DOCSLIB.ORG

Isolation of Cellulase Enzymes from Penicillium Spinulosum

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Isolation of Cellulase Enzymes from Penicillium Spinulosum Colorado State University-Pueblo Isolation of cellulase enzymes from Penicillium spinulosum Higgins, Nicole R. April 24, 2017 Senior Honors Thesis Abstract Cellulases are enzymes produced by select microorganisms and fungi that possess the ability to degrade cellulose, a particularly stable biopolymer found in plant cell walls. These enzymes are important to the organisms that produce them because they provide a carbon source that is inaccessible to most other organisms. They are also of particular interest in industry due to their potential to convert cellulose to biofuels that may serve as an alternative for non-renewable petroleum based fuels. Therefore, the isolation of cellulases with traits that make the production of biofuels on an industrial scale economically sustainable is of particular interest. This study aims to isolate cellulase genes from the fungus Penicillium spinulosum. Ultimately, the goal is to obtain the most efficient cocktail of enzymes for biofuel production whose gene sequences can be inserted into a genetically engineered bacterial vector for easy use in industrial applications. Therefore, this study also aims to determine the optimal conditions for the isolation of cellulases at all steps using techniques such as gradient PCR. In addition, a brief bioinformatics analysis of the primers developed for isolation of cellulases from P. spinulosum was conducted using NCBI BLAST to determine if any similarity with other cellulases or fungal organisms existed. BLAST search results indicated that the target cellulases are relatively novel. Introduction The cell walls in plants are primarily composed of cellulose, a biopolymer containing β-1,4- glycosidic linkages1. Structurally, cellulose is a homo-polysaccharide that arranges into unbranched chains of glucose molecules called microfibrils1. These microfibrils are difficult to degrade due to the β- 1,4-glycosidic linkages between each glucose molecule as well as the presence of hydrogen bonding between the cellulose fibers2. As a result, enzymes specialized for hydrolyzing the β-1,4-glycosidic linkages in cellulose have developed in many bacterial and fungal species3. These enzymes, termed cellulases, are produced by select microorganisms and fungi and provide such organisms with a carbon source that is unattainable by the majority of organisms. The general term cellulase encapsulates three distinct groups of hydrolytic enzymes capable of degrading cellulose: endoglucanases, exoglucanases, and β-glucosidases (BGL)3. Cellulolytic enzymes are classified into one of these three groups based on the method by which they cleave β-1,4-linkages as well as their specificity for particular substrates3. The first class of cellulases, endoglucanases, acts randomly on the cellulose chain to produce smaller oligosaccharides3. Exoglucanases, on the other hand, act more specifically by cleaving off units of cellobiose from the ends of cellulose chains3. Cellobiose is the smallest cleavable unit of cellulose that can be converted to glucose3. BGL, also known as cellobiase, is then responsible for hydrolyzing cellobiose to glucose2,3. Ultimately, these three groups of enzymes work synergistically in order to hydrolyze cellulose to glucose for use in metabolic processes2,3. It is important to note that the final conversion of cellobiose to glucose monomers by BGL is frequently the limiting step of the process3. This is due to the fact that BGL exists in a smaller ratio compared to the other two classes of cellulases3. In addition, BGL is inhibited by both its end product, glucose, as well as its substrate cellobiose 3. Apart from their importance in the metabolic processes of many microorganisms, cellulases have a number of important industrial roles including their potential to convert cellulose and its derived sugars to renewable biofuels that may provide a viable substitute for non-renewable petroleum based fuels. Biofuels produced via this mechanism (shown in Figure 1) are commonly referred to as lignocellulosic ethanol, named after dry plant matter termed lignocellulose2. The use of lignocellulose in the production of biofuels is appealing for several reasons. First of all, a large portion of this raw plant material is inedible and therefore its use for biofuel production would not negatively impact food production4. In addition, biomass has a low economic cost and has been reported as one of the largest available energy resources in the world with India alone possessing an annual availability of about 500 million metric tons2,5. It has been suggested that in the United States alone, approximately 30% of the current petroleum usage could be offset by switching to biofuels2. Finally, the use of fuels derived from lignocellulose has the potential to substantially reduce many of the negative effects of climate change due to the fact that their combustion releases lower levels of greenhouse gases in comparison to fossil fuel combustion4. Figure 1: General schematic of the biofuel production process using cellulase enzymes. However, isolation and production of cellulase enzymes remains a costly process3–5. In addition, the enzymatic efficiency of many commercially available enzymes is still too slow to maintain biofuel production on an industrial scale3–5. Therefore, developing more efficient methods to isolate cellulases, as well as identifying cellulases with traits that eliminate economic constraints, have become widespread areas of research. Ultimately, a successful transition to the use of biofuels on a commercial level will depend on the development of a “cellulase cocktail” that completely and efficiently hydrolyzes the intended biomass substrate in a cost-effective manner2,3. As previously stated, cellulases are produced by a number of microorganisms including fungi and both aerobic and anaerobic bacteria, however fungi are the most studied of these microorganisms3. The reasons for this are twofold, the first being that they tend to have a higher enzyme yield2,3. Furthermore, fungi secrete cellulase enzymes extracellularly making them more accessible than the majority of their bacterial counterparts2,3. In particular filamentous fungi, including those from the genera Penicillium, tend to produce higher levels of cellulase enzymes3. More importantly, such fungi produce all three types of cellulases giving them the ability to completely hydrolyze cellulose polymers3. More specifically, the fungus P. spinulosum, is a fast growing, non-pathogenic fungal species, making it an attractive choice for the study of cellulolytic enzymes. Ultimately, this study aims to isolate the gene sequence for cellulase enzymes from the genome of P. spinulosum as well as determine the optimal conditions under which this isolation is most efficient. Optimal conditions for the isolation the enzymes will be carefully considered at all steps in order to obtain the most efficient cocktail of enzymes for biofuel production. Methods Standard Growth Media Preparation Two 500 mL batches of standard growth media were prepared, one with glucose and the other with sucrose as a carbon source. In both cases, the sugar solutions were prepared separately from the salt solutions. Sugar solutions were prepared by adding 28.84 g glucose and 28.82 g sucrose to their respective 500 mL flasks and dissolving in 250 mL of distilled, deionized water. Salt solutions were prepared together in a 1 L flask by combining the remaining macronutrients and micronutrients in a 500 mL of distilled, deionized water according to the concentrations listed in Table 1. All of the micronutrients, except iron sulfate, were in solution. Each micronutrient was pipetted into the solution of macronutrients and iron sulfate at a final volume of 250 μL/L. After preparation, the salt solution was divided into two 250 mL portions and placed in 1 L flasks. All of the salt and sugar solutions were autoclaved and allowed to cool. Upon cooling, the sugar solutions were each aseptically combined with a salt solution by adding the sugar solution to the salt solution in a biosafety cabinet (Fig. 2). Table 1: Standard Growth Media Preparation Nutrients Constituents Concentrations Macronutrients mM g/L Carbon Glucose/Sucrose 320 57.6 Nitrogen NH4Cl 80 4.3 Phosphorus Na2HPO4 20 2.8 Sulfur Na2SO4 2 0.28 Buffer/Chelator Sodium Citrate 12.5 3.0 Citric Acid 12.5 2.2 Potassium K2CO3 3 0.42 Magnesium MgCO3 4 0.34 Sodium NaCl 20 1.2 Micronutrients Element, ppm Salt, mg/L . Iron FeSO4 7H2O 10 50 Zinc ZnCl2 5 10.4 . Manganese MnCl2 4H2O 5 18.0 . Molybdenum (NH4)6Mo7O24 4H2O 2 3.7 . Calcium CaCl2 5H2O 0.5 1.8 . Copper CuSO4 5H2O 0.4 1.6 Vanadium NH4VO3 0.2 0.46 Boron H3BO3 0.1 0.57 . Chromium Cr2(SO4)3 12H2O 0.1 0.93 . Nickel NiCl2 6H2O 0.1 0.40 . Cobalt CoCl2 6H2O 0.1 0.40 Sucrose (28.82 g) in Glucose (28.84 g) in 250 mL H O in 500 mL 250 mL H2O in 500 mL 2 flask (calc. for 500 flask (calc. for 500 mL) mL) 500 mL Sucrose Std. 500 mL Glucose Std. Growth Growth Salts in 250 mL H O in Salts in 250 mL H2O in 2 1 L flask (calc. for 1 L) 1 L flask (calc. for 1 L) Figure 2: Schematic Diagram of Standard Growth Media for P. spinulosum cultures. Spore Suspension A spore suspension to be used for current and future inoculations was prepared by adding sterile water to three Penicillium spinulosum cultures in petri dishes and scraping them with a sterile glass spreader. The spores were resuspended in an aqueous 0.12% (w/v) Tween 20 solution with 0.9% (w/v) NaCl to increase the density of the solution and suspend the spores. The Tween 20 is hydrophobic and therefore coats the spores to prevent them from germinating. Media Inoculation Each liquid media preparation was inoculated with 0.20 mL of the Penicillium spinulosum from the spore suspension using a sterile micropipette. The inoculated media flasks were then placed in the shaker and allowed to grow for approximately 1 week. Fungal DNA Extraction In order to extract DNA from the prepared cultures, the MasterPure Yeast DNA Purification kit was used6.
Load more

Recommended publications
  • Ergot Alkaloids Mycotoxins in Cereals and Cereal-Derived Food Products: Characteristics, Toxicity, Prevalence, and Control Strategies
    agronomy Review Ergot Alkaloids Mycotoxins in Cereals and Cereal-Derived Food Products: Characteristics, Toxicity, Prevalence, and Control Strategies Sofia Agriopoulou Department of Food Science and Technology, University of the Peloponnese, Antikalamos, 24100 Kalamata, Greece; [email protected]; Tel.: +30-27210-45271 Abstract: Ergot alkaloids (EAs) are a group of mycotoxins that are mainly produced from the plant pathogen Claviceps. Claviceps purpurea is one of the most important species, being a major producer of EAs that infect more than 400 species of monocotyledonous plants. Rye, barley, wheat, millet, oats, and triticale are the main crops affected by EAs, with rye having the highest rates of fungal infection. The 12 major EAs are ergometrine (Em), ergotamine (Et), ergocristine (Ecr), ergokryptine (Ekr), ergosine (Es), and ergocornine (Eco) and their epimers ergotaminine (Etn), egometrinine (Emn), egocristinine (Ecrn), ergokryptinine (Ekrn), ergocroninine (Econ), and ergosinine (Esn). Given that many food products are based on cereals (such as bread, pasta, cookies, baby food, and confectionery), the surveillance of these toxic substances is imperative. Although acute mycotoxicosis by EAs is rare, EAs remain a source of concern for human and animal health as food contamination by EAs has recently increased. Environmental conditions, such as low temperatures and humid weather before and during flowering, influence contamination agricultural products by EAs, contributing to the Citation: Agriopoulou, S. Ergot Alkaloids Mycotoxins in Cereals and appearance of outbreak after the consumption of contaminated products. The present work aims to Cereal-Derived Food Products: present the recent advances in the occurrence of EAs in some food products with emphasis mainly Characteristics, Toxicity, Prevalence, on grains and grain-based products, as well as their toxicity and control strategies.
    [Show full text]
  • Isolation of Fungal Cellulase Gene Transcript from Penicillium Spinulosum
    Isolation of fungal cellulase gene transcript from Penicillium spinulosum A Master’s Thesis Presented to the faculty of The College of Science and Mathematics Colorado State University – Pueblo In Partial Fulfillment of the requirements for the degree of Master of Science in Biochemistry By Srivatsan Parthasarathy Colorado State University – Pueblo May, 2018 ACKNOWLEDGEMENTS I would like to thank my research mentor Dr. Sandra Bonetti for guiding me through my research thesis and helping me in difficult times during my Master’s degree. I would like to thank Dr. Dan Caprioglio for helping me plan my experiments and providing the lab space and equipment. I would like to thank the department of Biology and Chemistry for supporting me through assistantships and scholarships. I would like to thank my wife Vaishnavi Nagarajan for the emotional support that helped me complete my degree at Colorado State University – Pueblo. III TABLE OF CONTENTS 1) ACKNOWLEDGEMENTS …………………………………………………….III 2) TABLE OF CONTENTS …………………………………………………….....IV 3) ABSTRACT……………………………………………………………………..V 4) LIST OF FIGURES……………………………………………………………..VI 5) LIST OF TABLES………………………………………………………………VII 6) INTRODUCTION………………………………………………………………1 7) MATERIALS AND METHODS………………………………………………..24 8) RESULTS………………………………………………………………………..50 9) DISCUSSION…………………………………………………………………….77 10) REFERENCES…………………………………………………………………...99 11) THESIS PRESENTATION SLIDES……………………………………………...113 IV ABSTRACT Cellulose and cellulosic materials constitute over 85% of polysaccharides in landfills. Cellulose is also the most abundant organic polymer on earth. Cellulose digestion yields simple sugars that can be used to produce biofuels. Cellulose breaks down to form compounds like hemicelluloses and lignins that are useful in energy production. Industrial cellulolysis is a process that involves multiple acidic and thermal treatments that are harsh and intensive.
    [Show full text]
  • Penicillium on Stored Garlic (Blue Mold)
    Penicillium on stored garlic (Blue mold) Cause Penicillium hirsutum Dierckx (syn. P.corymbiferum Westling) Occurrence P. hirsutum seems to be the most common and widespread species occurring in storage. This disease occurs at harvest and in storage. Symptoms In storage, initial symptoms are seen as water soaked areas on the outer surfaces of scales. This leads to development of the green-blue, powdery mold on the surface of the lesions. When the bulbs are cut, these lesions are seen as tan or grey colored areas. There may be total deterioration with a secondary watery rot. Penicillium sp. causing a blue-green rot of a garlic head Photo by Melodie Putnam Penicillium sp. on garlic clove Photo by Melodie Putnam Disease Cycle Penicillium survives in infected bulbs and cloves from one season to the next. Spores from infected heads are spread when they are cracked prior to planting. If slightly infected cloves are planted, they may rot before plants come up, or the seedlings may not survive. The fungus does not persist in the soil. Close-up of Penicillium sp. on garlic headad Air-borne spores often invade plants through Photo by Melodie Putnam wounds, bruises or uncured neck tissue. In storage, infection on contact is through surface wounds or through the basal plate; the fungus grows through the fleshy tissue and sporulation occurs on the surface of the lesions. Entire cloves may eventually be filled with spores. Susan B. Jepson, OSU Plant Clinic, 1089 Cordley Hall, Oregon State University, Corvallis, OR 97331-2903 10/23/2008 Management ● Cure bulbs rapidly at harvest ● Avoid wounds or injury to bulbs at harvest, and separate those with insect damage ● Plant cloves soon after cracking heads ● Eliminate infected seed prior to planting ● Store at low temperatures (40 F prevents growth and sporulation), with low humidity and good ventilation References Bertolini, P.
    [Show full text]
  • Identification and Nomenclature of the Genus Penicillium
    Downloaded from orbit.dtu.dk on: Dec 20, 2017 Identification and nomenclature of the genus Penicillium Visagie, C.M.; Houbraken, J.; Frisvad, Jens Christian; Hong, S. B.; Klaassen, C.H.W.; Perrone, G.; Seifert, K.A.; Varga, J.; Yaguchi, T.; Samson, R.A. Published in: Studies in Mycology Link to article, DOI: 10.1016/j.simyco.2014.09.001 Publication date: 2014 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Visagie, C. M., Houbraken, J., Frisvad, J. C., Hong, S. B., Klaassen, C. H. W., Perrone, G., ... Samson, R. A. (2014). Identification and nomenclature of the genus Penicillium. Studies in Mycology, 78, 343-371. DOI: 10.1016/j.simyco.2014.09.001 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. available online at www.studiesinmycology.org STUDIES IN MYCOLOGY 78: 343–371. Identification and nomenclature of the genus Penicillium C.M.
    [Show full text]
  • Penicillium Digitatum Metabolites on Synthetic Media and Citrus Fruits
    J. Agric. Food Chem. 2002, 50, 6361−6365 6361 Penicillium digitatum Metabolites on Synthetic Media and Citrus Fruits MARTA R. ARIZA,† THOMAS O. LARSEN,‡ BENT O. PETERSEN,§ JENS Ø. DUUS,§ AND ALEJANDRO F. BARRERO*,† Department of Organic Chemistry, University of Granada, Avdn. Fuentenueva 18071, Spain; Mycology Group, BioCentrum-DTU, Søltofts Plads, Technical University of Denmark, 2800 Lyngby, Denmark; and Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark Penicillium digitatum has been cultured on citrus fruits and yeast extract sucrose agar media (YES). Cultivation of fungal cultures on solid medium allowed the isolation of two novel tryptoquivaline-like metabolites, tryptoquialanine A (1) and tryptoquialanine B (2), also biosynthesized on citrus fruits. Their structural elucidation is described on the basis of their spectroscopic data, including those from 2D NMR experiments. The analysis of the biomass sterols led to the identification of 8-12. Fungal infection on the natural substrates induced the release of citrus monoterpenes together with fungal volatiles. The host-pathogen interaction in nature and the possible biological role of citrus volatiles are also discussed. KEYWORDS: Citrus fruits; Penicillium digitatum; quivaline metabolites; tryptoquivaline; P. digitatum sterols; phenylacetic acid derivatives; volatile metabolites INTRODUCTION obtained from a Hewlett-Packard 5972A mass spectrometer using an ionizing voltage of 70 eV, coupled to a Hewlett-Packard 5890A gas P. digitatum grows on the surface of postharvested citrus fruits chromatograph. producing a characteristic powdery olive-colored conidia and Preparation of TMS Derivatives and Analysis by GC-MS. TMS is commonly known as green-mold. This pathogen is of main derivatives were obtained according to the procedure previously concern as it is responsible for 90% of citrus losses due to described (7).
    [Show full text]
  • Fact-Sheet-Garlic-Post-Harvest-Final
    EXTENSION AND ADVISORY TEAM FACT SHEET APRIL 2020 | ©Perennia 2020 GARLIC STORAGE, POST-HARVEST DISEASES, AND PLANTING STOCK CONSIDERATIONS By Rosalie Gillis-Madden1, Sajid Rehmen1, P.D. Hildebrand2 1Perennia Food and Agriculture, 2Hildebrand Disease Management Once post-harvest disease manifests in garlic, there is the drying process and creating ideal conditions for fungal little to be done, so it is best to avoid creating conditions infection. Optimal curing temperatures are 24-29ºC or that are conducive to disease development in the first 75-85ºF. Using forced ambient air (5 ft3/min/ft3 of garlic) place. Disease management starts in the field by ensuring will significantly reduce drying time. Curing can take 10-14 appropriate harvest timing and good harvest practices. days, longer if the relative humidity is high. Stems can be either cut before or after curing, whichever optimizes labour efficiency on your farm. If curing in containers, Harvest make sure that they are open enough to allow for good To avoid post-harvest disease in your garlic, harvest timing airflow. Garlic should not be stacked more than three feet is important. While garlic should be left in the ground long high in a container to avoid bruising. Curing is complete enough to maximize yield, it is important that the cloves when two outer skins (scales) are dry and crispy, the neck is do not start to separate from the stem, as this will reduce constricted, and the centre of the cut stem is hard. marketability and storability. A wetter year will cause the cloves to separate quicker than is typically expected so as harvest nears, be sure to check your crop regularly.
    [Show full text]
  • Identification and Nomenclature of the Genus Penicillium
    available online at www.studiesinmycology.org STUDIES IN MYCOLOGY 78: 343–371. Identification and nomenclature of the genus Penicillium C.M. Visagie1, J. Houbraken1*, J.C. Frisvad2*, S.-B. Hong3, C.H.W. Klaassen4, G. Perrone5, K.A. Seifert6, J. Varga7, T. Yaguchi8, and R.A. Samson1 1CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands; 2Department of Systems Biology, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark; 3Korean Agricultural Culture Collection, National Academy of Agricultural Science, RDA, Suwon, Korea; 4Medical Microbiology & Infectious Diseases, C70 Canisius Wilhelmina Hospital, 532 SZ Nijmegen, The Netherlands; 5Institute of Sciences of Food Production, National Research Council, Via Amendola 122/O, 70126 Bari, Italy; 6Biodiversity (Mycology), Agriculture and Agri-Food Canada, Ottawa, ON K1A0C6, Canada; 7Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Közep fasor 52, Hungary; 8Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan *Correspondence: J. Houbraken, [email protected]; J.C. Frisvad, [email protected] Abstract: Penicillium is a diverse genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Other species are considered enzyme factories or are common indoor air allergens. Although DNA sequences are essential for robust identification of Penicillium species, there is currently no comprehensive, verified reference database for the genus. To coincide with the move to one fungus one name in the International Code of Nomenclature for algae, fungi and plants, the generic concept of Penicillium was re-defined to accommodate species from other genera, such as Chromocleista, Eladia, Eupenicillium, Torulomyces and Thysanophora, which together comprise a large monophyletic clade.
    [Show full text]
  • New Xerophilic Species of Penicillium from Soil
    Journal of Fungi Article New Xerophilic Species of Penicillium from Soil Ernesto Rodríguez-Andrade, Alberto M. Stchigel * and José F. Cano-Lira Mycology Unit, Medical School and IISPV, Universitat Rovira i Virgili (URV), Sant Llorenç 21, Reus, 43201 Tarragona, Spain; [email protected] (E.R.-A.); [email protected] (J.F.C.-L.) * Correspondence: [email protected]; Tel.: +34-977-75-9341 Abstract: Soil is one of the main reservoirs of fungi. The aim of this study was to study the richness of ascomycetes in a set of soil samples from Mexico and Spain. Fungi were isolated after 2% w/v phenol treatment of samples. In that way, several strains of the genus Penicillium were recovered. A phylogenetic analysis based on internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit 2 gene (rpb2) sequences showed that four of these strains had not been described before. Penicillium melanosporum produces monoverticillate conidiophores and brownish conidia covered by an ornate brown sheath. Penicillium michoacanense and Penicillium siccitolerans produce sclerotia, and their asexual morph is similar to species in the section Aspergilloides (despite all of them pertaining to section Lanata-Divaricata). P. michoacanense differs from P. siccitol- erans in having thick-walled peridial cells (thin-walled in P. siccitolerans). Penicillium sexuale differs from Penicillium cryptum in the section Crypta because it does not produce an asexual morph. Its ascostromata have a peridium composed of thick-walled polygonal cells, and its ascospores are broadly lenticular with two equatorial ridges widely separated by a furrow. All four new species are xerophilic.
    [Show full text]
  • Blues of the Midwest
    Blues of the Midwest CJ Bienert - The Cheese Shop of Des Moines, Des Moines Iowa Mark Johnson - Wisconsin Center for Dairy Research, Madison Wisconsin US Code of Federal Regulations 133.106 Blue ≤ 46% moisture ≥ 50 FDB (fat in solids portion) Must be aged at least 60 days No legal standard for -milk type used -color -flavor -body characteristics Milk Preparation • Blue cheese can be made from any milk • Roquefort is only made from sheep milk in France and aged in specific caves • Optional milk preparations: • Pasteurization of milk • Homogenization of milk or cream •Increases flavor development •Makes cheese whiter and smoother Make Procedure •Starter bacteria are added to produce acid •Rennet is added to clot the milk •Coagulum is cut into cubes Penicillium roquefortii •Added to milk or to curd after whey drainage •Different strains produce different flavor profiles and different colors PR1 PR4 PR3 PRG2 PR5 PRG1 Provided by: PRG3 Cooking/Curd Transfer/Hooping •Curd/whey is heated •From 94ºF to 100ºF •Whey is separated from curd •Salt and mold spores may be added to dry curd •Curd is placed in “hoops” and allowed to drain •Cheeses are brined or dry salted •Cheeses are pierced Brine System Ripening Process Mold Ripened Cheese Ripen all the way through, mold needs oxygen to grow Blue Ripening Room • Moderate air flow • ~95% humidity • 59º F • ~21-30 days to grow Penicillium roqueforti Cheese Ripening Full flavored, softer mature cheese Lactose Proteins Fat Fermentation Proteolysis Lipolysis Lactic acid Peptides Fatty acids Free amino-acids
    [Show full text]
  • Strong Effect of Penicillium Roqueforti Populations on Volatile and Metabolic Compounds
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 1/38 1 Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds 2 responsible for aromas, flavour and texture in blue cheeses 3 4 Authors: 5 Thibault CARON1,4, Mélanie LE PIVER4, Anne-Claire PÉRON3, Pascale LIEBEN3, René 6 LAVIGNE2, Sammy BRUNEL4, Daniel ROUEYRE4, Michel PLACE4, Pascal 7 BONNARME3, Tatiana GIRAUD1*, Antoine BRANCA1*, Sophie LANDAUD3*, Christophe 8 CHASSARD2* 9 1: Ecologie Systematique Evolution, Université Paris Saclay, CNRS, AgroParisTech, 91400 10 Orsay, France 11 2: Université Clermont Auvergne, INRAE, Vetagro Sup, UMRF, 20 Côte de Reyne, 15000 12 Aurillac, France 13 3: Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, 78850 Thiverval- 14 Grignon, France 15 4: Laboratoire Interprofessionnel de Production – SAS L.I.P., 34 rue de Salers, 15 000 16 Aurillac, France 17 *These authors jointly supervised the study 18 19 Corresponding author: Antoine Branca [email protected] 20 Running title: Penicillium roqueforti population impact on cheeses 21 Keywords: Roquefort cheese, fungi, Penicillium, domestication, volatile compounds 22 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.974352; this version posted March 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Analysis of Various Bioreactor Configurations for Heavy Metal Removal Using the Fungus Penicillium Ochro-Chloron
    ANALYSIS OF VARIOUS BIOREACTOR CONFIGURATIONS FOR HEAVY METAL REMOVAL USING THE FUNGUS PENICILLIUM OCHRO-CHLORON A Thesis Submitted to the Faculty of WORCESTER POLYTECHNIC INSITUTE in partial fulfillment of the requirements for the Degree of Master of Science in Biology by Eva Lotta Andersson May 1999 Approved: Dr. Alexander A. DiIorio, Major Advisor Dr. Theodore C. Crusberg, Thesis Advisor Dr. Ronald D. Cheetham, Head of Department Abstract Penicillium ochro-chloron (ATCC strain # 36741), a filamentous fungus with the capability for removing copper ions from aqueous solutions, was studied as a possible biological trap (biotrap) for remediation of heavy metal contaminants in industrial wastewaters. This research demonstrated that in shake flasks the fungus removed copper from surrogate wastewater with 100mg/L copper contamination by as much as 99%. These results did not translate to the bioreactor configuration of a packed bed column, as channeling occurred through the bed, shown by conductivity tracer studies. A fluidized bed configuration was studied and resulted in copper removal of 97%, with a capacity of 149 mg[Cu]/g dry weight biomass, under the conditions of 50% dissolved oxygen. For dissolved oxygen concentrations below the critical oxygen concentration for the fungus (20% saturation) there was minimal copper removal. Mixing studies in the fluidized bed reactor showed that the system was diffusion limited. Mathematical modeling using first order kinetics associated with diffusion limited reactions resulted in rate constants for Cu2+ uptake of approximately 0.031 h-1, which were dependent on the dissolved oxygen concentration. Modeling of the reaction with a second order kinetic equation showed that there are possibly factors regulating copper uptake besides oxygen.
    [Show full text]
  • Biosynthesis of Ergot Alkaloids by Penicillium Citrinum Through Surface Culture Fermentation Process
    Pakistan J. Zool., vol. 47(2), pp. 361-365, 2015. Biosynthesis of Ergot Alkaloids by Penicillium citrinum through Surface Culture Fermentation Process Memuna Ghafoor Shahid,1,* Safdar Ali1 and Muhammad Nadeem2 1Botany Department, GC University, Lahore 2FBRC, PCSIR Laboratories Complex, Lahore Abstract.- The present study deals with the production of ergot alkaloids from Penicillium sp. IIB and the optimization of culture conditions for enhanced production of ergot alkaloids. The strain of Penicillium citrinum was proved to be considerably useful for biotechnological purposes. The effect of temperature, pH, tryptophan, asparagine, different supplementary nitrogen and carbon sources and KH2PO4 on ergot alkaloids production was optimized during the study. Ergot alkaloids were produced between 23°C and 30°C incubation temperatures, with maximum yield (2.01 g/l) at 25°C. Maximum ergot alkaloid production was achieved at pH 5 (2 mg/l) with maximum incubation period of 21 days. Key words: Ergot alkaloids, culture conditions, Penicillium species. INTRODUCTION through which one can enhance the yield of ergot alkaloids in the fermentation medium. Ergot alkaloids are indole metabolic MATERIALS AND METHODS compounds with specific biological activities (Schardl et al., 2006). Human recognition of ergot Fungal strain alkaloids is as old as the civilization. The Penicillium citrinum was obtained from compounds have been widely used as drugs and in Institute of Industrial Biotechnology, GC medicines to cure different human diseases. Their University, Lahore and cultured on 4g MEA ecological role is weakly understood but they have medium (2g malt extract and 2g agar) and incubated been involved in activities against mammals, at 25°C for 15 days.
    [Show full text]