Unit 5.1 (Difficulty:Easy) Microbiological Analysis of Food

• Microbiological analysis involves detecting and identifying microorganisms and is critical in food analysis.

Food Preparation

• Food preparation requires ensuring the correct microbes are present.
• Streptobacillus lactis is used for cheese production.
• Acetobacter is used for vinegar production.

Food Spoilage

• Food spoilage involves controlling microbes to acceptable levels.
• Lactobacillus in milk at 2 x 10^5 CFUs/mL can cause spoilage.
• E. coli in bean sprouts at 5 x 10^3 CFUs/g can cause spoilage.
• CFU stands for colony-forming unit.

Foodborne Illnesses

• Infection occurs when bacteria colonize the gut.
• Intoxication occurs when bacteria produce a toxin.
• Intoxification occurs when bacteria colonize and produce a toxin.

Microorganisms

• Bacteria are single-celled prokaryotes; some E. coli strains cause foodborne illnesses.
• Yeast are single-celled eukaryotes, such as Saccharomyces species used in beer or wine.
• Mold is a structure formed by fungi, for example, Penicillium species.
• Viruses are infectious agents replicating in other organisms and can be spread through foods, such as Norovirus.
• Parasites can be animals or protozoa, for example, Giardia.

Growth Requirements - pH

• Most microorganisms are neutrophiles.
• Acidity inhibits the growth of most microorganisms.
• Bacteria typically grow at pH > 4.5 (at aw of 0.85).
• Yeasts typically grow at pH > 4.0 – 4.5.
• Molds typically grow at pH < 4.0.
• Important acidophiles include mold, yeasts, and lactic acid bacteria.
• Fruits, soft drinks, fermented milk products, sauerkraut, and pickles have good keeping qualities due to their low pH from organic acids.
• Organic acids minimize food spoilage.

Water Activity

• Water activity influences microbial growth.

Salinity

• Salinity and water activity are inter-related.

Temperature

• Psychrophiles are "cold-loving" and grow from -20°C to 20°C.
• Psychrotrophs are cold-tolerant, growing at 0°C but prefer 20°C – 40°C.
• Mesophiles have an optimum growth range of 20°C – 45°C and are used in fermentation for products like yoghurt, cheese, wine and beer; most human pathogens are mesophiles.
• Thermophiles are "heat-loving" and thrive between 50°C and 64°C.
• Extreme Thermophiles thrive between 65°C and 79°C.

Atmosphere

• Aerobic and anaerobic bacteria are identified by growing them in test tubes of thioglycollate broth.
• Obligate aerobes need oxygen to respire aerobically and gather at the top of the tube where oxygen concentration is highest.
• Obligate anaerobes are poisoned by oxygen and gather at the bottom of the tube where oxygen concentration is lowest.
• Facultative anaerobes grow with or without oxygen, metabolizing energy aerobically or anaerobically, and gather mostly at the top due to higher ATP production from aerobic respiration.
• Microaerophiles need oxygen but are poisoned by high concentrations, gathering in the upper part of the test tube.
• Aerotolerant organisms do not require oxygen as they metabolize energy anaerobically but are not poisoned by oxygen, and are evenly spread throughout the test tube.

Nutrients

• Microorganisms need specific nutritional sources including Carbon, Nitrogen, Sulfur, and Phosphorous

Growth Factors and Food Preservation

• pH: Acidifying foods with organic acids or acid-producing microorganisms lowers pH to preserve the food (e.g., pH < 4.6 for canned goods).
• Water activity: Storing food with water activity < 0.80 prevents microorganism growth.
• Salinity: Salting foods prevents the growth of microorganisms.
• Temperature: Heating foods to remove bacteria (pasteurization, cooking meat to 165°F) and storing food in cold conditions prevents growth; also consider maximum holding time for foods at room temperature.
• Atmosphere: Limiting oxygen prevents growth of some food spoilage organisms, important in canned goods.
• Nutritional requirements: Controlling a growth factor can manage food microorganisms, as food is generally a good source of the nutritional requirements.
• Spoilage of acid foods (pH 4.6 or lower) must be detected by cultural methods using acidified media (pH 5.0 – 5.5).
• pH isn't the best indicator for predicting microbial response for preservation, as it doesn't indicate changes in total acidity (buffering effects) or the type of organic acid produced.

Growth in Food

• Microorganisms in food can cause food spoilage and illness (food pathogens).
• Some microorganisms like bacteria/fungi are helpful in fermented food.
• Controlling microorganism growth using listed growth requirements are important, whether they are helpful or harmful.
• Food microbiologists must know which microorganisms are present and their required storage to prevent growth.
• Multiple preservation methods may be needed to address different organisms by targeting multiple growth factors.

Detecting, Identifying, and Counting Microorganisms

• Detection and identification involve culturing, molecular biology (genetic markers), specific immunological markers, and other specific tests.

Culture Methods

• Growing organisms on suitable media in a petri dish is done through a Traditional Approach.
• Different organisms have different growth requirements, as pathogenic organisms tend to be more fastidious, different media may be required to detect different organisms.

Defined vs Complex Media

• Defined media contains all known components
• Complex media uses "undefined" components like yeast extract or tryptone.

Selective vs Differential Media

• Selective media allows only selective bacteria to grow.
• Differential media allows most bacteria to grow but has the ability to distinguish certain characteristics, like blood agar that identifies bacteria that breakdown red blood cells.
• Bacteria typically takes 1 - 2 days to culture, while mold and yeast take longer
• Paper backed thin films of growth media (Petrifilm) represents a more recent innovation.

Biochemical Testing

• Each organism has unique biochemical reactions that aid in categorization and identification.
• Examples include gelatinase (hydrolysis of gelatin), IMViC (indole, methyl red, Voges-Proskauer, citrate utilization), and fermentation of lactose.

Enumerating

• Enumerating (counting)is achieved by growing microorganisms in a suitable medium at different dilutions
• Generating separate colonies and easily countable numbers on the plate is important for accurate enumeration.
• The number is expressed as cfu (colony forming units), assuming each colony arises from a single bacterium.

Molecular Biology (identification)

• Molecular Biology utilizes DNA.
• Molecular Biology probes for specific and unique parts of a genome, offering specific identification within hours.

Immunological

• Immunological analyses utilizes a unique molecular marker and requires an antibody to that marker.

Other Methods

• Measuring titratable acidity (TA) in milk is a quick measure of microbial activity
• Lactobacillus in milk produces lactic acid; low bacterial levels result in low acidity.
• Lactic acid mostly drives low acidity.
• Acidity increases as the population increases.