Food Microbial Ecology Lecture Notes 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  0.797 0.971 Multi-Domain Food: Mille-Feuille Cake The mille-feuille cake is a classic pastry that consists of layers of puff pastry and cream. The pastry has a water activity (aw) of 0.797, while the cream has a higher aw of 0.971. This creates a permissive environment for microbial growth in the cream, contrasted with the less permissive environment of the pastry. Over time, and throughout its shelf life, there can be exchanges between these components. As moisture migrates from the cream to the puff pastry, the once crisp layers can absorb moisture, becoming permissive and losing their quality, such as texture and crunchiness. This highlights the importance of understanding the dynamics between different components in multi-domain foods. 3  aw reduced aw Crunchiness→ Soften aw Intermediate-high aw Softness→Harden Multi-domain systems Regions with varying aw (water activity); Understanding Water Migration and Quality Preservation Water migration in food occurs when moisture moves from areas of higher water activity (aw) to those with lower aw. This process is driven by the difference in aw between food components, and the rate of migration can vary depending on the food's structure. To limit moisture migration and maintain product quality, several strategies can be implemented: 1.Match Components: Align the water activity of different components to prevent migration. 2.Adjust Water Activity: Lower the aw of components with high water activity or raise the aw of those with low water activity. 3.Retard Diffusion: Increase the viscosity within components to slow down the diffusion process. 4.Use Edible Barriers: Incorporate barriers that can reduce moisture exchange. 5.Separate Packaging: Employ different packaging for components to minimize moisture transfer. These methods are crucial, especially since changes in water activity can significantly affect food quality. 4 For instance, reduced aw can lead to: Loss of Crunchiness: The texture can soften as moisture is absorbed, resulting in a less desirable product. Additionally, maintaining the flow properties of powders and preventing caking involves several approaches: Drying: Reduce moisture content to low levels. Low Humidity Treatment: Process powders in controlled low humidity environments. High Moisture Barrier Packaging: Use packaging that limits moisture ingress. Low-Temperature Storage: Keep products at lower temperatures to slow down moisture absorption. In-Package Desiccation: Utilize desiccants within the packaging to absorb excess moisture. Agglomeration and Anti-Caking Agents: Incorporate techniques that prevent caking and improve flowability. By understanding and applying these solutions, we can effectively manage moisture migration and maintain the desired quality of food products. 4  Traditional Fruit Cake – make components to same water activity (flour, sugar, candied fruits, raisins, butter, eggs, nuts) Water activity Moisture Content (% db) Dough 0.857 24.5 Fruits (mixed) 0.862 52.2 Multi-domain systems Regions with varying aw (water activity); 5  https://handary.com/category/aboutus/?id=10061 Definition of Hurdle Technology Hurdle technology refers to a systematic approach in food preservation that combines multiple preservation methods to enhance the safety and shelf life of food products. By applying various barriers or "hurdles," such as temperature control, pH adjustments, moisture reduction, and the use of preservatives, this technology can effectively manage microbial growth and spoilage. The effects of hurdle technology can result in either killing or inhibiting the growth of microorganisms. The ultimate goal is to extend the shelf life of food by prolonging the period before critical thresholds are exceeded. These thresholds may include: Critical Limits for Specific Spoilage Organisms (SSOs): Levels at which spoilage becomes noticeable or unacceptable from a sensory perspective. Pathogen Thresholds: Levels at which pathogens can cause infection or release toxins, such as the infectious dose for certain microorganisms. By strategically applying these hurdles, food manufacturers can ensure that products remain safe and of high quality for a longer duration, minimizing the risk of spoilage and foodborne illness. 6  Microbial Growth – Hurdle Technology Clostridium botulinum in Fresh Pasta Bacterial spores may exist in fresh pasta products Pasteurization can’t kill spores Temperature abuse may allow bacterial growth and toxin production Water Activity of less than 0.95 inhibits toxin production Combination of water activity and pH inhibits toxin production even above minima limits Schebor, C and Chirife, J. (2000). A survey of water activity and pH values in fresh pasta packed under modified atmosphere manufactured in Argentina and Uruguay. Journal of Food Protection. 63(7):965-969. Example: Utilizing Hurdle Technology to Manage Clostridium botulinum Risk in Fresh Pasta In fresh pasta products, bacterial spores, including those of Clostridium botulinum, may be present. Since pasteurization alone cannot eliminate these spores, it is crucial to implement additional preservation strategies. 1.Temperature Control: Avoiding temperature abuse is essential. Maintaining pasta at safe temperatures prevents the growth of C. botulinum and reduces the risk of toxin production. 2.Water Activity (aw): Keeping the water activity below 0.95 effectively inhibits the production of toxins by C. botulinum. This can be achieved through methods such as drying or modifying the moisture content of the pasta. 3.pH Adjustment: Combining low water activity with a lower pH can further inhibit toxin production, even when the water activity is slightly above the minimum threshold. This can be done by incorporating acidic ingredients or using fermentation techniques. By applying these hurdles—temperature control, water activity management, and pH adjustments—we can significantly reduce the risk of Clostridium botulinum in fresh pasta, ensuring both safety and quality for consumers. 7  Aerobic Spore-Forming Bacterium Bacillus cereus Toxin Producer: Emetic Diarrheal At least 10^5–10^8 cfu/g Emetic Toxin: Resists 120°C for 90 minutes pH: 4.3 – 9 aw: 0.912 - 0.94 Temperature: 5 - 50 °C Clostrodium botulinum Anaerobic Spore-Forming Bacterium non proteolitic strains Toxin Producer with Neurological Action At least 10^5 cfu/g Toxin Inactivated at: 80°C in 10 minutes pH: 5 - 9 Process: 90°C per 10 min: vegetative forms aw: 0.97 Toxins of C. botulinum Temperature: 3 - 45 °C Additional Risks in Fresh Pasta: Spore-Forming Bacteria Fresh pasta is susceptible to various risks, particularly from spore-forming bacteria that can thrive even under refrigeration. Two notable examples include: 1.Bacillus cereus: 1. Toxin Production: Produces both emetic and diarrheal toxins. 2. Growth Conditions: Can grow at temperatures ranging from 5°C to 50°C. 3. Water Activity: Growth occurs at an aw of 0.912 to 0.94. 4. pH Range: Tolerates a pH of 4.3 to 9. 5. Heat Resistance: Emetic toxin is resistant to 120°C for 90 minutes. 2.Clostridium botulinum (non-proteolytic): 1. Toxin Production: Produces toxins with neurological effects. 2. Growth Conditions: Grows at temperatures as low as 3°C and up to 45°C. 3. Water Activity: Growth occurs at an aw of 0.97. 4. pH Range: Tolerates a pH of 5 to 9. 5. Heat Resistance: Toxin inactivated at 80°C for 10 minutes. These bacteria can proliferate in refrigerated environments, particularly at temperatures around 4°C. To mitigate these risks, we can seek additional limiting factors. Using Water Activity (aw) as a Control Point: Reducing the water activity is a viable 8 strategy. For instance, lowering the aw from 0.97 can be achieved through the addition of salt. This adjustment not only inhibits the growth of these spore-forming bacteria but also enhances the overall safety and shelf life of the fresh pasta. 8  Aim: aw tortellini filling < 0.97 Ingredients that Bind Water or Have Low Water Content: Breadcrumbs Salt Fats Fiber Proteins Etc. the use salt to control the aw of the filling Acidifying additives to maintain pH at values ≤ 4.5 Synergistic effect between salt, vegetable fiber, or other ingredients able to link water The amount of salt should be standardized according to the recipe! Objective: Creating Specific Recipes to Mitigate Risks of Clostridium botulinum in Fresh Pasta The primary goal is to develop recipes that effectively limit the risk of Clostridium botulinum through strategic ingredient selection and formulation. Feasibility of Recipe Development Creating recipes that address these risks is indeed feasible, provided we utilize ingredients that can bind water or maintain low water activity. Some key ingredients include: Breadcrumbs Salt Fats Fiber Proteins Research Insights Studies on various formulations (including spinach, meat, and tomato) indicate that sodium chloride (NaCl) can significantly affect water activity. For instance, to achieve an aw of 0.97 with Clostridium botulinum present, it requires a salt concentration of about 3.5 g per 100 g of product. In some cases, reaching this level necessitates adding approximately 5.5 g of salt, making it challenging to rely solely on water 9 activity reduction. Additional Considerations 1.Acidifying Additives: Incorporating acidifying agents to maintain pH at values ≤ 4.5 can further inhibit bacterial growth. 2.Synergistic Effects: There is a synergistic interaction between salt, vegetable fiber, and milk proteins that can enhance the effectiveness of moisture control and food safety. Implementation Challenges While the use of these ingredients is promising, several obstacles must be considered to enhance their effectiveness: Taste and Texture: High levels of salt or certain additives may impact the flavor and texture of the final product, necessitating careful formulation to maintain sensory qualities. Ingredient Interactions: Understanding how different ingredients interact, particularly in terms of moisture retention and microbial inhibition, is crucial for developing effective recipes. By strategically selecting and combining these ingredients, we can formulate fresh pasta recipes that effectively mitigate the risks posed by Clostridium botulinum and other spore-forming bacteria, thereby enhancing food safety and shelf life. 9 ISO 5807:1985 is an international standard that provides guidelines for the presentation of flowcharts and diagrams used in various fields, including information processing and food production. It defines symbols and conventions for representing processes, inputs, outputs, and flows in a clear and standardized manner. Key elements defined in ISO 5807 include: 1.Flowchart Symbols: It specifies different shapes for various functions, such as: 1. Parallelogram: Represents inputs and outputs (data). 2. Terminator (start end final product raw materials) 3. Rectangle: Indicates processes or operations. 4. Diamond: Used for decision points. 5. Arrows: Show the direction of flow. 6. Triangle: Wastes or byproducts 2.Clarity and Consistency: The standard emphasizes the importance of clear and consistent use of symbols to ensure that diagrams are easily understandable. 3.Usage Contexts: While it can be applied in numerous fields, it is particularly useful in technical documentation and process management. However in most part of cases only rectangles and arrows are used  Fish-loins line for the production of smoked fish products (simplified) thawing Gutting, filleting and preparation of slices Salt Salting aw -- Drying Smoke smoking aw -- Reduction in slices / trimming Smoked fillets Packaging Flowchart for Smoked Salmon Production 1.Input (Parallelogram) 1. Fresh Salmon 2.Operation (Rounded Rectangle) 1. Cleaning and Gutting 3.Operation (Rounded Rectangle) 1. Filleting 4.Input (Parallelogram) 1. Brine Solution (Salt, Sugar, Spices) 5.Operation (Rounded Rectangle) 1. Brining (Soaking in Brine) 6.Output (Parallelogram) 1. Excess Brine (Waste) 7.Operation (Rounded Rectangle) 1. Rinsing 8.Operation (Rounded Rectangle) 1. Drying (Air Drying) 9.Operation (Rounded Rectangle) 1. Smoking (Hot or Cold) 11 10. Output (Parallelogram) 1. Smoked Salmon 11.Waste (Triangle) 1. Trimmings and Byproducts The addition of salt and the smoking process are two steps that progressively reduce the water activity (aw) of the product, creating a less permissive environment for microbial growth. Definition of Semi-preserved Products: Semi-preserved products are food items that have been treated with brine, salt, or other preservatives to inhibit spoilage and extend shelf life, while maintaining some degree of moisture. These products typically undergo processes such as curing, marinating, or smoking, which enhance flavor and safety by reducing the water activity, thus limiting the growth of pathogenic and spoilage microorganisms. 11  Smoked products Low aw 0.92 es. Legislation Raw Limit for Listeria products And low limit for the Semi-preserved Fish product growth of Listeria By simply measuring the aw, it's possible to verify whether a product is at risk for Listeria monocytogenes growth or not. The limit indicated by the regulation EC 2073/2005 and its amendments specifies 0.92; below this threshold, products are not considered at risk. Semi-preserved seafood refers to products that have undergone partial preservation treatments but still require refrigeration for extended shelf life. These products are not fully sterilized and typically have a limited shelf life compared to fully preserved (canned or sterilized) seafood. The partial preservation methods may include smoking, salting, acidification, or mild heat treatment. Examples of semi-preserved seafood include: 1.Smoked salmon: Cold or hot smoked salmon fillets 2.Acidified seafood salad: A mix of various seafood items in an acidic dressing 3.Marinated tuna carpaccio: Thin slices of raw tuna marinated in citrus juices or vinegar 4.Gravlax: Scandinavian-style cured salmon with dill 5.Pickled herring: Herring preserved in vinegar or brine 6.Ceviche: Latin American dish of raw fish cured in citrus juices 7.Anchovies in oil: Salted and oil-packed anchovies 12 8. Cold-smoked trout: Lightly smoked trout fillets 9.Marinated mussels: Cooked mussels preserved in an acidic marinade 10.Seafood pâtés: Spreadable seafood products with added preservatives These products typically require refrigeration and have a shelf life ranging from a few days to several weeks, depending on the specific preservation method used and storage conditions. 12  pH pH = -log10 [H+] pH in Foods pH is a measure of acidity or alkalinity in a solution, including foods. It's defined as the negative logarithm (base 10) of the hydrogen ion concentration: pH = -log10 [H+] In foods: 1.pH Scale: Ranges from 0 (most acidic) to 14 (most alkaline), with 7 being neutral. 2.Importance: pH affects food safety, preservation, texture, and flavor. 3.Common ranges: 1. Most foods: pH 3.0 to 7.0 2. Acidic foods (pH < 4.6): Citrus fruits, vinegar, fermented products 3. Low-acid foods (pH > 4.6): Meat, fish, vegetables 4.Food safety: Low pH (high acidity) inhibits many pathogenic bacteria. 5.Preservation: Lowering pH is a common preservation technique (e.g., pickling). 6.Quality: pH influences enzyme activity, color changes, and texture in foods. 7.Natural variations: pH can vary within a food item and change during ripening or processing. Understanding and controlling pH is crucial in food processing, preservation, and safety management. 13 13  pH The pH of foods, the direct measurement of the presence of free hydrogen ions, is one of the factors capable of influencing the behavior of microorganisms in the most significant way Preservation of food by acidification (fermentation or addition of weak acids) is one of the oldest systems of food preservation The acidity of foods is mainly determined by the presence/ production/addition of organic acids (acetic> lactic> citric) Why organic acids? 14  Effect of organic acids Organic acids carboxyl RCOO- + H+ Ac. Acetic 4.75 pKa pH =4.5 RCOOH Cell OH- RCOOH pH =7 H+ neutral the cell membrane is RCOO- + H+ impermeable to hydrogen ions. Denaturation of proteins / DNA ATP ADP + pi ATPasi Actions at pH 5,5 The pKa of an acid corresponds to the pH at proton pumps H+ which the organic acid is 50% dissociated and 50% undissociated. The pKa (acid dissociation constant) is a chemical constant that measures the strength of an acid in an aqueous solution and indicates the pH value at which the acid is half-dissociated (ionized) and half-undissociated (non-ionized). In other words, pKa represents the ability of an acid to donate a proton (H+) in a solution. Acids with a lower pKa are considered stronger, while acids with a higher pKa are considered weaker. pKa is an important concept in chemistry and bio.chemistry and is used to understand the acidic and basic behaviors of molecules in solution. The effectiveness of organic acids as food preservatives is rooted in their unique ability to penetrate microbial cell membranes and disrupt internal cellular processes. A crucial factor in this process is the bacterial cell's tendency to be negatively charged, which allows only non-ionized compounds to penetrate. At low pH levels, typically around 4.5, organic acids like acetic acid exist predominantly in their undissociated form. This form is lipophilic, allowing it to easily pass through the cell membranes of microorganisms, a feat that ions and dissociated acids cannot accomplish. The type of acid present or added significantly influences the final effect of pH on microbial growth. For instance, Salmonella can proliferate at pH 4 in the presence of hydrochloric acid (HCl) or citric acid, but its growth is inhibited at higher pH levels (5.4 15 and 5.5) when acetic or propionic acids are used, respectively. Generally, acetic and lactic acids have stronger antimicrobial effects compared to citric, tartaric, malic, or succinic acids. Once inside the cell, where the pH is generally around 7.0, these organic acids encounter a more alkaline environment. This pH difference triggers the dissociation of the acids, releasing protons (H+) and their conjugate base. The sudden influx of protons leads to a decrease in intracellular pH, which can have severe consequences for the microorganism. This acidification can denature proteins and enzymes, disrupt vital metabolic pathways, and even damage DNA and other cellular components. Microbial proliferation is inhibited by both the action of free H+ ions and the undissociated organic acids that penetrate the cell. In response to this proton accumulation, the cell activates its defense mechanisms, primarily proton pumps (ATPases), to expel the excess H+ ions. However, this process is energy-intensive, requiring significant ATP consumption. As the cell diverts energy to maintain its internal pH, other essential cellular processes suffer, further compromising the microorganism's viability. The preservation effect is self-reinforcing: as the cell expels protons, it maintains the pH gradient across the membrane, allowing more undissociated acid to enter. This creates a cyclical process that continually challenges the microorganism's ability to maintain homeostasis. Organic acids often work synergistically with other preservation factors like low water activity or refrigeration, enhancing their overall antimicrobial efficacy. Moreover, different organic acids have varying pKa values and lipophilicity, affecting their effectiveness against different microorganisms and in different food matrices. This complex mode of action makes organic acids highly effective preservatives, particularly in acidic foods. Their ability to penetrate cells in the undissociated form and then dissociate intracellularly provides a unique and potent antimicrobial mechanism that is difficult for many microorganisms to overcome, thereby ensuring food safety and extending shelf life. The specific choice of organic acid and its concentration can be tailored to target particular microorganisms or to achieve desired preservation effects in different food systems. 15  Growth in acidic environment: adaptations Adaptations in Salmonella enterica Typhimurium Decarboxylation with production of amines Increase the pH Homeostatic response pH