9.3 Fermentation

9.3.1 Definition of Fermentation

Fermentation, in the strictest sense, means "the breakdown of carbohydrates under anaerobic (absence of oxygen) conditions." However, in a broader sense, fermentation is often used to describe the anaerobic and aerobic breakdown of carbohydrates and carbohydrate-like materials by microorganisms. In fermented foods, proteins and lipids may be hydrolyzed and metabolized by microorganisms involved in the fermentation process.

When we speak of fermented foods, we are referring to the foods which have been produced with the aid of microorganisms. The changes that occur, may not be solely to the carbohydrate component of the foods, but will also likely involve microbial induced changes to the proteins and lipids in the foods to create the desired colours, flavours and textures characteristic of fermented foods.

Most fermented foods are not genetically engineered (see the above explanation of genetic engineering and compare it to the definition of fermented foods).

9.3.2 Preservation principle

The basis of preservation of foods by fermentation is the encouragement of growth and metabolism of alcohol and acid-producing microorganisms to suppress the growth and metabolic activities of proteolytic and lipolytic, spoilage-causing microorganisms. This condition forms the basis of microbial antagonism that is the principle of preservation of foods by microorganisms specifically cultured for the production of fermented foods. Microorganisms, when cultured in foods, produce a variety of end products including acids and alcohols which act as antimicrobial agents.

Fermentation of foods leads to the formation of chemicals ( e.g. acetic acid, citric acid, lactic acid) that are commonly added to foods as preservatives. In this case, preservatives are formed in situ. In addition, other microorganisms, especially the bacteria that produce lactic acid, also produce as yet unidentified substances that have antimicrobial activity, particularly toward spoilage- and disease-causing microorganisms.
Foods in which acids are produced, especially when the pH is lowered to 4.6 or lower, will not support the growth of pathogenic bacteria such as Clostridium botulinum. Products such as sauerkraut, wine, yogurt and cheese are more stable forms of the low acid food materials that were used at the start of the fermentation process.

9.3.3 Benefits of Fermentation

Fermented foods in many cases can be more nutritious than the unfermented original materials.

This is particularly true for mould fermented foods where the moulds synthesize B-vitamins. Food products such as miso, and tempeh have higher levels of B-vitamins than the soybeans that are used to produce those fermented foods.
Microorganisms also liberate nutrients from parts of plants that are normally undigestible in the human gastrointestinal tract. The availability of minerals and vitamins that are usually biologically unavailable is thereby increased.
Fermentation can enhance the nutritional value of foods by microbial hydrolysis of cellulosic materials that are undigestible in the human digestive tract. This renders the fermented foods more digestible than their unfermented counterparts.

Fermented foods add variety to our food supply, which adds to our diet a group of nutritious products. When you review Table 9.1 you will note that microorganisms used in the production of fermented foods increase the variety of foods in our diet. A number of fermented foods listed in Table 9.1 may not be familiar to you as the list represents fermented foods produced throughout the world.

Table 9.1. Some examples of fermentations used in the food industry.

Microorganism	Food commodity	Fermented product
Lactic acid bacteria	Cucumbers Olives Cabbage milk Coffee cherries Vanilla beans Meat Dairy	Dill pickles, sour pickles Green olives, ripe olives Sauerkraut & Kimchi Kishk Coffee beans Vanilla Meat sausages (salami) Sour cream, yogurt
Lactic acid bacteria with propionic acid bacteria	Dairy products	Swiss, Emmenthaler, Gruyère cheeses
Lactic acid bacteria with mould	Vegetable products Dairy products	Tempeh, soy sauce Roquefort, Camembert, Brie, Blue cheeses
Acetic acid bacteria	grapes	vinegar
Yeasts	Malt Fruit Wines Rice Bread dough	Beer, ale, stout Wine, vermouth Brandy Saké bread
Yeast with lactic acid bacteria	Ginger plant beans	Ginger beer vermicelli
Mould	Soybeans	Miso, soy sauce

(Adapted from: Potter, N. and Hotchkiss, J.H. 1995. Food Science (5th ed), Ch. 12. Aspen Publishers., p. 265.)

You will note that several foods or food ingredients, such as dill pickles, olives, sauerkraut, coffee beans, vanilla, meat sausages and dairy products, are produced by fermentation with lactic acid bacteria. Lactic acid bacteria are used alone or in combination with other microorganisms in the production of a variety of foods.

Acetic acid bacteria oxidize ethanol to acetic acid. This phenomenon forms the basis for the production of food grade acetic acid (vinegar). If you have attempted to produce your own alcoholic beverages, you may have had the unfortunate experience of having what you hoped would be a great wine or beer turned to a fruit or barley vinegar by acetic acid bacteria contaminants in your fermentation vessels.
Yeasts are used widely in the production of alcoholic beverages and breads. Yeasts with lactic acid bacteria are used in the production of sourdough breads and pancakes and in the conversion of beans to vermicelli.
Moulds are used in the production of many foods, particularly in Asia, as well as in the production of well known cheeses such as Roquefort, Brie and Camembert.

9.3.4 Microbial Changes in Fermented Foods

Microbial changes in foods that undergo fermentation fall into certain categories as shown in Table 9.2. Conditions in the food to be fermented are controlled to favour growth and desirable metabolic activities of the microorganisms used in the fermentation process. As you can guess, control of conditions for production of fermented foods can be a complicated operation involving many variables.

Table 9.2 Microbial changes in foods undergoing fermentation

Raw material	Agent	Product
Lactose, glucose	Lactic acid bacteria	Lactic acid
Glucose, other fermentable carbohydrates	Yeasts	Ethanol
Ethanol	Acetic acid bacteria	Acetic acid
Lipids	Microbial lipases	Free fatty acids, aldehydes, ketones
Proteins	Microbial proteinases	Polypeptides, peptides, free amino acids, amines (cause textural and flavour changes in food)
Cellulose	Mould cellulases	Oligosaccharides, glucose

9.3.5 Factors Affecting Fermentation in Foods

Factors that are controlled in the production of fermented foods include: Starter cultures, Formation of Metabolites ( e.g Acids, Alcohol), Temperature, Oxygen and Salt.

Starter cultures

Modern food fermentation (biotechnology) employs the use of microbial cultures specifically selected and maintained for desirable trait(s): e.g.acid production; alcohol production; production of flavour compounds; production of specific enzymes; rate of growth. In current food fermentation practices, the starter cultures are grown under specific conditions and the harvested cells are added in a specific proportion to the food to be fermented. Cultures may be grown within the food processing plant or may be purchased as frozen or dehydrated cultures.

You may also have used starter cultures in your home:

If you have baked bread in your home, you may have used dehydrated yeast as your starter culture for the bread dough. The food industry uses dehydrated starter cultures in a similar manner, but on a much larger scale.
If you maintain a sourdough culture in your refrigerator, you use some of the techniques employed in industry to maintain the vigour of the culture, although in industry the numbers and types of organisms in the starter cultures are constantly monitored.
Similarly, if you have made yogurt in your home, you have probably used part of a previous batch of yogurt or a commercially produced yogurt as the source of the starter culture.

Formation of Metabolites

Metabolites are products resulted from metabolism by the organism. In fermentation, the desired microorganisms or more specifically the starter culture is grown to produce the desired metabolites. These compounds vary depending on the type and condition of growth of the microorganisms. The most common metabolites are acids and alcohols.

Acids

Foods can be preserved by the addition of acids to lower the pH or by the encouragement of growth of acid-producing microorganisms used in food fermentations. Milk and meat are treated in such a manner that the lactic acid-producing bacteria, added as a starter culture, will grow rapidly and produce lactic acid in sufficient quantities to suppress growth and metabolism of spoilage and disease-causing microorganisms. Cheese, yogurt, and fermented sausages must be stored under refrigeration with or without vacuum packaging to delay growth of the acid-tolerant psychrotrophic yeasts and moulds.

Alcohol

Alcohol, like acid, in sufficient concentrations functions as a preservative. Alcohol in wines and beer is not of sufficient concentration to inhibit growth of ethanol-oxidizing bacteria. Wines and beer must be further processed by pasteurization or filtration through membranes with pore diameters smaller than the spoilage-causing microorganisms. Fortified wines with alcohol contents above 20% do not require further preservation treatments.

Temperature

Temperature can be a very important factor in controlling the type of microorganism that grows during food fermentation. For example, temperature is a key factor in ensuring the sequential development of the desirable lactic acid-producing microflora in shredded cabbage during the fermentation of cabbage to sauerkraut. The development of a typical sauerkraut flavour requires the proper succession of lactic acid bacteria starting with Leuconostoc meserenteroides (which requires cool temperatures of about 21°C) and followed in sequence by Lactobacillus cucumeris (32°C) and Lactobacillus pentoaceticus (37°C). If you have made sauerkraut in your home, you have created conditions favourable to growth of these bacteria which are part of the normal microflora of cabbages. You will also have noted that sauerkraut has a much longer storage life than the cabbages from which it was produced but it will eventually become spoiled by acid-tolerant yeasts and moulds, particularly if good sanitary habits are not followed and storage temperature of the finished sauerkraut is too high. This is one of the reasons why sauerkraut is bottled and pasteurized to further extend its storage life.

Oxygen

Oxygen may be desirable or undesirable in fermentation processes. Microorganisms used in fermentation processes have different oxygen requirements for growth and fermentation activity. An example is baker's yeast (Saccharomyces cerevisae). This yeast will grow better under aerobic conditions; however, the yeast ferments sugars more rapidly under anaerobic conditions. Therefore, oxygen requirement conditions may differ and may change during the different steps of the fermentation process.

Salt

Adding salt to cabbage, olives and some meats favours the growth of lactic acid-producing bacteria while inhibiting the growth of normal spoilage- and disease-causing microorganisms. Salt also tends to draw moisture and the water-soluble nutrients from tissues making them available for use by the fermentative microorganisms. This important function is one of the purposes of using salt in sauerkraut, olive and pickle production. Salt also acts as a means of controlling the growth of undesirable microorganisms that may not be inhibited by the acid produced during the fermentation process. The acid and salt together produce a food system more inhibitory to disease- and spoilage-causing microorganisms than the salt or acid alone at the same concentrations.

How does a starter culture work?

Starter cultures are used in the production of fermented dairy products, meats, wines, beer and other alcoholic beverages. The starter culture, upon addition to the food to be fermented, begins to grow rapidly under the favorable conditions provided by the food processor and to produce desirable products of metabolism (acid, alcohol, flavour compounds, enzymes).

Of special concern to commercial users of starter cultures is the potential presence of microbial viruses (phage) in the food that could infect the culture and inactivate it. The fermentation industry constantly evaluates and selects starter cultures for their phage resistance and vigour in producing the desirable end products of metabolism. Likewise, the purity of starter cultures is maintained through sterile handling techniques and strict attention to processing plant sanitation.

Other fermented foods, such as olives, salt stock cucumbers and sauerkraut, are produced by creation of conditions favourable to growth of desirable microorganisms which are part of the normal microbial flora of the starting plant materials (cabbage, cucumbers, olives). Environmental conditions such as salt concentration and temperature are strictly controlled to maximize the growth of the desirable microorganisms while suppressing the growth of undesirable microorganisms.

Fermented foods, such as yogurt, contain in excess of one billion lactic acid bacteria per gram of product. Many people shudder at the thought of consuming bacteria, but in the cases of "the good" microorganisms, no harmful effects occur to the human body. In fact, there is some evidence that certain microorganisms used in food fermentation may have beneficial effects on the digestive function of the human body (Lesson 13)

9.3.6 The Technology of Fermentation

The production of Cheese will be used as example to demonstrate the technology of fermentation.

Figure 9.1 Scheme of Cheese Making Process

Want to learn more?
Figure 9.1 shows that milk is the starting ingredient in the cheese making process. Note that 10 kg of milk are needed to produce 1 kg of cheese. The proximate composition of cow's milk & Cheddar cheese is compared in Table 9.3. Which of the milk components are recovered in the cheese? Milk is composed of 88% water and approximately 3.3 g protein/100 g milk, with 2.7g being casein, the protein fraction involved in cheese curd formation. The remainder of the protein (0.6g) is whey protein. Milk fat partitions to the curd during cheesemaking. Most cheeses are rich in protein, fat, calcium and phosphorus, but low in lactose content. Individuals who are intolerant to lactose in foods can usually consume cheeses without experiencing gastrointestinal disturbances.

Table 9.3 Proximate composition of Cow's Milk and Cheddar Cheese, in grams per 100 grams edible portion (source: USDA Nutrient Database)

	Milk	Cheddar cheese
Water	88.32	36.75
Fat	3.25	33.14
Protein	3.22	24.90
Lactose	5.26	0.23
Ash (minerals)	0.69	3.93

Setting the milk

The lactic acid-producing culture is added to pasteurized milk at a concentration of 1% (v/v) to ensure that the starter culture is present in much larger numbers than other microorganisms in the pasteurized milk. This is done to ensure that the starter culture becomes the dominant portion of the microbial population in the milk.

Colouring agent is also added at this point. In Canada, annatto and ß-carotene are permitted for use as colourants for Cheddar cheese (see lesson 4: Division 8 and/or Table III of Division 16 in the Food and Drugs Act of Canada) http://laws-lois.justice.gc.ca/eng/regulations/C.R.C.,_c._870/page-160.html#docCont (Links to an external site.)

Once the inoculated milk becomes mildly acidic, rennet or enzyme is added. The combination of acid and rennet causes the caseins to coagulate and form a gel very much like that found in a carton of yogurt. The commercial rennin preparation is known as "rennet", which is obtained from the 4th stomach of the calf and contains rennin and other small amounts of other materials. Rennin (also called chymosin) is a pure enzyme.

The enzyme hydrolyzes a portion of the k-casein from the casein micelle, changing the micelles from a calcium-stable to a calcium-sensitive state that leads to the formation of the coagulum with the aid of the lactic acid produced by the starter culture. The whey is trapped within the three-dimensional network created by the aggregating casein micelles and forming a gel.

Curd cutting

The curd is cut into cubes to promote efficient removal of whey from the curd. During this phase, the lactic acid culture continues to produce lactic acid which also aids in expression of the whey from the casein curd by causing the aggregated micelles to aggregate even further.

Cooking

The cut curds are cooked at 38°C to accelerate lactic acid production and further expulsion of whey from the curd.

Draining whey and curd matting

The curd cubes settle and the whey is drained from the cheese vat. Matting of the curd leads to fusion of the curd pieces to form a rubbery slab. This fusion is promoted by attractive interactions between the casein micelles along the curd edges.

During matting and Cheddaring2, the lactic acid bacteria continue to produce lactic acid which aids in curd fusion and shrinkage, leading to further expulsion of the whey.

2 Cheddaring involves cutting the matted curd into blocks, turning the blocks every 15 minutes, and piling the blocks on one another. This process allows whey to be further squeezed from the curd.

Salting and milling

The matted, Cheddar curd is cut (milled) and salted. The functions of the salt are:

to draw the whey out of the curd;
to flavour the final cheese;
to inhibit growth of proteolytic and lipolytic spoilage-causing microorganisms that may be associated with the newly formed milled curd;
to provide conditions favourable to the proteolytic action of the rennet and of the proteinases of the lactic acid starter during ripening of the cheese.

Pressing

The curd is pressed and hooped before curing. During curing, the curds knit together such that the curd junctions cannot be seen in a good quality Cheddar cheese. If you have some Cheddar cheese in your refrigerator, cut a slice and see if you can see curd junctions in the slice. Gently bend the slice as you look at it.

If the curd has not completely knit together, the cheese will fracture along the junction lines. Cheese in which the junctions are clearly visible tends to have a crumbly rather than a smooth texture. During ripening, proteinases of the lactic acid bacteria and the rennet continue to hydrolyze the casein to produce peptides and free amino acids that contribute to the typical Cheddar cheese flavour.

Curing and ripening

Complex changes occur within the ripening of cheese that lead to desirable flavours and textures of the cheese.

After the pressing step, the cheese is placed in a cool room for 3-4 days. In order to prevent mould from growing on the surface of the cheese, the cheese is vacuum packed in flexible film or dipped in hot paraffin.

Curing and ripening takes place at 2°C and 85% RH. The ripening stage is continued for at least 60 days. However, as you will note with different kinds of Cheddar cheese, ripening may be continued for 12 months or more if peak flavour and aroma are desired.

Want to learn more?
Using the website below, find out if cheese produced from "raw milk" (referring to "unpasteurized" milk, and including heat-treated but not pasteurized milk) allowed for sale in Canada? http://healthycanadians.gc.ca/eating-nutrition/healthy-eating-saine-alimentation/safety-salubrite/milk-lait/raw-milk-lait-cru-eng.php?_ga=1.16692222.997077515.1449084569 Can you think of another function of the flexible film or dipped hot paraffin on the cheese? After reading about the different steps involved in cheese making, you are now ready to watch the video on Dairy processing: Cheese production. This will also help you to think about the answers to the following questions. What are the main consequences of cheese making that result in preservation, i.e. longer shelf life of cheese, compared to the starting milk? (hint: you should be able to identify 4 main factors that provide the basis of preservation of cheese) What are some of the variables during cheese making that can influence the shelf-life as well as sensory properties of cheese?

9.3.7 Descriptions of Some Common Cheeses

Cheeses are classified as soft, semisoft, hard and very hard, process cheese, and whey cheese. Variations in the cheese making process result in different cheeses, in terms of flavour, texture, appearance, and shelf-life. In addition to the lactic acid cultures, a secondary culture may be used to develop characteristic properties of some of these cheeses. Swiss, blue-veined and Camembert processes are described below.

Optional: If you are interested in more details of cheese making, follow this Link (Links to an external site.) to the ebook by Professor Arthur Hill at the University of Guelph.

Swiss cheese

The production of Swiss cheese (characterized as a hard-type cheese) begins much like that of cheddar cheese except that an extra ("secondary") bacterial culture, Propionibacterium shermanii, is added to the milk along with the lactic acid starter bacteria.

Note that multiple starters are used for production of Swiss cheese and that one of the starters, Streptococcus thermophilus, is heat-tolerant so that acid can still be produced at the higher cooking temperatures employed during production of Swiss cheese. Also note that another bacterium, Propionibacterium shermanii, is used as part of the starter culture since this organism produces propionic acid and carbon dioxide from lactic acid. The P. shermanii also produce the amino acid proline which imparts the sweet taste characteristic of Swiss cheese. The starter cultures are very important in the production of a high quality Swiss cheese.

Swiss Cheese: Note the characteristic eyes

The propionic acid contributes to the characteristic flavour of Swiss cheese while the carbon dioxide forms the holes (eyes) in the cheese during aging

Want to learn more?
The next time you are in the grocery store purchase a package of Swiss cheese and examine it for the presence of the eyes in the cheese. Look at the cheese closely to evaluate the shape and size of the eyes, and experience the odour and flavour of the cheese by putting some in your mouth. The starter cultures are very important in the production of a high quality Swiss cheese.

Blue-veined cheese

The blue-veined cheeses are characterized as semi-soft cheeses.

There are 4 varieties of blue-veined cheeses; three that are made from cow's milk:

Blue cheese (Denmark, U.S.)
Stilton (England)
Gorgonzola (Italy)
And one (perhaps the most famous) made from ewes' (sheep's) milk: Roquefort (Roquefort region of France)

The production procedure for the blue veined cheeses is similar to that of Cheddar cheese except that the curd is inoculated with a mould, Penicillium roquefortii, which grows within the hooped curd producing the characteristic flavour and colour of the blue veined cheeses. After it is hooped and pressed, the cheese curd is pierced in order to provide channels for the oxygen, required for mould growth, to enter the cheese.

Blue Cheese

Blue Cheese: Note sites of inoculation

The blue colour is due to the mould spores that are formed during growth of the mould along the lines where the cheese curd was pierced. Penicillium roquefortii is an active producer of the enzyme lipase which breaks down the milk fat into free fatty acids, aldehydes and ketones that contribute to the sharp, distinctive flavour characteristic of the blue veined cheeses. You may remember from earlier lessons that in most food preservation situations, great efforts are made to impede the action of lipolytic enzymes since lipolysis is generally considered to be a sign of spoilage. In the blue veined cheeses, however, lipolysis of the milk fat is a necessary part of proper flavour development. Part of the flavour of the blue cheeses is a musty, somewhat mouldy flavour that is contributed by the mould mycelia.

Want to learn more?
If you are a regular consumer of blue cheeses, slowly eat a piece of the cheese and savour its flavour. Is the flavour more intense in regions of the cheese where the mould growth was heaviest? Note the presence of the holes on the outer edge of the cheese where the curd was pierced to provide oxygen for the mould. If you have never tried a blue veined cheese, purchase a very small piece to evaluate the texture, odour and flavour of the cheese. You will note that part of the flavour of the blue cheeses is a musty, somewhat mouldy flavour that is contributed by the mould mycelia.

Camembert cheese

You may be a regular consumer of mould-ripened cheese such as Camembert and Brie (soft-type cheeses) or, if you are not, you have undoubtedly seen these types in the cheese counters at the local delicatessen. The curd is produced through the use of a lactic fermentation much like that used for Cheddar cheese. In this case the surface of the pressed cheese curd is also inoculated with spores of Penicillium camembertii (or Penicillium candidum*). Vigorous growth of the mould leads to the formation of a layer of mycelia that form the white, velvet-like coating on the outer layer of the cheese. The mould is highly proteolytic and the mould proteinases diffuse into the cheese curd, hydrolyzing the casein into long-chain peptides which do not contribute much flavour to the cheese.

Camambert cheese: The cheese surface is inoculated with mould spores. A layer of mycelia forms the white, velvet-like coating on the surface of the cheese

The hydrolysis of the casein leads to the formation of the creamy texture characteristic of a good quality Camembert cheese. If the Penicillium culture is too proteolytic or if curing takes place too long, bitter tasting short-chain peptides, free amino acids and ammonia are formed. A good quality Camembert cheese has a mild flavour, is not bitter, and has a characteristic creamy texture. With Camembert cheese, the mould should not contribute to a mouldy flavour to the cheese; a mouldy flavour is considered a quality defect.

*For the past few years in Normandy, France, the mould Penicillium candidum has been commonly used instead of P. camembertii. The reason for replacing the 'traditional' mould was that P. camembertii was sometimes responsible for causing 'blue moisture' on the cheese.

Want to learn more?
For a visual tour of how Camembert cheese is made in Normandy, please visit the Camembert website. If you have never eaten Camembert cheese, you should taste a small piece. Note the feel of the outer layer in your hands and in your mouth and evaluate the texture and flavour of the interior of the cheese in your mouth. With Camembert cheese, the mould should not contribute a mould flavour to the cheese; a mouldy flavour is considered a quality defect