GENERAL: Although most microorganisms are harmless to humans, some cause food spoilage while others cause of produce toxins that cause food poisoning. Humans have spent much time and effort in searching for ways to protect themselves from food poisoning and their food products from spoilage. Today, you will participate in two exercises which will demonstrate the efficacy of two types of food preservatives against some of the most common bacteria and fungi involved in food spoilage. (While it would be very interesting to illustrate the effects of microbial toxins, we will not be using any toxin-producing organisms in our exercises, for obvious reasons!) In one activity, spices are used as food preservatives. The use of spices dates back many centuries and is a time-honored method of preservation. The second exercise illustrates the use of chemical additives to preserve food. Sugar and salt, which are included in this list of chemical additives, have been used for thousands of years for this function. In more recent years, additional chemical preservatives have grown in importance, as more and more of the population lives in urban settings, away from the immediate areas of food production.

Note: Although none of the microorganism strains you are going to be working with are pathogenic, it is essential that you use the best aseptic technique possible. Before you start working, note where the disinfectant bottles and biohazard disposal bags are located.

OBJECTIVES: In the first exercise, you and your team will examine the inhibitory effect of three common spices (black pepper; chili powder and cloves) on the growth and/or spore formation by five food-spoilage microbes: three bacteria -- Bacillus cereus, Escherichia coli, and Pseudomonas fluorescens; and two fungi -- Penicillium notatum and Saccharomyces cerevisiae. In the second exercise, you will similarly test for the inhibitory qualities of four common food preservative chemicals (sodium benzoate, sodium nitrite, propionic acid and sorbic acid) against six microorganisms known to cause food spoilage: three bacteria -- Bacillus cereus, Micrococcus luteus and Pseudomonas fluorescens; and three fungi -- Penicillium chrysogenum, Rhizopus stolonifer and Saccharomyces cerevisiae.

EXERCISE 1

INTRODUCTION: Many uncooked foods have a collection of microbes associated with the source of the food. For example, most fruits have the yeast, Saccharomyces cerevisiae, on their surfaces. Soil on the ingredients can contain soil-inhabiting organisms such as Bacillus, Pseudomonas, or Penicillium. Subsequent handling, processing and cooking of the food may allow growth of these microflora, which can lead to food spoilage and/or food poisoning.

Considerable time and money is spent in food-processing factories to keep equipment clean and sanitary, because otherwise microorganisms are invariably transferred to food products. Both water and air used in food processing can be contaminated when they enter the processing plant. Pseudomonas and Escherichia species may be in the water supply, while Penicillium and Bacillus species are common air contaminants. The air filtration systems in processing plants do not remove microbial cells or spores.

Escherichia coli also may enter food if unsanitary conditions are practiced by food handlers. E. coli is a fecal coliform, one of the bacteria commonly found in the gut of warm-blooded animals and, therefore, is found in sewage. Although most strains of E. coli are harmless, some strains (i.e., E.coli O:157) can cause pathogenic conditions. Bacillus cereus causes spoilage of cooked meats, poultry, raw and cooked vegetables, and dessert dishes; it can also cause gastroenteritis. To prevent food spoilage by B. cereus, foods should be prepared shortly before serving and, if stored, should be cooled rapidly and then held under refrigeration. Penicillium notatum causes spoilage of eggs, cured meats, milk, and fresh fish. S. cerevisiae spoils many foods that contain a high sugar content.

Throughout history, spices have served many purposes. The Chinese recorded the use of spices in cooking as far back as 2000 BC The ancient Egyptians enjoyed spicy foods and used spices in their embalming and mummification processes as well as in incenses and perfumes. In the Bible, the Queen of Sheba brought spices as a gift to King Solomon, and one of the Magi brought spices to the infant Jesus.

By the Middle Ages, spices had been introduced to Europe. At that time, the European diet of cereal and root crops was rather bland, so the addition of spices greatly enhanced the flavor of these staples. The European desire for spices encouraged explorers to look for new routes to those countries where spices could be purchased. Marco Polo’s voyage in the 13th-14th centuries was in part to establish a spice trade, while Columbus’s voyage in search of a more direct route to the Spice Islands resulted in the discovery of the Americas.

The use of spices in medicinal practice has been reported as far back as 1000 BC in Indian medical literature. Although only a few spices are bactericidal (able to kill bacteria), some are bacteriostatic (prevent bacteria from reproducing). Early scientists believed that spices had germicidal properties, which was one of the reasons for adding them to foods. Spices were also used to preserve foods and mask the bad flavors resulting from spoiling.

In the Middle Ages, it was believed that pepper could ward off the Black Death. Black pepper was used as a remedy for aching legs in15th century England. Throughout history, pepper has been used medicinally as a stimulant, a carminative (a substance that relieves colic), as a digestive aid, and as a cure for diarrhea, cholera, body aches, and arthritis.

Pepper is the most popular spice in the world. Each American consumes about 4 ounces of black pepper annually. Black pepper is prepared from the berries of an evergreen climbing vine, Piper nigrum, native to the Malagar Coast of southwestern India. It is now cultivated in the tropic regions of both hemispheres. As the berries ripen, they turn from green to yellow to red. Black pepper is prepared when slightly underripe berries are dried. These dried peppercorns consist of dark outer hulls surrounding light-colored cores. This is why pepper is a mixture of light and dark particles.

Cloves have been used in medicine to aid digestion and relieve toothaches. Cloves are prepared from the dried flower buds of the clove plant, an evergreen plant indigenous to the Moluccas (Spice Islands). They are grown today in many other tropical countries as Tanzania, Brazil, Java and Sumatra. The clove buds are hand-picked and spread out to dry. It takes about 5,000 to 7,000 clove buds to produce a pound of the spice. Cloves possess greater bactericidal properties than any of the other spices.

Chili powder is an American innovation created in the southwestern United States in the 19th century. It is a blend of chili pepper as the basic ingredient plus varying amounts of cumin, oregano, garlic, salt, cloves, allspice and onion. It has become a basic ingredient in Mexican foods.

EQUIPMENT:

You will work in your basic teams of four (or three).

Each team will have:

One petri plate of each of the following media:
· Nutrient agar (for control plate)
· 2% black pepper agar
· 2% clove agar
· 2% chili powder agar

One culture plate or tube of ONE of the following organisms:
· Bacillus cereus
· Escherichia coli
· Penicillium notatum
· Pseudomonas fluorescens
· Saccharomyces cerevisiae

· Sterile swabs

PROCEDURE:

1. Label all plates with team identification, your test organism (found on the tube or plate of your organism) and the date.

2. For each plate, remove one sterile applicator from the package, being careful not to touch the cotton tip to any surface.

3. With the applicator, pick up some of the assigned cultured organism from the plate or tube. Streak the culture across the center of the control agar (no spices) plate, beginning and ending about 2 cm from the each edge. (See Figure 1) Discard the applicator stick in the biohazard bag.

4. Repeat steps 2 and 3 with the three "spice agar" plates. Remember to use a new applicator stick for each plate!

5. Place your inoculated plates in the containers designated by your instructor. They will be incubated at room temperature (22-25 degrees Celsius) for five days, then refrigerated to inhibit any further growth.

6. Your team should select a time in 7-8 days when they can all come in together to record the growth on the plates, to complete this lab exercise (Plates from Exercise 2 will be read at this time also.)

7. Record your results on Table 1 on your Data Sheet, following the instructions for culture growth interpretation printed there.

EXERCISE 2

INTRODUCTION: Food spoilage results in considerable annual economic loss to businesses and individuals. Many foods have a collection of microbes associated with the source of the food. Much money and labor is spent by food processing plants to keep the equipment and environment clean and sanitary, and to prevent transfer of microorganisms to the food.

In this part of today’s activities, you will be working with six microorganisms that can be found on food products or introduced into them via processing. The organisms contain three types of bacteria and three types of fungi. (See OBJECTIVES for the names of these microorganisms).

You have already read about B. cereus, P. fluorescens, and S. cerevisiae in Exercise 1. The new organisms in this exercise cause similar problems: Micrococcus luteus, a slow growing bacterium, is common in dust, soil, water and on the skin of humans and animals. Penicillium chrysogenum is found in air, water and soil. It causes the spoilage of many foodstuffs, but is extremely useful to humans, as it is the most effective producer of the antibiotic, penicillin. Rhizopus stolonifer is the common black mold found on bread. It also causes molds on some vegetables such as sweet potatoes. Its spores are common air contaminants. On the good side, it is used commercially in the production of steroids.

Smoking, freezing and drying have been used for food preservatives since the Neolithic Period. As the human race moved farther and farther away from farmlands, the need for chemical preservatives to protect food increased. Salt (sodium chloride) was used by primitive humans to prevent the spoilage of meat and fish. Sugar has been used for centuries to prepare jams and jellies, thereby preserving different fruits. Spices (as seen previously) are also widely used. Still, the food industry had a critical need for additional nontoxic food preservatives.

The use of food additives, including chemical preservatives, in this country is currently regulated by the federal government’s Food and Drug Administration (FDA). This organization’s efforts protect the public from unscrupulous business practices. For example, before government regulations, some dairy industries added formaldehyde to poor quality milk to disguise its deteriorating condition. In 1938, the Federal Food, Drug, and Cosmetic Act authorized the FDA to monitor the purity, safety, and wholesomeness of food products. Today, the FDA, as well as state agencies, regulate the addition of chemical additives to food products. The FDA publishes and enforces a list of accepted chemicals called the GRAS ("Generally Recognized and Safe" additives) list. This list specifies what chemicals and how much of each one can be added to a product. In order to add a new chemical to the list, extensive toxicity studies must be performed.

Before the use of chemical preservatives, mold was common in many foods, especially breads. The heat of baking destroys the microbes present in the ingredients, but baked goods are exposed to airborne microbes after baking. Packaging is not intended to be airtight; therefore, chemical preservatives are necessary to protect the baked goods until they are consumed. Besides the chemicals added directly to the foods, some are applied to wrapping materials to control contamination. Also, some chemicals are added to the water used to wash foods; a chemical residue remains on the food, which preserves it.

The observation that an acidic environment prevents the growth of some microbes suggested that acids could be used as food preservatives (i.e., vinegar). Organic acids (acids which contain the element carbon) and their salts are the most common food preservatives. Acids may be present naturally in some foods or they may be a by-product of microbial fermentation of the food product. They also may be added to foods to preserve them. Acetic (vinegar), ascorbic, citric, malic, tartaric, and phosphoric acids are all these types of acids. Sauerkraut and pickles, for example, are preserved by acids produced by bacterial fermentation. Sulfurous acid (sulfur dioxide in solution), propionic, sorbic, and benzoic acids and/or their salts are not naturally found in foods but may be added to them as prescribed by the FDA.

Benzoic acid was the first chemical preservative allowed in foods by the FDA, and it is still used widely in highly acidic foods such as ciders, salad dressings, soft drinks, margarine, and syrups. However, sodium benzoate is more soluble than benzoic acid and it preferred for this purpose. Most bacteria are inhibited solely by the low pH of benzoic acid, but molds and yeasts are not. However, benzoic acid does inhibit molds and yeasts by interfering with the microbe’s metabolism.

Propionic acid and its salts are effective against molds but have little or no effect on bacteria and yeasts. Since yeasts are not affected, propionates can be used in baked goods to prevent mold growth without inhibiting the yeast’s leavening activity. Propionates are also used to inhibit molds in cheeses, malt extracts, chocolates, fruits, vegetables, cakes, pies, jams and jellies. Wrapping paper and tubs for margarine are treated with propionic acid. Propionates interfere with the protein metabolism and plasma membrane permeability in microbes.

Sorbic acid inhibits yeasts and molds more than it does bacteria. It is used in the preservation of cheeses, baked goods, beverages, syrups, jams, jellies, canned and dried fruits, pickles and margarine. It is also used as a spray, dip, or coating on packaging materials. Sorbic acid is used because its inhibitory effect is selective; it inhibits undesirable microbes while not affecting other beneficial ones. For example, mold growth on cheeses can be retarded without inhibiting the molds necessary for maturing the cheese. It is believed that sorbic acid interferes with a microbe’s enzyme systems and permeability.

Although sodium nitrate is not an organic acid, it is used as a color stabilizer and spoilage inhibitor in cured meats such as frankfurters and lunch meats. Nitrites are particularly effective in inhibiting Clostridium botulinum, the causative agent of the frequently fatal form of food poisoning known as botulism. Sodium nitrite also enhances the flavor of meats, especially pork, although the chemical basis for this reaction is unknown. Nitrites are converted to nitrous acid in the stomach which, under certain conditions, can react with secondary and tertiary amines (elementary proteins formed during protein digestion) to form nitrosamines, which are carcinogens in animals other than humans. Because of this, the safety of nitrites has been reviewed and approved by the FDA, but its use in foods is likely to remain controversial.

EQUIPMENT:

You will work in your basic teams of four (or three).

Each team will have:

One petri plate of each of the following media:
· Nutrient agar (for control plate)
· Sodium benzoate agar
· Sodium nitrite agar
· Propionic acid agar
· Sorbic acid agar

One culture plate or tube of ONE of the following organisms:
· Bacillus cereus
· Micrococcus luteus
· Pseudomonas fluorescens
· Penicillium chrysogenum
· Rhizopus stolonifer
· Saccharomyces cerevisiae

· Sterile swabs

PROCEDURE:

1. Label all plates with team identification, your test organism (found on the tube or plate of your organism) and the date.

2. For each plate, remove one sterile applicator from the package, being careful not to touch the cotton tip to any surface.

3. With the applicator, pick up some of the assigned cultured organism from the plate or tube. Streak the culture across the center of the control agar (no chemical additives) plate, beginning and ending about 2 cm from the each edge. (See Figure 1) Discard the applicator stick in the biohazard bag.

4. Repeat steps 2 and 3 with the four "chemical additive" plates. Remember to use a new applicator stick for each plate!

5. Place your inoculated plates in the containers designated by your instructor. They will be incubated at room temperature (22-25 degrees Celsius) for seven days.

6. Your team should select a time in 7-8 days when they can all come in together to record the growth on the plates, to complete this lab exercise (Plates from Exercise 1 will be read at this time also.)

7. Record your results on Table 2 on your Data Sheet, following the instructions for culture growth interpretation printed there.

For Exercise 1:

1. Was your test organism inhibited by all three spices? Which spice was the most effective? Which one was the least effective?

2. Was there a difference in the appearance of your organism on the control plate and the test plates? If so, can you account for this difference?

3. List three reasons for the use of spices by humans.

For Exercise 2:

It’s lunch time, and you have selected the following items for your meal:

· A ham and cheese sandwich on whole wheat bread, with mustard
· A small tossed salad with French dressing
· A package of corn chips
· A can of soda

Identify at least one probable chemical preservative for each of the food items mentioned above, indicating which type(s) of microorganism(s) it protects against. (Use the back of the page for your answer, if necessary.)

(These exercises are based on kits designed by Carolina Biological Supply Company, Burlington, NC)

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