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Food Science
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FOOD 2420
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Shai Barbut
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Summer

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lFOOD*2420 Fundamental Food Microbiology UNIT 1 Chapter 1 History and Development of Food Microbiology Discovery of Microorganisms • The first person to see different types of microorganisms, especially bacteria, under and microscope was Anthony Leeuwenhoek. • He observed bacteria in saliva, rainwater, vinegar, and other materials; sketched the three morphological groups (spheroids or cocci, cylindrical rods or bacilli, and spiral or spirilla); also described some to be motile. • He called them animalcules, and between 1676 and 1683 he reported his observations to the newly formed leading scientific organization. WhereAre They Coming From • The emergence of maggots from dead bodies and spoiled flesh was thought to be due to spontaneous generation. • Maggots in spoiled meat and fish could only appear if flies were allowed to contaminate them. • The advocates of the spontaneous generation theory argued that the animalcules could not regenerate by themselves (biogenesis), but they were present in different things only through abiogenesis (spontaneous generation). • Louis Pasteur demonstrated that, in boiled infusion, bacteria could grow only if the infusions were contaminated with bacteria carried by dust particles in air. • His careful and controlled studies proved that bacteria were able to reproduce (biogenesis) and life could not originate by spontaneous generation. WhatAre Their Functions • The involvement of invisible organisms in many diseases in humans was suspected as early as the thirteenth century by Roger Bacon. • Many human diseases were transmitted from person to person by small creatures. • Putrefaction and fermentation were connected with the presence of the organisms derived from air. • Wine fermentation from grapes and souring of wine was caused by microorganisms. • Spoilage of meat and milk was associated with the growth of microorganisms. • Robert Koch isolated pure cultures of bacteria responsible for anthrax, cholera, and tuberculosis. • He also developed the famous Koch’s postulates to associate a specific bacterium as a causative agent for a specific disease. • Along with his associates, he developed techniques of agar plating methods to isolate bacteria in pure cultures and to determine microbial numbers in a sample, the Petri dish, staining methods for better microscopic observation of bacteria, and the use of steam to sterilize materials to grow bacteria. Development of Early Food Microbiology (BeforeA.D. 1900) • Fermentation was used extensively by many societies not only to preserve foods but also as a method to produce various types of desirable foods from milk, meat, fish, eggs, grains, fruits, and vegetables. Food Microbiology: Current Status • Specific methods were studied to prevent growth as well as to destroy the spoilage and pathogenic bacteria. • There was also some interest to isolate beneficial bacteria associated with food fermentation, especially dairy fermentation, and study their characteristics. • After the 1950s, food microbiology entered a new era. • Food Fermentation/Probiotics o Development of strains with desirable metabolic activities by genetic transfer among strains. o Development of bacteriophage-resistant lactic acid bacteria. o Metabolic engineering of strains for overproduction of desirable metabolites. o Effective methods to produce starter cultures for direct use in food processing. • Food Spoilage o Spoilage due to bacterial enzymes of frozen refrigerated foods with extended shelf life. • Foodborne Diseases o Methods to detect emerging foodborne pathogenic bacteria from contaminated foods. o Effective detection and control methods of foodborne pathogenic viruses. o Transmission potentials of prion diseases from food animals to humans. o Importance of environmental stress on the detection and destruction of pathogens. o Control of pathogenic parasites in food. • Miscellaneous o Application of hazard analysis of critical control points (HACCP) in food production, processing, and preservation. Food Microbiology and Food Microbiologists • This information is helping to develop methods for rapid and effective detection of spoilage and pathogenic bacteria, to develop desirable microbial strains by recombinant DNAtechnology, to produce fermented foods of better quality, to develop thermo stable enzymes in enzyme processing of food and food additives, to develop methods to remove bacteria from food and equipment surfaces, and to continue several control methods for effective control of spoilage and pathogenic microorganisms in food. Chapter 2 Characteristics of Predominant Microorganisms in Food Introduction • The microbial groups important in foods consist of several species and types of bacteria, yeasts, molds, and viruses. • Bacteria, yeasts, molds, and viruses are important in food for their ability to cause foodborne diseases and food spoilage and to produce food and food ingredients. • Many bacterial species some molds and viruses, but not yeasts, are able to cause foodborne diseases. • Most bacteria, molds, and yeasts, because of their ability to grow in foods (viruses cannot grow in foods), can potentially cause food spoilage. • Several species of bacteria, molds, and yeasts are considered safe or food grade, or both, and are used to produce fermented foods and food ingredients. • Among the four major groups, bacteria constitute the largest group. • Because of their ubiquitous present and rapid growth rate, even under conditions where yeasts and molds cannot grow, they are considered the most important in food spoilage and foodborne diseases. Classification of Microorganisms • Living cellular organisms were grouped originally in five kingdoms, in which bacteria belonged to prokaryotes (before nucleus) and the eukaryotic (with nucleus) molds and yeasts were grouped under fungi. • For the classification of yeasts, molds, and bacteria, several ranks are used after kingdom: divisions, classes, orders, families, genera (singular genus), and species. • The basic taxonomic group is the species. • Several species with similar characteristics form a genus. • Among eukaryotes, species in the same genus can interbreed. • This is not considered among prokaryotes, although conjugal transfer of genetic materials exists among many bacteria. • Several genera make a family, and the hierarchy follows the same procedure. • Among bacteria, a species is regarded as a collection of strains having many common features. • Astrain is the descendent of a single colony (single cell). • Among the strains in a species, one is assigned as the type strain, and is used as a reference strain while comparing the characteristics of an unknown isolate. • Evolutionary relationships among viruses, if any, are not known. Their classification system is rather arbitrary and based on the types of disease they cause (such as hepatitis virus, causing inflammation of the liver cells), nucleic acid content (RNAor DNA, single stranded or double stranded), and morphological structures. • In food, two groups of viruses are important: the bacterial viruses (bacteriophages) of starter culture bacteria and some foodborne pathogenic bacteria, and the human enteric pathogenic viruses associated with foodborne diseases. Nomenclature • The name has two parts (binomial name): the first part is the genus name and the second part is the specific epithet (adjective). • Both parts are Latinized; when written, they are italicized (or underlines), with the first letter of the genus written in a capital letter (e.g., Saccharomyces). Morphology and Structure of Microorganisms in Foods • Yeasts and Molds o Both are eukaryotic, but yeasts are unicellular whereas molds are multicellular. o Eukaryotic cells are generally much larger than prokaryotic cells. o Eukaryotic cells have rigid walls and thin plasma membranes. o The cell wall does not have peptidoglycan, is rigid, and is composed of carbohydrates. o The plasma membrane contains sterol. o The cytoplasm is mobile (streaming) and contains organelles (mitochondria, vacuoles) that are membrane bound. o Ribosomes are 80S type and attached to the endoplasmic reticulum. o The DNAis linear (chromosomes), contains histones, and is enclosed in a nuclear membrane. o Cell division is by mitosis (i.e. asexual reproduction); sexual reproduction, when it occurs, is by meiosis. o Molds are nonmotile, filamentous, and branched. o The cells wall is composed of cellulose, chitin, or both. o Yeasts are widely distributed in nature. o The cells are oval, spherical, or elongated. o They are nonmotile. o The cell wall contains polysaccharides (glycans), proteins, and lipids. • Bacterial Cells o Bacteria are unicellular in size, and have three morphological forms: spherical (cocci), rod shaped (bacilli), and curved (comma). o They can form associations such as clusters, chains (two or more cells), or tetrads. o They can be motile or nonmotile. o The genetic materials (structural and plasmid DNA) are circular, not enclosed in nuclear membrane, and do not contain basic proteins such as histones. o Bacterial cells are grouped as Gram-negative or Gram-positive. o Gram-negative cells have a complex cell wall containing an outer membrane (OM) and a middle membrane (MM). o The OM is composed of lipopolysaccharides (LPS), lipoprotein (LP), and phospholipids. o Gram-positive cells have a thick cell wall composed of several layers of peptidoglycan (mucopeptide; responsible for thick rigid structure) and two types of teichoic acids. o Some species also have a layer over the cell surface, called surface layer protein (SLP). o Because of the complexity in the chemical composition of the cell wall, Gram-positive bacteria are considered to have evolved before Gram-negative bacteria. • Viruses o Are regarded, as non-cellular entities. o Bacterial viruses (bacteriophages) important in food microbiology are widely distributed in nature. o They are composed of nucleic acids (DNAor RNA) and several proteins. o The proteins form the head (surrounding the nucleic acid) and tail. o Some viruses carry appendages or surface molecules for attachment to host cells. o Abacteriophage attaches itself to the surface of a host bacterial cell and inoculates its nucleic acid into the host cell. o Several pathogenic viruses have been identifies as causing foodborne diseases in humans. Important Microorganisms in Food • Important Mold Genera o Molds are important because they can grow even in conditions in which many bacteria cannot grow, such as low pH, low water activity (Aw), and high osmotic pressure. o Many types of molds are found in foods. o They are important spoilage microorganisms. o Many strains also produce mycotoxins and have been implicated in foodborne intoxication. o Some mycotoxins are carcinogenic or mutagenic and cause organ-specific pathology such as hepatotoxic (liver toxicity) or nephrotoxic (toxic or kidney). o Many are used in food bioprocessing. o Finally, many are used to produce food additives and enzymes. o Some of the most common genera of molds found in food are listed here.  Aspergillus – Members have septate hyphae and produce black-colored asexual spores on conidia. • Able to grow in lowAw and can grow in grains, causing spoilage. • Involved in spoilage of food such as jams, cured ham, nuts, and fruits and vegetables (rot).  Alternaria – Members are septate and form dark-colored spores on conidia. • They cause rot in tomatoes and rancid flavor in dairy products.  Fusarium – Many types are associated with rot in citrus fruits, potatoes, and grains. • They form cottony growth and produce septate, sickle shaped conidia.  Geotrichum – Members are septate and form rectangular arthrospores. • They grow, forming a yeast-like cottony, creamy colony. • Often grow on dairy products (dairy mold).  Mucor – They produce cottony colonies. • Some species are used in food fermentation and as a source of enzymes. • They cause spoilage of vegetables.  Penicillium – Many species cause fungal rot in fruits and vegetables. • They also cause spoilage of grains, breads, and meat.  Rhizopus – They cause spoilage of many fruits and vegetables. • Rhizopus stolonifer is the common black bread of mold. • Important Yeast Genera o Yeasts are important in food because of their ability to cause spoilage. o Many are also used in food bioprocessing. o Some are used to produce food additives. • Foodborne Protozoan Parasites o Are eukaryotic cells and some are associated with water and foodborne outbreaks. o Often wild animals, livestock’s, pets, or even humans carry these parasites and serve as source. o Contaminated soil and irrigation water are known causes for the contamination of fresh produce. o Members of these organisms generally require two hosts to complete their life cycle. o These organisms cause typical gastroenteritis with abdominal cramps and sometimes muscle ache, fever, and headache. • Important Viruses o Some are able to cause enteric disease, and thus, if present in a food, can cause foodborne diseases. o Hepatitis Aand Norwalk-lie or Noroviruses have been implicated in foodborne outbreaks. o In some countries where the level of sanitation is not very high, they can contaminate foods and cause disease. o Some bacterial viruses (bacteriophages) are used to identify some pathogens. o Bacteriophages are used to transfer genetic traits in some bacterial species or strains by a process called transduction. o Finally, some bacteriophages can be very important because they can cause fermentation failure. o Many lactic acid bacteria, used as starter cultures in food fermentations, are sensitive to different bacteriophages. o They can infect and destroy starter-culture bacteria, causing product failure. Important Bacterial Groups in Foods • Among the microorganisms found in foods, bacteria constitute major important groups. • Because of their rapid growth rate, ability to utilize food nutrients, and ability to grow under a wide range of temperatures, aerobiosis, pH, and water activity, as well as to better survive adverse situations, such a survival of spores at high temperatures. • LacticAcid Bacteria o They are bacteria that produce relatively large quantities of lactic acid from carbohydrates. • AceticAcid Bacteria o They are bacteria that produce acetic acid. • PropionicAcid Bacteria o They are bacteria that produce propionic acid and are used in dairy fermentation. • ButyricAcid Bacteria o They are bacteria that produce butyric acid in relatively large amounts. • Proteolytic Bacteria o They are bacteria that can hydrolyze proteins because they produce extracellular proteinases. • Lipolytic Bacteria o They are bacteria that are able to hydrolyze triglycerides because they produce extracellular lipases. • Saccharolytic Bacteria o They are bacteria that are able to hydrolyze complex carbohydrates. • Thermophilic Bacteria o They are bacteria that are able to grow at 50 degrees Celsius and above. • Psychrotrophic Bacteria o They are bacteria that are able to grow at refrigerated temperature (<=5 degrees Celsius). • Thermoduric Bacteria o They are bacteria that are able to survive pasteurization temperature treatment. • Halotolerant Bacteria o They are bacteria that are able to survive high salt concentrations (>=10%). • Aciduric Bacteria o They are bacteria that are able to survive at low pH (<4.0). • Osmophilic Bacteria o They are bacteria that can grow at a relatively higher osmotic environment than that is needed for other bacteria. • Gas-Producing Bacteria o They are bacteria that produce gas (CO2, H2, H2S) during metabolism of nutrients. • Slime Producers o They are bacteria that produce slime because they synthesize polysaccharides. • Spore Formers o They are bacteria having the ability to produce spores. • Aerobes o They are bacteria that require oxygen for growth and multiplication. • Anaerobes o They are bacteria that cannot grow in the presence of oxygen. • FacultativeAnaerobes o They are bacteria that are able to grow in both the presence and absence of oxygen. • Coliforms o They are used as an index of sanitation. • Fecal Coliforms o They are also used as an index of sanitation. Chapter 3 Sources of Microorganisms in Foods Introduction • The internal tissues of healthy plants (fruits and vegetables) and animals (meat) are essentially sterile. • Yet raw and processed (except sterile) foods contain different types of molds, yeasts, bacteria, and viruses. • Natural sources for foods of plant origin include the surfaces of fruits, vegetables, and grains, and the damaged tissues and the pores in some tubers (e.g. radish and onion). • Besides natural microorganisms, a food can be contaminated with different types of microorganisms coming from outside sources such as air, soil, sewage, water, feeds, humans, food ingredients, equipment, packages, and insects. Predominant Microorganisms in Different Sources • Plants (Fruits and Vegetables) o The inside tissue of foods plant sources are essentially sterile, except for a few porous vegetables (e.g., radishes and onions) and leafy vegetables (e.g., cabbage and Brussels sprouts). o Some plants produce natural antimicrobial metabolites that can limit the presence of microorganisms. o Fruits and vegetables harbor microorganisms on the surface; their type and level vary with soil condition, type of fertilizers, and water used, and air quality. o Pathogens, especially of enteric types can be present if the soil is contaminated with untreated sewage. o Diseases of the plants, damage of the surface (before, during, and after harvest), long delay between harvesting and washing, and unfavorable storage and transport conditions after harvesting and before processing can greatly increase microbial numbers as well as predominant types. • Animals, Birds, Fish, and Shellfish o Food animals and birds normally carry many types of indigenous microorganisms in the digestive respiratory, and urinogenital tracts, the teat canal in the udder, as well as in the skin, hooves, hair, and feathers. o Laying birds have been suspected of asymptomatically carrying Salmonella Enteritidis in the ovaries and contaminating the yolk during ovulation. o Prevention of food contamination from these sources needs to use of effective husbandry of live animals and birds, which include good housing, avoid overcrowding, and supply of uncontaminated feed and water. o Also, testing animals and birds for pathogens and culling the carriers are important in reducing the incidence of pathogenic microorganisms in foods. • Air o Microorganisms are present in dust and moisture droplets in the air. o They do not grow in dust, but are transient and variable, depending on the environment. o Generally, dry air with low dust content and higher temperature has a low microbial level. o Spores, molds, and cells of some Gram-positive bacteria, as well as yeasts, can be predominantly present in air. o If the surroundings contain a source of pathogens (e.g., animal and poultry farms or a sewage- treatment plant), different types of bacteria, including pathogens and viruses (including bacteriophages), can be transmitted via the air. • Soil o Soil, especially the type used to grow agricultural produce and raise animals and birds, contains several varieties of microorganisms. Because microorganisms can multiply in soil, their numbers can be very high. o Many types of molds, yeasts, and bacterial genera can enter foods fro the soil. o Soil contaminated with fecal materials can be the source of enteric pathogenic bacteria and viruses in food. o Different types of parasites can also get in food from soil. o Removal of soil (and sediments) by washing and avoiding soil contamination can reduce microorganisms in foods from this source. • Sewage o Sewage, especially when used as fertilizer in crops, can contaminate food with microorganisms. o This can be a major concern with organically grown food and many imported fruits and vegetables, in which untreated sewage and manure might be used as fertilizer. o Pathogenic parasites can also get in food from sewage. o It is better not to use sewage as fertilizer. o If used, it should be efficiently treated to kill the pathogens. • Water o Water is used to produce, process, and, under certain conditions, store foods. o Wastewater can be recycled for irrigation. o However, chlorine-treated potable water (drinking water) should be used in processing, washing, sanitation, and as an ingredient. o Although potable water does not contain coliforms and pathogens (mainly enteric types), it can contain other bacteria capable of causing food spoilage o To overcome the problems, many food processors use water, especially as an ingredient, that has a higher microbial quality than that of potable water. • Humans o Between production and consumption, foods come in contact with different people handling the foods. o They include not only people working in farms and food-processing plants, but also those handling foods at restaurants, catering services, retail stores, and at home. o Human carriers have been the source of pathogenic microorganisms in foods that later caused foodborne diseases, especially with ready-to-eat foods. • Food Ingredients o In prepared or fabricated foods, many ingredients or additives are included in different quantities. o Many of these ingredients can be the source of both spoilage and pathogenic microorganisms. • Equipment o Awide variety of equipment is used in harvesting, transporting, slaughtering, processing, and storing foods. o Depending on the environment (moisture, nutrients, and temperature) and time, microorganisms can multiply and, even from a low initial population reach a high level and contaminate large t volumes of foods. o Proper cleaning and sanitation of equipment at prescribed intervals are important to reduce microbial levels in food. • Miscellaneous o Foods might be contaminated with microorganisms from several other sources, namely packaging and wrapping materials, containers, flies, vermin’s, birds, house pets, and rodents. o Many types of packaging materials are used in food. Chapter 13 Starter Cultures and Bacteriophages Introduction • Means a selected strain of food-grade microorganisms of known and stable metabolic activities and other characteristics that is used to produce fermented foods of desirable appearance, body, texture, and flavor. • Some starter cultures are also used to produce food additives, as probiotics, and for drug delivery. • These starters were mixtures of unknown bacteria. • The bacteriological makeup of these starters (types and proportion of the desirable as well as undesirable bacteria) during successive transfers was continually susceptible to changes as a result of strain dominance among those present initially, as well as from the contaminants during handling. • This introduced difficulties in producing products of consistent quality and resulted in product failure due to bacteriophage attack of starter bacteria. History • Initial development of starter cultures resulted from the need and changes in the cheese industry. • They needed products of consistent quality and could not afford to have too many starter failures from phage attack. • Starter-culture producers developed single-strained cultures and supplied these in dried form to the cheese processors, who, in turn used them to produce mother cultures and bulk cultures. Starter-Culture Problems • StrainAntagonism o In mixed-strain cultures, in which a starter culture contains two or more strains, dominance of one over the others under a given condition can change the culture profile quickly. o Dominance can result from optimum growth environment or production of inhibitory metabolites (e.g., bacteriocins, acids, peroxides). o This can affect product quality and increase starter failure through phage attack. • Loss of a Desired Trait o Astrain carrying a plasmid-linked desired trait can lose the trait during storage, subculturing, and under some growth conditions. o Physical and chemical stress and long freezing can also result in loss of a trait. • Cell Death and Injury o The effective use of frozen and freeze-dried concentrated cultures, especially for direct use (such as DVS cultures), depends on two important characteristics: (1) cultures need to have large numbers of viable cells and (2) cells should have a short lag phase so that they can start multiplying very quickly. o The cells can be exposed to adverse physical and chemical environments (or stresses) that can reduce survival, growth, and metabolism. • Morphology and Characteristics o Bacteriophages are filterable viruses of bacteria widely distributed in the environment, especially in food fermentation environments. • Life Cycle 2 o Aphage cannot multiply by itself in food. Instead, it attaches (needs Ca + for adsorption) with its tail on the surface of a bacterial cell (specific host) and injects its DNAinside the cell cytoplasm. • Host Specificity o The phages are host specific, and there can be one specific host (strain) for a specific phage to several related strains for a phage. o Abacterial strain can also be the host of many different types of phages. o Abacterial strain can have restriction enzymes that can hydrolyze and destroy the DNAof a phage. o Aphage can be lytic or temperate. o All phages require Ca + for their adsorption on the cell surface of lactic cultures. • Control Methods o These include modifying the genetic makeup of a starter strain to inhibit phage adsorption, destroying phage DNAby restriction enzyme systems of cells, or aborting the phages before lysis. o By combining these traits through genetic manipulation, a strain that is resistant to several phages can be developed. o Current studies on genome analysis of lactic acid bacteria and bacteriophages win help develop phage-resistant starter strains Conclusion • Isolation and identification of microorganisms associated with food fermentation have helped the use of specific species and strains in pure culture for controlled fermentation. • These starter cultures are currently produced by commercial culture producers for use by food- processing companies directly to start fermentation of raw materials. • This has also helped to reduce product loss associated with culture failure notably from phage attack. Summary The Basics unit described how food microbiology developed as a science, differences between the main microbial types encountered in foods, how microbes are classified and the environments in which they are typically encountered. The opening section of the unit underlined the fact that man entered a world dominated by microbes. In order to survive humankind had to readily adapt to control spoilage and disease causing microbes. The findings of key scientists were fundamental in establishing research approaches, classification schemes and preservation methods. Microbes differ with respect to form, size and complexity. Viruses have a relatively simple structure and need a suitable host in order to replicate. Bacteria are more complex but also have a relatively simple structure compared to eukaryotic cells (yeast and molds). The different structural characteristics of microbes can be applied in devising classification schemes. Size, shape, physiology, DNAand Gram stain (in the case of bacteria) have all facilitated classification of microbes into distinct groups. Linnaeus defined a hierarchical system that is used today to classify microbes. The system sequentially defines microorganisms into smaller, more specific groups. The names donating the microbial types are from the Greek or Latin and hence are always written in italics (except for Salmonella serovars). Microbial names attempt to relate information about the microorganism in terms of cellular characteristic, habitat or initial source of isolation. Finally we learnt that through evolution microbes have adapted to different environmental niches. This is why certain microbes are typically associated with specific environments/foods. This information is used to predict the likely type of contamination that can be derived from an environmental source. The microbes we are dealing with in the food area can be divided into good, bad and ugly. In addition, the concept of HACCP which is widely used by the food industry today can help companies to reduce the risks associated with microbial, chemical and physical contamination of the food we consume. UNIT 2 Chapter 7 Microbial Metabolism of Food Components Introduction • Bacterial growth in food occurs though the metabolism of food components or nutrients mainly in the cytoplasm and cytoplasmic membrane (also in periplasmic space in Gram-negative bacteria) of cells. • The complete process involves transport of nutrients from the environment (for macro- molecules after their enzymatic breakdown) inside the cell through the cell wall and cell membrane, breakdown of nutrients to generate energy and active building blocks, synthesis of cell components from the building blocks (mainly macromolecules and structura1 and functional components) and release of unusable end products in the environment. Respiration and Fermentation During Growth • During growth in a food, microorganisms synthesize energy and cellular materials. • In a food system, the substrates are mainly the metabolizable carbohydrates, proteins, and lipids. • Microorganisms important in foods are heterotrophs (i.e., require organic carbon sources, substances more reduced than C02) and chemoorganotrophs (i.e., use organic compounds as electron donors to generate energy). • The energy-generating metabolic pathways also produce (from the substrates) many metabolic products that the microbial cells either use for the synthesis of cellular components or release into the environment. • Microbial spoilage of foods with the loss of acceptance qualities (e.g., flavor, texture, color, and appearance) is directly related to microbial growth and metabolism. • Toxin production in food by food-poisoning microorganisms also results from their growth in a food. • Many microbial metabolites are also important for their ability to produce desirable characteristics in fermented foods, such as texture, flavor, and long shelf life. • Microbial metabolic products are also used in foods for processing (enzymes), preservation (bacteriocins and acids), and improving texture (dextran) and flavor (diacetyl). • Plant foods are, in general, rich in carbohydrates; although some (e.g., nuts, lentils, and beans) are also rich in protein and some others (e.g., oilseeds) are rich in Lipids. • Foods of animal origin are rich in proteins and lipids, whereas some (e.g., meat and fish) are low in carbohydrates; others, such as milk, organ meats (liver), and mollusks (oysters), are rich in proteins as well as carbohydrates. • Microorganisms preferentially metabolize carbohydrates as an energy source over proteins and lipids. • Thus, microorganisms growing in a food rich in metabolizable carbohydrates utilize carbohydrates, but in a food low in metabolizable carbohydrates and rich in metabolizable proteins they metabolize proteins (after metabolizing the carbohydrates). • In a food rich in both carbohydrates and proteins, microorganisms usually utilize the carbohydrates first, then produce acids, and reduce the pH. Metabolism of Food Carbohydrates • Food carbohydrates comprise a large group of chemical compounds that include monosaccharides (tetroses, pentoses, and hexoses), disaccharides, oligosaccharides and polysaccharides. • Although carbohydrates are the most preferred source of energy production, microorganisms differ greatly in their ability to degrade individual carbohydrates. • Degradation of Monosaccharides o Five major pathways metabolize fermentable monosaccharides, and many microbial species have more than one pathway. o They are the Embden-Meyerhoff-Parnas (EMP) pathway, the hexose monophosphate shunt (HMS) or pathway, the Entner-Doudroff (ED) pathway, and two phosphoketolase (PK) pathways (pentose phosphoketolase and hexose phosphoketolase). o Pyruvic acid produced via these pathways is subsequently metabolized by microorganisms in several different pathways through fermentation, anaerobic respiration, and aerobic respiration. • Fermentation o Anaerobic and facultative microorganisms ferment monosaccharides by the five major pathways mentioned previously. • EMP Pathway o The EMP pathway is used by homofermentative lactic acid bacteria, Enterococcusfaecalis, Bacilllls spp., and yeasts. • HMP Pathway o The HMP pathway is also called the HMP shunt, pentose cycle, or Warburg-Dickens-Horecker pathway. o It is used by heterofennentative lactic acid bacteria, Bacillus spp., and Pseudomonas spp. • Synthesis of Polymers o Leuconostoc mesellteroides cells growing on sucrose hydrolyze the molecules and predominantly metabolize fructose for energy production. Glucose molecules are polymerized to form dextran (polymer of glucose). o Useful as food stabilizers and to give viscosity in some fermented foods; they can also cause quality loss in some foods. o Metabolism of food carbohydrates by microorganisms is undesirable when it is associated with spoilage. o Fermentation of carbohydrates is desirable in food bioprocessing and production of metabolites for use in foods (such as lactate and diacetyl). Metabolism of Food Proteins • Proteinaceous compounds present in foods include different types of simple proteins (e.g., albumin, globulin, zein, keratin, and collagen), conjugated proteins (e.g., myoglobin, hemoglobin, and casein), and peptides containing two or more amino acids.Amino acids urea, creatinine, trimethyl amine, and l others form the nonprotein nitrogenous (NPN) group. • Proteins and large peptides in a food are hydrolyzed to amino acids and small peptides by microbial extracellular proteinases and peptidases. • Small peptides are transported in the cell and converted to amino acids before being metabolized further. • Aerobic Respiration (Decay) o Many aerobic and facultative anaerobic bacteria can oxidize amino acids and use them as their sole source of carbon, nitrogen, and energy. • Fermentation (Putrefaction) o The products of microbial degradation of amino acids vary greatly with the types of microorganisms and amino acids and the redox potential of the food. o Some of the products are keto acids fatty acids, H2, C02, NH3, H2S, and amines. o Metabolic products of several amino acids are of special significance in food because many of them are associated with spoilage (foul smell) and health hazards. o They include indole and skatole from tryptophan, putrescine and cadaverine from lysine and arginine, histamine from histidine, tyramine from tyrosine, and suIfur-containing compounds (H2S, mercaptans, and sulfides) from cysteine and methionine. o Some of these sulfur compounds, as well as proteolytic products of proteinases and peptidases (both extra- and endocellular) of starter- culture microorganisms are important for desirable and undesirable (bitter) flavor and texture in several cheeses. o In addition to degradation (catabolism) of proteinaceous compounds of foods, the synthesis (anabolism) of several proteins by some foodborne pathogens while growing in foods is important because of the ability to produce proteins that are toxins. Metabolism of Food Lipids • The main lipids in food are mono-, di-, and triglycerides; free saturated and unsaturated fatty acids; phospholipids; sterols; and waxes, with the glycerides being the major lipids. • Microorganisms have low preference for metabolizing lipids. Being hydrophobic, lipids are difficult to degrade when present in mass. • Glycerides are hydrolyzed by extracellular lipases to release glycerol and fatty acids. • The fatty acids then can be transported inside the cells and metabolized by, B-oxidation to initially • Generate acetyl CoAunits before being utilized further. • Fatty acids if produced at a rapid rate, accumulate in the food. Conclusion • Microbial growth in food is accomplished through the metabolism of food nutrients, principally the metabolizable carbohydrates, proteins, and lipids. • Many types of end products are produced during metabolism of the nutrients, which, depending on the chemical nature, are associated with food spoilage, food poisoning, or production of fermented food. • Some are also used to improve texture and flavor of foods. • As long as the physical and nutritional environments are maintained, bacterial cells continue to multiply, generating energy and cellular materials through the metabolic processes. • However, a change in the environment can cause some species to shut down the cell multiplication cycle and trigger the sporulation cycle, in which a cell is differentiated as an endospore. Chapter 8 Microbial Sporulation and Germination Introduction • Microorganisms that are important in food normally divide by binary fission (or elongation, as in nonseptate molds). • In addition, molds, some yeast, and some bacteria can form spores. • In molds and yeasts, sporulation is associated with reproduction (and multiplication), whereas in bacteria it is a process of survival in an unfavorable environment. • In molds and yeasts, sporulation can occur by sexual and asexual reproduction. and sexual reproduction provides a basis for strain improvement for those that are used industrially. • In bacteria, sporulation occurs through differentiation and it provides a means to retain viability in a harsh environment. • Among the spores, bacterial spores have special significance in foods, because of their resistance to many processing and preservation treatments used in food. • Compared with bacterial spores, mold and yeast spores are less resistant to such treatments. • Spore formation in molds, yeasts, and bacteria is briefly discussed here. Mold Spores • Molds form large numbers of asexual spores and, depending on the type, can form conidia, sporangiaspores and arthrospores. • Conidia are produced on special fertile hyphae called conidiophores. • Aspergillus and Penicillium species form conidia. • Sporangiospores are formed in a sack (sporangium) at the tip of a fertile hypha (sporangiophores). • Mucor and Rhizopus species are examples of molds that form sporangiospores.Arthrospores. • Formed by the segmentation of a hypha, are produced by Geotrichum. • An asexual spore in a suitable environment germinates to form a hypha and resumes growth to produce the thallus. Yeast Spores • Yeasts important in food are divided into two groups: those that can produce sexual ascospores are designated as Ascomycetes (true yeasts), and those that do not form spores are called false yeasts. • Examples of some yeasts important in food that form ascospores are Saccharomyces, Kluyveromyces, Pichia, and Hansenula. • Species in the genera Candida, Torulopsis, and Rhodotorula do not form spores. Bacterial Spores • The ability to form spores is confined to only a few bacterial genera, namely the Gram- positive Bacillus, Alicyclobacillus, Clostridium, Sporolactobacillus, and Sporosarcina and the Gram- negative Desulfotomaculum species. • Among these, Bacillus, Alicyclobacillus, Clostridium, and Desulfotomaculum are of considerable interest in food, because they include species implicated in food spoilage and foodborne diseases. • Several Bacillus and Clostridium species are used to produce enzymes important in food bioprocessing. • Bacterial cells produce endospores (inside a cell and one spore per cell). During sporulation and until a spore emerges following cell lysis, a spore can be located terminal, central, or off-center, causing bulging of the cell. • The surface of a spore is negatively charged and hydrophobic. • The life cycle of spore forming bacteria has a vegetative cycle (by binary fission) and a spore cycle. • Sporulation o The transition from a normal vegetative cell cycle to sporulation in spore forming bacteria is triggered by the changes in the environmental parameters in which the cells are growing as well as a high cell number exists in the environment. o The environmental factors include reduction in nutrient availability (particularly carbon, nitrogen, and phosphorous sources) and changes in the optimum growthtemperature and pH. o A cell initiates sporulation only at the end of completion of DNA replication. o A triggering compound may be involved at that time for a cell to decide to either go through normal cell division or to initiate steps for sporulation. o The triggering compound is probably synthesized when nutrition depletion and other unfavorable conditions occur. o Adenosine bistriphosphate (Abt) could be one of the triggering compounds, as it is synthesized by sporeformers under carbon or phosphorous depletio. Sporulation events can be divided into about seven stages: • Termination of DNA replication, alignment of chromosome in axial filament, and formation of mesosome • Invagination of cell membrane near one end and completion of septum • Engulfment of prespore or forespore • Formation of germ cell wan and cortex, accumulation of cat, and synthesis of DPN • Deposition of spore coats • Maturation of spore: dehydration of protoplast, resistance to heat, and refractile appearance • Enzymatic lysis of wall and liberation of spore • The process isreversible before Stage 3. However, once the process has entered Stage 3, a cell is committed to sporulation. • Dormancy o Spores are formed in such a manner as to remain viable in unfavorable conditions. o This is achieved by increasing their resistance to extreme environments and reducing metabolic activity to dormancy. o Dehydration of the core and reduced molecular movement has been attributed to dormancy. o In a suitable environment, the dormancy of a spore can be ended through a series of biochemical reactions involved in spore activationgermination, outgrotth and growth. o Some spores may need a long time before they go through the sequences of germination, and are called superdormant spores. o They are quite common in Bacillus and Clostridium. o Superdormancy is thought to be the consequence of the inherent nature of a spore, spore injury, and environmental factors. o Injured spores need to repair their injury before they can germinate and outgrow. o During storage, they can germinate and outgrow and subsequently cause spoilage of a food, or, if a pathogen, a spore can make a food unsafe for consumption. • Activation o Spore activation before germination is accompanied by reorganization of macromolecules in the spores. o Spores can be activated in different ways, such as sub lethal heat treatment, radiation, high-pressure treatment with oxidizing or reducing agents, exposure to extreme pH, treatment with high pressure, and sonication. • Germination o Once the germination process starts, the dormant stage is irreversibly terminated. o Generally, germination is a metabolically degradative process. • Outgrowth o Outgrowth constitutes the biosynthetic and repair processes between the periods following germination of a spore and before the growth of a vegetative cell. o The events during this phase include swelling of the spore due to hydration and nutrient uptake; repair and synthesis of RNA, proteins, and materials for membrane and cell wall; dissolution of coats; cell elongation; and DNA replication. o The factors that can enhance the process include favorable nutrients, pH, and temperature. Importance of Spores in Food • Spore formation, especially by molds and some bacterial species, enables them to survive for a long time and provides a basis for the continuation of the species. • It also provides a means of their easy dissemination by dust and air in the environment. • In this manner, foods can be contaminated by their spores rather easily from various sources. • In a suitable food environment, spores germinate, grow, and produce undesirable (or desirable) effects. • Mold and yeast spores are relatively sensitive to heat, and their growth can also be prevented by storing foods in the absence of air. Conclusion • Spore formation by certain yeasts, molds, and bacterial species is a means of survival and continuation of the life process. • heir growth in food can be undesirable when they cause spoilage and produce toxins (except yeasts) in food and can be desirable in the processing of some foods. • Some bacterial species sporulate as a means of survival strategy under conditions of physical, chemical, or environmental stresses by genetically regulated processes. Chapter 16 Food Biopreservatives of Microbial Origin Introduction • Even in the early days of food fermentation, our ancestors recognized that fermented foods not only have delicate and refreshing tastes, but also have a longer shelf life and reduce the chances of becoming sick from food-borne diseases. • It is now known that the food-grade bacteria associated with food fermentation can produce several types of metabolites that have antimicrobial properties. Viable Cells of LacticAcid Bacteria as Preservatives • The process involves the addition of viable cells of mesophilic Lactococcus lactis, some Lactobacillus species, and Pediococcus species in high numbers to control spoilage and pathogenic bacteria during the refrigerated storage of a food at or below 5°C. • In the presence of mesophilic lactic acid bacteria, the growth of psychrotrophic spoilage and pathogenic bacteria is reported to be controlled. • Addition of cells of lactic acid bacteria in refrigerated raw milk also increased the yield of cheese and extended the shelf life of cottage cheese. • The inhibitory property can be because of the release of intracellular antimicrobial compounds, such as organic acids, bacteriocins, and hydrogen peroxide, from the cells by the non-metabolizing lactic acid bacteria. Organic, Diacetyl, Hydrogen Peroxide, and Reuterine as Food Preservatives • OrganicAcid o Acetic acid, its salts, and vinegar (which contains 5-40% acetic acid and many other compounds that give it the characteristic aroma) are used in different foods for inhibiting growth and reducing the viability of Gram-positive and Gram-negative bacteria, yeasts, and molds. o More effective against Gram-negative bacteria. o However, this effect is pH dependent and the bactericidal effect is more pronounced at low pH (below pH 4.5). o It is added to salad dressings and mayonnaise as an antimicrobial agent. o It is permitted to be used as a carcass wash. o Propionic acid and its salts are used in food as a fungistatic agent, but they are also effective in controlling growth and reducing viability of both Gram-positive and Gram-negative bacteria. o Propionic acid is used to control molds in cheeses, butter, and bakery products and to prevent growth of bacteria and yeasts in syrup, applesauce, and some fresh fruits. o Lactic acid and its salts are used in food more for flavor enhancement than for their antibacterial effect, especially when used above pH 5.0. o Growth of both Gram-positive and Gram-negative bacteria is reduced, indicating increased bacteriostatic action. o The antimicrobial effect of these three acids is considered to be due to their undissociated molecules. o The antimicrobial action of the undissociated molecules is produced by dissociation of the molecules in the cytoplasm following their entry through the membrane. o H+ released following dissociation initially reduces the trans membrane proton gradient and neutralizes the proton motive force, and then reduces the internal pH, causing denaturation of proteins and viability loss. • Diacetyl o Diacetyl is produced by several species of lactic acid bacteria in large amounts, particularly through the metabolism of citrate. o Several studies have shown that it is antibacterial against many Gram-positive and Gram- negative bacteria. o Gram-negative bacteria are particularly sensitive at pH 5.0 or below. o Diacetyl has an intense aroma, and thus its use is probably limited to some dairy-based products in which its flavor is not unexpected. o Diacetyl has an intense aroma, and thus its use is probably limited to some dairy-based products in which its flavor is not unexpected. • Hydrogen Peroxide o Hydrogen peroxide is permitted in refrigerated raw milk and raw liquid eggs (ca. 25 ppm) to control spoilage and pathogenic bacteria. o Its antibacterial action is attributed to its strong oxidizing property and its ability to damage cellular components, especially the membrane. o Because of its oxidizing property, it can produce undesirable effects in food quality, such as discoloration in processed meat, and thus has limited use in food preservation. • Reuterine o Some strains of Lactobacillus reuteri, found in the gastrointestinal tract of humans and animals, produce a small molecule, reuterine, which is antimicrobial against Gram-positive and Gram- negative bacteria. o It produces an antibacterial action by inactivating some important enzymes, such as ribonucleotide reductase. o However, reuterine is produced by the strains only when glycerol is supplied in the environment, which limits its use in food preservation. o The bacteriocins produced by many strains of lactic acid bacteria and some propionic acid bacteria are of special interest in food microbiology because of their bactericidal effect normally to different Gram-positive spoilage and pathogenic bacteria and under stressed conditions to different Gram-negative bacteria important in food. • Bacteriocin-Producing Strains o Bacteriocins of lactic acid bacteria are bactericidal to sensitive cells, and death occurs very rapidly at a low concentration. Chapter 17 Food Ingredients and Enzymes of Microbial Origin Introduction • Many microbial metabolites can be used as food additives to improve nutritional value, flavor, color, and texture. • Some of these include proteins, essential amino acids, vitamins, aroma compounds, flavor enhancers, salty peptides, peptide sweeteners, colors, stabilizers, and organic acids. • Many enzymes from bacteria, yeasts, molds, as well as from plant and mammalian sources, are currently used for the processing of foods and food ingredients. • Some examples are production of high-fructose corn syrups, extraction of juice from fruits and vegetables, and enhancement of flavor in cheese. • Recombinant DNAtechnology (or biotechnology) has opened up the possibilities of identifying and isolating genes or synthesizing genes encoding a desirable trait from plant and animal sources, or from microorganisms that are difficult to grow normally, clone it in a suitable vector (DNAcarrier), and incorporate the recombinant DNAin a suitable microbial host that will express the trait and produce the specific additive or enzyme economically. • In addition, metabolic engineering, by which a desirable metabolite can be produced in large amounts by a bacterial strain, is being used to produce food additives from new sources. • The metabolites can then be purified and used as food additives and in food processing. Microbial Proteins and FoodAdditives • Single-Cell Proteins (SPCs) o Molds, yeasts, bacteria, and algae are rich in proteins, and the digestibility of these proteins ranges from 65 to 96%. o Proteins from yeasts, in general, have high digestibility as well as biological values. In commercial production, yeasts are preferred. o The use of microbial proteins as food has several advantages over animal proteins. o Microbial proteins can be a good source of B-vitamins, carotene, and carbohydrates. o There are some disadvantages of using microbial proteins as human food. They are poor in some essential amino acids, such as methionine. o However, this can be corrected by supplementing microbial proteins with the needed essential amino acids. • AminoAcids o Proteins of most cereal grains are deficient in one or more of the essential amino acids, particularly methionine, lysine, and tryptophan. o To improve the biological values, cereals are supplemented with essential amino acids. o Supplementing vegetable proteins with essential amino acids has been suggested to improve the protein quality for people who either do not consume animal proteins (people on vegetarian diets) or do not have enough animal proteins (such as in some developing countries, especially important for children). • Nutraceuticals and Vitamins o There is a large market for vitamins, especially some B-vitamins and vitamins C, D, and E. o Vitamin C is now produced by yeast by using cheese whey. Microorganisms have also been a source of vitamin D. Many are capable of producing B-vitamins. • Flavor Compounds and Flavor Enhancers o Flavor compounds and enhancers include those that are associated directly with the desirable aroma and taste of foods and indirectly with the strengthening of some flavors. o Many microorganisms produce different types of flavor compounds, such as diacetyl (butter flavor by Leuconostoc), acetaldehyde (yogurt flavor by Lactobacillus acidophilus), some nitrogenous and sulfur-containing compounds (sharp cheese flavor by Lactococcus lactis), propionic acid (nutty flavor by dairy Propionibacterium). pyrazines (roasted nutty flavors by strains of Bacillus subtilis and Lac. lactis), and terpenes (fruity or flowery flavors by some yeasts and molds). o Several flavor enhancers are now used to strengthen the basic ftavors of foods. o Monosodium glutamate (MSG; enhances meat flavor) is produced by several bacterial species, such as COlynebacterium glutamicum and Micrococcus glutamicus. • Colours o Many bacteria, yeasts and molds produce different color pigments. t o This includes the red color pigment astaxanthine of a yeast species (Phafjia sp.). o This pigment gives the red color to salmon, trout, lobster, and crabs. • Exopolysaccharides (EPS) o Different polysaccharides are used in food systems as stabilizers and texturizers. o Strains of many lactic acid bacteria, such as Streptococcus thermophilus, Lab. rizamnoslIs, Lab. helveticus, Lab. casei, and Lac. lactis, produce many different types of exopolysaccharides (EPS) that contain units of glucose, galactose, rhamnose, mannose, and other carbohydrates. o Many of these strains are currently being used to produce fermented dairy products with better consistency and texture (in yogurt and buttermilk), to hold moisture in low fat-high moisture cheeses (in mozzarella cheese). • OrganicAcids o Several other organic acids and their salts are used in foods to improve taste (flavor and texture) and retain quality. o Ascorbic acid is also used in some foods as a reducing agent to maintain color (to prevent color loss by oxidation). o It also has an antibacterial action. Citric acid is used in many foods to improve taste and texture (in beverages) and stabilize color (in fruits). o It also has some antibacterial property. o Citric acid is produced by the mold Aspergillus niger. • Preservatives o Bacterial cells of lactic acid bacteria, several organic acids produced by them, and their bacteriocins can be used to control spoilage and pathogenic bacteria in foods. Microbial Enzymes in Food Processing • Many enzymes are used in the processing of food as food additives. • Use of specific enzymes instead of microorganisms has several advantages. • Aspecific substrate can be converted into a specific product by an enzyme through a single-step reaction. • Finally, by using recombinant DNAtechnology, the efficiency of enzymes can be improved and, by immobilizing, they can be recycled. • The main disadvantage of using enzymes is that if a substrate is converted to a product through many steps (such as glucose to lactic acid), microbial cells must be used for their efficient and economical production. • Enzymes Used o Among the five classes of enzymes, three are predominantly used in food processing: hydrolases, isomerases, and oxidoreductases (oxygenation or hydrogenation). • Alpha-Amylase, Glucoamylase, and Glucose Isomerase o Together, these three enzymes are used to produce high-fructose corn syrup from starch. o Alpha-Amylase is also used in bread-making to slow down staling (starch crystallization due to loss of water). • Catalase o Raw milk and liquid eggs can be preserved with H202 before pasteurization. • Cellulase, Hemicellulase, and Pectinase o Because of their ability to hydrolyze respective substrates, the use of these enzymes in citrus juice extraction has increased juice yield. • Invertase o Invertase can be used to hydrolyze sucrose to invert sugars (mixture of glucose and fructose) and increase sweetness. It is used in chocolate processing. • Lactase o Whey contains high amounts of lactose. Lactose can be concentrated from whey and treated with lactase to produce glucose and galactose. o It can then be used to produce alcohol. • Lipases o Lipases may be used to accelerate cheese flavor along with some proteases. • Proteases o They are used to tenderize meat, extract fish proteins, separate and hydrolyze casein in cheese- making (rennet), concentrate cheese flavor (ripening), and reduce bitter peptides in cheese (specific peptidases). • Enzyme Production by Recombinant DNATechnology o The enzymes that are currently used in food processing are obtained from bacteria, yeasts, molds, plants, and mammalian sources. o The supply of these enzymes can be limited and thus costly. o Also, molds grow slower than bacteria or yeast, and some strains can produce mycotoxins. o Involves separating specific mRNA (while growing on a substrate) and using the mRNAto synthesize cDNAby employing the reverse transcriptase enzyme. o The cDNA (double stranded) is cloned in a suitable plasmid vector, which is then introduced by transformation in the cells of a suitable bacterial strain (e.g., Esc. coli). o The transformants are then examined to determine the expression and efficiency of production of the enzyme. o This method has been successfully used to produce rennin (of calf) and cellulase (of molds) by bacteria. Rennin thus produced is used to make cheese. • Immobilized Enzymes o Enzymes are biocatalysts and can be recycled. o An enzyme is used only once when added to a substrate in liquid or solid food. o In contrast if the molecules of an enzyme are attached to a solid surface (immobilized), the enzyme can be exposed repeatedly to a specific substrate. o Enzymes can be immobilized by several physical, chemical, or mechanical means. o The techniques can be divided into four major categories. • Adsorption on a Solid Support o The technique involves adding an enzyme solution to the support (such as ion-exchange resins) and washing away the unattached molecules. o The association is very weak, and the molecules can be desorbed and removed. • Covalent Bonding o The enzyme molecules are covalently bound to a solid surface (such as porous ceramics) by a chemical agent. o The enzymes are more stable. • Entrapping o The enzyme molecules are enclosed in a polymeric gel (e.g., alginate) that has an opening for the substrate molecules to come in contact with the catalytic sites. o The enzymes are added to the monomer before polymerization. • Crosslinking o Crosslinking is achieved by making chemical connections between the enzyme molecules to form large aggregates that are insoluble. o This is a very stable system. o Immobilization can reduce the activity of an enzyme. o Substrate molecules may not be freely accessible to the immobilized enzymes. o The method may not be applicable if the substrate molecules are large. o Glucose isomerase can be immobilized, as its substrate is small glucose molecules. o The supporting materials can be contaminated with microorganisms that are difficult to remove and can be a source of contamination in food. • Thermostable Enzymes o The term thermostable enzymes is generally used for those enzymes that can catalyze reactions above 60°C. o There are several advantages of using thermostable enzymes in a process. o The rate of an enzyme reaction doubles for every 10°C increase in temperature; thus, production rate can be increased or the amount of enzyme used can be reduced. o At high temperatures, when an enzyme is used for a long time (as in the case of immobilized enzymes), the problems of microbial growth and contamination can be reduced. • Enzymes in Food Waste Treatment o Food industries generate large volumes of both solid and liquid wastes.
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