Food Microbiology

FoodMicrobiologySven-Olof EnforsKTH - BiotechnologyStockholm 2008S.-O. Enfors: Food microbiology

ContentChapt 1 Introduction...................................................................................1Chapt 2. The ecological basis of food spoilage ...........................................52.1 The microflora ........................................................................52.2 The physico-chemical properties .............................................82.3 Chemical reactions ................................................................15Chapt 3. Spoilage of different types of food .............................................22Chapt 4. Foodborne pathogens..................................................................384.1 Microbial food intoxications .................................................394.2 Foodborne microbial infections.............................................44Chapt 5. Food preservation.......................................................................515.1 Heat sterilisation and pasteurisation ......................................515.2 Chemical preservatives..........................................................655.3 Classification of preserved food ............................................65Chapt 6 Fermented foods .........................................................................736.1 Beer brewing.........................................................................746.2 Fermented milk products......................................................816.3 Fermented meat products .....................................................886.4 Fermented vegetables...........................................................89S.-O. Enfors: Food microbiology

1Chap 1IntroductionLiving organisms are usually classified as animals, plants, algae, protozoa,bacteria, archae or viruses. All viruses, archae, bacteria, and protozoa plus theunicellular algae and some fungi, so called micro-fungi, are collectively calledmicroorganisms. The microfungi can be further divided into yeast and molds, aclassification that is based on the cell morphology. Based on DNA analysis, thegroup previously called bacteria is further divided into eubacteria and archaeand today the word bacteria is usually used as synonym to eubacteria.Most microorganisms that we encounter in the normal spoilage of food belongto the eubacteria, here called “bacteria”, yeasts and molds. When it comes tofoodborne diseases, also viruses, some protozoa and archae, i.e. the “blue-greenalgae”, are involved.A full species name is composed of two parts: the genus name plus thespecification defining the species within that genus. sometimes these genera aregrouped into families. This is illustrated in Table 1.1. Note that the genus nameis spelt with leading capital letter, while the species name is spelled with lowercase letters: Eschericia coli, Penicillium chrysogenum. The family, genus, andspecies names should always be written with italic letters. It is common in foodmicrobiology literature that the full species name is not used since many specieswithin the same genus are discussed. Then, Bacillus sp. means one not definedBacillus species and Salmonella spp. means several not defined Salmonellaspecies.Table 1.1. Examples of family names, genus names and species namesFamily Genus SpeciesEnterobacteriacae Escherichia Escherichia coliSalmonellaSalmonella typhimuriumSalmonella entericaBacillacae Bacillus Bacillus subtilisBacillus cereusBacillus anthracisClostridiumClostridium botulinumBergey’s Manual of Determinative Bacteriology divides bacteria into 35 groups. Groups,families, and genera which are most relevant in food microbiology are listed in Table 2.1.In bacterial classification, the cell morphology, the relation to oxygen, and theGram staining reaction are important parameters. Most commonmorphological types are rods, cocci (spheric cells), and vibrioforms (short bentrods). The Gram reaction gives information about the cell envelope. Gramnegative cells have an outer membrane outside the cell wall which prevents thestaining. Obligate aerobes require molecular oxygen for their energymetabolism (aerobic respiration). Anaerobes have an alternative energymetabolism that does not need oxygen. It may either be anaerobic respirationS.-O. Enfors: Food microbiology

Introduction 2(with e.g. nitrate as electron acceptor) or fermentation. Oxygen is often toxicfor anaerobic cells. Facultative anaerobic cells use oxygen and aerobicmetabolism if oxygen is available but switch to anaerobic metabolism inabsence of oxygen. Microaerophilic cells require low concentrations ofoxygen, while normal air contact is inhibitory. Lactic acid bacteria (e.g.Lactobacillus and Lactococcus) have an obligately anaerobic metabolism butare still resistant to oxygen.Table 2.1. Some of the bacterial groups (according to Bergey’s Manual of DeterminativeBacteriology) which are commonly encountered in food microbiology.Group DescriptionFood related organismsnr2 Gram-neg., aerobic, mobile, vibrioformedCampylobacter4 Gran-neg., aerobic rods or cocci Pseudomonas, Shewanella, Legionella5 Gram-neg., facultatively anaerobicrodsFamily Enterobacteriacae(e.g. Escherichia, Enterobacter,Salmonella, Shigella, Yersinia, Erwinia)Vibrio17 Gram-pos. cocci Staphylococcus, Streptococcus,Lactococcus, Enterococcus, Micrococcus,Leuconostoc18 Gram-pos endospore formersaerobic or facultatively anaerobic: Bacillusobligate anaerobes:Clostridium19 Gram-pos, non-sporulating rods LactobacillusBrochothrixListeriaThere is a number of often used group names of microorganisms. Some foodrelated examples are:”Gram-negative psychrotrophic rods”: This includes the genera Pseudomonas,Achromobacter, Alcaligenes, Acinetobacter, and Flavobacterium.”Lactic acid bacteria” (LAB) includes the food related genera Lactobacillus,Lactococcus, Pediococcus och Leuconostoc.”Coliform bacteria” is not synonymous to E. coli but includes Escherichia coliand Enterobacter.S.-O. Enfors: Food microbiology

Introduction 3A special problem with the microbial taxonomy is that the names often are “datedependent” due to repeated re-classification of species. One example is thelactic acid bacteria which previously were called Streptococcus lactis,Streptococcus cremoris a.o. These so called “lactic streptococci” are nowreferred to a new genus and galled Lactococcus lactis, Lactococcus cremorisetc. Other previous Streptococcus spp. wich are associated with the intestinesare now called Enterococcus, while yet another group of the previousStreptococcus genus remain as Streptococcus. When it comes to pathogenicorganisms a further classification problem is that only some strains of a certainspecies may be pathogenic while other strains are harmless. An example isEscherichia coli to which species the feared EHEC (enterohaemorrhagic E. coli)belong. In such cases immunological or DNA analyses are required for properclassification.Streptococcus is a genus with species of very different impact for humans. Someof todays Lactococcus and Enterococcus were previously classified asStreptococcus. They were then referred to as the lactic group and the entericgroup of the streptococci, respectively. A classification of the old streptococciaccording to current nomenclature is:1. Lactococci (Lactococcus lacits, L. cremoris a.o.). These organisms are oftenused for fermentation of food.2. Enterococci (Enterococcus faecalis, E. faecium a.o.) are in most cases notpathogenic, but certain strains have been reported to cause serious infections.Such contradictions are due to the limitation in the current nomenclature which isbased on phenotypic properties. These organisms are common in the intestinalflora. The presence of enterococci in food is not considered to be a health risk perse, but it is used as an indication of bad hygiene and that constitutes a risk, sinceother organisms of faecal origin like Salmonella may be present. For this reasonenterococci (together with the coliforms) are called indicator bacteria.3. Hemolytic streptococci. There are two types of hemolytic streptococci, andthese organisms remain in the genus Streptococcus: α-hemolytic and ß-hemolytic.The α-hemolytic streptococci are named the viridans group and they are commonon mucous membranes in the mouth and respiratory tract and on the teeth. The ß-hemolytic streptococci are named the pyogenes group and among them there areserious pathogens involved in several diseases and wound infections. α-hemolyticorganisms produce a greenish discolorisation zone around the colonies on bloodagar while ß-hemolytic cells produce a clear zone.Lactic acid fermentation is to a large extent also employed for production offood, namely some of the fermented foods: cheese, yoghurt, fermentedsausages, and fermented vegetables like sauerkraut, pickles, olives, and others.S.-O. Enfors: Food microbiology

5Chapter 2The ecological basis of food spoilage2.1 The microfloraFood consists to a large extent of cells from plants or animals (meat, fish,fruits, vegetables) and biological material with this origin (milk, juice, fat,starch etc). When discussing the shelf life of food it must be done from anecological viewpoint. All biological material in Nature is degraded to simplemolecular components, eventually down to inorganic components. This iscalled mineralization and it is a integrated part of the carbon and nitrogencycles in Nature (Fig 2.1) which is a prerequisite for life on Earth. If theprocess is interrupted all nutrients would eventually be bound in deadbiological material. The circumstance that we select some part of thisbiological material for food purpose does not change the natural fate of thefood, namely microbial degradation. However, it means that our interest in along shelf-life of food is in conflict with the natural processes.CO 2 + N 2LightAnimals PlantsOrganicmateriaArchaeBacteriaFungiAlgaeProtozoaFig 2.1. Microorganisms,especially bacteria and fungi,account for the mainrecirculation of carbon andnitrogen to the atmospherefrom where it is adsorbed forgeneration of plants whichconstitute the original sourceof food.Dead organismsThe degradation of biological material is mainly catalysed by microorgansims,which together carry an enormously diversified metabolic capacity. This isillustrated in fig 2.2 which summarises the main paths of the biological energymetabolism.All energy is generated, with exception of photosynthesis, by oxidation(combustion) of reduced substances (energy sources). Higher organisms likeanimals and also some microorganisms make this by oxidation of reducedcarbon compounds, e.g. sugars. These compounds are oxidised in many stepsin which oxidised co-enzymes (e.g. NAD + ) constitute the oxidant, which thenbecomes reduced (e.g. NADH). These co-enzymes must be re-oxidised andeventually molecular oxygen in the air is used as the ultimate oxidant for this inthe respiration. The reduced compound or energy source is called electrondonor and the ultimate oxidant (oxygen) is called electron acceptor in thisS.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 6energy metabolism. The electron donor in this case ends up as carbon dioxidewhile the electron acceptor oxygen is reduced to water. This respiration processis also coupled to phosphorylation of ADP to ATP.Re-oxidation of co-enzymesS 2-ATPC redElectron donors (energy source)NH 3 S 2- Fe 2+H 2NAD +EthanolN 2H 2 ONAD +NADHO 2-Pyruvate NO 3SO 2- 4Electron acceptorsADPNADHCO 2 NO- 3 SO 24 - Fe 3+H 2 OFermentationRespirationFig 2.2. Summary of different types of energy metabolism. Common principle is that energyis derived by oxidation in several steps of a reduced compound (C, N, S, Fe, H 2 a.o.) bymeans of co-enzymes, here represented by NAD + . Re-oxidation of the reduced co-enzymecan be achieved with respiration, in which molecular oxygen, nitrate or nitrite, and sulphateare common oxidants (electron acceptors). An alternative to respiration is fermentation, inwhich a partially oxidised carbon compound from the metabolic path (e.g. pyruvate) is usedas electron acceptor for re-oxidation of the co-enzyme and then becomes reduced, in thiscase to ethanol.When oxygen is used as electron acceptor the process is called aerobicrespiration, while the use of alternative electron acceptors like nitrate, nitrite,sulphate etc. is called anaerobic respiration. Many facultatively anaerobicbacteria use oxygen if it is available but can switch to anaerobic respiration(e.g. nitrate respiration) or fermentative metabolism in absence of molecularoxygen. Of these respiration types, it is mainly the aerobic respiration andnitrate respiration that take place in food.Some microorganisms can use other reduced compounds than carboncompounds as energy source. Some examples are ammonia and nitrite whichare oxidised by nitrifying bacteria, and sulphide, ferrous iron, and hydrogengas. These reactions are very important in the environment but seem to playlittle role in the handling of food.One alternative type of energy metabolism which is common inmicroorganisms growing in food is fermentation, in which a reducedintermediate is used as electron acceptor in the re-oxidation of reduced coenzymes.There is a number of different fermentative metabolic pathways,S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 7named according to the dominating products, like ethanol fermentation, lacticacid fermentation, mixed-acid fermentation etc. Some of these reactions aredetrimental for the food while others are utilised in processing of food. Themain fermentative pathways and their role in food microbiology are furtherdiscussed in the section on degradation of carbohydrates.To increase the shelf-life of food means that the progress of the naturaldegradation path must be prevented or delayed. However, food spoilage is notexclusively a matter of microbial degradation. Other spoilage reactions aredehydration, oxidation of fat, and endogenous metabolism (over-maturation offruits and vegetables), but microbial metabolism is the most important type ofreaction that reduces the quality of food during storage.The common microbial food spoilage usually does not make the food unsafe oreven reduce its nutritional value, but it makes the product unpalatable. Thenegative perception of food which is severely contaminated by microorganismsis an important defence mechanisms for us, since the risk associated witheating food increases considerably if it is spoilt by microbial metabolism. Thisis due to the risk that some organisms among the spoilage flora may bepathogens.It is impossible to give a simple and yet comprehensive description of themicrobial spoilage of food since this is a very diversified process. What is saidin this booklet must be seen as typical and common cases, to avoid the use ofvery large lists of microbial names. When, for instance, it is stated below thatthe activities of Pseudomonas spp. limits the shelf-life of refrigerated freshmeat and fish, it means that most investigations - but not all- show thatPseudomonas species dominate the spoilage flora but there are usually anumber of other species involved, usually in the group "psychrotrophic, Gramnegativerods". Another problem is that it is not always sure that thedominating microflora is responsible for the main spoilage reactions. Anexample is that it may require 10 times more Achromobacter cells thanShewanella cells to make fresh fish unacceptable in taste. Another example isthe lactic acid bacteria of the homo-fermentative type which have a relativelylow impact on the spoilage due to the domination of lactic acid in the metabolicproducts.Most food raw materials have a primary flora of microorganisms whichorigins from the production environment. During the continuing processing ofthe raw material and additional contamination (or secondary) flora infects thefood. It may come from the air, especially from dust in the air, from processwater, process equipment, or from humans which handle the food. During thesubsequent storage of the product the different species develop differentlydepending on the environment. The primary plus initial contamination floraS.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 8usually is in the order of 10 3 cells/cm 2 of solid foodstuff if the quality is verygood (see table 2.1). Depending on the conditions for growth some of thesespecies will grow exponentially (see Fig 2.3) up to concentrations above 10 7 /cm 2 (or per gram). The finally dominating microflora may origin from theprimary or the contamination microflora. When the number of cells exceed 10 7to 10 8 cells/cm 2 (or per gram) the product usually develops bad smell and themicroflora is then called the spoilage flora. It is the nutritional (formicroorganisms) properties of the food and the environment (temperature,water activity, pH etc.) that determine which species will dominate thespoilage flora, their metabolic products and how fast this spoilage process willproceed. In the sections below the environmental parameters will be discussedand in Chapter 2.2 the most important chemical reactions of food spoilage arepresented.Table 2.1 Typical size of different food microfloras at goodproduction hygieneProductInternal tissues of healthy animals 0Plant surfacesFish skinEgg shellMilkMeatFish filletSpoilage flora on most food typesMicrobial concentrationPrimary flora ≈ 10 3 cells/ cm 2Contamination flora ≈ 10 3 cells / mlContamination flora ≈ 10 3 cells / cm 2≈ 10 7 - 108cells / cm 2 or gram2.2 The physico-chemical propertiesThe possibility of the food to serve as a substrate for microbial growth dependson a number of physical and chemical properties:- Temperature- Water activity (a w )- pH and buffer capacity- Oxygen concentration and transfer- Mechanical barriers- Metabolisable energy sources- Metabolisable nitrogen sources- Chemical inhibitorsTemperature. The temperature influences of course the rate of growth, andthereby the shelf-life of the product. But it has also an impact on selection ofspecies in the microflora. This is probably the explanation why reduction oftemperature in the refrigeration range (0-8°C) has such a dramatic influence onthe growth rate, as demonstrated by experimental data Fig 2.3. The organismsS.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 9growing at 20°C have an initial generation time of about 4.8 h, while thegeneration time at 0°C is about 25 h, and represents psycrotrophic organisms.10 Log(cfu)920°C10°C8°C4°C70°C5310Time (days)015Fig 2.3. Influence of temperature on the total bacterial count (colony forming units, cfu)on fresh meat. The dotted line indicate the typical level of spoilage. Note that the growthinitially is exponential.Microorganisms are usually classified in four groups according to theirrelationship to temperature. Fig 2.4 illustrates this. In general, the mesophileshave the highest maximum growth rate and an optimum temperature in therange of 30-40 °C .Relative growth ratePsychrotrophesPsychrophilesMesophilesThermophiles0 10 20 30 40 50 60 °CFig 2.4. Schematic illustration of the temperature dependence of the growth rate of differentclasses of microorganisms. There are no general and exact limits for the temperature ranges.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 10The psychrophiles have the lowest maximum growth rate, but can grow quitefast at refrigerator temperature. Thermophiles have an optimum above 40°Cand some can grow even above 100°C. The psychrotrophic organismsconstitute an important group in food microbiology. They grow well in the 20-35 °C range like the mesophiles but they can also grow relatively fast atrefrigerator temperature.The growth rate of microorganisms is expressed either with the generation time(t g , h) or with the specific growth rate constant (µ, h -1 ). The generation time is thetime needed to double the amount of cells. The specific growth rate expresses therate of cell formation per cell. The correlation between these parameters can bederived from a mass balance of the cell number:dNdt = µNwhere N is the number of cells, µ (h -1 ) is the specific growth rate and t (h) is time.Integration with N 0 cells at t = 0 and N t cells at time t, gives:ln(N t)ln(N 0) = µtAfter one generation time, t g , the cell number becomes 2N 0 . Insertion of this inthe equation above gives:ln(2N 0)ln(N 0) = µt gfrom which the correlation between generation time and specific growth rate isobtained:ln(2)µ = t ! 0.69gµWater activity (a w ). The water activity is one of the main parameters whichdetermine how fast and by which type of organisms the food is spoilt. Thewater activity of food can be determined as the water vapour pressure (p H2O ) ina w = p H 2 Op H2 O*a closed vessel in which the product is enclosed in relation to the water vapourpressure of purewater (p H2O *):For a water solution with low molecular weight compounds (e.g. salt or sugar)the water activity is approximately:n wa w!n w+ n SS.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 11wheren w = number of moles watern s = number of moles of dissolved moleculesSome common food components that reduce the water activity are:- Ions (e.g. salts)- Dissolved molecules (e.g. sugars)- Hydrophilic colloids (e.g. starch)- IceThe water activity is a measure of the availability of the water for themicroorganisms. It is not only the water concentration that determines thewater activity but also the capacity of the material to bind water. This isillustrated in Fig 2.5 which shows sorption isotherms for some materials withdifferent water binding capacity. Cellulose get a relatively high water activityand starch a lower water activity at the same water concentration.Water concentration (%)Fruit3020StarchMeat10Cellulose00 0.3 0.6 0.9Water activityRelative rateRelreaktionshastighetLipidoxidationLipolysisProteolysisFungi0 0.2 0.4 0.6 0.8 1Water activityBacteriaFig 2.5. Sorption isotherms for differentmaterials show that a w is not the same aswater concentrationFig 2.6. Schematic view of how thea w influences the rate of enzymereactions and microbial growth.Most biochemical reaction rates decline with declining water activity.However, the sensitivity to reduced water activity varies, as illustrated in Fig2.6. Among microorganisms, molds and yeasts are generally more resistant tolow water activity and many enzymes retain their activity at even lower wateractivity. But there are many exceptions to this rule. Three types ofmicroorganisms prefer reduced water activity. These are osmophilic (sugarpreferring) yeasts, xerophilic (drought preferring) fungi, and halophilic (saltpreferring) bacteria. These organisms not only grow faster than most otherorganisms at lower water activity, but they also prefer a reduced water activity.See further in Table 2.2.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 12Table 2.2 Examples of typical minimum water activity for growth of somemicroorganisms and corresponding a w in some foods.Organism Min a w Food examples Food a wMilk, fish, meat 0.99Pseudomonas 0.97E. coli 0.96 Sausage, 7% salt 0.96Clostridium 0.95Brochothrix0.94thermosphactaBacillus 0.93 Ham, 12% salt 0.93Lactobacillus 0.93StreptococcusLactococcusMicrococcus0.93Salmonella 0.91 Jam, 50% socker 0.91Hard cheese, breadHerring, 20% salt 0.87Staphylococcus 0.86Yeasts in general 0.85Molds in general 0.80Halophilic bacteria 0.75Grains w.10% water 0.7Xerophilic molds 0.65Osmophilic yeasts 0.60 Dried fruits, 15% water 0.6NoneDry milk, soups etc.Dry breadHalophilic = salt preferring; xerophilic = drought preferring;osmophilic = preferring high osmotic pressure (of sugar).< 0.5The water activity of food has a large impact on the rate of spoilage but also onthe type of spoilage since it exerts a selection pressure on the microflora. Manyof the common food spoiling microorganisms are very sensitive to reducedwater activity and the growth rate of these declines rapidly when the wateractivity drops below the optimum, which is close to 1 for Pseudomonas andEnterobacteriacae. Many conclusions can be drawn from Table 2.2.Pseudomonas, which dominate the spoilage of refrigerated fresh meat and fishdoes not create problems in sausages and salted herrings or if meat and fish isdried. Such products get a spoilage flora of more low-a w resistant organismslike lactic acid bacteria, molds and yeasts. The table also explains why moldsare the main problem during storage of cheese and bread, and why driedproducts like flour, grains, dry milk are not attacked by microorganisms at all,provided they are stored in a dry environment so they do not absorb water. It isalso obvious that the toxin producing Staphylococcus, which are commonlypresent on human hands, constitute a threat at "smörgåsbord" and other buffets.Note that the figures in Table 2.2 are collected from different sources. Theactual minimum a w for and organism depends on other parameters like pH,S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 13temperature, and nutritional conditions. Thus, such data are only approximateand indicative of relative sensitivities.pH is another parameter with large impact for the shelf-life of food. The pHinfluences both the growth rate and the type of organisms that will dominateduring storage. Most food products have pH below 7 (Table 2.3) and mostfood spoiling bacteria require a relatively neutral pH (Table 2.4), with theexception lactic acid bacteria wich grow well down to a pH in the range 4-5.In Nature there are many examples of bacteria that can grow at very low andvery high pH values, but these organisms are not relevant in foodmicrobiology. Comparing these tables give one reason why fruits and manyvegetables mainly are degraded by molds and sometimes yeasts.Table 2.3. Typical pH-values of common food productsShrimps 7Cabbage 5.5Fish 6.7 Potatoes 5.5Corn 6-7 Tomatoes 4.2Milk 6.5 Orange juice 4Melon 6.5 Yoghurt 3.5Butter 6.2 Apples ≈3Meat 5.1-6.4 Lemon ≈2Cheese 5.9Oysters 5-6Table 2.4. Generalised picture of pH ranges for microbial growthpH range pH optimumMost food spoilage bacteria 6 - 9 7±1Lactic acid bacteria 4-7Molds 2 - 11 5±1Yeasts 2.5 - 7 4-5Oxygen availability and the diffusion rate of oxygen are important parametersthat influence the type of metabolism. The rate of growth may be slower inanaerobic than in aerobic environments but on the other hand is the anaerobicmetabolism associated with much more detrimental products for the shelf-life.An exception to this is the lactic acid bacteria which have anaerobicmetabolism but usually produce less ill-smelling compounds than most otheranaerobic organisms. Anaerobic conditions are a prerequisite for growth of thedangerous pathogen Clostridium botulinum, and therefore special precautionsmust be taken when storing some types of food under anaerobic conditions.The mechanical structure may be important for the shelf-life of food. Onwhole meat bacteria grow only on the contaminated surface, where they dwellS.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 14on the exudate, i.e. the glucose and amino acid rich liquid which leaks fromdamaged cells and blood vessels. If the meat is minced this surface andexudate increase enormously which leads to much higher microbial activityand growth in the inner anaerobic parts of the minced meat. Fruits andvegetables are protected from microorganisms by the outer shell or skin and bythe gelatine-like pectins which cements adjoining plant cells together. Outsidethe skin/shell the water activity is low and there is a lack of nutrients forgrowth of the contaminating microflora. But if the product is mechanicallydamaged or if the organism can produce pectinases the nutrients becomeavailable and the spoilage rate increases. It is mainly molds that producepectinases, and this, together with the often low pH of these products, explainswhy this type of food often is spoilt by molds. Yeasts, which also grow well atlow pH, often come as a second infection after the initial mold attack. Erwiniais one of few bacterial genera with pectinase producing species which attackplant material.Antimicrobial substances. Many food raw materials, especially vegetables andother food with plant origin, contain antimicrobial compounds which hamperthe microbial growth. Some examples are listed in Table 2.5.Many microorganisms produce antimicrobial substances (antibiotics) and infood there is often growth of lactic acid bacteria, some of which produceantibiotics (Table 2.6). Nisin is a polypeptide antibiotic naturally produced infresh (unpasteurised) milk by Lactococcus lactis which belong to the normalflora transmitted during milking. Other antibiotics, like acidocin B and reuterinare mainly produced in processed milk if it is inoculated with the producingorganism.Table 2.5. Some examples of naturally occurring antimicrobial substances.FoodInibitorHorseradishAllyl isothiocyanateOnion and garlicAllicin and diallylthiosulphinic acidTomatoTomatinRadishRaphaninLingonberryBensoic acidOreganoEteric oilsTable 2.6. Antibiotic substances produced by lactic acid bacteriaAntibioticOrganismNisin (in milk)Lactococcus lactisSalvaricinLactococcus. salvaricusAcidocin B (fermented milk) Lactobacillus acidophilusReuterin (fermented milk) Lactobacillus reuteriiS.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 15Some definitions of antimicrobial compoundsAntibioticsProbioticsPrebioticsBacteriocinesMicrobial product with an antimicrobial (bactericide/fungicide or bacteristatic/fungistatic) activity and whichhave low toxicity to humans. If the latter is not added tothe definition most mycotoxins would also be classified asantibiotics.Microbial cultures, mainly lactic acid bacteria, which areconsumed for stabilisation of the intestinal microflora ofhumans or animals. They are believed to act byestablishing on the intestinal mucouse membrane andprevent, possibly by production of antibiotics, the growthof other disturbing organisms.Components (oligosaccharides) in the food that are notdigested in the intestines but are assumed to promote thebeneficial microflora.Bacterial proteins or peptides with bactericidal effectmainly on related species and strains.bactericide = bacteria killing; fungicide = fungi killing;bacteri/fungi-static = inhibiting growth of bacteria/fungi.2. 3 The chemical reactionsThe most important chemical reactions involving food components duringmicrobial spoilage of food are:- Degradation of N- compounds- Degradation of fat- Degradation of carbohydrates- Pectin hydrolysisDegradation of nitrogen compoundsThe dominating and usually the first reaction is oxidative deamination ofamino acids:amino acid + O 2deaminaseNH 3 + organic acidThis reaction is assumed to be the dominating spoilage reaction in refrigeratedfresh meat and fish. The amino acid is then used as energy source by splittingoff the amino group with an oxidative deaminase, which leaves the organicacid that enters the energy metabolism.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 16Proteolysis. One could expect that proteolysis should be a common spoilagereaction. However, most microorganisms do not secrete proteases and thosewho do, usually do not produce them until there is a lack of nitrogen source.In later stages of spoilage, however, proteases and peptidases may degrade theprotein:proteinasepeptidaseProteins peptides amino acidsMany peptides have strong taste, bitter or sweet, and this sometimescontributes to the spoilage. These reactions are also important for thedevelopment of characteristic tastes of many fermented products.Putrification is a set of anaerobic reactions with amino acids which results in amixture of amines (e.g. cadaverine, putrescine, histamine), organic acids, andstrong-smelling sulfur compounds like mercaptans and hydrogen sulphide:amino acidsAnaerobicmetabolismAminesOrganic acidsS-compoundsIndolMany of these compounds have terrible odour. Cadaverine, putrescine, andhistamine are formed by decarboxylation of lysine, ornithine, and histidine,respectively (Fig 2.6) While cadaverine and putrescine in food probably haveno health impacts, only spoil the food due to the odour, histidine causesintoxication problem since it may induce a serious anaphylactic shock. This isoften associated with microbial activity in histidine rich fishes of mackereltype, e.g. tuna fish.Putrification is typical for microbial degradation of meat and other protein richfoods at higher temperature (> 15°C). Bacillus and Clostridium species maythen grow fast and rapidly make the food toxic, but under refrigerationconditions these organisms are usually not active and under these conditionsthe oxidative deamination spoils the food before the putrification becomesdominating.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 17Fig 2.6. Histamine, cadaverine and other amines are formed by decarboxylation of aminoacids.Reduction of trimethylamine oxide (TMAO). Marine animals may contain highconcentrations of trimethylamine oxide, which is believed to have a function inprotecting proteins from denaturation at low temperatures, high pressure andhigh osmolarity. Certain microorganisms, like Pseudomonas and Shewanella,can utilise TMAO as electron acceptor in anaerobic respiration:CH 3H 3 C - N = OCH 3TMAO-reductaseH 3 C - NCH 3CH 3TMAOTMAThis results in formation of trimetylamin (TMA) which gives a typical "fishy"smelling. TMA can also be formed by enzymatic hydrolysis of lecithin.Degradation of fatWhen fat is degraded it becomes rancid and this rancidification depends onmany different reactions which are not all well known in detail. One attempt ofclassification is shown in Fig 2.7. The hydrolytic rancidification results in freefatty acids (FFA) and glycerol. Our organoleptic tolerance of free fatty acidsdepend on the type of the fatty acids, especially the carbon chain length. Up to15% FFA is said to be acceptable in beef, which has long fatty acids, whileonly up to 2% is acceptable in olive oil. If very short FFA are formed, e.g.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 18butyric acid from butter, only traces of the acids can be accepted. Thehydrolysis can be spontaneous but then at a very low rate, while it may proceedfast if lipolytic enzymes from the foodstuff or from the contaminatingmicroflora are present.Fig 2.7. Different types of rancidification reactions.The oxidative rancidification requires presence of oxygen. Autooxidativerancidification is catalysed by metal ions and is accelerated by light. In thisprocess peroxide radicals (ROO*) are produced and they react with other fattyacids to form instable hydroperoxides (R-OOH) which later on decompose toaldehydes and ketones which give the rancid taste (Fig 2).Fig 2.8. Autooxidation of a fatty acid (RH) results in aldehydes and ketones. Thechain reaction is initiated by a radical (R*) which is produced from the fatty acidunder catalysis of Fe 2+ and other metal ions and light. The radical reacts withmolecular oxygen to form a peroxide radical (ROO*). Antioxidants in food are usedto scavenge the peroxide radical that otherwise continuous the chain reaction byreacting with another fatty acid to produce a new radical (R*) and a hydroperoxide(R-OOH). The hydroperoxide is instable and decomposes to ketones or aldehydes.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 19ß-oxidation is the common metabolic route for degradation of fatty acids andeach cycle results in generation of one acetyl-CoA and a new fatty acid with 2C shorter C-chain (Fig 2.9). Some microorganisms have a side reaction in thelast step of the ß-oxidation cycle, by which very aromatic methyl ketones areformed and may contribute to bad taste (rancidity) of the food.Fig 2.9. Methyl ketones may be formed as by-products in the ß-oxidation of fatty acids.Lipoxydaser are common enzymes in plant and animal tissues and they are alsoproduced by some molds. The enzyme oxidises unsaturated fatty acids withcis-cis 1-4 pentadien configuration to hydroperoxides which decomposespontaneously to ill-tasting aldehydes and ketones. This configuration ispresent in linolic and linolenic acids in plants and in arachidonic acids inanimal tissues. To prevent this type of rancidification during storage somevegetables, e.g. frozen spinach and peas, are heat treated to inactivate the plantenzyme. However, these aldehydes and ketones are not always unwantedproducts in food. They are also important ingredients in certain types ofcheeses (see Chapter 6).Degradation of carbohydratesMicroorganisms growing on food mainly use various sugars as carbon- andenergy source. Under aerobic conditions the energy source is combusted tocarbon dioxide and water but under oxygen limiting or anaerobic conditionsmany species switch to fermentative metabolism which results in variousfermentation products (see Fig 2.10). The most common fermentativepathways are listed in Table 2.7.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 20Table 2.7. Common fermentation typesFermentation typeProductsAlcohol fermentation Ethanol, CO 2Homofermentative lactic acid fermentation Lactic acidHeterofermentative lactic acid fermentation Lactic acid, Acetic acid, Ethanol,CO 2Propionic acid fermentation Propionic acid, Acetic acid, CO 2Butyric acid fermentation Butyric acid, Acetic acid, CO 2 , H 2Mixed-acid fermentationLactic acid, Acetic acid, CO 2 , H 2 , Ethanol2,3-butanediol fermentationCO 2 , Ethanol, Butanediol, Formic acidOf these fermentation types, it is the butyric acid, mixed acid and butanediolfermentations which are most detrimental for the food taste. The mixed-acidand butanediol fermentations are typical for organisms in theEnterobacteriacae family. Butyric acid fermentation is common amongsaccharolytic Clostridium. Lactic acids is mainly produced by lactic acidbacteria but it proceeds also under aerobic conditions since these bacteria arerelatively indifferent towards oxygen although they always use thefermentative metabolism. A more detailed picture of the different fermentationpathways from glucose via the common intermediate pyruvate is shown in Fig2.10.NAD +NAD +GlucoseLactic acid fermentationATPEthanol fermentationLactateNADHNAD + Pyruvate Acetaldehyde EthanolNAD + acetylCoAAcet-NAD + Acetate Ethanol H 2 CO 2AcetateOxaloacetateAcetylCoA Formate AcetylCoA + CO 2+H 2ATPATPSuccinateAcetoin Mixed acid fermentationATPNAD +ButyrateAcetoneATPNAD +PropionatePropionic acidfermentationButandiolButandiolfermentationNAD +NAD+Butanol 2-propanolButyric acid fermentationFig 2.10 Summary of the six main fermentative pathways. The main end products areemphasised by frames. Sites of co-enzyme generation and ATP formation are indicated.S.-O. Enfors: Food microbiology

2. The ecological basis of food spoilage 21Pectin hydrolysisPectins are carbohydrate polymers mainly composed of partially methylatedpoly-α-(1,4)-D-galacturonic acid. They are present in all fruits and vegetableswhere they function as a glue between the plant cells which gives mechanicalrigidity. During ripening of fruits and berries indigenous pectinases aresynthesised or activated and start hydrolysing the pectins which makes thestructure soft. Also mechanical damages on fruits and vegetables activatepectinases and this opens for microbial attack. However, also somemicroorganisms produce and secrete pectinases. Many molds have thiscapacity and among bacteria plant pathogens in the genus Erwinia alsoproduce pectinases which serve as tools for the microbial invasion resulting insoft rot.Slime productionMicrobial spoilage of meat and fish sometimes results in a slimy surface layer,composed of microbial polysaccharides. Such polysaccharide slime can alsoappear as a result of microbial growth on vegetables, wine and vinager. Aspecial case of slime formation is the so called ropiness of bread which iscaused by B. subtilis which may survive the baking as spores and thengerminate and grow if the water activity is high and the temperature kept toohigh after the baking. The slime formation on cold-stored fresh meat usuallycomes after the meat has become unacceptable due to smelling. Some speciesof lactic acid bacteria produce polysaccharides and this is sometimes utilised invarious fermented milk products to give a higher viscosity (yoghurt, Swedishlångmjölk). However, the viscosity of yoghurt is mainly caused by proteinprecipitation due to low pH.S.-O. Enfors: Food microbiology

22Chapter 3. Spoilage of different types of foodFrom a microbiological viewpoint it is convenient to classify different typesof food according to the conditions they provide for microbial growth whichgives an indication of the food shelf-life. One such classification is shown inTable 3.1.Table 3.1 Food categories with different protection against microbial spoilage.Food properties Example ProtectionWater-richProtein-richRelatively neutral pHMeatFishMilkCooked foodNoneWater-richProtein-poorRelatively sourWater-poorFermented foodPreserved foodFruitsVegetablesRoot-fruitsGrainsFlourBreadSee Chapter 6Salted/driedPickledSmokedSterilisedPasteurisedLow pHInhibitorsMechanical structureLow a wOften low a w + low pHMicrobial competitorsMicrobial inhibitorsLow a wLow pHLow pH, low a w , inhibitorsNo microfloraSmall initial microfloraOften in combination withchemical preservatives3.1 Water and protein rich foodsFresh meat, fish and milk belong to this category. They have a water activityclose to 1, contain lots of energy sources and other nutrients for microbialgrowth, are relatively pH neutral and contain no or little microbial inhibitors.If not treated by preservation methods these food stuffs are spoilt by microbialactivity in a couple of days or shorter at room temperature. Therefore theseproducts are always stored at refrigerator temperatures to reduce the rate ofmicrobial growth.At a first look one would expect that eggs should belong to this category, butfor obvious reasons Nature has build a sophisticated system which keeps theS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 23egg protected from microbial attack for several weeks at room temperature.This is described in Fig 3.11.MeatAt the moment of slaughter, the animal's breathing and the aerobic respirationcease abruptly but the cells in the body tissues continue their metabolism forseveral hours and these reactions are important for the later microbialdevelopment. During the post mortem metabolism glucose is metabolisedthrough the glycolysis, but due to lack of oxygen, lactic acid is produced fromthe pyruvate. Glycolysis generates two ATP molecules per glucose molecule,which is much less than in the aerobic respiration but still enough to preventthe formation actomyosin complex in the muscle (See Fig 3.1). However, theformation of lactic acid reduces the tissue pH from neutral towards pH 5.5-6.Eventually the low pH inhibits the glycolysis and the ATP generation ceaseswhich results in formation of actomyosin from the components actin andmyosin which are kept dissociated by ATP. Formation of actomyosin resultsin muscle contraction and it is observed as rigor mortis.Fig 3.1. The post mortem glycolysis generated protons and ATP. The ATP forces theequilibrium between actin + myosin and the actomyosin towards the dissociated state.When pH has dropped too much the ATP generation through glycolysis ceases and theequilibrium shifts towards formation of the actomyosin complex, which results in musclecontraction, i.e. rigor mortis. After some time (Table 3.2) the actomyosin complex ishydrolysed by proteases (cathepsins and calpains).The time course of this most mortem metabolism and the final pH depends onthe animal species (Table 3.2). The final pH is considered important for theshelf-life. This pH is not only dependant on the animal species but also on thecondition of the animal before slaughtering. An animal that has been stressedhas a lower blood glucose level and the post mortem metabolism can thencease due to glucose limitation rather than pH inhibition and the result is ameat with higher pH. Since the dominating spoilage flora on refrigerated freshmeat is Pseudomonas (and other Gram negative psychrotrophic rods) andthese organisms are quite sensitive to pH below about 5.5-6, the final pH ofthe meat is considered important for the shelf-life.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 24Table 3.1. Typical pH of meat from different animals and lenth of rigor mortis.Animal type Rigor mortis final pHCow 10-20 h 6 - 5.5Swine 4-8 h 6Chicken 2-4 h 6.4 - 6Fish min-h (longer on ice) 6.8 - 6.4The meat contains many nutrients for the microorganisms (Table 3.3) whichonly grow on the exudate from damaged tissue. Furthermore, it is only on thesurface of meat the microorganisms grow, unless the meat has beenmechanically perforated or minced. Therefore, the microbial count isexpressed as cells/ cm 2 or cfu/ cm 2 , where cfu means colony forming units onagar plates.Table 3.2 .Example of microbial nutrients in meat exudateComponentConcentration g/KgLactic acid 9Creatine 5Inosine 3Carnosine 3Amino acids 3Glucose-6P 1Nucleotides 1Glucose 0.5Fresh meat is usually stored at refrigerator temperature which gives a shelflife around one week, however longer for beef, but this shelf life dependsstrongly on other factors like the hygiene during slaughter and handling of themeat. It is often assumed that also a low pH after rigor mortis is important.Under these conditions the microflora at the time of spoilage is dominated byGram negative psychrotrophic rods of the genera Pseudomonas,Achromobacter, Alcaligenes, Acinetobacter och Flavobacterium. Theseorganisms are often obligate aerobes. Many investigations reportPseudomonas, and especially P. fragi as common spoilage flora on freshcold-stored meat. There are also reports which state that this type ofmicroflora on meat is universal and not dependent on which animal the meatcomes from. The flora is always dominated by bacteria, only small amountsof yeasts and molds are developing under these conditions.During storage, the bacteria initially grow exponentially, sometimes after alag phase which is caused by a shift of domination microflora. The cellconcentration increases from about 10 3 cells/cm 2 on a meat of highesthygienic quality towards 10 7 - 10 8 cells /cm 2 . Then the spoilage becomesS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 25apparent through bad odour, and sometimes discolorisation and slimeformation. Typical growth curves on refrigerated pork and chicken are shownin Fig 3.2 It is apparent that the shelf life of such products depends on thegrowth rate, which is mainly determined by the temperature, and the initialamount of bacteria, which is strongly related to the hygiene during and afterslaughter.log N/cm 287654321slimeodourchickenkycklinggriskött porkslemodörFig 3.2. Example of microbialgrowth measured as "totalaerobic count" during storage offresh pork and chicken meat atrefrigerator temperature.0 2 4 6 8 10Days Tid (d)According to one hypothesis, the shelf-life of fresh meet depends on theavailability of glucose at the surface. As long as glucose is available, this isthe main energy source for the bacteria, but when it is exhausted, otherorganic compounds, e.g. amino acids provide the energy. When aminoacidsare used as energy source, ammonia is split off by oxidative deamination andproduces bad odour. This is supported by the data shown in Fig 3.3 whichshows how the glucose gradually is exhausted at the surface when themicroflora approaches the spoilage stage. It can also be an explanation of whymeat from stressed animals has a lower shelf-life, since short intensive stressbefore the slaughter may reduce the blood glucose concentration.400N*10 -7 =400Glucose (µg/g)02.76.332110 2800 20Distance from surface (mm)0Fig 3.3. Glucose concentrationgradients and microfloradevelopment during cold storing offresh meat. At N=32*10 7 cm -2 themeat was classified as spoilt and thiscoincides with glucose exhaustion atthe surface.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 26Carbon dioxide and vacuum packagesVacuum packaging of meat, both fresh and cured meat, dramatically prolongsthe shelf-life. It was originally believed that the main mechanisms of vacuumpackaging is that oxygen is removed and that this hampered the main spoilageflora. However, storing meat under nitrogen atmosphere does not improve theshelf-life. Fig 3.4 shows that the microflora develops slower, but thefermentative metabolism which dominates under anaerobic conditionsproduces more off-flavour, unless the dominating microflora is composed oflactic acid bacteria. The figure also shows that storing the meat under CO 2atmosphere significantly reduces the rate of microbial growth. When the CO 2packed meat was opened and subjected to air, the microbial growth rateimmediately increased.logN / cm298airLuftN 2Kväve765airLuftairLuftCO24CO 230 8 16 24 32Tid (dagar)Fig 3.4. Influence ofthe gas atmosphere onthe growth rate ofmicroorganisms onrefrigerated fresh porkmeat. Some of the CO 2stored samples wereopened and furtherexposed to air, asindicated in the CO 2 -plot.When the composition of the microflora was investigated under theseconditions it became clear that the atmosphere exerts a selecting pressure, seetable 3.4. In air the dominating microflora usually is Pseuomonas. Theseorganisms are obligate aerobes or use nitrate respiration in absence of oxygen.In nitrogen atmosphere different species from the Enterobacteriacae familydominate. These organisms possess a strong fermentation capacity with illtastingproducts from the mixed-acid fermentation or 1,3 butandiolfermentation pathways. The CO 2 not only reduces the rate of growth on themeat, but it also exerts a selective pressure which favours growth ofLactobacillus, which with their lactic acid fermentation have less impact onthe spoilage than the Pseudomonas .S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 27Table 3.4. Dominating spoilage flora on cold stored pork in different atmospheres.O2%N2%CO2%Pseudomonas EnterobacteriacaeAeromonas Brochothrix Lactobacillus20 80 +100 +80 20 +80 20 + +10 90 + +100 +The selective pressure of CO 2 is explained by the different inhibitory effectthis gas has on various microorganisms. Pseudomonas belongs to the mostCO 2 sensitive bacteria while lactic acid bacteria are very resistant to this gas.Most molds are very sensitive while yeasts are very resistant to CO 2 .Fig 3.5 Relative sensitivity ofmicroorganisms to inhibition of growthby carbon dioxide.When fresh meat is vacuum packed after slaughter, which is often the case formeat that is to be stored for tendering, CO 2 is released from the tissues duringthe first day and since the plastic film of the vacuum package has a low gaspermeability and the gas headspace is removed by the vacuum, the partialpressure of CO 2 raises rapidly and exerts a protecting function. Also the shelflifepromoting effect of vacuum packing of cured meat products is similar butin that case it is the metabolic activity of the microflora which produces theCO 2 . Table 3.5 lists some properties of bacteria which contribute to theselection pressure in vacuum packed fresh and cured meat.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 28Table 3.5 Some characteristics of the organisms that dominate thespoilage flora on cold-stored fresh and cured meat in differentatmospheres.OrganismPseudomonasEnterobacteriaceaeAeromonasBrochothrix thermosphactaLactobacillusPropertiesFast growingAerobicVery CO 2 -sensitiveSensitive to low a wFacultativeIntermediate CO 2 -sensitivityFacultativeIntermediate CO 2 -sensitivityFacultativeRelatively CO 2 resistantResistant to low a wVery CO 2 -resistantIndifferent to oxygenResistant to low a wThe inhibitory effect of CO 2 seems to be synergistic with low temperature instorage of meat as shown in Fig 3.6. This may partly be due to the increasingsolubility of CO 2 at declining temperature. Even if CO 2 dissolves in water andpartly is hydratized and dissociates to bicarbonate, it is the gaseous CO 2molecule which has the inhibitory effect. This also means that the effect isstrongly pH dependent and declines with increasing pH.Fig 3.6. Time needed to reach 10 6 cells cm -2 on pork meat stored at different temperaturesin air or in CO 2 .°CS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 29The antimicrobial effect of CO 2 on many spoilage organisms has been utilisedalso for direct packaging of food in gaseous atmosphere. These so called"controlled atmosphere" packages contain mainly carbon dioxide as growthinhibiting compound but also some oxygen to avoid anaerobic metabolismand decolourization of the haeme in meat.Vacuum packing of food is applied also for other reasons than to providemicrobial inhibition via CO 2 . One common reason for vacuum packing is toprevent oxidative rancidification or other oxidising reaction with molecularoxygen (e.g. peanuts), or to prevent evaporation of flavour compounds (e.g.coffe). When cheese is packed in vacuum tight plastic films it is likely that amold inhibiting CO 2 atmosphere develops, but on the other hand, molds areobligately aerobic so the lack of oxygen is also a mold-protecting mechanism.Fish.The post mortem metabolism is important also in the fish. An importantreaction is the degradation of ATP which results in a transient accumulationof inosine monophosphate (IMP). This compound contributes to the sensoricappreciation of "fresh fish" taste. IMP is also utilised as a flavour improvingadditive in the food industry, in analogy with the meat flavour enhancingeffect of glutamine.ATPATPaseADPMyokinaseAMPAMP-deaminaseIMPFig 3.7. During the post mortemmetabolism in the fish tissue inosinemonophosphate (IMP) is transientlyaccumulated.Inosinehypoxhantine + ribose-PPhosphomonoesteraseNucleoside phosphorylaseThis metabolism has been utilised to develop a "fish-freshness" biosensor inJapan (Fig 3.8). Since the absolute level of the IMP varies much between fishsorts and even between individuals, it is not sufficient to analyse only theconcentration of IMP. Instead the ratio IMP/(IMP + inosin + hypoxanthine) isused as a fish-freshness index. The enzymatic biosensor measures the oxygenconsumption catalysed by xanthine oxidase. If only xanthine oxidase ispresent in the analysis, the oxygen consumption represents the concentrationof hypoxanthine. If also the nucleotide phosphorylase is present, the oxygenconsumption represents the concentration of hypoxanthine + inosine. ByS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 30including also the 5'-nucleotidase the oxygen consumption also includes theIMP.OHIMPInosineHypoxanthineNN3 xan t h i ne ox id a seNOH2 nuc l e ot ideph o s p h o r y l a seNO1 5 ’ - nu c l e o ti d a seOHOHCH 2 - P OOHEnzym e r = analys3 = Hx2 3 = I + Hx1 2 3 = IMP + I + HxIndex =IMPIMP + I + HxFig 3.8. Principle of a "fish-freshness" biosensor based on analysis of the degradation ofIMP degradation. The oxygen consumption catalysed by xanthine oxidase is analyses withor without the enzymes nucleotide phosphorylase and 5'-nucleotidase and a index thatrepresents the concentration of IMP in relation to the sum of the metabolites is calculated.The microbial spoilage of refrigerated fresh fish has large similarities withthat of fresh meat. Pseudomonas is often dominating in the spoilage flora (Fig3.9). A similar organism, Shewanella putrifaciens (previously calledPseudomonas putrifaciens or Alteromonas putrifaciens) is another spoilageorganism specifically associated with marine fishes. It has the capacity toproduce both hydrogen sulfide from cysteine and trimetylamine (TMA) byanaerobic respiration with TMAO as electron acceptor. Due to this capacity toproduce bad odour the fish may be spoilt at 10 times lower total microflora ifShewanella putrifaciens dominates.Fig 3.9. Distribution ofspoilage organisms onrefrigerated fresh fish.Aeromonas is mainlyassociated with freshwaterfishes andShewanella withmarine fishes.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 31MilkMilk is a very good substrate for microbial growth. However, it is protectedby several antimicrobial mechanisms which favour the development of lacticacid bacteria if the temperature is not too low. The lactoperoxidase system isone of these antimicrobial systems in milk (Fig 3.10). Milk contains theenzyme lactoperoxidase and small concentrations of its substrate thiocyanate.The milk is contaminated with lactic acid bacteria during the milking. Thesebacteria are catalase negative and therefore the hydrogen peroxide, whichalways is produced as a by-product in the metabolism, is not removed bycatalase as in other microbial systems. Instead, the lactoperoxidase uses thehydrogen peroxide to oxidise the thiocyanate to hypothiocyanate. Thiscompound is strongly oxidising and reacts with sulfhydryl groups in transportproteins in the bacterial membrane, especially in Gram negative bacteria,while the lactic acid bacteria are relatively resistant. The lactoperoxidasesystem has been reported to have an antimicrobial function also in tears andother body-fluids.O 2oxidase catalaseH 2 O 2H 2 OLPthiocyanateSCN -OSCN -hypothiocyanateHO-S-proteinHS-proteinFig 3.10. The lactoperoxidase system. The lactoperoxidase in milk uses the hydrogenperoxide to oxidise thiocyanate to the strongly oxidising hypothiocyanate which oxidisestransport proteins in bacterial membranes. Especially Gram negative bacteria are sensitiveto the hypothiocyanate.When the milk leaves the udder it becomes infected by about 100 so callledudder cocci per milliliter. During the further handling in the cow house themilk is infected with several types of microorganisms as shown in Table 3.6Table 3.6 The initial milk contamination microfloraInfectionSourceE. coliFecesEnterococcusMicrococcusBacillus sporesAirMold sporesYeastsLactococcusLactobacillusMilking equipmentGram-negative rodsS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 32If the milk is stored at room temperature the "lactic streptococci", i.e.Lactococcus spp. will first dominate the microflora and protect it from mostof the other microorganisms by means of lactic acid production. EventuallyLactobacillus, which can grow at lower pH than the other bacteria (below 5)will dominate. This fermented milk is similar to yoghurt and it was previouslyproduced on the farms (Swedish filbunke). If the milk is stored furtherproteolytic molds will finally raise the pH and it will be further destroyed byputrification by Clostridium and Bacillus. These reactions do not take place inrefrigerated milk.When the milk is cooled after milking and stored refrigerated on the farm,psychrotrophic gram negative rods (Pseudomonas and similar) will dominate.These bacteria will not make it sour as does the lactic acid bacteria. If storedtoo long the milk is spoilt by ammonia, peptides and free fatty acids. Thispsychrotrophic microflora, which itself is very heat sensitive, is known toproduce comparatively heat resistant proteases and lipases which may createproblems in the later storage. When the milk reaches the dairy it is pasteurisedwhich efficiently eliminates the psychrotrophic Pseudomonas flora and mostother bacteria. However, some of the more heat resistant organisms, mainlyLactobacillus and Micrococcus will survive, and the bacterial endosporesfrom Bacillus are not influenced at all by the pasteurisation.After the pasteurisation the milk becomes re-infected with the dairyequipment microflora. This may restore the psychrotrophic Pseudomonasflora or at bad hygiene even the Enterobacteriacae flora. The final spoilage ofthe refrigerated milk therefore differs depending on the contamination flora.Members of the Enterobacteriacae family may spoil the milk withfermentation. Bacillus spores my germinate and spoil the milk by proteolysis.This is especially common in fatty products like cream. Also proteolysis andlipolysis by enzymes from the early Pseudomonas flora may contribute to thefinal spoilage of milk. However, the old days souring of milk by lactic acidbacteria is not the common fate of refrigerated pasteurised milk.EggThe egg is infected on the surface when the hen lays the egg. This flora isdominated by Pseudomonas, Staphylococcus, Micrococcus and fecalbacteria. It is not uncommon that the hen is infected with Salmonella andduring the 1990ths many reports on Salmonella infected egg yolks appearedin England. The surface microflora is usually not infecting the interior of theegg due to a number of defence mechanisms, which are illustrated in Fig 3.11.If this protection fails and the egg becomes invaded by bacteria it is usuallyPseudomonas fluorescens which dominates (80%). These infections can bedetected by illumination of the egg with UV-light.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 33Albumin: viscous, high pH(pH9,5), riboflavin + pyridoxincomplexingNoprotectionCon-albumin: Fe2+ complexingAvidin: Biotin complexingLysozyme: kills G+ bacteriaouter mucin layer1-10µm pores in shellinner keratinmembraneFig 3.11. The egg is protected against bacterial infections an multiple ways: The shell andthe two membranes provide mechanical hinders for the bacteria. The high pH in the eggwhite is non-optimal for many bacteria. The egg white contains several protectionmechanisms: Lysozyme ruptures cell walls of many bacteria. Albumin, conalbumin andavidin make several nutrients unavailable by strong complex formations.3.2 Fruits and vegetablesFruits and vegetables do have a high water activity but they develop anotherspoilage scenario than meat, fish and milk. Many of these products areprotected mechanically by the pectins which constitute a "glue" between thecells and gives rigidity. When fruits and berries ripen, endogeneous pectinasesstart to hydrolyse the pectin and this also makes the products more susceptibleto microbial attacks. Another common protection is the low pH of some ofthese products. This group of foods also has a much lower concentration offree amino acids and other nutrients than meat, fish, and milk. For thesereasons it is usually not the Pseudomonas and other spoilage bacteriamentioned above which dominate in the spoilage. Instead it is often pectinaseproducing organisms, which mostly means molds, that initiate the spoilage offruits and vegetables. In the later phase, when the pectinolytic organisms haveopened up the defence structure, also yeasts participate in the spoilage.One of few bacteria involved in spoilage of vegetables is the plant pathogenErwinia carotovora. This organism has been subject to studies of the corumsensing phenomenon which plays a central role in the ecology of manyorganisms. In this case the corum sensing is based on accumulation of N-acylated homoserine lactones (AHL) which accumulates around the cells (Fig3.12). When the concentration of AHL is high enough this compound inducesthe pectinase synthesis. The strategic advantage of not producing thepectinase constitutively is obvious, since the plants have their defencesystems which generate antimicrobial chemicals when the plant is attacked.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 34Only by delaying the pectinase synthesis until the number of bacteria is largeenough, can the hydrolysis of the pectins be fast and efficient enough. Oncethe pectinases have damaged the structure of the fruit/vegetable, otherorganisms follow and contribute to the soft rot. Due to the often low pH,molds and yeasts, rather than bacteria are common in the spoilage of theseproducts.plant cellpectinolyticbacteriaAHLAHLAHLAHLAHLFig 3.12. Erwinia carotovora utilises corum sensing to invade plants. They start byhydrolysing the protecting pectin layer with extracellular pectinases. When the plantrecognises a microbial attack it defends itself by producing antimicrobial ( )compounds. Instead of initiating this defence response at low concentration of Erwiniacells, they first accumulate acylated homoserine lactones (AHL) and when theconcentration is high enough this is a signal for induction of the pectinase ( )production. By delaying the attack until many bacterial cells have accumulated Erwiniagains increased virulence.It is estimated that only about 20% of the fruits and vegetables are spoilt bymicroorganisms. The endogenous metabolism of the products, which leads toover-maturation plays a major role for the spoilage. Furthermore, drying alsocontributes to the spoilage. To reduce and better control the endogenousmetabolism, fruits and to some extent also vegetables are stored in modifiedatmospheres (Controlled Atmosphere, CA-storage). Common principles are toincrease the CO 2 -concentration, which also has a microbial inhibition effect,and to reduce the oxygen concentration by adding nitrogen gas. Many fruitsproduce ethylene gas, which acts as a maturation hormone, and for someproducts absorption of the ethylene is included in the CA storage. Addition ofethylene or cessation of the absorption is then used to initiate the ripening.Table 3.7 gives an example of a modified atmosphere for fruits. The exactcomposition is optimised for each product.S.-O. Enfors: Food microbiology

3. Spoilage of different types of food 353.3 CerealsTable 3.6. Example of modified atmosphere for storage of fruitsO 2 : 0 - 5 %CO 2 : 2 - 10 %N 2 : 90-95 %Relative humidity: 90-95%Grains on the field usually have a primary microflora of 10 3 - 10 6 bacteria g -1 .Lactic acid bacteria, coliform bacteria and Bacillus spores dominate. Aweather dependent flora of fungal spores is also present. At humid conditionsthe mold spore count can be 10 5 g -1 . Different species of Aspergillus andPenicillium usually dominate. If the grains are soaked in water the bacterialflora will dominate. Regulations set a maximum water concentration of 13%for storage of grains for human food and then no significant microbial activityis expected due to the low water activity. If the water content exceed 15%mold growth begins. Even if the grains are kept dry enough according to theregulations, local humid zones may appear in the silos, e.g. due to watercondensation on walls. Under these conditions mold growth and mycotoxinformation may appear.During the milling of the grain most of the microflora follows the hull butsome microorganisms are transferred to the flour. Typical microbial countsare 10 2 -10 3 bacteria plus about 100 mold spores per gram sifted flour andabout ten times more in course flour. At correct dry storage of the flour thereis no microbial activity, but as soon as water is added a vigorous growthstarts.The surface of the bread becomes sterilised in the oven and a dry hard breadsurface protects the bread against mold growth. If the bread is cut beforepacking the surfaces are usually infected and if the bread is kept too moist in aplastic bag mold growth will spoil it. The inner part of a bread is usuallyheated to 95-99°C which means it is essentially sterile with respect tovegetative cells and mold spores. There is however a rare bakery problemcalled ropiness, which is caused by polysaccharide formation by Bacillussubtilis. The organism has then survived the baking in spore form and if thetemperature is kept at 30-45 °C too long and the bread has not become dryenough during the baking the B. subtilis spores germinate and grow very fastand produce the polysaccharides.During storage of the bread, spoilage is entirely caused by molds which havecontaminated the bread after the baking. To reduce the rate of mold growthpropionates are often used a preservatives in industrial baking. Dry bread(knäckebröd) is not subject to any microbial spoilage, provided it is storedS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 36dry. Under such conditions the very slow spoilage is eventually caused byrancidification.3.4 Preserved foodsThe spoilage of preserved food depends on the type of preservation. Ingeneral, if the preservation prevents microbial spoilage, the ultimate fate isusually spoilage by rancidification, which usually is a very slow process.Dried products. In the drying process the water activity is reduced to so lowlevels that no microorganisms are active. If the storage conditions are not dryenough, mold formation may occur, but otherwise the shelf-life is limited byrancidification processes, which depend very much on the fat composition ofthe product. During spray-drying the food is exposed temperatures that killthe most sensitive bacteria, but endospores, mold spores and more heatresistant vegetative bacteria as Enterococcus, Lactococcus, Micrococcus, andLactobacillus may survive. When such products (e.g. dry milk, soups, sauces,etc.) are reconstituted with water they are usually very susceptible to fastmicrobial spoilage and considerable risks for food poisoning.Table 3.8. Summary of common spoilage floras on different types of foodCured meat products are usually protected by the low water activity createdby salt addition. If the products are fermented they are also protected by thelactic acid and the competitive effects of the lactic acid bacteria. Theseproducts are often further protected with nitrite. A common bacterium invacuum packed cured meat products, is Brochothrix thermosphacta. Thisorganism is similar to Lactobacillus (CO 2 resistant and tolerant against lowa w ) which usually dominates vacuum packed meat products, but it is a severeS.-O. Enfors: Food microbiology

3. Spoilage of different types of food 37spoilage organism since it produces stinking metabolites. Also the low-a wtolerant Micrococcus and Lactobacillus are common in these products.Salted fish and fish preserves are also protected mainly by the low wateractivity and chemical preservatives. In salted fish products mainly halophilicstrains of Pediococcus, Micrococcus, and yeasts grow and they do this at avery low rate with slow spoiling. Usually these products are to be cold storedand the main shelf-time limitation is usually rancidification of the fat.Table 3.9.Properties of common food related organismsOrgansim Properties ProductsGramneg. rods:- Pseudomonas- ShewanellaputrefaciensLactic acid bacteria:-Lactobacillus-Lactococcus-PediococcusEnterococcusB. thermpsphactaGrampositive cocci:-Micrococcus-StaphylococcusSpore formers:-Bacillus-ClostridiumEnterobacteriaceae:-E.coli-Enterobacter m.fl.-ErwiniaMoldsYeasts- Psychrotropic- Aerobic- Sensitive to low aw- Sensitive to low pH- CO 2 -sensitive- H 2 S-producer- O2-indifferent- Resistant to low aw- Resistant to low pH- CO 2 -resistant- Facultative- Resistent to low aw- Resistant to low pH- Heat resistant- Lipo-/proteolytic- Extremt värmeresistenta- Mesofila- Starkt fermentativa- Strongly fermentative- Pektinase-active- Aerobic- CO 2 -sensitive- Pektinase-active- Resistant to low aw- Resistant to low pH- Lipo-/proteolytic- Facultative- CO 2 -resistant- Resistant to low aw- Resistant to low pHRefrigerated fresh:-Meat-Fish-MilkFishFish preservesVacuum packedFermented foodSmoked/salted/dried meat/fishPicklesFish preservesVacuum packedFermented foodSmoked/salted/dried meat/fishHeat sterilised foodReconstituted dried food.Pre-cookedMilk: B.cereusMilkPre-cooked foodVegetablesVegetablesFruitDried foodVegetablesFruitLow-pH preservesSweet productsS.-O. Enfors: Food microbiology

38Chapter 4.Foodborne pathogensMost cases of so called food poisoning are caused by microorganisms. Only afew per cent of the food poisoning cases are reported to be caused by toxic rawmaterials like toxic mushrooms or plants or contamination by toxic impuritieslike heavy metals. The remaining cases of food poisoning can be divided intomicrobial food intoxication, when microorganisms have produced toxins in thefood and microbial food borne infections, when pathogenic microorganisms inthe food are ingested and infect the human body.Intoxications and infections caused by microorganisms in food and wateraccount for a large number of fatal cases and large economic loss in thesociety. Food borne pathogens causes millions of death cases every year,especially in poor countries and it is especially children that are the victims. Itis difficult to estimate the true statistics behind the food borne diseases, sincemost cases are never confirmed by clinical analysis. This is especially true for"mild" but common diseases like Bacillus cereus intoxication and Clostridiumperfringens infections, since they are usually confirmed by analyses only inlarge outbreaks. On the other hand, statistics on the severe Clostridiumbotulinum intoxication is probably reflecting the true cases, at least in theindustrial world.Fig 4.1 Number of cases with food borne diseases reported to the Swedish Institute forInfectious Disease Control according to the law for report on certain diseases (Averagenumber per year during 1997-2005).

4. Foodborne pathogens 39Only some of the microbial food poisoning diseases are reported to authoritiesaccording to law. See Fig 4.1. Other sources of statistics that also includeorganisms that are not covered by obligatory reporting gives a similar picture,namely that Campylobacter, Salmonella and Norovirus (earlier calledcalicivirus) are among the most frequent causes of food borne illness, but italso shows that Clostridium perfringens and Staphylococcus often occur in theoutbreaks (Fig 4.2).CasesOutbreaksFig 4.2 Statistics of food borne diseases in Sweden for a 5 year period. Calicivirus =Norovirus .Source: Vår Föda, nr 5, 1999.4.1 Microbial food intoxicationsStaphylococcus aureus. The probably most common microbial foodintoxication is caused by certain strains of Staphylococcus aureus. Thisorganism is also known as a common pathogen causing infections in woundsand blood, but these infections are not considered to be transferred via food. S.aureus produces a series of toxins and other virulence factors (Table 4.1) but itis mainly the enterotoxins that cause food poisoning after ingestion of food onwhich S. aureus has grown and produced the enterotoxins.Table 4.1 Some virulence factors of S. aureusToxinsMembrane damaging toxins (several)Epidermolytic toxinToxic shock syndrom toxinPyrogenic exotoxinEnterotoxin ( 6 serotypes)ExoenzymesCoagulaseStaphylokinaseProteasesPhospholipaseLipaseHyaluronidase

4. Foodborne pathogens 40S. aureus is a common inhabitant on animals and humans where it grows onmucose membranes, for instance in the nose, even on healthy individuals, andit is frequently found in pus and wounds. The common source of foodcontamination is therefore human hands. This organism is very resistant to lowwater activity (Table 2.2) which means that they can grow on salted andrelatively dry products. It does not grow under refrigerator conditions, andwithout growth no toxin is produced. It has low competitive power comparedto many other bacteria, like lactic acid bacteria and Pseudomonas. For thisreason, a small amount of S. aureus is usually accepted in food ( e.g. 10 2 - 10 3g -1 ) before it is classified as not acceptable (Swedish: otjänligt) which meansthe product must be withdrawn from the market.The intoxications are associated with a large number of foods, often food thathas been cooked which eliminates competing microorganisms and food that ishandled by human hands: Chicken, ham, salads, pizza, kebab, sauses, paseriesetc. The enterotoxins are very heat stable and contaminated food may thereforestill be poisonous after re-heating when all vegetative cells have been killed.The disease caused when eating S. aureus enterotoxis is characterised by aviolent nausea with vomiting, diarrhoea and convulsions. It is one of the fewcases when the eating of infected food results in an almost immediate illness,within one or a couple of hours. The patient usually recovers in 1-2 days andthe disease is not associated with further complications.Bacillus cereus. This organism is a facultatively anaerobic endospore formerthat is ubiquitously present in Nature. Therefore vegetables are usuallycontaminated with this organism. It is also frequently present in milk, probablysince the dusty air in the barn contaminates the milk and the subsequentpasteurisation has no effect on the endospores, while most competing organismare killed. B. cereus produces three enterotoxins which cause relatively milddiarrhoeal illness with an incubation time of 6-24 hours, and an emetic toxin,cereluid. The haemolysins are inactivated in the stomach and this type ofdisease is actually an infection where the toxin is produced locally by B. cereusgrowing in the intestine. The cereluid is a heat stable protein and this disease isconsidered to be a true intoxication. It has a shorter incubation time, 0.5-6hours, and is especially associated with rice dishes. B. cereus is assumed to bea very common agent of food poisoning, but both diseases are usuallyproceeding fast with little complications and therefore isolated outbreaks arenormally not identified and the statistics becomes unsure. B. cereus is not socompetitive but after heating of a product the spores may become thedominating organisms and if the food after that is kept too long in thetemperature range 15-45°C the spores may germinate and grow and produce

4. Foodborne pathogens 41the toxin. Like most Bacillus this organism is typical mesofilic with respect totemperature, but certain strains are reported to be psychrotrophic and maygrow down to about 4 °C.Clostridium botulinum. The most well-known and feared microbialintoxication is botulism, which is caused by one of several toxins of Cl.botulinum. This organism is an obligately anaerobic spore forming bacteriumthat is very common in soil and water. The toxin is classified according toserotype A-F, where type A, B, E, and F are toxic to humans. Cl. botulinumtype E is commonly found on fishes and this toxin is relatively heat labile,destroyed by boiling, while type A is more heat resistant.The endospores make also heat treated food potentially dangerous sincesurviving spores may grow out. The botulin toxin is a very toxic protein that isproduced during growth of the vegetative cells in food. Cl. botulinum does notgrow at temperatures below 4°C, at pH below 4.5, or in presence of oxygen.The toxin acts as a neurotoxin paralysing the central nervous system. It is oneof the most potent toxins known with a lethal dose of about 10 -6 g. After anincubation time of 18-36 hours, the illness sometimes starts with nausea and isfollowed by the effects on the CNS caused by blocking of the acetyl cholinerelease at the nerve synapses: double-seeing, difficulties to swallow and finallyparalysing of the breathing. At this stage the mortality is high. In US statisticsduring 1950 - 1970 the number of fatal cases was almost as high as the numberof reported cases. After that an anti-toxin became available but mortality is stillconsiderable. Fortunately, the number of cases is low, in Sweden the average isless than one/year.The few cases of botulism in Sweden are associated with home preserved(marinaded or smoked) fish and home preserved meat. The precautions thatmust be taken to avoid botulism in association with food preservation are lowpH (often vinegar), high salt concentration and storage below 4°C. Incommercial preservation nitrate also plays an important role. This is furtherdescribed in Chapter 6.The so called infant botulism has another mechanism. It is caused by a Cl.botulinum infection of the intestines where the spores germinate, grow andproduce the toxin. This disease is only associated with babies under one yearage who have not obtained the normal competitive intestinal microflora, andthe infection origin has exclusively been honey which often (10%) containsspores of Cl. botulinum. For this reason authorities recommend not to givehoney to babies.

4. Foodborne pathogens 42Mycotoxins. Intoxications by fungal toxins, mycotoxins, are not found in thestatistics on food borne diseases. The reason is that these diseases, contrary tothe other diseases discussed here, do not cause acute symptoms. Most reportson mycotoxins describe their effects as cancerogenic or liver or kidneydamaging, with symptoms emerging long time after consumption of the food.An exception is patulin, which is associated with intestinal illness but it is alsoa suspected carcinogen.There are hundreds of mycotoxins described in the literature. Biochemicallythey are typical secondary metabolites produced by moulds. It means they aremainly produced late in or after the growth phase. Most mycotoxins areresistant to temperatures used in cooking. Fig 4.3 shows the chemical structureof some mycotoxins. For some of the mycotoxins (e.g. aflatoxins, ochratoxinsand patulin, there are regulatory concentration limits for food, based on TDIvalues (TDI=tolerable daily intake). TDI-values are usually in the range below1 mg/Kg body weight.Aflatoxin B1Ochratoxin AFig 4.3 Examples of mycotoxinsTable 4.2 lists some well-known mycotoxins, producing organisms and foodthey are typically associated with. The table demonstrates two characteristicsof mycotoxins: several species, even from different genus, may produce thesame mycotoxin and one mycotoxigenic organism may produce severalmycotoxins.There are several variants of chemically related aflatoxins. Aflatoxin B1 is themost commonly observed and most toxic of the aflatoxins and it is a stronglypotent carcinogen. In animal experiments daily intake of less that 100 ng/kgbody weight causes liver tumours. Aflatoxin M1 is found in milk and it is adegradation product of aflatoxin E.

4. Foodborne pathogens 43Table 4.2 Examples of mycotoxins, mycotoxigenic molds, and associated foodToxin Organism Associated food EffectAflatoxins Aspergillus flavus Nuts, figs, corn Liver cancerAsp. parasiticusOchratoxin APatulinPenicillinic acidAsp. ochraceusPenicillium viridicatumPc. exapnsumPc roquefortiPc. cyclopiumPc viridicatumGrains, coffee, wine,beansFruits, berrysPeasKidney/liverdamage, teratogenicDiarrhoeaZearalenon Fusarium graminearum Grains InfertilityRoquefortin Pc. roqueforti Breadµg aflatoxin/ kg breadzone Bread 1 Bread 2 Bread 31 >> 15 000 150-300 40-802 600 n.d 203 100 n.d n.d4 n.dFig 4.4 Analysis of aflatoxin distribution in three breads that were inoculated with A. flavusand incubated until a colony was formed (Vår Föda, 31, 390-399, 1979).The extremely high toxicity of aflatoxin and the fact that mould colonies oftengrow on bread raises the question about how far the toxin reaches from thefungal colony. In an investigation 3 breads were inoculated at the surface withan aflatoxin producing strain of Aspergillus flavus, as indicated in Fig 4.4.Samples were taken from 4 zones at different distances from the colony andanalysed for aflatoxin B1, B2, G1, and G2. The table in Fig 4.4 shows the sumof the aflatoxin concentrations in the zones after one week. The permitted levelin bread was 5 µg/Kg.While aflatoxins are mainly associated with nuts and figs, ochratoxins aregenerally found in food and especially in food that is consumed in largeamounts, like cereals. Ochratoxin is also spread via meat from animals fed ongrains. It has been shown to cause damages on liver and kidney and it is alsoteratogenic. The TDI is 14 ng/Kg body weight but due to expected but not

4. Foodborne pathogens 44proved cancerogenic effects the TDI value used by some authorities isconsiderably lower.Patulin was first studied as a potential antibioticum but is now classified as amycotoxin. The source in nature is fruits and berries, and it is frequently foundat very low concentrations in commercial fruit juices and jam.Several strains of P. roqueforti isolated from commercial blue cheeses havebeen shown in the laboratory to produce mycotoxins, among them PR toxinand roquefortine. This organism is also a common contaminant in many foodsand it is a predominant organism in silage where it is said to have a positiveeffect on the acceptance by cattle. Roquefortine C has been reported to have aneurotoxic effect and it is an inhibitor of Gram positive bacteria.The uncertainty of the real effects of consumption of mycotoxins with food hasresulted in the general recommendation to avoid mold infected food.Algal toxins. Planktonic algae called dinoflagellates are responsible fordifferent types of shellfish poisoning: Paralytic shellfish poisoning (PSP),diarrheic shellfish poisoning (DSP) and others. The PSP is observed asrespiratory paralysis within 0.5 - 2 hours after consumption of the toxic foodand it may get severe consequences if not treated. The DSP causes diarrheawithin 0.5 - 3 hours and lasts for 2-3 days with no after effects. These types ofpoisoning are associated with filter-feeding molluscs, like mussels, clams,scallops and oysters.Cyanobacteria, previously called blue-green algae, are involved in so calledalgal blooms, some of which may make the water toxic. Nodularia spumigenais one of the most common toxic cyanobacteria in algal blooms in the Balticsea. These intoxications are normally not associated with food or drinkingwater, however.4.2 Food borne infectionsFood borne diseases caused by microbial infection of the consumer is muchmore frequent than the intoxications caused by ingestion of microbial toxinsproduced in the food. Two of the most frequent diseases in the statistics (Fig4.1) namely Campylobacter and Salmonella are infections caused by eatingcontaminated food. The pathogens may then grow in the intestines and causethe disease. In general, this type of disease has an incubation time of one toseveral days, which often results in difficulties to identify the food that was

4. Foodborne pathogens 45causing the problem. The incubation times reported for infections varies muchwith the status of the individual and with the infecting dose. Also reportedminimal infectious doses are very unsure figures and depend on the conditionof the person. Mostly elderly people and children are much more sensitive thatgrown-up and healthy individual.In Table 4.3 common infections are grouped according to the probable sourceof contamination. Bacteria with fecal origin may enter the food from water orraw material that has been in contact with feces, which is the naturalenvironment for these organisms. Alternatively, the food has got this infectiondirectly from feces contaminated hands of someone handling the food. Most ofthe food pathogens of fecal origin belong to the family Enterobacteriaceae,which includes among others the genera Salmonella, Shigella, Yersinia andEscherichia belong to theTable 4.3 Classification of common food pathogens based on their probable sourceFecal origin Water origin Soil originCampylobacter Listeria monocytogenes Clostridium perfringensSalmonella Aeromonas hydrophila Bacillus cereus (diarrhoeal)ShigellaVibrio parahaemolyticusYersinia enterocoliticaPathogenic E. coliCampylobacter is a common inhabitant of intestines of many types of warmblooded animals without causing any symptoms in the animal. It is mainly thespecies C. jejuni that causes the food borne infections. It is a Gram negativebacterium and it is environmentally sensitive: it grows only in the rangebetween 25 and 42 °C, is microaerophilic, and very sensitive to drying,freezing and disinfectants. The food contamination source is often chicken, unpasteurisedmilk or water. Flies are also suspected to transmit the bacteria fromfeces to food. Several large outbreaks have been caused from municipal water,but mostly it is chickens that are associated with Campylobacter infections.The chicken (and also other types of meat) becomes contaminated from itsfeces during the slaughter and since the infectious dose is very small ( 500cells) infected meat can cause disease even if it has been stored so that nofurther growth has been possible. The only way to avoid this disease is toapply good hygiene in the food preparation and to heat the food enough to killthe cells, which requires 65 °C through all parts of the meat. The infectiongives diarrhoea and other typical gastroenteritis symptoms for about 2-5 daysbut sometimes reactive arthritis prolongs and complicates the disease.Salmonella. There are more than 2000 different serotypes of Salmonella andsome cause relatively mild diseases while other strains cause severe illness. S.

4. Foodborne pathogens 46typhi and S. paratyphi cause the most dangerous infections. Salmonella isfrequently found in poultry and swine. It is environmentally very resistantwhich explains why they are widely spread in Nature even if they grow mainlyin animals. The organisms are usually distributed via meat that is contaminatedwith feces during slaughter. Infected animal feed is another carrier ofSalmonella. It is also common in spices and vegetables, probably throughcontamination with infected water or soil.. The disease breaks out after 12-48hours and lasts for a couple of days, with some exceptions when there arecomplications with reactive arthritis or septicemia with subsequent infection oforgan systems. Humans may also become carriers of Salmonella withoutshowing any symptoms. The infective dose varies much but as little as 15-20cells has been reported. According to FDA the number of cases ofsalmonellosis is 2-4 millions/year in the US and the frequency is rapidlyincreasing. Especially S. enteritides is rapidly spreading in US and Europe.Shigella. Contrary to Salmonella these organisms are very host specific andgrow only in the intestines of humans and apes. The food borne infections aremostly caused by bad personal hygiene but also by vegetables that have beencontaminated with water containing human feces. Infected humans mayrecover and still be "healthy carrier" of the organisms. This, together with thevery low infectious dose (10 cells), also makes shigellosis (bacillus dysenteri)directly transferable between individuals. Shigella multiply intracellulary in theepitheleal cells which results in tissue destruction. Some strains produce shigatoxin which is similar to the toxin produced by EHEC. This protein, whenproduced by the bacteria in the infected human host cell, inhibits the proteinsynthesis and results in cell death with severe hemorrhage in the patient.Yersinia enterocolitica. There are three pathogenic Yersinia species. Y. pestis,Y. pseudotuberculosis and Y. enterocolitica. The latter is associated with foodborne infections, mainly from pork, since swine is a common reservoir of thisorganism. Also dogs and cats are frequent carriers of Y. enterocolitica whichgrows not only in the intestinal tract but also in mucous membranes in themouth and throat. This is one of few psychrotrophic pathogens which can growat high rate in the refrigerator, even down to 0°C. Most cases are associatedwith pork and vacuum packed meat products but also water and un-pasteurisedmilk have been involved in out-breaks. Vacuum packed meat products haveoften been heat treated, which removes most vegetative cells inclusive thequite heat sensitive Y. enterocolitica, and if the product then is re-infected andstored for long time in the refrigerator the product may cause infection. Thedisease breaks out 3-7 days after the infection and it lasts for 1-3 weeks. Thedisease is relatively rare in the statistics which partly may be due to the

4. Foodborne pathogens 47difficulties to isolate the bacteria. It is also assumed that only certain strains ofY. enterocolitica are pathogenic.Pathogenic E. coli. There are four enteropathogenic groups of E. coli. They areclassified according to serotype. The nomenclature is not strict, but a commonclassification is:EHECETECEPECEIECenterohemorrhagic E. colienterotoxigenic E. colienteropathogenic E. colienteroinvasive E. coliEHEC (enterohemorrhagic E. coli ) produces shiga-like toxins, also calledverotoxins. The most well-known EHEC are characterised and analysed as theserotype O157:H7, but these shiga-like toxins are produced also by other E.coli serotypes. Alternative names of EHEC are STEC ( shiga-toxin producingE. coli ) or VTEC (verotoxin producing E. coli). The natural reservoir ofEHEC is probably the intestines of cows, who are not themselves showing anysymptoms, and then the distribution occurs via fecal contamination. EHECinfections have been associated with hamburgers, un-pasteurised milk, water,and alfalfa sprouts. In some cases it has been assumed that humans keepingindoors in a cow-house can be infected directly from this environment. Thevery low infectious dose,10 cells, means that the bacteria do not need to growon food to make it infective. EHEC has unusually high resistance to low pH,and the cells can survive extended periods in sour products like juice, yoghurt,and fermented sausages, products that usually have been considered as safe inthis respect. The two shiga-like toxins are coded by genes (stx1 and stx2)which are located on lambda phages and integrated as inactive prophage genesin the bacterial genome. Only after induction, which can be by agents resultingin the SOS response or by iron limitation, does the prophage enter the lyticphase which induces the toxin production. The toxin kills the cells in theintestines and causes bloody diarrhoea. In severe cases, especially in children,the infection is spread to the kidney which may be permanently destroyed.ETEC, or enterotoxigenic E. coli causes a relatively mild gastroenteritis withwatery diarrhea, often called travelers' diarrhea. These infections are alsocommon among children in poor countries. Large infective doses (> 10 8 cells)are required and the incubation time is about 1 day. ETEC is not common incountries with good sanitary standards but when the water is contaminatedwith human feces there is a risk for ETEC infections in food.

4. Foodborne pathogens 48EPEC are strains of E. coli that cause the infantile diarrhea in newborn babies.It is not assumed to be food associated.EIEC invade the epithelial cells of the intestine, resulting in a mild form ofdysentery. It is not known if this is a food associated infection.Water and soil are reservoirs for several pathogenic bacteria that maycontaminate food: Listeria monocytogenes, Clostridium perfringens, Bacilluscereus, Aeromonas hydrophila, and Vibrio parahaemolyticus.Listeria monocytogenes is widely spread in nature both in water, soil, plants,and in animal intestines. It grows often in biofilms which are common in foodmanufacturing facilities. L. monocytogenes is therefore very common in food.Also humans are often carrying this organism in its intestinal flora without anysymptoms. Many strains are pathogenic to some extent. Listeriosis is notprimarily a gastroenterit but it is rather manifested as septicemia, meningitis,and cervicial infections in pregnant women which may result in spontaneousabortion. Sometimes the symptoms are preceded by gastrointestinal symptomslike nausea, vomiting, and diarrhea. The bacteria invade the human phagocyticcells and propagate intracellulary. In this way the infection is spread to organswith the blood. The organism is one of the psychrotrophic pathogens whichcan grow to dangerous concentrations also in a refrigerator. It is frequentlyfound in vacuum packed smoked or marinaded fish and in soft cheeses madeon un-pasteurized milk. Cooking at 70°C kills the bacteria. Food associatedwith Listeria outbreaks are often such food where the organism gets the chanceto grow during production and then is consumed without further heating, e.g.in soft cheeses, marinaded meat, and smoked fish. The infective dose isunknown. The reason why the number of identified disease cases in not largerwhile Listeria is often present in food is probably that only some strains arepathogenic and the pathogenecity factors are not known enough to be the goalfor analysis.Clostridium perfringens is a common spore forming soil bacterium whichmeans that vegetables often are contaminated. It can also grow in the intestinesof humans and animals without causing any symptoms. Many strains of Cl.perfringens produce enterotoxins. There are a number of facts that togethermakes this one of the most frequent diseases associated with large-scalecooking, especially with soups and casserols: 1) Being a common soilbacterium it is often added to food as spores in contaminating vegetables,2) The spores not only survives cooking at 100°C but even become activated togerminate, while most competing bacteria are killed. 3) The boiling alsoremoves the oxygen which otherwise prevent growth of this obligately

4. Foodborne pathogens 49anaerobic bacterium. 4) In rich media and optimal temperature it can growextremely fast (8 min doubling time at 45°C). If the cooling from thistemperature down to below 15°C is not fast enough, or if the food is kept warmat too low temperature (should be ≥ 60°C), conditions for growth of Cl.perfringens are excellent. The common form of perfringens poisoning ischaracterized by intense abdominal cramps and diarrhea that come within 8-24hours after consumption of foods with large numbers of cells and lasts forabout 24 hours. The infective dose is very large , over 10 8 cells.Bacillus cereus. This organism produces the toxin cereluid that act as foodintoxication causing vomiting. But many strains of B. cereus also produce oneor several of three enterotoxins: haemolysin BL, non-haemolytic enterotoxin,and cytokine K. These proteins do not survive the passage through thestomach, and therefore its is considered that the bacteria also can establishthemselves in the intestines and produce the enterotoxins there. This diarrhoealdisease is often associated with meat and vegetable dishes and sauces. Also thespores may germinate and grow in the intestines, so contaminated food maycause disease even after heating. This is assumed to be a very common sourceof mild illness, that is seldom investigated clinically, and therefore thestatistics is uncertain.Vibrio parahaemolyticus is widespread in marine environments and brackishwaters all over the world, especially in areas with warm climate. It isassociated with infections from fish, shellfish, shrimps and other seafood,especially raw food that has not been heat treated. The organism attaches itselfto the small intestine and secretes a toxin. The illness comes after about 24hours and lasts for a couple of days. All the common food poisoning symptomsmay be involved: diarrhea, abdominal cramps, nausea, vomiting, headache,and fever.Aeromonas hydrophila. This organism has only recently been recognised as afood pathogen and there is not much information available on this. However,in several cases it has been isolated from stools of patients with gastroenteritiswithout any other sign of infection. A. hydrophila is a common bacterium insoil and water, even in drinking water pipes. It grows well down to 5°C and itgrows in vacuum packed food. Shrimps, ham, sausages are examples of foodthat has been associated with these infections.Virus. There are several virus infections spread with food and water. Viralgastroenteritis is usually a mild illness characterized by nausea, vomiting,diarrhea, and fever. The infectious dose is not known but is presumed to below. These infections are either spread via contaminated water or food or

4. Foodborne pathogens 50through direct contacts between people. Norovirus is one of these viruses thatcause short but intensive gastroenteritis especially in children. It has previouslybeen named Calicivirus. The virus is present in the feces of infected persons. Itis assumed that only 10 virus particles is enough for an infection and this mayexplain why this disease also is very contagious and not only distributed viafood and water.

51Chapter 5. Food preservationThere are two main principles to preserve food from microbial spoilage (Fig5.1): Inactivate the microorganisms or create conditions which slow down thegrowth rate. The dominating method to inactivate microorganisms in food is byheat (sterilisation and pasteurisation) but to some extent also inactivation byirradiation is used and recently also exposure to high pressure is emerging as afood preservation method. The most important method to reduce the growthrate is by reducing the temperature (refrigeration or freezing) or by reducing thepH or water activity (drying or salting). Addition of chemical foodpreservatives is common. Sterilisation by filtration is important for productionof sterile liquids in the pharmaceutical industry, but this is hardly applicable inthe food industry.Inactivation of microorganisms:Inhibition of microorganisms:Removal of microorganisms:HeatIrradiationHydrostatic pressureChemical disinfectionCooling/FreezingLow a w , pHChemical preservativesFiltrationFig 5.1 Principles of preservation against microbial spoilage.5.1 Heat sterilisation and pasteurisationHeating is the most coming method for killing microorganisms in food. If thisis made with the goal to kill even endospores temperature in the range of 120°C or higher must be used and this can result in real sterilisation, i.e. also theendospores are killed. If the goal is to eliminate the majority of vegetative cells,temperatures in the range of 70-90°C are used and this is called pasteurisation,and it has no inactivating effect on endospores.Mechanisms of heat inactivation of microorganisms.Microorganisms may be classified in two groups with respect to heatsensitivity: 1) Bacterial endospores and 2) Vegetative cells and spores of othertypes, e.g. fungal spores. Endospore formation is mainly found in the generaBacillus and Clostridium, but also Sporosarcina, Desulfotomaculum, Sporolactobacillusand Thermoactinomyces may form endospores. Endospores areextremely resistant to heat, UV and ionising radiation, drying and chemical

5. Food preservation 52agents. It takes heat treatment in the range of 100°C and higher to inactivatethese spores. Note that other spores, e.g. fungal spores, may be quite resistant todrying but they are only slightly more resistant to heating than are thecorresponding vegetative cell. To inactivate these spores and vegetative cells ingeneral, heat treatment in the range of 50 to 90 °C (pasteurisation) may beefficient and it does not provide complete sterility since it has no effect on theendospores.Fig 5.2 shows the main structures of a bacterial endospore. The exactmechanisms behind the extraordinary resistance of bacterial endospores are notknown, though some information is available from mutants lacking differentcomponents in the spore: The spore has three distinctive structures: The core,containing the DNA, a few key enzymes and 2-10% dipicolinic acid (DPA) incomplex with Ca 2+ and the DNA. The core also contains some basic proteinsthat are quickly hydrolysed and serve as amino acid source during thegermination. The water content of the core is low, which together with DPA isassumed to contribute to the large thermal stability of the spore. The basicproteins contribute to the high UV radiation resistance. The surrounding cortexcontains negatively charged peptidoglucans and the water in the cortex is freelyexchangeable with surrounding water. The difference in water concentrationbetween the core and the cortex makes the spore refractile and gives it a lightappearance in a phase contrast microscope, while vegetative cells appear dark.The cortex is surrounded by a spore coat of proteins that confer the chemicalresistance to the spore. The size of a spore is somewhat smaller than thevegetative cell, as indicated in Fig 5.3Core: DNA, Ca-DPA, few ribosomes,key enzymes, no waterCortex: Negatively charged peptidoglucanesCoat: chemically resistant proteinsFig 5.2 Structure of a bacterial endospore.Transformation of a spore to a vegetative cell involves a number of reactions(Fig 5.3). The activation is a reversible reaction, which is poorly understood.Activation may be needed to make a spore competent for the next stage,germination.S.-O. Enfors: Food microbiology

5. Food preservation 53activationgerminationdormant spore activated spore germinated sporelysisoutgrowthsporulationvegetative cellgrowthFig 5.3 The endospore germination-sporulation cycle. The dormant spore may needactivation before the initiation of germination can take place. Activation is a reversibelraction and does not change the resistance or appearance of the spore. At the initiation ofgermination all resistance properties disappear and the spore then grows out to a vegatativecell which divides a number of times until harsh environmental conditions inducesporulation. The spore is eventually liberated by cell lysis.Agents which cause activation are, for instance, sub-lethal heat treatment, highpressure and extreme pH. Spores that are difficult to activate are called superdormant spores. It is difficult to differentiate between super dormant spores anddead spores, since it is only when the spore has been provoked to germinate thatit has been proven that it was not a dead spore. The activation reaction does notresult in any visible change of the spore structure or composition nor anyobservable metabolic reaction.An activated spore may be initiated to germinate by several chemicals likeamino acids, nucleotides etc. The initiation of germination is seen as a swellingof the spore and it is associated with migration of the Ca 2+ ions from the DPAcomplex in the core to the cortex where they neutralise the electronegativelyexpanded cortex, which shrinks and in this process water enters the core whichswells. The germination of one spore takes only a couple of minutes. In a phasecontrast microscope the appearance of the spore is changed from a brightreflecting structure of the ungerminated spore to a dark colour, like that of thevegetative cell, of the germinated spore. Germination of a whole sporepopulation can also be observed as a reduction of the absorbance in aspectrophotometer. The germination process of a whole population of sporesmay be completed as fast as within 15 minutes, but it may also take muchS.-O. Enfors: Food microbiology

5. Food preservation 54longer time. The initiation of the germination is characterised by a completeloss of all the resistance factors of the spore. Electron microscopy reveals thatthe germination is associated with an expansion of the core and a thinning ofthe surrounding cortex (Fig 5.3). During this phase a sequence of metabolicreactions and synthesis of enzymes is initiated.The last phase of the germination is called outgrowth. During this phase, whichtakes about one generation time, all the normal metabolic reactions are restoredand the spore is gradually converted to a vegetative cell.Heat inactivation of the endospore is believed to be a matter DNA damage, butalso heat denaturation of essential proteins in the core may be involved. Heatinactivation of vegetative cells involves quite different reactions, and it ismainly a matter of disorganisation of the cell membrane. This is indicated byseveral phenomena observed in the heat surviving fraction of a population, asfor instance the increased osmosensitivity and increased leakage of cellcomponents. Also DNA damages and denaturation of proteins may be observedduring heat killing of vegetative cells. The thermal resistance of vegetative cellsis also influenced by the level of its heat shock proteins, which participate in theprotection against thermal denaturation of proteins. Since the heat shockproteins may be induced by e.g. thermal (sub-lethal) chock and other stressagents, the thermal stability of a vegetative cell depends not only on theenvironment but also on its history. Also endospore stability depends onenvironmental factors like the composition of the medium during thesporulation. Metal ions like Ca 2+ and Mn 2+ are often required for the endosporeto aquire full heat resistance.Kinetics of heat inactivation of cellsHeat inactivation of spores as well as vegetative cells can be described with thesame mathematical model. Therefore the same methods of calculation may beemployed for sterilisation and pasteurisation.The rate of heat inactivation of a population is proportional to the number ofcells, N. If N represents the number of organisms in the total volume ofmedium to be sterilised, the rate of inactivation becomes:dNdt = "kN (1)where k (min -1 ) is the specific heat inactivation constant, also called the deathrate constant and t (min) is the time. Integration from time zero with the initialnumber of cells (No)!givesS.-O. Enfors: Food microbiology

5. Food preservation 55which can be rearranged to"ln N %$ ' = (kt# N 0 &(2)! ln( N) = ln(N 0) " kt(3)or toN = N 0e "kt (4)!for calculation of the number of surviving cells after a given time. Eq. 3 isillustrated in Fig 5.4. ! Experimentally determined inactivation curves often showdeviations from this model. Some examples of this are shown and explained inFig 5.4.Note that this first order kinetic model does not permit calculation of the timewhen the number of cells reaches zero, which is the time it takes to sterilise asample! However, when N is below one cell (N < 1) the sample is in practicesterile. An interpretation of this is that when N < 1 (i.e. ln(N)

5. Food preservation 56Fig 5.4. Heat inactivation curves. The left hand figure shows two inactivation curves withdifferent death rate constants. The right hand figure shows some deviations from themodel:1. This form may be caused by super-dormant spores, which are activated by the first heattreatment and do not germinate unless they get this treatment;2. This may be observed in samples that contain aggregates of cells, since analysis isusually made by viable count that gives number of colony forming units rather thannumber of cells. The viable count does then not decline until the last cell in an aggregate iskilled. This curve form can also be caused by an experimental artefact, if heat transfer isnot fast enough.3. Non-uniform heat resistance in the population, e.g. when the sample contains specieswith different thermal sensitivity. This is the expected curve for a mixed microflora.Eq. 5 can be only used to calculate the effect of a temperature change on therate of heat inactivation within a limited temperature range, where theinactivation is caused by the same reaction. The constants A and the activationenergy, ΔE, can be obtained from the logarithmic form of the Arrheniusequation:ln(k) = ln(A) " #E 1R T (6)which shows that a plot of the logarithm for the thermal death rate, k, againstthe reciprocal absolute ! temperature will have the slope ΔE/R and an interceptwith the ln(k) axis corresponding to ln(A). See Fig 5.5.1k (1/min)0.1Thiamine!E 92kJ/mole112 °C 96 °C0.012.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.91/Tspores!E 280kJ/moleFig 5.5 Arrhenius plots of inactivationof B. stearothermophilus spores andthiamine. Note that a temperatureincrease has a larger effect on the sporeinactivation rate than on the vitamininactivation rate.The heat treatment does not only cause cell death but also increased rates ofother chemical reactions, which may be beneficial or detrimental to the product.Examples are inactivation of vitamins and other nutrients, lipid oxidation, andso called Maillard reactions. The latter is a group of reactions involvingS.-O. Enfors: Food microbiology

5. Food preservation 57reducing sugars and amino groups and it is an important class of reaction infood processing, including both wanted and unwanted reactions, depending onthe situation. The principle reaction is shown in Fig 5.6.high T, low a w , high pHFig 5.6. A Maillard reaction is a reaction between reducing sugar and an amino acidfavoured by high temperature, low a w and high pH.Depending on which sugars and amino acids that are involved, the productsmay be have different taste, be toxic, and cause colourisation of the product. Alarge group of such mellanoidines are important for the organoleptic propertiesof food.Also chemical reactions like vitamin inactivation and Maillard reactions can bemodelled with first order kinetics. In analogy with eq. 2 :"ln C %$ ' = ( k Ct# C 0 &(7)where C and Co are the time dependent and the initial concentration of thecompound, respectively, ! and kc is the inactivation rate constant. Thetemperature dependence of these reactions also follows the Arrhenius equation(eq. 5) and can be characterised with the activation energy. Table 5.1 lists someexamples of activation energies for inactivation of endospores and for someother chemical reactions. There is a tendency that the activation energy for cellkilling is higher than the activation energy of most of the chemical reactions.This can be utilised to minimise the chemical reactions during sterilisation byapplying continuous sterilisation.S.-O. Enfors: Food microbiology

5. Food preservation 58Table 5.1 Examples of ∆E values for heatinactivation of spores and some chemicalreactions.Inactivation of ∆E (kJ mol -1 )B. subtilis spores 318B. stearothermophilus spores 283Cl. botulinum spores 343Folic acid 70d-panthotenyl alcohol 88Cyanocobalamin 97Thiamine HCl 92Maillard reactions≈125Calculation of sterilisation timeAccording to the model for heat inactivation, eq. 3 and Fig 5.4, it is not possibleto calculate the time needed to reach zero concentration of viable cells. Yet,when the cell number, N, in eq. 3 is below 1, the medium is sterile. If eq. 3 isused to calculate the time needed to reach e.g. 10 -3 cells, it means that there is aprobability that one batch of 1000 sterilised batches will be infected. The timeneeded to reach this probability of sterility does not only depend on the deathrate constant, k, but also on the initial number of organisms, No, as is obviousfrom Fig 5.4. Thus, a sterility criterion, ∇ (nabla) has to be defined:#" = ln N &0%$ N(f '(8)where Nf is the final number of organisms. Eq. 2 can now be used to estimatethe sterilisation time, ! F (min), needed to satisfy the sterility criterion ":F = " k !(9)This sterilisation time depends also on the temperature applied since k is afunction of temperature. The sterilisation time (FT ) required to satisfy the same!sterility criterion at another temperature (T °K) than the reference temperature(Tref °K) at which the sterilisation time Fref once has been assessed, can beobtained from eq. 9 written for the two sterilisation temperatures:" = F refk ref= F Tk T(10)!S.-O. Enfors: Food microbiology

5. Food preservation 59which, after substitution of k according to the Arrhenius model (eq 4), gives thewanted sterilisation time at a different temperature T °K:F T= F refe"#ER$ 1&% T ref" 1 'T)((11)Batch sterilisation!If the death rate constant, k, is known and constant during the sterilisation, eq. 9gives the answer for the sterilisation time required to satisfy the sterilitycriterion. However, in batch sterilisations the temperature is first increasing,then constant, and finally declining (See Fig 5.7). Since the thermal death rateconstant, k, depends on the temperature, this effect must be included in thecalculation. In the beginning of the sterilisation, when the temperature is low,the rate of sterilisation is low. We must then introduce a time dependent relativesterilisation dose ∇ (t)= ln(No/N(t)), during the sterilisation. The sterilitycriterion is then satisfied when ∇(t) = ∇. Combining eq. 9 with eq. 5 gives anexpression that shows how the sterilisation dose depends on time:#$ERT"(t) = t k(T) = t Ae(12)Since the temperature varies with time during batch sterilisation, the totalsterilisation dose is obtained as the integral of eq. 12!t"(t) = A % e #$E / RT dt0( )The batch sterilisation has reached the criterion on sterility when ∇t = ∇. Thiscan be calculated ! without knowledge of the constant A in equation 11.According to eq 4 and eq 8, the sterility criterion can be written(13)!" = F refAe #$E /RT refDivision of both sides of eq 13 by eq 14 gives the ratio between ∇(t) and ∇:t#$E / RT% ( e )dt"(t)" = 0F refe #$E / RT ref( )Fig 5.7 shows an example of a batch sterilisation temperature profile and thesterility according to eq.15. In this example the heating phase is relatively shortand it contributes ! only with some 20 per cent of the total sterilisation dose.Since cooling from the highest temperature first is very efficient, thecontribution to sterilisation from the cooling phase is very small. Note also that(14)(15)S.-O. Enfors: Food microbiology

5. Food preservation 60the exponential dependence of the sterilisation rate on the temperature meansthat good temperature control at the holding phase is important for the precisionof the sterilisation.Fig 5.7. Simulation of the progress of the sterilisation (∇t /∇) and inactivation of atemperature sensible compound (C/Co) during a batch sterilisation. The sterility wascalculated according to eq.15 and the concentration of compound according to eq. 7 and eq.5. Parameters: ∇ =20, A= 1035.8 sec -1 and 109 sec -1 for sterilisation and chemical reaction,respectively. ΔE= 282 kJ mole -1 and 92 kJ mole -1 , respectively. R= 8.31 J mol -1 °K -1 .Continuous HTST sterilisationDuring the low temperature part of the batch sterilisation, the rate ofsterilisation is relatively low, but other chemical reactions, like vitamininactivation, lipid oxidation and Maillard reactions may take place at aconsiderable rate, which is sometimes detrimental for the food quality. Aninteresting property of the sterilisation reaction is that it has a relatively highactivation energy, as pointed out in Table 5.1. The HTST sterilisation (HighTemperature Short Time) utilises the different sensitivity to temperature of thetwo reactions "sterilisation" and "chemical reaction", which was expressed asdifferent values of the activation energy. This is exemplified in Fig 5.7, whichshows how the rate constant for spore inactivation increases much faster with atemperature increase than does the rate constant of thiamine inactivation,because the former has a higher activation energy (slope of the curve).Therefore, the vitamin inactivation will be reduced if an increased sterilisationtemperature is combined with a reduced sterilisation time to give identicalsterility criterion, ∇. This effect of increased sterilisation temperature isdemonstrated in Fig 5.8.S.-O. Enfors: Food microbiology

5. Food preservation 61301FC / Co20F (min)C / Co100115 120 125 130 135 1400145TFig 5.8 Effect of sterilisationtemperature on a chemicalreaction in a continuoussterilisation. C/C o is the fractionof non-reacted chemicalcompound. F is the sterilisationtime according to eq. 9. C/C owas obtained from eq. 7 and thetemperature dependence of thetwo reaction rate constants, k,was obtained from the Arrheniusequation (eq. 5). Parameters asfor the batch sterilisation, Fig 5.7.The D-value and the Z-valueThe theory of heat sterilisation was developed in the food industry during thefirst part of the 20th century. It was then common to use logarithms with thebase of 10 and much literature on sterilisation, and especially constants on heatsensitivity and temperature dependency, are still based on this nomenclature,which uses a D-value and a Z-value to describe the inactivation rate and thetemperature sensitivity of the inactivation rate, respectively.The rate of heat inactivation according to eq. 2 can be written on a 10 log basisas"10 log N %$ ' = ( 1# N 0 & D t (16)where 1/D is the slope of the curve when the number of surviving cells (N) isplotted against time ! during heat exposure (Fig 5.9). The decimal reduction time(D, min), is the time needed to reduce the number of cells to one tenth of theprevious value.S.-O. Enfors: Food microbiology

5. Food preservation 62Fig 5.9 Inactivation curve plotted on 10 log basis showing the definition of the D-value.Solving eq. 2 for t = D and N = No/10 gives the correlation between theinactivation constant k and the D-value:D = ln(10)k (17)D-values for inactivation of spores as well as for inactivation of vegetative cellsare available in ! the literature and, if not found, can be determinedexperimentally by plotting the data as in Fig 5.4 or 5.9. Table 6.2 shows someexamples of D-values. For endospores D-values are often standardised tominutes at 121 °C but for vegetative cells lower temperatures, e.g. 60°C, areoften used.These D-values (and k) depend much on the environment in which the heatingis performed. As a general rule one may say that the heat resistance increaseswith reduced water activity but it decreases when the organism is subjected toother extreme conditions like extreme pH, toxic compounds etc. The effect ofthe water activity means that it may be very difficult to heat sterilise media withsuspended solids like starch, in which spores may stay relatively dry.Sterilisation of dry materials like glass and other equipment requires muchhigher temperature and/or prolonged heating. While water solutions mostly aresterilised by some 15 minutes at 120°C (steam sterilisation) the correspondingsterilisation of dry materials (dry heat sterilisation) may require about 4-6 hoursat 160°C or 1.5 h at 170°C to give similar effect. Data given in this chapterrefers to sterilisation in water solutions.S.-O. Enfors: Food microbiology

5. Food preservation 63Table 5.2 D-values (min) for heat inactivation of microorganismsOrganism D 121 D 60 D 50Endospores (general) 0.1-4Cl .botulinum 0.2Cl. thermosaccharolyticum 10-20Micrococcus spp. 5-20Streptococcus spp. 5-20Fungal spores 5-20Virus 1-10Mesophilic bacteria 1Psychrotrophic bacteria 1-5Psychrophilic bacteria

5. Food preservation 64In analogy with the calculation of sterilisation time at another temperature thanthe reference temperature (eq. 11), it is possible to show that this calculationcan be done based on the Z-value:F T= F ref10 (T ref "T )/ZThe Z-value for endospores is usually in about 10°C while it is lower, about5°C for inactivation of vegetative cells (pasteurisation).!(19)S.-O. Enfors: Food microbiology

5. Food preservation 655.2 Chemical preservationA number of chemical additives is used by the food industry to improvedifferent properties of the food. These additives are usually identified andreferred to by serial E-numbers. Table 5.3 lists different categories. Only theE200 series containing the chemical food preservatives will be described here.Table 5.3 E-numbers for different categories offood additivesColor additives E 100Preservatives E 200Antioxidants E 300Emulgators / thickening agents E 400Inorganic salts E 500Flavour improving agents E 600Sweeteners E 900Starch derivatives E 1400Table 5.5 on next page lists in detail all currently accepted preservatives inSweden. This list is not static and components may be removed or added andit varies somewhat from country to country. For each component there aredetailed specifications on maximum concentration and in which products thespecific preservative may be used.Week organic acids. A closer look on the list shows that a large part of thepreservatives are weak organic acids and their corresponding salts. It is theundissociated acid which is the active component in this category of foodpreservatives even if it often is a salt which is used. With this view, the list oforganic acid preservatives is reduced from 24 to 7 components, as shown inTable 5.4Table 5.4 Week organic acids used as food preservativesCode Active substance Application examplesE26- Acetic acid Antibacterial No conc. limitE28- Propionic acid Antimold. Only in bread and snuffE270 Lactic acid Antibacterial. No conc. limitE20- Sorbic acid Antimold/yeastE21- Benzoic acid Antimold/yeastE296E297Malic acidFumaric acidNo concentration limitSweets, desertsS.-O. Enfors: Food microbiology

5. Food preservation 66Table 5.5 Food preservatives and corresponding E-number.E200 Sorbic acidE232 Sodium ortophenylphenolE202 Potassium sorbateE234 NisinE203 Calcium sorbateE235 NatamycinE210 Benzoic acidE239 HexamethylenetetraamineE211 Sodium benzoateE242 DimethyldicarbonateE212 Potassium benzoateE249 Potassium nitriteE213 Calcium benzoateE250 Sodium nitriteE214 Parahydroxybenzoic acid ethylester E251 Sodim nitrateE215 Parahydroxybenzoic acid ethylester-Na E252 Potassium nitrateE216 Parahydroxybenzoic acid propylester E260 Acetic acidE217 Parahydroxybenzoic acid propylester-Na E261 Potassium acetateE218 Parahydroxybenzoic acid methylester E262 Sodium(hydrogen)acetateE219 Parahydroxybenzoic acid methylester-Na E263 Calcium acetateE220 Sulfur dioxideE270 Lactic acidE221 Sodium sulfiteE280 Propionic acidE222 Sodium hydrogensulfiteE281 Sodium propionateE223 Sodium disulfiteE282 Calcium propionateE224 Potassium disulfiteE283 Potassium propionateE226 Calcium sulfiteE284 Boric acidE227 Calcium hydrogensulfiteE285 Sodium tetraborateE228 Potassium hydrogensulfiteE290 Carbon dioxideE230 DiphenylE296 Malic acidE231 OrthophenylphenolE297 Fumaric acidSeveral organic acids, like acetic acid, are often good carbon/energy sourcesfor microorganisms. However, it is the dissociated ion, e.g. acetate, whichthen is taken up by active transport mechanisms, while the undissociated acidhas the inhibitory effect. This means that the food pH has a large influence onthe effect of this class of food preservatives. Table 5.6 shows the pK a valuesfor some of the most common food preservatives and the relationship betweenpH and concentration of acid.S.-O. Enfors: Food microbiology

5. Food preservation 67Table 5.6. pKa-values for some food preservatives and formula for the acid-baseequilibrium, showing the pH influence on concentration of undissociated acid.Acid pK aAcetic acid 4,8Propionic acid 4,9Lactic acid 4,3Sorbic cid 4,8Benzoic acid 4,2![ ][ ]pH = pK a+ log baseacidThe food preservative acids are all relatively week acids (Table 5.6), whichmeans that at the relatively low pH in most foods, a considerable part of thetotal acid/base system is present as undissociated acid. This is illustrated asillustrated in Fig 5.11 which shows a graphic illustration of the dissociation ofthe acid HA to the base A- and a proton (in H 3 O + )log Clog C tot0pK a0 14HA A -pHOH - H 3 O +Fig 5.11 Diagram illustrating the acid-base equilibrium for the dissociationC tot is the total concentration HA+A - . Not the logarithmic scale.HA " H + + A #The hypothesis that it is mainly the undissociated form of ! the acid which hasthe inhibitory effect is illustrated in the experiments the inhibitory effects ofseveral weak organic acids on E. coli shown in Fig 5.12. In these experimentsthe concentration of undissociated acid was varied either by varying the totalconcentration or by varying the medium pH (see also Fig 5.11). The left panelin Fig 5.12 shows that the growth rate depends on the concentration ofundissociated acetic acid irrespectively whether the concentration was variedby total concentration or pH. The middle panel shows that this resulted inS.-O. Enfors: Food microbiology

5. Food preservation 68reduced intracellular pH from 7.4 at max growth rate to about 6.2 when thegrowth rate was as lowest. The right panel shows that the growth rate declineswith the intracellular pH irrespectively of which organic acid was used toreduce the intracellular pH.10080Growth rate (%)60402000 0.1 0.2 0.3 6.2 6.6 7 7.4 6.2 6.6 7 7.4Undissociated acid (mM) Intracellular pH Intracellular pH= pHo varied / Total conc. 2.5 mM= Total conc. varied / pHo 5.0Propionic acidCinnamic acidSorbic acidBencoic acidFig 5.12 The growth rate of E. coli depends on the intracellular pH which is reduced by theconcentration of undissociated acid. See text above for further explanation.Possible mechanisms behind these effects of undissociated acids areillustrated in Fig 5.13.Fig 5.13 Two mechanisms contributing to the inhibitory effects of undissociated organicacids. Left: The undissociated acid HA diffuses through the cell membrane and dissociatesin the cytoplasm (pH ≈ 7.4), which reduces the pH. Protons are pumped out on expense ofATP. Right: The reduced pH gradient caused by reduced intracellular pH reduces thedriving potential for ATP regeneration in respiration.S.-O. Enfors: Food microbiology

5. Food preservation 69At least two mechanisms may contribute. Only the non-polar undissociatedacid can diffuse through the cytoplasmic membrane. In the relatively high pHin the cytoplasm the acid dissociates and reduces the pH. The cell tries tocontrol the intracellular pH by pumping out protons, which costs energy inform of ATP. Too high diffusion rate of undissociated acid into the celltherefore uncouples energy from growth. It can not be ruled out that inhibitionof enzymatic reactions in the declining cytoplasmic pH also plays a role. Asecond mechanism, relevant for respirating organisms, may be that thedeclining intracellular pH also diminishes the proton gradient over the cellmembrane which is needed for the ATP generation in respiration.Parabens. The different forms of p-hydroxybenzoic acid esters are calledparabens. The inhibitory mechanism of some of the parabens is inhibition ofthe phosphotransferas system for uptake of sugars but other mechanisms arealso likely.Sulfites. A third group of chemical food preservatives is the various forms ofsubstances which form SO 2 . When hydrogensulfites, sulfates or disulfites aredissolved in water dissociation reactions results in small concentrations ofdissolved gaseous sulfur dioxide SO 2 which probably is the activecomponents. Also this compound then diffuses into the cell where it alsoreduces the pH but other more specific interactions with enzymes probablyconstitute the main inhibiting mechanism.Nitrites and nitrates is a group of salts which in water solution generate smallamounts of nitrous acid HNO 2 . It is likely that also these compounds enter thecell by diffusion. The nitrite and nitrate ions per se have no preservativeeffect. Also these ions are common nutrients for microorganisms, e.g. indenitrification reactions. The hypothesis that it is the undissociated nitrousacid rather than nitrite which exerts the inhibition is illustrated in Table 5.7,which shows that irrespectively of the nitrite concentration it is theconcentration of undissociated acid which determines the inhibition.Table 5.7. Maximum concentration of nitrite for growth of Staphylococcus aureusat different pH and corresponding concentration of undissociated nitrous acid.pH-Max concentration of NO 2 Concentration of HNO 2 (ppm)for growth (ppm)6,9 3500 1,005,8 300 1,065,7 250 1,115,2 80 1,13S.-O. Enfors: Food microbiology

5. Food preservation 70The exact mechanisms of nitrous acid inhibition in the cells is not known, butit generates the gas nitric oxide, NO, which is strongly toxic through reactionwith sulfhydryl containing enzymes. The use of nitrate and nitrite as foodpreservative has been controversial. Firstly, nitrate has probably an effect onlythrough its partial conversion to nitrite, a reaction which may be catalysed bymany bacteria through nitrate respiration, which generates an unknownamount of nitrite. Secondly, nitrite can generate the cancerogenicnitrosamines when heated in sour environment. On the other hand, nitrite is anefficient inhibitor of germination of Cl. botulinum spores, and for his reason itis used to increase the safety in products where these spores occur, likepreserved meat and fish products.The fear for botulism in preserved food has resulted in some standards forfood preservation. In chemical preservation it is generally assumed that eitherof the following criteria is sufficient to prevent growth of Cl. botulinum:pH>4.5; NaCl > 8%; or acetic acid > 2.5%.In practice a combination of chemical preserving factors is often used. Fig5.14 shows an example of how much nitrite is needed for the prevention ofthe growth of Cl. botulinum at different pH and salt concentrations.Fig 5.14. 3D diagram showing combined effect of sodium nitrite, salt and low pH on theinhibition of Clostridium botulinum. Areas with absence of a cube symbol means thatgrowth is prevented.S.-O. Enfors: Food microbiology

5. Food preservation 715.3 Classification of preserved foodsPreserved food products are classified in categories which demand specificstorage conditions. See Table 5.8.Fully preserved food. To this category belongs heat sterilised canned food.The product must be hermetically contained, usually in glass or metal cans.Canned soups, ham, vegetables and fruits belong to this group. The sterilityshall guarantee that these products have a shelf-life of minimum one year atroom temperature. In reality the shelf-life is usually longer. Since it is notlimited by microbial growth, it is usually rancidifiaction or other chemicalreactions which limits the shelf-life. To reduce oxidation reactions, theseproducts may have been supplemented with antioxidants. The concept“commercially sterile” is sometimes used in food sterilisation. It means thatthe processing has eliminated all endospores which can germinate and growout at room temperature, but there may still be a few extremely heat resistantspores of thermophilic Clostridium spp. These cells will not spoil the productunder the storage condition “room temperature”.Table 5.8. Classification of food preserves and storage conditionsCold-stored preserves is another type of preserved food, which may also becanned or packed in plastics. They are either pasteurised, rather than heatsterilised and they are usually further preserved by chemical preservatives.The shelf-life demand is minimum half a year at refrigerator temperature. Foreach product the maximum storage temperature should be indicated on thepackage. Fish preserves are common in this category and the storageS.-O. Enfors: Food microbiology

5. Food preservation 72temperature is usually maximum 4°C. The shelf-life is usually limited byrancidification and/or by slow growth of lactic acid bacteria, especiallyPediococcus and yeasts.Frozen products is a large category of preserved food, which is usuallypreserved only by the low temperature which shall be below -18°C. Theshelf-life of these products are usually limited by rancidification. To reducethe activity of certain endogenous enzymes in the product, vegetables andfruits are sometimes blanched, i.e. subjected to a short heat treatment, beforethe freezing.S.-O. Enfors: Food microbiology

73Chapter 6. Fermented foodsAll food raw materials are contaminated by microorganisms, which take part inthe mineralisation of organic materials in Nature. Therefore, Man had early tolearn to live with microbially infected food. The microbial reactions mostlyresulted in spoilage of the food. However, Man learnt to handle some foods inways that extended their shelf-life. These preservation methods were mainlybased on drying or fermentation. Food fermentations are still used to produce socalled fermented food, but today preservation is not the main objective of thefermentation, but it is rather the specific taste and texture that is the goal of thefermentation. Food fermentation is applied to a all main types of food, as meat(sausage), milk (cheese and yoghurt), grains (beer and bread), fruit juice (wine)and vegetables (sauerkraut and pickles). In Africa and Eastern Asia many othertypes of food fermentation are applied. For a European, the most well-known ofthese products is soy sauce, which is produced by fermentation of soy, sometimessupplemented with rice. Table 6.1 lists the main types of fermented food in theWestern world together with the main biochemical reactions employed in thesefermentations.In most food fermentation the basis of fermentation control is inoculation andadjustment of the oxygen concentration and the water activity:1) Inoculation with a microflora. In traditional fermentations the inoculum was acontamination from earlier production via the equipment or addition of someproduct that had already been fermented. Some processes still rely on thespontaneous natural microflora. This method is now gradually replaced by the useof pure starter cultures, as the production becomes more industrial, sinceinoculation increases the control and reproducibility of the process.2) Adjustment of the oxygen concentration. Ethanol fermentations are inhibited byoxygen and therefore require un-aerated conditions. Lactic acid bacteria areindependent on the oxygen concentration, but since anaerobic metabolism ofcompeting organisms is slower than aerobic metabolism, also lactic acidfermentation is favoured by anaerobic conditions. Acetic acid fermentationsrequire oxygen. Also moulds, which are important producers of hydrolyticenzymes in some food fermentation, are obligately aerobic organisms.3) Reduction of water activity. Several food fermentation processes are controlledby reduction of the water activity by addition of salt. This is the case infermentation of meat, fish, vegetables and soy sauce (the lactic acid stage). Thebackground to this is that lactic acid bacteria, which are active in thesefermentations, are relatively resistant to reduced water activity and therefore arefavoured in this environment. In sausage fermentation the salt is mixed with theminced meat and in the other cases the raw material is placed in a salt brine.

6. Fermented foods 74Table 6.1 Fermented foods, their raw materials and main biochemical reactionsRaw material Products Main type of reactionMeat Sausages Lactic fermentationFish Sour herring Enzymatic hydrolysis and lacticfermentationMilk Cheese Enzymatic hydrolysis and lacticfermentation and (sometimes mold)fermentationYoghurtFermented milkLactic (thermophilic) fermentationLactic (mesophilic) fermentationButter Lactic fermentation 1)Vegetables Sauerkraut Lactic fermentationPicklesLactic fermentationCereals Bread Ethanol fermentationBeerSoy sauceEnzymatic hydrolysis and ethanolfermentationEnzymatic hydrolysis by moulds, lactic andethanol fermentationFruits Wine Ethanol (and malo-lactic) fermentationCiderVinegarCocoaCoffeeOlivesEthanol fermentationEthanol and acetic acid fermentationEthanol and acetic acid fermentationMicrobial pectin hydrolysisLactic fermentation1) In some countries the cream is fermented before the churning of butter to providediacetyl as aroma compound.6.1 Beer brewingProduction of beer by ethanol fermentation of grains dates back to at least 4000BC, when it was applied in Egypt. In the ancient beer production lactic acidfermentation probably played a role, and certain beer types are still produced withmixed cultures of yeast and lactic acid bacteria. Hops was introduced as an aromacompound and preservative during the 15th century. Around 1840 the lager typeof beer was introduced in Bavaria, characterised by slow fermentation at lowtemperature (below 10 °C) and maturation before bottleing.

6. Fermented foods 75The beer brewing process is outlined in Fig 6.1. It contains a large number ofbiochemical reactions. The raw materials of beer are malt, sometimes suppliedwith other grains called adjunct, hops and water. Yeast, either Saccharomycescerevisiae or Saccharomyces uvarum, is added as a biocatalyst and sometimesalso additional enzymes of microbial origin are added to improve the enzymaticreactions.Fig 6.1 Summary of the beer production process.Malting. The first stage of beer production is the malting of barley. The barleyshould be of low nitrogen type, as opposite to the fodder barley. The grains arefirst soaked in water in a steeping process during about two days to raise thewater content to 45%, which initiates sprouting of the grains. The grain content ofgiberellic acid is important for the resulting germination. This germinationinvolves respiration, and the grains must be aerated to provide oxygen and removethe carbon dioxide. Since the reaction is exothermic cooling must also be providedand the grains are mechanically turned to provide homogeneous conditions.During the malting process many of the barley enzymes are activated and start to

6. Fermented foods 76hydrolyse the grain: Hemicellulases, proteinases, α- and ß- amylases. Roots alsodevelop from the grain during the germination, which may take about 4-6 days tobe completed.The germination and the emerging enzymatic reactions are interrupted by thekilning, in which the temperature is gradually raised to 65-85 °C. During thekilning, the high temperature results in Maillard reactions between reducingsugars and amino-groups, that colour the malt, darker the higher the temperature isused. This is the main way of controlling the beer colour. Maillard reactionproducts also contribute to the taste of the malt and the beer. During the kilningthe water content is reduced so the malt can be stored for later use. Thus, malt canbe considered as a package of hydrolytic enzymes, notably α- and ß-amylases,packed with the enzyme substrates, mainly starch. The last stage of the malting isthe removal of the rootlets which, like most other by-products from the beerproduction, are used as fodder. Malt is not always produced by the brewer, butoften obtained from specialised malting companies.Table 6.2 Composition of barley grains beforeand after maltingCompound % in barley % inmaltStarch 64 59Sugar 2.5 9ß-glucans 9 7Cellulose 5 5Amino acids andpeptides 0.5 1.5Mashing. The malt is milled, coarsely to facilitate the later separation of the husk.The milled malt is mixed with hot water to extract starch and enzymes from thegrains in the mashing process at about 65 °C. Some brewers supply additionalstarchy materials, adjuncts, that are cheaper than malt, like maize, wheat or rice.Even sugar may be used. This also reduces the protein concentration of the wort,which may be an advantage if the malt is too protein rich, since proteins maycause problems with precipitations in the beer. On the other hand, the use ofstarchy adjuncts requires higher enzyme activity in the malt.Starch is composed of amylose, that is a straight chain of α-1,4-linked polyglucose,and amylopectin which besides α-1,4 bonds also contains branchingpoints with α-1,6 bindings (Fig 6.2). During starch hydrolysis α-amylaserandomly hydrolyses α-1,4 bonds between the glucose units in the starch, whichresults in smaller poly-glucose molecules called dextrins. Thus, hydrolysis by α-

6. Fermented foods 77amylase gradually reduces the mean molecular weight and the viscosity of thestarch solution but little fermentable sugar is produced in this reaction.Fig 6.2. Hydrolysis of amylopectin to dextrins, maltotriose and maltose by α-amylase and ß-amylase. Both enzymes hydrolyse at the α-1,4 site leaving thebranching α-1,6 sites in low molecular weight dextrins. Oligosaccharides larger thanmaltotriose are not fermented by the yeast.The ß-amylase hydrolyses α-1,4 bindings two glucose units from the nonreducingterminal of amylopectin, amylose or dextrin to produce the disaccharidemaltose, which is the main fermentable sugar in the wort (Table 6.4). Thus, thelonger the mashing continues the higher becomes the concentration of fermentablesugar. However, these enzymes can not hydrolyse the branching points (α-1,6bonds) of the amylopectin and therefore small branched dextrins are left. Thesedextrins are not fermentable and they remain in the beer and contribute tosweetness and viscosity of the product.Additional enzymes like proteases or ß-glucanases, may also be added to improvethe proteolysis or the ß-glucan hydrolysis. Pullulanase, a debranching enzyme thathydrolyses α-1,6 bonds in the amylopectin, may also be used to increase theconcentration of fermentable sugar from the starch.

6. Fermented foods 78Table 6.3 Temperature and pH optimaof the main malt enzymesEnzyme pH Temperatureα-amylase 5.7 70ß-amylase 5.5 60ß-glucanase 5.1 57proteinase 4-5 40-50These enzymes have different temperature optima (Table 6.3). During themashing different temperature programmes can therefore be used to control thehydrolysis of the macromolecules. The proteolysis should furnish the wort withamino acids for the growth of the yeast during the fermentation but it should alsodegrade proteins that would otherwise precipitate in the beer. Likewise, the ß-glucanolysis is important to reduce later precipitations and it yieldsoligosaccharides. The main reaction during mashing is the degradation of starch tofermentable sugars and non-fermentable dextrins. A typical composition of thewort is shown in Table 6.4Table 6. 4. Components of starch hydrolysis in wort.Product % of total starchMaltose 51Maltotriose 12Glucose 9 FermentableFructose 2Sucrose 2Maltotetrose 3 Non-fermentableDextrins 21The enzymatic hydrolysis is interrupted by boiling of the wort for 1-2 hours. pHhas then dropped from 5.8 to 5.4. Before this, the husks and precipitated proteinsare removed from the wort and hops are added. It is the dried non-fertilized femaleflower of Humulus lupulus that is used. Today also pelleted hops and even hopsextract is used by the brewer. During the subsequent wort boiling, aromaticcompounds are extracted from the hops, some unwanted aroma compounds areevaporated, all enzymatic activity ceases and the wort becomes essentially sterile.Hops contain two main types of flavour compounds: humulones (the so calledalpha acids) and lupulones (called beta acids).Fig 6.3 Basic structureof the humulones of hops.

6. Fermented foods 79The molecules isomerise during the wort boiling which makes them more watersoluble and more bitter. Negatively charged tannins are also extracted from thehops and they form precipitate with proteins. After the wort boiling the hopsresiduals are separated off together with the precipitated proteins and used asfodder. The so clarified wort is cooled and inoculated with yeast.Fermentation. The fermentation process is performed in a batch according toeither of two principles. In top fermentation Saccharomyces cerevisiae is used.This yeast flotates to the top when the fermentation has ceased due to lack offermentable sugar. The bottom fermentation processes utilise Saccharomycesuvarum (carlsbergensis) which sediments to the bottom after the fermentation.Bottom fermentation is typical for lager beer and pilsner and it is performed atlow temperature: 5-10 °C for about one to two weeks, until all visiblefermentation has ceased. Top fermentations is applied to produce the beers of ale,stout and porter type and this fermentation is made at higher temperature, around20°C, which results in more ester production.N*10 -6 /mL60E (%)5EtAc (mg/L)50NEEtAc00 50 100 150Time (hrs)Fig 6.4 Progress of a lager beer fermentation at 10°C. N = yeast cellnumber; E = ethanol concentration; EtAc = concentration of ethyl acetate.00During the fermentation, the yeast biomass concentration increases about fourtimes (Fig 4.4). Cells separated from the beer after fermentation are partly used toinoculate next batch and partly used as fodder. To permit growth of the yeastduring the conditions in the wort, oxygen must be available for synthesis of cellmembrane constituents. Therefore the wort is saturated with oxygen from airbefore inoculation. This oxygen is quickly consumed by the cells and then theprocess is strictly anaerobic. From this time in the process much effort is focusedon keeping the beer free from oxygen since the shelf-life is strongly reduced by

6. Fermented foods 80oxidations in the beer. All fermentable carbohydrates (Table 6.4) are convertedduring the fermentation to biomass carbon dioxide, ethanol and other organiccompounds that contribute to the taste. Since the yield coefficient for ethanol frommaltose is about 0.5 g/g, the final alcohol concentration can be predicted from theconcentration of wort used to start the fermentation. However, it depends also onthe extent of the starch hydrolysis to fermentable sugars. To make a low-caloricbeer there is only one way: reduce the wort concentration, since most of theenergy of the sugar is preserved in the ethanol. Depending on the extent of starchhydrolysis, the low caloric beer can either be a low alcohol beer with a normalalcohol to dextrin ratio or a low dextrin beer with normal alcohol content.Ethanol is a major contributer to the taste of beer, but minor quantities of organicacids, higher alcohols, esters and other aroma compounds are also produced andmake important contributions to the taste of the beer. However, also less pleasantcompounds are produced and for this reason a post-fermentation process isincluded. One of these unwanted compounds is diacetyl. It is not produceddirectly by the yeast cells, but α-acetolactate is secreted by the cells during thelater phase of the primary fermentation (see the ethyl acetate curve in Fig 6.4) andthen spontaneously decarboxylated to diacetyl.The main fermentation results in a "green" beer which must be matured in a postfermentationprocess at 0 - 10 °C before use. Lager beer is generally matured fora longer period, 2 weeks to 2 months at a temperature close to 0 °C, while ale isstored at higher temperature for a much shorter period of time. Many lesscharacterised reactions takes place during the post- fermentation. One of theproducts from the main fermentation, α-acetolactic acid, spontaneouslydecarboxylates to diacetyl, which is considered unpleasant in beer. However,during the late stage of the fermentation, and further during the post fermentation,this diacetyl is resorbed by the remaining yeast cells, and the concentration ofremaining diacetyl is sometimes used as a measure of the post-fermentationprogress. A problem in this process is that it is the decarboxylation of the α-acetolactate that is the rate limiting step. New technology has been developed toachieve the postfermentation by means of an accelerated decarboxylation inducedby continuous heat treatment in a heat exchanger followed by diacetyl removal byimmobilised yeast in a packed bed column. In this way, the post fermentationreactions can be accomplished with about 2 hours residence time during whichalmost all diacetyl is resorbed by the cells.After the post-fermentation the beer is clarified by centrifugation or filtration. Toreduce effects of microbial infections, the beer is often pasteurised or sometimessterile filtered. It is mainly other yeasts and lactic acid bacteria that can interferewith beer during storage, due to the low pH, the alcohol content and the highpartial pressure of carbon dioxide. As long as these infections can be avoided the

6. Fermented foods 81shelf life of some 3-6 months is mainly limited by oxidation reactions. To reducethese reactions ascorbic acid is commonly added as an anti-oxidant in beer.6.2 Fermented milk productsLactic acid bacteria is a group of species that are characterised by fermentation ofsugar to lactic acid. The group is divided into two categories, homofermentativeand heterofermentative lactic acid bacteria, depending on whether the metabolismyields mainly lactic acid (homofermentative) or also considerable amounts ofacetic acid, ethanol and carbon dioxide is formed (heterofermentative). Thisclassification is not strict, since cultivation conditions may influence the productpattern. Table 6.5 lists typical representatives of lactic acid bacteria in thesegroups.Table 6.5 Lactic acid bacteria classification according tothe product patternHomofermentative HeterofermentativeLactococcus spp . (all) Leuconostoc spp .(all)Pediococcus spp. (all) Lactobacillus spp. (some)Lactobacillus spp . (some)The lactic acid bacteria play an important role in fermentation of food. Table 6.1shows that they are involved in fermentation of milk, meat, fish and vegetables. Inthese cases the lactic acid fermentation plays an important role to stabilise theproduct against microbial spoilage. The mechanism of this food preservationeffect is not at all generally known. It is well known, however, that many lacticacid bacteria, when grown in mixed culture in the laboratory, are verycompetitive. This competitiveness has been ascribed a number of factors likeproduction of inhibitors and resistance against low pH and low water activity (a w )as depicted in Table 6.6.Table 6.6 Competition advantages associated with lactic acid bacteriaAntagonistic productsLactic acidAcetic acidHydrogen peroxideAntibiotics, e.g. nisin and reuterinProperties of the bacteriaUbiquitous on food raw materials (inoculum)Oxygen indifferentRelatively fast growingTolerant to carbon dioxideTolerant to low pHTolerant to low a w

6. Fermented foods 826.2.1 Fermented milk and yoghurt.Fermentation of milk with lactic acid bacteria is probably the oldest method topreserve milk. It is widely used all over the world, probably because it has beenthe safest way to consume milk. Milk that is not quickly fermented with lacticacid bacteria soon becomes infected with a number of potentially pathogenicbacteria. Only lately has it become possible to store non-fermented milk safely forseveral days in refrigerators. Milk is fermented with lactic acid bacteria in manydifferent ways in different countries. Here only two types of fermentation will beconsidered: a mesophilic fermentation employing a mixture of Lactococcus spp.and yogurt, that is a thermophilic fermentation employing Lactobacillus spp. aswell. These two types are summarised in Table 6.7 and Fig 6.4.Note that the Lactococcus genus in older literature is called Streptococcus. Onlythe so called lactic streptococci are re-named Lactococcus. Streptococcus of theenteric, viridans and pyogenes types are still classified in the Streptococcus genus.The mesophilic fermentation employs two types of Lactococcus spp.; theacidifiers Lactococcus lactis and Lactococcus cremoris, which arehomofermentative and have the task to quickly reduce pH and produce lactic acid,and the heterofermentative aroma bacteria Lactococcus diacetilactis andLeuconostoc cremoris, which are slow fermenters but produce diacetyl, which is adesired aroma contributor in dairy products. The species mentioned in Table 6.7are used by Swedish dairies, but many variants of this concept may be utilised.The American fermented buttermilk, Swedish filmjölk, Danish ymer and Finnishvilli belong to this category . Villi is, however, also inoculated with a surfacegrowing mould, Geotrichium candidum, that contributes to the flavour and thesurface crust.Lactobacillus spp. are generally slower to initiate the lactic acid fermentation, butthey are more resistant to low pH. Reduction of pH inhibits the glycolysis in allstarter organisms but Lactococcus spp stop the fermentation at about pH 4.5,while the Lactobacillus fermentation continues to pH 3.9. Thus, pH in theLactococcus fermented milk is higher than in yogurt.Table 6.7 Examples of two starter cultures forfermentation of milkMesophilic (20°C) Thermophilic (44°C)"Filmjölk"YogurtLactococcus lactisLactococcus cremorisLactococcus diacetilactisLeuconostoc cremorisLactococcus thermophilusLactobacillus bulgaricus

6. Fermented foods 83Another difference between the two types of fermented milk is the consumption oflactose. The starter culture is inoculated to a concentration of about 10 6 -10 7cells/ml which grow to about 10 8 -10 9 cells/ml. For this purpose lactose is used asthe energy source. The organisms of the yogurt starter culture do hydrolyselactose to glucose and galactose, but only glucose is consumed leaving thegalactose. Since the total biomass produced is similar or even higher in yoghurt,the result is that yoghurt has lower concentration of lactose than the commonmesophilically fermented milk (Fig 6.5).This may be of significance in many partsof the world, since adults generally do not accept too much lactose. The so calledlactose intolerance among adults, expressed as abdominal pains and diarrhoeabecause of inability to hydrolyse the lactose in the intestines, is unevenlydistributed over the world. Generally, North Europeans and the white populationin America have a large tolerance to lactose while Asians and Africans generallyhave very low lactose tolerance.Many alternative species of lactic acid bacteria are used for fermentation of milk,sometimes with the claim to give a more healthy product. The basis of theseproperties would be that the cells colonise the intestine. Examples of such starterorganisms are Lactobacillus acidophilus, which grow very slowly compared toother starter bacteria, and Bifidobacterium spp., which is frequently isolated fromthe gastrointestinal tract. Other fermented milk types, like kefir and koumisscontain yeast species, e.g. Candida spp and Saccharomyces spp , which contributeto the flavour by production of alcohols and esters in very small quantities.Fig 6.5 Schematic presentation of the lactose consumption in a fast thermophilic yoghurtfermentation and mesophilic 'filmjölk' fermentation with a Lactococcus based starter culture.

6. Fermented foods 846.2.2 Cheese. Like beer production, manufacturing of cheese is a combination ofenzymatic and microbial processes and the origin of the product dates back toprehistoric times. The main steps of hard cheese production is outlined in Table6.8. However, the variety of cheeses available on the market is reflected by a largenumber of process variations. Only some common features and examples fromtwo main types of hard cheeses and the mould fermented cheeses will be treated.The milk selected for cheese production is pasteurised (with some exceptions) atfor instance 72°C for 15 seconds. It is extremely important that it is antibiotic free,since the starter cultures used are very sensitive to antibiotics. Especiallypenicillin, which is often used to treat mastitis, may accidentally be present in themilk. Lactic acid bacteria are extremely sensitive to penicillin. Antibiotics in themilk may delay the lactic fermentation and it gives the opportunity forClostridium spores to germinate. Especially Cl. tyrobutyricum is a problem and aspore concentration below 10 spores per 100 ml milk is required. Clostridialgrowth in cheese may cause excessive gas production, butyric acid off-flavour andeven health hazards. Thus, special quick-test kits have been developed to analysethe presence of antibiotics in the milk before cheese production.Table 6.8 Main stages of cheese productionActionMain purposePasteurisation Inactivate pathogenic andcompeting organismsFermentationAddition of rennetCutting and pressingof curd, wheyseparationStoringReduce pH.Produce lactic acidProduce cells for later functionHydrolyse and precipitatecasein to a curdFormation of the cheeseMaturation of the cheeseCow's milk contains about 87% water. The main ingredients of the dry matter areshown in Table 6.9. Cheese is composed mainly of the caseins, except for part ofthe κ-casein that is removed by enzymatic hydrolysis, the fat and part of the salts.

6. Fermented foods 85Table 6.9. Main ingredients of cow's milkComponent Concentration (%)Water 87Lactose 5Fat 3.8Protein 3.4Caseins 2.8α- 1.7β- 0.6γ- 0.1κ- 0.4Whey proteins 0.6albuminsglobulinsSalts 0.9Calcium-Citrates-The milk is inoculated with starter cultures that have much concordance withthose used to produce fermented milk. Two main types may be distinguished forhard cheese production: The Emmentaler and Gruyère type of cheese is based onthermophilic Lactobacillus and Propionibacterium mixture while the Cheddarand Gouda type is based on a mesophilic Lactococcus mixture (Table 6.10). thepurpose of the fermentation is to initiate the casein precipitation by reduction ofpH and to provide cells which are entrapped in the precipitated curd to take part ofthe later maturation process.Also the soft cheeses like Camembert, Brie, Roquefort, Stilton and Gorgonzolaare started with Lactococcus mixtures but they are also inoculated with a mouldspecies before the maturation and the action of these organisms takes place duringthe maturation(Table 6.11). Since moulds are obligate aerobes, they grow only onthe surface, unless the cheese is perforated by holes.Table 6.10 Examples of starter cultures for cheese productionCheddar / GoudaEmmentaler / GruyèreLactococcus cremoris Lactococcus. thermophilusLactococcus lactisLactobacillus helveticusLactococcus diacetylactis Lactobacillus lactisLeuconostoc spp.Lactobacillus bulgaricusPropionibacterium shermaniiThe declining pH during the fermentation contributes to precipitation of casein atits isoelectric point 4.6, as in the case of milk fermentation. However, proteases

6. Fermented foods 86are also added to the milk during cheese manufacturing, and these enzymescontribute to an efficient precipitation of the main part of the casein. The majorprotease preparation is calf-rennet, which is an enzyme extract from youngcalves. The proteases of rennet are mainly chymosin (rennin) and pepsin. Whenthe calf grows older the proportion of pepsin increases, which makes the extractless useful for cheese production, since pepsin hydrolysis is too extensive whichreduces the curd yield. A relative lack of calf rennet has provoked thedevelopment of microbial proteases for cheese production. Such microbial rennetis in extensive use in some countries. Calf chymosin has been cloned to a yeast,Kluyveromyces sp., to produce chymosin in bioreactors. The process has beenscaled up and introduced on the market.Table 6.11 Mould species used for maturation cheesesCheese type Example Mold specie (example)White moulded cheeses CamembertBriePenicillium camembertiBlue-vein cheesesRoquefortGorgonzolaStiltonPenicillium roquefortiThe casein is present in milk as colloidal micelles of very complex structure asillustrated in Fig 6.6. The micelles with a diameter around 100 nm, are composedof submicelles. The inner part of the submicelle is composed of α- and ß- caseinswhich interact by their hydrophobic parts and via Ca 2+ ions also between theirhydrophilic parts. The stabilisation of the submicelle is achieved by a surfacelayer of κ-casein which is divided into a very hydrophilic part, turned outside, anda hydrophobic part turned inwards the submicelle.The main action of the rennet enzymes is a selective cleavage in the regionbetween the hydrophobic and hydrophilic parts of κ-casein. This removes thehydrophilic surface layer from the micelle which, by hydrophobic interactions,start to aggregate and precipitate as the cheese curd. Thus, the hydrophilic parts ofthe κ-casein make up the whey proteins together with the globulins and thealbumins. The whey also contains the lactic acid and most of the remaininglactose. It is important that the cells and some of the rennin enzymes and a littlelactose are entrapped in the curd, since they form the basis for the maturationprocess.

6. Fermented foods 87Fig 6.6 Schematic illustration of the composition of a casein submicelle in milk. The caseinmolecules have characteristic hydrophobic (dotted) and hydrophilic (white) regions. ß- caseinforms chains which are interlinked by hydrophobic interaction. α-casein binds to thehydrophobic areas of this chain and Ca 2+ ions stabilises the complex by ionic bindings betweenthe hydrophilic parts. Finally, the submicelle increases its hydrophilicity of the surface byattracting κ-casein units which bind their hydrophobic ends inwards against the hydrophobicsites. Chymosin and pepsin act by specific hydrolysis in the region between hydrophilic andhydrophobic parts of κ-casein, thus exposing a hydrophobic surface. The degraded micellesstart to interact by hydrophobic binding and precipitate as a cheese curd.The precipitated curd is cut in pieces, separated from the whey, washed andpressed etc., according to different procedures for the different types of cheeses.During the subsequent storing for some months, a large number of biochemicalreactions takes place to give the product its special texture and taste. First thestarter culture cells resume a slow growth, since the pH, that had declined to stopthe glycolysis during the initial fermentation, is increased after removal of most ofthe lactic acid with the whey. During this stage heterofermentative lactic acidbacteria or propionic bacteria produce gas that is entrapped in the cheese to givethe characteristic holes. Heterofermentative lactic acid metabolism also results indiacetyl formation which is important for the flavour, as is the lactic acid and insome cases propionic acid and other microbial products of the primarymetabolism.Eventually the microorganisms die and lyse, thus releasing proteases and lipases.These enzymes, together with the traces of the rennet proteases and the lowactivity extracellulary cell bound proteases of the lactic acid bacteria induce a veryslow proteolysis and lipolysis that produce peptides and fatty acids to contributeto the flavour. Furthermore, in mould inoculated cheeses, extracellular proteasesand lipases gradually diffuse from the mycelium to slowly soften and mature thecheese. Moulds also produce lipoxydases, enzymes that catalyse oxidativedegradation of fatty acids which results in methylketones. Among these

6. Fermented foods 88degradation product are 2-heptanone and 2-nonanone considered to be especiallyimportant for the cheese flavour.6.3 Fermented meat productsIn Europe, fermented meat is mainly found in some types of sausage, like thesalami and many other of the hard, often smoked sausages. Meat is normallycontaminated by an aerobic psychrotrophic flora dominated by Pseudomonas spp.that normally spoil the meat by growth on the surface. When meat is minced thisflora is mixed into the product and it is furnished by a surplus of nutrients fromthe damaged meat cells. Thus, minced meat is extremely sensitive to microbialspoilage. However, since long time ago, Man learnt that if the minced meat wassalted and stuffed in a gut it did not develop the unpleasant odour but stabilisedand could be used as food for very long time. This is still the basic procedure inproduction of fermented sausages.There are several mechanisms that stabilise the meat in a fermented sausage:Addition of salt reduces the water activity to prevent the Pseudomonas spp. todevelop. These organisms are especially sensitive to reduced water activity, whilelactic acid bacteria are especially tolerant in this respect. It is also essential thatlactic acid bacteria are present in the mixture so that lactic acid is quicklyproduced and pH declines. This prevents organisms of the Enterobacteriaceaefamily, Clostridium spp. and Bacillus spp., which are normally present at lowconcentrations, to develop. Traditional formulations often included garlic orspices with antimicrobial compounds that further increased the stability. Toincrease the safety with respect to the dangerous Clostridium botulinum , alsonitrite is added nowadays, since the undissociated acid, HNO 2 , is known to bevery efficient in preventing endospore germination. Actually, the name botulinumcomes from Latin botulus = sausage, since botulism was formerly oftenassociated with infected sausages. The package of the sausage, originally gutsfrom animals but nowadays often synthetic materials, also protects the meat frominfection during the storage. It is quite common that the surface becomes coveredwith certain species of mould during the storage. This growth of moulds is evenutilised for the processing in certain case, like the production of Salami.In the traditional procedures the inoculum was obtained either automatically fromthe not too clean vessels used to mince and mix the meat. Some formulations alsocontain milk or other sources of lactic acid bacteria. It is also common to mix oldproduct into the fresh unfermented mixture to inoculate the meat. Today it hasbeen common practice to use starter cultures to make the process more safe andreproducible. Lactobacillus plantarum, Pediococcus spp , Lactococcus spp. andin some cases even Micrococcus spp are used as starter cultures for fermentation

6. Fermented foods 89of sausages. After the fermentation the sausages are often smoked, which furthercontributes to the preservation of the product.6.4 Fermented vegetablesVegetables are not fermented to a large extent in Europe. Some common productsare sauerkraut, that is fermented cabbage, pickles, that is a mixture of fermentedvegetables and fermenter cucumber. The history of fermented vegetables is,however, probably as old as that of the other fermented foods. It is documentedthat large scale fermentation was applied to furnish the workers with food duringconstruction of the Great Chinese Wall during the third century BC.The microbiology of fermented vegetables is not so well documented as that ofbeer, wine or cheese production. It is also just recently that starter cultures hasbeen adopted. The common method is still to place the vegetables in a 3-6% saltbrine and wait till the natural flora starts the fermentation. This takes some timesince the lactic acid bacteria are present only at very low concentrations.Meanwhile, the main flora that may be Enterobacteriaceae members and Bacillusspp. are retarded by the salt and they gradually die. Typically, this fermentationstarts with Leuconostoc mesenteroides and is followed by Lactobacillus brevis,that can ferment pentoses and Lactobacillus plantarum that is the main acidproducer. Also Pediococcus cerevisiae is commonly found in fermentedvegetables. After the main fermentation a slow post-fermentation by yeasts iscommonDuring the fermentation, that may take a couple of weeks, the low concentrationof fermentable sugars is further reduced, which is part of the stabilisation of theproduct against other microorganisms. Lactic acid is also produced atconcentrations determined by the available sugar concentration. 1-2% lactic acidis achievable. The product obtains special characteristics not only by the taste ofthe acid produced, but also by the effect of the low pH that makes the vegetablecrispy. Another important function of the fermentation is that it may inactivatesome of the plant enzymes, like pectinases, that otherwise would hydrolysepectins to soften the product.