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Book Review |
Dean, College of Agriculture
California State Polytechnic University, Pomona
Lactoferrin: Natural, Multifunctional, Antimicrobial. A.S. Naidu. Boca Raton, FL: CRC Press, 2000.
This monograph of 86 pages is well written by one of the leading researchers in the field. He has contributed significantly to altering the perceived function of lactoferrin (LF) from that of an iron delivery protein for mammalian infants to that of a natural antimicrobial agent protecting the infant from pathogenic infections. This text reviews all aspects of LF: purification, characterization, antimicrobial effects and chemical modification in order to enhance its activity.
Lactoferrin is an iron-binding glycoprotein found in milk, but also in a variety of exocine secretions such as saliva, tears, seminal fluid, mucins and granules of neutrophils. In addition, LF can bind other metals as well. LF can bind two ferrous ions with two carbonate ions, providing the major structure function relationship. Interaction of LF with specific targets on the microbial surface causes an array of outcomes either to the advantage of the host (microbial blocking effects) or the microorganism (iron-acquisition and pathogenesis).
The most common methods for isolation and purification of LF include size exclusion chromatographic separation on CM-Sephadex columns and affinity chromatography on Cibachron Blue F3G-A-Sepharose columns, on Heparin agarosecross linked columns and on single stranded DNA-agarose columns. Iron saturation can be affected by dialysis against buffered acidic solutions. Apo-LF is prepared by dialysis against pH 4.0 acetate buffer, while holo-LF is prepared by dialysis against a large excess of citric acid, followed by raising the pH to 7.0 and addition of excess bicarbonate and iron. Human and bovine LF have specific differences in physico-chemical properties, such as IEP, absorption spectra, glycosylation, proteae sensitivity and iron-binding affinity. These characteristics are related to its varied biologic functions.
LF may be expected to provide diverse antimicrobial functions in different biological fluids. Structural characteristics and spatial orientation of the molecule are critical in defining the functionality of an antimicrobial. The environmental characteristics of viscosity, pH and ionic strength and the changing presence of other physiologic proteins affect LF thereby causing a broad-spectrum of antimicrobial activities. Antimicrobial activities include stasis, cidal, adhesion-blockade, cationic, synergistic and opsonic mechanisms against different bacteria (gram-positive and Gram-negative, rods and cocci, and aerobes and anaerobes), DNA and RNA viruses, a variety of yeasts, fungi and parasites. In addition, LF expresses anti-inflammatory and immunomodulatory properties that enhance effectiveness of its antimicrobial action.
LF can interfere with microbial activity by binding to the surface via an array of specific and non-specific interactions, either promoting host defense (elimination) or microbial virulence (iron acquisition by pathogens). LF binding sites have been identified, including porins, lipopolysaccharides and DNA. In vivo LF exhibits multifunctional activities. It exhibits a regulatory role in various physiologic pathways. Although iron binding is an important molecular property, a number of cellular functions are independent of this metal binding property.
LF has found its way into infant formula and health foods in SE Asia. It is used as a therapeutic and prophylactic agent for control of intestinal illnesses. Recent advances in LF research have elucidated structure-function relationships, antimicrobial mechanisms and cost effective technologies for large scale protein isolation and biotechnology.
A number of efficacy studies and clinical trials are ongoing in various laboratories with over 100 patents filed on this molecule in the last ten years. LF is emerging as one of the leading natural microbial blocking agents in food safety and preservation.
Everyone interested in natural antimicrobial agents will enjoy reading this treatise. It is thorough, well researched (over 370 references), well written and easily read. The style and format can serve as a model to evaluate other antimicrobials. Graduate students, researchers and teachers alike will benefit from reading this text.
Food Hygiene, Microbiology and HACCP, 3rd ed. S.J. Forsythe and P.R. Hayes. Gaithersburg, MD: Chapman & Hall Food Science Book, Aspen Publishers, Inc, 1998.
This volume is the continuation of P.R. Hayes’ original writing, but is updated by S.J. Forsythe. The intent is to inform and educate the reader about hygienic practices needed in food manufacture. It includes discussions of HACCP, food microbiology and methodologies used to detect the microbes. The purpose is to maintain perspective on food safety issues, even as the guidelines and regulations continue to change. In the revision, new techniques, such as genetic HACCP using gene analysis to detect and identify the sources of contamination, are discussed to encourage needed changes in industry applications. The book is well written, informative, well referenced to critical citations and worthy of adding to one’s reference shelf once read.
A few highlights of the book are presented here for the reader’s consideration. The first section of the book provides the fundamental principles of microbiology—the study of microscopic organisms, their distribution, harmful and beneficial characteristics and their adaptation to their environment. Although bacteria are the most important in relation to our food supply, fungi have a significant role and the impact of viruses is just beginning to be understood. Characteristics and types of bacteria, fungi, viruses and prions were presented to form the basis for the materials presented in the book’s chapters. The adaptability of these organisms enables them to live in extreme environmental conditions and thereby cause unexpected problems in the food industry.
Next, a discussion on bacterial food poisoning is presented. Many developed countries publish the incidence of food poisoning, yet only 1% of cases are actually reported. The term "food poisoning" covers a variety of illnesses expressed by the gastrointestinal tract as a result of the consumption of contaminated foods or drinks. Bacterial food poisoning is caused by multiplication of the organism in the food which induces illness following consumption of the food. In food borne infections, the food merely acts as a carrier for the causative organism. In addition, bacterial food poisoning can be divided into the infection type and the toxin type.
An infection food poisoning is characterized by an acute gastroenteritis following ingestion of food in which bacterial multiplication has occurred; the ingested bacteria continue to grow within the host body to produce typical symptoms. Salmonellae are principally responsible for this type of food poisoning in which endotoxins are released as the bacterial cells disintegrate. The toxin type (intoxication) is a genuine food poisoning since a poisonous substance, an enterotoxin, is present in the food as a result of the bacterial growth in the food prior to consumption. The toxin also causes an acute gastroenteritis, but results directly from the toxin, not the producing organism. Examples of bacteria toxin food poisoning include Clostridium perfringes and Staphylococus aureus.
Each of the critical microbial agents related to food poisoning are well described, identifying the disease, common food sources and control measures. Bacterial agents characterized as causing food poisoning include Salmonella enteriditis, Campylobacter jejuni, Staphlococcus aureus, Bacillus cereus, Vibrio parahaemolyticus, Clostridium botulinum, Listeria monocytogenes and Clostridium perfringens. Other microbial agents are presented, but in briefer form. Mycotoxicoses are caused by ingestion of poisonous metabolites (mycotoxins) produced by fungi in foods; for example, Aspergillus flavus produces aflatoxin and Fusarium species produce fumonisin. Viral food poisoning occurs by consumption of food containing viruses, such as infective hepatitis A, rotavirus and small round structural viruses. These organisms do not replicate in the food, but are carried into the intestinal tract. Animal toxins are primarily of marine origin and animal parasites carried by pork, beef and fish.
Another section, presenting a thorough review of food spoilage, discusses a variety of causes including insect damage, physical injury, indigenous enzymes, chemical spoilage such as rancidity and the activity of microorganisms—bacteria, yeasts and molds. The shelf life of food depends on all of these factors and the rate of deterioration. All foods—meats and cured meats, fish and shellfish, dairy products, eggs, vegetables and fruits, cereal-based products, beer and wine, sauerkraut—are affected. Processing methods, such as canning, freezing, dehydrating and irradiating, contribute to food preservation, but spoilage occurs when flaws exist in the application of the methods.
To ensure food production meets statutory, corporate and consumer standards and the quality of incoming materials and ingredients from the suppliers, process control and line sanitation are all maintained, microbiological testing must be meticulously carried out. Needed analyses include estimation of the number of microorganisms and the number of indicator organisms, along with the number of food spoilage organisms, examining for food poisoning or food-borne pathogenic microorganisms and examination of the metabolic products of microorganisms. The chapter on microbiological examining methods, placing emphasis on sampling plans and techniques, sample treatment and examination methods used, is very thorough.
Two chapters discuss in detail factory design, layout and construction. To prevent many of the problems older plants have, the proper design of new plants should prevent problems and enhance processing quality. Of course remodeling of older plants can benefit the quality of production. In addition, design of equipment should account for ease of cleaning, maintenance and inspection. The equipment should protect the food from both external and internal contamination while still performing the function for which it was designed.
A chapter describes Hazard Analysis Critical Control Point (HACCP) to eliminate the risks of food consumption and reduce the incidence of food poisoning outbreaks. Total Quality Management (TQM) is being added as well as the integration of HACCP with Quality Assurance programs (ISO 9000) to enhance food safety. Quality Control was the term used previously to oversee the controllable factors that determined the quality of the product up to the point it was delivered to the consumer. Quality Assurance has expanded this effort to include evaluation of raw material and final products, design of the factory, process line layout, design of the machinery, packaging, storage and distribution. HACCP is an approach to hygienic food production by prevention of problems, using relative risks for hazards, but does not cover the quality of the product. Monitoring and verification procedures are then established to maintain the production of a hygienically acceptable product by controlling the key steps in the production process where the hazards have been identified. Implementation of a HACCP plan (seven principles) provides a mechanism to ensure that product safety is continuously achieved. Inclusion of TQM is a continual activity led by management whereby everyone accepts personal responsibility for safety and quality in providing a uniform-quality product. All food companies need to embrace these activities.
The operations of cleaning and disinfection are essential parts of food production, and the efficiency with which these operations are performed greatly affects final product quality. The most effective sanitation systems cannot overcome basic deficiencies in equipment and factory design, and if design faults exist sanitation cannot be achieved. Cleaning depends on the nature of the soil or contaminant to be removed, the type of surface to be cleaned, the materials used for cleaning, the degree of water hardness and the standard of cleanliness required.
To achieve successful sanitation, personnel must be trained in hygiene practices, including hand washing, use of bactericidal soaps and use of gloves. Outdoor clothing and jewelry must be left in lockers; hair must be restrained and habits like smoking restricted. Pre-employment medical examinations and health monitoring of employees prevents transmission of communicable diseases. Education of employees about the need for hygiene practices assures better compliance.
World-wide food safety programs and legislative controls are converging. Although differences remain, common standards and methods of analysis are being implemented. Yet microbiological standards continue to vary significantly for specific foods depending on the country of origin.
Everyone involved in sanitation practices, HACCP programs and ISO 9000 implementation should read this book from cover to cover. It reads easily and contains exceptional information on food hygiene and HACCP practices. Students interested in QC/QA and food safety practices should also read this book. It will give readers an overall perspective of what needs to be achieved, how to do it and how to assess the result.
Microbial Foodborne Diseases: Mechanism of Pathogenesis and Toxin Synthesis. Jeffrey W. Cary, John E. Linz and Deepak Bhatnagar. Lancaster, PA: Technomic Publishing Company, 2000.
This volume is a commendable update on foodborne diseases written by well respected researchers in microbiology and pathogenesis. The comprehensive presentation is a logical progression of material, timely in the information included. The text is divided into five sections, containing 16 chapters, covering bacterial foodborne pathogens, toxigenic fungi and marine dinoflagellates, protozoan pathogens and viral and virus-like foodborne pathogens. Importantly, it covers the entire field in a systematic review of their hazards, the molecular basis of the biosynthesis of toxins and their mechanisms of action and discusses intervention procedures. The knowledge derived from these studies provides the means by which we can successfully intervene and assure a safer food supply.
Section I contains six chapters on Gram Negative Foodborne Bacterial Pathogens, discussing the molecular biology of Salmonella pathogenesis, Shigella infections, molecular pathogenesis of Escherichia coli infections, virulence determinants of Yersinia enterocolitica, molecular pathogenesis of Vibrio infections, and molecular mechanisms governing Campylobacter pathogenicity. Each chapter contains an in depth coverage of molecular mechanisms in toxin production and pathogenicity. Brief highlights of these chapters are provided for the interested reader.
Salmonellae are successful pathogens infecting animals and humans. Ubiquity in the environment and the ability to colonize in food animals make diseases caused by Salmonellae one of most important and difficult to eradicate. Molecular studies have demonstrated sophisticated strategies used by Salmonellae to engage host cell functions to promote bacterial uptake, inflammation and fluid secretions. Bacterial proteins responsible for enteropathogenesis as well as their host targets are being characterized. Another advance is the molecular genetics of Salmonella virulence. Diverse Salmonella-specific DNA regions contain essential virulence functions for adherence, invasion, elicitation of enteropathogenesis, intramacrophage survival and intracellular proliferation. A variety of interactions exist between Salmonellae and host eukaryotic cells. Different cell types stimulate varied responses in the invading bacteria, resulting in different protein synthesis affecting pathogenesis.
Pathogenic mechanisms of Shigella infection and the role played by the host, in particular by the immune system, is poorly understood. Immunity against LPS is considered protective, but it is not known to what extent the immune response engendered by Shigella infection contributes to tissue destruction and to what extent it is protective. Importantly, blocking inflammatory mediators during Shigellosis in animal models has been successful, but there are no data for the human condition. The most appealing alternative is to prevent disease altogether. Creation of a safe, cheap vaccine against the three major Shigella strains (S. flexneri 2a, S. sonnei and S. dysenteriae 1) could reduce morbidity by 70% to 80%.
The ability of E. coli to cause disease is also due to the presence of virulence genes that define pathotypes of the species. Commonly, E. coli pathogenesis includes the presence of mobile genetic elements such as plasmids, pathogenicity islands and bacteriophages, which encode virulence factors. The ability to produce pili, which are often involved in adherence to host tissues, and the expression of extracellular effector molecules, such as enterotoxins, hemolysin or proteins secreted through type III secretion systems, affect host functions.
Heating of seafood is the only effective means of preventing infections by the non-cholera Vibrio spp., whose contamination in the environment is not due to human waste. Genetic determination of V. cholerae virulence mechanisms may lead to development of a safe and efficacious vaccine. Need exists to use genetic tools to characterize virulence in the pathogenesis, such as a lack of markers for the Vibrio spp., which are required for discriminatory tests.
Section II contains three chapters on Gram Positive Foodborne Bacterial Pathogens also describing the action, genetics and synthesis of Clostridium perfringens enterotoxin, the mechanism of toxin synthesis and pathogenesis of Clostridium botulinum, and the determinants of pathogenesis of Listeria monocytogenes. Brief highlights of these chapters are provided for the interested reader.
C. perfringens type A food poisoning is the second most common form of food poisoning in the US. This illness is most serious for the elderly and debilitated. C. perfringens is widespread in both soil and the gastrointestinal tract of humans and animals, providing ample opportunity to contaminate food. C. perfringens can grow at up to 50 C and has the ability to form heat-resistant endospores. Rapid doubling time allows easy food contamination and C. perfringens foodborne disease. C. perfringens enterotoxin (CPE) is a protein toxin, highly active on human gastrointestinal tract. CPE is considered the virulence factor responsible for the gastrointestinal symptoms of C. perfringens type A food poisoning. Interestingly the CPE gene is only present in less than 5% of the C. perfringens population.
C. botulinum produces seven distinct neurotoxins (ATX) types A to G. The ATX inhibit release of acetylcholine at the neuromuscular junctions and synapses, and cause botulism in humans and animals. ATXs bind to other components forming complexes called progenitor toxins. Genes for toxins A to G have been cloned. The ATX toxins are zinc-binding proteins with endopeptidase activity. The mechanism of pathogenesis and toxin synthesis in C. botulinum has been described. The ATXs and the progenitor toxins’ genes have been sequenced and cloned.
L. monocytogenes serotype 4b is the most common form of Listeriosis associated with pathogenesis. The most susceptible population groups include pregnant women, newborn infants, elderly and immunocompromised. The organism is found in soil and decaying vegetation. It grows well at refrigeration temperatures, making cold-stored foods like dairy products prime targets. L. monocytogenes is a faculative, intracellular pathogen. Successful adaptions include invasion of host cells, intracellular multiplication and direct cell-to-cell spread. Surface components influence uptake. Pathogenesis is affected by genetic mechanisms of virulence including determinants such as hemolysin, LLO, Prfa, internalin and the like. These factors appear to be shared by all L. monocytogenes species. Distinct lineages within the species may differ in their genetic and antigenetic endowment and expression of important properties related to pathogenesis and/or adaptive physiology.
Section III contains two chapters on fungal toxins (Aflatoxin and Fusarium) and one chapter on marine toxins (PSP). Aspergillus flavus and A. parasiticus are fungal contaminants found in corn, peanuts, cottonseed and tree nuts. Aflatoxins continue to receive attention from the food industry because aflatoxin B1 is extremely toxic to man and animal, but also because they are the most carcinogenic of all known natural compounds in several animal models. AB1 is a hepatocarcinogen in rats and trout at 1 µg/kg. The most prevalent aflatoxins produced by A. flavus are aflatoxin B1 and B2, while A. parasiticus produces two additional aflatoxins G1 and G2. The incidence of the aflatoxin in food and feed is ubiquitous throughout the world, and both therefore are carefully regulated. A very comprehensive review of the molecular biology of aflatoxin biosynthesis by Aspergillus is presented. Knowledge about gene regulation is essential to prevent AF or ST contamination of food or feed crops. Natural plant extracts may be effective, such as caffeine, black pepper extract and neem extracts, as inhibitors of AF synthesis and of formation of peroxidation products from plant lipids which stimulate AF synthesis. Oils of cassia, clove and cinnamon inhibit fungal growth.
Fusarium is a genus of filamentous fungi that include plant pathogens, saprophytes and opportunistic human and animal pathogens. Fusarium mycotoxins are found in food and feed. Epidemiological data indicate widespread occurrence around the world and correlation of some diseases to consumption of contaminated food. The harmful effects of fumonisin and trichothecene have been demonstrated in vitro and in several animal species. The chapter examines the toxicity and biosynthesis of trichothecenes and fumonisins production by Fusarium. Trichothecenes are a family of more than 60 sesquiterpenoid compounds sharing the tricyclic trichothecene structure having an epoxide bond. The occurrence of contamination of grains, such as corn, wheat, barley and rye, has cost the agricultural community billions of dollars. Fumonisins are a group of mycotoxins associated with esophageal cancer related to contaminated corn in South Africa. These toxins are aminopolyols with a core structure consisting of a 20-carbon backbone with an amino group at C-2, and one to three hydroxyl groups. The structure mimics sphingosine and may interrupt sphingosine metabolism as part of its toxicity. Fumonisin B1 is the most abundant fumonisin mycotoxin. The toxins cause liver cancer in rats, pulmonary edema in swine, leukoencephalomalacia in equines and kidney and liver damage in rats and sheep. In the rat, toxicity may be expressed through apoptosis. Genetic mapping and molecular characterization of genes and their location as related to toxin biosynthesis are ongoing. Further studies should be directed to reduce production of these mycotoxins in the grain crops or increase the plant resistance to growth of these fungi. Such intervention should decrease the mechanisms by which these toxins induce disease in humans and animals.
Section IV provides two chapters on parasitic protozoa (Toxoplasma, Entamoeba histolytica and Cryptosporidium parvum). These chapters characterize factors governing pathogenicity of protozoan parasites. E. histolytica and C. parvum are pathogenic are pathogenic protozoa transmitted by the fecal-oral route. Infection occurs by ingestion-resistant cyst forms of the parasites found in contaminated food and water. Both parasites require a host to complete their life cycles and do not multiply in the environment alone. Cryptosporidiosis is emerging as a human disease, initially identified as a disease of farm animals. Although seen in Third World countries, it is recognized as one of the leading causes of waterborne disease outbreaks in the United States. The chapter discusses lifecycles, disease, epidemiology, detection, vaccine development and pathogenesis. The health threat is increased by the resistance of the organism to normal food processing and water treatment. In addition, very little is currently known about the molecular determinants of pathogenicity.
Section V concludes with chapters on human caliciviruses and on prion diseases with thorough discussion of the epidemiology and pathogenicity of Norwalk and other human calciviruses. These viruses are very resistant to chlorine levels used in normal water treatment methods, low pH and heat (60 degrees C for 30 minutes). Outbreaks of foodborne disease have been associated with consumption of uncooked and cooked shellfish, ice, bakery products, various salads, cold foods and hot foods, resulting in viral gastroenteritis. Water contamination is serious in private wells, small communities, cruise ships and ground water contamination. Needs are many, including better systems to detect or prevent contamination. To date it remains difficult to cultivate these viruses, thereby hampering studies to discover the mechanisms of viral growth and pathogenicity.
Prion diseases, transmissible spongiform encephalopathies, related to mad cow disease and Creutzfeldt-Jakob disease, have been well publicized. Prions are proteinaceous material suspected as the mechanism of transmission of these diseases from animal to human. This chapter is significant for its discussion of what is known and what is speculated in the molecular biology of these diseases. For example, BSE is transmittable to humans; wild-type mice inoculated with prions from humans suffering from nvCJD developed the disease in the same time period as those inoculated with BSE prions and demonstrated the same lesion profiles; human PrP can be converted by bovine PrP (BSE) into the proteinaceous K-resistant state. Several models are discussed to identify differences between virus or nucleic acid participating replication and the proteinaceous replication model. None have been totally eliminated.
The book is highly recommended. Everyone interested in microbial pathogens, toxins, interventions and food safety should read this volume. The water and food industries must be aware of these developing problems and work with scientists to minimize and eliminate human exposure. The authors and the editors have produced an excellent reference volume that could easily be used for an upper division or graduate course on mechanisms of foodborne pathogenesis. The book is well balanced in style and scientific content and reads very well.
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