Food Microbial Ecology PDF

Food Microbial ecology Luca Fasolato Università degli Studi di Padova Dipartimento di Biomedicina Comparata e Alimentazione 1  Summary Factors affecting Microbial growth Ecophysiology...

Food Microbial ecology Luca Fasolato Università degli Studi di Padova Dipartimento di Biomedicina Comparata e Alimentazione 1  Summary Factors affecting Microbial growth Ecophysiology of MOs in food Intrinsic factors: pH and aW Redox potential 2  Microbial Food ecology Study of the interactions between the chemical, physical and structural characteristics of a niche and the composition of its specific population. Habitat: the abiotic characteristics that distinguish a certain environment Microbial population: groups of individuals (cells) of the same species or genotypes in a given area Microbial community: set of populations in a given habitat (functions and interactions) Niche: biological community with its own metabolic activity that develops in a specific habitat 3  What does the food microbial ecology study? ❖The food microorganisms and the problems that their proliferation can generate, as a danger and as a depreciation of quality ❖The correlations that exist between food substrate and all the resident microbial forms (present in food in consequence of contamination) during the process and commercial life stages up to consumption. 4 In reality, microorganisms do not exist to deteriorate food or cause diseases; they originate from the environment and play vital roles in their propagation and reproduction within food. By utilizing nutrients, microorganisms carry out metabolic reactions that, although incidental, can lead to changes in food quality.Simplifying to the core, the role of food microbiology can be summarized in two key points: assessing the quantity and types of microorganisms present in a given food item, which requires an understanding of the food itself to anticipate which organisms are likely to be present, and identifying those that should not naturally occur.It is also important to consider the correlations between the food substrate and the resident microbial forms, which are present due to contamination, throughout the processing and commercial life stages up to consumption. The proliferation of these microorganisms can generate issues related to both food safety and product quality. 4 Microorganisms in food 5  Intrinsic factor of Food  pH  activity water aw  Redox(Eh)  biological structure  Nutrients Antimicrobial substances Preoceses Extrinsic factors factors -Temperature Contamination and -Atmosphere of foods -.Humidity. Implicit factors Microbial growth in the food environment is conditioned by: intrinsic factors of food (pH, water activity and nutrients), factors extrinsic to food (temperature, humidity, gas, microbial community). Conditions may vary within the food, creating micro-environments with pH gradients, nutrients, etc. 6  Intrinsic and extrinsic factors: preservation for food safety By modifying and controlling the intrinsic and extrinsic factors of the food environment, it is possible to modify and preserve a food and keep it 'safe' for the consumer THE COLD CHAIN It is the combination of these factors that can affect storage time, shelf life, and safety, with temperature being one of the most important factors. 7  Factors affecting growth Lethal Sub-Lethal Sub-optimal Optimal Sub-Lethal conditions conditions conditions conditions Lethal contitions conditions Opt Growth rate Min max Range or intensity of a Factor (as pH, temperature ecc) Growth range Stress Adaptation Lethal stress: cells cannot Sub-lethal Stress (need resuscitation) adapt VBNC viable but nonculturable cells The Growth Rate-Stress Factor Relationship: Understanding Microbial Responses The graph illustrates the relationship between growth rate and various stress factors. It delineates different vital intervals: 1.Normal Growth Range: Where optimal growth occurs. 2.Initial Adaptation Phase: Growth rate remains minimal or null as microorganisms adjust. 3.Sublethal Stress Zones: Bacteria cannot grow but must adapt and recover vital mechanisms. Cells under sublethal stress often fail to replicate on selective media. 4.Viable But Non-Culturable (VBNC) State: Requires extended resuscitation periods, typically only in culture broths. 5.Lethal Zone: Beyond extreme stress values, microbial death occurs. Key Concepts: Cross-Resistance: Microorganisms growing in suboptimal conditions may develop resistance to multiple stressors (e.g., thermal resistance leading to dehydration resistance). Cardinal Values: Represent the maximum and minimum levels of a factor beyond which the bacterium may die. Intrinsic Factors: Each has an optimal value for microbial growth. For comprehensive understanding, it's crucial to determine the minimum, optimal, and maximum growth 8 values for the microorganism under study across various stress factors. This multifaceted approach to microbial growth dynamics is essential for effective food safety protocols and preservation strategies 8  Intrinsic Factors The intrinsic factors characterize the food and derive from its composition and structure. The effect on growth derives from the interaction of factors with each other. pH Water activity Redox potential (ability of a substrate to release or acquire electrons) Food components Nutrients, Added additives antimicrobial 9  Water activity In a biological system as a food -Free water: Easely removable /weak Binding water: molecular bonds Linked ar associated with proteins ions etc.. Water activity represents a fraction of the water present in the food, available for microbial proliferation and degradative reactions (as enzymatic activities). It does not correspond to moisture content. Water is essential for all living organisms, but not all water in food is available for microbial growth. As the amount of available water decreases, growth slows down. Chemical and enzymatic reactions are also influenced by the presence of water. Bound water does not freeze or evaporate and is tightly linked to the solid components of the system (proteins, solutes, sugars, etc.). Water bound to proteins is not engaged in bonds with other molecules and is retained in interstitial spaces solely due to surface tension. Weakly incorporated water in macrostructures is not the constitutive water of the food. Increasing the concentration of solutes (e.g., through dehydration of cod or by adding molecules such as salts and sugars) renders free water unusable and draws water from the bacterial cell through osmosis. Hydrophilic colloids, like agar, can make water unavailable. An additional factor to consider is that crystallization during freezing makes microbial development impossible. Free water tends to migrate from areas of high water activity (aw) to those of low aw. For example, honey (aw ≈ 0.6) stored in air humidity (aw ≈ 0.7) will tend to absorb water from the air. Both bound and free water contribute to the total water value in food. Understanding these water dynamics is crucial for food preservation and safety. The manipulation of water availability through various processing techniques (dehydration, freezing, addition of solutes) is a fundamental strategy in controlling microbial growth and ensuring food stability. 10  Water activity: water available 0→1 aw = P / P0 The ratio between the vapor pressure of water sorunding a product (P) in equilibrium and the vapor pressure of pure water (P0) in the same conditions. The water activity does not coincide with the moisture which includes both free water and water linked to food components Water activity (aw) refers to the measure of the availability of water in a substance or solution for chemical reactions or microbial growth. It is a dimensionless quantity that ranges from 0 to 1, where 0 indicates completely dry conditions (no water available), and 1 represents pure water. In other words, water activity quantifies the relative amount of water vapor pressure present in a substance compared to the vapor pressure of pure water under the same conditions. Water activity is an important parameter in various fields, including food science, microbiology, and materials science. In the context of food, it is used to assess the potential for microbial growth and chemical reactions, as well as the shelf life and stability of products. Low water activity can inhibit the growth of most microorganisms, while high water activity can promote spoilage and the growth of pathogenic bacteria. 11  Growth at low aw Growth behaviors at Optimal levels of aw Log of microorganisms Growth at Sub-Optimal levels of aw Time Microbial Response to Water Activity Reduction Microorganisms typically do not die when water activity (aw) is moderately reduced. Instead, several adaptations occur: 1.Extended Initial Adaptation Phase: The lag phase is prolonged as microorganisms adjust to the new environment. 2.Altered Growth Pattern: Growth proceeds, but at a slower rate and with more difficulty. 3.Lower Maximum Population: The final microbial concentration is lower compared to growth under optimal conditions. 4.Increased Survival Time: Paradoxically, these stressed microorganisms may survive longer than those grown under optimal conditions. 5.Enhanced Stress Resistance: Once adapted to low aw, microorganisms often exhibit increased resistance to additional stressors. The growth curve in low aw conditions typically shows: An extended lag phase, reflecting the initial stress response A slower growth rate during the logarithmic phase A lower maximum population in the stationary phase A potentially extended stationary phase This adaptive response highlights the remarkable resilience of microorganisms. It underscores the importance of considering not just growth inhibition, but also potential stress-induced adaptations when designing food preservation strategies. Understanding 12 these dynamics is crucial for effective microbial control in food systems with reduced water activity 12  Growth at low aw opt Microbial growth typically occurs between 0.999 and 0.620 Aw. Each microbial species has an optimal aw and a minimum Min Max aw for growth. 0.65 0.999 Decreasing aw leads to a prolonged duplication phase and Order of sentitivity an increased death rate (the opposite is true for thermal treatments). Gram - > Gram+> yeasts > mold> spore The aw range increases at the optimal temperature. For example, S. aureus is a halotolerant pathogen that If many nutrients are present, the aw range is broader. can grow at an Aw of 0.83 But at this level no toxins are released Prof. Leistner in 1995, as “an intelligent combination of hurdles which secures the microbial safety and stability as well as the organoleptic and nutritional quality and the economic viability of food products” Water activity (aw) plays a crucial role in microbial growth and survival, with different microorganisms exhibiting varying levels of sensitivity to reduced water availability. The order of sensitivity to decreasing aw generally follows a pattern from most sensitive to least sensitive: Gram-negative bacteria, Gram-positive bacteria, yeasts, molds, and finally, bacterial spores. This hierarchy reflects the diverse adaptations microorganisms have developed to cope with water stress. Microbial growth typically occurs within a range of aw from 0.999 to 0.620, but each species has its own optimal and minimum aw for growth. As aw decreases, microorganisms face increasing challenges. For instance, Staphylococcus aureus, a halotolerant pathogen, can grow at an aw as low as 0.83, demonstrating its remarkable adaptability. However, it's important to note that while S. aureus can survive at this low aw, it cannot produce toxins under these conditions, illustrating the complex relationship between growth and virulence factors. The effects of reduced aw on microbial growth are multifaceted. As aw decreases, microorganisms experience a prolonged duplication phase and an increased death rate. This response is notably different from their reaction to thermal stress, where higher temperatures typically accelerate metabolic processes. The relationship between aw and microbial growth is further complicated by other environmental factors. For example, at a microorganism's optimal growth temperature, the range of tolerable aw values broadens, allowing for 13 growth under more varied water conditions. Similarly, the presence of abundant nutrients can expand the aw range in which microorganisms can grow, highlighting the interconnected nature of environmental factors in microbial ecology. Understanding these dynamics is essential for effective food preservation strategies and safety measures, as manipulating aw in conjunction with other factors like temperature and nutrient availability can provide powerful tools for controlling microbial growth in various food systems. 13  Hygrophiles: 0.995-0.980 Aw with longer latency phases. Xerophiles: Less than 0.85-0.65 Aw for molds → Low Aw → Xerotolerance Osmophiles: 0.62-0.65 Aw in osmotic compounds such as glucose → Osmotolerance Halophiles: High concentrations of salt (NaCl) → Halotolerance Extreme halophiles (12% of salt) Halobacterium salinarum (mould/ yeasts) Classification according to aw Water activity (aw) is a crucial parameter in food science and microbiology, providing valuable insights into product stability, perishability, and safety. It serves as an essential tool for monitoring critical control points throughout the food production chain, including processing and smoking stages. This concept is particularly significant when interpreting microbiological criteria for pathogens like Listeria. Microorganisms can be categorized based on their sensitivity to aw changes, reflecting their diverse adaptations to water availability. Hygrophiles, which thrive in high aw environments (0.995-0.980), represent the majority of food-related microorganisms, including both pathogens and spoilage organisms. These microbes may exhibit extended latency phases when exposed to lower aw conditions. At the other end of the spectrum are xerophiles, adapted to survive in dried substrates with aw values below 0.85-0.65. This group includes many molds that have developed xerotolerance, allowing them to persist in low-moisture environments. Osmophiles prefer substrates rich in sugars, capable of growth at aw levels as low as 0.62-0.65. Their osmotolerance enables them to thrive in high-sugar foods. Halophiles represent another specialized group, requiring high salt concentrations for growth. Their halotolerance allows them to multiply in environments with elevated sodium chloride levels. It's important to note that while some bacteria are tolerant to these extreme conditions, they generally prefer environments with higher aw for optimal growth. The aw values associated 14 with each group are characteristic of specific species within that category. Understanding these microbial preferences and tolerances is crucial for developing effective preservation strategies and ensuring food safety across various product types and storage conditions. This knowledge allows food scientists and manufacturers to manipulate aw as part of a multi-hurdle approach to food preservation, targeting specific microbial groups and enhancing overall product stability and safety. 14  Mechanisms of Growth at Low Water Activity and Solute Tolerance ❖ Growth at low water activity (Aw): To counter osmosis, an increase in solute concentration inside the cell occurs by: Transferring inorganic ions from the external environment to the inside of the cell. Synthesizing or concentrating an organic solute: compatible solute. Compatible solutes: Sugars, alcohols, amino acids or their derivatives, potassium ions mannitol from KCl (Archaea and extreme halophilic bacteria), synthesized or accumulated from the environment. ❖ Tolerance to high solute concentrations: Defined by the ability to produce or accumulate compatible solutes. ❖ Most common compatible solutes: Proline (Staphylococcus) Glycine betaine Glycine betaine, a highly soluble derivative of glycine (Halophilic bacteria and cyanobacteria) non-reactivity with macromolecules, Ectoine (extreme halophiles), aspartic acid high polarity and hydrophilicity, low toxicity, stabilizing effect on proteins Glycerol (xerophilic yeasts and molds, and halophilic green algae) and membranes, small molecular size, and ability to retain water (colligative properties). How do organisms grow in low water activity conditions? When an organism grows in a medium with low water activity, it can obtain water from the surrounding environment only by increasing the concentration of solutes within itself. The increase in intracellular solute concentration can be achieved by transferring inorganic ions from the external environment into the cell, or by synthesizing or concentrating an organic solute. Some organisms are known to possess both mechanisms. The solute used to regulate water activity within the cytoplasm must not inhibit the cell's biochemical processes; compounds of this type are called compatible solutes. Several compounds are known to act as compatible solutes for microorganisms: these include highly water- soluble sugars, alcohols, amino acids or their derivatives, except for extreme halophilic Archaea and some species of extreme halophilic Bacteria, which use potassium ions (from KCl). Compatible solutes can be synthesized directly by microorganisms or, as in the case of betaine (trimethylglycine) and potassium, can be accumulated following uptake from the environment. The concentration of compatible solutes within the cell is regulated by the concentration of extracellular solutes, and in each organism, the maximum amount of compatible solutes produced or accumulated is a genetically controlled characteristic; these factors determine the ability of various microorganisms to adapt to environments where water has different activities. The classification of a given microorganism as non-halotolerant, halotolerant, halophilic, or extremely halophilic is thus defined, at least to some extent, by its genetically determined ability to produce or accumulate compatible solutes. The properties of compatible solutes, which allow them to increase intracellular concentration without interfering with cellular mechanisms, are determined by a combination of chemical and physical characteristics, including: 1.Non-reactivity with macromolecules 2.Polarity and hydrophilicity 3.Low toxicity 4.Stabilizing effect on proteins and membranes 5.Small molecular size 6.Ability to retain water (colligative properties) Compatible solutes are small, polar, and chemically inert molecules that stabilize proteins and membranes, maintaining osmotic balance without interfering with cellular processes. These traits make them ideal for survival in extreme environments. Gram- positive cocci belonging to the genus Staphylococcus are notably halotolerant bacteria (indeed, a common isolation procedure involves using media containing 7.5% NaCl) and use the amino acid proline as a compatible solute. Betaine is a derivative of the amino acid glycine in which the protons of the amino group are replaced with three methyl groups. This substitution results in a permanent positive charge on the N atom, increasing the compound's solubility. Betaine is a widely distributed compatible solute, especially among halophilic bacteria and cyanobacteria. Some extreme halophiles produce a different compatible solute called ectoine, which is a cyclic derivative of aspartic acid. Marine algae produce various glycosides but, with rare exceptions, these compounds are not accumulated in large quantities within the cell, as these organisms are not strongly halophilic. Xerophilic yeasts and halophilic green algae primarily produce glycerol as a compatible solute. 15  Water activity: aw xerophiles: microorganisms capable of growing at extremely low aw values of 0.75 or even lower halophiles: microorganisms that require the presence of salt to grow, including some bacteria with extreme.habitats; Halotolerant: microorganisms that have optimal growth at low salt concentrations but are able to grow even at high concentrations (for example Staphylococcus aureus); osmophiles: microorganisms that have a privileged development at high concentrations of sugars (for example some species of yeasts, such as Zygosaccharomyces) 16  Salt and Water activity: aw 3% 7% 16% 22% Percentage of salt Growth rate of Microorganisms Non -halophiles Halotolerant Moderates halophiles Extremophile halophiles 0.96 0.90 0.86 aw NaCl concentration (M) Salt has a certain toxic effect as it combines with the protoplasm (present anions). C. perfringens (0.95 sucrose; 0.97 NaCl; 0.93 Glycerol) As can be seen from the graph, increasing concentrations of salt lead to increasingly prohibitive levels of water activity (aw). Some microorganisms like E. coli are sensitive to very low levels of salt (about 3%), while others that are halotolerant, such as S. aureus, can tolerate large amounts of salt up to 10%. Moderate halophiles like Vibrio require salt for growth, while among extreme halophiles, we find rare cases in foods. This latter group includes bacteria responsible for the "red of stockfish," specifically orange-red pigmented bacteria, namely Halococcus spp. and Halobacterium spp. These bacteria are strongly proteolytic and produce unpleasant odors from the substrate. The type of solute used to reduce aw in food is essential in determining whether growth is reduced or not. For example, in Clostridium perfringens, the minimum growth limits vary depending on whether sucrose, salt, or glycerol are used as solutes. In glycerol, the microorganism can replicate even at aw close to 0.93, while salt proves inhibitory at an aw of 0.97. 17  Salt vs glucose and Water activity: aw Concentrations of NaCl and Glucose at Various Levels of aw (at 25°C) aw % w/w NaCl % w/w glucose 1.00 0.00 0.00 0.99 1.74 8.90 0.98 3.43 15.74 0.96 6.57 28.51 0.94 9.38 37.83 0.92 11.90 43.72 0.90 14.18 48.54 0.88 16.28 53.05 0.86 18.18 58.45 18 Glycerol, often indicated on labels as E422, is commonly used to reduce water activity (aw) in various products. Compared to salt and sucrose, glycerol exhibits intermediate behavior: a 22% aqueous solution of glycerol results in an aw value of approximately 0.95, while a sodium chloride solution achieves around 0.86, and a sucrose solution reaches roughly 0.98. Among sugars, there are notable differences between sucrose, glucose, invert sugar, and glucose syrup (39-42 DE). To obtain an aqueous solution with the same aw value (0.9), one would need to use 58% sucrose, 48% glucose, 41% invert sugar, or 33% glucose syrup. This demonstrates that different ingredients have varying effects on water activity, which must be considered when developing new formulations. For example, when using sugars, it is essential to adhere to a specific level of sweetness, account for their solubility, and understand their influence on other parameters, such as the Freezing Point Depression Factor (FPDF). Additionally, starches, proteins, and hydrocolloids also impact water activity, albeit through different mechanisms. They can act as "water traps" 18 (Water Holding Capacity, WHC) or "binders" (Water Binding Capacity, WBC), thereby limiting water availability and migration. At 25°C, the water activity of various concentrations of salt and sugar is documented. In terms of weight, salt is more effective than sugar; however, comparisons should ideally be made on a molar basis since aw depends on the number of ions or molecules present in the water. Moreover, salt dissociates into two ions and has a lower molecular weight, enhancing its efficacy. 18  Water activity: aw Water content critical limit Water activity 0.70 > 0.95: easily alterable Food 0.95: can be stored for a few days types/food