This document examines the effect of centrifugal pretreatment, pH, and water activity on the growth and protease production of Pediococcus acidilactici. It finds that centrifugation at 3000 rpm, a pH of 6.5, and a water activity of 0.99 each favor growth and protease production individually. However, when combined their effect is lower protease production. The results support the use of P. acidilactici as a starter culture in meat fermentation as it shows high protease production under conditions similar to fresh meat.
Proteases are protein-degrading enzymes that catalyses hydrolytic reaction in which protein molecules are degraded into peptides and amino acids. Thermostable alkaline proteases are of particular great interest for industrial application because they are stable and active at temperature above 60-70˚C. Thermophiles are found in wide array of environment such as mushroom compost material, nest, hay, wood chips, grains, soil, manure, coal mines etc. Alkaline proteases are most important industrial enzymes and they occupy about 60% of total enzyme market. From the soil samples, eight different fungal species were isolated through soil dilution plate method. In the present study, two fungi Aspergillus nidulans and Aspergillus glaucus from mushroom compost and two fungi Aspergillus terrus, and Aspergillus fumigates from cow manure, showing alkaline protease activity, were isolated. The zones of clearance were observed in Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species of fungi isolated from cow manure and mushroom compost. The best enzyme production was observed in Aspergillus terrus (1.005 ± 0.057 IU/mg protein) obtained from cow manure and the minimum enzyme activity was observed with Aspergillus glaucus (0.278 ± 0.026 IU/mg protein). However, more studies are required to assess the potential of Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species. Key-words- Alkaline protease, Thermophiles, Zone of clearance, Trichloroacetic acid
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
Evaluation of antioxidant activities of Cyperusrotundus (Ethanolic extract an...iosrphr_editor
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).
PRODUCTION AND OPTIMIZATION OF PECTINASE BY BACILLUS SP. ISOLATED FROM VEGETA...SUS GROUP OF INSTITUTIONS
Microbial enzymes have shown tremendous potential for different applications. Over the years due to their remarkable features enzymes have occupied the centre stage of all the biochemical and industrial processes. Pectinases are a group of enzymes responsible for the hydrolysis of pectic materials found in plants and are important industrial enzymes. In the present study, pectinase is produced from Bacillus sp. that was isolated from vegetable waste dump soil samples. A total of five isolates showed pectinase production and designated as PPB1 to PPB5. The screened isolates were used as a source of pectinase production using cassava waste as a substrate. Isolate PPB5 showed maximum enzyme activity of 0.641 IU/ml. Pectinase activity was optimized for various parameters like incubation time, temperature, pH, different carbon and nitrogen sources. Enzyme activity was observed maximum at 96 hr of incubation, 35°C temperature and at pH 6. The best carbon was found to be glucose. Among organic and inorganic nitrogen sources yeast extract and ammonium nitrate was founded to be better than other nitrogen sources. Among the five isolates, the isolate PPB5 showed maximum activity at all optimum conditions. This isolate is best producer and can be used in future for further pectinase production.
Analysis of Cow’s Urine for Detection of Lipase Activity and Anti-Microbial P...IOSR Journals
There is enormous amount of literature in Ayurveda stating the importance of cow’s urine for all purposes, including its importance in our daily life. This research targets on the antimicrobial activity of urine and its biochemical content that can be the key potential factor for its usage as a medicine.
A detailed biochemical analysis of cow’s urine was done to understand its antibacterial/antifungal properties along with lipase activity which could make it a potentially effective anti-cancer agent.
Various micro-organismal plating techniques were applied using Nutrient and Potato Dextrose Agar as the medium for bacterial and fungal growth, to study the inhibitory activity of cow’s urine on these organisms. Thin layer chromatography, Volumetric analysis, Spectrophotometric analysis and Tributyrin tests were conducted on cow’s urine sample to analyze the lipase activity present within the urine content.
Statistical comparisons, of the spectra obtained from the Spectrophotometric analysis of urine sample, were made with those already analyzed on various types of lipase activity detections from previous researches, and similarities were observed in both studies to ascertain the lipase factor potential within cow’s urine sample.
The tests proved that cow’s urine was highly effective in inhibiting bacterial and fungal growth and also a potential natural source of lipase enzyme.
Proteases are protein-degrading enzymes that catalyses hydrolytic reaction in which protein molecules are degraded into peptides and amino acids. Thermostable alkaline proteases are of particular great interest for industrial application because they are stable and active at temperature above 60-70˚C. Thermophiles are found in wide array of environment such as mushroom compost material, nest, hay, wood chips, grains, soil, manure, coal mines etc. Alkaline proteases are most important industrial enzymes and they occupy about 60% of total enzyme market. From the soil samples, eight different fungal species were isolated through soil dilution plate method. In the present study, two fungi Aspergillus nidulans and Aspergillus glaucus from mushroom compost and two fungi Aspergillus terrus, and Aspergillus fumigates from cow manure, showing alkaline protease activity, were isolated. The zones of clearance were observed in Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species of fungi isolated from cow manure and mushroom compost. The best enzyme production was observed in Aspergillus terrus (1.005 ± 0.057 IU/mg protein) obtained from cow manure and the minimum enzyme activity was observed with Aspergillus glaucus (0.278 ± 0.026 IU/mg protein). However, more studies are required to assess the potential of Aspergillus nidulans, Aspergillus glaucus, Aspergillus terrus, and Aspergillus fumigatus species. Key-words- Alkaline protease, Thermophiles, Zone of clearance, Trichloroacetic acid
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
Evaluation of antioxidant activities of Cyperusrotundus (Ethanolic extract an...iosrphr_editor
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).
PRODUCTION AND OPTIMIZATION OF PECTINASE BY BACILLUS SP. ISOLATED FROM VEGETA...SUS GROUP OF INSTITUTIONS
Microbial enzymes have shown tremendous potential for different applications. Over the years due to their remarkable features enzymes have occupied the centre stage of all the biochemical and industrial processes. Pectinases are a group of enzymes responsible for the hydrolysis of pectic materials found in plants and are important industrial enzymes. In the present study, pectinase is produced from Bacillus sp. that was isolated from vegetable waste dump soil samples. A total of five isolates showed pectinase production and designated as PPB1 to PPB5. The screened isolates were used as a source of pectinase production using cassava waste as a substrate. Isolate PPB5 showed maximum enzyme activity of 0.641 IU/ml. Pectinase activity was optimized for various parameters like incubation time, temperature, pH, different carbon and nitrogen sources. Enzyme activity was observed maximum at 96 hr of incubation, 35°C temperature and at pH 6. The best carbon was found to be glucose. Among organic and inorganic nitrogen sources yeast extract and ammonium nitrate was founded to be better than other nitrogen sources. Among the five isolates, the isolate PPB5 showed maximum activity at all optimum conditions. This isolate is best producer and can be used in future for further pectinase production.
Analysis of Cow’s Urine for Detection of Lipase Activity and Anti-Microbial P...IOSR Journals
There is enormous amount of literature in Ayurveda stating the importance of cow’s urine for all purposes, including its importance in our daily life. This research targets on the antimicrobial activity of urine and its biochemical content that can be the key potential factor for its usage as a medicine.
A detailed biochemical analysis of cow’s urine was done to understand its antibacterial/antifungal properties along with lipase activity which could make it a potentially effective anti-cancer agent.
Various micro-organismal plating techniques were applied using Nutrient and Potato Dextrose Agar as the medium for bacterial and fungal growth, to study the inhibitory activity of cow’s urine on these organisms. Thin layer chromatography, Volumetric analysis, Spectrophotometric analysis and Tributyrin tests were conducted on cow’s urine sample to analyze the lipase activity present within the urine content.
Statistical comparisons, of the spectra obtained from the Spectrophotometric analysis of urine sample, were made with those already analyzed on various types of lipase activity detections from previous researches, and similarities were observed in both studies to ascertain the lipase factor potential within cow’s urine sample.
The tests proved that cow’s urine was highly effective in inhibiting bacterial and fungal growth and also a potential natural source of lipase enzyme.
Production of Pectinase by Aspergillus niger Cultured in Solid State Media - IJBInnspub Net
Solid state fermentation was carried out with 7 fungal strains, obtained from different sources. Among 7 isolates Aspergillus niger,IM-6 was found as effective pectinase producer.Maximum enzymatic activity (142.44U/gm) was observed after 7 days incubation at 40˚C temperature in 750 ml conical flask. In this study 1.69% (NH4)2SO4 was used as nitrogen source, although peptone as a nitrogen source showed better result but use of peptone was not cost effective. As a substrate, wheat bran and potato starch showed good result (85.54U/gm) in solid state culture. Addition of 9.68% pectin was found to increase the enzyme production as 116.57U/gm. Pectinase production was optimum in 60% moisture (98.34U/gm). Aeration showed positive effects on pectinase production (136.86U/gm) at 750 ml flask than 1000 ml flask. Thus the wild strain Aspergillus niger IM-6 has outstanding pectinase producing capability at 40◦C in 60% initial moisture content for 7 days of incubation in solid state fermentation. Get the full articles at: http://www.innspub.net/volume-1-number-1-february-2011-3/
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
Optimization of l asparaginase production by aspergillus terreus mtcc 1782 us...eSAT Journals
Abstract Enzymes are the biocatalysts synthesized by living cells. They are Complex protein molecules that bring about chemical reactions concerned with life. They are protein in nature, colloidal and thermolabile in character, and specific in their action. L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an anticancer agent. The present work deals with production of extracellular L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour under solid state fermentation Process parameters like Incubation time(96 h), Temperature (300 C), Moisture content (70% v/w), pH of the medium(8.0), Inoculum Age (5 days), Inoculum volume (1 ml), carbon source (1.5% w/v glucose), nitrogen source ( 2% w/v ammonium sulphate), and metal salts ( 0.1% w/v Magnesium sulphate) were optimized and giving an overall yield of 273.3 U/gds of maximum L-asparaginase activity after optimization. The observation made in this study hold great promise for scale up production of L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour as substrate under solid state fermentation. Index terms: L-asparaginase, Aspergillus terreus, Bajra seed flour, Solid state fermentation, Optimization
Commercial Application of Anoectochilus formosanus: Immunomodulating ActivitiesCây thuốc Việt
Anoectochilus formosanus is an important ethnomedicinal plant of Taiwan. We investigated the effect of oral administration of A. formosanus effective fraction (AFEF) on the innate immune response in mice. Male BALB/c mice were treated orally for 2 weeks with 500, 1000 and 1500 mg/kg of AFEF. Primary peritoneal macrophage harvest from mice that administered with AFEF (500 –1500 mg/kg) was directed to activate phagocytosis. AFEF significantly increased interferon-production from lymph node cells by ConA stimulation for 48 hours in AFEF (1500 mg/kg) treated group. AFEF might be the active fraction in activation of innate immunity.
Alkaline Protease - One of the class of protease enzyme.
An extracellular enzyme.
Performs proteolysis, that is, protein catabolism by hydrolysis of the peptide bonds.
Active at alkaline pH 8 to 12 and at temperature 30⁰-80⁰C.
Molecular weight is about 20,000 to 45,000 Dalton.
The structure is determined by X-ray crystallography.
EC (Enzyme Commission) Number: 3.4.21–24.99
In 1971, Japanese scientist Koki Horikoshi first reported the production of alkaline protease from bacteria.
Microbial Production Of Alkaline Proteases And Evaluation Of Its Performances...Shafkat Shamim Rahman
A high alkaline protease producing bacterial strain was isolated and identified a local soil sample. The organism was gram positive and forms spore during adverse condition in the growth medium. After various tests it was suggested and the features agreed with the description of Bacillus subtilis. It was also identified as B. subtilis with 99.9% identity by API 50 CHB. The enzyme hydrolyses a number of proteins including azocasein which suggests that it is an extracellular alkaline protease. The experimentally determined isoelectric point was 5.1 and the optimal enzyme activity was at 60°C and at pH 8.5. The esterase preferentially hydrolyzed short-chain fatty acids. Native enzyme preparations typically showed a Michaelis constant (Km) and Vmax of 0.40mM and 12,200 U mg)-1, respectively. This microbial enzyme was partially purified by ammonium sulfate fractionation, dialysis, DEAE cellulose chromatography and electrophoretic analysis. Enzyme purity was tested by SDS-PAGE. Quantitative estimation has shown that 40mL of culture supernatant could dehair 2×1 cm of leather completely in 9 hours. In future the tanneries will use a combination of chemical and enzymatic processes. In practical applications, protease is a useful enzyme for promoting the hydrolysis of proteins and showing significant industrial applications.
Screening and Production of Protease Enzyme from Marine Microorganism and Its...iosrjce
Marine sediment samples were collected from the Gulf of Mannar, Mandapam coast to screen for
protease producing microbes. Among the five isolates screened only two isolates showed maximum proteolytic
activity with the zone of 21mm and 19mm respectively. Biochemical characterization of the isolates were
performed and identified as strain P2 belonged to Bacillus subtilis and strain P5 belonged to Bacillus
licheniformis. Both the strains have the ability to tolerate 7%Nacl concentration. The amount of protease
produced was expressed in microgram of tyrosine released under standard assay conditions. The total protein
content of crude enzyme extracts of Bacillus subtilis and Bacillus licheniformis were quantified which revealed
21.2mg/ml for strain P2 and 22.4mg/ml of protein content was presented by strain P5. The proteolytic bacteria
gave an optimum performance were both strains exhibited the enzymes stable at PH
7. In the present study
Bacillus subtilis showed a remarkable activity at 40ºC where as Bacillus licheniformis exhibited maximum
activity at 50ºC. Studies pertaining to carbon sources starch and lactose were utilized by Bacillus subtilis and
Bacillus licheniformis and maximum production was achieved. Among the different nitrogen sources tested
yeast extract induced maximum proteolytic activity where as ammonium sulphate was found to be the best
nitrogen sources for protease production. The crude enzyme was efficient to remove hair dye and blood stain by
Bacillus subtilis and Bacillus licheniformis
Bioprocess development for enhanced spore production in shake flask and pilot...iosrjce
Bacillus thuringiensis subsp. Israelensis (Bti), has proven to be a safe and effective larvicide for controlling
mosquito and black fly larvae. The effect of cultivation and bioprocess development on Bti growth and sporulation was
investigated in shake flask level and batch cultivation in the semi-industrial scale 16-L stirred tank bioreactor. For
industrial production of biocontrol microorganism, it is necessary to obtain high cell mass and spore production in a short
time with low cost cultivation media. In this study, the new composition of production media was optimized which composed
of (g L
-1
): glucose, 10; yeast extract, 30; KH2PO4, 5; K2HPO4, 5; MgSO4. 7H20, 0.005; MnSO4.H2O, 0.03; FeSO4, 0.01;
CaCl2.7 H2O, 0.05; NaH2PO4, 1.5; NH4H2PO4, 1.5. The maximal cell dry mass and spore production, Sporemax for shake
flask study were 4.26 gL-1
at 36 h and 3.29106
spore mL-1
, respectively. Furthermore, studies of the cultivation conditions
under controlled and uncontrolled pH in the 16L-bioreactor was performed. The growth of Bti under uncontrolled pH
cultivation showing decreased of glucose and total protein concentration in the media was correlated with the vegetative cell
growth and sporulation. The maximal cell dry mass and Sporemax for uncontrolled pH bioreactor were 4.14 gL-1
at 36h and
3.7106
spore mL-1
, respectively. The maximal cell dry mass and spore production, Sporemax for controlled pH bioreactor
were 3.36 gL-1
at 26 h and 3.23 106
spore mL-1
, respectively. In conclusion, batch cultivation in 16-L bioreactor with the
new optimized production medium under uncontrolled pH condition increased of the cell dry mass and number of spores up to 23 % and 47 % , respectively
Kinsenoside isolated from Anoectochilus formosanus suppresses LPS-Stimulated ...Cây thuốc Việt
In the present study, we reported that kinsenoside, a major component of Anoectochilus formosanus, inhibited inflammatory reactions in mouse peritoneal lavage macrophages and protects mice from endotoxin shock. In LPSstimulated mouse peritoneal lavage macrophages, kinsenoside inhibited the inflammatory mediators, such as nitric oxide, TNF-!, IL-1", monocyte chemoattractant protein 1, and macrophage migration inhibitory factor production. Furthermore, kinsenoside decreased the formation of a nuclear factor .BYDNA complex and nuclear p65 and p50 protein levels. Kinsenoside inhibited nuclear factor .B translocation through both I.B!-dependent and -independent pathway. In contrast, it stimulated anti-inflammatory cytokine IL-10 generation and enhanced the mRNA expression of IL-10 and suppressor of cytokine signaling 3 in the same cells induced by LPS. In an animal model, both pretreatment and posttreatment of kinsenoside increased the survival rate of ICR mice challenged by LPS (80 mg/kg, i.p.). Pretreatment with kinsenoside decreased serum levels of TNF-!, IL-1", IL-10, monocyte chemoattractant protein 1, and migration inhibitory factor at 1 h after sublethal dose of LPS (40 mg/kg, i.p.) in mice. In contrast, kinsenoside enhanced serum IL-10 level at 24 h after LPS injection in mice. In conclusion, kinsenoside inhibited the production of inflammatory mediators and enhanced antiinflammatory cytokine generation. Therefore, kinsenoside can alleviate acute inflammatory hazards.
The Effects of Grape Seed Powder and Extract on Antimicrobial of Fermented Tu...Agriculture Journal IJOEAR
— In this study, the effects of the grape seed powders and extracts of from two different grape cultivars (Razakı and Siyah Gemre) on the quality characteristics of Turkish sausage were investigated during the ripening period. Some characteristics of Turkish sausage including phenolic content, radical scavenging, hydrogen peroxide scavenging, Fe +2 chelating activity, Inhibition of Escherichia coli, Staphylococcusaureus, Candida albicans were studied. In fermented sausage, grape seed powders and extracts demonstrated the greatest inhibitory activity against Staphylococcus aureus. Furthermore, the results showed that Siyah Gemre grape seed extract was able to reduce Staphylococcusaureus populations by 42 CFU/g, while the population of Escherichia Coli was reduced by 590 CFU/g. Siyah Gemre grape seed extract was able to reduce Candida albicans populations by 880 CFU/g. Also this study demonstrated that grape seed extracts were more effective than grape seed powders. Our results suggest that the use of grape seed extract is a feasible alternative as antibacterial and antioxidant agents to prevent the deterioration of foods by bacteria and oxidation. Keywords— Grape seed powder and extract, Razakı and Siyah Gemre antimicrobial, antioxidant, natural preservative.
Production of Pectinase by Aspergillus niger Cultured in Solid State Media - IJBInnspub Net
Solid state fermentation was carried out with 7 fungal strains, obtained from different sources. Among 7 isolates Aspergillus niger,IM-6 was found as effective pectinase producer.Maximum enzymatic activity (142.44U/gm) was observed after 7 days incubation at 40˚C temperature in 750 ml conical flask. In this study 1.69% (NH4)2SO4 was used as nitrogen source, although peptone as a nitrogen source showed better result but use of peptone was not cost effective. As a substrate, wheat bran and potato starch showed good result (85.54U/gm) in solid state culture. Addition of 9.68% pectin was found to increase the enzyme production as 116.57U/gm. Pectinase production was optimum in 60% moisture (98.34U/gm). Aeration showed positive effects on pectinase production (136.86U/gm) at 750 ml flask than 1000 ml flask. Thus the wild strain Aspergillus niger IM-6 has outstanding pectinase producing capability at 40◦C in 60% initial moisture content for 7 days of incubation in solid state fermentation. Get the full articles at: http://www.innspub.net/volume-1-number-1-february-2011-3/
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
Optimization of l asparaginase production by aspergillus terreus mtcc 1782 us...eSAT Journals
Abstract Enzymes are the biocatalysts synthesized by living cells. They are Complex protein molecules that bring about chemical reactions concerned with life. They are protein in nature, colloidal and thermolabile in character, and specific in their action. L-asparaginase (L-asparagine amido hydrolase, E.C.3.5.1.1) is an extra cellular enzyme that has received considerable attention since it is used as an anticancer agent. The present work deals with production of extracellular L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour under solid state fermentation Process parameters like Incubation time(96 h), Temperature (300 C), Moisture content (70% v/w), pH of the medium(8.0), Inoculum Age (5 days), Inoculum volume (1 ml), carbon source (1.5% w/v glucose), nitrogen source ( 2% w/v ammonium sulphate), and metal salts ( 0.1% w/v Magnesium sulphate) were optimized and giving an overall yield of 273.3 U/gds of maximum L-asparaginase activity after optimization. The observation made in this study hold great promise for scale up production of L-asparaginase from Aspergillus terreus MTCC 1782 using Bajra seed flour as substrate under solid state fermentation. Index terms: L-asparaginase, Aspergillus terreus, Bajra seed flour, Solid state fermentation, Optimization
Commercial Application of Anoectochilus formosanus: Immunomodulating ActivitiesCây thuốc Việt
Anoectochilus formosanus is an important ethnomedicinal plant of Taiwan. We investigated the effect of oral administration of A. formosanus effective fraction (AFEF) on the innate immune response in mice. Male BALB/c mice were treated orally for 2 weeks with 500, 1000 and 1500 mg/kg of AFEF. Primary peritoneal macrophage harvest from mice that administered with AFEF (500 –1500 mg/kg) was directed to activate phagocytosis. AFEF significantly increased interferon-production from lymph node cells by ConA stimulation for 48 hours in AFEF (1500 mg/kg) treated group. AFEF might be the active fraction in activation of innate immunity.
Alkaline Protease - One of the class of protease enzyme.
An extracellular enzyme.
Performs proteolysis, that is, protein catabolism by hydrolysis of the peptide bonds.
Active at alkaline pH 8 to 12 and at temperature 30⁰-80⁰C.
Molecular weight is about 20,000 to 45,000 Dalton.
The structure is determined by X-ray crystallography.
EC (Enzyme Commission) Number: 3.4.21–24.99
In 1971, Japanese scientist Koki Horikoshi first reported the production of alkaline protease from bacteria.
Microbial Production Of Alkaline Proteases And Evaluation Of Its Performances...Shafkat Shamim Rahman
A high alkaline protease producing bacterial strain was isolated and identified a local soil sample. The organism was gram positive and forms spore during adverse condition in the growth medium. After various tests it was suggested and the features agreed with the description of Bacillus subtilis. It was also identified as B. subtilis with 99.9% identity by API 50 CHB. The enzyme hydrolyses a number of proteins including azocasein which suggests that it is an extracellular alkaline protease. The experimentally determined isoelectric point was 5.1 and the optimal enzyme activity was at 60°C and at pH 8.5. The esterase preferentially hydrolyzed short-chain fatty acids. Native enzyme preparations typically showed a Michaelis constant (Km) and Vmax of 0.40mM and 12,200 U mg)-1, respectively. This microbial enzyme was partially purified by ammonium sulfate fractionation, dialysis, DEAE cellulose chromatography and electrophoretic analysis. Enzyme purity was tested by SDS-PAGE. Quantitative estimation has shown that 40mL of culture supernatant could dehair 2×1 cm of leather completely in 9 hours. In future the tanneries will use a combination of chemical and enzymatic processes. In practical applications, protease is a useful enzyme for promoting the hydrolysis of proteins and showing significant industrial applications.
Screening and Production of Protease Enzyme from Marine Microorganism and Its...iosrjce
Marine sediment samples were collected from the Gulf of Mannar, Mandapam coast to screen for
protease producing microbes. Among the five isolates screened only two isolates showed maximum proteolytic
activity with the zone of 21mm and 19mm respectively. Biochemical characterization of the isolates were
performed and identified as strain P2 belonged to Bacillus subtilis and strain P5 belonged to Bacillus
licheniformis. Both the strains have the ability to tolerate 7%Nacl concentration. The amount of protease
produced was expressed in microgram of tyrosine released under standard assay conditions. The total protein
content of crude enzyme extracts of Bacillus subtilis and Bacillus licheniformis were quantified which revealed
21.2mg/ml for strain P2 and 22.4mg/ml of protein content was presented by strain P5. The proteolytic bacteria
gave an optimum performance were both strains exhibited the enzymes stable at PH
7. In the present study
Bacillus subtilis showed a remarkable activity at 40ºC where as Bacillus licheniformis exhibited maximum
activity at 50ºC. Studies pertaining to carbon sources starch and lactose were utilized by Bacillus subtilis and
Bacillus licheniformis and maximum production was achieved. Among the different nitrogen sources tested
yeast extract induced maximum proteolytic activity where as ammonium sulphate was found to be the best
nitrogen sources for protease production. The crude enzyme was efficient to remove hair dye and blood stain by
Bacillus subtilis and Bacillus licheniformis
Bioprocess development for enhanced spore production in shake flask and pilot...iosrjce
Bacillus thuringiensis subsp. Israelensis (Bti), has proven to be a safe and effective larvicide for controlling
mosquito and black fly larvae. The effect of cultivation and bioprocess development on Bti growth and sporulation was
investigated in shake flask level and batch cultivation in the semi-industrial scale 16-L stirred tank bioreactor. For
industrial production of biocontrol microorganism, it is necessary to obtain high cell mass and spore production in a short
time with low cost cultivation media. In this study, the new composition of production media was optimized which composed
of (g L
-1
): glucose, 10; yeast extract, 30; KH2PO4, 5; K2HPO4, 5; MgSO4. 7H20, 0.005; MnSO4.H2O, 0.03; FeSO4, 0.01;
CaCl2.7 H2O, 0.05; NaH2PO4, 1.5; NH4H2PO4, 1.5. The maximal cell dry mass and spore production, Sporemax for shake
flask study were 4.26 gL-1
at 36 h and 3.29106
spore mL-1
, respectively. Furthermore, studies of the cultivation conditions
under controlled and uncontrolled pH in the 16L-bioreactor was performed. The growth of Bti under uncontrolled pH
cultivation showing decreased of glucose and total protein concentration in the media was correlated with the vegetative cell
growth and sporulation. The maximal cell dry mass and Sporemax for uncontrolled pH bioreactor were 4.14 gL-1
at 36h and
3.7106
spore mL-1
, respectively. The maximal cell dry mass and spore production, Sporemax for controlled pH bioreactor
were 3.36 gL-1
at 26 h and 3.23 106
spore mL-1
, respectively. In conclusion, batch cultivation in 16-L bioreactor with the
new optimized production medium under uncontrolled pH condition increased of the cell dry mass and number of spores up to 23 % and 47 % , respectively
Kinsenoside isolated from Anoectochilus formosanus suppresses LPS-Stimulated ...Cây thuốc Việt
In the present study, we reported that kinsenoside, a major component of Anoectochilus formosanus, inhibited inflammatory reactions in mouse peritoneal lavage macrophages and protects mice from endotoxin shock. In LPSstimulated mouse peritoneal lavage macrophages, kinsenoside inhibited the inflammatory mediators, such as nitric oxide, TNF-!, IL-1", monocyte chemoattractant protein 1, and macrophage migration inhibitory factor production. Furthermore, kinsenoside decreased the formation of a nuclear factor .BYDNA complex and nuclear p65 and p50 protein levels. Kinsenoside inhibited nuclear factor .B translocation through both I.B!-dependent and -independent pathway. In contrast, it stimulated anti-inflammatory cytokine IL-10 generation and enhanced the mRNA expression of IL-10 and suppressor of cytokine signaling 3 in the same cells induced by LPS. In an animal model, both pretreatment and posttreatment of kinsenoside increased the survival rate of ICR mice challenged by LPS (80 mg/kg, i.p.). Pretreatment with kinsenoside decreased serum levels of TNF-!, IL-1", IL-10, monocyte chemoattractant protein 1, and migration inhibitory factor at 1 h after sublethal dose of LPS (40 mg/kg, i.p.) in mice. In contrast, kinsenoside enhanced serum IL-10 level at 24 h after LPS injection in mice. In conclusion, kinsenoside inhibited the production of inflammatory mediators and enhanced antiinflammatory cytokine generation. Therefore, kinsenoside can alleviate acute inflammatory hazards.
The Effects of Grape Seed Powder and Extract on Antimicrobial of Fermented Tu...Agriculture Journal IJOEAR
— In this study, the effects of the grape seed powders and extracts of from two different grape cultivars (Razakı and Siyah Gemre) on the quality characteristics of Turkish sausage were investigated during the ripening period. Some characteristics of Turkish sausage including phenolic content, radical scavenging, hydrogen peroxide scavenging, Fe +2 chelating activity, Inhibition of Escherichia coli, Staphylococcusaureus, Candida albicans were studied. In fermented sausage, grape seed powders and extracts demonstrated the greatest inhibitory activity against Staphylococcus aureus. Furthermore, the results showed that Siyah Gemre grape seed extract was able to reduce Staphylococcusaureus populations by 42 CFU/g, while the population of Escherichia Coli was reduced by 590 CFU/g. Siyah Gemre grape seed extract was able to reduce Candida albicans populations by 880 CFU/g. Also this study demonstrated that grape seed extracts were more effective than grape seed powders. Our results suggest that the use of grape seed extract is a feasible alternative as antibacterial and antioxidant agents to prevent the deterioration of foods by bacteria and oxidation. Keywords— Grape seed powder and extract, Razakı and Siyah Gemre antimicrobial, antioxidant, natural preservative.
Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic...Mushafau Adebayo Oke
Oke, M. A., Annuar, M. S. M., and Simarani, K. (2016). "Enhanced endoglucanase production by Bacillus aerius on mixed lignocellulosic substrates." BioResources, 11(3), 5854-5869.
An enrichment lesson for upper elementary school students on the importance of following directions and the real life consequences that arise from not following directions. Ties directly into following reading and listening directions in the classroom.
The Role of Cell Wall-Degrading Enzymes in the Development of Anthracnose Dis...Agriculture Journal IJOEAR
— The ability of Colletotrichumtruncatum CP2 in producing pectinolytic and cellulolytic enzymes was evaluated by shake flask fermentations. The results of enzymatic activity experiment indicated that PG was the first cell wall-degrading enzymes detected and the activities obtained were higher (0.24±0.10 U/mL) than other enzymes, which appeared later and in lower amount. After the cell wall was degraded by the action of PG, further degradation of the cell wall was affected by pectin methylesterases, pectin lyase, pectate lyase and cellulases. The disparity in enzymatic activity at different intervals may suggest their specific role for pathogenesis at proper timings.
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
ABSTRACT- In this present study, the biotransformation of phenol to L-tyrosine was studied with resting cells of
Citrobacter freundii MTCC 2424 containing high tyrosine phenol lyase activity. Different process parameters leading to
synthesis of L-tyrosine were optimized. The L-tyrosine formed from biotransformation reactions was detected and
quantified by HPLC technique. The maximum L-tyrosine conversion 69% (6.49g/l) was obtained with ammonium
chloride 0.25M, phenol 0.1M, and sodium pyruvate 0.2M in borate buffer (0.1M) of pH 8.5 at 35°C temperature for
45min of incubation. Higher phenol concentration was found to be inhibitory for biotransformation due to phenol
inactivation of catalyst.
Key-words- Citrobacter freundii MTCC 2424, L-tyrosine, Tyrosine phenol lyase, Biotransformation
Isolation and Characterization of Thermostable Protease Producing Bacteria fr...IOSR Journals
This study is a search for potential thermostable protease producing strain. Among nine protease
producing strains screened from soap industry effluent, one was selected as promising thermostable protease
producer and identified as Bacillus subtilis. The activity of the protease produced by this organism is stable up
to 70ºC. The optimum yield was achieved after 48 hours of culture, at 65ºC with the pH 8.0. The maximum
protease activity was observed at 65ºC and at pH 8.0.
An Investigation Into The Mechanisms Underlying Enhanced Biosulphidogenesis I...iosrjce
Anthropogenic activities like mining, processes of metallurgy and other chemical industries lead to
the discharge of a high amount of sulphate into the environment that causes serious problems to human health.
This paper illustrates the employment of thermophilic sulphate reducing bacteria for biosulphidogenesis. Two
different species have been isolated from hot water spring of Vajreshwari and Ganeshpuri,Thane, Maharashtra,
INDIA.The mechanism involved in biosulphidogenesis includes production of specific protein as well as
liberation of some extracellular polymeric compound (EPS) e.g. proteins, carbohydrate, acids etc. that are
produced during the microbial cell metabolism. These compounds plays an important role in the faster
reduction of sulphate and decrease in production rate of sulphide.The isolate was found to be of genus
Bacillusand type strain was found to be subtilis Zankar and licheniformis Sonali. The strain sequence were
deposited in NCBI database with accession number KJ939324 and KJ939325 respectively. The result highlights
the potential use of these organism in biosulphidogenesis.
Undergraduate study done in an attempt to expedite yeast growth to fit the busy biology undergraduate schedule. The yeast were growth at various time/temp/rotation increments and were monitored for growth with a spectrometer. This information is now being used by our team of graduate students on a more in depth sudy using yeast speroplasts to study apoptosis mechanisms.
Hepatoprotective Effect of Cestrum parqui L. aerial parts and Phytochemical ...Jing Zang
This study deals with the investigation of hepatoprotective effect of 70% methanolic extract from Cestrum parqui aerial parts and determination of the bioactive components of the plant. The hepatoprotective effect of Cestrum parqui methanol extract (100, 500, 1000 mg/kg) was analysed on carbon tetrachloride (CCl4)-induced acute liver injury. The administration of a single dose of 40% CCl4 (1ml/kg b.w.) causes an increase in the activities of serum alanine aminotransferase (ALT) and aspirate aminotransferase (AST) enzymes and so pretreated orally of a dose from Cestrum parqui methanol extract (100, 500, 1000 mg/kg) and silymarin (200 mg/kg) for three consecutive days prior to The administration of a single dose of CCl4 significantly prevented the increase in the activities of these enzymes. Histological analysis showed that Cestrum parqui methanol extract at doses of 500 and 1000 mg/kg and silymarin reduced the incidence of liver lesions including vacuole formation, neutrophil infiltration and necrosis of hepatocytes induced by CCl4. The extract cause a negative result on the antioxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRd) and decreased malondialdehyde (MDA) level in liver, as compared to those in the CCl4-treated group and this suggests that the hepatoprotective activity of the extract is due to the antioxidant effect of the extract. Phytochemical analysis of the methanol extract from Cestrum parqui aerial parts showed that it contained different phytoconstituents, flavonoids, tannins, saponins, alkaloids, terpenes and carbohydrates.
Effcet of Vernonia Amygdalina.Del Ethanolic Extract Fraction on Serum Prolact...iosrjce
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) is a double blind peer reviewed International Journal edited by the International Organization of Scientific Research (IOSR). The journal provides a common forum where all aspects of Agricultural and Veterinary Sciences are presented. The journal invites original papers, review articles, technical reports and short communications containing new insight into any aspect Agricultural and Veterinary Sciences that are not published or not being considered for publication elsewhere.
ABSTRACT- Live microorganisms, have beneficial effects on their host’s health, are called as probiotics. There are various possible sources to isolate
these bacteria. In this studyp harmaceutical probiotic sachet is used as isolation source. The purpose of this study is to search the potentiality of
probiotic bacteria and investigate the probiotic properties of isolates.9 different samples of 3 brands of sachet were used for isolation of bacteria.
Isolates were examined according to their probiotic properties. The probiotic characteristics like pH and Bile tolerance, Antagonistic activity and
Antibiotic susceptibility of isolated bacteria Such as Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium bifidum was done. Bile
Tolerance and pH tolerance was determined with the help of the help of coefficient of growth inhibition if their coefficient of growth inhibition is less
than 0.5 the organism was considered as the pH and Bile tolerance. The Strains of Lactobacillus acidophilus and Lactobacillus rhamnosus and Bifidobacterium
bifidum show best result at the pH Acidic to Neutral (5 to 7) and show a bile tolerance from 1-4 % bile. All the isolated bacteria show
the maximum inhibition against Staphyloccocus aureus and minimum against Salmonella typhi by Lactobacillus Strains but Bifidobacterium show
minimum against Escheria coli. Most isolates show resistance toward antibiotics. From this study it can be concluded that pharmaceutical probiotic
products used in the study were showing satisfactory quality and potential probiotic strain.
Key words- Probiotic, Lactobacillus, Bifidobacterium, Sachet
Effects of pretreatment of single and mixed lignocellulosic substrates on pro...Mushafau Adebayo Oke
A mixed substrate (MS) comprising oil palm empty fruit bunch (EFB), oil palm frond (OPF), and rice husk (RH) was evaluated for endoglucanase production by Bacillus aerius S5.2. Effects of sulphuric acid, sodium hydroxide, N-methylmorpholine-N-oxide (NMMO), and hydrothermal pretreatments on endoglucanase production were investigated. Endoglucanase production by B. aerius on the untreated (0.677 U/mL) and pretreated MS (0.305 – 0.630 U/mL) was generally similar, except that the acid (0.305 U/mL) and hydrothermal (0.549 U/mL) pretreatments that were more severe consequently produced significantly lower titres. Alkali pretreatment supported the highest enzyme production (0.630 U/mL) among all pretreatments that were studied. When endoglucanase production on the alkali-pretreated MS and single substrates (SS) was compared, alkali-pretreated EFB produced a titre (0.655 U/mL) similar to the MS, and this was significantly higher than titres recorded on OPF (0.504 U/mL) and RH (0.525 U/mL). Lower enzyme production was found to be consistent with higher pretreatment severity and greater removal of amorphous regions in all the pretreatments. Furthermore, combining the SS showed no adverse effects on endoglucanase production.
Mixed Feedstock Approach to Lignocellulosic Ethanol Production—Prospects and ...Mushafau Adebayo Oke
Lignocellulosic ethanol is a promising alternative to fossil-derived fuels because lignocellulosic biomass is abundant, cheap and its use is environmentally friendly. However, the high costs of feedstock supply and the expensive processing requirements of lignocellulosic biomass hinder the development of the lignocellulosic biorefinery. Lignocellulosic ethanol production so far, has been based mainly on single feedstocks while the use of mixed feedstocks has been poorly explored. Previous studies from alternative applications of mixed lignocellulosic biomass (MLB) have shown that their use can bring about significant cost savings when compared to single feedstocks. Although laboratory-scale evaluations have demonstrated that mixed feedstocks give comparable or even higher ethanol yields compared to single feedstocks, more empirical studies are needed to establish the possibility of achieving significant cost savings in terms of pre-biorefinery logistics. In this review, some potential benefits of the use of MLB for ethanol production are highlighted. Some anticipated limitations of this approach have been identified and ways to surmount them have been suggested. The outlook for ethanol production from MLB is promising provided that revolutionary measures are taken to ensure the sustainability of the industry.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...
Effect of centrifugal pretreatment, p h and water activity on the production of protease by pediococcus acidilactici
1.
2.
3. EFFECT OF CENTRIFUGAL PRETREATMENT, pH AND WATER ACTIVITY ON
THE PRODUCTION OF PROTEASE BY Pediococcus acidilactici
Mushafau A. OKE1
and Mutiat B. ODEBISI1
1. Department of Microbiology, University of Ilorin, Ilorin, Nigeria.
ABSTRACT
Pediococcus acidilactici and other lactic acid bacteria (LAB) have wide applications in the food
industry due to the beneficial characteristics they impact on fermented foods. The proteolytic
system of LAB contributes to their use as starter cultures. The proteolytic system of the pediococci
has not been extensively researched like that of other LAB. This work was therefore aimed at
studying the effect of centrifugation, pH and water activity on the growth and production of
protease enzyme by Pediococcus acidlactici. The organism was grown in MRS broth and
subjected to a pretreatment of 2000 rpm and 3000rpm centrifugation speed, pH 4.5 and 6.5 and
water activity levels of 0.99, 0.97, 0.95 and 0.93. The effect of these treatments on growth and
protease production were determined separately and in combination. Centrifugation speed of 3000
rpm, pH 6.5 and water activity of 0.99 favoured growth and protease production by the organism.
However, lower protease production was observed when the three treatments were combined. The
results support the use of P. acidilactici as starter culture in meat fermentations.
Keywords: Lactic acid bacteria, Pediococcus acidilactici, protease, food fermentation.
INTRODUCTION
Pediococcus acidilactici belongs to a group of organisms known as lactic acid bacteria (LAB).
They are gram-positive, non-endospore forming, usually non-motile and metabolise glucose by
lactic acid fermentation (Maczulak, 2011). Other members of the group include Streptococcus,
Enterococcus, Lactococcus, Lactobacillus and Leuconostoc (Willey et al., 2009). LAB have
received much attention owing to their role in diary and food industry like baking, brewing, cheese
manufacturing and meat tenderization (Hans, 1993). They are particularly important in food
fermentation and their proteolytic system is considered to be one of the major processes involved
in texture and flavor development (Fadda et al., 2001).
The inability of LAB to synthesize many of the amino acids required for protein synthesis
necessitates the active functioning of a proteolytic system in those environments where protein
constitutes the main nitrogen source (Pritchard and Coolbear, 1993). The proteolytic system is
composed of proteinases which initially cleave the protein to peptides, peptidases which cleave
the peptides thus formed into smaller peptides and amino acid transport systems which are
involved in the cellular uptake of small peptides and amino acids (Law and Haandrikman, 1997).
Several environmental factors have been found to affect the growth and metabolism of P.
acidilactici but most of the studies have been mainly on bacteriocin production by the organism
(Calderon-Santoyo, 2001; Guerra and Pastrana, 2002; Vasquez et al., 2003). Few reports exist
about the proteases of the genus Pediococcus compared to other LAB genera (Llorente-Bousquets
et al., 2008). Due to the importance of P. acidilactici in the fermentation of vegetables, meat and
other fermented foods, this work was designed to determine the effects of centrifugation, water
activity and pH on the growth and protease production by the organism.
EXPERIMENTAL
4. Source of organism: Pediococcus acidilactici used in this work was obtained from the culture
collection stock of the Department of Microbiology, University of Ibadan. Pure cultures of the
organism were stored on MRS agar slants at 4o
C.
Effect of centrifugal pretreatment on growth and protease production
A loopful of the organism was inoculated into each of 2 McCartney bottles containing 10ml of
sterile MRS broth. These were incubated at 350
C for 24 hours after which they one of the bottles
was centrifuged at 2000 rpm and the other at 3000 rpm for 5 minutes. The bottles were tapped
slightly and shaken gently to disperse the settled cells. From each bottle, an inoculum load of 2.4
x 105
cfu/ml was taken and inoculated into 10ml of sterile MRS broth. These were then incubated
at 350
C for 48 hours after which the cruse enzyme was obtained by centrifugation at 2000 rpm for
15 minutes. The supernatant was obtained for protease assay and the cells were plated out for
growth determination.
Effect of pH on growth and protease production
Using either 0.1M NaOH or 0.1M HCl where necessary, 2 sets of McCartney bottles each
containing 10ml of MRS broth were set at pH 4.5 and 6.5. The bottles were sterilized and allowed
to cool. Each of the bottles was inoculated with 2.4 x 105
cfu/ml of the organism. Each set was
incubated for 24 hours and 48 hours. At the end of each interval, aliquots of the culture were
plated out. The cultures wee centrifuged at 2000 rpm for 5 minutes and the supernatants were
obtained for protease assay.
Effect of water activity (aw) on growth and protease production
Four sets of McCartney bottles containing 10ml of MRS broth each were adjusted to aw of 0.99,
0.97, 0.95 and 0.93 using NaCl (humectant) concentrations of 1.2%, 4.3%, 7.5% and 10.6%
respectively following the method of Troller and Stinson (1981). The bottles were sterilized,
cooled and then inoculated with the organism (2.4 x 105
cfu/ml). They were incubated for 24 hours
at 350
C and then plated out. Supernatants were obtained as described earlier and used for protease
assay.
Enzyme production under optimal conditions
P. acidilactici was grown in MRS broth for enzyme production under the treatments that gave the
highest protease production i.e. 3000 rpm centrifugation speed, pH 6.5 and aw of 0.99. The
organism was grown in sterile broth for 24 hours. The culture was centrifuged for 15 minutes at
3000 rpm. Inoculums from this culture was then used to inoculate 250ml of sterile MRS broth
which had been adjusted to a pH of 6.5 and aw of 0.99. This was incubated at 350
C for 24 hours
and at the end, it was centrifuged at 10000 rpm for 20 minutes. The supernatant (crude enzyme)
was obtained for and protease assay.
Protease assay
Protease assay was done using a modification of the method of Kunitz (1947). One percent casein
solution was prepared in 0.1M citrate phosphate buffer (pH 5.5) and was heat-denatured at 1000
C
for 15 minutes in a water bath and allowed to cool. The reaction mixture consisted of 1 ml
substrate (1% casein) thoroughly mixed with 0.5ml of the enzyme extract. Incubation was for 1
hour at 370
C after which the reaction was terminated by adding 3ml of cold (20
C) 10%
trichloroacetic acid (TCA). The reaction tubes were allowed to stand for 1 hour at 20
C to allow
the undigested protein to precipitate. Control tubes contained 0.5ml of enzyme extract incubated
for 1 hour at 370
C before adding 3ml of cold TCA and 1ml of 1% casein. The reaction mixtures
were centrifuged at 10000 rpm for 5 minutes at 40
C. Optical density readings of the carefully
decanted supernatant fluids were measured with a spectrophotometer at 660nm wavelength
5. against a blank containing the control. One unit of protease activity was defined as the amount of
enzyme that released 1 µg tyrosine per ml per minute from 1 mg casein under the specified assay
conditions.
RESULTS AND DISCUSSION
Effect of centrifugal pretreatment on growth and protease production
Table 1 shows the results of the effect of centrifugation on P. acidilactici.
Table1: Effect of centrifugation on growth and protease production by P. acidilactici.
Centrifugation speed (rpm) Growth (cfu/ml) Protease production
(units/ml)
2000 4.2 x 107
87.63
3000 4.8 x 107
103.98
The highest protease activity was observed at 3000 rpm probably because the higher
centrifugation speed dislodged cell surface-associated proteinases hence causing their release into
the surrounding medium. Some LAB are known to have proteinases located at the outer surface
of the cell (Pritchard and Coolbear, 1993). Centrifugation is known to have several effects on
bacterial cells such as affecting the hydrophobicity and electrophoretic mobility (Pembrey, 1991),
destabilization of the envelope (Gilbert, 1991) and alteration of cellular surface macromolecules
(Tsunada et al., 2003) which can be stripped off or compressed during centrifugation. An
important application of this finding is in the use of centrifugation as a pretreatment step for P.
acidilactici starter culture preparation for meat fermentations.
Effect of pH on growth and protease production
From the results in table 2, it can be observed that the growth of the organism at pH 4.5 and 6.5
at 24 hours was the same (1.4 x 108
cfu/ml). However, after 48 hours, higher growth was recorded
at pH 6.5. according to Harvey (1965), LAB grow best when the medium is near neutral pH and
growth rate declines as the extracellular medium becomes more acidic.
Table 2: Effect of pH on growth and protease production by P. acidilactici
Growth (cfu/ml) Protease production (units/ml)
pH 24 hours 48 hours 24 hours
4.5 1.4 x 108
3.2 x 105
71.57
6.5 1.4 x 108
4.1 x 105
82.37
The highest protease activity was recorded at pH 6.5 (82.37 units/ml) while the lowest was at pH
4.5 (71.57 units/ml). Other researchers have also found pH 6.5 to favour the growth and
metabolism of P. acidilactici. This pH has been found to favour cell mass, acid and pediocin
production after 16 hours of growth in TGE broth (Biswas et al., 1991) and it was also found to
favour higher biomass production in MRS broth containing 5.0% sugar cane strap molasses as
carbon source by P. acidilactici (Sant’ Anna and Torres, 1998).
Effect of water activity (aw) on growth and protease production
Growth of the organism showed a gradual decline as aw level was reduced (table 3). The highest
growth (6.5 x 108
cfu/ml) was at aw 0.99 while the lowest was at aw of 0.93.
6. Water activity (aw) Growth (cfu/ml) Protease production
(units/ml)
0.99 6.5 x 108
71.57
0.97 5.4 x 108
0
0.95 2.7 x 108
0
0.93 2.4 x 108
0
A similar trend was observed by Troller and Stinson (1981) in a test to determine the effect of
reduced water activity levels on growth and metabolite production by some LAB using NaCl,
glycerol and sucrose as humectants. They recorded the highest growths at aw of 0.998 and the
lowest growth at 0.95.
Protease production was only detected at aw of 0.99 and the growth slowed down while enzyme
production was halted. According to Braun et al. (1999), a decrease in temperature, pH or water
activity is usually associated with a loss of enzyme activity. Furthermore, Willey et al. (2009)
stated that microorganisms differ greatly in their ability to adapt to low aw and different organisms
present varying responses (in terms of growth and metabolite production) to reduced aw. The
organism has thus shown a promising feature in meat fermentation due to its favourable protease
production at aw 0.99 which is the aw of fresh meat (Ray, 2005).
Enzyme production under optimal conditions
The protease production observed under combination of the optimal conditions was 63.39 unit/ml.
this value is lower than that recorded when the treatments were treated separately thus suggesting
that a combination of the treatments would not favour protease production.
In conclusion, this study has shown that a centrifugal pretreatment of 3000 rpm supports protease
production by the organism. This could be applicable in preparation of P. acidilactici as starter
culture in the fermentation of meat and other products. Also the organism would do well in
initiating the hydrolysis of meat proteins in meat fermentation so as to enhance the qualities of
the product. This is as a result of its high protease production at physicochemical conditions
similar to that of fresh meat.
REFERENCES
Biswas, S.R.; Ray, P.; Johnson, M.C. and Ray, B. (1991). Influence of growth conditions on the
production of a bacteriocin, pediocin AcH by Pediococcus acidilactici H. Appl. Environ.
Microbiol.57: 1265-1267.
Braun, P.; Fehlaber, K.; Klug, C. and Kropp, K. (1999). Investigations into the activity of enzymes
produced by spoilage-causing bacteria: a possible basis for improved shelf-life estimation. Food
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Calderon-Santoyo, M.; Mendoza-Garcia, P.G.; Garcia-Alvarado, M.A. and Escudero-Abarca, B.I.
(2001). Effect of physical factors on the production of bacteriocin from Pediococcus acidilactici
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Fadda, S.; Viguelo, G. and Oliver, G. (2001). Meat Protein Degradation by Tissue and Lactic
Acid Bacteria Enzymes. In: Spencer, J.F.T. and Spencer, A.L.R. (eds). Food Microbiology
Protocols. Humana Press, Inc. Totora, New Jersey.
Gilbert, P. (1991). Centrifugation injury of gram-negative bacteria. Journal of Antimicrobial
Chemotherapy 27: 550-551.
7. Guerra, N.P. and Pastrana, L. (2002). Modeling the influence of pH on the kinetics of both nisin
and pediocin production and characterization of their functional properties. Process Biochemistry
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