antioxidant activity of pomegranate peel powder (PPP) and whey powder (WP) was evaluated, their hepatoprotective effect of each alone or in combination (PPWP) at equal levels was also evaluated in Wistar rats against carbon tetrachloride (CCL4) induced liver injury.
The hepatoprotective activity was assessed using various biochemical parameters and histopathological studies.
Detection of the Antibacterial Activity of Bioactive Peptide Isolated from Fe...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Effect of estradiol -17 β on arachidonic acid metabolism in sheep uterus: in ...iosrjce
The effect of estradiol-17 β on Arachidonic acid (AA) metabolism in non-pregnant sheep uterus was
studied under in vitro conditions. On incubation of uterine slices with estradiol-17β, the levels of prostaglandins
were altered but not Lipoxygenase (LOX) products. Based on their analysis on conventional TLC technique, the
Cyclooxygenase (COX) products PGF2α, 6-keto PGF1α and PGE2 were shown to be altered over an incubation
period of 0 to 120 minutes. The LOX products, HPETEs and HETEs did not show any change upon incubation
with estradiol-17β. This study gives a preliminary understanding of role of estradiol on AA metabolism.
Biochemical and pharmacological study of biologically active preparation of p...inventionjournals
Our aim was to perform some biochemical and pharmacological studies of bioactive bovine placental preparation via digestion of cow placenta using enzyme contained in swine stomach. Amino acid compositions and contents in biologically active preparation of placenta, obtained by digestion of cow placenta with enzyme contained in swine stomach were measured by HPLC technique and it was found that contents of such amino acids as glycine, proline and lysine were highest and 9 essential amino acids, including valine, histidine, methionine, lysine, threonine, arginine, phenylalanine, leucine and isoleucine were measured. In pharmacological study, acute toxicity (LD50) of the preparation and effect of the preparation on immune response to sheep erythrocyte were investigated in white mice, weighing 18 to 20 g each. The study revealed acute toxicity (LD50) of the preparation was 60 ml per kg. Spleen index of the first and second experimental group animals treated by the preparation during both provoked and unprovoked immune responses increased by 1.2 to 3.09 times as compared to that of negative control animals, while splenocyte count elevated by 1.2 to 2.2 times than negative control animals. Higher contents of essential amino acids of the biologically active preparation of cattle placenta shows its biologically higher nutritive value, as well as pharmacological study reveals the preparation has minimal toxicity and higher effect to stimulate immune responses.
This method confirms the presence of the tranquilizer azaperone and its metabolite azaperol in swine liver tissue using gas chromatography/mass spectrometry (GC/MS). Swine liver is ground and extracted using acetonitrile and sodium chloride buffer. Solid phase extraction is used to isolate azaperone and azaperol, which are then eluted and analyzed by GC/MS. The method was validated by analyzing fortified tissue samples containing 10 ppb of both compounds and incurred tissue from pigs dosed with azaperone, confirming the presence of the parent drug and metabolite.
CYP2A6_HPLC_PK_2015 New Simple Method for Coumarin in Liver Cytochrome of RatsWael Ebied
This document describes the development and validation of a new liquid chromatography method for determining coumarin and its 7-hydroxy metabolite as a marker of cytochrome P450 2A6 activity in rats. The method uses a C18 column and isocratic mobile phase to separate coumarin, 7-hydroxycoumarin, and an internal standard. The method was validated and showed good linearity, precision, and accuracy. This validated method was then applied to study the effect of resveratrol, sulforaphane, and thymoquinone on hepatic CYP2A6 activity in spontaneously hypertensive rats.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Hepatoprotective activity of aqueous extract of Hibiscus Sabdariffa on alcoho...Bhavana Gundavarapu
The aim of present study was to investigate the Hepatoprotective activity of aqueous extract of Hibiscus sabdariffa (Malvaceace) leaves in albino rats on alcohol induced hepatotoxic activity. .
Strategie nutraceutiche per ridurre l'infiammazione.CreAgri Europe
I polifenoli estratti dalle olive sono in grado di ridurre l'infiammazione mediata da TNF alfa. Esiste inoltre un effetto sinergico nella riduzione dello stato infiammatorio tra idrossitirosolo e glucosammina.
- inglese - testo scientifico -
This study examined the secretion and intracellular trafficking of apolipoprotein A-V (apoA-V) using transfected cell lines. The key findings were:
1) Only about 20% of newly synthesized apoA-V was secreted from cells within 3 hours, with about 65% undergoing degradation before secretion.
2) When cells were treated with oleic acid to stimulate triglyceride synthesis, apoA-V secretion decreased while cell-associated apoA-V increased and moved to cytosolic lipid droplets.
3) Expression of apoA-V in hepatoma cells inhibited triglyceride secretion by 50% and reduced the diameter of secreted VLDL particles, but did not impact apolip
This study investigated the effects of antioxidants such as alpha-lipoic acid, vitamin C, and resveratrol on the overexpression of cytochrome P450 2E1 in streptozotocin-induced diabetic rat tissues. Real-time PCR and RT-PCR analysis showed that administration of vitamin C to diabetic rats had the most significant effect in reducing CYP2E1 overexpression, while administration of vitamin C together with alpha-lipoic acid caused the second most decrease. Administration of alpha-lipoic acid alone resulted in lower reduction of CYP2E1 expression compared to vitamin C and the combination of vitamins.
Purification optimization and characterization of protease from Bacillus va...Vaibhav Maurya
This presentation is a research work carried out by me in B.Tech 8 semester. and gives an idea about purification, optimization and characterization of protease from Bacillus Valismortis
This document summarizes various experiments analyzing mitochondrial ribosome interacting proteins and overexpression of EF-Tu and EF-G proteins in E. coli. Figure 1 shows a SDS-PAGE gel with labeled mitochondrial ribosome subunit samples. The second section provides criteria for database analysis of mitochondrial ribosome associated proteins. Figures 2-4 show SDS-PAGE gels and western blots analyzing overexpression of EF-Tu and purification of EF-Tu and EF-G. Figures 7-8 again examine overexpression of EF-Tu and EF-G using SDS-PAGE and western blot.
1) The document discusses measuring enzyme activity, which is important for understanding cellular functions and diagnosing diseases. Enzyme activity is determined by measuring the rate of substrate conversion to product.
2) Common methods to measure enzyme activity include spectrophotometric, fluorescence, manometric, and electrode methods. Spectrophotometric methods are often used to monitor the appearance of product that absorbs light.
3) Factors that affect enzyme activity include substrate and enzyme concentration, temperature, pH, and inhibitors. Activity is typically measured under conditions where less than 20% of substrate is consumed to determine the initial reaction rate.
Isolation and analytical characterization of local malaysian leech saliva ext...Younis I Munshi
This study isolated and characterized proteins and peptides from the saliva of local Malaysian leeches. A modified extraction method was used to obtain leech saliva without harming the leeches. The saliva extract was found to contain high concentrations of proteins using UV and Bradford assays. Gel electrophoresis revealed over 25 protein and peptide bands between 3.7-80 kDa. RP-HPLC showed over 30 distinct peaks in the saliva extract. Some proteins matched known proteins from related leech species, while others were potentially novel biologically active compounds. This research provides a basis for further investigation of proteins in Malaysian leech saliva that may have applications in medicine.
Glutathione S-transferase enzymes (GSTs) play central roles in phase II detoxification of both xenobiotics and endogenous compounds in almost all living organisms. The enzyme was extracted and partially purified from wheat leaves through a procedure including ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures yielded a 7.14-fold purification with 71% recovery. Optimum activity conditions-pH, temperature and ionic strength-of the enzyme were determined. Its some kinetic properties such as Vmax, KM, and kcat were calculated for GSH and CDNB substrates. The kcat/KM values of the enzyme were 603.5 for GSH and 385.3 for CDNB. The native molecular weight of the enzyme was estimated to be 52 kDa based on its mobility in gel filtration column.
1) The study investigated the potential protective effects of honey against paracetamol-induced liver toxicity in rats.
2) Administration of paracetamol caused significant increases in liver enzymes and oxidative stress markers along with liver damage.
3) Pretreatment with honey or the reference drug silymarin prevented the paracetamol-induced increases in liver enzymes and oxidative stress and reduced liver damage based on histological examination.
This project involves the structural analysis of various starch debranching enzymes from Bacillus species and their production. The objectives are to perform comparative sequence and structural analysis of active sites, analyze activity of debranching enzymes from Bacillus, and optimize production of these enzymes. Experiments will include culturing Bacillus organisms, qualitative and quantitative analysis of amylase and pullulanase activity, protein purification, DNA isolation, sequence alignment and structural modeling of enzymes. Preliminary results show highest amylase and pullulanase activity in Bacillus strains 121 and 1790.
This study assessed the enzymatic hydrolysis of proteins from the microalgae Chlorella pyrenoidosa and Spirulina sp. LEB 18 to produce protein hydrolysates. Three commercial proteases were used under different conditions to hydrolyze the microalgae proteins. The highest degrees of hydrolysis for Spirulina and Chlorella, respectively, were 55.31% and 52.9% and were obtained with 4 hours of reaction time using Protemax N200 protease. Statistical analysis showed that enzyme concentration, substrate concentration, and reaction time significantly affected the degree of hydrolysis. The results indicate it is possible to obtain protein hydrolysates with varying degrees of hydrolysis from microalgae
This study investigated whether BAR704, a potent and selective FXR agonist, protects against liver fibrosis in a mouse model. Mice were administered carbon tetrachloride to induce liver fibrosis over 8 weeks. Treatment with BAR704 reversed biochemical and histological signs of fibrosis, reducing markers of liver damage and collagen deposition. BAR704 also decreased expression of profibrogenic genes and normalized levels of genes related to endothelial function. The results suggest that selective FXR agonists like BAR704 may be an attractive treatment for liver fibrosis.
1) BAR501, a selective agonist of Gpbar1, promoted adipose tissue browning and autophagy in mice fed a high fat diet, improving their lipid metabolism and reducing features of non-alcoholic steatohepatitis (NASH).
2) In mice, BAR501 treatment attenuated weight gain, insulin resistance, liver steatosis and fibrosis induced by the high fat diet. It also increased the expression of browning and autophagy markers in white adipose tissue.
3) In cultured adipocytes, BAR501 treatment induced autophagy and the expression of genes related to browning and adipogenesis.
ANTIOXIDANT ACTIVITY AND HEPATOPROTECTIVE EFFECTOF POMEGRANATE PEEL AND WHEY...Anurag Raghuvanshi
The antioxidant activity of pomegranate peel powder (PPP) and whey powder (WP) was evaluated, their hepatoprotective effect of each alone or in combination (PPWP) at equal levels was also evaluated in Wistar rats against carbon tetrachloride (CCL4) induced liver injury.
The hepatoprotective activity was assessed using various biochemical parameters and histopathological studies.
Detection of the Antibacterial Activity of Bioactive Peptide Isolated from Fe...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Effect of estradiol -17 β on arachidonic acid metabolism in sheep uterus: in ...iosrjce
The effect of estradiol-17 β on Arachidonic acid (AA) metabolism in non-pregnant sheep uterus was
studied under in vitro conditions. On incubation of uterine slices with estradiol-17β, the levels of prostaglandins
were altered but not Lipoxygenase (LOX) products. Based on their analysis on conventional TLC technique, the
Cyclooxygenase (COX) products PGF2α, 6-keto PGF1α and PGE2 were shown to be altered over an incubation
period of 0 to 120 minutes. The LOX products, HPETEs and HETEs did not show any change upon incubation
with estradiol-17β. This study gives a preliminary understanding of role of estradiol on AA metabolism.
Biochemical and pharmacological study of biologically active preparation of p...inventionjournals
Our aim was to perform some biochemical and pharmacological studies of bioactive bovine placental preparation via digestion of cow placenta using enzyme contained in swine stomach. Amino acid compositions and contents in biologically active preparation of placenta, obtained by digestion of cow placenta with enzyme contained in swine stomach were measured by HPLC technique and it was found that contents of such amino acids as glycine, proline and lysine were highest and 9 essential amino acids, including valine, histidine, methionine, lysine, threonine, arginine, phenylalanine, leucine and isoleucine were measured. In pharmacological study, acute toxicity (LD50) of the preparation and effect of the preparation on immune response to sheep erythrocyte were investigated in white mice, weighing 18 to 20 g each. The study revealed acute toxicity (LD50) of the preparation was 60 ml per kg. Spleen index of the first and second experimental group animals treated by the preparation during both provoked and unprovoked immune responses increased by 1.2 to 3.09 times as compared to that of negative control animals, while splenocyte count elevated by 1.2 to 2.2 times than negative control animals. Higher contents of essential amino acids of the biologically active preparation of cattle placenta shows its biologically higher nutritive value, as well as pharmacological study reveals the preparation has minimal toxicity and higher effect to stimulate immune responses.
Conceição et al, 2006. orpotrin a novel vasoconstrictor peptide from the veno...pryloock
1. Researchers isolated and characterized a novel peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi.
2. Using RP-HPLC and mass spectrometry techniques, they purified a peptide from the venom which was shown to strongly constrict blood vessels when applied to mice cremaster muscle tissue during intravital microscopy experiments.
3. Through de novo amino acid sequencing with mass spectrometry, the researchers determined the peptide's sequence to be HGGYKPTDK, which does not match any known bioactive peptides but aligns with residues 97-105 of creatine kinase, suggesting it may be produced from limited proteolysis of creatine kinase.
Kinetic study of free and immobilized protease from Aspergillus sp.IOSR Journals
In the present investigation partially purified alkaline protease from Aspergillus sp. As#6 and As#7 strains were entrapped in calcium alginate beads and characterized using casein as a substrate. Temperature and pH maxima of protease from As#6 strain showed no changes before and after immobilization and remained stable at 450C and pH 9, respectively. However km value was slightly shifted from 4.5mg/ml to 5 mg/ml. Proteases from As#7 strain showed shifting in pH optima to a more alkaline range (10.0) as compared with free enzyme (9.0). Optimum temperature for protease from As#7 strain showed changes after immobilization and shifted from 650C to 850C. However there was no significant effect on Km value but Vmax of immobilized protease from As#7 strain was also shifted from 200U/ml to 370U/ml. Immobilized protease from As#6 strain was reused for 3 cycles with 22% loss in its activity whereas immobilize protease from As#7 strain was reused for 3 cycles with 17% loss in its activity. Protease from As#7 strain has a higher affinity for the substrate and higher proteolysis activity than protease from As#6 strain. The present work concludes that Aspergillus As#7 strain may be a good source of industrial protease
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
This document summarizes a study examining the anti-proliferative effects of Arisaema intermedium lectin (AIL) on human colon cancer cells. AIL was purified from Arisaema intermedium tubers and its effects were tested on HCT-15 colon cancer cells. MTT assays showed AIL inhibited the growth of HCT-15 cells in a dose-dependent manner, with 53% inhibition at 100 μg/mL AIL. Further experiments found AIL treatment led to DNA fragmentation, changes in nucleic acid content, cell morphology changes, and decreased cell viability, indicating AIL induces apoptosis in HCT-15 cells. The results suggest AIL has anti-cancer properties and works through an apoptotic
Clinical chemistry uses chemical processes to measure levels of chemical components in the blood. It is very useful for the early diagnostic of disease and for monitoring organ function. The most common specimens used in clinical chemistry are blood and urine. Table 1 shows the common blood tests and measurable items using UV/Vis spectrophotometers.In this application note, the cholesterol level in human serum was determined by the enzymatic method using the LAMBDA™ 465 UV/Vis Spectrophotometer and UV Lab™ software.
This document summarizes a study that screened eight Monascus strains for their ability to produce lovastatin, an alternative to statin drugs. Strain M. purpureus Went (JCM 6934) produced the highest amount of lovastatin at 84.85 ppm. Angkak rice fermented with the local Philippine strain M. purpureus UPLB-MNH-MCC 2108 was found to also contain lovastatin. This showed that lovastatin is a characteristic component of angkak rice. A preliminary study found very low levels of the toxin citrinin in cookies made with angkak rice, showing it is safe for human consumption.
The document describes a nutritional supplement developed to address vitamin and mineral deficiencies in diabetic patients. The supplement contains dried eggs and yeast (60%), vitamins B1 and nicotinamide, chromium chelate, selenium, and lecithin. Testing in alloxan-induced diabetic rats found the supplement, incorporated into animal feed at 5%, improved metabolic processes, antioxidant activity, lipid profiles, and carbohydrate metabolism compared to controls. The supplement aims to safely provide nutrients commonly deficient in diabetics.
The document summarizes a student project to develop two assays: 1) A reducing end sugar assay to measure amylase activity using PAHBAH reagent in the presence of metal ions at low temperature. This assay detects glucose produced from starch hydrolysis using a colorimetric method. 2) An ELISA assay to estimate protein concentration. The student expresses gratitude to supervisors and colleagues who provided support and guidance during the project.
In vitro and in vivo evaluation of positively charged liposaccharide derivati...Adel Abdelrahim, PhD
This document describes a study evaluating positively charged liposaccharide derivatives as oral absorption enhancers for delivering anionic drugs. Positively charged liposaccharide derivatives were synthesized and combined with the anionic model drug piperacillin through ion pairing. The conjugates were evaluated in vitro and in vivo to assess antimicrobial activity, plasma stability, permeability across Caco-2 cell monolayers, and oral absorption. Results showed that ion pairing the liposaccharide derivatives with piperacillin improved permeability in Caco-2 cells without altering antimicrobial activity, indicating potential as oral absorption enhancers.
Zina Al-Saffar 2016- BACT control biosenor_final articleZina Al-Saffar
This document compares measurements of alkaline phosphatase (ALP) enzyme activity from a biosensor called BACTcontrol to standard microbial analysis methods for assessing water quality. The study analyzed four types of water samples and found that BACTcontrol correlated well (R2 = 0.83-0.99) with cell counts from flow cytometry, ATP measurements, and microscopic counts using viability stains for the same type of water. However, correlations decreased when comparing different water types. BACTcontrol did not correlate with traditional heterotrophic plate counts. The study concludes that BACTcontrol can detect significant microbial changes in water and trigger alarms to automatically take samples for further laboratory analysis.
Study on Fructooligosaccharide (Fos) Production By Enzyme Pectinex Ultra Sp-l...IJAEMSJORNAL
Fructooligosaccharide (FOS) is a new alternative sweetener with its characteristics such as low energy and safety for people with diabetes. Pectinex Ultra SP-L which has fructosyltransferase, catalyzes the reaction to produce short chain fructooligosaccharides. The research was conducted to enhance the high FOS content in process of FOS production by immobilized enzyme. Results achieved by Empirical planning Design Expert 7.0 - central composite method (CCD) identified at optimum conditions for process of immobilized enzyme with alginate 3.3 (%), ratio of enzyme: alginate was 0.79 (w/w), CaCl2 3.75 (%). Efficiency of loading protein reached 73.32 (%). Reaction conditions: 60 (oC), shaking velocity 90 (rpm), the initial sucrose concentrations of 50 (%), pH 5.75. When produced in 20 (h), the reaction obtained the highest level of FOS with column reaction system. The FOS for producted by immobilized enzyme achieved 47.87 (%), of which 1-kestose obtained 37.06 (%), the remaining was 10.81 (%) including nystose and fructofuranosylnystose. Efficiency of producing FOS by using immobilized enzyme compared to the free enzyme was high up to 89.49 (%).
This document describes research to develop mathematical models that predict the time required to inactivate Salmonella in orange juice using high-pressure processing (HPP). Models were developed for both Australian Valencia orange juice (pH 4.3) and navel orange juice (pH 3.7) relating time to inactivation to pressure level (300-600 MPa) and initial Salmonella inoculation level (3-7 log CFU/ml). The models can help juice processors select HPP conditions to achieve at least a 5-log reduction of Salmonella, as required by the FDA. Validation studies showed pressure-treated Salmonella may survive if conditions allow for growth, highlighting the importance of refrigerated storage. These predictive models allow
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This document describes the isolation and purification of cellulase enzymes from the fungus Aspergillus terreus. The intracellular cellulase was purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration chromatography. This purification scheme achieved a 270-fold purification with a 22.11% yield. Tests including PAGE, SDS-PAGE, immunodiffusion, and isoelectric focusing confirmed the homogeneity of the purified enzyme. The purified cellulase showed optimal activity between pH 4-7 and temperatures of 40-50°C.
Protective effects of commelina benghalensis linn (root) extract on ethanol i...IJSIT Editor
The present study was undertaken to investigate the protective effect and possible mechanism of
alcoholic (AlE) and aqueous extract (AqE) from Commelina benghalensis root (CB) on EtOH-induced hepatic
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ALP, bilirubin, protein, lipid profile (Cholesterol, triglyceride, VLDL and HDL) and level of antioxidants
together with histopathological examination. Liv 52® was used as a reference hepatoprotective agent
(5ml/kg-1b.w.). AlE and AqE (200 mg/kg-1b.w.) on oral administration decreased the level of AST, ALP, ALT,
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GSH and CAT) in rats being treated with ethanol (EtOH). Pentobarbitone -induced sleeping time study was
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These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
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These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
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Histololgy of Female Reproductive System.pptxAyeshaZaid1
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Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
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2. sequenced by tandem spectrometry (MS/MS). The potential
ACE-inhibitory activity of these peptides is discussed in relation
to the structure of the peptides.
MATERIALS AND METHODS
Samples and Preparation of Sample Extracts. Fermented milks
(FM-1 to FM-15) and fresh cheeses (FC-1 to FC-4) were purchased
on the Spanish market. Water-soluble extract (WSE) of fermented
products (solid or liquid) was obtained by centrifugation at 12000g for
20 min at 5 °C and by filtration through Whatman no. 40 filter. Most
fermented milks were made from skim or half-skim milk with skim
milk powder or other milk protein fractions and different lactic acid
strains. LF-13 and the fresh cheeses were prepared by using rennet.
Two liquid fermented milks (FM-5 and FM-8) were selected to simulate
gastrointestinal digestion. FM-5 was prepared from half-skimmed milk,
sugars, and different lactic acid bacteria including Lactobacillus casei,
and FM-8 contained skim milk, powder skim milk, sugars, and different
lactic acid bacteria.
Measurement of ACE-Inhibitory Activity. ACE-inhibitory activity
was measured by the spectrophotometric assay of Cushman and Cheung
(11) as modified by Kim et al. (12). Briefly, 20 µL of each sample
was added to 0.1 mL of 0.1 M potasium phosphate buffer (pH 8.3)
containing 0.3 M NaCl and 5 mM hippuryl-histidyl-leucine (Sigma
Chemical Co., St. Louis, MO). ACE (5 milliunits) (EC 3.4.15.1, 5.1
units/mg, Sigma) was added, and the reaction mixture was incubated
at 37 °C for 30 min. The reaction was terminated by the addition of
0.1 mL of 1 M HCl. The hippuric acid formed was extracted with ethyl
acetate, heat-evaporated at 95 °C for 10 min, redissolved in distilled
water, and measured spectrophotometrically at 228 nm. The activity
of each sample was tested in triplicate.
The ACE-inhibitory activity of products with ACE-inhibitory indices
>50% was also calculated as the protein concentration needed to inhibit
50% of the original ACE activity (IC50), and 1 unit of ACE-inhibitory
activity was expressed as the potency showing 50% ACE inhibition
under these conditions.
Total protein content of the WSEs was determined according to the
Kjeldahl method. Aminic nitrogen was measured using the Cd-
nynhidrin method according to Doi et al. (13). The peptidic nitrogen
content was calculated as the total minus aminic nitrogen.
Analysis by On-line RP-HPLC-MS/MS. RP-HPLC separations of
the WSEs were performed on an Agilent HPLC system connected on-
line to an Esquire-LC quadrupole ion trap instrument (Bruker Daltonik
GmbH, Bremen, Germany). The HPLC system was equipped with a
quaternary gradient pumping system, an in-line degasser, a variable-
wavelength absorbance detector set at 220 nm, and an automatic injector
(all 1100 series, Agilent Technologies, Waldbronn, Germany). The
column used in these experiments was a 250 mm × 4.6 mm Widepore
C18 column (Bio-Rad, Richmond, CA). The injection volume was 50
µL. Solvent A was a mixture of water/trifluoroacetic acid (1000:0.37
v/v), and solvent B contained acetonitrile/trifluoroacetic acid (1000:
0.27 v/v). Peptides were eluted with a linear gradient of solvent B in
A going from 0 to 45% in 60 min at a flow rate of 0.8 mL/min. The
flow was split postdetector by placing a T-piece (Valco, Houston, TX)
connected with a 75 µm i.d. peek outlet tube of an adjusted length to
give ∼20 µL/min of flow directed into the mass spectrometer via the
electrospray interface. Nitrogen was used as nebulizing and drying gas
and operated with an estimated helium pressure of 5 × 10-3
bar. The
capillary was held at 4 kV. Spectra were recorded over the mass/charge
(m/z) range of 100-2500. About 25 spectra were averaged in the MS
analyses and about 5 spectra in the MS(n) analyses. The signal threshold
to perform auto MS(n) analyses was 10000 (i.e., 5% of the total signal),
and the precursor ions were isolated within a range of 4.0 m/z and
fragmented with a voltage ramp going from 0.35 to 1.4 V. Using data
analysis (version 3.0; Bruker Daltoniks), the m/z spectral data were
processed and transformed to spectra representing mass values.
BioTools (version 2.1; Bruker Daltoniks) was used to process the MS-
(n) spectra and to perform peptide sequencing.
Simulation of Gastrointestinal Digestion. Two fermented milks
(FM-5 and FM-8) were selected to simulate gastrointestinal digestion.
These hydrolysates were prepared from an aqueous solution of product
(0.7 wt % protein/vol). Hydrolysis was carried out according to the
method of Alting et al. (14). The samples were first hydrolyzed with
pepsin (EC 3.4.4.1; 1:60000, 3400 units/mg) (Sigma) (20 mg/g of
protein) for 90 min at 37 °C at a pH of 2.0 followed by hydrolysis
with Corolase PP (40 mg/g of protein) (Ro¨hm, Darmstadt, Germany)
at pH 7-8 and 37 °C for 240 min. Corolase PP is a proteolytic enzyme
preparation from pig pancreas glands that besides trypsin and chymo-
trypsin contains numerous amino- and carboxylpeptidase activities.
Hydrolysis was carried out in a thermally controlled water bath under
constant stirring. Aliquots were withdrawn after hydrolysis with pepsin,
the pH was raised to 7-8 with 1 M NaOH, and they were heated at 95
°C for 10 min in a water bath. During hydrolysis with Corolase PP,
samples were also taken at 30, 120, and 240 min. The enzyme was
inactivated by heating at 95 °C for 10 min, followed by cooling to
room temperature. Each sample was stored at -20 °C until further
analysis.
After the last samples had been taken, aliquots and the remaining
reaction mixtures were centrifuged at 10000g for 30 min, and the
supernatants were subjected to ultrafiltration through a hydrophilic 3000
Da cutoff membrane (Centripep, Amicon, Inc., Beverly, MA). The
permeates were freeze-dried and kept at -20 °C until use.
RESULTS AND DISCUSSION
ACE-Inhibitory Activity of Commercial Fermented Prod-
ucts. In a first experiment, the ACE-inhibitory activity of the
WSEs of several commercial fermented products was screened.
Because the ACE-inhibitory activity of these products can be
related to their peptide content, the peptidic nitrogen of these
products was also calculated. Figure 1 shows the ACE inhibition
percentage versus peptidic nitrogen expressed as milligrams of
nitrogen per 100 g of product. Most of the fermented products
(12 from a total of 19) showed low ACE-inhibitory indices
(<50%) and a peptide content that ranged between 39 and 150
mg of nitrogen/100 g of product. Only five products exhibited
ACE-inhibitory indices >50%. Four of them contained levels
of peptidic nitrogen similar to those of most of the fermented
products analyzed in this study (i.e., values lower than 150 mg/
100 g), but their ACE-inhibitory indices were significantly
higher, ranging between 66 and 80%. The other product, which
corresponded to a fresh cheese sample, showed a moderate
ACE-inhibitory index (60%) and a relatively high proteolysis
degree (533 mg of peptidic nitrogen/100 g). The ACE-inhibitory
activity of the two fermented milks (FM-14 and FM-15) and
Figure 1. Values of the ACE-inhibitory indices versus peptide content
expressed as milligrams of peptidic nitrogen per 100 g of product for the
water-soluble extracts obtained from commercial fermented milks (FM)
and fresh cheeses (FC).
ACE-Inhibitory Activity in Fermented Products J. Agric. Food Chem., Vol. 52, No. 6, 2004 1505
3. the two fresh cheeses (FC-1 and FC-2) with ACE-inhibitory
indices >50% was also calculated as IC50, that is, the concentra-
tion needed to inhibit 50% the original ACE activity. The IC50
values of FM-14, FM-15, FC-1, and FC-2 were 61.4 µg of
peptidic nitrogen/mL (91 ACE-inhibitory units/mL), 16.9 µg
of peptidic nitrogen/mL (270 ACE-inhibitory units/mL), 22.4
µg of peptidic nitrogen/mL (330 ACE-inhibitory units/mL), and
69.1 µg of peptidic nitrogen/mL (138 ACE-inhibitory units/
mL), respectively. Interestingly, among these four fermented
products, the fermented milks FM-14 and FM-15 corresponded
to fermented products elaborated with milk of caprine origin,
whereas the rest of the samples considered in this study were
of bovine origin. Specifically, FM-14 corresponded to a Kefir-
type product made from caprine milk cultured with several
species of lactic and acetic acid bacteria and yeast. A Kefir-
like fermented milk prepared with different lactic acid bacteria
and yeast had previously been reported to exhibit an antihy-
pertensive effect in spontaneously hypertensive rats, although
it showed weak in vitro ACE inhibition (15). It has been
postulated that fermented milk produced by mixing several types
of microbes might contain a wider variety of functional
substances than milk cultured with a single strain (15). In fact,
Calpis sour milk, which is fermented with Lactobacillus
helVeticus and Sacharomyces cereVisiae, had been the source
of two potent ACE-inhibitory peptides (3). Although the ACE-
inhibitory indices found in this screening are moderate, they
are higher than that previously reported for a commercial yogurt,
for which the activity of the extract was <10% (10).
Hydrolysis under Simulated Gastrointestinal Conditions.
To evaluate whether these fermented products may act as a
natural source of ACE-inhibitory peptides produced by gas-
trointestinal enzymes, two products with low ACE-inhibitory
indices (FM-5 and FM-8) were subjected to a two-stage
hydrolysis process that simulates physiological digestion. During
the pepsin-catalyzed part of the simulated physiological diges-
tion, the ACE-inhibitory index of these two products increased
sharply compared with the nonhydrolyzed products. The activity
decreased gradually as digestion with Corolase PP progressed,
but this reduction was less pronounced for FM-5 than for FM-8
(Figure 2). As a result, after simulated physiological digestion,
the ACE-inhibitory activity of FM-5 was higher than that
observed before digestion and higher than the activity found in
the digested FM-8 sample. Similar activity values were found
for FM-8 before and after hydrolysis (31 and 34%, respectively).
Because it has been suggested that small peptides make a
considerable contribution to the ACE-inhibitory activity of
protein hydrolysates (16) and short peptide sequences are good
candidates to play a physiological antihypertensive role in vivo,
ACE-inhibitory activity and the peptide content of permeates,
obtained following ultrafiltration of the hydrolysates, were also
assayed. The ACE-inhibitory activities of the 3 kDa permeates
of FM-5 and FM-8 and their peptide contents throughout the
simulated physiological digestion are shown in Figure 3. The
inhibitory potency and the evolution of the ACE-inhibitory
activity of the permeates during simulated digestion followed
the same trend as that observed for the total hydrolysate.
However, it has to be noted that the activity of the permeates
at the end of digestion was higher than that observed prior to
digestion for both fermented products. This can be explained
by low ACE-inhibitory activity found in the permeates of the
undigested products, which was approximately half the activity
measured in the total undigested products. This result suggests
that the ACE-inhibitory activity found in the undigested product
was partially caused by peptides of masses >3 kDa. The peptide
contents of FM-5 and FM-8 notably increased during the pepsin-
catalyzed part of the digestion and after the first 30 min of
hydrolysis with Corolase PP. Although the final ACE-inhibitory
activity of the FM-5 permeate is higher than that found for FM-
8, the peptide content of FM-5 over the digestion process was
always lower than that of FM-8. This fact may indicate the
presence of peptide sequences with higher ACE-inhibitory
activity or higher concentration of active peptides in the
hydrolysate of FM-5.
Identification of Peptides after Simulated Physiological
Digestion. The 3 kDa permeate from FM-5 was subjected to
RP-HPLC coupled on-line to a mass spectrometer in order to
identify the peptide sequences obtained at the end of the
simulated physiological digestion. In our case, the mass
spectrometer was a quadrupole ion trap capable of multiple
stages of mass analysis from a single precursor ion. As an
example, Figure 4 shows the MS/MS spectrum of a single
charged ion with m/z 652.4 and the amino acid sequence of the
identified peptide with the major fragment ions. Major fragment
ions were observed at m/z 243.9 and 440.2 that corresponded
Figure 2. Evolution of the ACE-inhibitory indices of two fermented milks
(FM-5 and FM-8) during simulated gastrointestinal digestion. Each point
in the curve corresponds to an aliquot withdrawn during hydrolysis.
Figure 3. ACE-inhibitory indices (lines) and peptide contents (bars)
expressed as milligrams of peptidic nitrogen per 100 g of product of the
3 kDa permeates from two fermented milks (FM-5 and FM-8) during
simulated gastrointestinal digestion.
1506 J. Agric. Food Chem., Vol. 52, No. 6, 2004 Herna´ndez-Ledesma et al.
4. to the y′′-type ions adjacent to the amino acid proline. This
amino acid is associated with very abundant y′′-type ions, which
are often easily identifiable because of their intensity (17). In
some cases, some different charged ions from the same peptide
were fragmented, allowing us to acquire additional information
for peptide sequencing. All peptides of the total ion chromato-
gram with a signal >10000 units were considered for peptide
sequencing. Only a few detected masses and the corresponding
fragmentation spectra obtained by MS/MS could not be matched
with any peptide fragment originated by milk protein hydrolysis
and, thus, probably peptides from other origins are also present
in this ultrafiltered hydrolysate.
A total of 56 peptide fragments were identified, of which 24
corresponded to β-casein fragments (Table 1), 19 to Rs1- and
Rs2-casein fragments (Table 2), and 5 to κ-casein fragments; 7
were derived from the β-lactoglobulin sequence (Table 3). The
dipeptide, RL (peptide 44, Table 2), could be originated from
hydrolysis of various protein fractions. Of many ACE-inhibitory
peptides, structure-activity correlation indicates that the C-
terminal tripeptide residues play a predominant role in competi-
tive binding to the active site of ACE. Among the most favorable
C-terminal amino acids are aromatic amino acids, as well as
the imino acid proline, whereas ACE only weakly binds peptides
that have terminal dicarboxylic amino acids (18). It has to be
stressed that several of the peptides identified in this study share
the characteristic of having hydrophobic residues, such as
leucine, phenylalanine, or valine, at C-terminal positions. Other
peptides had lysine as the C-terminal residue (see, for instance,
peptides 5-7 in Table 1). Structure relationship data suggest
that many ACE-inhibitory peptides derived from milk proteins
contain a positively charged amino acid as the C-terminal
residue, which contributes substantially to the inhibitory potency
(19).
Among the identified peptides, β-casein f(108-113) (peptide
7 in Table 1) had been previously identified by Pihlanto-Leppa¨la¨
et al. as an ACE inhibitor in a casein sample fermented with
lactic acid starters (16). This peptide showed moderate ACE-
inhibitory activity with an IC50 value of 423 µg/mL. Moreover,
some β-casein-derived peptides share some C-terminal residues
with different ACE-inhibitory peptides previously described in
the literature. For instance, β-caseins f(193-201) and f(196-
201) (peptides 19 and 20 in Table 1) share the three terminal
positions with the tripeptide GPV, which has been proved to
have a potent in vitro ACE-inhibitory activity (IC50 ) 1.2 µg/
mL, 4.67 µM) (20). Although the activity of di- or tripeptides
with ACE-inhibitory activity may not always be strictly
extrapolated to longer peptides (21), the structural similarity of
the C-terminal region may allow a similar activity to be
Figure 4. Tandem MS spectrum of ion m/z 652.4. Following sequence interpretation and database searching, the MS2 spectrum was matched to
β-casein f(170−175). The sequence of this peptide is displayed with the fragment ions observed in the spectrum. Fragment ions are labeled according
to the nomenclature proposed by Roepstorff et al. (29).
ACE-Inhibitory Activity in Fermented Products J. Agric. Food Chem., Vol. 52, No. 6, 2004 1507
5. anticipated. In the same manner, peptides β-casein f(202-209)
and β-casein f(203-209) (peptides 22 and 23 in Table 1) are
included in a previously described active sequence (IC50 ) 21
µg/mL) from β-casein obtained by hydrolysis of casein with
Lactobacillus helVeticus CP790 proteinase. This casein hydroly-
sate had demonstrated not only in vitro ACE-inhibitory activity
but also antihypertensive effect in spontaneously hypertensive
rats after oral administration (22).
The MS/MS data permitted us to identify several peptides
that belong to different genetic variants of β-casein. This is the
case of β-casein f(63-68), for which our results agreed with
the variant A1 or peptide β-casein f(115-119), where we found
the fragment corresponding to the A2 variant. The amino acid
changes due to the genetic variants of β-casein lead to a
discrepancy between the two sequences found in this study and
the same regions previously reported in the literature with ACE-
inhibitory activity (peptides 3 and 10 in Table 1) (23).
Formation of the peptides of interest during simulated
gastrointestinal digestion could easily be followed by analysis
of the 3 kDa permeates by HPLC-MS and extraction of the
characteristic ion of the peptide of interest. As an example,
Figure 5 shows the total ion current chromatogram correspond-
ing to digested FM-5 and the extracted ion chromatograms of
the molecular ion corresponding to β-casein f(170-175) (m/z
652.4). This β-casein fragment was also included in two
previously described sequences, which exhibited potent ACE-
inhibitory activity (Table 1) (22). This peptide was absent in
the undigested product or after the pepsin-catalyzed part of the
digestion (Figure 5B), but it was detected after 30 min of
hydrolysis with Corolase PP (Figure 5C). The concentration
Table 1. β-Casein-Derived Peptides Identified in the 3 kDa Permeate Obtained from FM-5 after Simulated Physiological Digestion
obsd mass calcd massa protein fragmentb sequence ACE-inhibitory peptidesc IC50 (ref) peptide formationd
1 674.4 674.32 β-CN f(1−5) RELEE 2, 3, 4, 5
2 512.3 512.28 β-CN f(49−52) IHPF DKIHPF 193.9 µg/mL (5) 4, 5
3 633.4 633.32 β-CN A1 f(63−68) PGPIHN YPFPGPIPN (β−CN A2) 14.8 µg/mL (23) 5
4 753.4 753.44 β-CN f(81−87) PVVVPPF 3, 4, 5
5 872.5 872.48 β-CN f(98−105) VKEAMAPK 3, 4, 5
6 645.4 645.32 β-CN f(100−105) EAMAPK 3, 4, 5
7 747.4 747.36 β-CN f(108−113) EMPFPK EMPFPK 423 µg/mL (16) 3, 4, 5
8 603.4 603.29 β-CN f(114−118) YPVEP 3, 4, 5
9 750.4 750.36 β-CN f(114−119) YPVEPF 3, 4, 5
10 587.3 587.30 β-CN A2 f(115−119) PVEPF MPFPKYPVQPF (β−CN A1) nre (23) 5
11 1104.4 1103.57 β-CN f(124−133) SLTLTDVENL 3, 4, 5
12 688.5 688.43 β-CN f(134−139) HLPLPL HLPLP 23.6 µg/mL (24) 3, 4, 5
13 551.4 551.37 β-CN f(135−139) LPLPL LPLP 315.6 µg/mL (24) 3, 4, 5
14 664.4 664.45 β-CN f(135−140) LPLPLL 3, 4, 5
15 673.4 673.34 β-CN f(157−162) FPPQSV 3, 4, 5
16 520.2 520.25 β-CN f(164−168) SLSQS 2, 3, 4, 5
17 651.4 651.40 β-CN f(170−175) VLPVPQ SKVLPVPQ
PPQSVLSLSQSKVLPVPQ
39 µg/mL (22)
25 µg/mL (22)
3, 4, 5
18 503.3 503.24 β-CN f(179−182) PYPQ 3, 4, 5
19 1000.4 1000.52 β-CN f(193−201) YQEPVLGPV GPV 1.2 µg/mL (20) 3, 4, 5
20 580.5 580.36 β-CN f(196−201) PVLGPV GPV 1.2 µg/mL (20) 3, 4, 5
21 685.5 685.39 β-CN f(202−207) RGPFPI 3, 4, 5
22 897.5 897.54 β-CN f(202−209) RGPFPIIV LLYQQPVLGPVRGPFPIIV 21 µg/mL (22) 3, 4, 5
23 741.4 741.44 β-CN f(203−209) GPFPIIV LLYQQPVLGPV RGPFPIIV 21 µg/mL (22) 3, 4, 5
24 529.4 529.29 β-CN f(203−207) GPFPI 3, 4, 5
a Monoisotopic mass. b β-Casein sequence according to ref 25. c Previously described ACE-inhibitory peptides that share at least three C-terminal residues with those
found in this study. d 1, undigested product; 2, digestion with pepsin for 90 min; 3, digestion with pepsin and Corolase PP for 30 min; 4, digestion with pepsin and Corolase
PP for 120 min; 5, digestion with pepsin and Corolase PP for 240 min. e Not reported.
Table 2. Rs1- and Rs2-Casein-Derived Peptides Identified in the 3 kDa
Permeate Obtained from FM-5 after Simulated Physiological Digestion
obsd mass calcd massa protein fragmentb sequence
25 399.3 399.26 Rs1-CN f(1−3) RPK
26 493.4 493.30 Rs1-CN f(4−7) HPIK
27 678.4 678.35 Rs1-CN f(8−13) HQGLPQ
28 906.4 906.46 Rs1-CN f(8−15) HQGLPQEV
29 904.5 904.47 Rs1-CN f(24−31) FVAPFPEV
30 488.3 488.23 Rs1-CN f(27−30) PFPE
31 507.4 507.28 Rs1-CN f(90−93) RYLG
32 633.4 633.35 Rs1-CN f(104−108) YKVPQ
33 484.4 485.25 Rs1-CN f(107−110) PQLE
34 525.3 525.26 Rs1-CN f(125−129) EGIHA
35 644.4 644.32 Rs1-CN f(133−138) EPMIGV
36 520.2 520.25 Rs1-CN f(146−149) YPEL
37 449.3 449.24 Rs1-CN f(150−152) FRQ
38 374.3 374.18 Rs1-CN f(155−157) QLD
39 910.4 910.46 Rs1-CN f(163−169) AWYYVPL
40 788.5 788.44 Rs2-CN f(99−105) LYQGPIV
41 1023.5 1023.62 Rs2-CN f(111−119) QVKRNAVPI
42 398.3 399.21 Rs2-CN f(115−118) NAVP
43 527.4 527.30 Rs2-CN f(118−122) PITPT
44 287.1 287.20 various fragments RL
a Monoisotopic mass. b Rs1- and Rs2-casein sequence according to refs 25 and
26.
Table 3. κ-Casein- and β-Lactoglobulin-Derived Peptides Identified in
the 3 kDa Permeate Obtained from FM-5 after Simulated Physiological
Digestion
obsd mass calcd massa protein fragmentb sequence
45 495.3 495.23 κ-CN f(18−21) FSDK
46 574.3 573.35 κ-CN f(20−24) DKIAK
47 469.3 469.29 κ-CN f(26−29) IPIQ
48 548.4 548.30 κ-CN f(61−65) YAKPA
49 655.4 655.37 κ-CN f(106−111) MAIPPK
50 443.9 444.30 β-Lg f(1−4) LIVT
51 506.3 506.25 β-Lg f(5−8) QTMK
52 273.1 273.18 β-Lg f(40−41) RV
53 516.4 517.28 β-Lg f(44−47) EELK
54 374.3 374.22 β-Lg f(83−85) KID
55 576.2 575.24 β-Lg f(130−134) DEALE
56 516.4 517.24 β-Lg f(155−158) QLEE
a Monoisotopic mass. b κ-Casein and β-lactoglobulin sequence according to refs
27 and 28.
1508 J. Agric. Food Chem., Vol. 52, No. 6, 2004 Herna´ndez-Ledesma et al.
6. of this peptide increased with hydrolysis time to reach its
maximum concentration after 240 min of hydrolysis with
Corolase PP (Figure 5E). The same procedure was followed
with other β-casein-derived sequences, and the results are
summarized in Table 1. Except for the β-casein peptides f(49-
52), f(63-68), and f(115-119) (peptides 2, 3, and 10 in Table
1) that appeared with longer incubation times with Corolase
PP, most peptides found at the end of the simulated digestion
were formed after 30 min of incubation with this enzyme. Only
two peptides, β-casein f(1-5) and β-casein f(164-168) (pep-
tides 1 and 16), which were detected after hydrolysis with
pepsin, survived the treatment with the pancreatic extract.
In conclusion, this paper demonstrates the presence of
moderate ACE-inhibitory activity in most of the fermented
products considered. However, few commercial products showed
potent activity, which could be related in some products to the
origin of the milk. The ACE-inhibitory activity of these
commercial products remained stable or increased after simu-
lated gastrointestinal digestion. The successful strategy, by MS/
MS sequencing and extraction of the ion of interest, allowed
us to unambiguously identify peptides in the final digest of one
selected product and to follow their formation during simulated
physiological digestion. Most peptides found at the end of the
simulated gastrointestinal digestion were formed during incuba-
tion with the pancreatic extract. Our results suggest that
physiological digestion may promote the formation of active
peptides from the proteins and oligopeptides present in com-
mercial dairy products. Some of the identified peptides showed
marked structural similarities with previously described ACE
inhibitors.
ACKNOWLEDGMENT
We acknowledge Ro¨hm Enzyme GmbH for providing the
enzymatic preparation (Corolase PP).
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Received for review September 3, 2003. Revised manuscript received
January 13, 2004. Accepted January 21, 2004. This work has received
financial support from Projects AGL 2000-1480 and CAL01-046-C2.
B.H.-L. was the recipient of a fellowship from Instituto Danone, Spain.
JF034997B
1510 J. Agric. Food Chem., Vol. 52, No. 6, 2004 Herna´ndez-Ledesma et al.