Food and Industrial Microbiology

Introduction

The relationship between foods and microorganisms has been present throughout history. Foods not only provide nourishment for humans, but they also offer nutrients for microbes. The existence of various microorganisms can either spoil or preserve foods by means of fermentation. These microorganisms are utilized to convert raw foods into delicious fermented products like yogurt, cheese, sausages, tempeh, pickles, wine, beer, and other alcoholic beverages.

Identification and management of pathogens and spoilage organisms play a critical role in food microbiology due to the potential for disease transmission through food. Throughout every stage from production to consumption, microorganisms have the ability to affect both the quality of food and human health.

History of microorganisms in food

The documentation regarding the awareness of microorganisms in food is not exact. The year 1674 is known as the birth year of microbiology when Leeuwenhoek analyzed microorganisms in a sample of lake water. After approximately 100 years, microbiology became recognized as a science. Various experiments conducted by scientists offered indirect evidence of the presence of microorganisms in food and their origins. The period before microbiology/bacteriology became established as a science is referred to as the prescientific era, which includes both a period of gathering food and a period of producing food.

During the food gathering period, which lasted from approximately 3 million years ago until 8000-10000 years ago, humans mainly ate meat and eventually included plant foods in their diet. It was also during this time that humans acquired the skill of cooking food. The food producing period began around 8000-10000 years ago and is ongoing, but it encountered several obstacles regarding prepared foods. Some of these challenges encompassed inadequate storage resulting in food spoilage, instances of food poisoning due to prepared meals, and the spread of diseases through food.

Even though there was no scientific understanding of food preservation methods at that time, certain techniques such as using oils, snow, and smoking meats were employed. One of the earliest individuals to propose the impact of microorganisms on food spoilage was Monk Kircher, who made references to worms in decaying bodies and spoiled milk. Additionally, Spallanzani’s observations revealed that heated meat infusion, when sealed in a flask, remained uncontaminated and devoid of microorganisms. Likewise, Pasteur’s experiments refuted the notion of spontaneous generation while showcasing the concept of preserving food through heat.

The importance of food safety lies in the spoilage of food and the presence of food poisoning organisms. Total quality of food now includes not only nutritional balance but also microbiological safety. This chapter will cover the general principles of microbial food spoilage, detection and enumeration of spoilage and food poisoning microorganisms. Additionally, it will discuss the spoilage of significant foods and two characteristics of important food poisoning organisms that are crucial for maintaining food safety.

Food Spoilage and General Principles Underlying Spoilage of Food

Food spoilage encompasses any alteration that makes food unsuitable for human consumption, and can occur due to various factors, including:

• insect damage;
• physical injury due to freezing, drying, burning, pressure, drying, radiation etc;
• activity of indigenous enzymes in plant and animal tissues;
• chemical changes not induced by microbial or naturally occurring enzymes. These changes usually involved O2, light and other than microbial spoilage, are the most common cause of spoilage e. g. xidative rancidity of fats and oils and the discoloration of cured meats;
• growth and activity of microorganisms- bacteria, yeasts and molds.

Even if it is not spoiled, an inedible food may have a substantial amount of bacteria that could cause food poisoning. The presence of microorganisms in food can be determined by different indicators such as changes in color, the formation of gas pockets and swelling, softening and mushiness in texture, alterations in odor and flavor, or the emergence of slime. It is important to consider the properties of plant and animal tissues when dealing with food since they can influence the growth of microorganisms.

It is essential for food microbiologists to comprehend how plants and animals, which are consumed as food, have the ability to defend against microorganisms. Understanding the origins of microorganisms and the factors that impact their growth is crucial in this field. This understanding plays a significant role in utilizing microorganisms for fermentation, producing single cell protein, and preserving food by identifying conditions that hinder their growth.

Microbial media contains a variety of food products with different purposes. These include malt extracts, peptone, tryptone, tomato juice, sugar, and starch. The addition of these components is essential for promoting the growth of microorganisms. It is crucial because if they are unable to utilize a key component in the food material, it puts them at a disadvantage compared to other microorganisms that can. The growth requirement varies among organisms; molds have the lowest requirement followed by yeasts, gram-negative bacteria, and gram-positive bacteria. Many food microorganisms obtain energy from sugars, alcohols, and amino acids while others can use complex carbohydrates like starches and cellulose as an energy source.

There is a smaller amount of microorganisms that can use fats as an energy source. Heterotrophic microorganisms primarily depend on amino acids for nitrogen sources, although proteins, peptides, and nucleotides can also fulfill this role. Generally, microorganisms first utilize simple compounds like amino acids if they are present before resorting to fats and polysaccharides. Certain microorganisms may need small quantities of B vitamins, which are plentiful in natural foods for those incapable of synthesizing them.

In general, gram negative bacteria and molds can produce most or all of their necessary substances. As a result, these two categories of organisms can be present on foods that are deficient in B vitamins. Fruits typically contain lower levels of B vitamins compared to meats, and they also have a lower pH and positive Eh, which is why molds rather than bacteria cause their spoilage.

The water content of foods such as bread and cheese, which may seem dry, is usually over 35%. The state of water in food can be described by its water activity. Water activity is the ratio between the food’s vapor pressure and the vapor pressure of pure water under similar conditions. It is commonly measured as Equilibrium Relative Humidity (ERH) using the formula: Water Activity (aW) = ERH / 100. Water activity plays a crucial role in determining food safety, stability, and quality.

The use of water activity is widespread in various applications, such as preserving food stability, minimizing browning reactions and lipid oxidation, prolonging the efficacy of enzymes and vitamins in food, and improving physical attributes like texture. Most foods have a water activity range between 0.2 (for dry foods) to 0.99 (for fresh and moist foods). Water activity is measured on a scale from 0 (completely dry) to 1.00 (pure water).

According to regulations, a food with a water activity value of 0.5 or lower is considered safe. This is because most bacteria, including Clostridium botulinum, cannot grow when the water activity is below 0.91. However, Staphylococcus aureus can be inhibited by a water activity value of 0.91 under anaerobic conditions or 0.86 under aerobic conditions.

In general, molds and yeasts need a minimum water activity value of 0.80 to grow. Therefore, dry foods like bread are typically spoiled by molds rather than bacteria.

The water activity requirement decreases for microorganisms in the following order: Bacteria > Yeast > Mold. Microbiological growth cannot occur if the water activity is below 0.60. This is why dried foods like milk powder, cookies, and biscuits are more stable and safe than moist or semi-moist foods. Factors that affect the water activity requirements of microorganisms include solute type, medium nutritional value, temperature, oxygen supply, pH, and inhibitors.

Table 4 displays the water activity and susceptibility to spoilage caused by microorganisms for certain foods. The presence of water is essential for microorganisms as it facilitates important biochemical reactions. To reduce the water activity in food and prevent microbial growth, various methods can be employed such as adding solutes or hydrophilic colloids, cooking, drying, dehydration (e.g., egg powder, pasta), or concentration (e.g., condensed milk). These techniques ensure the microbiological stability and safety of the food. Controlling the water activity allows for preservation of numerous types of food.

These include Dried or Low Moisture Foods, which have less than 25% moisture and a final water activity between 0.0 and 0.60, such as dried egg powder, milk powder, crackers, and cereals. These products can be kept at room temperature without any secondary method of preservation. They are shelf stable and will not spoil as long as the moisture content remains low.

Leuconostoc carnosum and Leuconostoc gelidum create CO2 and a small amount of lactic acid, resulting in the build-up of gas and liquid in the package. Enterobacter, Serratia, Proteus, and Hafnia species, which are facultative anaerobes, metabolize amino acids during meat growth to generate amines, ammonia, methylsulfides, and mercaptans, leading to putrefaction. Some strains also produce a small quantity of H2S, causing the meat to turn green. Shewanella putrefaciens is capable of growing in both aerobic and anaerobic conditions and metabolizes amino acids, particularly cysteine, to produce large amounts of methylsulfides and H2S. These substances, along with their unpleasant odors, negatively impact the natural color of meats. Furthermore, the presence of H2S oxidizes myoglobin into a form of metmyoglobin, resulting in a greenish discoloration.

To prevent spoilage of fresh meats, various methods are utilized such as storage at low temperatures (~ 0 to 1°C), modified atmosphere packaging, and vacuum packaging. Additionally, several techniques have been employed to decrease the initial microbial load and hinder the growth of Gram-negative rods. These methods include the incorporation of small quantities of organic acids to lower the meat’s pH (slightly above pH 5.0), drying the meat surfaces (to reduce aW), and a combination of these factors along with lower storage temperature. Ready-to-Eat Meat Products encompass both high heat-processed and low heat-processed uncured and cured meat products. High heat-processed cured and uncured meats undergo heat treatment to ensure their commercial sterility.

The survival of thermophilic spores in low heat-processed uncured meats relies on temperature abuse. If the products are not exposed to temperature abuse, only Bacillus and Clostridium spp. spores, along with certain highly thermoduric species such as Lactobacillus viridescens, Enterococcus, and Micrococcus, can endure. Other microorganisms may contaminate the products during post-heat handling due to equipment, personnel, and air. Furthermore, microbial contamination can also arise from the incorporation of spices and other ingredients in the products.

Certain products can be spoiled by anaerobic bacteria, such as psychrotrophic facultative anaerobic and obligate anaerobic bacteria. This spoiling process is characterized by the production of gas and accumulation of purge, resulting in a color change from brown to pink to red and an unpleasant odor. Psychrotrophic Clostridium spp. are responsible for this color change and odor formation. Additionally, vacuum-packaged and gas-packaged products can be spoiled by psychrotrophic Lactobacillus and Leuconostoc spp. during storage. Serratia liquifaciens growth can lead to the breakdown of amino acids, resulting in a flavor similar to ammonia. Unpackaged cooked products may undergo putrefaction due to the growth and degradation of proteins by Gram-positive bacteria with proteolytic properties.

During long-term storage of products, yeasts and molds can grow, leading to off-flavour, discoloration, and sliminess. Additionally, H2O2-producing lactic acid bacteria can cause a green to gray discoloration in the products. The spoilage in milk and milk products is caused by various microorganisms present in raw milk. Cow’s milk has an average composition consisting of 3.2% protein, 4.8% carbohydrates, 3.9% lipids, and 0.9% minerals. It also contains free amino acids that act as a nitrogen source along with casein and lactalbumin. Microorganisms with lactose-hydrolyzing enzymes (such as lactase or β-galactosidase) have an advantage over those unable to metabolize lactose because it is the main carbohydrate found in milk. Microbial lipases can break down milk fat and release volatile fatty acids such as butyric, capric, and caproic acids.

Table 9 shows the different spoilage defects found in milk and milk products. The spoilage of raw milk due to microbes can happen when lactose, proteins, unsaturated fatty acids, and triglycerides degrade. If raw milk is promptly refrigerated after milking and kept for a few days, the primary culprits for spoilage are Gram-negative psychrotrophic rods such as Pseudomonas, Alcaligenes, Flavobacterium spp., and specific coliforms.

Pseudomonas and related species cannot metabolize lactose but can break down protein compounds in milk, resulting in changes to its flavor, such as bitterness, fruitiness, or an unpleasant taste. On the other hand, the growth of lactose-positive coliforms leads to milk curdling and sourness due to the production of lactic, acetic, and formic acids as well as CO2 and H2.

Alcaligenes spp and certain coliforms produce thick polysaccharides that make milk slimy or rope-like. If raw milk is not refrigerated promptly, it may experience excessive growth of mesophiles like Lactococcus, Lactobacillus, Enterococcus, Bacillus, Pseudomonas Proteus coliforms. This further causes changes in the milk such as souring and curdling. Yeast and mold growth are not typically expected under normal conditions.

Pasteurized milk contains different types of bacteria that can resist heat, such as Micrococcus, Enterococcus, Lactobacillus, Streptococcus, and Corynebacterium. It also has spores of Bacillus and Clostridium that can survive the pasteurization process. There is a risk of post-pasteurization contamination by coliforms, Pseudomonas, Alcaligenes, and Flavobacterium. Therefore, the shelf life of pasteurized milk in the fridge mainly depends on the growth of these cold-loving contaminants. The spoilage pattern in pasteurized milk is similar to raw milk. Flavor defects caused by their growth can be detected when their population reaches around 106 cells/ml.

Bacillus cereus, a type of Bacillus spp., has been linked to the spoilage of refrigerated pasteurized milk, particularly when there are low levels of contaminants after pasteurization. The psychrotrophs in Bacillus produce rennin-like enzymes that can cause sweet curdling of milk at a higher pH than needed for acid curdling. Ultrahigh temperature-treated milk (heated to 150°C for a few seconds) is a commercially sterile product that may only have viable spores of certain thermophilic bacteria. This type of milk does not spoil at room temperature but can spoil if exposed to high temperatures, similar to canned foods.

Evaporated milk, condensed milk, and sweetened condensed milk are different types of concentrated dairy products that are prone to limited microbial spoilage during storage. These products undergo adequate heat treatments to eliminate both vegetative microorganisms and spores of certain molds and bacteria. Evaporated milk comprises whole milk with 7.5% milk fat and 25% total solids. It is packaged in sealed cans and heated for commercial sterility. In appropriate processing conditions, only thermophilic spores of spoilage bacteria like B. coagulans from the Bacillus species can cause milk coagulation.

Condensed milk is typically condensed and contains approximately 10 to 12% fat and 36% total solids. The milk has an initial low heat treatment, similar to pasteurization temperature, and is then evaporated under partial vacuum (around 50°C). Consequently, only thermoduric microorganisms can grow and result in spoilage. Other microorganisms can also contaminate the product throughout the condensing process. Sweetened condensed milk has around 8.5% fat, 28% total solids, and 42% sucrose. The milk is initially heated to a high temperature (80 to 100°C) and then condensed at approximately 60°C under vacuum and packaged. It is vulnerable to spoilage due to the proliferation of osmophilic yeasts like Torula spp, leading to gas formation.

If there is enough head space and oxygen in the containers, molds like Penicillium and Aspergillus may grow on the surface, which can contaminate the product after heat treatment. Butter is made up of 80% milk fat and can be either salted or unsalted. The microbial quality of butter depends on the quality of cream used and the sanitary conditions during processing. Bacteria like Pseudomonas, yeasts such as Candida, and molds such as Geotrichum can grow on the surface, leading to flavor defects like putrid, rancid, or fishy taste, as well as discoloration. In unsalted butter, coliforms, Enterococcus, and Pseudomonas can thrive in the water portion and cause flavor defects.

Spoilage of fruits and vegetables Vegetables

Microorganisms in vegetables primarily come from sources such as soil, water, air, and other environmental sources. These microorganisms can also include plant pathogens. Fresh vegetables contain a considerable amount of carbohydrates (5% or more) and low levels of proteins (around 1 to 2%), except for tomatoes which have a high pH. Damaged or cut vegetables provide a favorable environment for microorganisms to grow at a faster rate. Factors such as the presence of air, high humidity, and higher temperatures during storage increase the likelihood of spoilage. Common spoilage defects are caused by molds from the genera Penicillium, Phytophthora, Alternaria, Botrytis, and Aspergillus. Bacterial species from the genera Pseudomonas, Erwinia, Bacillus, and Clostridium are also significant contributors to spoilage.

Microbial vegetable spoilage is commonly referred to as rot, and it is often accompanied by changes in appearance such as black rot, gray rot, pink rot, soft rot, and stem-end rot (Table 11). Various methods, including refrigeration, vacuum or modified atmosphere packaging, freezing, drying, heat treatment, and chemical preservatives, are utilized to minimize microbial spoilage of vegetables. Table 11 provides a list of typical defects found in fruits and vegetables, such as bacterial soft rot, gray mold rot, rhizopus soft rot, blue mold rot, alternaria rot, pink mold rot, green mold rots, watery soft rot, brown rot, downy mildew, sliminess or souring, black rot/smut/black mold, and anthracnose. Fresh fruits contain a high amount of carbohydrates (at least 10%), very low protein content (less than 0%), and have a pH level of 4.5 or lower.

The spoilage of fruits and fruit products by microorganisms is limited to molds, yeasts, and aciduric bacteria such as lactic acid bacteria, Acetobacter, and Gluconobacter. Similar to fresh vegetables, fresh fruits are susceptible to rot caused by various types of molds including Penicillium, Aspergillus, Alternaria, Botrytis, Rhizopus, and others. Mold spoilages are classified as black rot, gray rot, soft rot, brown rot, and others based on their appearance (Table 11). Fermentation of certain fruits like apples, strawberries, citrus fruits, and dates is caused by yeasts like Saccharomyces, Candida, Torulopsis, and Hansenula.

Bacterial spoilage in berries and figs is caused by the growth of lactic acid and acetic acid bacteria. To prevent spoilage, fruits and fruit products can be preserved through methods such as refrigeration, freezing, drying, reducing aW, vacuum packaging, and heat treatment. Fermented vegetable and fruit products, such as pickles/cucumber and sauerkraut, are produced in large quantities. In salt stock pickles with approximately 15% salt, yeasts and halophilic bacteria can grow if the acidity level is not sufficient. To ensure the product is pathogen-free, dill pickles with low salt (4.6/low acid) content undergo a heating process to destroy heat-resistant spores of the pathogenic bacteria Clostridium botulinum.

Some spoilage bacteria are able to survive heat treatment and are known as commercially sterile foods. Another group of foods with a low pH or high acid content undergo heat treatment to eliminate vegetative cells and some spores. While a low pH can prevent the germination and growth of C. botulinum, certain aciduric thermophilic spoilage bacteria can still germinate and grow under higher temperatures during storage. Additionally, some spores of thermoduric mesophilic spoilage bacteria (including pathogenic ones) can withstand the heating process, but they are inhibited by the low pH.

Both nonmicrobial and microbial factors contribute to the spoilage of canned food. Nonmicrobial spoilage results from chemical and enzymatic reactions, leading to hydrogen production (hydrogen swell), C02 release, browning, corrosion of cans, and liquification, gelation, and discoloration of products due to enzymatic reactions. On the other hand, microbial spoilage can be attributed to three primary causes:

• inadequate cooling after heating or high-temperature storage, allowing germination and growth of thermophilic spore formers;
• inadequate heating, resulting in survival and growth of mesophilic microorganisms;
• leakage (microscopic) in the cans, allowing microbial contamination from outside following heat treatment and their growth.

Thermophilic Sporeformers

Low-acid foods like corn, beans, and peas can be affected by thermophilic sporeformers if the cans are exposed to temperatures of 43°C and higher, even for a brief period. This can lead to three different types of spoilage.

• Flat Sour Spoilage In this type of spoilage, the cans do not swell but the products become acidic due to growth of facultative anaerobic Bacillus stearothermophilus. The organism ferments carbohydrates to produce acids without gas.
• Thermophilic Anaerobe (TA) Spoilage This type of spoilage occurs due to the growth of anaerobic Clostridium thermosaccharolyticum which leads to the production of large quantities of H2 and CO2 gas and swelling of cans.
• Sulfide Stinker Spoilage Gram-negative anaerobic sporeformer Desulfotomaculum nigrificans is responsible for this type of spoilage.

The spoilage is characterized by containers that are flat but contain darkened products with the odor of rotten eggs, which is caused by H2S produced by the bacterium. Insufficient heating during processing leads to spoilage due to the survival of mainly spores of Clostridium and some Bacillus spp. These spores can germinate and grow, causing spoilage. The main concern is the growth of C. botulinum bacteria, which can produce toxins. Spoilage can occur either from the breakdown of carbohydrates or proteins. Certain species of Clostridium ferment carbohydrates to produce volatile acids, H2, and CO2 gas, resulting in swelling of cans. Proteolytic species metabolize proteins and produce foul-smelling H2S, mercaptans, indole, ammonia, as well as CO2 and H2 gas (which also cause swelling of cans).
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Spoilage Due to Container Leakage

Leakage of containers during transport permits various microorganisms to enter the can, leading to the growth of microorganisms in the food and causing spoilage based on the type of microbe. If contaminated with pathogens, the product becomes unsafe. Major Food Borne Infections/Intoxications Caused by Bacteria: What is food borne disease? Safe, nutritious foods are crucial for human health and well-being. Nevertheless, food-borne diseases pose a global issue. Foodborne disease refers to any illness resulting from consuming food contaminated with one or more disease-causing agents.

The World Health Organization (WHO) estimates that approximately 1.5 billion instances of food-borne diseases result in around 3 million deaths annually, causing up to $40 billion in healthcare expenses and job-related absences. These diseases are caused by various microorganisms such as bacteria, parasites, viruses, fungi, and their byproducts, as well as non-microbial toxic substances. A total of over 250 different food-borne illnesses have been identified. Although these diseases present with diverse symptoms, they generally manifest in the gastrointestinal tract, resulting in common signs such as nausea, vomiting, abdominal cramps, and diarrhea.

The primary causes of foodborne illnesses are as follows:

• Improper holding temperature during processing.
• Inadequate cooling during storage.
• Contaminated equipments and utensils.
• Food from unsafe source.
• Poor personal hygiene.
• Adding contaminated ingredients to cooked foods.

Approximately 30 grams or milliliters of food containing toxins produced by 106 to 107 cells per gram (ml) for a normal healthy individual is enough to induce symptoms. The main symptoms include salivation, nausea, vomiting, abdominal cramps, and diarrhea. Additional symptoms encompass sweating, chills, headache, and dehydration. To identify the cause of food poisoning outbreaks, enumeration techniques are employed using selective differential media such as Baird-Parker agar and G. C. Gioletti Cantoni broth to determine the quantity of viable cells. Furthermore, several biochemical tests such as hemolysis, coagulase, and thermonuclease reactions are conducted. Immunological tests like ELISA have also been developed for this purpose.