Module 9. Microbial process strategies

Lesson 31

31.1 Introduction

Traditional fermented food is not only the staple food for most of developing countries but also the key healthy food for developed countries. As the healthy function of these foods are gradually discovered, more and more high throughput biotechnologies are being used to promote the old and new industry. As a result, the microflora, manufacturing processes and product healthy function of these foods were pushed forward. The application and progress of the high throughput biotechnologies into traditional fermented food industries like fermented milk products (yogurt, cheese), fermented sausages, fermented vegetables (kimchi, sauerkraut), fermented cereals (sourdough) and fermented beans (tempeh, natto) are gaining momentum. Given the further promotion by high throughput biotechnologies, the middle and/or down-stream process of traditional fermented foods would be optimized and the process of industrialization of local traditional fermented food having many functional factors but in small quantity would be accelerated. Fermented foods may be defined as foods which are processed through the activities of microorganisms but in which the weight of the microorganisms in the food is usually small. The influence of microbial activity on the nature of the food, especially in terms of flavor and other organoleptic properties is profound. A few fermented foods of economic and nutritional importance are listed in Table 31.1.

Table 31.1 Some fermented foods of economic and nutritional importance


31.2 Fermented Vegetables

Like the fermentation of other foods, vegetables have been preserved by fermentation from time immemorial by lactic bacterial action. A wide range of vegetables and fruits including cabbages, olives, cucumber, onions, peppers, green tomatoes, carrots, okra, celery, and cauliflower have been preserved. Only sauerkraut and cucumbers will be discussed, as the same general principles apply to the fermentation of all vegetables and fruits. In general they are fermented in brine, which eliminates other organisms and encourages the lactic acid bacteria.

31.2.1 Sauerkraut

Sauerkraut is produced by the fermentation of cabbages, Brassica oleracea, and has been known for a long time. Specially selected varieties which are mild-flavored are used. The cabbage is sliced into thin pieces known as slaw and preserved in salt water or brine containing about 2.5% salt. The slaw must be completely immersed in brine to prevent it from darkening. Kraut fermentation is initiated by Leuconostoc mesenteroides, a heterofermentative lactic acid bacterium (i.e., it produces lactic acid as well as acetic acid and CO2). It grows over a wide range of pH and temperature conditions. CO2 creates anaerobic conditions and eliminates organisms which might produce enzymes which can cause the softening of the slaw and also encourages the growth of other lactic acid bacteria. Gram negative coliforms and pseudomonads soon disappear, and give way to a rapid proliferation of other lactic acid bacteria, including Lactobacillus brevis, which is heterofermentative, and the homofermentative Lactobacillus plantarum; sometimes Pediococcus cerevisiae also occurs. Compounds which contribute to the flavor of sauerkraut begin to appear with the increasing growth of the lactics. These compounds include lactic and acetic acids, ethanol, and volatile compounds such as diacetyl, acetaldehyde, acetal, isoamyl alcohol, n-hexanol, ethyl lactate, ethyl butarate, and isoamyl acetate. Besides the 2.5% salt, it is important that a temperature of about 15°C be used. Higher temperatures cause a deterioration of the kraut.

31.2.2 Cucumbers (pickling)

Cucumber (Cucumis sativus) is eaten raw as well after fermentation or pickling. Cucumbers for pickling are best harvested before they are mature. Mature cucumbers are too large, ripen easily and are full of mature seeds. Cucumbers may be pickled by dry salting or by brine salting .

Dry salting is also generally used for cauliflower, peppers, okra, and carrots. It consists of adding 10 to 12% salt to the water before the cucumbers are placed in the tank. This prevents bruising or other damage to the vegetables.

Brine salting is more widely used. A lower amount of salt is added, between 5 and 8% salt being used. Higher amounts were previously used to prevent spoilage. During the primary fermentation lasting two or three days, most of the unwanted bacteria disappear allowing the lactics and yeasts to proliferate. In the final stages, after 10 to 14 days, Lactobacillus plantarum and L. brevis, followed by Pediococcus, are the major organisms.

31.3 Fermented Foods From Cereals and Beans

31.3.1 Idli

Idli is a popular fermented breakfast and hospital food which has been eaten in South India for many years. It is prepared from rice grains and the seeds of the leguminous mung grain, Phaeseolus mungo, or from black gram (udad dhal), Vigna mungo, which are also known as dal. When the material contains Bengal grain, Circer orientium, the product is known as khaman. It has a spongy texture and a pleasant sour taste due to the lactic acid in the food. It is often embellished with flavoring ingredients such as cashew nuts, pepper and ginger. Production of idli

The seeds of the dahl (black gram) are soaked in water for 1-3 hours to soften them and to facilitate decortication, after which the seeds are mixed and pounded with rice in a proportion of three parts of the beans and one of rice (Figure 31.1). The mixture is allowed to ferment overnight (20-22 hours). In the traditional system the fermentation is spontaneous and the mixture is leavened up to approximately 2 or 3 times. The organisms involved in the acidification have been identified as Streptococcus faecalis, and Pediococcus spp. The leavening is brought about by Leuconostoc mesenteroides, although the yeasts, Torulopsis candida and Trichosporon pulluloma have also been found in traditional Idli. The fermented batter is steamed and served hot. Idli is highly nutritious, being rich in nicotinic acid, thiamine, riboflavin, and methionine.


Fig. 31.1 Flow diagram for production of idli (Okofer et al., 2007)

31.3.2 Production of beer

Barley beers can be divided into two broad groups: top-fermented beers and bottom fermented beers. This distinction is based on whether the yeast remains at the top of brew (top-fermented beers) or sediments to the bottom (bottom-fermented beers) at the end of the fermentation. Raw materials for brewing

The raw materials used in brewing are: barley, malt, adjuncts, yeasts, hops, and water.

a) Brewer’s yeasts

Yeasts in general will produce alcohol from sugars under anaerobic conditions, but not all yeasts are necessarily suitable for brewing. Brewing yeasts besides producing alcohol, are able to produce from wort sugars and proteins in a balanced proportion of esters, acids, higher alcohols, and ketones which contribute to the peculiar flavor of beer.

b) Brewery Processes

The processes involved in the conversion of barley malt to beer may be divided into the following:

1. Malting

2. Cleaning and milling of the malt

3. Mashing

4. Mash operation

5. Wort boiling treatment

6. Fermentation

7. Storage or lagering

8. Packaging Malting

The purpose of malting is to develop amylases and proteases in the grain. These enzymes are produced by the germinated barley to enable it to break down the carbohydrates and proteins in the grain to nourish the germinated seedling before its photosynthetic systems are developed enough to support the plant.

a. Cleaning and milling of malt

The purpose of milling is to expose particles of the malt to the hydrolytic effects of malt enzymes during the mashing process. The finer the particles, greater the extract from the malt.

b. Mashing

Mashing is the central part of brewing. It determines the nature of the wort, hence the nature of the nutrients available to the yeasts and therefore the type of beer produced. The purpose of mashing is to extract as much as possible the soluble portion of the malt and to enzymatically hydrolyze insoluble portions of the malt and adjuncts. The aqueous solution resulting from mashing is known as wort. The wort is boiled for 1-1½ hours in a stainless steel kettle. When corn syrup or sucrose is used as an adjunct it is added at the beginning of the boiling. Hops are also added, some before and some at the end of the boiling. Hops are the dried cone-shaped female flower of hop-plant Humulus lupulus. The importance of hops in brewing lies in its resins which provide the precursors of the bitter principles in beer and the essential (volatile) oils which provide the hop aroma.

The purpose of boiling is as follows.

(a) To concentrate the wort,

(b) To sterilize the wort

(c) To inactivate any enzymes

(d) To extract soluble materials from the hops

(e) To precipitate protein, which forms large flocs because of heat denaturation and complexing with tannins extracted from the hops and malt husks. Unprecipitated proteins form hazes in the beer, but too little protein leads to poor foam head formation.

(f) To develop color in the beer; some of the color in beer comes from malting but the bulk develops during wort boiling. Color is formed by several chemical reactions including caramelization of sugars, oxidation of phenolic compounds, and reactions between amino acids and reducing sugars.

(g) Removal of volatile compounds: volatile compounds such as fatty acids which could lead to rancidity in the beer are removed.

c. Fermentation

The cooled wort is pumped or allowed to flow by gravity into fermentation tanks and yeast is inoculated or ‘pitched in’ at a rate of 7-15 x 106 yeast cells/ml, usually collected from a previous brew. The progress of fermentation is followed by wort specific gravity. During fermentation the gravity of the wort gradually decreases because yeasts are using up the extract. However, alcohol is also being formed. As alcohol has a lower gravity than wort the reading of the special hydrometer (known as a saccharometer) is even lower. °Brix is used in the sugar industry, whereas Balling (United States) and °Plato (continental Europe) are used in the brewing industry.

d. Lagering

During lagering secondary fermentation occurs. Yeasts are sometimes added to induce this secondary fermentation, utilizing some sugars in the green beer. The secondary fermentation saturates the beer with CO2. Packaging

The beer is transferred to pressure tanks from where it is distributed to cans, bottles and other containers. The beer is not allowed to come in contact with oxygen during this operation; it is also not allowed to lose CO2 or to become contaminated with microorganisms. To achieve these objectives, the beer is added to the tanks under a CO2, atmosphere, bottled under a counter pressure of CO2 and all the equipment is cleaned and disinfected regularly. Beer defects

The most important beer defect is the presence of haze or turbidity, which can be of biological or physico-chemical origin. Biological turbidities are caused by spoilage organisms and arise because of poor brewery hygiene (i.e. poorly washed pipes) and poor pasteurization. Spoilage organisms in beer must be able to survive the following stringent conditions found in beer: low pH, the antiseptic substances in hops, pasteurization of beer, and anaerobic conditions.

Yeasts and certain bacteria are responsible for biological spoilage because they can withstand these. Wild or unwanted yeasts which have been identified in beer spoilage are spread into many genera including Kloeckera, Hansenula, and Brettanomyces, but Saccharomyces spp appear to be commonest, particularly in top-fermented beers. These include Sacch. cerevisiae var. turblidans, and Sacch. diastaticus. The latter is important because of its ability to grow on dextrins in beer, thereby causing hazes and off flavors. Among the bacteria, Acetobacter, and the lactic acid bacteria, Lactobacillus and Streptococcus are the most important. The latter are tolerant of low pH and hop antiseptics and are microaerophilic hence they grow well in beer. Acetobacter is an acetic acid bacterium and produces acetic acid from alcohol thereby giving rise to sourness in beer. Lactobacillus pastorianus is the typical beer spoiling lactobacilli, in top-fermented beers, where it produces sourness and a silky type of turbidity. Streptococcus damnosus (Pediococcus damnosus, Pediococcus cerevisiae) is known as ‘beer sarcina’ and gives rise to ‘sarcina sickness’ of beer which is characterized by a honey-like odor.

31.3.3 Wines

Wine is by common usage defined as a product of the “normal alcoholic fermentation of the juice of sound ripe grapes”. Nevertheless any fruit with a good proportion of sugar may be used for wine production. If the term is not qualified then it is regarded as being derived from grapes, Vitis vinifera. The production of wine is simpler than that of beer in that no need exists for malting since sugars are already present in the fruit juice being used. This however exposes wine making to greater contamination hazards Processes in wine making

a. Crushing of grapes

Selected ripe grapes are crushed to release the juice which is known as ‘must’, after the stalks which support the fruits have been removed. These stalks contain tannins which would give the wine a harsh taste if left in the must. The skin contains most of the materials which give wine its aroma and color. Grape juice has an acidity of 0.60-0.65% and a pH of 3.0-4.0 due mainly to malic and tartaric acids with a little citric acid.

b. Fermentation

(i) Yeast used: The grapes themselves harbor a natural flora of microorganisms (the bloom) which in previous times brought about the fermentation and contributed to the special characters of various wines. Yeasts are then inoculated into the must. The yeast which is used is Saccaromyces cerevisiae var, ellipsoideus (synonyms: Sacch. cerevisiae, Sacch. ellipsoideus, Sacch, vini.).

Wine yeasts have the following characteristics: (a) growth at the relatively high acidity (i.e., low pH) of grape juice; (b) resistance to high alcohol content (higher than 10%); (c) resistance to sulfite.

c. Control of fermentation

(a) Temperature: Heat is released during the fermentations. It has been calculated the temperature of a must containing 22% sugar would rise 52°F (11°C) if all the heat were stopped from escaping. If the initial temperature were 60°F (16°C) the temperature would be 100°F (38°C) and fermentation would halt while only 5% alcohol has been accumulated. For this reason the fermentation is cooled and the temperature is maintained at around 24°C with cooling coils mounted in the fermentor.

(b) Yeast nutrition: Yeasts normally ferment the glucose preferentially although some yeasts e.g. Sacch. elegans prefer fructose. Most nutrients including macro- and micro-nutrients are usually abundant in must; occasionally, however, nitrogeneous compounds are limiting. They are then made adequate with small amounts of (NH4)2 SO4 .

(c) Oxygen: As with beer, oxygen is required in the earlier stage of fermentation when yeast multiplication is occurring. In the second stage when alcohol is produced the growth is anaerobic and this forces the yeasts to utilize such intermediate products as acetaldehydes as hydrogen acceptors and hence encourage alcohol production.

(d) Flavor development: Although some flavor materials come from the grape most of it come from yeast action and has been shown to be due to alcohols, esters, fatty acids, and carbonyl compounds, the esters being the most important. Diacetyl, acetoin, fuel oils, volatile esters, and hydrogen sulfide have received special attention.

d. Ageing and Storage

The fermentation is usually over in three to five days. At this time ‘pomace’ formed from grape skins (in red wines) will have risen to the top of the brew. At the end of this fermentation the wine is allowed to flow through a perforated bottom if pomace had been allowed. When the pomace has been separated from wine and the fermentation is complete or stopped, the next stage is ‘racking’. The wine is allowed to stand until a major portion of the yeast cells and other fine suspended materials have collected at the bottom of the container as sediment or ‘lees’. It is then ‘racked’, during which process the clear wine is carefully pumped. The wine is then transferred to wooden casks (100-1,000 gallons), barrels (about 50 gallons) or tanks (several thousand gallons). The wood allows the wine only slow access to oxygen. Water and ethanol evaporate slowly leading to air pockets which permit the growth of aerobic wine spoilers e.g. acetic acid bacteria and some yeasts. The casks are, therefore regularly topped up to prevent the pockets. In modern tanks made of stainless steel the problem of air pockets is tackled by filling the airspace with an inert gas such as carbon dioxide or nitrogen. During ageing desirable changes occur in the wine. These changes are due to a number of factors:

e. Clarification

The wine is allowed to age in a period ranging from two years to five years, depending on the type of wine. At the end of the period some will have cleared naturally. For others artificial clarification may be necessary. The addition of a fining agent is often practiced to help clarification. Fining agents react with the tannin, acid, protein or with some added substance to give heavy quick-settling coagulums. The usual fining agents for wine are gelatin, casein, tannin, egg albumin, and bentonite.

f. Packaging

Before packing in bottles the wine from various sources is sometimes blended and then pasteurized. In some wineries, the wine is not pasteurized, rather it is sterilized by filtration. In many countries the wine is packaged and distributed in casks.

g. Wine defects

The most important cause of wine spoilage is microbial; less important defects are acidity and cloudiness. Factors which influence spoilage by bacteria and yeasts include the following (a) wine composition, specifically the sugar, alcohol, and sulfur dioxide content; (b) storage conditions e.g. high temperature and the amount of air space in the container; (c) the extent of the initial contamination by microorganism during the bottling process. When proper hygiene is practiced bacterial spoilage is rare. When it does occur the microorganisms concerned are acetic acid bacteria which cause sourness in the wine. Lactic acid bacteria especially Leuconostoc, and sometimes Lactobacillus also spoil wines. Various spoilage yeasts may also grow in wine. The most prevalent is Brettanomyces, slow growing yeasts which grow in wine causing turbidities and off-flavors. Other wine spoilage yeasts are Saccharomyces oviformis, which may use up residual sugars in a sweet wine and Saccharomyces bayanus which may cause turbidity and sedimentation in dry wines with some residual sugar. Pichia membanaefaciens is an aerobic yeast which grows especially in young wines with sufficient oxygen. Other defects of wine include cloudiness and acidity.

Last modified: Saturday, 3 November 2012, 10:50 AM