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Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth.

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1 Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth

2 As our foods are of plant and/or animal origin, it is worthwhile to consider those characteristics of plant and animal tissues that affect the growth of microorganisms. The plants and animals that serve as food sources have all evolved mechanisms of defense against the invasion and proliferation of microorganisms, and some of these remain in effect in fresh foods.

3 INTRINSIC PARAMETERS The parameters of plant and animal tissues that are an inherent part of the tissues are referred to as intrinsic parameter. These parameters are as follows: 1. pH 2. Moisture content 3. Oxidation–reduction potential (Eh) 4. Nutrient content 5. Antimicrobial constituents 6. Biological structures

4 1. pH It has been well established that most microorganisms grow best at pH values around 7.0 (6.6–7.5), whereas few grow below 4.0. Sensitivity to acidity: Bacteria > yeasts> molds



7 pH minima and maxima of microorganisms should not be taken to be precise boundaries, as the actual values are known to be dependent on other growth parameters. For example, the pH minima of certain lactobacilli have been shown to be dependent on the type of acid used, with citric, hydrochloric, phosphoric, and tartaric acids permitting growth at a lower pH value than acetic or lactic acids.



10 With respect to temperature, the pH of the substrate becomes more acid as the temperature increases. salt Concentration of salt has a definite effect on pH growth rate curves, where it can be seen that the addition of 0.2 M NaCl broadened the pH growth range of Escherichia coli. However, when the salt content exceeds this optimal level, the pH growth range is narrowed. Young cells are more susceptible to pH changes than older or resting cells. When microorganisms are grown on either side of their optimum pH range, an increased lag phase results.

11 Some food such as fruits and vinegar fall below the point at which bacteria normally grow. It is a common observation that fruits generally undergo mold and yeast spoilage, and this is due to the capacity of these organisms to grow at pH values <3.5, which is considerably below the minima for most food-spoilage and all food- poisoning bacteria. Most vegetables have higher pH values than fruits, and, consequently, vegetables should be subject more to bacterial than fungal spoilage.


13 Most of the meats and sea foods have a final ultimate pH of about 5.6 and above. This makes these products susceptible to bacteria as well as to mold and yeast spoilage. With respect to the keeping quality of meats, it is well established that meat from fatigued animals spoils faster than that from rested animals and that this is a direct consequence of final pH attained upon completion of rigor mortis. Upon the death of a well-rested meat animal, the usual 1% glycogen is converted to lactic acid, which directly causes a depression in pH values from about 7.4 to about 5.6, depending on the type of animal.


15 Some foods such as fruit are characterized by “inherent acidity” or “natural acidity”. The natural or inherent acidity of foods, especially fruits, may have evolved as away of protecting tissues from destruction by microorganisms. Others owe their acidity or pH to the actions of certain microorganisms. This type is referred to as “biological acidity” and is displayed by products such as fermented milks (e.g yoghourt) and pickles.

16 Some foods are better able to resist changes in pH than others. Those that tend to resist changes in pH are said to be “buffered”. Meatsmore highly buffered Meats are more highly buffered than vegetables. Contributing to the buffering capacity of meats are their various proteins.

17 Vegetables are generally low in proteins and, consequently, lack the buffering capacity to resist changes in their pH during the growth of microorganisms Although acidic pH values are of greater use in inhibiting microorganisms, alkaline values in the range of pH 12–13 are known to be destructive, at least to some bacteria. For example, the use of CaOH 2 to produce pH values in this range has been shown to be destructive to Listeria monocytogenes and other foodborne pathogens.

18 Effects of adverse pH on microorganisms: enzymes  Disruption of cellular enzymes transport of nutrients  Disruption of the transport of nutrients into the cell key cellular compounds as DNA  Disruption of such key cellular compounds as DNA and ATP require neutrality. morphology  The morphology of some microorganisms can be affected by pH. more sensitive to toxic agents  An adverse pH makes cells much more sensitive to toxic agents.

19 internal pH It appears that the internal pH of almost all cells is near neutrality. When microorganisms are placed in environments below or above neutrality, their ability to proliferate depends on their ability to bring the environmental pH to a more optimum value or range. When most microorganisms grow in acid media, their metabolic activity results in the medium or substrate becoming less acidic, whereas those that grow in high pH environments tend to effect a lowering of pH.


21 pH 4.0 The amino acid decarboxylases that have optimum activity at around pH 4.0 and almost no activity at pH 5.5 cause a spontaneous adjustment of pH toward neutrality when cells are grown in the acid range. When amino acids are decarboxylated, the increase in pH occurs from the resulting amines. Bacteria such as Clostridium acetobutylicum raise the substrate pH by reducing butyric acid to butanol, whereas Enterobacter aerogenes produces acetoin from pyruvic acid to raise the pH of its growth environment. pH 8.0 When grown in the alkaline range, a group of amino acid deaminases that have optimum activity at about pH 8.0 and cause the spontaneous adjustment of pH toward neutrality as a result of the organic acids that accumulate.

22 2. Moisture content The preservation of foods by drying is a direct consequence of removal or binding of moisture, without which microorganisms do not grow. It is now generally accepted that the water requirements of microorganisms should be described in terms of the water activity (aw) in the environment. at the same temperature Aw is defined by the ratio of the water vapor pressure of food substrate to the vapor pressure of pure water at the same temperature: aw = p/p o Where p is the vapor pressure of the solution and p o is the vapor pressure of the solvent (usually water). This concept is related to relative humidity (RH) in the following way: RH = 100 × aw.

23  When salt is employed to control aw, an extremely high level is necessary to achieve aw values below 0.80.  Pure water  Pure water has an aw of 1.00 22% NaCl solution  22% NaCl solution (w/v) has an aw of 0.86 Saturated solution of NaCl  Saturated solution of NaCl has an aw of 0.75.



26 above 0.99 The water activity (aw) of most fresh foods is above 0.99.

27 bacteria than fungi In general, bacteria require higher values of aw for growth than fungi Gram-negativehaving higher requirements Gram-negative bacteria having higher requirements than Gram positives. yeasts and molds wider pH range Just as yeasts and molds grow over a wider pH range than bacteria, the same is true for aw.

28 halophiles halophiles = “salt-loving” Xerophilic Xerophilic = “dry-loving” Osmophilic= Osmophilic= “preferring high osmotic pressures”



31 Certain relationships have been shown to exist among aw, temperature, and nutrition: growreduced as the aw is lowered  At any temperature, the ability of microorganisms to grow is reduced as the aw is lowered. range of aw is greatest at the optimum temperature  Second, the range of aw over which growth occurs is greatest at the optimum temperature for growth. nutrientsincreases the range of aw  The presence of nutrients increases the range of aw over which the organisms can survive.

32 Effects of Low aw The general effect of lowering aw below optimum is:  Adverse effects cell membrane  Adverse effects on the functioning of the cell membrane, which must be kept in a fluid state disruption of nutrient transportation  Disruption of nutrition of microorganisms (due to disruption of nutrient transportation through an aqueous environment)  Increase the length of the lag phase  Increase the length of the lag phase of growth  Decrease the growth rate  Decrease size of final population pHtemperature Aw is influenced by other environmental parameters such as pH and temperature: pH and temperature made unfavorableminimum aw for growth was higher  When both the pH and temperature of incubation were made unfavorable, the minimum aw for growth was higher.

33 strategyprotection intracellular accumulation of compatible solutes In general, the strategy employed by microorganisms as protection against osmotic stress is the intracellular accumulation of compatible solutes. The three most common compatible solutes in most bacteria are:  Carnitine  Glycine betaine  Proline  Osmophilicyeastspolyhydricalcohols  Osmophilic yeasts accumulate polyhydric alcohols to a concentration commensurate with their extracellular aw.

34 3. Oxidation–reduction (O/R) potential ---(symbol =Eh) Definition: ease with which the substrate loses or gains electrons. When electrons are transferred from one compound to another, a potential difference is created between the two compounds and expressed as millivolts (mV). loses electronsis oxidized When an element or compound loses electrons, the substrate is oxidized, gains electronsreduced When a substrate that gains electrons becomes reduced. gives up electrons reducing agent. A substance that readily gives up electrons is a good reducing agent. takes up electrons oxidizing agent A substance that readily takes up electrons is a good oxidizing agent.

35 more highly oxidized more positive The more highly oxidized a substance, the more positive will be its electrical potential. more highly reduced more negative The more highly reduced a substance, the more negative will be its electrical potential. oxidant and reductant is equal zero When the concentration of oxidant and reductant is equal, a zero electrical potential exists.

36 positive Eh Aerobic microorganisms: require positive Eh values (oxidized) for growth. negative Eh Anaerobes: require negative Eh values (reduced). Microaerophiles: Aerobic bacteria actually grow better under slightly reduced conditions such as lactobacilli and campylobacters.

37 The O/R potential of a food is determined by the following: O/R potential of the original food  The characteristic O/R potential of the original food. resistance to change  The poising capacity (resistance to change in potential of the food) atmosphere about the food  The oxygen tension of the atmosphere about the food. access that the atmosphere  The access that the atmosphere has to the food. (Ehmore negative alkaline conditions  pH: (Eh tends to be more negative under progressively alkaline conditions) decrease  Microbial activity: Microorganisms decrease the Eh of their environments during growth:  As aerobes growO 2 depleted  As aerobes grow, O 2 in the medium is depleted, resulting in the lowering of Eh.  By production of metabolic H 2 Slower Eh  By production of metabolic such as H 2 S (lower Eh to −300 mV)

38 Reducing substances in some foods: meats  –SH groups in meats fruits and vegetables  Ascorbic acid and reducing sugars in fruits and vegetables Eh of some food: Plant foods +300 to 400 Plant foods, especially plant juices, tend to have Eh values of from +300 to 400 mV. Cheeses −20 to around −200 Cheeses of various types have been reported to have Eh values on the negative side, from −20 to around −200 mV. Solid meats −200 minced meats+200 Solid meats have Eh values of around −200 mV; in minced meats, the Eh is generally around +200 mV.

39 Change in pH of meat pre and post slaughter: after death  Eh of muscle after death is +250 mV, at which time clostridia failed to multiply.  At 30 hours postmortem  At 30 hours postmortem, the Eh had fallen to about 30 mV in the absence of bacterial growth.  When bacterial growth  When bacterial growth was allowed to occur, the Eh fell to about -250 mV.  Thus, anaerobic bacteria do not multiply until the onset of rigor mortis because of the high Eh in pre-rigor meat.



42 4. Nutrient content Microorganisms to grow and function normally require the following: 1. Water 2. Energy 3. Nitrogen 4. Vitamins 5. Minerals

43 water The importance of water to the growth and welfare of microorganisms is obvious. the other four groups With respect to the other four groups of substances requirement: Molds < Gram-negative bacteria < Yeasts < Gram-positive bacteria. energy As sources of energy, microorganisms may utilize:  Sugars  Alcohols  Amino acids (Also as nitrogen source) Complex carbohydrates Complex carbohydrates such as starches and cellulose are also used by some microorganisms as sources of energy by first degrading these compounds to simple sugars Fats Fats are also used by few microorganisms as sources of energy.

44 simple compounds before complex compounds In general, simple compounds such as amino acids will be utilized by almost all organisms before any attack is made on the more complex compounds such as high-molecular- weight proteins. The same is true of polysaccharides and fats. The same is true of polysaccharides and fats.

45 require B vitamins Microorganisms may require B vitamins in low quantities, and almost all natural foods have an abundant quantity for those organisms that are unable to synthesize their essential requirements. Gram-positivethe least synthetic In general, Gram-positive bacteria are the least synthetic and must therefore be supplied with one or more of these compounds before they will grow. Gram-negative bacteria and molds able to synthesize The Gram-negative bacteria and molds are able to synthesize most or all of their requirements. Gram-negative bacteria and molds Thus, Gram-negative bacteria and molds may be found growing on foods low in B vitamins.

46 fruitsmolds Whys fruits spoilage by molds more than bacteria? 1) Fruits have lower B vitamins (not suitable for most bacteria) 2) Fruits have lower pH (not suitable for most bacteria) 3) Fruits have positive Eh (not suitable for most bacteria)

47 5. Antimicrobial constituents plant species Some plant species are known to contain essential oils that possess antimicrobial activity. Among these are: cloves  Eugenol in cloves garlic  Allicin in garlic mustard  Allyl isothiocyanate in mustard carrot phytoalexin 6-methoxy- mellein  Antilisterial compounds in carrot (not exactly determined yet but probably: phytoalexin 6-methoxy- mellein)

48 milk Cow’s milk contains several antimicrobial substances, including:  Lactoferrin (a iron-binding glycoprotein)  Conglutinin  A rotavirus inhibitor in raw milk (susceptible to pasteurization)  Casein  Free fatty acids  Lysozyme  Lactoperoxidase system.

49 Lactoperoxidase system in milk inhibitory system bovine milkthree components This is an inhibitory system that occurs naturally in bovine milk, and it consists of three components: 1) Lactoperoxidase 2) Thiocyanate (SCN−) 3) H 2 O 2 All three components are required for antimicrobial effects.

50 Activation of Lactoperoxidase system: lactoperoxidaseenough The quantity of lactoperoxidase in bovine milk is enough. H 2 O 2 and thiocyanate lower than requested But the quantity of H 2 O 2 and thiocyanate maybe lower than requested. adding thiocyanate and H 2 O 2 hypothiocyanate So, lactoperoxidase system in raw milk is activated by adding thiocyanate and H 2 O 2 and finally hypothiocyanate be generated. shelf life 5 days compared to 48 hours Then, shelf life was extended to 5 days compared to 48 hours for controls.

51 Gram-negative psychrotrophs sensitive Gram-negative psychrotrophs such as the pseudomonads are sensitive. moreeffective at 30◦C than at 4◦C The system was more effective at 30◦C than at 4◦C. where refrigeration is uncommon The lactoperoxidase system can be used to preserve raw milk in countries where refrigeration is uncommon.



54 Eggs:  Lysozyme  Conalbumin (appears to be the inhibitory substance in raw egg white that inhibits Salmonella enteritidis) Fruits, vegetables, tea, molasses: hydroxy-cinnamic acid derivatives  The hydroxy-cinnamic acid derivatives (p-coumaric, ferulic, caffeic, and chlorogenic acids)


56 6. Biological structures natural covering of some foods such as The natural covering of some foods provides excellent protection against the entry and subsequent damage by spoilage organisms such as:  Outer covering of fruits  Shell of nuts  Hide of animals  Shells of eggs  Skin covering of fish

57 EXTRINSIC PARAMETERS are not substrate dependent The extrinsic parameters of foods are not substrate dependent. properties of the storage environment They are those properties of the storage environment that affect both the foods and their microorganisms. As follows: 1. Temperature of storage 2. Relative humidity of environment 3. Presence and concentration of gases 4. Presence and activities of other microorganisms

58 1. Temperature of storage several groups based on their temperature It is customary to place microorganisms into several groups based on their temperature requirements for growth:

59 psychrotrophs Most psychrotrophs of importance in foods belong to the genera:  Pseudomonas  Enterococcus  Shewanella  Brochothrix  Flavobacterium  Lactobacillus  Psychrobacter Psychrotrophsgrow well at refrigerator spoilage5–7◦C Psychrotrophs grow well at refrigerator temperatures and cause spoilage at 5–7◦C of meats, fish, poultry, eggs,... psychrotrophs PseudomonasEnterococcus The psychrotrophs found most commonly on foods are those that belong to the genera Pseudomonas and Enterococcus.

60 Enterococcus faecalis 0 ◦ C to >40 ◦ C Some organisms such as Enterococcus faecalis can grow over a range from 0 ◦ C to >40 ◦ C. Most thermophilic bacteria of importance in foods belong to the genera:  Bacillus  Paenibacillus  Clostridium  Geobacillus  Alicyclobacillus  Thermoanaerobacter

61 Molds have wider ranges of temperature Molds have wider ranges of temperature than bacteria (like pH, osmotic pressure and nutrient content). moldsgrow at refrigerator AspergillusCladosporium Thamnidium Many molds are able to grow at refrigerator temperatures, notably some strains of Aspergillus, Cladosporium, and Thamnidium. Yeasts not within the thermophilic range. Yeasts grow over the psychrotrophic and mesophilic temperature ranges but generally not within the thermophilic range.

62 refrigerator temperatures this is not always best Although it would seem desirable to store all foods at refrigerator temperatures or below, this is not always best for the maintenance of desirable quality in some foods. bananas keep better if stored at 13–17◦C than at 5–7◦C. For example, bananas keep better if stored at 13–17◦C than at 5–7◦C. vegetables such potato as are favored by temperatures of about 10◦C. A large number of vegetables such potato as are favored by temperatures of about 10◦C.

63 Temperature he most important parameter that affects the spoilage Temperature of storage is the most important parameter that affects the spoilage of highly perishable Foods. The rate of spoilage of fresh poultry :  at 10◦C is about twice that at 5◦C  a15◦C is about three times that at 5◦C


65 2. Relative humidity of environment is important both The RH of the storage environment is important both from: aw  The standpoint of aw within foods growthsurfaces  The growth of microorganisms at the surfaces When foods with low aw values are placed in environments of high RH, the foods pick up moisture until equilibrium has been established. Likewise, foods with a high aw lose moisture when placed in an environment of low RH. Relationship between RH and temperature: The higher the temperature, the lower the RH, and vice versa.

66 , surface spoilage deep spoilage Surface spoilage: In the refrigerator, surface spoilage occurred before deep spoilage occurs in food such as improperly wrapped beef cuts. Why?  High RH of the refrigerator  Meat-spoilage biota is generally aerobic (deep sections of meat have often low O 2 ).

67 3. Presence and concentration of gases CO 2 Carbon dioxide (CO 2 ) is the single most important atmospheric gas that is used to control microorganisms in foods. O 2 and O 3 (Ozone) are important in modified atmosphere packaged (MAP) foods. O 3 :  has antimicrobial properties.  strong oxidizing agent.  should not be used on high-lipid-content foods since it would cause an increase in rancidity.  is recognized GRAS (generally recognized as safe) in Australia, France, and Japan and USA.

68 4. Presence and activities of other microorganisms organisms produce substances inhibitory or lethal Some foodborne organisms produce substances that are either inhibitory or lethal to others;  These include:  Antibiotics  Bacteriocins  Hydrogen peroxide  Organic acids  …

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